Oncology/Hematology
Erythrocyte Rheology Takeshi Shiga, Nobuji Maeda, and Kazunori Kon I. INTRODUCTION
Theflowproperties of erythrocytes are important in blood circulation, since the erythrocytes occupy nearly half of blood volume. According to the Hagen-Poiseuille law, the flow volume of liquid in a rigid tube is expressed as follows: (Flow) fXJ(Viscosity)-‘*(Pressure difference).(Radius)4*(Length)-’ Therefore, the blood viscosity causes the resistance to flow in blood vessels. The blood viscosity increases with an augmentation of the hematocrit (volume fraction of erythrocytes in blood). Furthermore, as is well known, blood is a non-Newtonian fluid, that is, at a high shear rate blood viscosity decreases due to the deformation of erythrocytes, but at a low shear rate it increases due to the aggregation of erythrocytes. Such macroscopic viscosity is important for the blood flow in large vessels, but it may not be a direct measure for the blood flow in small vessels, because of F&raeus-Lindqvist effect (decrease of the blood viscosity due to the axial streaming of erythrocytes and the formation of circumferential plasma layer). The oxygen delivery in the microcirculation depends on the flow properties of erythrocytes and the oxygen binding of intracellular hemoglobin. Here again, cellular deformation is important, since the erythrocytes must deform to pass through vessel sections narrower than themselves. When the flow rate decreases, the erythrocytes tend to form aggregates depending on the plasma composition. Therefore, erythrocyte deformability and erythrocyte aggregation, as well as capillary hematocrit, are the main determinants of capillary passage and oxygen supply to tissues. The mechanical properties of erythrocytes are greatly influenced by the membrane, especially by its cytoskeletal structure, by hemoglobin concentration and by cell shape. The growth of erythrocyte aggregates depends mainly on interaction between the membrane surface and some plasma proteins. Therefore, the rheological characteristics of erythrocytes (and blood) must be understood on the basis of the biochemical structure and its regulation of the entire erythrocyte. The unique feature of mammalian erythrocytes is the lack of protein synthesis, thus the functional proteins in erythrocytes are gradually denaturing during their lifespan. After a long trip in circulation, the aged erythrocytes are removed from circulation by the reticuloendothelial system. The aging process greatly affects the properties of individual erythrocytes, that is, the cellular deformability decreases due to biochemical deterioration and morphological alteration. Although many excellent reviews’-‘3 on these subjects have
appeared recently, we would like to focus our attention on the biochemical mechanism of erythrocyte rheology. To begin with, the biochemical structure of erythrocytes (Section II); the process of deformation (Section III) and the kinetics of aggregation (Section IV) are discussed extensively (especially the method of analysis and the mechanisms of the phenomena); and, finally, the alteration of rheological properties with erythrocyte aging is summarized (Section V).
II. BIOCHEMICAL ERYTHROCYTES
STRUCTURE
OF
A schematic model of an erythrocyte membrane is shown in Figure 1. In the plane of the phospholipid bilayer containing cholesterol, various membrane proteins are laterally drifting, and oligo- or polysaccharides are attached on the surface of certain proteins. Underneath the bilayer plane the network of cytoskeletal proteins are extended in two dimensions. Biochemical and biophysical studies of erythrocyte membrane have been carried out extensively, thus the structure of erythrocyte membrane is one of the well-known biomembranes.5~6*‘4-2’ The cytoplasm of erythrocytes is rich in proteins, mainly hemoglobin, which carries oxygen, and the water content is about 70%. In this section, we describe its structure and interactions within the cellular constituents briefly, especially in connection with the rheology and/or the mechanical properties of human erythrocytes. A. Chemical Constituents and Structure of the Membrane The mechanical properties of erythrocyte membrane are mainly determined by the cytoskeletal network, but the roles of the phospholipid bilayer cannot be ignored. 7. Lipid Bilayer The constituents of phospholipids in the erythrocyte membrane vary with the animal species. The phospholipid composition of human erythrocytes depends partly on nutritional
T. Shiga received a M.D. degree and a Dr. Med. Sci. degree from the Osaka University School of Medicine in Osaka, Japan. Dr. Shiga is currently a Professor in the Department of Physiology in the School of Medicine at Osaka University in Osaka, Japan. N. Maeda received a M.D. degree and a Dr. Med. Sci. degree from Nara Medical College in Kashihara, Nara, Japan. Dr. Maeda is currently a Professor in the Department of Physiology in the School of Medicine at Ehime University in Ehime, Japan. K. Kon received a B.Sci. degree from the Science University of Tokyo in Tokyo, Japan; a M.Sci. degree from the University of Hokkaido in Sapporo, Japan; and a Dr. Med. Sci. degree from the University of Osaka in Osaka, Japan. Dr. Kon is currently an Associate Professor in the Department of Physiology at the Ehime College of Health Science in Ehime, Japan.
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Critical 3eviews In ATP, etc.33 and may be stabilized by membrane proteins.34-36 Therefore, the variation in phospholipid composition modifies the interactions with various constituents inside the erythrocyte membrane, but the resultant change in mechanical properties is not well known. c. ACYL-CHAIN COMPOSlTlON The fluidity of the lipid bilayer greatly depends on the interactions among neighboring acyl-chains of phospholipids. 37-39The physicochemical characteristics of the bilayer may be roughly related to the molar ratio of unsaturated- to saturated-acyl-chains. FIGURE 1. A model of erythrocyte membrane. (Left) The interaction among membrane proteins and their electrophoretic mobilities in polyacrylamide gel containing sodium dodecyl sulfate are shown (see text). Numbers in the membrane model correspond to the electrophoretic band. Abbreviations are as follows: Ad, adducin; Ca, calcium; CaM, calmodulin; GPA, glycophorin A; G3-PD, glucose 3-phosphate dehydrogenase; Hb, hemoglobin. The black belt on F-actin (oligomer of G-actin, band 5) is tropomyosin.
habits. It is known that the transport activity of membrane depends on lipid environment. *OThe mechanical and rheological properties of the lipid bilayer may be closely related to the dynamics of membrane structure,22~23which may be relatively represented by the ease of molecular motion within a membrane( “membrane fluidity’ ‘) . Some important relationships between lipid composition and the fluidity of membrane are summarized below. a. CHOLESTEROUPHOSPHOLlPID
RATION
In the human erythrocyte membrane, the molar ratio of cholesterohphospholipid is about one. Cholesterol shows dual effects on the lipid bilayer. ***it fluidizes the membrane below the transition temperature, but it stiffens the bilayer above this temperahue; futihermote, the transition temperature itself shifts with the cholesterol content. It is known that the activity of membrane proteins is modulated by the cholesterohphospholipid molar ratio. Rogausch” and Rogausch and Distleti5 have shown the increased blood viscosity of cholesterol-fed rabbits, and Coopeti6 and Cooper et al.*’ have discussed the relation between the fluidity and the stability of the erythrocyte membrane (and pathogenesis). However, as shown in a later section (Section III.B.3), the direct effect of cholesterol on erythrocyte rheology is rather smal1,28 contrary to the expectation based on the decreased fluidity induced by cholesterol loading. b. PHOSPHOLIPID COMPOSITION
Some phospholipids (phosphatidyl-choline and sphingomyelin) preferentially locate in the outer half of the lipid bilayer, while some others (phosphatidyl-serine and phosphatidylethanolamine) are rich in the inner half.23*29Divalent cations (such as Ca”) interact with phosphatidyl-serine and phosphatidyl-ethanolamine to form clusters of these phospholipids30,31or to modify the transmembrane distribution of these lipids. 32 The phospholipid distribution is influenced by 10
d. LIPID-PROTEIN INTERACTION
Membrane proteins interact with lipid bilayer in their hydrophobic domain.*5*34~40 The motion of lipids around such proteins is more or less restricted (called “amntlar lipids”); therefore, in the presence of membrane proteins the membrane fluidity is no longer uniform, but the membrane is composed of many microplates or microdomains. The microdomains of the erythrocyte membrane are not precisely recognized, and their relevance to the rheological properties has not been discussed. We have shown that an androstane spin label in glutaraldehyde-tteated erythrocyte membrane is present in different domains, such as fluid domain, less fluid domain, and solid plaque (although this is an extreme ~ase).~*‘~~The temporal and spatial fluctuation of the membrane microdomain structure may be important for erythrocyte functions, thus further studies are needed. 8. PROTECTlON AGAINST OXlDATlVE STRESS In some in vitro studies, the peroxidation or oxidation of the erythrocyte membrane induces hemolysis.44 Not only the unsaturated acyl-chains, but also sulfhydryl groups in proteins are the targets of the oxidative stresses.45.46However, the circulating erythmcytes possess protective enzyme systems against such an oxidative process (see Section II.B.2); thus, in certain diseases of low antioxidative activity massive hemolysis may be induced by the mechanical forces. Furthermore, specific antibody binding to the membrane protein (e.g., band 3) may accelerate the phagocytosis of oxidatively stressed erythrocytes .47 2. Membrane Ptvtehs and Cytoskereta! Structure A wide variety of membrane proteins are recognized in erythrocytes. A routine method for semiquantitative separation is polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS). The proteins in the ghost membrane am separated roughly according to their molecular weight (see Figure 1). a. CLASSIFICATION OF MEMBRANE PROTEINS
According to their location, the membrane proteins are classified into two groups: the integral proteins and the peripheral proteins. The integral proteins penetrate through the
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Oncology/Hematology lipid bilayer and are divided into two classes: (1) the proteins that cross the bilayer with a single segment (i.e., a helix of some 20 amino acids), e.g., glycophorins; (2) the proteins that form a channel with multiple spanning segments and serve as the transport proteins, e.g., band 3 protein (anion channel). These proteins can hardly be removed from bilayer without destroying the bilayer structum (e.g., with nonionic detergents). The peripheral proteins are also divided into two classes: (1) one locates in either the outer or inner half of the lipid bilayer, most of which are receptors and enzymes, e.g., ace@choline receptor, glyceraldehyde-3-phosphate dehydrogenase; (2) the others, which interact with the cytoplasmic half of the bilayer and form cytoskeletal network, and thus called ‘ ‘cytoskeletal proteins’ ’ . b. CYTOSKELETAL STRUCTURE The cytoskeletal network underneath the lipid bilayer is responsible for the mechanical properties of the cells and the maintenance of cell shape. Several major proteins are involved in constructing the network.5~6,‘4.21.48-5’ A proposed scheme of the erythrocyte cytoskeleton is shown in Figure 1. The individual characteristics of the membrane proteins are as follows.’ The main component of the cytoskeleton is spectrin heterodimer (0~and l3) of about 100 nm in length.52 The heterodimers associate at one end (head) to form a tetramer. The other end (tail) binds to a sort of knot, composed of a short actin filament (consisting of 12 to 15 Gactins,53.54with tropomyosins” and band 4.9 proteins56) and band 4.1 proteins5’ (which are phosphatidyl-serine binding protein 58s9).One knot ties several spectrin bundles. Besides the direct interaction between spectrin and lipid bilayer, the linking between the cytoskeletal network and lipid bilayer (involving integral proteins) is made as follows: (1) the band 4.1 protein binds to glycophorin A and/or C, which spans the lipid and is surrounded by triphosphoinositides;62 (2) bilayet”9*60p6’ the ankyrin (acylated with fatty acid, which turns over)63-66ties the band 3 protein (anion transporter) with spectrin (at about 20 nm from the center of the spectrin tetramer;63 (3) the band 4.1 protein may also bind with band 3 protem6’ at low levels of triphosphoinositides. The assembling process of the spectrin network, particularly with avian erythrocytes during erythropoiesis, has been studied in detail by Lazarides.51 It is still unclear how the biconcave disk shape is maintained by the cytoskeletal network. The deficiency or abnormality of some cytoskeletal proteins leads to abnormal shape and/or increased fragility, such as elliptocytosis, spherocytosis, These days, the cDNA structures or the amino acid etc. 19~so*68-70 sequences of some proteins are known, thus the relationship between the abnormal loci and the erythrocyte abnormality can be revealed in the near future.
3. Carbohydrates On the outer surface of the erythrocyte membrane, some carbohydrates are attached to the membrane proteins. The sac-
charide chain bound to band 3 protein serves as ABO-type antigens. Saccharides are also attached to glycophorins. The sialic acid in these chains, together with carboxyl groups of acidic amino acids in membrane proteins located on the outer surface, gives tbe surface negative charges of the membrane. The electrostatic repulsion between negatively charged surfaces is an important character to prevent the erythrocyte aggregation. When sialic acids are removed with neuraminidase, the electrophoretic mobility (toward cathode) decreases proportionally to the number of sialic acid” (see Section 1V.C. 1.d). B. Intracellular Constituents Human erythrocytes have no intracellular structure, such as nucleus, mitochondria, etc. Therefore, the content of the cell interior is often regarded as a homogeneous protein-rich solution, mostly hemoglobin. Besides hemoglobin, the erythrocyte contains various enzymes and other proteins. Reticulocytes, present in about 1% in the circulating blood, are direct precursors of mature erythrocytes and contain various amounts of RNA and ribosome. About 70% (by weight) of an erythrocyte is water.72.73 Assuming the average volume of erythrocytes is some 90 pm3 (corresponding to a sphere of 2.8 p,rn radius of which the surface area is about 100 pm2). The average surface area of erythrocytes (140 pm2)74 is about 1.4 times greater than the calculated value for a sphere of the same volume (for cell shape, see Figure 20). Therefore, the biconcave disk-shaped erythrocytes can take up water without hemolysis, and the cell volume may be increased by 1.5 to 1.8 times in hypotonic environment without destruction (the outflow of interior contents due to the destruction or rupture of the membrane is “hemolysis”). Furthermore, such flatness (diameter of 7 to 8 km and thickness of 1 to 2 p,rn) serves to allow extreme deformation by external forces, e.g., the erythrocytes can be folded up. 1. Hemoglobin The main role of hemoglobin is the transport of oxygen by the association-dissociation of oxygen with the Fe(II)-protoporphyrin of a tetrameric hemoglobin molecule. The oxygen supply to tissues is regulated by pH, pCO,, the concentration of 2,3-diphosphoglycerate (2,3-DPG), and temperature. The transport of CO, is also closely linked with the proton movement in hemoglobin molecule, with the aid of carbonic anh ydrase .
Another important property of intracellular hemoglobin is its contribution to internal viscosity, which determines the rheological properties of erythrocytes. The intracellular viscosity may be represented by mean corpuscular hemoglobin concentration (MCHC), which is calculated from the hemoglobin content and the cell volume. The viscosity increases nonlinearly with increasing hemoglobin concentration.75-77 Therefore, at a high intracellular hemoglobin concentration, the deformation of erythrocytes is suppressed and their passage 1990
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Critical Reviews In through the capillaries may be retarded. Further, the increased internal viscosity reduces the convection of hemoglobin molecules and the diffusion of oxygen in erythrocytes. It should be noted that the intracellular hemoglobin concentration differs from one erythrocyte to another, depending on the cell age (see Section V. 3 .e); the intracellular hemoglobin concentration tends to increase with the concomitant decrease of surface area and volume of erythrocytes during aging.78*79 In some hemoglobin diseases, typically sickle cell anemia, the pathologic hemoglobin (e.g., Hb S) polymerizes inside the erythrocytes to form straight rods upon deoxygenation, then the sickling of the cell occurs.8o In other hemoglobinopathies, when the oxidation and denaturation of hemoglobin (and other components) proceed, Heinz bodies are formed and the mechanical properties of erythrocytes are affected.81*82The formation of Heinz bodies occurs in diseases where the antioxidative capacity is decreased by either congenital or postnatal causes. The rheological properties of erythrocytes are greatly impaired in these abnormalities. Hemoglobin molecules interact with the membrane components, such as band 3 protein.83**4The membrane-bound hemoglobin shows an increase in oxygen affinity,*’ and it may affect the convection of intracellular hemoglobin and the mechanical properties of membrane to some extent. 2. Enzymes and Other Proteins Eqthrocytescontain many enzymes. Unlike the other cells, however, enzymes of aerobic glycolysis are not included in human erythrocytes. Glycolysis (of glucose) ends up at the level of pyruvate and/or lactate, and 2 mol of ATP are generated from 1 mol of glucose without the consumption of oxygen. Antioxidative systems are well equipped, for example, the renewal system of reduced glutathione, glutathione peroxidase, superoxide dismutase, and catalase. If one of these systems suffers from dysfunction, (1) the oxidation of hemoglobin8’~82 and membrane proteins,*6-88and (2) the peroxidation of lipids may develop. The activity of enzymes declines gradually with cell aging; thus the activity of certain enzymes is used as a marker of cell age.89 Density gradient centrifugation has been applied for fractionating erythrocytes; high-density fraction contains aged cells, and low-density fraction is rich in young cells and reticulocytes. However, if the enzymatic activities decrease steeply during maturation of reticulocytes as pointed out recently,% the distinction between the aging process of mature erythrocytes and the maturing process of reticulocytes becomes important. A recent study on rabbits does not detect a decline of enzymatic activity of glycolysis.9* Therefore Clark” has written “a lack” of adequate cell age-associated markers, although a counter argument is presented by Piomelli.92 On the other hand, studies of reticulocytes have just become accessible with the use of recombinant erythropoietin; clinical trials show a considerable reticulocytosis upon administration of erythro-
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poietin,93*94as well as animal studies95 (e.g., we observe some 15% reticulocytes with erythropoietin-injected rats). More investigations, including the methods of separation, on the distinction between reticulocyte maturation and erythrocyte aging are expected in the near future. Other than enzymes, the regulator proteins, e.g., calciumbinding proteins (calmodulin96*97and adducin98,99)are present in human erythrocytes. 3. Ions and Water Content The contents of inorganic ions are regulated by various transport proteins in erythrocyte membranes. Since the membrane is permeable to water (though the actual pathway is not well defined at the molecular level), recently the role of band 3 and band 4.5 proteins is noticed,‘W.‘O’ the control of the concentration of various ions is important for the maintenance of cell volume and cell shape, and, consequently, the intracellular viscosity (hemoglobin concentration). The water content in erythrocytes is osmotically regulated, particularly by intracellular K + and extracellular Na’ ; thus the escape of K + induces the loss of water and the increase of intracellular viscosity, then the deformability decreases (see Section IlI.B.2). C. Cell Shape and Biochemical Structure The shape of normal erythrocytes is flat and biconcave disk-like. The biochemical basis to maintain such a shape is not yet fully understood, but, instead, the causes of shape alteration have been studied extensively. The shape change of normal erythrocytes is usually divided into two categories. One is “transformation”, which is induced by the chemicals and by metabolic depletion, and the other is “deformation’ ’ , which is induced by external physical forces. 1. Transformation of Etythrocytes The typical transformation is classified into two distinct shapes:102.103 (1) echinocytosis, or externalization, is characterized by slight prominences of membrane; (2) stomatocytosis, or internalization, is made conspicuous by the caving of the membrane to form a cup-shape, then to create small holes, as shown in Figure 2. However, at the extreme, these shape changes end up in spherocytosis. a. TRANSFORMATION DUE TO METABOLIC DEPLETION Echinocytic transformation occurs during blood storage in acid-citrate-dextrose or citrate-phosphate-dextrose at 4°C; as Nakao et al.‘” have noticed in 1962, the cell shape and the ATP content are closely related. Further, the following impairments are noticed in erythrocytes during blood storage:‘05 (1) ATP-depleted echinocytes show a short survival time after transfusion, (2) the oxygen affinity increases due to the reduced 2,3-DPG, (3) the blood viscosity increases with development of echinocytosis, and (4) the contents of inorganic ions change. Therefore, the methods of rejuvenation have been studied ex-
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-2 FIGURE 2. Morphological changes of erythrocytes. Stages of “transformation” to echinocytes (upper array) and stomatocytes (lower array) are shown. The numbers are used for quantitative expression of the degrees of transformation. (From Kon, K., Maeda, N., and Shiga, T., J. Physiol. (London), 339, 573, 1983. With permission.)
tensively. lo5 At the present stage, organic phosphates (ATP, 2,3-DPG) can be regenerated by incubation with certain substrates (inosine, pyruvate, phosphate, etc.), or even pyridoxal phosphate is useful for recovery of oxygen transport activity,‘06**07 but rheological properties cannot be restored by either of the organic phosphates. Echinocytes induced by metabolic depletion can be restored to discocytes, when the intracellular ATP is regenerated. However, Feo and Mohandas”’ have found no direct relation between ATP content and cell shape. On the other hand, the phosphorylation of several membrane proteins may intervene between ATP level and the cytoskeletal structure,‘Og although no direct interaction between the membrane components and ATP has been demonstrated. Further studies are needed on these points. b. TRANSFORMATION
half induces the external protrusion of the entire bilayer. On the contrary, the cationic chemicals tend to be incorporated into the inner half, and the expansion of the inner area caves the bilayer in. With an excess of chemicals, the transformation ends up with a spherical shape (called either sphero-echinocytes or sphero-stomatocytes). On the other hand, Nelson et al.‘l3 have suggested the possible role of calmodulin on the basis of transformation due to calmodulin inhibitors. Bums and Gratzer114 have demonstrated that trifluoperazine-induced stomatocytosis takes place at low concentrations of intracellular calcium, where the activity of calmodulin on the membrane is presumably suppressed. Our study’15 suggests that some calmodulin inhibitors can bind to calmodulin at low concentrations where they do not induce stomatocytosis. In order to study the chemically induced transformation further, the amounts of chemicals in the inner and outer halves of the lipid bilayer and in the cytoplasm (free or bound to some proteins) should be determined separately.
DUE TO CHEMICALS
In general, echinocytosis is induced by anionic reagents (e.g., lysolecithin, dehydroepiandrosterone sulfate, etc.), while stomatocytosis appears with cationic reagents (e.g., chlorpromazine, trifluoperazine, etc.). The velocity of transformation has been measured with the light-scattering stoppedflow method,“O,ll’ but it depends on the reagents and their concentration. Sheetz and Singerllz have proposed the “bilayer couple” hypothesis as an explanation, taking into account the asymmetric distribution of the reagent and the resultant differences in the surface area, between the inner and outer halves of lipid bilayer. The anionic chemicals are preferentially incorporated into the outer layer, and the expansion of the outer
In the in vivo capillaries, of which the diameter is sometimes smaller than that of erythrocyte, the cells must deform themselves in order to pass through. In the larger vessels, erythrocyte deformation is also important to reduce hydrodynamic resistance: actually, at high shear rates the blood (or suspension) viscosity decreases due to cell deformation. It is necessary to make a theoretical model in order to analyze erythrocyte deformation in shear flow. Two models 1990
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Critical Reviews In are available: (1) the fluid droplet model’16*1’7and (2) the microcapsule mode1.1’8-‘20In the fluid droplet model, a uniform shear stress is acting on the entire membrane, but in the microcapsule model the stresses differ from one point to another. In either model, the membrane rotates around the cytoplasm (called “tank-tread” movement),“6 and the cell interior is treated as a continuous fluid. The theories of erythrocyte deformation and motion in capillaries are now progressing’*’ and further developments are awaited. The deformation of erythrocytes is easily observed when external forces are applied on erythrocytes as follows: (1) under uniform shear stress the biconcave disk-shaped cells are elongated and deformed into flat ellipsoids;‘** (2) the cells can be folded back, when the cells are caught on a fine spider-thread in a ~trearn;‘*~(3) a portion of the cell membrane can be stretched into a tongue-like shape by aspiration with a micropipette; (4) the whole erythrocyte can be aspirated into a glass capillary (which is narrower than the diameter of erythrocyte),‘*’ or filtered through narrow pores.126 When the applied external forces are physically defined and the degree of the deformation is measurable, the degree can be used as a quantitative measure of the “deformability” (i.e., “the readiness to deform by physical force”). It is also important, when the applied forces are suddenly ceased, that the cell shape can be restored into a biconcave disk after a short recovery time.
III. ERYTHROCYTE DEFORMABILITY The deformation of erythrocytes is easily induced by external forces. The deformation is important with respect to (1) the passage of erythrocyte through narrow capillary, and (2) the reduction of blood viscosity under high shear rates. Therefore, quantitative measurements of erythrocyte deformability have been made in many diseases and plenty of evidence of the above phenomena is collected. In this section, the following problems will be discussed: (1) the advantages and disadvantages of various methods for deformability measurements and (2) the biochemical factors influencing deformability. The alterations of deformability in health and diseases will be discussed in Section V. A. Measurements of Deformabllity The term “deformability” is commonly used without exact definition. In the literature, the meaning of deformability sometimes differs from one method to another. Some methods apply a strong force on the whole cells, thus the entire cell shape is changed (“cellular” deformation). Some other methods make a small shape change in a very limited portion of the cell surface, for example, by aspiration with a micropipette (“membrane” deformation). Another important methodological distinction concerns the velocity of deformation and/or restoration: many methods are static and stationary, i.e., the deformation is quantified under a constant force lasting a certain period of time; on the other hand, some methods concern the time-re14
solved processes of deformation/restoration, either by changing the force stepwise or by applying an alternate force. It is desirable to define the physical nature of applied external forces and to measure the degree of deformation. Although the erythrocyte is so small and its shape is so complex that it is hard to apply the defined force and to express the shape change, several methods have been developed. It may be convenient to classify the various methods into three categories in a practical sense: (1) the cellular deformation in uniform shear flow, (2) the readiness of filtering in small pores, and (3) the physical properties of the cell membrane. Measurements (1) and (2) reflect the properties of whole cells, either as individual cells or as a mass, thus the “cellular deformability” is determined, whereas measurement (3) concerns only the viscoelasticity of the erythrocyte membrane. Furthermore, the methods for analysis of time-dependent phenomena are developed. 7. Deformability of the Whole Etythrocyte Under a defined external force the erythrocytes can deform, thus the “deformability” (the ease of deformation by an external force) can be quantitatively measured, if the force and the degree of deformation are determined. The shear force may be the first choice as a physically defined force, since it is easily controlled and acts on the whole cell body. The “rheoscope” of S&mid-Schonbein et al.127,128and the “ektacytometer” of Bessis and Mohandas129*‘30are the representative instruments; some other methods have been developed, but are not widely employed, because the manipulation and/or construction of the apparatus seem to be difficult. The filtration method will be described in the next section, since the physical forces applied on cells cannot be defined and the processes of shape change cannot be followed. 8. MEASUREMENT OF THE DEFORMATION UNDER UNIFORM SHEAR STRESS
Application of a steady shear stress to erythrocytes is carried out by modifying the cone-plate viscometer or the coaxial double cylinder viscometer. The disk-shaped etythrocytes are deformed into flat ellipsoids under high shear stress. In order to observe such deformation, the erythrocytes are suspended in a viscous solution containing dextran (molecular weight 40,000) or arabino-galactan (molecular weight 30,000). The representative methods are as follows. i. Rheoscope, Flash Photography of the Deformed Erythrocytes A transparent cone (rotating) is mounted on an inverted microscope (Figure 3). The shear stress produced in the coneplate viscometer is the same at any spatial position, and the flash photographs of flowing deformed cells can be easily obtained (Figure 4). In order to quantify the degree of deformation, the lengths of the long axis (L) and the short axis (S) of individual cells are measured one by one on the enlarged photographs, and either the “ellipticity” (S/L) or the “elon-
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r Pm
Viscometer
0
FIGURE 3. A schematic diagram of rheoscope. The erythrocytes, suspended in viscous solution (at the final hematocrit of about 0.2%), are poured into the gap between a 0.8” cone and the plate. The cone rotates on the glass plate. The photographs are taken using a flash lamp of 2 t~s duration. (From Suda, T., Maeda, N., Shimizu, D., Kamitsubo, E., and Shiga, T., Biorheology, 19, 555, 1982. With permission.)
gation or deformation index” ([L - S]/[L + S]) are adopted as an index of deformation. The frequency distribution of the indices for normal, individual erythrocytes is Gaussian. The index varies with the applied shear stress, the product of the shear rates, and the viscosity of the suspending medium. Figure 5 shows the plots of index to the shear stress and the effect of the hardening of cells by oxidative crosslinking of spectrin,‘3’ i.e., the deformation index decreases at every shear stress as the crosslinking proceeds. The advantage of this method is the monitoring of individual cells, i.e., the frequency distribution of index can be obtained and, if abnormally hard cells are present, normal and abnormal cells can be distinguished on photographs. However, the measurement of lengths on photographs is time-consuming and tedious, and thus may not be used at the hospital bedside (the diffractometric measurement of deformation in next item may serve for this purpose). The original design of the rheoscope is composed of a rotating cone and a fixed plate,12’ thus the cells are rapidly flowing and hardly viewed directly. Later the counterrotating apparatus is introduced, then the position of cells at the central plane does not move and the “tank-treading motion”“6*‘32 of the membrane can be monitored (see Section III.A.4.iv). The other application of the rheoscope is to obtain the frequency response of the erythrocyte deformation, under oscillatory varying shear ~tress’~~,‘~~(see Section III.A.4.iii). ii. Ektacytometer,
Diffractometty
of the Deformed
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FIGURE 4. Flash photographs of deformed erythrocytes, observed with rheoscope. Measured in 20% Dextran T-40 (18.6 cP) at the shear rate of 0, 75, and 750 s-’ at 25°C. The erythrocytes were deformed to flat ellipsoids, of which the long axes aligned along the flow line. Flash photographs were taken on Kodak Tri-X film. (From Suda, T., Maeda, N., Shimizu, D., Kamitsubo, E., and Shiga, 19, 555, 1982. With permission.) T., Biorheology,
medium and simultaneously a laser beam is projected through a window (rigorously saying, the shear stress is not uniform and has a slight gradient). The diffraction of the laser beam changes with the shape and size of the particles, thus of the deformed cells.‘3~‘29~‘30 When the cells deform ellipsoidally under high shear stress, the contour of the light diffraction is
Elythrocytes
Within a narrow space between coaxial double cylinders, the shear stress is applied to erythrocytes suspended in viscous 1990
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% Crosslinking FIGURE 5. E@rocyte deformation decreased by crosslinking of spectrin with diamide. Measured at the shear rates of 150 and 750 SX’ (in 20% Dextran T-40) at 25”C, with rheoscope. The percentage of crosslinking is expressed by the percent loss of spectrin beta subunit. The deformability is expressed by the deformation index, (L - S)/(L + S), in which L and S are the long and short radii of ellipsoidally deformed cells, respectively. The deformability decreased with a progress in crosslinking. (From Maeda, N., Kon, K., Imaizumi, K., Sekiya, M., and Shiga, T., Biochim. Biophys. ACZU, 735, 104, 1983. With permission.)
elongated ellipsoidally and is quantified to deduce the “deformation index”. The advantage of this method is real-time measurement and the simplicity of handling. However, since the diffraction reflects the averaged deformation of the whole cell fraction, the monitoring of individual cells is impossible and the concomitant abnormal cell (with abnormal shape) may be ignored. Moreover, in order to obtain the diffraction pattern, the reflection coefficient of the medium has to be high (i.e., the viscosity inevitably becomes high). Another application of the ektacytometer is to combine it with an apparatus for continuous modification of osmolarity. 13.135Both the mechanical deformation and stability are relatively quantified, thus this “osmotic gradient ektacytometry” seems to be useful for studies of hemolytic anemias. b. MEASUREMENT IN PARALLEL-PLATE FLOW CHANNEL
A laminar stream in a thin rectangular flow channel is sometimes useful for studies of erythrocyte flow behavior.
16
The “deformation-orientation” behavior of erythrocytes has been studied with phospholipid spin-labeled cells that are flowed in a flat channel. A parameter, reflecting the orientation of the membrane surface to the flow and deformation of disk-shaped erythrocytes, is calculated from the electron spin resonance spectra. 136.137 This method is successfully applied to study erythrocyte behavior at high hematocrit,‘3s.139 but cannot be used to figure out the degree of deformation because (1) the applied shear stress varied with the depth in the flow channel and (2) the deformation is accompanied with the orientation of cells along the stream line. Erythrocytes in isotonic buffered saline frequently adhere to the material surface (e.g., glass, methacrylate, etc.). When the channel is filled with erythrocyte suspension and the cells are allowed to settle, a portion of the sedimented cells attaches to the surface of the channel. Applying a laminar flow, the erythrocytes adhered with one point of their membrane are deformed into a tear-dropletlike shape by the wall shear stress (Figure 6), and the degree of deformation may be quantified by comparing the extended length (L) with the original length (LO) of the cells with a microscope. This method is essentially “uniaxial loading of shear stress to point-attached erythand the rheoscope may be used for this rocytes “, 140.141 purpose (as shown in Figure 6) instead of the flow channel. Hochmuth et a1.14’ have observed three types of erythrocyte stretching in shear flow: whole cell stretching (as described above), cell evagination, and long tether stretching (i.e., the formation of thin process, of which the mechanics is discussed by Evans and Hochmuth14*). The disadvantage of the method may be the uncertainty in the size and nature of the “point” attachment, which cannot be determined with the microscope. The electron microscopic feature of the point is very complex (see Figure 3 of Reference 143), and the binding force of individual cells to the surface varies widely.lW
0
75
750
se?
FIGURE 6. Deformation of point-attached erythrocytes. An application of the rheoscope, for the measurement of the elongation of erythrocytes, which are point-attached to a polymethylmethacrylate plate, by wall shear force. (From Shiga, T., Sekiya, M., Maeda, N., and Oka, S., J. Colloid Interface Sci., 107, 194, 1985. With permission.)
Volume 10, Issue 1
Oncology/Hematology 3.
The combination of a rectangular flow channel and a laser beam, to monitor the light diffraction, has been tested.L4s The results are similar to those obtained with the ektacytometer. In this system, the velocity profile in the flow channel is parabolic, i.e., the velocity of flowing erythrocytes (thus the degree of deformation) is not uniform along the crossing laser beam. Therefore, some sort of averaged diffraction pattern is obtained.
Taken all together, a flow channel is simple to construct and easy to calculate the wall shear stress, thus it is adequate to monitor the phenomena at the wall, but inadequate to observe the entire depth of suspension. c. MEASUREMENT WITH VISCOMETER The viscosity of an erythrocyte suspension, in a nonaggregating medium (e.g., isotonic phosphate or HEPES buffered saline containing 1% albumin) at a given hematocrit (e.g., 20 to 40%), decreases as the applied shear rate increases. ‘4,~Such shear thinning is induced by the cellular deformation to reduce the hydrodynamic resistance; thus the ratio of suspension viscosity at different shear rates may give a measure of cellular deformation.8”47 In addition, at high shear rates (>200 per second) the intracellular viscosity determines the suspension viscosity, but at low shear rates the membrane properties and the cell shape dominate.‘48
laboratory to another. Therefore, for the sake of international standardization, a guideline has been suggested recently,“* including the preparative methods of human erythrocytes (i.e., blood collection, washing of erythrocytes, recommended buffers, etc.). The use of whole blood (or diluted blood) tends to be avoided, because the leukocytes, platelet microaggregates, or even erythrocyte aggregates interfere with the filtration of erythrocyte. At any rate, the pores are gradually clogged by the cells and the filter is usually disposed of after a single measurement (the filter may be washable and be reused, depending on the material’59). The advantage of the filtration technique is just its simplicity; every laboratory may be able to construct the apparatus at low cost. The disadvantages are summarized as follows: (1) the force against cells cannot be defined and kept constant during the passage; (2) the preparation of the erythrocyte suspension (of certain hematocrit, 5 to 15%) may be troublesome, especially for the perfect removal of leukocytes;‘@’ (3) at the moment, no standard suspension is available, and the comparison of data among many laboratories (or in literature) is not easy; and (4) some sort of averaged values is obtained, but it is strongly affected by the presence of concomitant rigid cells (if any). 16’ b. PASSAGE OF SINGLE ERYTHROCVTES THROUGH A PORE
2. Measurement of Filterability, the Ability to Pass Through Narrow Pore(s) In order to express the ability of the erythrocyte to pass through capillaries of diameter 5 to 8 pm (often narrower than the cell diameter), it is reasonable to quantify the ease of cell passage through the small pore(s).149 Two types of experimental arrangement are distinguished; one is to measure the passage of erythrocytes as whole blood or suspension, and the other is to test the individual cells one by one.
Each erythrocyte must enter into a narrow capillary; thus the ability to enter into and to pass through a small pore should give a practical measure of deformability. Many techniques have been designed with great expectation, but none of these is widely employed, perhaps because each technique is either too elaborate to construct or too artistic to manipulate. The advantage of the method is to obtain the statistical population of erythrocytes. The common disadvantage is the difficulty of defining (or controlling) the external force and the deformed shape. Some of the representative methods are briefly described below.
a. FILTRATION THROUGH MICROSIEVE
1.
most general method is to pass the erythrocyte suspension through a Nuclepore membrane filter, usually with round pores of 5 to 3 p,m diameter of lo-pm thickness. Either the passage time of a certain volume of suspension under a given pressure or the filtered volume during a certain period is measured.‘50-‘53When the pore density is known, the mean passage time of a single erythrocyte may be calculated.‘s4 The time course of the pressure drop (difference) across the filter gives a measure of erythrocyte deformability. 15s.‘s6The determinant factors for the filtration vary with the diameter of the pores, i.e., at a pore diameter of about 5 Frn, both the cell shape and the intracellular viscosity are dominant.1s7 Because the filtration method is simple, many variations are developed; for example, the diameter of pores is varied from 3 to 5 km, the applied pressure depends on the researchers, and the detailed design of the apparatus differs from one The
2.
3. 1990
It is possible to aspirate the individual erythrocytes with a micropipette (of 3 to 5 pm in diameter) by a negative pressure and to monitor the passage by the change in electric potential across the pipette.‘62 The readiness of entering into the narrow orifice and/or passing through a certain length in the pore’63 gives the measure of the erythrocyte deformability. However, the combination with high electric potential gradient may influence the cell entry times through the electrophoretic mechanism. I64 Using a single pore of the microsieve (e.g., Nuclepore membrane), a “single erythrocyte rigidometer” has been constructed165 that measures optically the passage times of individual cells through a pore (5p,m diameter, 10 km thickness) one by one. Kiesewetter et a1.165have compared the values obtained by this method with those by other instruments. “Resistive pulse spectrosc~py”,~~~~~~’which is an out17
Critical Reviews In
4.
growth of Coulter counter, measures the sizes (actually the change in electric resistance) of flowing cells, of which the shapes deform to elongated ellipsoid under high flow rate. The difference in the population patterns between the apparent sizing at a less-deformed state (under low flow rate) and that of an elongated state (under high flow rate) gives a parameter of deformation. “Cell transit-time analyzer” employs “oligopore” filter (e.g., 30 pores of diameter 4.5 urn and length 21 pm, polycarbonate membrane) through which a lOO-kHz AC current and a hydrostatic pressure (e.g., 8 cm H,O) are given. ‘68.‘69From changes in the electrical conductance of the filter due to erythrocyte passage through the pores, the transit times of individual cells are measured and the distribution of transit times for a large number of cells are obtained.
3. Measurement of Mechanical Properties of Membrane Mechanical properties of the erythrocyte membrane can be quantified: the static membrane properties are determined by analyzing the relation between the applied force and the resultant deformation of membrane, while the dynamic properties can be measured by observing the time-dependent process of deformation-restoration. Evans and Skalak”O have reviewed extensively the mechanics and thermodynamics of biomembrane, and recently Hochmuth and Waugh9 and EvansL2 have reviewed the methods for the measurements. Sometimes it is difficult to distinguish the membrane properties from the cellular properties, because the cytoskeletal components are interacting with cell contents (especially hemoglobin). In this section, the measurements largely concerned with membrane, i.e., micropipette aspiration techniques, are briefly described. a. MlCROPlPElTE ASPIRATION TECHNIQUE
Mitchison and Swann’71~‘72introduced a capillary suction technique to measure the mechanical properties of sea urchin egg membrane, and Rand and Burton’24have applied this method to erythrocytes. At present, the micropipette aspiration technique has been extended and employed in many laboratories. The static membrane properties, such as membrane elastic modulus Q.L)or extensional (shear) rigidity, of human erythrocytes can be determined as follows. A portion of erythrocyte membrane is aspirated by a negative pressure (P) through a micropipette (inner diameter of 2R, usually 1 pm), and the length (L) of the extended tongue inside the pipette is measured under a microscope (as a function of P). Using the l-urn micropipette, L/R increases linearly with increasing P-R (in the range 1 < L/R C 3). A theory of Evans and La Celle173 is approximated as follows: P . R/p = C, . (L/R) + C, where p, is the membrane elastic modulus and C, and C, are 18
constants (according to Chien et a1;174C, = 2.45, C, = -0.603). The membrane elastic modulus is independent of intracellular hemoglobin concentration (33 to 44 g/dl), and the typical value for normal erythrocytes is 9 ( + 1.7) x 10e3 dyn/ cm according to Evans et al.175 In addition, using a video recording system, it is possible to determine the time course of membrane restoration after expelling the cell from the pipette. Following the Kelvin-Voigt model, i.e., elastic and viscous elements are combined in parallel, the time constant for shape recovery (t,) is related to the ratio of the membrane surface viscosity (q,,,) and the membrane elastic modulus: t. = “I),,&. The time constant of the shape recovery (the disappearance of the extended tongue) is about 0.3 s according to Evans and Hochm~th,~‘~ to the estimate of the membrane surface viscosity is obtained as low3 dyns/cm. Further, the time constant (t,) is associated not only with the membrane, but with the intracellular hemoglobin concentration (or intracellular viscosity, -qi)and the thickness of erythrocyte (d) as follows:L2,‘75
When the negative pressure applied by the micropipette is further increased, the cell aspiration proceeds, and at a certain critical pressure the cell shape changes to form buckling or wrinkles. This critical pressure provides a measure of the bending elastic modulus (B):‘**“’ B = C.R3.P where a constant (C) depends on the inner diameter (R) of micropipette and is 0.005 to 0.012. The bending elastic modulus is 1.8 x lo- ‘* dyn-cm, according to Evans et al. L75 4. Dynamic Measurement of Cellular Deformability Many methods described above are static measurements of deformation, i.e., the strain of erythrocyte(s) is balanced with the applied external force. However, flowing erythrocytes in large arteries are always exposed to the pulsatile pressure wave, thus the relation between the blood viscosity and the erythrocyte deformability may not hold as it does in a viscometer. Moreover, the erythrocytes in capillaries change their shape accordingly to the geometry of the intracapillary paths. Therefore, we need a way to determine the time-dependent processes of quickly and repeatedly changing deformation and restoration. Several methods are developed for the quantitative measurement of the time-dependent process of whole erythrocytes. 1.
Using a rheoscope, we have applied oscillatory shear stress to flowing erythrocytes.‘33.‘34 As shown in Figure 7, the speed of cone rotation is modulated to produce alternate shear stress. The shapes of flowing erythrocytes change in response to the applied shear stress at low
Volume 10, Issue 1
Oncology/Hematology Oscillatory
oL-
0 102030
‘t . dyn km2
Phase
FIGURE 7. Erythrocyte deformation under stationary and oscillatory shear stresses. Measured in 14% Dextran T-40 (12 cP) at 25”C, with rheoscope. The deformability is expressed by the deformation index (see Figure 5). (Left) Deformation under stationary (uniform) shear stress: the deformation index increased with the applied shear stress (9 to 32 dyn/cmz). (Right) Deformation under oscillatory shear stress: a cone-plate rheoscope is modified to apply oscillatory shear stress (varied in the range 10 to 32 dyn/cm2, as shown in the bottom curve) by modulating the frequency of cone rotation (0.5 to 2.4 Hz). Each symbol shows the deformation under oscillatory shear stress (flash photographs are taken at desired moment of oscillation by adjusting the trigger timing): open circle, 0.6 Hz; half-closed circle (with broken line), 2.0 Hz; closed circle (with dotted line), 2.7 Hz. A solid line is the deformation index obtained under uniform shear stress. Note that above 2 Hz the deformation could not follow the increase in shear stres~.‘~‘~‘”
2.
3.
4
‘.
frequency and under low amplitude of oscillation. However, when the oscillation frequency exceeds a certain value (e.g., 2 Hz, in the oscillatory range of 10 to 30 dyn/cm2), the shape change cannot accommodate the change in shear force. Such retardation in deformation becomes evident in the increasing phase of the applied shear stress. In contrast, the shape restoration in the decreasing phase is a much faster process (e.g., within 0.1 s; see above). The phenomena of oscillatory deformation also strongly depend on the intracellular viscosity and the membrane properties and are more sensitive to the changes of these determinants than similar phenomena in the static measurement with the rheoscope under a constant shear stress. The whole erythrocyte aspirated in 4-pm micropipette can be folded without extension and then the cell is rapidly expelled from the tip. The time constant of unfolding (t;), i.e., the restoration of the width of the folded cell, is monitored. ‘75According to Evans, l2 4 is approximated by tf = (q;d)/(BK,,,*), where qi is the intracellular viscosity, d is the cell diameter, B is the membrane bending modulus (Section III.3.a.ii), and K,,, is a curvature of the fold. For the normal erythrocytes (MCHC of 32 to 34 g/ dl), the time constant of unfolding is 0.25 ( 2 0.09) s, and the value is strongly dependent on the intracellular hemoglobin concentration. The measurement of the extensional shape recovery has been introduced by Hochmuth et al.17s The erythrocyte, of which a portion is point-attached to the cover glass,
is extended end to end by an aspirating pipette at diametrically opposed position, so the cell shape becomes ellipsoid with narrow edges at both ends. When the aspirating site of cell is quickly released, the cell shape recovers to a biconcave disk shape within 0.3 s. The time constant of shape recovery (t,) is about 0.10 s for normal erythrocytes and is strongly dependent on both the intracellular hemoglobin concentration and the membrane properties. Further, Nash and Meisselman’79 have demonstrated prolonged t, for aged erythrocytes in spite of equal membrane elastic modulus Q.L)for young and aged cells. That is, the increased t, is not explainable by elevated intracellular viscosity (qi), but the membrane viscosity increases perhaps due to hemoglobin-membrane interaction when hemoglobin concentration is increased. Sutera et al. la0have also shown the decreased t, of projected cell length (observed with a rheoscope, after abrupt cessation of shearing) for aged erythrocytes. The tank-treading motion of a membrane can be observed with a counterrotating rheoscope.“6~‘n’~‘82 When an erythrocyte is subjected to countershear stresses (without change in the position), the membrane rotates around the cell interior, as visualized by the movement of a small particle (e.g., Latex of 0.8 pm diameter) attached on the cell surface. The tank-treading frequency of normal erythrocytes (5 to 40 per second) varies linearly with the applied shear rate, i.e., the ratio of frequency/shear rate is about 0.2 (in the range 20 to 180 per second of shear rate) and is strongly dependent on cell age.“’
B. Determinant Factors of Cellular Deformability In spite of the variety of deformation measurements, the major determinant factors for cellular deformability are unanimously agreed upon and summarized into three categories: 1. 2. 3.
Cell shape, or surface area-to-volume ratio Intracellular viscosity, or hemoglobin concentration Membrane stiffness, or membrane flexibility
However, reflecting the diversity in the measurement of deformation, the contribution of these factors to the cellular deformability differs from one method to another. Further, as will be discussed later in detail (see Section V.3), these factors vary with cell age. Dealing with the whole population of erythrocytes, one should keep in mind the considerable heterogeneity in terms of deformability. At the moment, the molecular basis of deformability has not yet been established, thus only representative cases are discussed here. 7. Cell Shape or Surface Area-to-Volume Ratio Distinct and extreme examples are the hereditary abnormal erythrocytes, such as elliptocytosis, spherocytosis, etc., caused by abnormality or defect in one of the cytoskeletal proteins. I9 1990
19
Critical Reviews In These erythrocytes are usually fragile and easily hemolyzed in circulation to induce anemia. The biconcave disk shape of normal erythrocytes is important for maintenance of deformability. This shape has greater surface area/volume ratio than a sphere. The sphere is minimum in the ratio and cannot be folded, while the biconcave disk is easily distorted and bent or folded. However, quantitative study of the relation between the cell geometry and deformability is hard. 1.
2.
3.
The modification of water content, for example, is inadequate. When the erythrocytes are exposed to a hypertonic (or hypotonic) solution, the dehydration (or hydration) inside the cell really modifies the surface area/ volume ratio, but simultaneously the intracellular hemoglobin concentration increases (or decreases). Therefore, two factors, the shape and the intracellular viscosity, cannot be separated. Certain trials to alter the surface area/volume ratio have been made. ‘83.*84The incubation of biconcave cells with lysophosphatidyl-choline induces a partial loss of membrane. After washing with albumin to remove the lysophospholipid, the cell returned to a biconcave disk shape, but the deformability decreased,lE4 perhaps due to decreased surface area-to-volume ratio. Certain chemicals can induce the transformation of erythrocytes. Rheological properties of echinocytes are impaired, i.e., the increased suspension viscosity,‘85-‘87 the increased resistance to extension,‘88 the decreased deformation under high shear stress. ‘22,‘86Further, we have shown the decreased rate of oxygen egress. lE9 A recent work’88,‘90 suggests that the normal biconcave disk represents an optimal shape for the flow in viva, since stomatocytosis impairs cell passage through the microvessels (due to the decrease in cell filterability) and echinocytosis affects the flow in large vessels (due to the increase in blood viscosity).
tration is 35 g/dl, the viscosity is about 10 CP (at 25°C),75~77 which is much higher than that of plasma. Morse I119’*i92has applied the spin probe method (with TEMPAMINE) for determining the intracellular viscosity, using electron spin resonance. However, the viscosity values obtained are about one third of the values estimated from viscosity of the hemoglobin solution. Endre et a1.‘93,‘94 have applied the nuclear magnetic resonance (NMR) measurement of T,, with ‘3C-glutathione in erythrocytes. Here again, low viscosity value is obtained. The discrepancy comes from the fact that the spin probe or NMR probe senses the viscosity of its very environment (thus called “microviscosity”), which differs from the bulk viscosity of hemoglobin solution. Hermann and Miiller’95 have recently shown that, above a hemoglobin concentration of 6 r&f, the viscosity of a hemoglobin solution shows rapid increase compared with the microviscosity measured with a spin probe (TEMPONE) . b. MAINTENANCE
OF WATER
CONTENT
IN ERYTHROCYTES
regulation of inorganic ions, pH, and cell volume in erythrocytes is related to the activities of many transporters and to extra- and intracellular environments. The interrelationship between the water movement across the membrane and the concentration of individual ions is too complex and is still in too early a stage to summarize. Here, we show a few examples following: The
Effect of phosphate ion is important for in vitro studies, since incubation without phosphate in the medium tends to reduce ATP and 2,3-DPG. Keidan et al. ‘96 have recommended, for rheological studies of human erythrocytes, the use of phosphate-buffered saline (around 50 mmolll of phosphate) or HEPES-buffered saline (5 to 40 mmol/l of HEPES) of osmolality 290 mOsm/kg pH 7.4. Bookchin et al. 19’have shown that phosphate entry causes alkalinization of intracellular pH and dehydration of erythrocytes .
2. Intracellular Viscosity Since hemoglobin is the most abundant protein inside the erythrocytes, its concentration is an important factor for deformability; for example, at very high shear stress, the erythrocyte may be approximated as a droplet of viscous liquid. The intracellular viscosity can be estimated (1) in vitro by the determination of hemoglobin concentration or (2) in situ by means of the magnetic resonance methods, although the values obtained from these methods do not agree. a. MEASUREMENT
OF INTRACELLULAR
VISCOSITY
In general, hemoglobin solutions prepared from hemolyzed human erythrocytes have Newtonian flow properties, but the presence of cell debris in the solution causes non-Newtonian flow behavior.76 When the intracellular hemoglobin concen-
20
When the intracellular calcium content increases, the calcium-activated K-channel induces potassium loss and dehydration, known as the Gardos effect.‘98*‘99 The effect of intracellular calcium is discussed later (Section III.B.3.c). The imbalance of sodium/potassium is also noticed in erythrocytes from spontaneous hypertensive rats, as well as in erythrocytes from the patients of essential hypertension.200-202 In these cases, decreased activity of Na-K countertransport results in Na overload and K depletion. In addition, the decreased membrane fluidity is detected with spin label technique for the erythrocytes of hypertensive patients. *03 In connection with this, it is known that an increase in membrane cholesterol affects the activity of Na-K ATPase. 2w The situation is similar to some
Volume 10, Issue 1
Oncology/Hematology the changes in the erythrocyte functions are measured by various means. The results are summarized in Figure 8. Briefly, with a doubling of the cholesterol contents in membrane
sorts of hyperlipidemia or obesity, where the membrane fluidity decreases and the activity of the Na-K pump is reduced. Therefore, awaited. 4.
further
detailed
studies
on these points
1.
are
The effect of pH has been studied by varying the pH of the incubation media. It is recognized that the erythrocyte shape is altered with pH: at high pH the cells are flattened, while at low pH the cells tend to become spheric and small in diameterSzo5 At low pH the deformability decreases, the membrane elastic modulus increases, and the filtration time prolongs.206
3. hf8n?br8ne Vi8co8/88ticity
2. 3. 4.
8nd Biochemical
5.
The decrease of membrane fluidity, as measured by the fatty acid spin-label method, corresponding to the temperature decrease of some 5°C (from 36 to 31°C for example)28,42 The decrease in the diffusion rate of oxygen in lipid bilayer of erythrocyte membrane209 The decrease in the velocity of oxygen egress of lo%, as measured with a stopped flow apparatus2’*‘lo The decrease in the velocity of cell aspiration into a some 20% reduction of narrow orifice,2o8 i.e., deformability The decrease in the suspension viscosity of 10% ‘OS
Structure On the other hand, Chabanel et a12” have detected no significant change in the membrane elastic modulus and the extensional recovery time with cholesterol-loaded erythrocytes, in spite of the decreased membrane fluidity of the outer membrane leaflet. These quantitative studies show that in spite of a considerable modification in the membrane fluidity upon cholesterol-loading up to 200%, the changes in rheological functions are some 10% or none. The alteration of lipid composition, even in diseases of lipid metabolism such as LCAT (lecithin-cholesterol acyltransferase)-deficiency, does not double the cholesterol content.2’2 Therefore, the contribution of the lipid bilayer to the mechanical properties of erythrocytes is not so great when compared with the modification or abnormality of the cytoskeletal protein network.
The mechanical properties of a membrane are an important determinant factor for deformability. However, as depicted in Section II, the erythrocyte membrane is a complex sheet, composed of the lipid bilayer and the cytoskeletal proteins. At present, the states of membrane cytoskeletal network appear to be more essential for the cellular deformability than the structure of the lipid bilayer. Although the lipids and proteins are interacting, perhaps it is convenient to discuss the effects on the deformability separately, as follows: 1. 2. 3.
The contribution of the lipid bilayer The role of the static structure of the cytoskeleton The regulation of the dynamic structure of the cytoskeleton
a. CONTRIBUTION OF THE LIPID BILAYER STRUCTURE TO CELLULAR DEFORMABILITY The dynamic structure and the state of the lipid bilayer
b. ROLE OF CYTOSKELETAL STRUCTURE
depend on the composition of the lipids, as proven by various techniques. In artificial membranes, the mechanical and rheological properties are greatly influenced by the lipid composition. However, with human erythrocyte membrane, only a few experimental studies are carried out to reveal “to what extent the deformability is altered as a function of a moditication in lipid composition and structure”, although much evidence for the decreased membrane stability of erythrocytes with the abnormality of lipid metabolism is reported. Cooper207 has shown the decreased deformability in cholesterol-rich erythrocytes. We have artificially modified the cholesterol content of the human erythrocyte membrane and obtained specimens with minute alterations in the energetics (ATP, ADP, AMP, etc.), 2,3-DPG, intracellular pH, and cell shape.28~20s In order to deplete or augment the membrane cholesterol, washed human erythrocytes are incubated with DPPC (dipalmitoyl-phosphatidyl-choline) vesicles containing an appropriate amount of cholesterol, according to the method of molar ratio is Cooper et al. 27 The cholesterobphospholipid modified in the range of 0.6 to 2.0 (normally about l), and
The abnormality of major cytoskeletal proteins in some hereditary diseases frequently induces a shape change of erythrocytes, such as elliptocytosis, spherocytosis, poikilocytosis, etc., and the stability and/or deformability are impaired, thus hemolytic anemia is developed. This subject has been reviewed extensively in this journal by Zail.” Recently, Waugh 213has studied the changes in the membrane elastic modulus and the membrane surface viscosity in the cells of patients having a variety of cytoskeletal disorders. His conclusion suggests that the membrane elastic modulus is proportional to the density of spectrin, but the reduction in the membrane surface viscosity is variable with the degree of membrane abnormality. Further studies are awaited. The loss or decrease of cellular deformability occurs from acquired causes. Mostly, the oxidative degradation or polymerization of membrane constituents leads to the abnormal mechanical properties of erythrocytes. The extreme model for decreased cellular deformability are malondialdehyde (MDA)““ or glutaraldehyde21s-treated erythrocytes, due to the crosslinking of membrane proteins (MDA is a degradation product of
1990
21
Critical Reviews In
I
Membranecholesterol
x2
Awl-chain motion
/
Decreased \ 02 diffusion in membrane
,L
Deformability
x 0.8
I
r-4 Increased
RGURE 8. Impairments of the rheological and oxygen-transporting functions of cholesterol-loaded erythkytes (see text).
arachidonic acid and an oxidative reagent). Similarly, diamide (diazene dicarboxylic acid bis(N,N-dimethyl amide)) is employed as a reagent for oxidative crosslinkage of sulfhydryl groups. *16Diamide treatment is suitable for the model studies, because (1) it is easy to control the degree of crosslinkage; (2) the crosslinkage is induced first in spectrin, then in other proteins; and (3) the linkage can be recovered at least partly by reducing reagent (e.g., dithiothreitol) depending on the degrees of polymerization. 13’On the other hand, MDA and glutaraldehyde are hard to handle, since they crosslink all the proteins through the react@ with amino groups, including hemoglobin, to form insoluble aggregates. We have also studied the quantitative relationships among the degrees of spectrin crosslinkage, the suspension viscosity, and the deformability. I31With an increase in the spectrin crosslinkage (measured by a decrease in the electrophoretic peak of band 2, spectrin l3 subunit), the suspension viscosity (at hematocrit of 40%) increases linearly with the percentage of crosslinkage, and the deformability determined with a highshear rheoscope decreases, as shown in Figure 9. It should be noted that such crosslinkage is partially reversible. The above example of diamide treatment (of low concentration) does not affect the intracellular viscosity and the cell shape, thus giving a quantitative relation between spectrin crosslinkage and the cellular deformability. That is, at high shear stress (e.g., 150 dyn/cm2) the change in deformability is hardly detected below 20% of spectrin crosslinkage, but at low shear stress (e.g., 15 dyn/cm2) the deformability steeply decreases by a subtle crosslinkage. Such spectrin crosslinkage is hardly detected in normal 22
erythrocytes, even in fractionated aged erythrocytes, since (1) the electrophoretic analysis of crosslinkage is not sensitive enough to detect subtle crosslinkage and (2) the highly crosslinked cells, if any, may already be removed in viva during their passage in the spleen. However, subtle crosslinkage may contribute to the decreased deformability of in vivo aged cells (see Section V.3). In addition, it may be noteworthy to comment on the protein-lipid interaction here. The molecular motion of the membrane lipids near the integral membrane proteins, called annular lipids, is suppressed. In contrast, the interaction from cytoskeletal peripheral proteins to membrane lipid may be detected, but very small; for example, we have detected a subtle decrease in membrane fluidity induced by spectrin crosslinkage, with a fatty acid spin label. c. REGULATION OF CYTOSKELETAL DYNAMIC STRUCTURE The deformability may be reversibly controlled by dynamic change or fluctuation in the cytoskeletal architecture, for example, the phosphorylation of spectrin, the regulation of ATP content (production and utilization).*” At present, not much study has been published. Here, we show briefly a regulatory mechanism through intracellular calcium as an example. The calcium content inside the erythrocytes can be artificially modified in a wide range by incubation with a Ca-ionophore (such as A23187) and a given amount of calcium in an appropriate buffer. After incubation the erythrocytes are washed and the total calcium is determined with an atomic absorption spectrometer. It should be noted that calcium loading in an isotonic NaCl solution induces the loss of potassium and water
Volume 10, Issue 1
Oncology/Hematology
2.5” v % Crosslinking FIGURE 9. Suspension viscosity and deformation of spectrin-crosslinked erythrocytes. The crosslinking is expressed by the percent loss of the spectrin beta subunit in diamide-treated erythrocytes. The suspension viscosity, measured at a hematocrit of 40% at 37°C. increased with the progress in crosslinking. The deformability, measured in 20% Dextran T-40 (18.6 cP) at 20°C with rheoscope and expressed by the deformation index (see Figure 5). decreased with the progress in crosslinking. (From Maeda, N., Kon, K., Imaizumi, K., Sekiya, M., and Shiga, T., Eiochim. Biophys. Acru. 735, 104, 1983. With permission.)
(known as the Gardos effect)r9’ due to Ca-activated K-channel and thus the deformability greatly decreases to induce the undeformable cellsL99(Figure 10). However, (1) the dehydration is avoided in a K-containing medium; (2) the energy charge and ATP content are restored by further incubation with glucose; and (3) minor differences of intracellular hemoglobin concentration can be adjusted in the medium of appropriate osmolarity, thus, finally, we can compare the deformation of cells at a similar level of intracellular viscosity and cell shape. ‘I5 The deformability of calcium-depleted or -loaded cells measured with the rheoscope are compared as shown in Figure 11. A change of deformability in two states is apparently recognized: erythrocytes of high calcium content are less deformable than those of low calcium content. When calmodulin inhibitors, such as trifluoperazine or w-7 (N-(&uninohexyl)-5chloro- 1-naphthalene sulfonamide) are added in the medium, the decreased deformability of calcium-loaded cells is restored, while the inhibitors show no effect on the deformability of low-calcium cells. The phenomena are explained below:‘i5 1. 2. 3.
With an increase in calcium content inside the cells, intracellular free Ca+ + increases The calcium-bound calmodulin augments The calcium-bound calmodulin binds to the cytoskeletal network (as to interfere with the spectrin-actin-band 4.1 binding)Z’8*2*9and somehow rigidifies the mechanical properties of the cytoskeletal network
4. 5.
Calmodulin inhibitors suppress the action of calciumcalmodulin The other calcium-binding protein(s) may contribute to the phenomena
In this study, the level of intracellular free Ca’ + is unknown, but it must be closely related to the total calcium content. Although no experiment covering other possible mechanisms is available (e.g., protein phosphorylation),‘@’ the work on the calcium effect provides an example suggesting the regulatory processes of deformability through the reversible change of dynamic cytoskeletal structure.
IV. ERYTHROCYTE
AGGREGATION
Erythrocyte aggregation is the reversible adhesion of adjacent erythrocytes, induced by the bridging of intercalated macromolecules. As a result, 1-dimensional aggregates, socalled ‘ ‘rouleaux’ ’ , or 3-dimensionally branched and piled aggregates are formed. The physiological importance of erythrocyte aggregation in circulation is its tendency to increase the blood viscosity in low shear flow220and to disturb the passage in capillary circulation tbrougb the formation of s1udge.22’When the blood flow falls into stasis, erythrocyte aggregation is pronounced. The erythrocyte aggregates retard the blood flow, and the aggregation is further accelerated. Therefore, a vicious circle may develop in some cases.222-224Since the process of erythrocyte aggregation involves a balance between the aggre1990
23
Critical Reviews In
CCa Ii
H
10jm FIGURE 10. Deformation of calcium-loaded erythrocytes: appearance of undeformed erythrocytes due to dehydration. Calcium is loaded in isotonic Na phosphate-buffered saline by using CaCI, and Ca-ionophore (A23187). The top flash photograph shows the deformation of normal cells observed at a shear stress of 140 dyn/cm* at 25°C. The middle and bottom photographs show calcium-loaded cells under the same shear stress; the number of undeformable cells were increased with an increase of the intracellular calcium content. The total calcium in erythrocytes, [Cal,, is expressed with micromoles per liter of packed cells. (From Shiga, T., Sekiya, M., Maeda, N., Kon, K., and Okazaki, M., Biochim. Biophys. Acta, 814, 289, 1985. With permission.)
gating force and the disaggregating force,zzo the imbalance between the two forces may induce circulatory disturbances, for example, (1) the stasis of blood flow decreases the disaggregating force and (2) the increased amount of bridging macromolecules (such as fibrinogen, immunoglobulins, etc.) increases the aggregating force. In this section, the following problems are discussed: (1) the mechanism of erythrocyte aggregation, (2) the measure24
ment of erythrocyte aggregation with its advantages and disadvantages, and (3) various factors influencing erythrocyte aggregation. The alterations of the erythrocyte aggregation in various diseases will be summarized in Section V.
A. Mechanism of Erythrocyte Aggregation Erythrocyte aggregation is induced by the bridging of in-
Volume 10, Issue 1
Oncology/Hematology
0.4 -
in 12 CP
Dextran
l-r 5.
0.3 r;
PI
The studies by Brooks and Seaman227*228on the bridging of macromolecules between adjacent erythrocyte surfaces support the above mechanism; the adsorption of macromolecules such as dextran expands the electric double layer on the erythrocyte surface and the resultant increase of the zeta potential leads to erythrocyte aggregation by the macromolecules.
376 s-’
0.2 -
brings the adjacent areas into sufficiently close range for further bridging to proceed, thus the deformation of the erythrocyte membrane facilitates the progression of bridging and results in a maximum area of erythrocyte surface bridged by the macromolecules The flow facilitates collisions among the erythrocytes, but the high shear stress acts as a disaggregating force.
2. Rate of Etythrocyte Aggregation and the Shape
of Aggregates L
0
1 10
I 20
[Cali (uM/I
I 30
I 40
The aggregating force in macromolecules is counteracted by the disaggregating force. Chien and Jan225 have summarized the forces as follows: the macromoiecular bridging force is opposed by the mechanical shearing force, the electrostatic repulsive force, and the bending resistance of erythrocyte membrane. Therefore, the rate and degree of erythrocyte aggregation is influenced by the physicochemical properties of bridging macromolecules, the character of erythrocytes, and the physical and chemical environments in flow. From the point of view of energy balance, the aggregating energy provided by macromolecular bridging must overcome the disaggregating energy of electrostatic repulsion and mechanical shearing in order to form stable erythrocyte aggregates. The net aggregation energy is largely stored in the erythrocyte membrane as a change in strain energy, which can be computed from the elastic properties of the erythrocyte membrane.229.230 The changes in membrane strain energy are related to the shape change of erythrocytes (concave or convex at the end) in rouleaux,230 which agree with the computed cell shape based on the adhesion theory.229 Therefore, the shape of individual cells in rouleaux depends on the species of macromolecules and their concentration.
I 50
packed cells)
FIGURE 11. Relation between erythrocyte deformability and intracellular calcium content. After correction of intracellular’ viscosity, i.e., equalizing the mean corpuscular hemoglobin concentration by varying the osmolality of suspending media, the deformation of erythrocytes is measured in Dextran T40-containing HEPES-buffered saline of 12 CP at 25°C by rheoscope (the calcium content in normal cells is 16 to 18 pmol/l of packed cells). The deformability is expressed by the deformation index (see Figure 5). Calcium-depleted cells are obtained by treatment with Ca-ionophore and EGTA, and calcium-loaded cells are prepared by incubation with Ca-ionophore and Ca-EGTA, in K+HEPES-buffered saline. All erythrocytes deformed, but two states were apparent, i.e., low calcium cells were more deformable, while high calcium cells were less deformable. (From Murakami, J., Maeda, N., Kon, K., and Shiga, T., Biochim. Biophys. Actn, 863, 23, 1986. With permission.)
tercalated macromolecules at both ends between of two adjacent erythrocytes.
the surfaces
3. Nature of Macromolecular Bridging on
Etythrocyte Surface
7. Macromolecular Bridging Between Erythrocyte Surfaces On the dynamics of rouleau formation of erythrocytes, model is presented by Chien and Jan:225~226 1. 2. 3.
4.
Bridging of macromolecules at their specific sites between erythrocytes induces erythrocyte aggregation, but such sites are not yet identified. Evidence for specific binding sites of bridging macromolecules on the erythrocyte surface is presented in an experiment using dextran and polyglutamic acid.23’ That is, dextran-induced erythrocyte aggregation is inhibited by nonbridging small dextran, but not by small polyglutamic acid, while polyglutamic acid-induced erythrocyte aggregation is inhibited by nonbridging small polyglutamic acid, but not by small dextran. This fact suggests that the binding site of dextran on the erythrocyte surface is different from that of polyglutamic acid. Erythrocyte-binding site on fibrinogen mol-
a
One end of the macromolecule is already adsorbed on the surface of an erythrocyte The other end attaches to another erythrocyte when two erythrocytes are close together The bridging distance must be far enough to reduce the electrostatic repulsion between negatively charged erythrocytes Such bridging of macromolecules over a limited area 1990
25
Critical Reviews In ecule has recently been suggested to be located in a limited
area of the molecule (see Section IV.C.2.e.ii).232 Any quantitative method to differentiate binding molecules and bridging molecules should be devised for the quantification of bridging molecules leading to rouleaux . On the other hand, an osmotic stress theory was proposed recently by Evans and Needham on the basis of a model experiment for dextran-induced adhesion of giant bilayer vesicles.234 Further evidence on the rouleau formation of erythrocytes is needed for this theory. B. Measurements of Erythrocyte Aggregation Several methods have been developed for quantitative and semiquantitative measurements of erythrocyte aggregation. It may be convenient to classify the various methods into four categories: 1. 2. 3. 4.
Direct measurement in uniform shear flow Indirect measurement through viscometry Indirect estimate from erythrocyte sedimentation rate Others
1. Direct Measurement in Uniform Shear FIow In order to obtain several kinetic parameters, the timecourse measurements of erythrocyte aggregation under controlled shear rate are performed using a viscometer (with coneplate or coaxial cylinder) combined with optical means. SchmidSchiinbein and co-workers235*236 have measured the change in light transmission during erythrocyte aggregation (Ht = 45%), in a gap between the cone and plate of a viscometer at a controlled shear rate (1 to 460 s- ‘) and have proposed some empirical parameters. Usami and Chien237have measured the light reflection of an erythrocyte suspension (Ht = 45%) in a Couette flow using a coaxial cylinder and have introduced the “reflectometric aggregation index” (a comparison of reflectometric reading at 200 s- ’ and at a low shear rate). Recently, Donner et a1.238have developed an instrument composed of a Couette viscometer with coaxial cylinders and have measured the intensity of backscattered light for erythrocyte suspension at a shear rate of 5 to 500 SK’, and several kinetic parameters on erythrocyte aggregation are determined with a microcomputer. Sacks et a1.239have quantified the degree of erythrocyte aggregation under a constant shear rate with a rheoscope, classifying the shape of aggregates into five classes by the microscopic observation. Since the light transmission and light reflection do not necessarily provide the same information,243 all optical measurements should be standardized. Shiga et al. 240have constructed an apparatus for determining the averaged velocity of rouleau formation, combining a rheoscope, a video camera, an image analyzer, and a computer. The process of erythrocyte aggregation is successively analyzed in a constant shear rate and expressed by the time courses of the growth in the projected area of particles. The velocity of
26
rouleau formation is estimated by the computed increment of area/count, i.e., the growth rate of linear rouleau, of which the process is simulated by a kinetic model of linear polymerization.241 Murata and Secomb242 recently presented a mathematical model for rouleau formation in constant shear flow. Their prediction for the relationship between the average size of rouleaux and the applied shear rate well explain the experimental data of Shiga et al.24o A common advantage of these methods is a well-controlled shear rate during aggregation. Some apparatus are, however, not applicable for whole blood: the hematocrit an&or the number of cells in the suspension must be reduced, in order to count the number of particles. 240Theoretical analysis of erythrocyte aggregation241.242can be applied to the method of Shiga et a1.240 2. lndirecf Measurement by Viscometry When the erythrocytes aggregate, the viscosity of the erythrocyte suspension at low shear rates increases more than that at high shear;220thus the measurement of the viscosity of erythrocyte suspension is suitable for the estimation of erythrocyte aggregation. In addition to time- and/or shear rate-dependency of viscosity, dynamic analysis assessing erythrocyte aggregation is performed by viscometry, when a time-dependent shear rate (or oscillatory shear rate) is applied;244,X5these analyses inform viscoelastic behavior of erythrocyte aggregates and thixotropic behavior of blood. The viscometric method may reflect the process of erythrocyte aggregation in well-controlled shear flow. The hematocrit should be adjusted precisely in all methods. 3. indirect Estimate from Erythrocyte Sedimentation Rate The erythrocyte sedimentation rate (ESR) correlates to the readiness of erythrocyte aggregation.*& The maximum sedimentation rate (after the development of erythrocyte aggregates) is usually adopted, but must be corrected for the variation of hematocrit and the viscosity of media.247,248Several factors concern erythrocyte sedimentation and are theoretically treated.220.249*250 for example, the aggregation and dispersion in the gedimentation process, the behavior of sedimenting aggregates and counterflow of the medium, and the incline of the sedimentation tube. The rate of erythrocyte packing under accelerated gravitation using a centrifugal apparatus was measured by Rampling and Sits.251 Furthermore, Bull and Brailsfordz2 have developed an instrument, the Zetafuge’? the zigzag sedimentation of erythrocytes is induced in a near vertically oriented capillary tube by subjecting to four cycles of dispersion and compaction with a controlled centrifuge. The zeta sedimentation ratio (ZSR), an index of erythrocyte sedimentation,252*253is expressed as a percentage of hematocrit divided by zetacrit (the final level of
Volume 10, Issue 1
Oncology/Hematology erythrocyte compaction in such specified centrifugation). The ZSR is independent of hematocrit (this is superior to ESR)2s2 and provides a good assessment of disease activity because of linear response to changes of fibrinogen or gamma globulins.“2*2” The amount of blood and/or the time for the measurement is reduced. The phenomenon should be interpreted by Oka’szso theory on the erythrocyte sedimentation in inclined tube. The measurement of the erythrocyte sedimentation is still useful in clinic. However, the shear force cannot be controlled, and the growing process of erythrocyte aggregates cannot be measured. 4. Others Some methods observe the process of erythrocyte aggregation after an abrupt stop of high shear flow. Dintenfass et al.247*255 have taken microphotographs of blood between parallel plates of 12.5 pm slit at certain time intervals after the abrupt stop of the flow, and the shape of aggregates is analyzed by parameters reflecting particle size and surface/volume ratio. The apparatus has been employed in the space laboratory by Dintenfass.“6zz57Tomita et al. 258have measured the light transmission of whole blood in a transparent vinyl tube of 0.26 cm I.D.: the exponential decay after the abrupt stop of rapid flow at a wall shear stress of 500 s-’ is observed. Both methods can be applied for clinical use with whole blood, but the shear rate during aggregation is not controlled, and the effect of sedimentation of particles is not taken into account. Chien et al.259 have counted the erythrocyte aggregates formed in a hemocytometer under natural sedimentation and introduced a measure of erythrocyte aggregation, “microscopic aggregation index”. That is, the total number of erythrocytes (obtained in a Ringer solution) divided by the number of cell units (i.e., the total number of single erythrocytes and aggregates obtained in a macromolecule-containing medium). Chien et alz60 have measured the shear stress for disaggregation of rouleau in a parallel-plate flow channel under microscopic observation: the shear stress required to cause 50% separation (the disaggmgation force) of two-cell rouleaux formed in 4 g/d1 dextran with molecular weight of 74,500 is 0.25 dyn/ cm2. The echogenicity to ultrasound scanning increases with an increase in the erythrocyte aggregation.26’ Some of these methods well observe the shape and size of erythrocyte aggregates, but the shear rate is uncontrolled; thus a kinetic analysis of erythrocyte aggregation cannot be carried out by these methods. C. Determinant Factors of Erythrocyte Aggregation The counteraction of the aggregating force (i.e., the attractive force caused by macromolecular bridging between erythrocytes) and the disaggregating force (mainly due to the electrostatic repulsive force caused by the surface negative
charges of erythrocytes) is essential for erythrocyte aggregation. The factors affecting erythrocyte aggregation may be conveniently divided into four categories: 1. 2. 3. 4.
Erythrocytes Bridging macromolecules molecules Physical environment Chemical environment
and coexistent nonbridging
1. Eryfhrocyfes In this category, the influence of the number of erythrocytes (or hematocrit) in the suspension and the general features of erythrocytes on the erythrocyte aggregation is discussed. Although various physical and chemical changes in the erythrocyte environment alter the characteristics of erythrocytes, these factors are discussed later (see Section IV.C.3 and 4). a. NUMBER OF ERYTHROCYTES
(OR HEMATOCRIT)
When the number of erythrocytes in the suspension is increased, the erythrocyte aggregation is accelerated due to the increased frequency of collisions among the celIs.240,24’ In contrast, concerning erythrocyte sedimentation of whole blood, the higher the hematocrit, the slower the sedimentation rate, and vice versa.X7*248 The ZSR is independent of hematocritX2.253 (see Section IV.B.3). b. CELL SHAPE
When two erythrocytes come into contact, a larger contact area creates more stable aggregates. Thus, the curvature of erythrocyte surface is an important factor in erythrocyte aggregation. The shape transformation of erythrocytes affects the aggregation,240 since the formation of stable erythrocyte aggregates is geometrically impeded. Furthermore, the shape transformation alters the topography of negative charge Anionic amphophiles, themselves, increase the negsites. 262*263 ative charge on the erythrocyte membrane and diminish erythrocyte aggregation. 264 c.
DEFORMABILITY
OF ERYTHROCYTES
When the deformability of erythrocytes decreases, the propagation of point-to-point contact to form surface-to-surface contact between erythrocytes is impeded (see Section 1V.A. 1). The oxidative crosslinking of cytoskeletal proteins increases the bending stiffness of the membrane;265thus, flexible contacts among erythrocytes are suppressed. The necessity of flexible structure of spectrin network on erythrocyte aggregation has been evaluated using diamide [diazene dicarboxylic acid bis(N,N-dimethylamide)]. We have shown that the inhibition of erythmcyte aggregation is detectable in erythrocytes in which 5% of the total spectrin is crosslinked.13’ As described above (see Section III.B.3.b), the deformability of erythrocytes decreases evidently at 5% crosslinkage of spectrin. 13’
1990
27
Critical Reviews In d. SURFACE CHARGE OF ERYTHROCYTES AND CONFORMATION OF MEMBRANE SURFACE
Sialic
Studies on the electrophoretic behavior of human erythrocytes in a wide pH rangezM have revealed the presence of groups of pK N 2.6 and pK N 3.4, probably from the carboxyl groups of sialic acid and of acidic amino acid residues, respectively. 267These negative charges on the erythrocyte surface cause an electrostatic repulsive force among erythrocytes. The electrostatic repulsive force varies with the surface potential of erythrocytes, represented by the zeta potential that is calculated by the Helmholtz-Smoluchowski equation. When the viscosity and the dielectric constant of the fluid medium are constant, the zeta potential is proportional to the electrophoretic mobility of erythrocytes. The electrophoretic mobility varies proportionally to the content of sialic acid in erythrocytes.71.268 When the quantity of sialic acid in the erythrocyte surface is reduced by hydrolysis with neuraminidase, the electrostatic repulsive force between erythrocytes decreases225*269 and erythrocyte aggregation is accelerated.71.225 Increased erythrocyte aggregation is observed in patient of acquired sialic acid deficiency in erythrocyte membrane.27o The phenomena may be partly related to the increased surface-to-surface affinity of neuraminidase-treated erytbmcytes (in dextran and in plasma). 271 The contribution of sialic acid to erythrocyte aggregation has been quantitatively evaluated using fibrinogen7’ and dextrat? as the bridging macromolecules. Our systematic study of erythrocytes with various contents of sialic acid’] is shown in Figure 12. A 10% increase in fibrinogen concentration from 0.3 g/d1 (physiological concentration) accelerates the rate of aggregation 18%, whereas a 10% decrease in sialic acid content from 0.8 FmoYml erythrocytes accelerates the rate only 6%. Therefore, the contribution of the bridging force of fibrinogen to the rate of erythrocyte aggregation is greater than the repulsive force due to the negative charge of sialic acid in erythrocyte surface. The interaction of macromolecules on the surface of erythrocytes may induce a conformational change of surface proteins. For example, dextran induces a conformational change of glycophorin,272*273but has a minimal effect on membrane surface viscosity.274 8. BLOOD GROUPS The effect of ABO blood groups may be considered for the response in erythrocyte aggregation to various drugs.275 The erythrocyte aggregation induced by various immunoglobulin preparations (for clinical use; immunoglobulin G and the fragments produced by treatment with pepsin or plasmin) is distinctly different among blood groups in spite of the complete removal of blood type-specific globulins.276 Erythrocytes from 0 + type are more resistant to aggregation than those from other typesn6 Interaction with fibrinogen may also be different among blood groups. *” The reason(s) for this phenomenon and the relevance to in vivo circulation are unknown.
28
I
o-2 Slallc
I
I
acid
I
0.4 0.6 08 acid (pmoles/ml RBC)
0.1 0.2 0.3 Fibrinogen (g/d\)
0.4
FIGURE 12. Effect of sialic acid content (in erythrocyte membrane) and fibrinogen concentration (in suspending medium) on the velocity of erythrocyte aggregation. Measured in isotonic phosphate-buffered saline containing fibrinogen and 5 g/d1 albumin (pH 7.4) at the shear rate of 7.5 s-’ at 25°C. The velocity increased with the increase in fibrinogen concentration and with the decrease in sialic acid content. (From Maeda, N., Imaizumi, K., Sekiya, M., and Shiga, T., Biochim. Biophys. Acra, 776, 151, 1984. With permission.)
2. Bridging Macromolecules and Coexisting Nonbridging Molecules The concentration, the molecular size, the molecular conformation, and the electric charge of bridging macromolecules are important determinants in erythrocyte aggregation. In addition, coexisting molecules may impede the bridging of macromolecules, either directly through competition at the same binding site on the erythrocyte surface, or indirectly (noncompetitively) through a stereochemical impediment.
a. CONCENTRATION OF MACROMOLECULES
When the concentration of macromolecules in the erythrocyte suspension is increased, erythrocyte aggregation is accelerated225*23’~232 due to the predominant effect of increased macromolecular bridging. However, it is observed for many bridging macromolecules (i.e., dextran, fibrinogen, etc.) that further increase of macromolecular concentration suppresses The suppressed erythrocyte the erythrocyte aggregation. 225*23’ aggregation at high concentration of bridging macromolecules is interpreted as follows: 1.
2.
3.
Shear stress is increased by the augmentation of bulk viscosity due to the macromolecules added and erythrocyte aggregation is prevented physically.225,231 The increased bridging (and/or binding) of macromolecules causes a local reduction of the effective ionic strength on the surface of erythrocytes due to the volume exclusion effect.*‘* Since the reduction of ionic strength in the suspending medium increases the electrostatic repulsive force, erythrocyte aggregation is inhibited.**’ The increased concentration of macromolecules increases
Volume 10, Issue 1
Oncology/Hematology the number of bound molecules on the etythrocyte surface,279but the number of bridging macromolecules must be decreased by substituting the one binding end of bridging molecules for the nonbridging macromolecules (which bind the erythrocyte surface only at one end of the molecule) .280
c. MOLECULAR CONFORMATION The molecular conformation may be one of the important factors affecting the shape of erythrocyte aggregates. We have recently found that
1.
Figure 13 shows the shear-rate dependency of the relation between the velocity of erythrocyte aggregation and the dextran concentration. Although the velocity of erythrocyte aggregation decreases with an increasing shear rate, the dextran concentration giving the maximum velocity of erythrocyte aggregation is constant. *‘OThis result shows that the binding isotherm and/or bridging behavior of macromolecules is not altered by the shear force.
2.
3.
Pullulan, a linear polysaccharide, accelerates the erythrocyte aggregation more strongly than dextran (with same molecular weight), a branched polysaccharide280 F(ab’),, a product of immunoglobulin G (Ig G) hydrolyzed by pepsin, accelerates the erythrocyte aggregation more than Ig G282in spite of the lower molecular weight, probably due to the different molecular properties, that is, F(ab’), is more flexible at the hinge region than Ig G and more positively charged than Ig G Fibrinogen, a fibrous molecule, tends to form the ldimensional rouleaux,241v284 while Ig G, a globular molecule, preferably produces 3-dimensional aggregates 240.241.284
d. ELECTRIC CHARGE OF MACROMOLECULES
Positively charged macromolecules, such as polylysine, bridge easily between negatively charged erythrocytes, presumably by ionic interaction. *” Negatively charged macromolecules, such as polyglutamic acid (with a molecular weight higher than 50,000)23’ or heparin (at higher concentration than 1 g/d&*= interact at the sites of nonionic and/or positively charged regions of the erythrocyte surface. Polyglutamic acid with a molecular weight of 20,000 does not induce erythrocyte aggregation, but polylysine with a molecular weight of 9000 still strongly aggregates erythrocytes .231
-5
-4
-3
8. BRIDGING MACROMOLECULES AND COEXISTING
-2
MOLECULES
log [dextran, Ml
The bridging macromolecules interact at the two ends with two adjacent erythrocytes. However, small molecules, which cannot bridge between erythrocytes, but can bind to the erythrocyte surface, occasionally impede the bridging of macromolecules.
flGURE 13. Dependency of the velocity of erythrocyte aggregation on (1) the molecular weight of dextran, (2) the concentration of dextran, and (3) the shear rate. Measured in isotonic phosphate-buffered saline (pH 7.4) containing dextran (T-500, 494,000 in molecular weight; T70,70,400, T-40,42,500; T-10, 10,300) and 0.5 gkll albumin at 25°C. (From Seike, M., J. Jpn. Sm. BloodTransfusion,34,420, 1988. With permission.)
i. Plasma Proteins
Fibrinogen, Ig G, and albumin contribute to erythrocyte aggregation.
b. MOLECULAR SIZE
Fibrinogen (and related compounds) - Fibrinogen is a fibrous molecule with a molecular weight of 340,000 with trinodular structure. The molecular length is 47.5 nm, and the diameter of the terminal nodules (D-domain) is 6.5 nm. The number of fibrinogen molecules bound to a single erythrocyte in the physiological concentration is about 20,000.286 With an increase in the concentration of fibrinogen, erythrocyte aggregation is accelerated.283~287 As shown in Figure 14, we have suggested that the erythrocyte binding site in a fibrinogen molecule leading to erythrocyte aggregation is mainly in the carboxy-terminal region of the A-alpha chain near the D-domain (residue No. 207-303)
Among similar molecular species, the larger molecules are more effective in erythrocyte aggregation than the smaller molecules. These phenomena have been observed for artificial macromolecules such as dextran,22s~231polyglutamic acid,23’ and polylysine.231*28’The increased aggregation with the increased size of macromolecule is due to (1) the increase in the number of bonds per macromolecules on the erythrocyte surface and (2) the decrease in electrostatic repulsive forces due to the increased intercellular distance.225*226 Various high-molecular-weight plasma proteins (fibrinogen, alphq-macroglobulin, immunoglobulins) accelerate the erythrocyte aggregation depending on the molecular size.282~i83 1990
29
Critical Reviews In AJ chain C-terminal
extension
Termi nai domal n
Central domain HMW(native)-Fibrinogen Protease
? 1e
by-d 6-b Fragment
LMW-Fibrinogen t
[Neurami
n i dose
&&&.
\
Fragment
Desialylated
X
Fibrinogen
Fragment
D
Y
Fmgment
Fragment
D
E
FIGURE 14. Schematic diagram of fibrinogen molecule and the related compounds produced by enzymatic hydrolysis. An erythrocyte-binding site in the fibrinogen molecule (marked with an arrow) is proposed as the cause of erythrocyte aggregation. (From Maeda, N., Seike, M., Kume, S., Takaku, T., and Shiga, T., Eiochim. Biophys. Acta, 904, 81, 1987. With permission.)
and partly in the carboxy-terminal region of the gamma chain (residue No. 375-41 1).232This conclusion comes from the following evidence 1.
2. 3.
The low-molecular-weight fibrinogen (mol wt = 305,ooO, partial degradation product of fibrinogen in plasma) has the same effectiveness in erythrocyte aggregation as native, high-molecular-weight fibrinogen (mol wt = 340,000)~3* Sialic acid in the fibrinogen molecule does not contribute to the interaction with erythrocytes232 Among fibrinogen degradation products (FgDP) produced by plasmin, FgDP-X (mol wt = 250,000) and FgDP-Y (mol wt = 150,ooO) still induce erythrocyte aggregation with decreased rate, but FgDP-D (mol wt = 90,000) and FgDP-E (mol wt = 50,000) do notu2~288~289
Immunoglobulin G (and the related compounds) - Ig G is a Y-shaped molecule with a molecular weight of 150,000. The molecular length is about 20 nm, and the dimensions of the cylindrical Fc and Fab portions are 4.0 nm (in diameter) x 4.5 nm (in length) and 3.5 nm (in diameter) x 6.0 rmr (in length), respectively. Ig G is important together with fibrinogen in accelerating erythrocyte aggregation by the nonimmunolVolume
ogical binding on erythrocyte surface. The number of Ig G molecules nonimmunologically bound to a single erythrocyte at a physiological concentration is about 400,000.2W On the other hand, autologous Ig G immunologically (or specifically) binds to erythrocytes, and the number of Ig G is about 200 SectionV.3.f.). Thenonspecific per erythrocyte 291*291a(alsosee binding of Ig G, which can induce the erythrocyte aggregation, differs from the immunological (or specific) binding of Ig G. F(ab’), (molecular weight 92,ooO, a degradation product of Ig G hydrolyzed by pepsin) remarkably accelerates erythrocyte aggregation.282 Fab and Fc (molecular weight 45,000 and 50,000, respectively; the degradation products of Ig G produced by plasmin) cannot induce erythrocyte aggregation themselves,282~292 but can inhibit the Ig G-induced erythrocyte aggregation.276*292 Inhibition by Fab is more effective than that by Fc,*~*probably because Fab is more positively charged than Fc . Fab and Fc also inhibit the erythrocyte aggregation induced by F(ab’)2.292 In dealing with Ig G and the related compounds (as well as fibrinogen) in the study of erythrocyte aggregation, immunologically specific agglutinin should be removed.276~282~292 serum albumin weight Albumin 66,000) ellipsoidal protein a dimension 15 nm length and nm in This molecule binds to
10, Issue 1
Oncology/Hematology the erythrocyte surface [the number of bound molecules adsorbed on a single erythrocyte in the physiological concentration is about 5 X 1W (293)], but is too short to bridge between erythrocyte by overcoming the electrostatic repulsion, thus the erythrocyte aggregation is not induced by itself. However, albumin shows the opposite effect on the erythrocyte aggregation induced by Ig G and fibrinogen, as shown in Figure 15.
I
Low
albumin
High
albumin
FIGURE 16. A model for the opposite effect of albumin on the fibrinogen- and Ig G-induced erythrocyte aggregation. Fibrinogen, Ig G, and albumin molecules anz expressed by hatched, dotted, and vacant frames, respectively. (From Maeda, N. and Shiga, T., Biochim. Biophys. Actu. 855, 127, 1986. With permission.)
r
I 1
I
1
I
2
3
4
Albumin
ment for the bridging of Ig G by albumin adsorbed on the erythrocyte surface.
(g/d11
FIGURE 15.
Effect of Ig G and fibrinogen on the velocity of the erythrocyte aggregation under various concentrations of albumin. Measured at a shear stress of 0.1 dyn/cm2 at 25°C. Albumin showed an opposite effect on erythrocyte aggregation: with Ig G it suppressed the velocity, while with fibrinogen it accelerated. (From Maeda, N. and Shiga, T., Biochim. Biophys. Acra, 855, 127, 1986. With permission.)
The erythrocyte aggregation induced by Ig G is inhibited by albumin, while that induced by fibrinogen is accelerated by albumin.283 An explanation for the opposite effect of albumin for Ig G- and fibrinogen-induced erythrocyte aggregation is schematically shown in Figure 16. In the physiological concentration, albumin, Ig G, and fibrinogen occupy the area of about 70,8, and 0.8 km2 on the erythrocyte surface, respectively (as roughly calculated from the data cited above). Thus the increase of albumin may prevent the bridging of Ig G spatially, but it may not interfere with the binding of fibrinogen for the sake of the small occupied area. When the erythrocyte surface is fully covered with albumin, the distance between erythrocytes is at least 30 nm (= 15 nm x 2). The molecular length of Ig G is less than 30 nm, while that of fibrinogen is more than 30 run, thus bridging by Ig G is prevented by albumin, but that by fibrinogen is not. In the physiological concentration of albumin, the inhibition by albumin becomes effective with an increase in the concentration of Ig G. The inhibitory effect of albumin for Ig Ginduced erythrocyte aggregation may be the physical impedi-
ii. Artificial Macromolecules
Some neutral polysaccharides, such as dextran, hydroxyethyl starch, etc., are clinically used as plasma substitutes for the supplement of colloid osmotic pressure in the circulatory blood.294 Polysaccharides - The critical molecular weight of dextran in leading to erythrocyte aggregation is about 40,000. The erythrocyte aggregation induced by dextran with a molecular weight above 70,000 is inhibited by dextran with the molecular weight below 40,000, by disaccharides, and even by monosaccharides.231*280 The mechanism of inhibition may be (1) the competition of bridging macromolecules and nonbridging molecules at the same binding site23’ and (2) the decrease of effective ionic strength in the contact surface between erythrocytes (due to the volume exclusion effect) and in the suspending medium. 225*295 PolygMamic acid - Erythrocyte aggregation is induced even by negatively charged polyglutamic acid (at least, of the molecular weight above 50,000). Erythrocyte aggregation induced by polyglutamic acid with a molecular weight of 50,000 is inhibited by polyglutamic acid with a molecular weight of 8000, but is accelerated by that of 20,000.23’ The reason(s) for the inverse effect of these nonbridging polyglutamic acids on the polyglutamic acid-induced erythrocyte aggregation is not yet known. 3. Physical Environments
a. SHEAR RATE The
1990
mechanics of the encounter of two blood cells in a 31
Critical Reviews In uniform shear flow has been analyzed by Goldsmith et al.*% With an increase in the shear rate, the collision frequency among cells increases. Therefore, at low shear rates the erythrocyte aggregation may be accelerated with an increase in the shear rate.““’ However, further increases of the shear rate prevent cell-to-cell adhesion and disperse the aggregates, due to increased shear stress.23’~240Such shear rate dependence of rouleau formation, (1) the increase of the velocity of rouleau formation with increasing shear rate in the low shear region and (2) the decrease of the velocity with further increase of shear rate, have been theoretically predicted.242 In oscillatory flow, even as low as 0.001 Hz, the motion accelerates erythrocyte aggregation. 297
L 0
I
/
I
20
30
40
Temperature
(“C)
FIGURE 17. Temperature dependency of the velocity of erythrocyte aggregation in autologous plasma. Measured in 70% autologous plasma + 30% isotonic phosphate-buffered saline (pH 7.4), at the shear rate of 7.5 s-’ at 25°C: different symbols from different donors. At 15 to 18”C, the velocity became minimum.w.“8
b. VISCOSITY
Macromolecules increase the viscosity of a suspending medium at high concentration. The increased viscosity inhibits the erythrocyte aggregation due to increased shear stress. When macromolecules are added to the suspending medium, the correction for the increased shear stress must be made.23’ c. TEMPERATURE With an increase in the temperature, the erythrocyte aggregation induced by various macromolecules (fibrinogen, Ig G, dextran, and polyglutamic acid) is accelerated.284*298The erythrocyte sedimentation rate increases at high temperature. 247 The temperature dependency of erythrocyte aggregation in autologous plasma is shown in Figure 17. The rate of erythrocyte aggregation is minimal around 15 to 18°C and then increases both below and above this temperature.284*298As shown in Figure 18, the shape of erythrocyte aggregates is 1-dimensional rouleaux above 15 to 18”C, while those aggregates below 15 to 18°C are 3-dimensional.284*298The temperature-dependent shape difference of erythrocyte aggregates in plasma is similar to those induced by fibrinogen.284 The erythrocyte aggregates induced by Ig G are 3-dimensional in all temperature ranges.284 The temperature dependency of the shape of erythrocyte aggregates is interpreted by the following terms:
1
10
creases with a decrease in the temperature, probably due to decreased membrane fluidity22.43and decreased mobility of membrane proteins3@ and lipids.30s The membrane elastic modulus and the membrane surface viscosity increase with decreasing temperature.306 At low temperature, the increased binding force of macromolecules to erythrocytes induces the 3-dimensional aggregates, together with the difficulty in transition from face-torim to face-to-face adhesion due to the decreased surface area to volume ratio and the decreased deformability. The temperature dependency of erythrocyte aggregation may be important for understanding the microcirculation in hypothermic tissues (e.g., for the exposure to a cold environment, under hypothermic anesthesia, under extracorporeal perfusion) or hyperthermic tissues (e.g., in metabolically active tissues, for exposure to a hot environment). 4. Chemical Environments
1.
2.
3.
32
With a decrease in temperature, the viscosity of suspending medium increases; thus the increased shear stress inhibits erythrocyte aggregation. 284 Properties of macromolecules - The reversible conformational change of macromolecules with temperature is observed for fibrinogen299 and Ig G;300thus the interaction of these macromolecules with erythrocyte surfaces may change, although fibrinogen binding is independent of temperature. 286 Properties of erythrocytes - With a decrease in temperature, the diameter of erythrocytes decreases and the thickness increases, but the cell volume does not change;284~301 thus the surface area to volume ratio decreases. The erythrocyte deformability302*303also de-
Mechanical
shearing force -
a. pH
With an increase in pH of the suspending medium, erythrocyte aggregation is accelerated.20s*240~298 The erythrocyte aggregates induced by fibrinogen are l-dimensional rouleaux in alkaline pH, but are 3dimensional aggregates in acidic pH.20s.298 The facilitation of erythrocyte aggregation in alkaline pH may be interpreted as follows: 1.
2.
- With increasing pH, the diameter of erythrocytes increases, the thickness decreases, and the cell volume decreases.20s*30**~7 The increased surface area to volume ratio of erythrocytes accelerates erythrocyte aggregation. &u&ace charge of erythrocytes - No change in the electrophoretic mobility of erythrocytes (thus, in the surface Shape of erythrocytes
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Oncology/Hematology
Plasma
Fibrinogen
.-
18OC
FIGURE 18. Microphotographs of etythrocyte aggregates formed in different media and at different temperatures. Aggregates formed after 10 min under a shear rate of 7.5 s-’ in 0.4 g/dl fibrinogen + 5 g/d1 albumin (fibrinogen), in 0.4 g/d1 Ig G + 5 g/d1 albumin (Ig G), and in 70% autologous plasma + 30% isotonic phosphate-buffered saline (plasma). Ig G tended to form 3-dimensional aggregates, whereas fibrinogen induced linear rouleaux except below 15°C. (From Maeda, N., Seike, M., and Shiga, T., Biochem. Biophys. Am, 904, 319, 1987. With permission.)
3.
4.
charge) is observed in the pH range of 3.0 to 9.5 at ionic strength 0.145. 266 Therefore, the electrostatic repulsive force due to the negative charge of the erythrocyte surface is constant in the physiological pH range. Eryrhrocyre deformability - The elastic property of the erythrocyte membrane, i.e., the membrane elastic modulus, is altered below pH 6.0.206~308 Interaction of macromolecules with erythrocyte su$ace - The binding of fibrinogen increases with decreasing pH below 6.5.286
b. IONIC STRENGTH
AND IONIC COMPOSITION
The decrease of ionic strength in the suspending medium expands the electric double layer of the surface of the erythrocyte membrane and increases the electrostatic repulsive force 225.278
Divalent cations, compared with monovalent cations, reduce the thickness of the electric double layer, and the decreased electrostatic repulsive force between erythrocyte surface accelerates erythrocyte aggregation.278 c. OSMOLALITY With decreasing osmotic pressure in phosphate-buffered saline from 285 to 190 mOsm, the velocity of erythrocyte aggregation decreases about half.298.3w On the other hand, with increasing the osmotic pressure to 400 mOsm, the velocity increases about twice, but it decreases in hypertonic media above 400 mOsm.298.309 The erythrocyte aggregation induced by fibrinogen is essentially l-dimensional rouleaux, but is 3dimensional below 200 mOsm.298 The increased erythrocyte
Altogether, the change of erythrocyte aggregation with pH is mainly due to the shape change of erythrocytes and partly due to the changes of erythrocyte deformability and macromolecular binding. The physiological importance of such pH dependency may be found in the microcirculatory flow through metabolically active tissues (with lowered pH): the decreased erythrocyte aggregation in a low pH facilitates the blood flow in such tissues.
1990
33
Critical Reviews In aggregation with increasing osmolality is due to several causes as follows:
Table 1 Symptoms of Hyperviscosity Syndromes
1.
Cardiovascular symptoms
2. 3. 4.
The shape change from spherical to disk-like (i.e., the increased surface area to volume ratio)298~30’ The increased defonnabilitymO’ The decreased electrostatic repulsive force due to the increased ionic strength225.278.295 The increased binding of fibrinogen to erythrocytes (above isotonicity)286
Concerning the effect of cell volume on the erythrocyte aggregation, Nash et al. 3’o have observed that the erythrocyte aggregation increases with a decrease in the cell volume up to 80% of normal (without changing either the osmolality or ionic strength of suspending medium by using valinomycin), but the aggregation decreases with further decrease of cell volume. The decreased aggregation above 400 mOsm in saline298 or above 600 mOsm in plasma3” may be mainly due to the decreased deformability, since the intracellular hemoglobin concentration increases under hypertonic condition. The phenomena may be important for understanding the blood flow under hypertonic tissues (e.g., renal medulla).
Retinal symptoms
Hemorrhagic symptoms Neurological symptoms
Decreased pulse pressure, peripheral edema, Cardiac inSUffICienCy(in prOgre88cdState) Sausage-shaped venous distension, flameshaped hemorrhage, capillary microaneurysms (retinopathy) Mucous membrane bleeding (from nose, gum, etc.) Headaches, vertigo, nystagmus, impaired hearing, etc.
Table 2 Causes of Hyperviscosity Syndromes CPuseS
Increasednumber of erythrocytes Decreased erythrocyte deformability
Accelerated erythrocyte aggregation
V. ERYTHROCYTE RHEOLOGY IN HEALTH AND DISEASES The rheological properties of erythrocytes are impaired in various diseases and in vivu aging. In this section, only brief consideration is given to (1) hyperviscosity syndrome, (2) abnormality in erythrocyte deformability, and (3) decline of deformability during in vivo aging of erythrocytes. A. Hyperviscosity Syndrome Increase of blood viscosity may induce circulatory disturbances either locally or systematically.312-3’5Typical symptoms summarized in Table 1 mostly concern the impeded flow in venous systems and are independent of the primary cause of the disease. These symptoms are caused by (1) increase in peripheral circulatory resistance, (2) hindrance in blood passage through the microvessels, and (3) stasis in the venous circulation due to the increased blood viscosity. The rheological causes for these symptoms are summarized in Table 2, that is, (1) an increased number of erythrocytes, (2) a decreased erythrocyte deformability, (3) an accelerated erythrocyte aggregation, and (4) others.8~223~224~31s B. Abnormality in the Erythrocyte Deformability The following various causes are considered here as typical examples: 1. 2.
The abnormality of cytoskeletal proteins The abnormal hemoglobins
Accelerated coagulation or agglutination Others (with various causes)
3. 4.
Disorders Polycythemia Hemoconcentration (dehydration), etc. Hereditary disorders with abnormal cell shape Unstable hemoglobinopathy Sickle cell anemia Formation of Heinz body, etc. Hyperfibrinogenemia Multiple myeloma Macroglobulinemia Cryoglobulinemia, etc. Hypercoagulability Incompatible blood transfusion, etc. Diabetes mellitus Myocardial infarction Raynaud phenomenon, etc.
The morphological abnormality of erythrocytes The formation of Heinz bodies
Although there are many diseases accompanying impaired erythrocyte rheology and leading to hemolytic anemia, 19*316*317 only a few cases are described. 7. AbnormaMy in the Cytoske/eta/ Proteins The genes of human alpha- and beta-spectrins are located at the chromosomes lq22-25 and 14~32, respectively. The gene of band 4.1 protein is located at the chromosome lp3436. The abnormality of DNA in these genes sometimes (but not always) induces an abnormality of corresponding protein, which often results in a morphological alteration of erythrocytes through abnormal cytoskeletal architecture and/or impaired interaction among cytoskeletal proteins, e.g., elliptocytosis, spherocytosis, pyropoikilocytosis, etc. These cells are poorly deformable, and thus are easily destroyed in circulation to induce anemia. These days, as summarized in a recent review in this journal, l9 hereditary abnormalities in cell shape are being clarified rapidly. However, it is too early to
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Oncology/Hematology draw any conclusions concerning the critical lesions in polypeptides that cause shape alteration, hemolysis, etc. Thus, more fundamental data of molecular pathology are needed.
4.
2. Abnorm8l Hemoglobins Intravascular infarction is induced in hemoglobin S disease, when the sickling of erythrocyte occurs upon deoxygenation or the irreversible sickle cells are formed.318The decreased deformability of the sickled erythrocytes is noticed. The increased membrane elastic modulus of the erythrocyte, the prolonged extensional recovery time, and the increased membrane viscosity are also observed, especially for the irreversibly sickled cells.3’9 In some cases of abnormal hemoglobin, e.g., hemoglobin K61n,320 decreased filterability is reported. If hemoglobin molecules are unstable, the resultant denatured hemoglobin forms Heinz bodies in the cell and the deformability is impeded.
5.
These changes are common features for dense erythrocytes fractionated by density gradient centrifugation. It is reasonable that metabolic activity in erythrocytes decreases in some extent during aging in circulation, and that the decreased activity accelerates above changes. However, contradictory results are sometimes reported on the age-associated changes (e.g., ATP content does not vary during aging). One should take the following points into consideration to interpret the results: 1.
3. Formation of Heinz Bodies Heinz bodies are small inclusions of dense masses (diameter of 0.3 to 2 km) attached to the membrane and stained with May-Grtinwald-Giemsa. Heinz bodies are composed of denatured proteins, mostly hemoglobin (or globin) and are observed in many hereditary diseases, for example, in thalassemias, in unstable hemoglobinopathies, and in hereditary defects of hexose monophosphate shunt (under oxidative stress). The denatured hemoglobin is usually bound to the membrane, thus the deformability is greatly impaired.81J’2~321
2.
3. 4. 5. 6.
4. Abnormality in Erythrocyte shape The other diseases inducing a shape alteration of erythrocytes are sometimes accompanied by decreased deformability. Figure 19 shows the schizocytes in acquired hemolytic anemia and the appearance of undeformable cells under high shear stress (observed with a rheoscopic flash photograph). In some cases of disturbances in lipid metabolism, acanthocytosis is induced. However, the deformability of these cells is not always impaired, although hemolytic anemia occurs.
2. 3.
The method adopted for cell fractionation (e.g., according to cell density, cell size, or surface charge) The media used for cell fractionation (such as albumin, high-molecular-weight polysaccharide, phthalate, or Percoll) The percentage of fractionation of cells The animals used for the experiment (different survivals in different animals) The enzymes and metabolites analyzed (in association with aging) The amount of reticulocytes
One should be especially careful with the concomitant veticulocytes in the preparation to deal with the age-dependent phenomena, since the decay of most enzyme activities is very rapid in reticulocytes (and perhaps in very young erythrocytes), but becomes very slow in mature cells.w.322 The rheological impairments in aged erythrocytes are induced by complex reasons (not a sole cause). The decreased deformability of aged cells is observed by rheoscopy,199.323.324 filterability,325.326and micropipette aspiration.78*163~179~327 The impaired deformability of aged cells originates from increased membrane viscosity79*‘79.328 and increased intracellular viscosbut the membrane elastic modulus does not vary durity, ‘79*329 ing aging. 79~179,328 We briefly describe the major disturbances one by one.
C. Decline of Deformability During In Viva Aging of Etythrocytes Aged erythrmytes are removed from circulation by phagocytosis in the reticuloendothelial system, especially in spleen. The decreased deformability and the increased adhesiveness of erythrocytes are major determinant factors for the removal of aged cells in spleen. The following changes are induced in erythrocytes during aging: 1.
ulated perhaps by intracellular calcium ion and by protein phosphorylation The condensation of hemoglobin, i.e., increased intracellular viscosity, due to the decrease of surface area and the loss of water The appearance of senescent antigenic sites and the increased binding of Ig G on the surface of aged erythrocytes
1. Ch8ngeS in Metabolic Components
Activity 8nd lntr8cellul8r
During erythrocyte aging, many enzymes (and transport proteins) show decreased activity; thus the rate of glycolysis is decreased, and the concentrations of various metabolites and decreased 2,3-DPG (thus increased inorganic ions change..3*4*89 oxygen affinity of the aged erythrocytes),78~163~330 increased methemoglobin content, 33l increased Na+ and decreased K+,330 increased Ca+ + ‘99*332 (with the decreased calmodulin activ-
The decrease in surface area and volume due to the loss of membrane components The oxidative modification of membrane components (both lipids and proteins) to stiffen the membrane Modification of the cytoskeletal dynamic structure, reg1990
35
Critical Reviews In
FIGURE19. Deformation of erythrocytes from autoimmune hemolytic anemia, observed with a rheoscope. (Top) Abnormal cells observed with a scanning electron microscope. (Bottom) Cell deformation at a shear stress of 70 dyn/cm* with rheoscope: undeformable cell shown by arrow (autoantibody of warm type is detected). (From our unpublished data.) .& W33*334
&&
&+
+,332.335 decreased
ATp,78.330.332.336
and so on. The creatine level in erythrocytes, which is higher in young cells than in aged cells, correlates well with erythrocyte survival in circulation.337 2. Loss of Membrane Components When the loss of membrane components exceeds the loss of intracellular proteins, the surface area to volume ratio inevitably decreases. As shown in Figure 20, the shape of the aged cells, separated by density gradient centrifugation, shows a
36
smaller diameter, smaller volume, and more spherical appearof ance than that of the young cells. 78.79.163.199.338 The mount membrane lipids certainly decreases during in vivo aging.78*89*339 However, there is apparent disagreement about the quantity of total proteins. 43*78In general, it has been believed that both lipids and membrane proteins are decreased during aging, but we have observed only a minute loss of proteins. The discrepancy concerning the amount of membrane proteins may ~ arise from the differences in fractionation of erythrocytes and i preparation of ghosts (we discarded the
Volume 10,
1
Oncology/Hematology
FIGURE
20.
Young erythmcytes(top) and aged erythrocytes
a scanning electron microscope. gradient centrifugation.78~19
The human erythrocytes
fraction, although less than 1% of total cells);” in methods quantifying membrane phospholipids and proteins; and in the conversion of the measured values to the contents of phospholipids and total protein (the amounts of proteins are often expressed by “albumin-equivalent weight”, of which no standard is given; the amounts of phospholipids are rather arbitrarily converted from the values of phosphate assay, without analyzing the composition of phospholipids). The decreased membrane fluidity of the in vivo aged eryth-
(bottom), observed with were fractionated by density
rocytes is observed by the electron spin resonance using fatty The alteration of protein-lipid interaction acid spin label. 43*78~163 may contribute to decreased fluidity, thus to increased membrane viscosity.79”79 3. Peroxidative Alterations of Membrane
Components Erythrocytes contain glutathione and possess many antioxidative enzymes, for example, glutathione peroxidase, cat1990
37
Critical Reviews In alase, and superoxide dismutase. However, the activity of the enzymes decreases during in viva aging,89*340*341 thus the ability of antioxidation diminishes gradually.342 Although the crosslinkage of membrane proteins is not well proven in aged erythrocytes, the extractability of spectrin (with 1 nN EDTA, pH 8) decreases,343 suggesting the modification of cytoskeletal networks due to aging. Increase of hemoglobinbound spectrin,344 decreased ratio of protein 4.lb to protein 4 . la ,415*Nincrease in carboxymethylation347and racemization~ of membrane proteins are demonstrated in aged cells. Some of these modifications contribute to the increased membrane viscosity of aged cells in part. There is some doubt about the contribution of MDA in polymerization of membrane proteins in aged cells349*350 as a major oxidative reagent, since the thiobarbituric acid reactivity is not altered during erythrocyte aging. On the other hand, exposure of erythrocytes to MDA as model studies well resembles the changes in lipids, e.g., the detection of phospholipid adducts of MDA and the increase of phosphatidyl-choline (also the decrease of phosphatidyl-serine).46 The increased ratio of alpha-tocopherol to phospholipids (especially arachidonic may protect cells from lipid peroxiacid) in aged cells351*352 dation to some extent. Further studies on the lipid-protein interaction, as well as searches for oxidative influences on cytoskeletal structure, are necessary. 4. Retention of Calcium and Changes in Cytoskeletal Structure Intracellular calcium content increases with erythrocyte aging,lw primarily because of the declined ability of the calcium extrusion during aging. Moreover, a report showed the enhancement of calcium permeability under high shear flo~.~” The entering calcium is extruded by calcium pump at the cost of ATP,355 and the transport is activated by Ca-calmodulin356 that is formed with an increase in the calcium content. Model studies to increase the intracellular calcium with ionophore have been carried out and the decreased deformability was unanimously observed in calcium-loaded cells.199 However, as calcium-loading progresses, Ca+ +-dependent Kchannel is activated; consequently a loss of potassium and water occurs357 (called the Gardos effect’98), and further ATP depletion and echinocytic transformation appear. 199.357-359 In addition, upon calcium overloading, the crosslinkage of membrane proteins (yielding glutamine-lysine bond)360 develops by the activation of transglutaminase. We have carried out a careful calcium-loading to fractionated erythrocytes (without shape change and ATP depletion)“s*‘99 and demonstrated that (1) calcium retention occurs easily with aged cells, but scarcely with young cells ;I9 (2) with an increase in the intracellular calcium, the cell volume decreases, thus the deformability is reduced;‘99 (3) after correcting the cell volume (and, consequently, the intracellular viscosity) by modifying the osmolality of medium, the deformability apparently changes between two states”’ (see Figure 1 l), i.e., high calcium creates less de38
formable cells and low calcium, more deformable cells; and (4) the decreased deformability of calcium-loaded erythrocytes can be restored perfectly with some calmodulin inhibitors. ’ l5 In a high calcium state, calcium-bound calmodulin is increased and the complex interacts with spectrin, band 4.1 protein, and/ or actins,96*1’4*218*2L9~361 then the properties of the cytoskeletal network are modified in such a way that the membrane stiffness increases. Calmodulin inhibitors prohibit such interactions through binding with the calcium-calmodulin complex.362*363 It is almost certain that the concentration of intracellular calcium has a key role for the regulation of the cytoskeletal dynamic structure, thus for the control of deformability. However, (1) the determination of intracellular free Ca+ + is hard when compared with that of total calcium; (2) the amount of total calcium in erythrocytes”.364,365is now becoming disputable; (3) the role of Mg+ + with ATP must be considered for the Ca+ + extrusion;366and (4) the distribution of calcium inside the erythrocyte367 must be studied further. 5. Condensation of Hemoglobin As the surface area decreases due to a loss of membrane components, but the intracellular proteins are preserved, the condensation of hemoglobin inevitably results with aging. The decreased activities of ion channels and ion pumps accelerate the dehydration. The increased intracellular hemoglobin concentration with cell age makes it possible to fractionate these erythrocytes by density gradient centrifugation.336*368-370 The increase in the intracellular hemoglobin concentration greatly affects the deformability of erythrocytes, as discussed above (see Section III.B.2). 6. Modification of Surface Structure During in vivo aging, the surface structure is (chemically) modified, as clarified by glycosylation of membrane proteins,37’ decreased number of insulin receptor,372-374decreased activity of acetylcholine esterase,89r37’decreased ouabain bindthe induction of senescent antigens,378-380and ing sites, 376,377 the resultant increase of Ig G (antibody) binding to such antigens. 381-384 The recognition of senescent erythrocytes by phagocytes is important in the destruction of these cells in the reticuloendothelial system. However, (1) the biochemical changes in erythrocytes to induce the senescent antigens (i.e., due to the destruction of band 3385and/or the desialation of glycophorin A3*6), (2) the biochemical structure of the senescent antigens appeared on the erythrocyte surface, and (3) the mechanism of the recognition of senescent erythrocytes by phagocytes are not known at the moment. 7. Changes in Adhesiveness In addition to the recognition of aged erythrocytes by phagocytes, adhesion of these erythrocytes to endothelial cells and the impeded passage due to their decreased deformability is an important moment in the trapping in the meshwork of splenic pulp, thus in the determination of the lifespan of individual
Volume 10, Issue 1
Oncology/Hematology 6. Marchesi, V. T., Stabilizing infrastructure of cell membranes, Annu. Rev. Cell Biol., 1, 531, 1985. 7. Fknoett, V., Cohen, C. M., Lux, S. E., and Palek, J., Eds., Membrane Skeletons and Cyroskeleral-Membrane Associations. Alan R.
erythrocytes. As a mechanism of erythrocyte destruction, the protective role of sialic acid has been studied extensively: upon removal of sialic acid, (1) the adhesiveness to endothelial cells is increased,387 (2) the survival time of erythrocytes in circulation is decreased,388 and (3) a new antigenic site may be During in vivo aging of human erythrocytes, exposed. 386,389 the total content of sialic acid (with some carbohydrates) decreases,390-392but the density of sialic acid on the erythrocyte surface is unaltered,393*394i.e., the effective negative charge per unit area of membrane surface remains constant.393 The exact role of sialic acid on erythrocyte survival is still unknown. These subjects are still open for investigation.
Liss, New York, 1986. 8. ChIen, S., Red cell deformability and its relevance to blood flow, Annv. Rev. Physiol., 49, 177, 1987. 9. Hochmuth, R. M. and Waugk, R. E., Erytbrocyte membrane elasticity and viscosity, Annu. Rev. Physiol., 49, 209, 1987.
10. Chasis, J. A. and Shohet, S. B., Red cell biochemical anatomy and membrane properties, Annu. Rev. Physiol., 49, 237, 1987. 11. Clark, M. R., Senescence of red blood cells: progress and problems, Physiol. Rev., 68, 503, 1988. 12. Evans, E., Deformability and adhesivity properties of blood cells and membrane vesicles: direct methods, in Red Cell Membranes, Shohet,
VI. SUMMARY 13.
Two main subjects of erythrocyte rheology, deformation and aggregation, are discussed in detail, on the basis of biochemical structure. The close relationship between the life span (or cell aging) and the rheology of individual erythrocytes is also briefly described. A currently important problem is emphasized, that is, the molecular aspect of the dynamic cytoskeletal structure and the mechanism of its regulation. This concerns not only the rheological function and the survival of circulating erythrocytes, but also the pathophysiology of abnormal erythrocytes.
14.
15. 16.
17.
S. B. and Mohandas, N., Eds., Churchill Livingstone, New York, 1988, 271. Mohandas, N., Measurement of cellular deformability and membrane material properties of red cells by ektacytometry, in Red Cell Membranes, Shohet, S. B. and Mohandas, N., Eds., Churchill Livingstone, New York, 1988, 299. Tsukita, S., Tsukita, S., Ishikawa, H., Sato, S., and Nakao, M., Electron microscopic study of reassociation of spectrin and actin with the human erythrocyte membrane, J. Cell Biol., 90, 70. 1981. Cohen, C. M., The molecular organization of the red cell membrane skeleton, Semin. Hemarol., 20, 141, 1983. Schrier, S. L., Ed., The red blood cell membrane. in Clinics in Haemarology, Vol. 14, Saunders, London, 1985, 1. Pollard, T. D. and Cooper, J. A., Actin and a&n-binding proteins. A critical evaltion of mechanisms and functions, Annu. Rev. Biochem.,
55, 987, 1986. 18. Jay, D. and Cantley, L., Structural aspects of the red cell anion exchange protein, Annu. Rev. Biochem., 55, 5 11, 1986. 19. ZaII, S., Clinical disorders of the red cell membrane skeleton, Crir. Rev. Oncol. Hematol., 5, 397, 1986. 20. Deuticke, B. and Haest, C. W. M., Lipid modulation of transport proteins in vertebrate cell membranes, Annu. Rev. Physiol., 49, 221, 1987. 21. Liu, S. C., Derick, L. H., and Palek, J., Visualization of the hexagonal lattice in the erythrocyte membrane skeleton, J. Cell Biol., 104,
ACKNOWLEDGMENTS The authors are indebted to Professors Syoten Oka, Itiro Tyuma, and the late Takehiko Azuma for their encouragement. The financial support from the Ministry of Education, Culture and Science of Japan, and the Ehime Health Foundation are acknowledged. We wish to thank Miss Misuzu Sekiya and Mr. Andrew W. Winden for their help in the preparation of the manuscript.
527, 1987. 22. Quinn, P. J. and Chapman, D., The dynamics of membrane stmcture, Crir. Rev. B&hem., 8, 1, 1980. 23. Schwartz, R. S., Chiu, D. T.-Y., and Lubm, B., Plasma membrane phospholipid organization in human erythrocytes, Curr. Top. Hematel., 5, 63, 1985. 24. Rogausch, H., Red cell deformability and adaptation in cholesterolfed guinea pigs, Pjluegers Arch., 373, 39, 1978. 25. Rogausch, H. and Distler, E., Erythrocyte rheology in cholesterolfed rabbits, Inr. J. Microcirc. Clin. Exp., 5, 27. 1986. 26. Cooper, R. A., Abnormalities of cell-membrane fluidity in the pathogenesis of disease, N. Engl. J. Med., 297. 371, 1977. 27. Cooper, R. A., Leslie, M. H., Fischkoff, S., Shinitzky, M., and Shattil, S. J., Factors influencing the lipid composition and fluidity of red cell membrane in vitro: production of red cells possessing more than two cholesterol per phospholipid, Biochemistry, 17, 327, 1978. 28. Shiga, T., Maeda, N., Suds, T., Kon, K., and Sekiya, M., Influences of cholesterol on red cell deformability, Clin. Hemorheol., 2,
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tial mechanisms of shape change in the senescent red cell, in Red Cell Shape - Physiology, Parhology, Ulrrasnucrure, Bessis, M., Weed, R. I., and Leblond, P. F., Eds., Springer-Verlag, New York, 1973, 69. 333. Ekholm, J. E., Shukla, S. D., and Hanahan, D. J., Change in cytosolic calmodulin activity of density (age) separated human erythrocytes toward membrane Ca*+/Mg ‘+ ATPase, Biochem. Biophys. Res. Commun., 103, 407, 1981. 334. Mown, C. M., Penn&on, J. T., Fairbanks, V. F., and Burgert, E. O., Erytbrocytic calmodulin correlates with red cell age, Br. J. Haematol., 51, 261, 1982. 335. Watson, W. S., Lyon, T. D. B., and Hilditch, T. E., Red cell magnesium as a function of cell age, Merabolism, 29, 397, 1980. 336. Luthra, M. G., Fridman, J. M., and Sears, D. A., Studies of density fractions of normal human erythrocytes labeled with iron-59 in vivo, J. Lab. Clin. Med., 94, 879, 1979. 337. Fehr, J. and Knob, M., Comparison of red cell creatine level and reticulocyte count in appraising the severity of hemolytic processes, Blood, 53, 966, 1979. 338. Nash, G. B. and Wyard, S. J., Changes in surface area and volume measured by micropipette aspiration for erythrocytes aging in vivo, Biorheology, 17, 479, 1980. 339. Prankerd, T. A. J., The aging of red cells, J. Physiol. (London), 143, 325, 1958. 340. Perona, G., Guidt, G. C., Piga, A., Cellerino, R., Menna, R., and Zatti, M., In vivo and in vitro variations of human erythrocyte glutathione peroxidase activity as result of cells aging, selenium availability and peroxide activation, Br. J. Haemarol., 39, 399, 1978. 341. Magnani, M., Piatti, E., Seraflni, N., Palma, F., Dacha, M., and Fornaini, G., The age-dependent metabolic decline of the red blood cell, Mech. Aging Dev., 22, 295, 1983. 342. Jozwlak, Z. and Jasnowska, B., Changes in oxygen-metabolizing enzymes and lipid peroxidation in human erythrocytes as a function of age of donor, Mech. Aging Dev.. 32, 77, 1985. 343. Maeda, N., Kon, K., and Shiga, T., Functional changes of human aged erythrocytes and the membrane structure, J. Physiol. Sot. Jpn., 44, 322, 1982. 344. Snyder, L. M., Garver, F., Liu, S. C., Leb, L., Trainor, J., and Fortter, N. L., Demonstration of haemoglobin associated with isolated, purified spectrin from senescent human red cells, Br. J. Haematol., 61, 415, 1985. 345. Sauberman, N., Fortier, N. L., Fairbanks, G. F., O’Connor, R. J., and Snyder, L. M., Red cell membrane in hemolytic disease. Studies on variables affecting electrophoretic analysis, Biochim. Biophys. Acta. 556, 292, 1979. 346. Ravindranath, Y., Brohn, F., and Johnson, R. M., Erythrocyte age-dependent changes of membrane protein 4.1: studies in transient erythroblastopenia, Pediarr. Res., 21, 275, 1987. 347. Barber, J. R. and Clarke, S., Membrane protein carboxyl methylation increases with human erythrucyte age, J. Biol. Chem., 258, 1189, 1983. 348. Bnmauer, L. S. and Clarke, S., Age-dependent accumulation of protein residues which can be hydrolyzed to o-aspartic acid human erythrocytes, J. Biol. C&m., 261, 12538, 1986. 349. Ho&stein, P. and Jatn, S. K., Association of lipid peroxidation and polymerization of membrane proteins with erythrccyte aging, Fed. Proc., Fed. Am. Sot. f&p. Biol., 40, 183, 1981. 350. Jain, S. K. and Hochstetn, P., Polymerization of membrane components in aging red blood cells, Biochem. Biophys. Res. Commun., 92, 247, 1980. 351. Burton, G. W., Cheng, S. C., Webb, A., and Ingold, K. U., Vitamin E in young and old human red blood cells, Biochim. Biophys. Acm, 860, 84, 1986. 352. Kay, M. M. B., Bosman, G. J. C. G. M., Shapiro, S. S., Bendich,
1980. 373. Dons, R. F., Corash, L. M., and Gorden, P., The insulin receptor is an age-dependent integral component of the human erythrocyte mem-
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Critical Reviews In brane, J. Biol. Chem., 256, 2982, 1981. 374. Baumann, G. and MacCart, J. G., Kinetics of cell age-dependent decline of insulin receptors in human red cells, Am. J. Physiol., 247, E667, 1984. 375. Trautscb, C., Tanner& C., and Maretzki, D., Disproportional loss of membrane constituents in the course of erythrocyte aging, Acre Biol. Med. Ger., 40, 743, 1981. 376. Joiner, C. IL and Lauf, P. K., Ouabain binding and potassium transport in young and old populations of human red cells, Memb. Biochem., 1, 187, 1978. 377. Hentachel, W. M., Wu, L. L., Tobin, G. O., Anstall, IL B., Smith, J. B., WiUiams, R. R., and Ash, K. O., Erythrocyte cation transport activities as a function of cell age, Clin. Chim. Acfu, 157, 33, 1986. 378. Kay, M. M. B., Goodman, S. R., Sorensen, K., Wbitfield, C. F., Wong, P., Zaki, L., and Rudloff, V., Senescent cell antigen is immunologically related to band 3, Proc. Nurl. Acud. Sci. U.S.A., 80, 1631, 1983. 379. Lutz, H. U., Flepp, R., and Stringaro-Wipf, G., Naturally occurring autoantibodies to exoplasmic and cryptic regions of band 3 protein, the major integral membrane protein of human red blood cells, J. Immwwl., 133, 2610, 1984. 380. Low, P. S., Waugb, S. M., Zinke, K., and Drenckhahn, D., The role of hemoglobin denaturation and band 3 clustering in red blood cell aging, Science, 227, 531, 1985. 381. Kay, M. M. B., Mechanism of removal of senescent cells by human macrophages in situ, Proc. Nad. Acad. Sci. U.S.A., 72, 3521, 1975. 382. Kay, M. M. B., Role of physiologic autoantibody in the removal of senescent human red cells, J. Suprumol. Srrucr., 9, 555, 1978. 383. Alderman, E. M., Fudenberg, H. H., and Lovins, R. E., Isolation and characterization of an age-related antigen present on senescent human red blood cells, Blood, 58, 341, 1981. 384. Singer, J. A., Jennings, L. K., Jackson, C. W., Dockter, M. E., Morrison, M., and Walker, W. S., Fxythrocytehomeostasis: antibody-mediated recognition of the senescent state by macmphages, Proc. Natl. Acad. Sci. U.S.A., 83, 5498, 1986. 385. Kay, M. M. B., Eoaman, G. J. C. G. M., Johnson, G. J., and Beth, A. H., Band-3 polymers and aggregates, and hemoglobin precipitates in red cell aging, Blood Cells, 14, 275, 1988. 386. Aminoff, D., The role of sialoglycoconjugates in the aging and sequestration of red cells from circulation, Blood Cells, 14, 229, 1988. 387. Dbermy, D., Shneon, J., Wautier, M.-P., Boivin, P., and Wautier, J.-L., Role of membrane sialic acid content in the adhesiveness of aged erythrocytes to human cultured endotheliaf cells, Biochim. Biophys. Acta. 904, 201, 1987. 388. Bocci, V., Determinants of erytbrocyte aging: a reappraisal, Br. J. Haematol., 48, 515, 1981. 389. Danon, D., Marikovsky, Y., and Fiihler, H., Surface charge of old, transformed, and experimentally deteriorated erythrocytes, Ann. N.Y. Acad. Sci., 416, 149, 1983. 390. Baxter, A. and Beeley, G., Surface carbohydrates of aged erythrocytes, Biochem. Biophys. Res. Commun., 83, 466, 1978. 391. Cboy, Y. M., Wang, S. L., and Lee, C. Y., Changes in surface carbohydrates of erytbrocytes during in vivo aging, Biochem. Biophys. Res. Commun., 91, 410, 1979. 392. Bladier, D., Gattegno, L., Fabia, F., Perret, G., and Cornillot,
P., Individual variations of the seven carbohydrate components of human erythrocyte membrane during aging in vivo, Carbohydr. Res., 83, 371, 1980. 393. Luner, S. J., Szklarek, D., Knox, R. J., Seaman, G. V. F., Jowfowicz, J. Y., and Ware, B. R., Red cell charge is not a function of cell age, Nature (London), 269, 719, 1977. 394. Lutz, H. U. and Fehr, J., Total sialic acid content of glycophorins during senescence of human red blood cells, J. Biol. Chem., 254, 11177, 1979.
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Volume 10, Issue 1
Erythrocyte Rheology Takeshi Shiga, Nobuji Maeda, and Kazunori Kon I. INTRODUCTION
Theflowproperties of erythrocytes are important in blood circulation, since the erythrocytes occupy nearly half of blood volume. According to the Hagen-Poiseuille law, the flow volume of liquid in a rigid tube is expressed as follows: (Flow) fXJ(Viscosity)-‘*(Pressure difference).(Radius)4*(Length)-’ Therefore, the blood viscosity causes the resistance to flow in blood vessels. The blood viscosity increases with an augmentation of the hematocrit (volume fraction of erythrocytes in blood). Furthermore, as is well known, blood is a non-Newtonian fluid, that is, at a high shear rate blood viscosity decreases due to the deformation of erythrocytes, but at a low shear rate it increases due to the aggregation of erythrocytes. Such macroscopic viscosity is important for the blood flow in large vessels, but it may not be a direct measure for the blood flow in small vessels, because of F&raeus-Lindqvist effect (decrease of the blood viscosity due to the axial streaming of erythrocytes and the formation of circumferential plasma layer). The oxygen delivery in the microcirculation depends on the flow properties of erythrocytes and the oxygen binding of intracellular hemoglobin. Here again, cellular deformation is important, since the erythrocytes must deform to pass through vessel sections narrower than themselves. When the flow rate decreases, the erythrocytes tend to form aggregates depending on the plasma composition. Therefore, erythrocyte deformability and erythrocyte aggregation, as well as capillary hematocrit, are the main determinants of capillary passage and oxygen supply to tissues. The mechanical properties of erythrocytes are greatly influenced by the membrane, especially by its cytoskeletal structure, by hemoglobin concentration and by cell shape. The growth of erythrocyte aggregates depends mainly on interaction between the membrane surface and some plasma proteins. Therefore, the rheological characteristics of erythrocytes (and blood) must be understood on the basis of the biochemical structure and its regulation of the entire erythrocyte. The unique feature of mammalian erythrocytes is the lack of protein synthesis, thus the functional proteins in erythrocytes are gradually denaturing during their lifespan. After a long trip in circulation, the aged erythrocytes are removed from circulation by the reticuloendothelial system. The aging process greatly affects the properties of individual erythrocytes, that is, the cellular deformability decreases due to biochemical deterioration and morphological alteration. Although many excellent reviews’-‘3 on these subjects have
appeared recently, we would like to focus our attention on the biochemical mechanism of erythrocyte rheology. To begin with, the biochemical structure of erythrocytes (Section II); the process of deformation (Section III) and the kinetics of aggregation (Section IV) are discussed extensively (especially the method of analysis and the mechanisms of the phenomena); and, finally, the alteration of rheological properties with erythrocyte aging is summarized (Section V).
II. BIOCHEMICAL ERYTHROCYTES
STRUCTURE
OF
A schematic model of an erythrocyte membrane is shown in Figure 1. In the plane of the phospholipid bilayer containing cholesterol, various membrane proteins are laterally drifting, and oligo- or polysaccharides are attached on the surface of certain proteins. Underneath the bilayer plane the network of cytoskeletal proteins are extended in two dimensions. Biochemical and biophysical studies of erythrocyte membrane have been carried out extensively, thus the structure of erythrocyte membrane is one of the well-known biomembranes.5~6*‘4-2’ The cytoplasm of erythrocytes is rich in proteins, mainly hemoglobin, which carries oxygen, and the water content is about 70%. In this section, we describe its structure and interactions within the cellular constituents briefly, especially in connection with the rheology and/or the mechanical properties of human erythrocytes. A. Chemical Constituents and Structure of the Membrane The mechanical properties of erythrocyte membrane are mainly determined by the cytoskeletal network, but the roles of the phospholipid bilayer cannot be ignored. 7. Lipid Bilayer The constituents of phospholipids in the erythrocyte membrane vary with the animal species. The phospholipid composition of human erythrocytes depends partly on nutritional
T. Shiga received a M.D. degree and a Dr. Med. Sci. degree from the Osaka University School of Medicine in Osaka, Japan. Dr. Shiga is currently a Professor in the Department of Physiology in the School of Medicine at Osaka University in Osaka, Japan. N. Maeda received a M.D. degree and a Dr. Med. Sci. degree from Nara Medical College in Kashihara, Nara, Japan. Dr. Maeda is currently a Professor in the Department of Physiology in the School of Medicine at Ehime University in Ehime, Japan. K. Kon received a B.Sci. degree from the Science University of Tokyo in Tokyo, Japan; a M.Sci. degree from the University of Hokkaido in Sapporo, Japan; and a Dr. Med. Sci. degree from the University of Osaka in Osaka, Japan. Dr. Kon is currently an Associate Professor in the Department of Physiology at the Ehime College of Health Science in Ehime, Japan.
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Critical 3eviews In ATP, etc.33 and may be stabilized by membrane proteins.34-36 Therefore, the variation in phospholipid composition modifies the interactions with various constituents inside the erythrocyte membrane, but the resultant change in mechanical properties is not well known. c. ACYL-CHAIN COMPOSlTlON The fluidity of the lipid bilayer greatly depends on the interactions among neighboring acyl-chains of phospholipids. 37-39The physicochemical characteristics of the bilayer may be roughly related to the molar ratio of unsaturated- to saturated-acyl-chains. FIGURE 1. A model of erythrocyte membrane. (Left) The interaction among membrane proteins and their electrophoretic mobilities in polyacrylamide gel containing sodium dodecyl sulfate are shown (see text). Numbers in the membrane model correspond to the electrophoretic band. Abbreviations are as follows: Ad, adducin; Ca, calcium; CaM, calmodulin; GPA, glycophorin A; G3-PD, glucose 3-phosphate dehydrogenase; Hb, hemoglobin. The black belt on F-actin (oligomer of G-actin, band 5) is tropomyosin.
habits. It is known that the transport activity of membrane depends on lipid environment. *OThe mechanical and rheological properties of the lipid bilayer may be closely related to the dynamics of membrane structure,22~23which may be relatively represented by the ease of molecular motion within a membrane( “membrane fluidity’ ‘) . Some important relationships between lipid composition and the fluidity of membrane are summarized below. a. CHOLESTEROUPHOSPHOLlPID
RATION
In the human erythrocyte membrane, the molar ratio of cholesterohphospholipid is about one. Cholesterol shows dual effects on the lipid bilayer. ***it fluidizes the membrane below the transition temperature, but it stiffens the bilayer above this temperahue; futihermote, the transition temperature itself shifts with the cholesterol content. It is known that the activity of membrane proteins is modulated by the cholesterohphospholipid molar ratio. Rogausch” and Rogausch and Distleti5 have shown the increased blood viscosity of cholesterol-fed rabbits, and Coopeti6 and Cooper et al.*’ have discussed the relation between the fluidity and the stability of the erythrocyte membrane (and pathogenesis). However, as shown in a later section (Section III.B.3), the direct effect of cholesterol on erythrocyte rheology is rather smal1,28 contrary to the expectation based on the decreased fluidity induced by cholesterol loading. b. PHOSPHOLIPID COMPOSITION
Some phospholipids (phosphatidyl-choline and sphingomyelin) preferentially locate in the outer half of the lipid bilayer, while some others (phosphatidyl-serine and phosphatidylethanolamine) are rich in the inner half.23*29Divalent cations (such as Ca”) interact with phosphatidyl-serine and phosphatidyl-ethanolamine to form clusters of these phospholipids30,31or to modify the transmembrane distribution of these lipids. 32 The phospholipid distribution is influenced by 10
d. LIPID-PROTEIN INTERACTION
Membrane proteins interact with lipid bilayer in their hydrophobic domain.*5*34~40 The motion of lipids around such proteins is more or less restricted (called “amntlar lipids”); therefore, in the presence of membrane proteins the membrane fluidity is no longer uniform, but the membrane is composed of many microplates or microdomains. The microdomains of the erythrocyte membrane are not precisely recognized, and their relevance to the rheological properties has not been discussed. We have shown that an androstane spin label in glutaraldehyde-tteated erythrocyte membrane is present in different domains, such as fluid domain, less fluid domain, and solid plaque (although this is an extreme ~ase).~*‘~~The temporal and spatial fluctuation of the membrane microdomain structure may be important for erythrocyte functions, thus further studies are needed. 8. PROTECTlON AGAINST OXlDATlVE STRESS In some in vitro studies, the peroxidation or oxidation of the erythrocyte membrane induces hemolysis.44 Not only the unsaturated acyl-chains, but also sulfhydryl groups in proteins are the targets of the oxidative stresses.45.46However, the circulating erythmcytes possess protective enzyme systems against such an oxidative process (see Section II.B.2); thus, in certain diseases of low antioxidative activity massive hemolysis may be induced by the mechanical forces. Furthermore, specific antibody binding to the membrane protein (e.g., band 3) may accelerate the phagocytosis of oxidatively stressed erythrocytes .47 2. Membrane Ptvtehs and Cytoskereta! Structure A wide variety of membrane proteins are recognized in erythrocytes. A routine method for semiquantitative separation is polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS). The proteins in the ghost membrane am separated roughly according to their molecular weight (see Figure 1). a. CLASSIFICATION OF MEMBRANE PROTEINS
According to their location, the membrane proteins are classified into two groups: the integral proteins and the peripheral proteins. The integral proteins penetrate through the
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Oncology/Hematology lipid bilayer and are divided into two classes: (1) the proteins that cross the bilayer with a single segment (i.e., a helix of some 20 amino acids), e.g., glycophorins; (2) the proteins that form a channel with multiple spanning segments and serve as the transport proteins, e.g., band 3 protein (anion channel). These proteins can hardly be removed from bilayer without destroying the bilayer structum (e.g., with nonionic detergents). The peripheral proteins are also divided into two classes: (1) one locates in either the outer or inner half of the lipid bilayer, most of which are receptors and enzymes, e.g., ace@choline receptor, glyceraldehyde-3-phosphate dehydrogenase; (2) the others, which interact with the cytoplasmic half of the bilayer and form cytoskeletal network, and thus called ‘ ‘cytoskeletal proteins’ ’ . b. CYTOSKELETAL STRUCTURE The cytoskeletal network underneath the lipid bilayer is responsible for the mechanical properties of the cells and the maintenance of cell shape. Several major proteins are involved in constructing the network.5~6,‘4.21.48-5’ A proposed scheme of the erythrocyte cytoskeleton is shown in Figure 1. The individual characteristics of the membrane proteins are as follows.’ The main component of the cytoskeleton is spectrin heterodimer (0~and l3) of about 100 nm in length.52 The heterodimers associate at one end (head) to form a tetramer. The other end (tail) binds to a sort of knot, composed of a short actin filament (consisting of 12 to 15 Gactins,53.54with tropomyosins” and band 4.9 proteins56) and band 4.1 proteins5’ (which are phosphatidyl-serine binding protein 58s9).One knot ties several spectrin bundles. Besides the direct interaction between spectrin and lipid bilayer, the linking between the cytoskeletal network and lipid bilayer (involving integral proteins) is made as follows: (1) the band 4.1 protein binds to glycophorin A and/or C, which spans the lipid and is surrounded by triphosphoinositides;62 (2) bilayet”9*60p6’ the ankyrin (acylated with fatty acid, which turns over)63-66ties the band 3 protein (anion transporter) with spectrin (at about 20 nm from the center of the spectrin tetramer;63 (3) the band 4.1 protein may also bind with band 3 protem6’ at low levels of triphosphoinositides. The assembling process of the spectrin network, particularly with avian erythrocytes during erythropoiesis, has been studied in detail by Lazarides.51 It is still unclear how the biconcave disk shape is maintained by the cytoskeletal network. The deficiency or abnormality of some cytoskeletal proteins leads to abnormal shape and/or increased fragility, such as elliptocytosis, spherocytosis, These days, the cDNA structures or the amino acid etc. 19~so*68-70 sequences of some proteins are known, thus the relationship between the abnormal loci and the erythrocyte abnormality can be revealed in the near future.
3. Carbohydrates On the outer surface of the erythrocyte membrane, some carbohydrates are attached to the membrane proteins. The sac-
charide chain bound to band 3 protein serves as ABO-type antigens. Saccharides are also attached to glycophorins. The sialic acid in these chains, together with carboxyl groups of acidic amino acids in membrane proteins located on the outer surface, gives tbe surface negative charges of the membrane. The electrostatic repulsion between negatively charged surfaces is an important character to prevent the erythrocyte aggregation. When sialic acids are removed with neuraminidase, the electrophoretic mobility (toward cathode) decreases proportionally to the number of sialic acid” (see Section 1V.C. 1.d). B. Intracellular Constituents Human erythrocytes have no intracellular structure, such as nucleus, mitochondria, etc. Therefore, the content of the cell interior is often regarded as a homogeneous protein-rich solution, mostly hemoglobin. Besides hemoglobin, the erythrocyte contains various enzymes and other proteins. Reticulocytes, present in about 1% in the circulating blood, are direct precursors of mature erythrocytes and contain various amounts of RNA and ribosome. About 70% (by weight) of an erythrocyte is water.72.73 Assuming the average volume of erythrocytes is some 90 pm3 (corresponding to a sphere of 2.8 p,rn radius of which the surface area is about 100 pm2). The average surface area of erythrocytes (140 pm2)74 is about 1.4 times greater than the calculated value for a sphere of the same volume (for cell shape, see Figure 20). Therefore, the biconcave disk-shaped erythrocytes can take up water without hemolysis, and the cell volume may be increased by 1.5 to 1.8 times in hypotonic environment without destruction (the outflow of interior contents due to the destruction or rupture of the membrane is “hemolysis”). Furthermore, such flatness (diameter of 7 to 8 km and thickness of 1 to 2 p,rn) serves to allow extreme deformation by external forces, e.g., the erythrocytes can be folded up. 1. Hemoglobin The main role of hemoglobin is the transport of oxygen by the association-dissociation of oxygen with the Fe(II)-protoporphyrin of a tetrameric hemoglobin molecule. The oxygen supply to tissues is regulated by pH, pCO,, the concentration of 2,3-diphosphoglycerate (2,3-DPG), and temperature. The transport of CO, is also closely linked with the proton movement in hemoglobin molecule, with the aid of carbonic anh ydrase .
Another important property of intracellular hemoglobin is its contribution to internal viscosity, which determines the rheological properties of erythrocytes. The intracellular viscosity may be represented by mean corpuscular hemoglobin concentration (MCHC), which is calculated from the hemoglobin content and the cell volume. The viscosity increases nonlinearly with increasing hemoglobin concentration.75-77 Therefore, at a high intracellular hemoglobin concentration, the deformation of erythrocytes is suppressed and their passage 1990
11
Critical Reviews In through the capillaries may be retarded. Further, the increased internal viscosity reduces the convection of hemoglobin molecules and the diffusion of oxygen in erythrocytes. It should be noted that the intracellular hemoglobin concentration differs from one erythrocyte to another, depending on the cell age (see Section V. 3 .e); the intracellular hemoglobin concentration tends to increase with the concomitant decrease of surface area and volume of erythrocytes during aging.78*79 In some hemoglobin diseases, typically sickle cell anemia, the pathologic hemoglobin (e.g., Hb S) polymerizes inside the erythrocytes to form straight rods upon deoxygenation, then the sickling of the cell occurs.8o In other hemoglobinopathies, when the oxidation and denaturation of hemoglobin (and other components) proceed, Heinz bodies are formed and the mechanical properties of erythrocytes are affected.81*82The formation of Heinz bodies occurs in diseases where the antioxidative capacity is decreased by either congenital or postnatal causes. The rheological properties of erythrocytes are greatly impaired in these abnormalities. Hemoglobin molecules interact with the membrane components, such as band 3 protein.83**4The membrane-bound hemoglobin shows an increase in oxygen affinity,*’ and it may affect the convection of intracellular hemoglobin and the mechanical properties of membrane to some extent. 2. Enzymes and Other Proteins Eqthrocytescontain many enzymes. Unlike the other cells, however, enzymes of aerobic glycolysis are not included in human erythrocytes. Glycolysis (of glucose) ends up at the level of pyruvate and/or lactate, and 2 mol of ATP are generated from 1 mol of glucose without the consumption of oxygen. Antioxidative systems are well equipped, for example, the renewal system of reduced glutathione, glutathione peroxidase, superoxide dismutase, and catalase. If one of these systems suffers from dysfunction, (1) the oxidation of hemoglobin8’~82 and membrane proteins,*6-88and (2) the peroxidation of lipids may develop. The activity of enzymes declines gradually with cell aging; thus the activity of certain enzymes is used as a marker of cell age.89 Density gradient centrifugation has been applied for fractionating erythrocytes; high-density fraction contains aged cells, and low-density fraction is rich in young cells and reticulocytes. However, if the enzymatic activities decrease steeply during maturation of reticulocytes as pointed out recently,% the distinction between the aging process of mature erythrocytes and the maturing process of reticulocytes becomes important. A recent study on rabbits does not detect a decline of enzymatic activity of glycolysis.9* Therefore Clark” has written “a lack” of adequate cell age-associated markers, although a counter argument is presented by Piomelli.92 On the other hand, studies of reticulocytes have just become accessible with the use of recombinant erythropoietin; clinical trials show a considerable reticulocytosis upon administration of erythro-
12
poietin,93*94as well as animal studies95 (e.g., we observe some 15% reticulocytes with erythropoietin-injected rats). More investigations, including the methods of separation, on the distinction between reticulocyte maturation and erythrocyte aging are expected in the near future. Other than enzymes, the regulator proteins, e.g., calciumbinding proteins (calmodulin96*97and adducin98,99)are present in human erythrocytes. 3. Ions and Water Content The contents of inorganic ions are regulated by various transport proteins in erythrocyte membranes. Since the membrane is permeable to water (though the actual pathway is not well defined at the molecular level), recently the role of band 3 and band 4.5 proteins is noticed,‘W.‘O’ the control of the concentration of various ions is important for the maintenance of cell volume and cell shape, and, consequently, the intracellular viscosity (hemoglobin concentration). The water content in erythrocytes is osmotically regulated, particularly by intracellular K + and extracellular Na’ ; thus the escape of K + induces the loss of water and the increase of intracellular viscosity, then the deformability decreases (see Section IlI.B.2). C. Cell Shape and Biochemical Structure The shape of normal erythrocytes is flat and biconcave disk-like. The biochemical basis to maintain such a shape is not yet fully understood, but, instead, the causes of shape alteration have been studied extensively. The shape change of normal erythrocytes is usually divided into two categories. One is “transformation”, which is induced by the chemicals and by metabolic depletion, and the other is “deformation’ ’ , which is induced by external physical forces. 1. Transformation of Etythrocytes The typical transformation is classified into two distinct shapes:102.103 (1) echinocytosis, or externalization, is characterized by slight prominences of membrane; (2) stomatocytosis, or internalization, is made conspicuous by the caving of the membrane to form a cup-shape, then to create small holes, as shown in Figure 2. However, at the extreme, these shape changes end up in spherocytosis. a. TRANSFORMATION DUE TO METABOLIC DEPLETION Echinocytic transformation occurs during blood storage in acid-citrate-dextrose or citrate-phosphate-dextrose at 4°C; as Nakao et al.‘” have noticed in 1962, the cell shape and the ATP content are closely related. Further, the following impairments are noticed in erythrocytes during blood storage:‘05 (1) ATP-depleted echinocytes show a short survival time after transfusion, (2) the oxygen affinity increases due to the reduced 2,3-DPG, (3) the blood viscosity increases with development of echinocytosis, and (4) the contents of inorganic ions change. Therefore, the methods of rejuvenation have been studied ex-
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Oncology/Hematology
-2 FIGURE 2. Morphological changes of erythrocytes. Stages of “transformation” to echinocytes (upper array) and stomatocytes (lower array) are shown. The numbers are used for quantitative expression of the degrees of transformation. (From Kon, K., Maeda, N., and Shiga, T., J. Physiol. (London), 339, 573, 1983. With permission.)
tensively. lo5 At the present stage, organic phosphates (ATP, 2,3-DPG) can be regenerated by incubation with certain substrates (inosine, pyruvate, phosphate, etc.), or even pyridoxal phosphate is useful for recovery of oxygen transport activity,‘06**07 but rheological properties cannot be restored by either of the organic phosphates. Echinocytes induced by metabolic depletion can be restored to discocytes, when the intracellular ATP is regenerated. However, Feo and Mohandas”’ have found no direct relation between ATP content and cell shape. On the other hand, the phosphorylation of several membrane proteins may intervene between ATP level and the cytoskeletal structure,‘Og although no direct interaction between the membrane components and ATP has been demonstrated. Further studies are needed on these points. b. TRANSFORMATION
half induces the external protrusion of the entire bilayer. On the contrary, the cationic chemicals tend to be incorporated into the inner half, and the expansion of the inner area caves the bilayer in. With an excess of chemicals, the transformation ends up with a spherical shape (called either sphero-echinocytes or sphero-stomatocytes). On the other hand, Nelson et al.‘l3 have suggested the possible role of calmodulin on the basis of transformation due to calmodulin inhibitors. Bums and Gratzer114 have demonstrated that trifluoperazine-induced stomatocytosis takes place at low concentrations of intracellular calcium, where the activity of calmodulin on the membrane is presumably suppressed. Our study’15 suggests that some calmodulin inhibitors can bind to calmodulin at low concentrations where they do not induce stomatocytosis. In order to study the chemically induced transformation further, the amounts of chemicals in the inner and outer halves of the lipid bilayer and in the cytoplasm (free or bound to some proteins) should be determined separately.
DUE TO CHEMICALS
In general, echinocytosis is induced by anionic reagents (e.g., lysolecithin, dehydroepiandrosterone sulfate, etc.), while stomatocytosis appears with cationic reagents (e.g., chlorpromazine, trifluoperazine, etc.). The velocity of transformation has been measured with the light-scattering stoppedflow method,“O,ll’ but it depends on the reagents and their concentration. Sheetz and Singerllz have proposed the “bilayer couple” hypothesis as an explanation, taking into account the asymmetric distribution of the reagent and the resultant differences in the surface area, between the inner and outer halves of lipid bilayer. The anionic chemicals are preferentially incorporated into the outer layer, and the expansion of the outer
In the in vivo capillaries, of which the diameter is sometimes smaller than that of erythrocyte, the cells must deform themselves in order to pass through. In the larger vessels, erythrocyte deformation is also important to reduce hydrodynamic resistance: actually, at high shear rates the blood (or suspension) viscosity decreases due to cell deformation. It is necessary to make a theoretical model in order to analyze erythrocyte deformation in shear flow. Two models 1990
13
Critical Reviews In are available: (1) the fluid droplet model’16*1’7and (2) the microcapsule mode1.1’8-‘20In the fluid droplet model, a uniform shear stress is acting on the entire membrane, but in the microcapsule model the stresses differ from one point to another. In either model, the membrane rotates around the cytoplasm (called “tank-tread” movement),“6 and the cell interior is treated as a continuous fluid. The theories of erythrocyte deformation and motion in capillaries are now progressing’*’ and further developments are awaited. The deformation of erythrocytes is easily observed when external forces are applied on erythrocytes as follows: (1) under uniform shear stress the biconcave disk-shaped cells are elongated and deformed into flat ellipsoids;‘** (2) the cells can be folded back, when the cells are caught on a fine spider-thread in a ~trearn;‘*~(3) a portion of the cell membrane can be stretched into a tongue-like shape by aspiration with a micropipette; (4) the whole erythrocyte can be aspirated into a glass capillary (which is narrower than the diameter of erythrocyte),‘*’ or filtered through narrow pores.126 When the applied external forces are physically defined and the degree of the deformation is measurable, the degree can be used as a quantitative measure of the “deformability” (i.e., “the readiness to deform by physical force”). It is also important, when the applied forces are suddenly ceased, that the cell shape can be restored into a biconcave disk after a short recovery time.
III. ERYTHROCYTE DEFORMABILITY The deformation of erythrocytes is easily induced by external forces. The deformation is important with respect to (1) the passage of erythrocyte through narrow capillary, and (2) the reduction of blood viscosity under high shear rates. Therefore, quantitative measurements of erythrocyte deformability have been made in many diseases and plenty of evidence of the above phenomena is collected. In this section, the following problems will be discussed: (1) the advantages and disadvantages of various methods for deformability measurements and (2) the biochemical factors influencing deformability. The alterations of deformability in health and diseases will be discussed in Section V. A. Measurements of Deformabllity The term “deformability” is commonly used without exact definition. In the literature, the meaning of deformability sometimes differs from one method to another. Some methods apply a strong force on the whole cells, thus the entire cell shape is changed (“cellular” deformation). Some other methods make a small shape change in a very limited portion of the cell surface, for example, by aspiration with a micropipette (“membrane” deformation). Another important methodological distinction concerns the velocity of deformation and/or restoration: many methods are static and stationary, i.e., the deformation is quantified under a constant force lasting a certain period of time; on the other hand, some methods concern the time-re14
solved processes of deformation/restoration, either by changing the force stepwise or by applying an alternate force. It is desirable to define the physical nature of applied external forces and to measure the degree of deformation. Although the erythrocyte is so small and its shape is so complex that it is hard to apply the defined force and to express the shape change, several methods have been developed. It may be convenient to classify the various methods into three categories in a practical sense: (1) the cellular deformation in uniform shear flow, (2) the readiness of filtering in small pores, and (3) the physical properties of the cell membrane. Measurements (1) and (2) reflect the properties of whole cells, either as individual cells or as a mass, thus the “cellular deformability” is determined, whereas measurement (3) concerns only the viscoelasticity of the erythrocyte membrane. Furthermore, the methods for analysis of time-dependent phenomena are developed. 7. Deformability of the Whole Etythrocyte Under a defined external force the erythrocytes can deform, thus the “deformability” (the ease of deformation by an external force) can be quantitatively measured, if the force and the degree of deformation are determined. The shear force may be the first choice as a physically defined force, since it is easily controlled and acts on the whole cell body. The “rheoscope” of S&mid-Schonbein et al.127,128and the “ektacytometer” of Bessis and Mohandas129*‘30are the representative instruments; some other methods have been developed, but are not widely employed, because the manipulation and/or construction of the apparatus seem to be difficult. The filtration method will be described in the next section, since the physical forces applied on cells cannot be defined and the processes of shape change cannot be followed. 8. MEASUREMENT OF THE DEFORMATION UNDER UNIFORM SHEAR STRESS
Application of a steady shear stress to erythrocytes is carried out by modifying the cone-plate viscometer or the coaxial double cylinder viscometer. The disk-shaped etythrocytes are deformed into flat ellipsoids under high shear stress. In order to observe such deformation, the erythrocytes are suspended in a viscous solution containing dextran (molecular weight 40,000) or arabino-galactan (molecular weight 30,000). The representative methods are as follows. i. Rheoscope, Flash Photography of the Deformed Erythrocytes A transparent cone (rotating) is mounted on an inverted microscope (Figure 3). The shear stress produced in the coneplate viscometer is the same at any spatial position, and the flash photographs of flowing deformed cells can be easily obtained (Figure 4). In order to quantify the degree of deformation, the lengths of the long axis (L) and the short axis (S) of individual cells are measured one by one on the enlarged photographs, and either the “ellipticity” (S/L) or the “elon-
Volume 10, Issue 1
Oncology/Hematology
r Pm
Viscometer
0
FIGURE 3. A schematic diagram of rheoscope. The erythrocytes, suspended in viscous solution (at the final hematocrit of about 0.2%), are poured into the gap between a 0.8” cone and the plate. The cone rotates on the glass plate. The photographs are taken using a flash lamp of 2 t~s duration. (From Suda, T., Maeda, N., Shimizu, D., Kamitsubo, E., and Shiga, T., Biorheology, 19, 555, 1982. With permission.)
gation or deformation index” ([L - S]/[L + S]) are adopted as an index of deformation. The frequency distribution of the indices for normal, individual erythrocytes is Gaussian. The index varies with the applied shear stress, the product of the shear rates, and the viscosity of the suspending medium. Figure 5 shows the plots of index to the shear stress and the effect of the hardening of cells by oxidative crosslinking of spectrin,‘3’ i.e., the deformation index decreases at every shear stress as the crosslinking proceeds. The advantage of this method is the monitoring of individual cells, i.e., the frequency distribution of index can be obtained and, if abnormally hard cells are present, normal and abnormal cells can be distinguished on photographs. However, the measurement of lengths on photographs is time-consuming and tedious, and thus may not be used at the hospital bedside (the diffractometric measurement of deformation in next item may serve for this purpose). The original design of the rheoscope is composed of a rotating cone and a fixed plate,12’ thus the cells are rapidly flowing and hardly viewed directly. Later the counterrotating apparatus is introduced, then the position of cells at the central plane does not move and the “tank-treading motion”“6*‘32 of the membrane can be monitored (see Section III.A.4.iv). The other application of the rheoscope is to obtain the frequency response of the erythrocyte deformation, under oscillatory varying shear ~tress’~~,‘~~(see Section III.A.4.iii). ii. Ektacytometer,
Diffractometty
of the Deformed
10
100
FIGURE 4. Flash photographs of deformed erythrocytes, observed with rheoscope. Measured in 20% Dextran T-40 (18.6 cP) at the shear rate of 0, 75, and 750 s-’ at 25°C. The erythrocytes were deformed to flat ellipsoids, of which the long axes aligned along the flow line. Flash photographs were taken on Kodak Tri-X film. (From Suda, T., Maeda, N., Shimizu, D., Kamitsubo, E., and Shiga, 19, 555, 1982. With permission.) T., Biorheology,
medium and simultaneously a laser beam is projected through a window (rigorously saying, the shear stress is not uniform and has a slight gradient). The diffraction of the laser beam changes with the shape and size of the particles, thus of the deformed cells.‘3~‘29~‘30 When the cells deform ellipsoidally under high shear stress, the contour of the light diffraction is
Elythrocytes
Within a narrow space between coaxial double cylinders, the shear stress is applied to erythrocytes suspended in viscous 1990
15
Critical Reviews In
% Crosslinking FIGURE 5. E@rocyte deformation decreased by crosslinking of spectrin with diamide. Measured at the shear rates of 150 and 750 SX’ (in 20% Dextran T-40) at 25”C, with rheoscope. The percentage of crosslinking is expressed by the percent loss of spectrin beta subunit. The deformability is expressed by the deformation index, (L - S)/(L + S), in which L and S are the long and short radii of ellipsoidally deformed cells, respectively. The deformability decreased with a progress in crosslinking. (From Maeda, N., Kon, K., Imaizumi, K., Sekiya, M., and Shiga, T., Biochim. Biophys. ACZU, 735, 104, 1983. With permission.)
elongated ellipsoidally and is quantified to deduce the “deformation index”. The advantage of this method is real-time measurement and the simplicity of handling. However, since the diffraction reflects the averaged deformation of the whole cell fraction, the monitoring of individual cells is impossible and the concomitant abnormal cell (with abnormal shape) may be ignored. Moreover, in order to obtain the diffraction pattern, the reflection coefficient of the medium has to be high (i.e., the viscosity inevitably becomes high). Another application of the ektacytometer is to combine it with an apparatus for continuous modification of osmolarity. 13.135Both the mechanical deformation and stability are relatively quantified, thus this “osmotic gradient ektacytometry” seems to be useful for studies of hemolytic anemias. b. MEASUREMENT IN PARALLEL-PLATE FLOW CHANNEL
A laminar stream in a thin rectangular flow channel is sometimes useful for studies of erythrocyte flow behavior.
16
The “deformation-orientation” behavior of erythrocytes has been studied with phospholipid spin-labeled cells that are flowed in a flat channel. A parameter, reflecting the orientation of the membrane surface to the flow and deformation of disk-shaped erythrocytes, is calculated from the electron spin resonance spectra. 136.137 This method is successfully applied to study erythrocyte behavior at high hematocrit,‘3s.139 but cannot be used to figure out the degree of deformation because (1) the applied shear stress varied with the depth in the flow channel and (2) the deformation is accompanied with the orientation of cells along the stream line. Erythrocytes in isotonic buffered saline frequently adhere to the material surface (e.g., glass, methacrylate, etc.). When the channel is filled with erythrocyte suspension and the cells are allowed to settle, a portion of the sedimented cells attaches to the surface of the channel. Applying a laminar flow, the erythrocytes adhered with one point of their membrane are deformed into a tear-dropletlike shape by the wall shear stress (Figure 6), and the degree of deformation may be quantified by comparing the extended length (L) with the original length (LO) of the cells with a microscope. This method is essentially “uniaxial loading of shear stress to point-attached erythand the rheoscope may be used for this rocytes “, 140.141 purpose (as shown in Figure 6) instead of the flow channel. Hochmuth et a1.14’ have observed three types of erythrocyte stretching in shear flow: whole cell stretching (as described above), cell evagination, and long tether stretching (i.e., the formation of thin process, of which the mechanics is discussed by Evans and Hochmuth14*). The disadvantage of the method may be the uncertainty in the size and nature of the “point” attachment, which cannot be determined with the microscope. The electron microscopic feature of the point is very complex (see Figure 3 of Reference 143), and the binding force of individual cells to the surface varies widely.lW
0
75
750
se?
FIGURE 6. Deformation of point-attached erythrocytes. An application of the rheoscope, for the measurement of the elongation of erythrocytes, which are point-attached to a polymethylmethacrylate plate, by wall shear force. (From Shiga, T., Sekiya, M., Maeda, N., and Oka, S., J. Colloid Interface Sci., 107, 194, 1985. With permission.)
Volume 10, Issue 1
Oncology/Hematology 3.
The combination of a rectangular flow channel and a laser beam, to monitor the light diffraction, has been tested.L4s The results are similar to those obtained with the ektacytometer. In this system, the velocity profile in the flow channel is parabolic, i.e., the velocity of flowing erythrocytes (thus the degree of deformation) is not uniform along the crossing laser beam. Therefore, some sort of averaged diffraction pattern is obtained.
Taken all together, a flow channel is simple to construct and easy to calculate the wall shear stress, thus it is adequate to monitor the phenomena at the wall, but inadequate to observe the entire depth of suspension. c. MEASUREMENT WITH VISCOMETER The viscosity of an erythrocyte suspension, in a nonaggregating medium (e.g., isotonic phosphate or HEPES buffered saline containing 1% albumin) at a given hematocrit (e.g., 20 to 40%), decreases as the applied shear rate increases. ‘4,~Such shear thinning is induced by the cellular deformation to reduce the hydrodynamic resistance; thus the ratio of suspension viscosity at different shear rates may give a measure of cellular deformation.8”47 In addition, at high shear rates (>200 per second) the intracellular viscosity determines the suspension viscosity, but at low shear rates the membrane properties and the cell shape dominate.‘48
laboratory to another. Therefore, for the sake of international standardization, a guideline has been suggested recently,“* including the preparative methods of human erythrocytes (i.e., blood collection, washing of erythrocytes, recommended buffers, etc.). The use of whole blood (or diluted blood) tends to be avoided, because the leukocytes, platelet microaggregates, or even erythrocyte aggregates interfere with the filtration of erythrocyte. At any rate, the pores are gradually clogged by the cells and the filter is usually disposed of after a single measurement (the filter may be washable and be reused, depending on the material’59). The advantage of the filtration technique is just its simplicity; every laboratory may be able to construct the apparatus at low cost. The disadvantages are summarized as follows: (1) the force against cells cannot be defined and kept constant during the passage; (2) the preparation of the erythrocyte suspension (of certain hematocrit, 5 to 15%) may be troublesome, especially for the perfect removal of leukocytes;‘@’ (3) at the moment, no standard suspension is available, and the comparison of data among many laboratories (or in literature) is not easy; and (4) some sort of averaged values is obtained, but it is strongly affected by the presence of concomitant rigid cells (if any). 16’ b. PASSAGE OF SINGLE ERYTHROCVTES THROUGH A PORE
2. Measurement of Filterability, the Ability to Pass Through Narrow Pore(s) In order to express the ability of the erythrocyte to pass through capillaries of diameter 5 to 8 pm (often narrower than the cell diameter), it is reasonable to quantify the ease of cell passage through the small pore(s).149 Two types of experimental arrangement are distinguished; one is to measure the passage of erythrocytes as whole blood or suspension, and the other is to test the individual cells one by one.
Each erythrocyte must enter into a narrow capillary; thus the ability to enter into and to pass through a small pore should give a practical measure of deformability. Many techniques have been designed with great expectation, but none of these is widely employed, perhaps because each technique is either too elaborate to construct or too artistic to manipulate. The advantage of the method is to obtain the statistical population of erythrocytes. The common disadvantage is the difficulty of defining (or controlling) the external force and the deformed shape. Some of the representative methods are briefly described below.
a. FILTRATION THROUGH MICROSIEVE
1.
most general method is to pass the erythrocyte suspension through a Nuclepore membrane filter, usually with round pores of 5 to 3 p,m diameter of lo-pm thickness. Either the passage time of a certain volume of suspension under a given pressure or the filtered volume during a certain period is measured.‘50-‘53When the pore density is known, the mean passage time of a single erythrocyte may be calculated.‘s4 The time course of the pressure drop (difference) across the filter gives a measure of erythrocyte deformability. 15s.‘s6The determinant factors for the filtration vary with the diameter of the pores, i.e., at a pore diameter of about 5 Frn, both the cell shape and the intracellular viscosity are dominant.1s7 Because the filtration method is simple, many variations are developed; for example, the diameter of pores is varied from 3 to 5 km, the applied pressure depends on the researchers, and the detailed design of the apparatus differs from one The
2.
3. 1990
It is possible to aspirate the individual erythrocytes with a micropipette (of 3 to 5 pm in diameter) by a negative pressure and to monitor the passage by the change in electric potential across the pipette.‘62 The readiness of entering into the narrow orifice and/or passing through a certain length in the pore’63 gives the measure of the erythrocyte deformability. However, the combination with high electric potential gradient may influence the cell entry times through the electrophoretic mechanism. I64 Using a single pore of the microsieve (e.g., Nuclepore membrane), a “single erythrocyte rigidometer” has been constructed165 that measures optically the passage times of individual cells through a pore (5p,m diameter, 10 km thickness) one by one. Kiesewetter et a1.165have compared the values obtained by this method with those by other instruments. “Resistive pulse spectrosc~py”,~~~~~~’which is an out17
Critical Reviews In
4.
growth of Coulter counter, measures the sizes (actually the change in electric resistance) of flowing cells, of which the shapes deform to elongated ellipsoid under high flow rate. The difference in the population patterns between the apparent sizing at a less-deformed state (under low flow rate) and that of an elongated state (under high flow rate) gives a parameter of deformation. “Cell transit-time analyzer” employs “oligopore” filter (e.g., 30 pores of diameter 4.5 urn and length 21 pm, polycarbonate membrane) through which a lOO-kHz AC current and a hydrostatic pressure (e.g., 8 cm H,O) are given. ‘68.‘69From changes in the electrical conductance of the filter due to erythrocyte passage through the pores, the transit times of individual cells are measured and the distribution of transit times for a large number of cells are obtained.
3. Measurement of Mechanical Properties of Membrane Mechanical properties of the erythrocyte membrane can be quantified: the static membrane properties are determined by analyzing the relation between the applied force and the resultant deformation of membrane, while the dynamic properties can be measured by observing the time-dependent process of deformation-restoration. Evans and Skalak”O have reviewed extensively the mechanics and thermodynamics of biomembrane, and recently Hochmuth and Waugh9 and EvansL2 have reviewed the methods for the measurements. Sometimes it is difficult to distinguish the membrane properties from the cellular properties, because the cytoskeletal components are interacting with cell contents (especially hemoglobin). In this section, the measurements largely concerned with membrane, i.e., micropipette aspiration techniques, are briefly described. a. MlCROPlPElTE ASPIRATION TECHNIQUE
Mitchison and Swann’71~‘72introduced a capillary suction technique to measure the mechanical properties of sea urchin egg membrane, and Rand and Burton’24have applied this method to erythrocytes. At present, the micropipette aspiration technique has been extended and employed in many laboratories. The static membrane properties, such as membrane elastic modulus Q.L)or extensional (shear) rigidity, of human erythrocytes can be determined as follows. A portion of erythrocyte membrane is aspirated by a negative pressure (P) through a micropipette (inner diameter of 2R, usually 1 pm), and the length (L) of the extended tongue inside the pipette is measured under a microscope (as a function of P). Using the l-urn micropipette, L/R increases linearly with increasing P-R (in the range 1 < L/R C 3). A theory of Evans and La Celle173 is approximated as follows: P . R/p = C, . (L/R) + C, where p, is the membrane elastic modulus and C, and C, are 18
constants (according to Chien et a1;174C, = 2.45, C, = -0.603). The membrane elastic modulus is independent of intracellular hemoglobin concentration (33 to 44 g/dl), and the typical value for normal erythrocytes is 9 ( + 1.7) x 10e3 dyn/ cm according to Evans et al.175 In addition, using a video recording system, it is possible to determine the time course of membrane restoration after expelling the cell from the pipette. Following the Kelvin-Voigt model, i.e., elastic and viscous elements are combined in parallel, the time constant for shape recovery (t,) is related to the ratio of the membrane surface viscosity (q,,,) and the membrane elastic modulus: t. = “I),,&. The time constant of the shape recovery (the disappearance of the extended tongue) is about 0.3 s according to Evans and Hochm~th,~‘~ to the estimate of the membrane surface viscosity is obtained as low3 dyns/cm. Further, the time constant (t,) is associated not only with the membrane, but with the intracellular hemoglobin concentration (or intracellular viscosity, -qi)and the thickness of erythrocyte (d) as follows:L2,‘75
When the negative pressure applied by the micropipette is further increased, the cell aspiration proceeds, and at a certain critical pressure the cell shape changes to form buckling or wrinkles. This critical pressure provides a measure of the bending elastic modulus (B):‘**“’ B = C.R3.P where a constant (C) depends on the inner diameter (R) of micropipette and is 0.005 to 0.012. The bending elastic modulus is 1.8 x lo- ‘* dyn-cm, according to Evans et al. L75 4. Dynamic Measurement of Cellular Deformability Many methods described above are static measurements of deformation, i.e., the strain of erythrocyte(s) is balanced with the applied external force. However, flowing erythrocytes in large arteries are always exposed to the pulsatile pressure wave, thus the relation between the blood viscosity and the erythrocyte deformability may not hold as it does in a viscometer. Moreover, the erythrocytes in capillaries change their shape accordingly to the geometry of the intracapillary paths. Therefore, we need a way to determine the time-dependent processes of quickly and repeatedly changing deformation and restoration. Several methods are developed for the quantitative measurement of the time-dependent process of whole erythrocytes. 1.
Using a rheoscope, we have applied oscillatory shear stress to flowing erythrocytes.‘33.‘34 As shown in Figure 7, the speed of cone rotation is modulated to produce alternate shear stress. The shapes of flowing erythrocytes change in response to the applied shear stress at low
Volume 10, Issue 1
Oncology/Hematology Oscillatory
oL-
0 102030
‘t . dyn km2
Phase
FIGURE 7. Erythrocyte deformation under stationary and oscillatory shear stresses. Measured in 14% Dextran T-40 (12 cP) at 25”C, with rheoscope. The deformability is expressed by the deformation index (see Figure 5). (Left) Deformation under stationary (uniform) shear stress: the deformation index increased with the applied shear stress (9 to 32 dyn/cmz). (Right) Deformation under oscillatory shear stress: a cone-plate rheoscope is modified to apply oscillatory shear stress (varied in the range 10 to 32 dyn/cm2, as shown in the bottom curve) by modulating the frequency of cone rotation (0.5 to 2.4 Hz). Each symbol shows the deformation under oscillatory shear stress (flash photographs are taken at desired moment of oscillation by adjusting the trigger timing): open circle, 0.6 Hz; half-closed circle (with broken line), 2.0 Hz; closed circle (with dotted line), 2.7 Hz. A solid line is the deformation index obtained under uniform shear stress. Note that above 2 Hz the deformation could not follow the increase in shear stres~.‘~‘~‘”
2.
3.
4
‘.
frequency and under low amplitude of oscillation. However, when the oscillation frequency exceeds a certain value (e.g., 2 Hz, in the oscillatory range of 10 to 30 dyn/cm2), the shape change cannot accommodate the change in shear force. Such retardation in deformation becomes evident in the increasing phase of the applied shear stress. In contrast, the shape restoration in the decreasing phase is a much faster process (e.g., within 0.1 s; see above). The phenomena of oscillatory deformation also strongly depend on the intracellular viscosity and the membrane properties and are more sensitive to the changes of these determinants than similar phenomena in the static measurement with the rheoscope under a constant shear stress. The whole erythrocyte aspirated in 4-pm micropipette can be folded without extension and then the cell is rapidly expelled from the tip. The time constant of unfolding (t;), i.e., the restoration of the width of the folded cell, is monitored. ‘75According to Evans, l2 4 is approximated by tf = (q;d)/(BK,,,*), where qi is the intracellular viscosity, d is the cell diameter, B is the membrane bending modulus (Section III.3.a.ii), and K,,, is a curvature of the fold. For the normal erythrocytes (MCHC of 32 to 34 g/ dl), the time constant of unfolding is 0.25 ( 2 0.09) s, and the value is strongly dependent on the intracellular hemoglobin concentration. The measurement of the extensional shape recovery has been introduced by Hochmuth et al.17s The erythrocyte, of which a portion is point-attached to the cover glass,
is extended end to end by an aspirating pipette at diametrically opposed position, so the cell shape becomes ellipsoid with narrow edges at both ends. When the aspirating site of cell is quickly released, the cell shape recovers to a biconcave disk shape within 0.3 s. The time constant of shape recovery (t,) is about 0.10 s for normal erythrocytes and is strongly dependent on both the intracellular hemoglobin concentration and the membrane properties. Further, Nash and Meisselman’79 have demonstrated prolonged t, for aged erythrocytes in spite of equal membrane elastic modulus Q.L)for young and aged cells. That is, the increased t, is not explainable by elevated intracellular viscosity (qi), but the membrane viscosity increases perhaps due to hemoglobin-membrane interaction when hemoglobin concentration is increased. Sutera et al. la0have also shown the decreased t, of projected cell length (observed with a rheoscope, after abrupt cessation of shearing) for aged erythrocytes. The tank-treading motion of a membrane can be observed with a counterrotating rheoscope.“6~‘n’~‘82 When an erythrocyte is subjected to countershear stresses (without change in the position), the membrane rotates around the cell interior, as visualized by the movement of a small particle (e.g., Latex of 0.8 pm diameter) attached on the cell surface. The tank-treading frequency of normal erythrocytes (5 to 40 per second) varies linearly with the applied shear rate, i.e., the ratio of frequency/shear rate is about 0.2 (in the range 20 to 180 per second of shear rate) and is strongly dependent on cell age.“’
B. Determinant Factors of Cellular Deformability In spite of the variety of deformation measurements, the major determinant factors for cellular deformability are unanimously agreed upon and summarized into three categories: 1. 2. 3.
Cell shape, or surface area-to-volume ratio Intracellular viscosity, or hemoglobin concentration Membrane stiffness, or membrane flexibility
However, reflecting the diversity in the measurement of deformation, the contribution of these factors to the cellular deformability differs from one method to another. Further, as will be discussed later in detail (see Section V.3), these factors vary with cell age. Dealing with the whole population of erythrocytes, one should keep in mind the considerable heterogeneity in terms of deformability. At the moment, the molecular basis of deformability has not yet been established, thus only representative cases are discussed here. 7. Cell Shape or Surface Area-to-Volume Ratio Distinct and extreme examples are the hereditary abnormal erythrocytes, such as elliptocytosis, spherocytosis, etc., caused by abnormality or defect in one of the cytoskeletal proteins. I9 1990
19
Critical Reviews In These erythrocytes are usually fragile and easily hemolyzed in circulation to induce anemia. The biconcave disk shape of normal erythrocytes is important for maintenance of deformability. This shape has greater surface area/volume ratio than a sphere. The sphere is minimum in the ratio and cannot be folded, while the biconcave disk is easily distorted and bent or folded. However, quantitative study of the relation between the cell geometry and deformability is hard. 1.
2.
3.
The modification of water content, for example, is inadequate. When the erythrocytes are exposed to a hypertonic (or hypotonic) solution, the dehydration (or hydration) inside the cell really modifies the surface area/ volume ratio, but simultaneously the intracellular hemoglobin concentration increases (or decreases). Therefore, two factors, the shape and the intracellular viscosity, cannot be separated. Certain trials to alter the surface area/volume ratio have been made. ‘83.*84The incubation of biconcave cells with lysophosphatidyl-choline induces a partial loss of membrane. After washing with albumin to remove the lysophospholipid, the cell returned to a biconcave disk shape, but the deformability decreased,lE4 perhaps due to decreased surface area-to-volume ratio. Certain chemicals can induce the transformation of erythrocytes. Rheological properties of echinocytes are impaired, i.e., the increased suspension viscosity,‘85-‘87 the increased resistance to extension,‘88 the decreased deformation under high shear stress. ‘22,‘86Further, we have shown the decreased rate of oxygen egress. lE9 A recent work’88,‘90 suggests that the normal biconcave disk represents an optimal shape for the flow in viva, since stomatocytosis impairs cell passage through the microvessels (due to the decrease in cell filterability) and echinocytosis affects the flow in large vessels (due to the increase in blood viscosity).
tration is 35 g/dl, the viscosity is about 10 CP (at 25°C),75~77 which is much higher than that of plasma. Morse I119’*i92has applied the spin probe method (with TEMPAMINE) for determining the intracellular viscosity, using electron spin resonance. However, the viscosity values obtained are about one third of the values estimated from viscosity of the hemoglobin solution. Endre et a1.‘93,‘94 have applied the nuclear magnetic resonance (NMR) measurement of T,, with ‘3C-glutathione in erythrocytes. Here again, low viscosity value is obtained. The discrepancy comes from the fact that the spin probe or NMR probe senses the viscosity of its very environment (thus called “microviscosity”), which differs from the bulk viscosity of hemoglobin solution. Hermann and Miiller’95 have recently shown that, above a hemoglobin concentration of 6 r&f, the viscosity of a hemoglobin solution shows rapid increase compared with the microviscosity measured with a spin probe (TEMPONE) . b. MAINTENANCE
OF WATER
CONTENT
IN ERYTHROCYTES
regulation of inorganic ions, pH, and cell volume in erythrocytes is related to the activities of many transporters and to extra- and intracellular environments. The interrelationship between the water movement across the membrane and the concentration of individual ions is too complex and is still in too early a stage to summarize. Here, we show a few examples following: The
Effect of phosphate ion is important for in vitro studies, since incubation without phosphate in the medium tends to reduce ATP and 2,3-DPG. Keidan et al. ‘96 have recommended, for rheological studies of human erythrocytes, the use of phosphate-buffered saline (around 50 mmolll of phosphate) or HEPES-buffered saline (5 to 40 mmol/l of HEPES) of osmolality 290 mOsm/kg pH 7.4. Bookchin et al. 19’have shown that phosphate entry causes alkalinization of intracellular pH and dehydration of erythrocytes .
2. Intracellular Viscosity Since hemoglobin is the most abundant protein inside the erythrocytes, its concentration is an important factor for deformability; for example, at very high shear stress, the erythrocyte may be approximated as a droplet of viscous liquid. The intracellular viscosity can be estimated (1) in vitro by the determination of hemoglobin concentration or (2) in situ by means of the magnetic resonance methods, although the values obtained from these methods do not agree. a. MEASUREMENT
OF INTRACELLULAR
VISCOSITY
In general, hemoglobin solutions prepared from hemolyzed human erythrocytes have Newtonian flow properties, but the presence of cell debris in the solution causes non-Newtonian flow behavior.76 When the intracellular hemoglobin concen-
20
When the intracellular calcium content increases, the calcium-activated K-channel induces potassium loss and dehydration, known as the Gardos effect.‘98*‘99 The effect of intracellular calcium is discussed later (Section III.B.3.c). The imbalance of sodium/potassium is also noticed in erythrocytes from spontaneous hypertensive rats, as well as in erythrocytes from the patients of essential hypertension.200-202 In these cases, decreased activity of Na-K countertransport results in Na overload and K depletion. In addition, the decreased membrane fluidity is detected with spin label technique for the erythrocytes of hypertensive patients. *03 In connection with this, it is known that an increase in membrane cholesterol affects the activity of Na-K ATPase. 2w The situation is similar to some
Volume 10, Issue 1
Oncology/Hematology the changes in the erythrocyte functions are measured by various means. The results are summarized in Figure 8. Briefly, with a doubling of the cholesterol contents in membrane
sorts of hyperlipidemia or obesity, where the membrane fluidity decreases and the activity of the Na-K pump is reduced. Therefore, awaited. 4.
further
detailed
studies
on these points
1.
are
The effect of pH has been studied by varying the pH of the incubation media. It is recognized that the erythrocyte shape is altered with pH: at high pH the cells are flattened, while at low pH the cells tend to become spheric and small in diameterSzo5 At low pH the deformability decreases, the membrane elastic modulus increases, and the filtration time prolongs.206
3. hf8n?br8ne Vi8co8/88ticity
2. 3. 4.
8nd Biochemical
5.
The decrease of membrane fluidity, as measured by the fatty acid spin-label method, corresponding to the temperature decrease of some 5°C (from 36 to 31°C for example)28,42 The decrease in the diffusion rate of oxygen in lipid bilayer of erythrocyte membrane209 The decrease in the velocity of oxygen egress of lo%, as measured with a stopped flow apparatus2’*‘lo The decrease in the velocity of cell aspiration into a some 20% reduction of narrow orifice,2o8 i.e., deformability The decrease in the suspension viscosity of 10% ‘OS
Structure On the other hand, Chabanel et a12” have detected no significant change in the membrane elastic modulus and the extensional recovery time with cholesterol-loaded erythrocytes, in spite of the decreased membrane fluidity of the outer membrane leaflet. These quantitative studies show that in spite of a considerable modification in the membrane fluidity upon cholesterol-loading up to 200%, the changes in rheological functions are some 10% or none. The alteration of lipid composition, even in diseases of lipid metabolism such as LCAT (lecithin-cholesterol acyltransferase)-deficiency, does not double the cholesterol content.2’2 Therefore, the contribution of the lipid bilayer to the mechanical properties of erythrocytes is not so great when compared with the modification or abnormality of the cytoskeletal protein network.
The mechanical properties of a membrane are an important determinant factor for deformability. However, as depicted in Section II, the erythrocyte membrane is a complex sheet, composed of the lipid bilayer and the cytoskeletal proteins. At present, the states of membrane cytoskeletal network appear to be more essential for the cellular deformability than the structure of the lipid bilayer. Although the lipids and proteins are interacting, perhaps it is convenient to discuss the effects on the deformability separately, as follows: 1. 2. 3.
The contribution of the lipid bilayer The role of the static structure of the cytoskeleton The regulation of the dynamic structure of the cytoskeleton
a. CONTRIBUTION OF THE LIPID BILAYER STRUCTURE TO CELLULAR DEFORMABILITY The dynamic structure and the state of the lipid bilayer
b. ROLE OF CYTOSKELETAL STRUCTURE
depend on the composition of the lipids, as proven by various techniques. In artificial membranes, the mechanical and rheological properties are greatly influenced by the lipid composition. However, with human erythrocyte membrane, only a few experimental studies are carried out to reveal “to what extent the deformability is altered as a function of a moditication in lipid composition and structure”, although much evidence for the decreased membrane stability of erythrocytes with the abnormality of lipid metabolism is reported. Cooper207 has shown the decreased deformability in cholesterol-rich erythrocytes. We have artificially modified the cholesterol content of the human erythrocyte membrane and obtained specimens with minute alterations in the energetics (ATP, ADP, AMP, etc.), 2,3-DPG, intracellular pH, and cell shape.28~20s In order to deplete or augment the membrane cholesterol, washed human erythrocytes are incubated with DPPC (dipalmitoyl-phosphatidyl-choline) vesicles containing an appropriate amount of cholesterol, according to the method of molar ratio is Cooper et al. 27 The cholesterobphospholipid modified in the range of 0.6 to 2.0 (normally about l), and
The abnormality of major cytoskeletal proteins in some hereditary diseases frequently induces a shape change of erythrocytes, such as elliptocytosis, spherocytosis, poikilocytosis, etc., and the stability and/or deformability are impaired, thus hemolytic anemia is developed. This subject has been reviewed extensively in this journal by Zail.” Recently, Waugh 213has studied the changes in the membrane elastic modulus and the membrane surface viscosity in the cells of patients having a variety of cytoskeletal disorders. His conclusion suggests that the membrane elastic modulus is proportional to the density of spectrin, but the reduction in the membrane surface viscosity is variable with the degree of membrane abnormality. Further studies are awaited. The loss or decrease of cellular deformability occurs from acquired causes. Mostly, the oxidative degradation or polymerization of membrane constituents leads to the abnormal mechanical properties of erythrocytes. The extreme model for decreased cellular deformability are malondialdehyde (MDA)““ or glutaraldehyde21s-treated erythrocytes, due to the crosslinking of membrane proteins (MDA is a degradation product of
1990
21
Critical Reviews In
I
Membranecholesterol
x2
Awl-chain motion
/
Decreased \ 02 diffusion in membrane
,L
Deformability
x 0.8
I
r-4 Increased
RGURE 8. Impairments of the rheological and oxygen-transporting functions of cholesterol-loaded erythkytes (see text).
arachidonic acid and an oxidative reagent). Similarly, diamide (diazene dicarboxylic acid bis(N,N-dimethyl amide)) is employed as a reagent for oxidative crosslinkage of sulfhydryl groups. *16Diamide treatment is suitable for the model studies, because (1) it is easy to control the degree of crosslinkage; (2) the crosslinkage is induced first in spectrin, then in other proteins; and (3) the linkage can be recovered at least partly by reducing reagent (e.g., dithiothreitol) depending on the degrees of polymerization. 13’On the other hand, MDA and glutaraldehyde are hard to handle, since they crosslink all the proteins through the react@ with amino groups, including hemoglobin, to form insoluble aggregates. We have also studied the quantitative relationships among the degrees of spectrin crosslinkage, the suspension viscosity, and the deformability. I31With an increase in the spectrin crosslinkage (measured by a decrease in the electrophoretic peak of band 2, spectrin l3 subunit), the suspension viscosity (at hematocrit of 40%) increases linearly with the percentage of crosslinkage, and the deformability determined with a highshear rheoscope decreases, as shown in Figure 9. It should be noted that such crosslinkage is partially reversible. The above example of diamide treatment (of low concentration) does not affect the intracellular viscosity and the cell shape, thus giving a quantitative relation between spectrin crosslinkage and the cellular deformability. That is, at high shear stress (e.g., 150 dyn/cm2) the change in deformability is hardly detected below 20% of spectrin crosslinkage, but at low shear stress (e.g., 15 dyn/cm2) the deformability steeply decreases by a subtle crosslinkage. Such spectrin crosslinkage is hardly detected in normal 22
erythrocytes, even in fractionated aged erythrocytes, since (1) the electrophoretic analysis of crosslinkage is not sensitive enough to detect subtle crosslinkage and (2) the highly crosslinked cells, if any, may already be removed in viva during their passage in the spleen. However, subtle crosslinkage may contribute to the decreased deformability of in vivo aged cells (see Section V.3). In addition, it may be noteworthy to comment on the protein-lipid interaction here. The molecular motion of the membrane lipids near the integral membrane proteins, called annular lipids, is suppressed. In contrast, the interaction from cytoskeletal peripheral proteins to membrane lipid may be detected, but very small; for example, we have detected a subtle decrease in membrane fluidity induced by spectrin crosslinkage, with a fatty acid spin label. c. REGULATION OF CYTOSKELETAL DYNAMIC STRUCTURE The deformability may be reversibly controlled by dynamic change or fluctuation in the cytoskeletal architecture, for example, the phosphorylation of spectrin, the regulation of ATP content (production and utilization).*” At present, not much study has been published. Here, we show briefly a regulatory mechanism through intracellular calcium as an example. The calcium content inside the erythrocytes can be artificially modified in a wide range by incubation with a Ca-ionophore (such as A23187) and a given amount of calcium in an appropriate buffer. After incubation the erythrocytes are washed and the total calcium is determined with an atomic absorption spectrometer. It should be noted that calcium loading in an isotonic NaCl solution induces the loss of potassium and water
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2.5” v % Crosslinking FIGURE 9. Suspension viscosity and deformation of spectrin-crosslinked erythrocytes. The crosslinking is expressed by the percent loss of the spectrin beta subunit in diamide-treated erythrocytes. The suspension viscosity, measured at a hematocrit of 40% at 37°C. increased with the progress in crosslinking. The deformability, measured in 20% Dextran T-40 (18.6 cP) at 20°C with rheoscope and expressed by the deformation index (see Figure 5). decreased with the progress in crosslinking. (From Maeda, N., Kon, K., Imaizumi, K., Sekiya, M., and Shiga, T., Eiochim. Biophys. Acru. 735, 104, 1983. With permission.)
(known as the Gardos effect)r9’ due to Ca-activated K-channel and thus the deformability greatly decreases to induce the undeformable cellsL99(Figure 10). However, (1) the dehydration is avoided in a K-containing medium; (2) the energy charge and ATP content are restored by further incubation with glucose; and (3) minor differences of intracellular hemoglobin concentration can be adjusted in the medium of appropriate osmolarity, thus, finally, we can compare the deformation of cells at a similar level of intracellular viscosity and cell shape. ‘I5 The deformability of calcium-depleted or -loaded cells measured with the rheoscope are compared as shown in Figure 11. A change of deformability in two states is apparently recognized: erythrocytes of high calcium content are less deformable than those of low calcium content. When calmodulin inhibitors, such as trifluoperazine or w-7 (N-(&uninohexyl)-5chloro- 1-naphthalene sulfonamide) are added in the medium, the decreased deformability of calcium-loaded cells is restored, while the inhibitors show no effect on the deformability of low-calcium cells. The phenomena are explained below:‘i5 1. 2. 3.
With an increase in calcium content inside the cells, intracellular free Ca+ + increases The calcium-bound calmodulin augments The calcium-bound calmodulin binds to the cytoskeletal network (as to interfere with the spectrin-actin-band 4.1 binding)Z’8*2*9and somehow rigidifies the mechanical properties of the cytoskeletal network
4. 5.
Calmodulin inhibitors suppress the action of calciumcalmodulin The other calcium-binding protein(s) may contribute to the phenomena
In this study, the level of intracellular free Ca’ + is unknown, but it must be closely related to the total calcium content. Although no experiment covering other possible mechanisms is available (e.g., protein phosphorylation),‘@’ the work on the calcium effect provides an example suggesting the regulatory processes of deformability through the reversible change of dynamic cytoskeletal structure.
IV. ERYTHROCYTE
AGGREGATION
Erythrocyte aggregation is the reversible adhesion of adjacent erythrocytes, induced by the bridging of intercalated macromolecules. As a result, 1-dimensional aggregates, socalled ‘ ‘rouleaux’ ’ , or 3-dimensionally branched and piled aggregates are formed. The physiological importance of erythrocyte aggregation in circulation is its tendency to increase the blood viscosity in low shear flow220and to disturb the passage in capillary circulation tbrougb the formation of s1udge.22’When the blood flow falls into stasis, erythrocyte aggregation is pronounced. The erythrocyte aggregates retard the blood flow, and the aggregation is further accelerated. Therefore, a vicious circle may develop in some cases.222-224Since the process of erythrocyte aggregation involves a balance between the aggre1990
23
Critical Reviews In
CCa Ii
H
10jm FIGURE 10. Deformation of calcium-loaded erythrocytes: appearance of undeformed erythrocytes due to dehydration. Calcium is loaded in isotonic Na phosphate-buffered saline by using CaCI, and Ca-ionophore (A23187). The top flash photograph shows the deformation of normal cells observed at a shear stress of 140 dyn/cm* at 25°C. The middle and bottom photographs show calcium-loaded cells under the same shear stress; the number of undeformable cells were increased with an increase of the intracellular calcium content. The total calcium in erythrocytes, [Cal,, is expressed with micromoles per liter of packed cells. (From Shiga, T., Sekiya, M., Maeda, N., Kon, K., and Okazaki, M., Biochim. Biophys. Acta, 814, 289, 1985. With permission.)
gating force and the disaggregating force,zzo the imbalance between the two forces may induce circulatory disturbances, for example, (1) the stasis of blood flow decreases the disaggregating force and (2) the increased amount of bridging macromolecules (such as fibrinogen, immunoglobulins, etc.) increases the aggregating force. In this section, the following problems are discussed: (1) the mechanism of erythrocyte aggregation, (2) the measure24
ment of erythrocyte aggregation with its advantages and disadvantages, and (3) various factors influencing erythrocyte aggregation. The alterations of the erythrocyte aggregation in various diseases will be summarized in Section V.
A. Mechanism of Erythrocyte Aggregation Erythrocyte aggregation is induced by the bridging of in-
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0.4 -
in 12 CP
Dextran
l-r 5.
0.3 r;
PI
The studies by Brooks and Seaman227*228on the bridging of macromolecules between adjacent erythrocyte surfaces support the above mechanism; the adsorption of macromolecules such as dextran expands the electric double layer on the erythrocyte surface and the resultant increase of the zeta potential leads to erythrocyte aggregation by the macromolecules.
376 s-’
0.2 -
brings the adjacent areas into sufficiently close range for further bridging to proceed, thus the deformation of the erythrocyte membrane facilitates the progression of bridging and results in a maximum area of erythrocyte surface bridged by the macromolecules The flow facilitates collisions among the erythrocytes, but the high shear stress acts as a disaggregating force.
2. Rate of Etythrocyte Aggregation and the Shape
of Aggregates L
0
1 10
I 20
[Cali (uM/I
I 30
I 40
The aggregating force in macromolecules is counteracted by the disaggregating force. Chien and Jan225 have summarized the forces as follows: the macromoiecular bridging force is opposed by the mechanical shearing force, the electrostatic repulsive force, and the bending resistance of erythrocyte membrane. Therefore, the rate and degree of erythrocyte aggregation is influenced by the physicochemical properties of bridging macromolecules, the character of erythrocytes, and the physical and chemical environments in flow. From the point of view of energy balance, the aggregating energy provided by macromolecular bridging must overcome the disaggregating energy of electrostatic repulsion and mechanical shearing in order to form stable erythrocyte aggregates. The net aggregation energy is largely stored in the erythrocyte membrane as a change in strain energy, which can be computed from the elastic properties of the erythrocyte membrane.229.230 The changes in membrane strain energy are related to the shape change of erythrocytes (concave or convex at the end) in rouleaux,230 which agree with the computed cell shape based on the adhesion theory.229 Therefore, the shape of individual cells in rouleaux depends on the species of macromolecules and their concentration.
I 50
packed cells)
FIGURE 11. Relation between erythrocyte deformability and intracellular calcium content. After correction of intracellular’ viscosity, i.e., equalizing the mean corpuscular hemoglobin concentration by varying the osmolality of suspending media, the deformation of erythrocytes is measured in Dextran T40-containing HEPES-buffered saline of 12 CP at 25°C by rheoscope (the calcium content in normal cells is 16 to 18 pmol/l of packed cells). The deformability is expressed by the deformation index (see Figure 5). Calcium-depleted cells are obtained by treatment with Ca-ionophore and EGTA, and calcium-loaded cells are prepared by incubation with Ca-ionophore and Ca-EGTA, in K+HEPES-buffered saline. All erythrocytes deformed, but two states were apparent, i.e., low calcium cells were more deformable, while high calcium cells were less deformable. (From Murakami, J., Maeda, N., Kon, K., and Shiga, T., Biochim. Biophys. Actn, 863, 23, 1986. With permission.)
tercalated macromolecules at both ends between of two adjacent erythrocytes.
the surfaces
3. Nature of Macromolecular Bridging on
Etythrocyte Surface
7. Macromolecular Bridging Between Erythrocyte Surfaces On the dynamics of rouleau formation of erythrocytes, model is presented by Chien and Jan:225~226 1. 2. 3.
4.
Bridging of macromolecules at their specific sites between erythrocytes induces erythrocyte aggregation, but such sites are not yet identified. Evidence for specific binding sites of bridging macromolecules on the erythrocyte surface is presented in an experiment using dextran and polyglutamic acid.23’ That is, dextran-induced erythrocyte aggregation is inhibited by nonbridging small dextran, but not by small polyglutamic acid, while polyglutamic acid-induced erythrocyte aggregation is inhibited by nonbridging small polyglutamic acid, but not by small dextran. This fact suggests that the binding site of dextran on the erythrocyte surface is different from that of polyglutamic acid. Erythrocyte-binding site on fibrinogen mol-
a
One end of the macromolecule is already adsorbed on the surface of an erythrocyte The other end attaches to another erythrocyte when two erythrocytes are close together The bridging distance must be far enough to reduce the electrostatic repulsion between negatively charged erythrocytes Such bridging of macromolecules over a limited area 1990
25
Critical Reviews In ecule has recently been suggested to be located in a limited
area of the molecule (see Section IV.C.2.e.ii).232 Any quantitative method to differentiate binding molecules and bridging molecules should be devised for the quantification of bridging molecules leading to rouleaux . On the other hand, an osmotic stress theory was proposed recently by Evans and Needham on the basis of a model experiment for dextran-induced adhesion of giant bilayer vesicles.234 Further evidence on the rouleau formation of erythrocytes is needed for this theory. B. Measurements of Erythrocyte Aggregation Several methods have been developed for quantitative and semiquantitative measurements of erythrocyte aggregation. It may be convenient to classify the various methods into four categories: 1. 2. 3. 4.
Direct measurement in uniform shear flow Indirect measurement through viscometry Indirect estimate from erythrocyte sedimentation rate Others
1. Direct Measurement in Uniform Shear FIow In order to obtain several kinetic parameters, the timecourse measurements of erythrocyte aggregation under controlled shear rate are performed using a viscometer (with coneplate or coaxial cylinder) combined with optical means. SchmidSchiinbein and co-workers235*236 have measured the change in light transmission during erythrocyte aggregation (Ht = 45%), in a gap between the cone and plate of a viscometer at a controlled shear rate (1 to 460 s- ‘) and have proposed some empirical parameters. Usami and Chien237have measured the light reflection of an erythrocyte suspension (Ht = 45%) in a Couette flow using a coaxial cylinder and have introduced the “reflectometric aggregation index” (a comparison of reflectometric reading at 200 s- ’ and at a low shear rate). Recently, Donner et a1.238have developed an instrument composed of a Couette viscometer with coaxial cylinders and have measured the intensity of backscattered light for erythrocyte suspension at a shear rate of 5 to 500 SK’, and several kinetic parameters on erythrocyte aggregation are determined with a microcomputer. Sacks et a1.239have quantified the degree of erythrocyte aggregation under a constant shear rate with a rheoscope, classifying the shape of aggregates into five classes by the microscopic observation. Since the light transmission and light reflection do not necessarily provide the same information,243 all optical measurements should be standardized. Shiga et al. 240have constructed an apparatus for determining the averaged velocity of rouleau formation, combining a rheoscope, a video camera, an image analyzer, and a computer. The process of erythrocyte aggregation is successively analyzed in a constant shear rate and expressed by the time courses of the growth in the projected area of particles. The velocity of
26
rouleau formation is estimated by the computed increment of area/count, i.e., the growth rate of linear rouleau, of which the process is simulated by a kinetic model of linear polymerization.241 Murata and Secomb242 recently presented a mathematical model for rouleau formation in constant shear flow. Their prediction for the relationship between the average size of rouleaux and the applied shear rate well explain the experimental data of Shiga et al.24o A common advantage of these methods is a well-controlled shear rate during aggregation. Some apparatus are, however, not applicable for whole blood: the hematocrit an&or the number of cells in the suspension must be reduced, in order to count the number of particles. 240Theoretical analysis of erythrocyte aggregation241.242can be applied to the method of Shiga et a1.240 2. lndirecf Measurement by Viscometry When the erythrocytes aggregate, the viscosity of the erythrocyte suspension at low shear rates increases more than that at high shear;220thus the measurement of the viscosity of erythrocyte suspension is suitable for the estimation of erythrocyte aggregation. In addition to time- and/or shear rate-dependency of viscosity, dynamic analysis assessing erythrocyte aggregation is performed by viscometry, when a time-dependent shear rate (or oscillatory shear rate) is applied;244,X5these analyses inform viscoelastic behavior of erythrocyte aggregates and thixotropic behavior of blood. The viscometric method may reflect the process of erythrocyte aggregation in well-controlled shear flow. The hematocrit should be adjusted precisely in all methods. 3. indirect Estimate from Erythrocyte Sedimentation Rate The erythrocyte sedimentation rate (ESR) correlates to the readiness of erythrocyte aggregation.*& The maximum sedimentation rate (after the development of erythrocyte aggregates) is usually adopted, but must be corrected for the variation of hematocrit and the viscosity of media.247,248Several factors concern erythrocyte sedimentation and are theoretically treated.220.249*250 for example, the aggregation and dispersion in the gedimentation process, the behavior of sedimenting aggregates and counterflow of the medium, and the incline of the sedimentation tube. The rate of erythrocyte packing under accelerated gravitation using a centrifugal apparatus was measured by Rampling and Sits.251 Furthermore, Bull and Brailsfordz2 have developed an instrument, the Zetafuge’? the zigzag sedimentation of erythrocytes is induced in a near vertically oriented capillary tube by subjecting to four cycles of dispersion and compaction with a controlled centrifuge. The zeta sedimentation ratio (ZSR), an index of erythrocyte sedimentation,252*253is expressed as a percentage of hematocrit divided by zetacrit (the final level of
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Oncology/Hematology erythrocyte compaction in such specified centrifugation). The ZSR is independent of hematocrit (this is superior to ESR)2s2 and provides a good assessment of disease activity because of linear response to changes of fibrinogen or gamma globulins.“2*2” The amount of blood and/or the time for the measurement is reduced. The phenomenon should be interpreted by Oka’szso theory on the erythrocyte sedimentation in inclined tube. The measurement of the erythrocyte sedimentation is still useful in clinic. However, the shear force cannot be controlled, and the growing process of erythrocyte aggregates cannot be measured. 4. Others Some methods observe the process of erythrocyte aggregation after an abrupt stop of high shear flow. Dintenfass et al.247*255 have taken microphotographs of blood between parallel plates of 12.5 pm slit at certain time intervals after the abrupt stop of the flow, and the shape of aggregates is analyzed by parameters reflecting particle size and surface/volume ratio. The apparatus has been employed in the space laboratory by Dintenfass.“6zz57Tomita et al. 258have measured the light transmission of whole blood in a transparent vinyl tube of 0.26 cm I.D.: the exponential decay after the abrupt stop of rapid flow at a wall shear stress of 500 s-’ is observed. Both methods can be applied for clinical use with whole blood, but the shear rate during aggregation is not controlled, and the effect of sedimentation of particles is not taken into account. Chien et al.259 have counted the erythrocyte aggregates formed in a hemocytometer under natural sedimentation and introduced a measure of erythrocyte aggregation, “microscopic aggregation index”. That is, the total number of erythrocytes (obtained in a Ringer solution) divided by the number of cell units (i.e., the total number of single erythrocytes and aggregates obtained in a macromolecule-containing medium). Chien et alz60 have measured the shear stress for disaggregation of rouleau in a parallel-plate flow channel under microscopic observation: the shear stress required to cause 50% separation (the disaggmgation force) of two-cell rouleaux formed in 4 g/d1 dextran with molecular weight of 74,500 is 0.25 dyn/ cm2. The echogenicity to ultrasound scanning increases with an increase in the erythrocyte aggregation.26’ Some of these methods well observe the shape and size of erythrocyte aggregates, but the shear rate is uncontrolled; thus a kinetic analysis of erythrocyte aggregation cannot be carried out by these methods. C. Determinant Factors of Erythrocyte Aggregation The counteraction of the aggregating force (i.e., the attractive force caused by macromolecular bridging between erythrocytes) and the disaggregating force (mainly due to the electrostatic repulsive force caused by the surface negative
charges of erythrocytes) is essential for erythrocyte aggregation. The factors affecting erythrocyte aggregation may be conveniently divided into four categories: 1. 2. 3. 4.
Erythrocytes Bridging macromolecules molecules Physical environment Chemical environment
and coexistent nonbridging
1. Eryfhrocyfes In this category, the influence of the number of erythrocytes (or hematocrit) in the suspension and the general features of erythrocytes on the erythrocyte aggregation is discussed. Although various physical and chemical changes in the erythrocyte environment alter the characteristics of erythrocytes, these factors are discussed later (see Section IV.C.3 and 4). a. NUMBER OF ERYTHROCYTES
(OR HEMATOCRIT)
When the number of erythrocytes in the suspension is increased, the erythrocyte aggregation is accelerated due to the increased frequency of collisions among the celIs.240,24’ In contrast, concerning erythrocyte sedimentation of whole blood, the higher the hematocrit, the slower the sedimentation rate, and vice versa.X7*248 The ZSR is independent of hematocritX2.253 (see Section IV.B.3). b. CELL SHAPE
When two erythrocytes come into contact, a larger contact area creates more stable aggregates. Thus, the curvature of erythrocyte surface is an important factor in erythrocyte aggregation. The shape transformation of erythrocytes affects the aggregation,240 since the formation of stable erythrocyte aggregates is geometrically impeded. Furthermore, the shape transformation alters the topography of negative charge Anionic amphophiles, themselves, increase the negsites. 262*263 ative charge on the erythrocyte membrane and diminish erythrocyte aggregation. 264 c.
DEFORMABILITY
OF ERYTHROCYTES
When the deformability of erythrocytes decreases, the propagation of point-to-point contact to form surface-to-surface contact between erythrocytes is impeded (see Section 1V.A. 1). The oxidative crosslinking of cytoskeletal proteins increases the bending stiffness of the membrane;265thus, flexible contacts among erythrocytes are suppressed. The necessity of flexible structure of spectrin network on erythrocyte aggregation has been evaluated using diamide [diazene dicarboxylic acid bis(N,N-dimethylamide)]. We have shown that the inhibition of erythmcyte aggregation is detectable in erythrocytes in which 5% of the total spectrin is crosslinked.13’ As described above (see Section III.B.3.b), the deformability of erythrocytes decreases evidently at 5% crosslinkage of spectrin. 13’
1990
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Critical Reviews In d. SURFACE CHARGE OF ERYTHROCYTES AND CONFORMATION OF MEMBRANE SURFACE
Sialic
Studies on the electrophoretic behavior of human erythrocytes in a wide pH rangezM have revealed the presence of groups of pK N 2.6 and pK N 3.4, probably from the carboxyl groups of sialic acid and of acidic amino acid residues, respectively. 267These negative charges on the erythrocyte surface cause an electrostatic repulsive force among erythrocytes. The electrostatic repulsive force varies with the surface potential of erythrocytes, represented by the zeta potential that is calculated by the Helmholtz-Smoluchowski equation. When the viscosity and the dielectric constant of the fluid medium are constant, the zeta potential is proportional to the electrophoretic mobility of erythrocytes. The electrophoretic mobility varies proportionally to the content of sialic acid in erythrocytes.71.268 When the quantity of sialic acid in the erythrocyte surface is reduced by hydrolysis with neuraminidase, the electrostatic repulsive force between erythrocytes decreases225*269 and erythrocyte aggregation is accelerated.71.225 Increased erythrocyte aggregation is observed in patient of acquired sialic acid deficiency in erythrocyte membrane.27o The phenomena may be partly related to the increased surface-to-surface affinity of neuraminidase-treated erytbmcytes (in dextran and in plasma). 271 The contribution of sialic acid to erythrocyte aggregation has been quantitatively evaluated using fibrinogen7’ and dextrat? as the bridging macromolecules. Our systematic study of erythrocytes with various contents of sialic acid’] is shown in Figure 12. A 10% increase in fibrinogen concentration from 0.3 g/d1 (physiological concentration) accelerates the rate of aggregation 18%, whereas a 10% decrease in sialic acid content from 0.8 FmoYml erythrocytes accelerates the rate only 6%. Therefore, the contribution of the bridging force of fibrinogen to the rate of erythrocyte aggregation is greater than the repulsive force due to the negative charge of sialic acid in erythrocyte surface. The interaction of macromolecules on the surface of erythrocytes may induce a conformational change of surface proteins. For example, dextran induces a conformational change of glycophorin,272*273but has a minimal effect on membrane surface viscosity.274 8. BLOOD GROUPS The effect of ABO blood groups may be considered for the response in erythrocyte aggregation to various drugs.275 The erythrocyte aggregation induced by various immunoglobulin preparations (for clinical use; immunoglobulin G and the fragments produced by treatment with pepsin or plasmin) is distinctly different among blood groups in spite of the complete removal of blood type-specific globulins.276 Erythrocytes from 0 + type are more resistant to aggregation than those from other typesn6 Interaction with fibrinogen may also be different among blood groups. *” The reason(s) for this phenomenon and the relevance to in vivo circulation are unknown.
28
I
o-2 Slallc
I
I
acid
I
0.4 0.6 08 acid (pmoles/ml RBC)
0.1 0.2 0.3 Fibrinogen (g/d\)
0.4
FIGURE 12. Effect of sialic acid content (in erythrocyte membrane) and fibrinogen concentration (in suspending medium) on the velocity of erythrocyte aggregation. Measured in isotonic phosphate-buffered saline containing fibrinogen and 5 g/d1 albumin (pH 7.4) at the shear rate of 7.5 s-’ at 25°C. The velocity increased with the increase in fibrinogen concentration and with the decrease in sialic acid content. (From Maeda, N., Imaizumi, K., Sekiya, M., and Shiga, T., Biochim. Biophys. Acra, 776, 151, 1984. With permission.)
2. Bridging Macromolecules and Coexisting Nonbridging Molecules The concentration, the molecular size, the molecular conformation, and the electric charge of bridging macromolecules are important determinants in erythrocyte aggregation. In addition, coexisting molecules may impede the bridging of macromolecules, either directly through competition at the same binding site on the erythrocyte surface, or indirectly (noncompetitively) through a stereochemical impediment.
a. CONCENTRATION OF MACROMOLECULES
When the concentration of macromolecules in the erythrocyte suspension is increased, erythrocyte aggregation is accelerated225*23’~232 due to the predominant effect of increased macromolecular bridging. However, it is observed for many bridging macromolecules (i.e., dextran, fibrinogen, etc.) that further increase of macromolecular concentration suppresses The suppressed erythrocyte the erythrocyte aggregation. 225*23’ aggregation at high concentration of bridging macromolecules is interpreted as follows: 1.
2.
3.
Shear stress is increased by the augmentation of bulk viscosity due to the macromolecules added and erythrocyte aggregation is prevented physically.225,231 The increased bridging (and/or binding) of macromolecules causes a local reduction of the effective ionic strength on the surface of erythrocytes due to the volume exclusion effect.*‘* Since the reduction of ionic strength in the suspending medium increases the electrostatic repulsive force, erythrocyte aggregation is inhibited.**’ The increased concentration of macromolecules increases
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Oncology/Hematology the number of bound molecules on the etythrocyte surface,279but the number of bridging macromolecules must be decreased by substituting the one binding end of bridging molecules for the nonbridging macromolecules (which bind the erythrocyte surface only at one end of the molecule) .280
c. MOLECULAR CONFORMATION The molecular conformation may be one of the important factors affecting the shape of erythrocyte aggregates. We have recently found that
1.
Figure 13 shows the shear-rate dependency of the relation between the velocity of erythrocyte aggregation and the dextran concentration. Although the velocity of erythrocyte aggregation decreases with an increasing shear rate, the dextran concentration giving the maximum velocity of erythrocyte aggregation is constant. *‘OThis result shows that the binding isotherm and/or bridging behavior of macromolecules is not altered by the shear force.
2.
3.
Pullulan, a linear polysaccharide, accelerates the erythrocyte aggregation more strongly than dextran (with same molecular weight), a branched polysaccharide280 F(ab’),, a product of immunoglobulin G (Ig G) hydrolyzed by pepsin, accelerates the erythrocyte aggregation more than Ig G282in spite of the lower molecular weight, probably due to the different molecular properties, that is, F(ab’), is more flexible at the hinge region than Ig G and more positively charged than Ig G Fibrinogen, a fibrous molecule, tends to form the ldimensional rouleaux,241v284 while Ig G, a globular molecule, preferably produces 3-dimensional aggregates 240.241.284
d. ELECTRIC CHARGE OF MACROMOLECULES
Positively charged macromolecules, such as polylysine, bridge easily between negatively charged erythrocytes, presumably by ionic interaction. *” Negatively charged macromolecules, such as polyglutamic acid (with a molecular weight higher than 50,000)23’ or heparin (at higher concentration than 1 g/d&*= interact at the sites of nonionic and/or positively charged regions of the erythrocyte surface. Polyglutamic acid with a molecular weight of 20,000 does not induce erythrocyte aggregation, but polylysine with a molecular weight of 9000 still strongly aggregates erythrocytes .231
-5
-4
-3
8. BRIDGING MACROMOLECULES AND COEXISTING
-2
MOLECULES
log [dextran, Ml
The bridging macromolecules interact at the two ends with two adjacent erythrocytes. However, small molecules, which cannot bridge between erythrocytes, but can bind to the erythrocyte surface, occasionally impede the bridging of macromolecules.
flGURE 13. Dependency of the velocity of erythrocyte aggregation on (1) the molecular weight of dextran, (2) the concentration of dextran, and (3) the shear rate. Measured in isotonic phosphate-buffered saline (pH 7.4) containing dextran (T-500, 494,000 in molecular weight; T70,70,400, T-40,42,500; T-10, 10,300) and 0.5 gkll albumin at 25°C. (From Seike, M., J. Jpn. Sm. BloodTransfusion,34,420, 1988. With permission.)
i. Plasma Proteins
Fibrinogen, Ig G, and albumin contribute to erythrocyte aggregation.
b. MOLECULAR SIZE
Fibrinogen (and related compounds) - Fibrinogen is a fibrous molecule with a molecular weight of 340,000 with trinodular structure. The molecular length is 47.5 nm, and the diameter of the terminal nodules (D-domain) is 6.5 nm. The number of fibrinogen molecules bound to a single erythrocyte in the physiological concentration is about 20,000.286 With an increase in the concentration of fibrinogen, erythrocyte aggregation is accelerated.283~287 As shown in Figure 14, we have suggested that the erythrocyte binding site in a fibrinogen molecule leading to erythrocyte aggregation is mainly in the carboxy-terminal region of the A-alpha chain near the D-domain (residue No. 207-303)
Among similar molecular species, the larger molecules are more effective in erythrocyte aggregation than the smaller molecules. These phenomena have been observed for artificial macromolecules such as dextran,22s~231polyglutamic acid,23’ and polylysine.231*28’The increased aggregation with the increased size of macromolecule is due to (1) the increase in the number of bonds per macromolecules on the erythrocyte surface and (2) the decrease in electrostatic repulsive forces due to the increased intercellular distance.225*226 Various high-molecular-weight plasma proteins (fibrinogen, alphq-macroglobulin, immunoglobulins) accelerate the erythrocyte aggregation depending on the molecular size.282~i83 1990
29
Critical Reviews In AJ chain C-terminal
extension
Termi nai domal n
Central domain HMW(native)-Fibrinogen Protease
? 1e
by-d 6-b Fragment
LMW-Fibrinogen t
[Neurami
n i dose
&&&.
\
Fragment
Desialylated
X
Fibrinogen
Fragment
D
Y
Fmgment
Fragment
D
E
FIGURE 14. Schematic diagram of fibrinogen molecule and the related compounds produced by enzymatic hydrolysis. An erythrocyte-binding site in the fibrinogen molecule (marked with an arrow) is proposed as the cause of erythrocyte aggregation. (From Maeda, N., Seike, M., Kume, S., Takaku, T., and Shiga, T., Eiochim. Biophys. Acta, 904, 81, 1987. With permission.)
and partly in the carboxy-terminal region of the gamma chain (residue No. 375-41 1).232This conclusion comes from the following evidence 1.
2. 3.
The low-molecular-weight fibrinogen (mol wt = 305,ooO, partial degradation product of fibrinogen in plasma) has the same effectiveness in erythrocyte aggregation as native, high-molecular-weight fibrinogen (mol wt = 340,000)~3* Sialic acid in the fibrinogen molecule does not contribute to the interaction with erythrocytes232 Among fibrinogen degradation products (FgDP) produced by plasmin, FgDP-X (mol wt = 250,000) and FgDP-Y (mol wt = 150,ooO) still induce erythrocyte aggregation with decreased rate, but FgDP-D (mol wt = 90,000) and FgDP-E (mol wt = 50,000) do notu2~288~289
Immunoglobulin G (and the related compounds) - Ig G is a Y-shaped molecule with a molecular weight of 150,000. The molecular length is about 20 nm, and the dimensions of the cylindrical Fc and Fab portions are 4.0 nm (in diameter) x 4.5 nm (in length) and 3.5 nm (in diameter) x 6.0 rmr (in length), respectively. Ig G is important together with fibrinogen in accelerating erythrocyte aggregation by the nonimmunolVolume
ogical binding on erythrocyte surface. The number of Ig G molecules nonimmunologically bound to a single erythrocyte at a physiological concentration is about 400,000.2W On the other hand, autologous Ig G immunologically (or specifically) binds to erythrocytes, and the number of Ig G is about 200 SectionV.3.f.). Thenonspecific per erythrocyte 291*291a(alsosee binding of Ig G, which can induce the erythrocyte aggregation, differs from the immunological (or specific) binding of Ig G. F(ab’), (molecular weight 92,ooO, a degradation product of Ig G hydrolyzed by pepsin) remarkably accelerates erythrocyte aggregation.282 Fab and Fc (molecular weight 45,000 and 50,000, respectively; the degradation products of Ig G produced by plasmin) cannot induce erythrocyte aggregation themselves,282~292 but can inhibit the Ig G-induced erythrocyte aggregation.276*292 Inhibition by Fab is more effective than that by Fc,*~*probably because Fab is more positively charged than Fc . Fab and Fc also inhibit the erythrocyte aggregation induced by F(ab’)2.292 In dealing with Ig G and the related compounds (as well as fibrinogen) in the study of erythrocyte aggregation, immunologically specific agglutinin should be removed.276~282~292 serum albumin weight Albumin 66,000) ellipsoidal protein a dimension 15 nm length and nm in This molecule binds to
10, Issue 1
Oncology/Hematology the erythrocyte surface [the number of bound molecules adsorbed on a single erythrocyte in the physiological concentration is about 5 X 1W (293)], but is too short to bridge between erythrocyte by overcoming the electrostatic repulsion, thus the erythrocyte aggregation is not induced by itself. However, albumin shows the opposite effect on the erythrocyte aggregation induced by Ig G and fibrinogen, as shown in Figure 15.
I
Low
albumin
High
albumin
FIGURE 16. A model for the opposite effect of albumin on the fibrinogen- and Ig G-induced erythrocyte aggregation. Fibrinogen, Ig G, and albumin molecules anz expressed by hatched, dotted, and vacant frames, respectively. (From Maeda, N. and Shiga, T., Biochim. Biophys. Actu. 855, 127, 1986. With permission.)
r
I 1
I
1
I
2
3
4
Albumin
ment for the bridging of Ig G by albumin adsorbed on the erythrocyte surface.
(g/d11
FIGURE 15.
Effect of Ig G and fibrinogen on the velocity of the erythrocyte aggregation under various concentrations of albumin. Measured at a shear stress of 0.1 dyn/cm2 at 25°C. Albumin showed an opposite effect on erythrocyte aggregation: with Ig G it suppressed the velocity, while with fibrinogen it accelerated. (From Maeda, N. and Shiga, T., Biochim. Biophys. Acra, 855, 127, 1986. With permission.)
The erythrocyte aggregation induced by Ig G is inhibited by albumin, while that induced by fibrinogen is accelerated by albumin.283 An explanation for the opposite effect of albumin for Ig G- and fibrinogen-induced erythrocyte aggregation is schematically shown in Figure 16. In the physiological concentration, albumin, Ig G, and fibrinogen occupy the area of about 70,8, and 0.8 km2 on the erythrocyte surface, respectively (as roughly calculated from the data cited above). Thus the increase of albumin may prevent the bridging of Ig G spatially, but it may not interfere with the binding of fibrinogen for the sake of the small occupied area. When the erythrocyte surface is fully covered with albumin, the distance between erythrocytes is at least 30 nm (= 15 nm x 2). The molecular length of Ig G is less than 30 nm, while that of fibrinogen is more than 30 run, thus bridging by Ig G is prevented by albumin, but that by fibrinogen is not. In the physiological concentration of albumin, the inhibition by albumin becomes effective with an increase in the concentration of Ig G. The inhibitory effect of albumin for Ig Ginduced erythrocyte aggregation may be the physical impedi-
ii. Artificial Macromolecules
Some neutral polysaccharides, such as dextran, hydroxyethyl starch, etc., are clinically used as plasma substitutes for the supplement of colloid osmotic pressure in the circulatory blood.294 Polysaccharides - The critical molecular weight of dextran in leading to erythrocyte aggregation is about 40,000. The erythrocyte aggregation induced by dextran with a molecular weight above 70,000 is inhibited by dextran with the molecular weight below 40,000, by disaccharides, and even by monosaccharides.231*280 The mechanism of inhibition may be (1) the competition of bridging macromolecules and nonbridging molecules at the same binding site23’ and (2) the decrease of effective ionic strength in the contact surface between erythrocytes (due to the volume exclusion effect) and in the suspending medium. 225*295 PolygMamic acid - Erythrocyte aggregation is induced even by negatively charged polyglutamic acid (at least, of the molecular weight above 50,000). Erythrocyte aggregation induced by polyglutamic acid with a molecular weight of 50,000 is inhibited by polyglutamic acid with a molecular weight of 8000, but is accelerated by that of 20,000.23’ The reason(s) for the inverse effect of these nonbridging polyglutamic acids on the polyglutamic acid-induced erythrocyte aggregation is not yet known. 3. Physical Environments
a. SHEAR RATE The
1990
mechanics of the encounter of two blood cells in a 31
Critical Reviews In uniform shear flow has been analyzed by Goldsmith et al.*% With an increase in the shear rate, the collision frequency among cells increases. Therefore, at low shear rates the erythrocyte aggregation may be accelerated with an increase in the shear rate.““’ However, further increases of the shear rate prevent cell-to-cell adhesion and disperse the aggregates, due to increased shear stress.23’~240Such shear rate dependence of rouleau formation, (1) the increase of the velocity of rouleau formation with increasing shear rate in the low shear region and (2) the decrease of the velocity with further increase of shear rate, have been theoretically predicted.242 In oscillatory flow, even as low as 0.001 Hz, the motion accelerates erythrocyte aggregation. 297
L 0
I
/
I
20
30
40
Temperature
(“C)
FIGURE 17. Temperature dependency of the velocity of erythrocyte aggregation in autologous plasma. Measured in 70% autologous plasma + 30% isotonic phosphate-buffered saline (pH 7.4), at the shear rate of 7.5 s-’ at 25°C: different symbols from different donors. At 15 to 18”C, the velocity became minimum.w.“8
b. VISCOSITY
Macromolecules increase the viscosity of a suspending medium at high concentration. The increased viscosity inhibits the erythrocyte aggregation due to increased shear stress. When macromolecules are added to the suspending medium, the correction for the increased shear stress must be made.23’ c. TEMPERATURE With an increase in the temperature, the erythrocyte aggregation induced by various macromolecules (fibrinogen, Ig G, dextran, and polyglutamic acid) is accelerated.284*298The erythrocyte sedimentation rate increases at high temperature. 247 The temperature dependency of erythrocyte aggregation in autologous plasma is shown in Figure 17. The rate of erythrocyte aggregation is minimal around 15 to 18°C and then increases both below and above this temperature.284*298As shown in Figure 18, the shape of erythrocyte aggregates is 1-dimensional rouleaux above 15 to 18”C, while those aggregates below 15 to 18°C are 3-dimensional.284*298The temperature-dependent shape difference of erythrocyte aggregates in plasma is similar to those induced by fibrinogen.284 The erythrocyte aggregates induced by Ig G are 3-dimensional in all temperature ranges.284 The temperature dependency of the shape of erythrocyte aggregates is interpreted by the following terms:
1
10
creases with a decrease in the temperature, probably due to decreased membrane fluidity22.43and decreased mobility of membrane proteins3@ and lipids.30s The membrane elastic modulus and the membrane surface viscosity increase with decreasing temperature.306 At low temperature, the increased binding force of macromolecules to erythrocytes induces the 3-dimensional aggregates, together with the difficulty in transition from face-torim to face-to-face adhesion due to the decreased surface area to volume ratio and the decreased deformability. The temperature dependency of erythrocyte aggregation may be important for understanding the microcirculation in hypothermic tissues (e.g., for the exposure to a cold environment, under hypothermic anesthesia, under extracorporeal perfusion) or hyperthermic tissues (e.g., in metabolically active tissues, for exposure to a hot environment). 4. Chemical Environments
1.
2.
3.
32
With a decrease in temperature, the viscosity of suspending medium increases; thus the increased shear stress inhibits erythrocyte aggregation. 284 Properties of macromolecules - The reversible conformational change of macromolecules with temperature is observed for fibrinogen299 and Ig G;300thus the interaction of these macromolecules with erythrocyte surfaces may change, although fibrinogen binding is independent of temperature. 286 Properties of erythrocytes - With a decrease in temperature, the diameter of erythrocytes decreases and the thickness increases, but the cell volume does not change;284~301 thus the surface area to volume ratio decreases. The erythrocyte deformability302*303also de-
Mechanical
shearing force -
a. pH
With an increase in pH of the suspending medium, erythrocyte aggregation is accelerated.20s*240~298 The erythrocyte aggregates induced by fibrinogen are l-dimensional rouleaux in alkaline pH, but are 3dimensional aggregates in acidic pH.20s.298 The facilitation of erythrocyte aggregation in alkaline pH may be interpreted as follows: 1.
2.
- With increasing pH, the diameter of erythrocytes increases, the thickness decreases, and the cell volume decreases.20s*30**~7 The increased surface area to volume ratio of erythrocytes accelerates erythrocyte aggregation. &u&ace charge of erythrocytes - No change in the electrophoretic mobility of erythrocytes (thus, in the surface Shape of erythrocytes
Volume 10, Issue 1
Oncology/Hematology
Plasma
Fibrinogen
.-
18OC
FIGURE 18. Microphotographs of etythrocyte aggregates formed in different media and at different temperatures. Aggregates formed after 10 min under a shear rate of 7.5 s-’ in 0.4 g/dl fibrinogen + 5 g/d1 albumin (fibrinogen), in 0.4 g/d1 Ig G + 5 g/d1 albumin (Ig G), and in 70% autologous plasma + 30% isotonic phosphate-buffered saline (plasma). Ig G tended to form 3-dimensional aggregates, whereas fibrinogen induced linear rouleaux except below 15°C. (From Maeda, N., Seike, M., and Shiga, T., Biochem. Biophys. Am, 904, 319, 1987. With permission.)
3.
4.
charge) is observed in the pH range of 3.0 to 9.5 at ionic strength 0.145. 266 Therefore, the electrostatic repulsive force due to the negative charge of the erythrocyte surface is constant in the physiological pH range. Eryrhrocyre deformability - The elastic property of the erythrocyte membrane, i.e., the membrane elastic modulus, is altered below pH 6.0.206~308 Interaction of macromolecules with erythrocyte su$ace - The binding of fibrinogen increases with decreasing pH below 6.5.286
b. IONIC STRENGTH
AND IONIC COMPOSITION
The decrease of ionic strength in the suspending medium expands the electric double layer of the surface of the erythrocyte membrane and increases the electrostatic repulsive force 225.278
Divalent cations, compared with monovalent cations, reduce the thickness of the electric double layer, and the decreased electrostatic repulsive force between erythrocyte surface accelerates erythrocyte aggregation.278 c. OSMOLALITY With decreasing osmotic pressure in phosphate-buffered saline from 285 to 190 mOsm, the velocity of erythrocyte aggregation decreases about half.298.3w On the other hand, with increasing the osmotic pressure to 400 mOsm, the velocity increases about twice, but it decreases in hypertonic media above 400 mOsm.298.309 The erythrocyte aggregation induced by fibrinogen is essentially l-dimensional rouleaux, but is 3dimensional below 200 mOsm.298 The increased erythrocyte
Altogether, the change of erythrocyte aggregation with pH is mainly due to the shape change of erythrocytes and partly due to the changes of erythrocyte deformability and macromolecular binding. The physiological importance of such pH dependency may be found in the microcirculatory flow through metabolically active tissues (with lowered pH): the decreased erythrocyte aggregation in a low pH facilitates the blood flow in such tissues.
1990
33
Critical Reviews In aggregation with increasing osmolality is due to several causes as follows:
Table 1 Symptoms of Hyperviscosity Syndromes
1.
Cardiovascular symptoms
2. 3. 4.
The shape change from spherical to disk-like (i.e., the increased surface area to volume ratio)298~30’ The increased defonnabilitymO’ The decreased electrostatic repulsive force due to the increased ionic strength225.278.295 The increased binding of fibrinogen to erythrocytes (above isotonicity)286
Concerning the effect of cell volume on the erythrocyte aggregation, Nash et al. 3’o have observed that the erythrocyte aggregation increases with a decrease in the cell volume up to 80% of normal (without changing either the osmolality or ionic strength of suspending medium by using valinomycin), but the aggregation decreases with further decrease of cell volume. The decreased aggregation above 400 mOsm in saline298 or above 600 mOsm in plasma3” may be mainly due to the decreased deformability, since the intracellular hemoglobin concentration increases under hypertonic condition. The phenomena may be important for understanding the blood flow under hypertonic tissues (e.g., renal medulla).
Retinal symptoms
Hemorrhagic symptoms Neurological symptoms
Decreased pulse pressure, peripheral edema, Cardiac inSUffICienCy(in prOgre88cdState) Sausage-shaped venous distension, flameshaped hemorrhage, capillary microaneurysms (retinopathy) Mucous membrane bleeding (from nose, gum, etc.) Headaches, vertigo, nystagmus, impaired hearing, etc.
Table 2 Causes of Hyperviscosity Syndromes CPuseS
Increasednumber of erythrocytes Decreased erythrocyte deformability
Accelerated erythrocyte aggregation
V. ERYTHROCYTE RHEOLOGY IN HEALTH AND DISEASES The rheological properties of erythrocytes are impaired in various diseases and in vivu aging. In this section, only brief consideration is given to (1) hyperviscosity syndrome, (2) abnormality in erythrocyte deformability, and (3) decline of deformability during in vivo aging of erythrocytes. A. Hyperviscosity Syndrome Increase of blood viscosity may induce circulatory disturbances either locally or systematically.312-3’5Typical symptoms summarized in Table 1 mostly concern the impeded flow in venous systems and are independent of the primary cause of the disease. These symptoms are caused by (1) increase in peripheral circulatory resistance, (2) hindrance in blood passage through the microvessels, and (3) stasis in the venous circulation due to the increased blood viscosity. The rheological causes for these symptoms are summarized in Table 2, that is, (1) an increased number of erythrocytes, (2) a decreased erythrocyte deformability, (3) an accelerated erythrocyte aggregation, and (4) others.8~223~224~31s B. Abnormality in the Erythrocyte Deformability The following various causes are considered here as typical examples: 1. 2.
The abnormality of cytoskeletal proteins The abnormal hemoglobins
Accelerated coagulation or agglutination Others (with various causes)
3. 4.
Disorders Polycythemia Hemoconcentration (dehydration), etc. Hereditary disorders with abnormal cell shape Unstable hemoglobinopathy Sickle cell anemia Formation of Heinz body, etc. Hyperfibrinogenemia Multiple myeloma Macroglobulinemia Cryoglobulinemia, etc. Hypercoagulability Incompatible blood transfusion, etc. Diabetes mellitus Myocardial infarction Raynaud phenomenon, etc.
The morphological abnormality of erythrocytes The formation of Heinz bodies
Although there are many diseases accompanying impaired erythrocyte rheology and leading to hemolytic anemia, 19*316*317 only a few cases are described. 7. AbnormaMy in the Cytoske/eta/ Proteins The genes of human alpha- and beta-spectrins are located at the chromosomes lq22-25 and 14~32, respectively. The gene of band 4.1 protein is located at the chromosome lp3436. The abnormality of DNA in these genes sometimes (but not always) induces an abnormality of corresponding protein, which often results in a morphological alteration of erythrocytes through abnormal cytoskeletal architecture and/or impaired interaction among cytoskeletal proteins, e.g., elliptocytosis, spherocytosis, pyropoikilocytosis, etc. These cells are poorly deformable, and thus are easily destroyed in circulation to induce anemia. These days, as summarized in a recent review in this journal, l9 hereditary abnormalities in cell shape are being clarified rapidly. However, it is too early to
Volume 10, Issue 1
Oncology/Hematology draw any conclusions concerning the critical lesions in polypeptides that cause shape alteration, hemolysis, etc. Thus, more fundamental data of molecular pathology are needed.
4.
2. Abnorm8l Hemoglobins Intravascular infarction is induced in hemoglobin S disease, when the sickling of erythrocyte occurs upon deoxygenation or the irreversible sickle cells are formed.318The decreased deformability of the sickled erythrocytes is noticed. The increased membrane elastic modulus of the erythrocyte, the prolonged extensional recovery time, and the increased membrane viscosity are also observed, especially for the irreversibly sickled cells.3’9 In some cases of abnormal hemoglobin, e.g., hemoglobin K61n,320 decreased filterability is reported. If hemoglobin molecules are unstable, the resultant denatured hemoglobin forms Heinz bodies in the cell and the deformability is impeded.
5.
These changes are common features for dense erythrocytes fractionated by density gradient centrifugation. It is reasonable that metabolic activity in erythrocytes decreases in some extent during aging in circulation, and that the decreased activity accelerates above changes. However, contradictory results are sometimes reported on the age-associated changes (e.g., ATP content does not vary during aging). One should take the following points into consideration to interpret the results: 1.
3. Formation of Heinz Bodies Heinz bodies are small inclusions of dense masses (diameter of 0.3 to 2 km) attached to the membrane and stained with May-Grtinwald-Giemsa. Heinz bodies are composed of denatured proteins, mostly hemoglobin (or globin) and are observed in many hereditary diseases, for example, in thalassemias, in unstable hemoglobinopathies, and in hereditary defects of hexose monophosphate shunt (under oxidative stress). The denatured hemoglobin is usually bound to the membrane, thus the deformability is greatly impaired.81J’2~321
2.
3. 4. 5. 6.
4. Abnormality in Erythrocyte shape The other diseases inducing a shape alteration of erythrocytes are sometimes accompanied by decreased deformability. Figure 19 shows the schizocytes in acquired hemolytic anemia and the appearance of undeformable cells under high shear stress (observed with a rheoscopic flash photograph). In some cases of disturbances in lipid metabolism, acanthocytosis is induced. However, the deformability of these cells is not always impaired, although hemolytic anemia occurs.
2. 3.
The method adopted for cell fractionation (e.g., according to cell density, cell size, or surface charge) The media used for cell fractionation (such as albumin, high-molecular-weight polysaccharide, phthalate, or Percoll) The percentage of fractionation of cells The animals used for the experiment (different survivals in different animals) The enzymes and metabolites analyzed (in association with aging) The amount of reticulocytes
One should be especially careful with the concomitant veticulocytes in the preparation to deal with the age-dependent phenomena, since the decay of most enzyme activities is very rapid in reticulocytes (and perhaps in very young erythrocytes), but becomes very slow in mature cells.w.322 The rheological impairments in aged erythrocytes are induced by complex reasons (not a sole cause). The decreased deformability of aged cells is observed by rheoscopy,199.323.324 filterability,325.326and micropipette aspiration.78*163~179~327 The impaired deformability of aged cells originates from increased membrane viscosity79*‘79.328 and increased intracellular viscosbut the membrane elastic modulus does not vary durity, ‘79*329 ing aging. 79~179,328 We briefly describe the major disturbances one by one.
C. Decline of Deformability During In Viva Aging of Etythrocytes Aged erythrmytes are removed from circulation by phagocytosis in the reticuloendothelial system, especially in spleen. The decreased deformability and the increased adhesiveness of erythrocytes are major determinant factors for the removal of aged cells in spleen. The following changes are induced in erythrocytes during aging: 1.
ulated perhaps by intracellular calcium ion and by protein phosphorylation The condensation of hemoglobin, i.e., increased intracellular viscosity, due to the decrease of surface area and the loss of water The appearance of senescent antigenic sites and the increased binding of Ig G on the surface of aged erythrocytes
1. Ch8ngeS in Metabolic Components
Activity 8nd lntr8cellul8r
During erythrocyte aging, many enzymes (and transport proteins) show decreased activity; thus the rate of glycolysis is decreased, and the concentrations of various metabolites and decreased 2,3-DPG (thus increased inorganic ions change..3*4*89 oxygen affinity of the aged erythrocytes),78~163~330 increased methemoglobin content, 33l increased Na+ and decreased K+,330 increased Ca+ + ‘99*332 (with the decreased calmodulin activ-
The decrease in surface area and volume due to the loss of membrane components The oxidative modification of membrane components (both lipids and proteins) to stiffen the membrane Modification of the cytoskeletal dynamic structure, reg1990
35
Critical Reviews In
FIGURE19. Deformation of erythrocytes from autoimmune hemolytic anemia, observed with a rheoscope. (Top) Abnormal cells observed with a scanning electron microscope. (Bottom) Cell deformation at a shear stress of 70 dyn/cm* with rheoscope: undeformable cell shown by arrow (autoantibody of warm type is detected). (From our unpublished data.) .& W33*334
&&
&+
+,332.335 decreased
ATp,78.330.332.336
and so on. The creatine level in erythrocytes, which is higher in young cells than in aged cells, correlates well with erythrocyte survival in circulation.337 2. Loss of Membrane Components When the loss of membrane components exceeds the loss of intracellular proteins, the surface area to volume ratio inevitably decreases. As shown in Figure 20, the shape of the aged cells, separated by density gradient centrifugation, shows a
36
smaller diameter, smaller volume, and more spherical appearof ance than that of the young cells. 78.79.163.199.338 The mount membrane lipids certainly decreases during in vivo aging.78*89*339 However, there is apparent disagreement about the quantity of total proteins. 43*78In general, it has been believed that both lipids and membrane proteins are decreased during aging, but we have observed only a minute loss of proteins. The discrepancy concerning the amount of membrane proteins may ~ arise from the differences in fractionation of erythrocytes and i preparation of ghosts (we discarded the
Volume 10,
1
Oncology/Hematology
FIGURE
20.
Young erythmcytes(top) and aged erythrocytes
a scanning electron microscope. gradient centrifugation.78~19
The human erythrocytes
fraction, although less than 1% of total cells);” in methods quantifying membrane phospholipids and proteins; and in the conversion of the measured values to the contents of phospholipids and total protein (the amounts of proteins are often expressed by “albumin-equivalent weight”, of which no standard is given; the amounts of phospholipids are rather arbitrarily converted from the values of phosphate assay, without analyzing the composition of phospholipids). The decreased membrane fluidity of the in vivo aged eryth-
(bottom), observed with were fractionated by density
rocytes is observed by the electron spin resonance using fatty The alteration of protein-lipid interaction acid spin label. 43*78~163 may contribute to decreased fluidity, thus to increased membrane viscosity.79”79 3. Peroxidative Alterations of Membrane
Components Erythrocytes contain glutathione and possess many antioxidative enzymes, for example, glutathione peroxidase, cat1990
37
Critical Reviews In alase, and superoxide dismutase. However, the activity of the enzymes decreases during in viva aging,89*340*341 thus the ability of antioxidation diminishes gradually.342 Although the crosslinkage of membrane proteins is not well proven in aged erythrocytes, the extractability of spectrin (with 1 nN EDTA, pH 8) decreases,343 suggesting the modification of cytoskeletal networks due to aging. Increase of hemoglobinbound spectrin,344 decreased ratio of protein 4.lb to protein 4 . la ,415*Nincrease in carboxymethylation347and racemization~ of membrane proteins are demonstrated in aged cells. Some of these modifications contribute to the increased membrane viscosity of aged cells in part. There is some doubt about the contribution of MDA in polymerization of membrane proteins in aged cells349*350 as a major oxidative reagent, since the thiobarbituric acid reactivity is not altered during erythrocyte aging. On the other hand, exposure of erythrocytes to MDA as model studies well resembles the changes in lipids, e.g., the detection of phospholipid adducts of MDA and the increase of phosphatidyl-choline (also the decrease of phosphatidyl-serine).46 The increased ratio of alpha-tocopherol to phospholipids (especially arachidonic may protect cells from lipid peroxiacid) in aged cells351*352 dation to some extent. Further studies on the lipid-protein interaction, as well as searches for oxidative influences on cytoskeletal structure, are necessary. 4. Retention of Calcium and Changes in Cytoskeletal Structure Intracellular calcium content increases with erythrocyte aging,lw primarily because of the declined ability of the calcium extrusion during aging. Moreover, a report showed the enhancement of calcium permeability under high shear flo~.~” The entering calcium is extruded by calcium pump at the cost of ATP,355 and the transport is activated by Ca-calmodulin356 that is formed with an increase in the calcium content. Model studies to increase the intracellular calcium with ionophore have been carried out and the decreased deformability was unanimously observed in calcium-loaded cells.199 However, as calcium-loading progresses, Ca+ +-dependent Kchannel is activated; consequently a loss of potassium and water occurs357 (called the Gardos effect’98), and further ATP depletion and echinocytic transformation appear. 199.357-359 In addition, upon calcium overloading, the crosslinkage of membrane proteins (yielding glutamine-lysine bond)360 develops by the activation of transglutaminase. We have carried out a careful calcium-loading to fractionated erythrocytes (without shape change and ATP depletion)“s*‘99 and demonstrated that (1) calcium retention occurs easily with aged cells, but scarcely with young cells ;I9 (2) with an increase in the intracellular calcium, the cell volume decreases, thus the deformability is reduced;‘99 (3) after correcting the cell volume (and, consequently, the intracellular viscosity) by modifying the osmolality of medium, the deformability apparently changes between two states”’ (see Figure 1 l), i.e., high calcium creates less de38
formable cells and low calcium, more deformable cells; and (4) the decreased deformability of calcium-loaded erythrocytes can be restored perfectly with some calmodulin inhibitors. ’ l5 In a high calcium state, calcium-bound calmodulin is increased and the complex interacts with spectrin, band 4.1 protein, and/ or actins,96*1’4*218*2L9~361 then the properties of the cytoskeletal network are modified in such a way that the membrane stiffness increases. Calmodulin inhibitors prohibit such interactions through binding with the calcium-calmodulin complex.362*363 It is almost certain that the concentration of intracellular calcium has a key role for the regulation of the cytoskeletal dynamic structure, thus for the control of deformability. However, (1) the determination of intracellular free Ca+ + is hard when compared with that of total calcium; (2) the amount of total calcium in erythrocytes”.364,365is now becoming disputable; (3) the role of Mg+ + with ATP must be considered for the Ca+ + extrusion;366and (4) the distribution of calcium inside the erythrocyte367 must be studied further. 5. Condensation of Hemoglobin As the surface area decreases due to a loss of membrane components, but the intracellular proteins are preserved, the condensation of hemoglobin inevitably results with aging. The decreased activities of ion channels and ion pumps accelerate the dehydration. The increased intracellular hemoglobin concentration with cell age makes it possible to fractionate these erythrocytes by density gradient centrifugation.336*368-370 The increase in the intracellular hemoglobin concentration greatly affects the deformability of erythrocytes, as discussed above (see Section III.B.2). 6. Modification of Surface Structure During in vivo aging, the surface structure is (chemically) modified, as clarified by glycosylation of membrane proteins,37’ decreased number of insulin receptor,372-374decreased activity of acetylcholine esterase,89r37’decreased ouabain bindthe induction of senescent antigens,378-380and ing sites, 376,377 the resultant increase of Ig G (antibody) binding to such antigens. 381-384 The recognition of senescent erythrocytes by phagocytes is important in the destruction of these cells in the reticuloendothelial system. However, (1) the biochemical changes in erythrocytes to induce the senescent antigens (i.e., due to the destruction of band 3385and/or the desialation of glycophorin A3*6), (2) the biochemical structure of the senescent antigens appeared on the erythrocyte surface, and (3) the mechanism of the recognition of senescent erythrocytes by phagocytes are not known at the moment. 7. Changes in Adhesiveness In addition to the recognition of aged erythrocytes by phagocytes, adhesion of these erythrocytes to endothelial cells and the impeded passage due to their decreased deformability is an important moment in the trapping in the meshwork of splenic pulp, thus in the determination of the lifespan of individual
Volume 10, Issue 1
Oncology/Hematology 6. Marchesi, V. T., Stabilizing infrastructure of cell membranes, Annu. Rev. Cell Biol., 1, 531, 1985. 7. Fknoett, V., Cohen, C. M., Lux, S. E., and Palek, J., Eds., Membrane Skeletons and Cyroskeleral-Membrane Associations. Alan R.
erythrocytes. As a mechanism of erythrocyte destruction, the protective role of sialic acid has been studied extensively: upon removal of sialic acid, (1) the adhesiveness to endothelial cells is increased,387 (2) the survival time of erythrocytes in circulation is decreased,388 and (3) a new antigenic site may be During in vivo aging of human erythrocytes, exposed. 386,389 the total content of sialic acid (with some carbohydrates) decreases,390-392but the density of sialic acid on the erythrocyte surface is unaltered,393*394i.e., the effective negative charge per unit area of membrane surface remains constant.393 The exact role of sialic acid on erythrocyte survival is still unknown. These subjects are still open for investigation.
Liss, New York, 1986. 8. ChIen, S., Red cell deformability and its relevance to blood flow, Annv. Rev. Physiol., 49, 177, 1987. 9. Hochmuth, R. M. and Waugk, R. E., Erytbrocyte membrane elasticity and viscosity, Annu. Rev. Physiol., 49, 209, 1987.
10. Chasis, J. A. and Shohet, S. B., Red cell biochemical anatomy and membrane properties, Annu. Rev. Physiol., 49, 237, 1987. 11. Clark, M. R., Senescence of red blood cells: progress and problems, Physiol. Rev., 68, 503, 1988. 12. Evans, E., Deformability and adhesivity properties of blood cells and membrane vesicles: direct methods, in Red Cell Membranes, Shohet,
VI. SUMMARY 13.
Two main subjects of erythrocyte rheology, deformation and aggregation, are discussed in detail, on the basis of biochemical structure. The close relationship between the life span (or cell aging) and the rheology of individual erythrocytes is also briefly described. A currently important problem is emphasized, that is, the molecular aspect of the dynamic cytoskeletal structure and the mechanism of its regulation. This concerns not only the rheological function and the survival of circulating erythrocytes, but also the pathophysiology of abnormal erythrocytes.
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ACKNOWLEDGMENTS The authors are indebted to Professors Syoten Oka, Itiro Tyuma, and the late Takehiko Azuma for their encouragement. The financial support from the Ministry of Education, Culture and Science of Japan, and the Ehime Health Foundation are acknowledged. We wish to thank Miss Misuzu Sekiya and Mr. Andrew W. Winden for their help in the preparation of the manuscript.
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