Volume 21, number 2

MOLECULAR & CELLULARBIOCHEMISTRY

November 1, 1978

THE U L T R A S T R U C T U R E OF DEFECTIVE H U M A N PLATELETS

James G. WHITE and Jonathan M. G E R R A R D From the Departments o[ Pediatrics and Laboratory Medicine and Pathology, University o[ Minnesota Health Sciences Center, Minneapolis, Minnesota 55455. (Received March 30, 1978)

Summary

Much of our current knowledge about the physiology of hemostasis has come from intensive study of platelets from patients with inherited and acquired bleeding disorders or an increased risk of thrombotic disease. Appreciation of the role of plasma proteins in platelet stickiness, of platelet surface membrane glycoproteins in aggregation, of the substances stored in platelet organelles in cell-cell interaction, vascular injury and atherosclerosis, and of endoperoxides and thromboxanes in platelet intercellular communication have resulted largely from investigations on various types of defective platelets. While the techniques of physiology and biochemistry have generated critical details about abnormal platelets, electron microscopy and ultrastructural cytochemistry have provided an improved morphological framework in which to integrate the new discoveries. The present review has attempted to correlate physiological, biochemical and ultrastructural concepts as they relate to the current understanding of inherited platelet disorders.

I. In~oducfion

The past twenty years has witnessed a sharp increase of interest in human blood platelets and a rapid accumulation of knowledge concerning their biochemistry, structure, physiology and pathology. Factors responsible for the surge of platelet research included the realization that

platelets are not only critical for hemostasis, but are involved in other aspects of host defense 1-3, in the evolution of thrombotic disease 4 and in the pathogenesis of atherosclerosis5'6. In addition, marked improvements in methods for separating platelets from whole blood, maintaining their unaltered discoid state in vitro, examining their interactions in response to chemical and physical stimuli, and studying their basic biochemistry and physiology made it possible to carry out definitive investigations on this, the smallest of the peripheral blood cells. Development of better procedures for preserving platelet fine structure and localizing specific chemical subunits by ultrastructural cytochemistry provided a markedly improved morphological framework for integrating the new discoveries about platelet biochemistry and physiology. The synthesis of information coming from the application of many sophisticated disciplines has resulted in a new understanding of platelet structural physiology and pathology. Much of our current knowledge has come from intensive study of platelets from patients with inherited bleeding disorders 7. Appreciation of the role of plasma proteins in platelet stickiness 8, of the platelet surface membrane glycoproteins in aggregation 9'1°, of the substances stored in platelet organelles in cell-cell interaction, vascular injury, chemotaxis, thrombosis and atherosclerosis 1~'12, and of the endoperoxides and thromboxanes 13 in platelet intercellular communication have resulted

Dr. W. Junk b.v. Publishers - The Hague, The Netherlands

109

largely from investigations on various types of defective platelets. For that reason it is worthwhile to examine periodically the current state of knowledge regarding abnormal platelets. Investigations in this laboratory have been mainly concerned with the ultrastructural physiology of normal and abnormal platelets. Therefore, the present discussion will emphasize the fine structure of defective cells. Yet, from the inception of our studies we have realized the importance of combining the information gained from electron microscopy with biochemistry and physiology, and the new findings will be presented in that context. The subject of abnormal platelets has been reviewed in several recent publications 14-19, and the reader is referred to these sources for a more comprehensive treatment. In this report we will limit our discussion to disorders in which new findings have developed in the past few years.

development of specific relationships between structure and function 17-19. The peripheral zone consists of the membranes and closely associated structures providing the surface of the platelet and walls of the tortuous channels making up the SSCS (Figs. 4A,4B). An exterior coat, or glycocalyx, rich in glycoproteins provides the outermost covering of the peripheral zone. Its chemical constituents provide the receptors for stimuli triggering platelet activation and the substrates for adhesion-aggregation reactions. The middle layer of the peripheral zone is a typical unit membrane. It is rich in asymmetrically distributed phospholipids which provide an essential

H. Platelet Structural Physiolog? A brief description of normal platelet structure and function is reasonable, prior to presentation of abnormalities observed in patients with bleeding disorders. Unaltered platelets have a characteristic discoid form in circulating blood and in anticoagulated samples of platelet-rich plasma prepared for in vitro study (Figs. 1A,1B). In contrast to leukocytes, which are generally covered with villous projections and surface folds, platelets in the quiescent state have smooth surface contours, not unlike the red blood cell. Closer inspection of the unruffled surfaces, however, reveals random dimples which are the sites of communication between the surface-connected open canalicular system (SCCS) or, more simply, open canalicular system. Thin sections of well preserved platelets also demonstrate the lentiform shape and smooth surface contours of the cells (Figs 2A,2B,3A,3B). The complex internal structure, however, belies the simplicity suggested by outward appearance. We have sought to bring order to the structural complexity of platelets by dividing its anatomy into three regions. The basis for the divisions selected was by no means arbitrary, and had as its major purpose the 110

Figs. 1A,1B. Discoid platelets. The lentiform shape of blood ' platelets is well preserved in samples fixed in glutaraldehyde and critically point dried for study in the scanning electron microscope. The indentations ( ~") apparent on the otherwise smooth surfaces of the platelets indicate sites where channels of the surface connectedopen canalicular system communicate with the cell exterior. Mag. l a x 10,000; 1Bx 20,000.

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Figs. 2A,2B. Discoid platelets. The diagram in 2A summarizes the ultrastructural features observed in thin sections of discoid platelets cut in the equatorial plane. Components of the peripheral zone include the exterior coat (EC), trilaminar unit membrane (CM), and submembrane area containing specialized filaments (SMF) which form the wall of the platelet and line channels of the surface connected canalicular system (CS). The matrix of the platelet interior is the sol-gel zone containing actin microfilaments, structural filaments, the circumferential band of microtubules (MT), and glycogen (Gly). Formed elements embedded in the sol-gel zone include mitochondria (M), granules (G), and dense bodies (DB). Collectively they constitute the organelle zone. The membrane systems include the surface connected canalicular system (CS) and the dense tubular system (DTS) which serve as the platelet sarcoplasmic reticulum. Figure 2B is a platelet sectioned in the equatorial plane which reveals most of the structures indicated on the diagram. The membrane complex (MC) is a specialized association of the DTS and CS. Mag. 2B × 22,000.

surface for interaction with c o a g u l a n t proteins and a critical fatty acid substrate, arachidonic acid, r e q u i r e d for prostaglandin synthesis. T h e

Figs. 3A,3B. Discoid platelets. The diagram in 3A summarizes the structures observed in platelets on cross section and 3B demonstrates an example of a cross-sectioned platelet. The designations for the structural features are presented in the legend of the previous illustration. An occasional Golgi apparatus (GZ) is also found in platelets. Mag. 3B × 41,500.

area lying just inside the unit m e m b r a n e represents the third c o m p o n e n t of the peripheral zone. It is closely linked to the unit m e m b r a n e and translates signals received on the outside surface into chemical messages and physical alterations required for platelet activation. T h e sol-gel zone is the matrix of the platelet cytoplasm. It contains several fiber systems in various states of p o l y m e r i z a t i o n which s u p p o r t the discoid shape of u n a l t e r e d platelets and p r o v i d e a contractile system involved in shape change, p s e u d o p o d extension, internal c o n t r a c tion and secretion. E l e m e n t s of the contractile system a p p e a r to be m a j o r c o m p o n e n t s , since they constitute a p p r o x i m a t e l y 5 5 % of the total platelet protein. T h e organelle zone consists of granules, electron dense bodies, peroxisomes, 111

Figs. 4A,4B. Membrane systems. The surface connected open canalicular system of channels (CS) burrows into the cytoplasm of the platelet in a serpentine fashion. Communications with the cell surface are evident in 4A and 4B. Elements of the dense tubular system (DTS) are randomly dispersed in the cytoplasm, though some of its channel are always associated with the circumferential band of microtubules. In one or two areas of the cytoplasm in each platelet channels of the CS and DTS form close associations referred to as the membrane complex (MC). Mags. 4A x 42,000; 4B x 37,000. l y s o s o m e s , m i t o c h o n d r i a a n d m a s s e s , as well as d i s c r e t e p a r t i c l e s of g l y c o g e n r a n d o m l y disp e r s e d in t h e c y t o p l a s m (Figs. 2 A , 2B, 3 A , 3B, 5 A , 5B, 6 A , 6B). It serves in m e t a b o l i c p r o c e s ses a n d t h e s t o r a g e of e n z y m e s , n o n - m e t a b o l i c a d e n i n e n u c l e o t i d e s , s e r o t o n i n , a v a r i e t y of p r o t e i n c o n s t i t u e n t s a n d c a l c i u m d e s t i n e d for s e c r e t i o n . I n the p a s t we h a v e i n c l u d e d e l e m e n t s of t h e d e n s e t u b u l a r s y s t e m w i t h o t h e r c o n s t i t u e n t s of t h e o r g a n e l l e z o n e 1~-19. H o w e v e r , r e c e n t studies h a v e d e m o n s t r a t e d t h a t p l a t e l e t 112

Fig. 5A. OrganeUe zone. Platelet dense bodies (DB) are the storage sites for serotonin, the non-metabolic pool of adenine nucleotides and calcium. They are referred to as dense bodies because they are inherently electron opaque as showin in this example of a whole mounted platelet viewed without fixation or negative staining. The opacicty of dense bodies in human platelets is due to their content of calcium. Mag. x 22,600. Fig. 5B. Organelle zone. The platelet in 5B is from a sample of platelet rich plasma (PRP) incubated with latex particles (L) and stained during fixation for acid phosphatase activity. An intact lysosome (Ly) containing dense reaction product is evident in one platelet. In another a lysosome has fused with an ingested latex particle to form a phagolysosome (PL). Most platelet granules (G) do not contain acid phosphatase activity. Mag. x 41,500.

membrane systems d e s e r v e a p l a c e of t h e i r o w n in p l a t e l e t a n a t o m y 2° (Figs. 4 A , 4B, 7 A , 7B, 8 A , 8B). T h e d e n s e t u b u l a r s y s t e m (DTS) h a s b e e n s h o w n to b e t h e site w h e r e c a l c i u m i m p o r t a n t for t r i g g e r i n g c o n t r a c t i l e e v e n t s is s e q u e s t e r e d . A l s o , it is t h e site w h e r e e n z y m e s

Fig. 6A. Organelle zone. One population of platelet granules contains platelet factor 4 and fibrinogen. The cells in 6A are from a sample of washed platelets treated with thrombin in the presence of EDTA. Granule associated fibrinogen has been extruded into the surface connected canalicular system and converted to fibrin (F). Mag. × 36,500. Fig. 6B. Organelle zone. The platelets in 6B are from a sample incubated at high pH according to the procedure of Breton-Gorius to demonstrate catalase activity. A peroxisome containing the reaction product of catalase is apparent in each platelet. Granules (G) are unreactive. Mag. x 38,000. i n v o l v e d in p r o s t a g l a n d i n s y n t h e s i s a r e l o c a l i z e d . T h e o p e n c a n a l i c u l a r s y s t e m p r o v i d e s access to the i n t e r i o r for p l a s m a b o r n e s u b s t a n c e s a n d an e g r e s s r o u t e for p r o d u c t s of t h e r e l e a s e r e a c t i o n . T o g e t h e r with e l e m e n t s of t h e d e n s e t u b u l a r s y s t e m , c h a n n e l s of t h e o p e n c a n a l i c u l a r s y s t e m form specialized membrane complexes which c l o s e l y r e s e m b l e t h e r e l a t i o n s h i p s of t r a n s v e r s e t u b u l e s a n d s a r c o t u b u l e s in e m b r y o n i c m u s c l e cells. O t h e r m e m b r a n o u s e l e m e n t s a r e p r e s e n t 4

Fig. 7A. Cytochemistry of membrane systems. The platelet in 7A was fixed in glutaraldehyde and osmium solution containing lanthanum. Electron dense tracer coats the surface of the cell and lines each channel of the surface connected canalicular system (CS). Microtubules (MT), a mitochondrion (M) and granules (G) are barely visible in this unstained section, but a dense body (DB) is prominent. Mag. x 41,500. Fig. 7B. Cytochemistry of membrane systems. This platelet is from a sample of PRP incubated for peroxidase activity. Enzyme reaction product is specifically localized to channels of the dense tubular system (DTS) and none is present in the surface connected canalicular system (CS). Mag. x 30,000. in o c c a s i o n a l p l a t e l e t s , such as a G o l g i a p p a r a t u s , b u t t h e y do n o t a p p e a r to s e r v e m a j o r r o l e s in p l a t e l e t s t r u c t u r a l p h y s i o l o g y . S t i m u l a t i o n of p l a t e l e t s b y a v a r i e t y of s u b s t a n c e s in v i t r o a n d b y v a s c u l a r d a m a g e in vivo r e s u l t s in a series of b i o c h e m i c a l , p h y s i o l o g i c a l and morphological events which are essential for t h e n o r m a l h e m o s t a t i c f u n c t i o n of t h e cell. A g e n t s which a c t i v a t e p l a t e l e t s i n t e r a c t with 113

Fig. 8A. Cytochemistry of membrane systems. The cell in 8A is from a sample of glutaraldehyde fixed platelets incubated in a solution containing lead ions. Lead has selectively deposited in channels of the dense tubular system (DTS). None is present in the surface connected system (CS). Note the proximity of lead filled channels to open channels in the membrane complex (MC). The same procedure has been used to demonstrate calcium binding sites in the sarcoplasmic reticulum of muscle. Mag. x 41,500. Fig. 8B. Membrane complex. The organization of the membrane complex (MC) and the fenestrated nature of the platelet sarcoplasmic reticulum is probably best seen in freeze fractured cells. In this example a channel of the surface connected canalicular system (CS) spreads out after entering the cell. A "swiss-cheese" appearance is created by fenestrations in the channel, and an element of the dense tubular system (DTS) projects through each of them. Mag. x 120,000.

four separable events may occur. In the microcirculation after injury to the vessel wall the first reaction is adherence of the platelets to the site of damage (Process 1). This process may occur without further platelet changes or may be accompanied by the full range of platelet responses. When platelets are exposed to epinephrine in vitro the initial reaction is development of platelet-platelet stickihess (Process 2). Most other aggregating agents cause contraction and/or granule labilization prior to the development of platelet-platelet stickiness. However, in the presence of the inhibitor, cytochalasin B, A D P can cause platelet-platelet stickiness without other physical changes 21. Platelets treated in vitro with phorbol myristate acetate show, as their initial response, labilization of the platelet granules 22'23 (Process 3). Granule membranes are fused with membranes of the surface connected open canalicular system and the granule contents extruded into the canaliculi. This process is similar to granule secretion in other systems, except that in most cells the granule contents are directly extruded to the outside. In the platelet granule contents are deposited in channels inside the cell. Deposition of granule contents within channels of the SCCS necessitates a fourth event (Process 4), internal contraction, which squeezes the granule contents out of the platelet 24. Thus, shape change and pseudopod extension, the centralization of granules and their final expulsion are manifestations of the platelet internal contractile process, though under some circumstances shape change and pseudopod extension can occur without internal contraction 25. Many examples could be put forward to show the complicated interdependence of the four fundamental platelet responses. The particular value in dissecting them into four categories is that it enables a logical classification and an improved understanding of platelet defects.

II!. Platelet Disorders

A. receptors on the cell surface and lining channels of the open canalicular system. Depending on the nature of the specific stimulus and the particular conditions employed, one or more of 114

General

Defective platelets can lead to hemorrhagic disorders on the one hand or t o thrombotic disorders on the other. This consideration leads naturally to an initial division of defective

platelets into: 1) Hypoactive platelets associated with a bleeding tendency and 2) Hyperactive platelets associated with a thrombotic disorder. Prior attempts at classification of platelet disorders have emphasized the former. Although knowledge of specific defects in the second category (thrombotic disorders) is still sparse, it is time to recognize such abnormalities and urge the beginning of their classification. Dissection of platelet function into separable processes allows further divisions within each category. All disorders of platelet function should relate to defects of: 1) Adhesion to an injured vessel wall 2) Platelet-platelet stickiness 3) Granule labilization or 4) Internal contraction. As simplistic as this classification system may seem, it is useful because it combines platelet structural physiology and pathology into the same framework. Therefore, each advance in our understanding of basic platelet structural physiology contributes to our knowledge of defects responsible for abnormal function, and clarification of fundamental abberations involved in the pathogenesis of platelet disorders elucidates features of normal platelet behavior.

connective tissue) may represent a third. Identification of these abnormalities have provided evidence that platelet glycoproteins, von Willebrand factor and vessel wall connective tissue are critical components of this process. a) Bernard-Soulier syndrome The Bernard-Soulier syndrome (BSS) is an autosomal recessively inherited bleeding disorder characterized by thrombocytopenia, prolonged bleeding time and giant platelets which are not aggregated by bovine fibrinogen or ristocetin26'27 (Fig. 9A). Adhesion of the giant platelets to subendothelium is defective and the cells are not retained by glass bead columns. Recent studies have suggested that BSS

B. Disorders associated with hypoactive platelets 1. Defects in platelet adhesion to an injured vessel wall The critical first step in the hemostatic response to vessel damage is the adhesion of platelets to the injured region. Studies of balloon catheter trauma and other injuries which remove the lining endothelium have enabled a much improved understanding of the process 11. Conditions of flow, albumin, red cells and calcium levels all alter the degree of platelet adhesion. Elevation of platelet cyclic AMP levels can markedly impair platelet adhesion, but the effect of cyclic AMP is non-specific as it inhibits all platelet functions. To identify a specific defect in platelet adhesion to the injured vessel wall, the capacity to develop platelet-platelet stickiness, undergo granule labilization, contraction and secretion must be demonstrated to be intact. Such evidence now exists for two disorders, the Bernard-Soulier syndrome (a defect in platelet surface glycoprotein) and von Willebrand's disease (absence or marked decrease of the plasma protein-von Willebrand factor). The Ehlers-Danlos syndrome (a defect in vessel wall

Fig. 9A. Bernard-Souliersyndrome.Platelets from patients with this disorder are usuallyseveral times larger than normal platelets. However, they contain a normal complement of organelles includinggranules, mitochondria,membrane systemsand dense bodies (DB). Mag.x 15,000. Fig. 9B. Thrombasthenia.Thrombasthenicplatelets have no distinguishingmorphologicalfeatures. They are entirely comparable to normal platelets. Mag.x 20,000. 115

platelets have an abnormal surface membrane which lacks a specific glycoprotein different from that reported missing in thrombasthenic cells 28. Morphological studies in the past indicated that the giant platelets lacked granules or contained a disorganized system of microtubules 29'3°. A recent ultrastructural investigation has suggested that, despite variations in the number of granules per cell, prominence and possible hypertrophy of the surface connected open canalicular system and dense tubular system, and some vacuolization, there are no ultrastructural features that are characteristic of BSS platelets 31. b) von Willebrand's Disease Adherence of platelets to an injured blood vessel is markedly decreased in yon Willebrand's disease s. However, the abnormality is not a primary platelet disorder since it can be corrected by normal plasma or cryoprecipitate (Fig. 9B). The deficiency in yon Willebrand's disease appears to be due to the absence or marked decrease of yon WiUebrand factor, a protein associated with the factor VIII molecule. Platelets from patients with von Willebrand's disease, possibly with a few exceptions, undergo normal reactions of plateletplatelet stickiness, granule labilization and internal contraction and have an entirely normal morphological appearance. c) Ehlers-Danlos syndrome Patients with Ehlers-Danlos syndrome frequently have a prolonged bleeding time, although platelet function is usually normal. Defective adhesion of the platelets to the vessel wall may be due to abnormal collagen 32, though this has not been definitely established.

2. Defects in platelet-platelet stickiness The process or processes whereby platelets become sticky and clump together is at present little understood. The presence of Ca ++ outside the platelet is known to be critical, since the calcium chelators, E D T A and EGTA, inhibit this process. An oxidation-reduction reaction may also be important since NBT and vitamin E reduce platelet stickiness 33. GARTNER et al. 34 have recently suggested that the process of platelet-platelet stickiness involves an interaction resembling that of a lectin with its receptor. 116

The only well-characterized inherited defect of platelet-platelet stickiness is thrombasthenia. a) Thrombasthenia Platelets from patients with thrombasthenia provide the classic example of a defect in plateletplatelet adhesion 3s. The content of serotonin, adenine nucleotides, and hydrolytic enzymes in platelets from these patients is normal. Some have been found deficient in enzymes required for glycolysis36, but most appear to have normal platelet metabolism. Platelet fibrinogen is reduced to ~ normal levels in thrombasthenic platelets 37. When stimulated by collagen or thrombin they synthesize prostaglandin endoperoxides, undergo shape change and secrete in the same manner as normal cells. Despite normal responses of the organeUe and sol-gel zones to stimulation, the thrombasthenic platelets are abnormal because they fail to become sticky. As a result, aggregation is abnormal and the patients have markedly prolonged bleeding times. A basic defect in thrombasthenic platelets is their inability to attach to one another when stimulated by aggregating agents. Whether added exogenously or derived through extrusion of the storage pool from the abnormal platelets, the nucleotide, ADP, fails to transform the external surface of the cells to a sticky state. Some workers have suggested that the exterior coat of thrombasthenic platelets is deficient in actomyosin believed essential for aggregation 3s, and immunoperoxidase staining techniques were employed to demonstrate that the exterior coat was deficient in contractile protein ag. Defects in the ability of thrombasthenic cells to spread on glass surfaces were noted in early studies, and abnormal granules, vacuoles and bizarre platelet shapes were reported. However, the early ultrastructural findings have not been substantiated in recent investigations 17-19. Almost simultaneously NtJRDEN and CAEN9 and PHILLIPS1°, employing techniques which selectively label carbohydrate groups localized on the outer surface, demonstrated that platelets from thrombasthenic patients are deficient in membrane glycoproteins, II and III. The discovery points out the importance of surface membrane glycoproteins in adhesion-aggregation reactions of platelets. Electron microscopic techniques specific for surface membrane glycoproteins have not been developed sufficiently to localize these exterior

components on the platelet surface with confidence. The presence of the molecular defect on the outside surface of the cell, however, confirms previous ultrastructural reports which failed to detect morphological abnormalities inside thrombasthenic platelets 17-~9. Preliminary studies by GERaARD e t a P ~ have carried the work by NURDEN and CAEN and PHILLIPS a step further. They have shown that thrombasthenic platelets with reduced levels of GP III are also deficient to the same extent in another protein, a-actinin. Molecular weight determinations suggested that GP III and aactinin were similar, and that each constituted about 3% of the total normal platelet protein. A specific antiserum prepared against muscle a-actinin revealed a strongly positive reaction with isolated and purified surface membrane GP III from normal cells, a slight interaction with GP II, but none with purified GP I and IV. These findings suggest that GP III and a-actinin are a single transmembrane protein in platelets. If the preliminary studies prove to be accurate, then GP III-t~-actinin will be the first transmembrane protein described in platelets. The exposed part of the molecule is important in cell-cell association and the transmembrane portion may anchor the contractile apparatus in the sol-gel zone. As a result, the zone of adhesion between aggregated platelets may closely resemble the Z line in skeletal muscle in which actin filaments are anchored by a-actinin. 3. Defects in platelet organelles The recognition that storage organelles serve an important function in platelet physiology has resulted in large measure from the study of abnormal cells 17-19. Early workers appreciated the possibility that the organelles might be secreted by platelets, but limitations in technology prevented morphological or biochemical characterization of the process. Improvements in biochemical and ultrastructural methods resuited in the identification of several types of storage organelles including granules, dense bodies, lysosomes and peroxisomes. Dense bodies, granules and lysosomes appear to be involved in the secretory process, while peroxisomes containing catalase are not discharged following platelet activation. Abnormalities of the organelles include reduction in their mem-

bers, decreases in their content of stored molecules, or defects in the process of their labilization and secretion. a) Reduced levels of secretory products 1) Decreased or absent dense bodies a. Hermansky-Pudlak syndrome The Hermansky-Pudlak syndrome (HPS) is a recessively inherited autosomal disease in which the triad of tyrosinase positive oculocutaneous albinism, accumulation of ceroid-like material in reticuloendothelial cells of bone marrow and other tissues and a hemorrhagic diathesis due to defective platelets are constantly associated 42-46. About 50 cases of this syndrome have been reported in the world literature. In previous reviews of ultrastructural defects in congenital disorders of platelet function it was suggested that HPS is the first disorder in which an abnormality detectable in the electron microscope could be correlated directly with a specific biochemical deficiency, impaired platelet function in vitro and clinical bleeding problems in patients ls'19. The population of electron dense bodies in HPS platelets was greatly reduced and in some cases virtually absent (Figs. 10A,10B). Biochemical analysis revealed that HPS platelets had very low levels of serotonin and a marked reduction in the non-metabolic pool of adenine nucleotides. However, earlier studies had shown that neither serotonin nor adenine nucleotides was responsible for the inherent opacity of platelet dense bodies, but that a concentration of heavy metal, such as calcium, impaired passage of the electron beam 47. Subsequent studies have shown that normal platelet dense bodies are rich in calcium 48, and that HPS platelets contain significantly less calcium than normal cells49. HPS platelets develop the same sequential changes as normal platelets when stimulated by aggregating agents, including shape change, internal transformation and molding of cell surfaces together in tightly packed small aggregates s°. Due to the marked deficiency in ADP, the amount of nucleotide secreted by activated HPS cells is insufficient to bring uninvolved platelets into large aggregates and sustain the platelet-platelet association long enough to establish irreversible aggregation. As a result, HPS platelets do not develop second waves of aggregation when exposed to concentrations of ADP, epinephrine and thrombin which cause 117

Figs. 10A,10B. Hermansky-Pudlak syndrome (HPS). The high frequency of electron dense bodies ( 1") storing serotonin, adenine nucleotides and calcium in normal platelets shown in 5A contrasts sharply with the virtual absence of dense bodies in the HPS platelets in 5B. The marked decrease in dense bodies in HPS platelets corresponds closely to the reduction in chemical constituentsof the storage pool and defectivefunction of the cells. Mags. 10Ax 25,000; 10B x 20,000. irreversible clumping of normal cells on the platelet aggregometer. However, they will form irreversible aggregates if exposed to a high concentration of exogenous ADP. Thus, the functional defect is due to inadequate secretion rather than inability of HPS cells to become sticky, aggregate or contract. The fundamental defect responsible for the failure of HPS platelets to form dense bodies is still not known with certainty. However, it is most likely tied to the inherited enzymatic defect which also affects pigmentation (even though the patients possess the tyrosinase en118

zyme required for melanin synthesis) and accumulation of ceroid and lipofuchsin in macrophages throughout the body 51. HPS platelets can take up serotonin as rapidly as normal platelets but reach saturation quickly and discharge the accumulated 5-hydroxytryptamine which normal cells retain s2. The catabolism of accumulated serotonin may relate more closely to the paucity of opague storage organelles in HPS platelets than the possibility that they are unable to form the specific type of storage organelle. Studies in this laboratory have shown that resting, unstimulated HPS platelets contain 2-10 times the normal level of thiobarbituric acid (TBA) reactive substance found in normal platelets 53. The TBA reaction can be used either to measure M D A or the presence of accumulated lipid peroxides in the cell. Since the analysis was done on resting HPS and normal platelets, the results indicate an accumulation of lipid peroxides in the cell rather than a defect in prostaglandin synthesis per se. HPS platelets synthesize normal amounts of endoperoxides and thromboxanes in response to AA, further arguing against a defect in PG synthesis 54'55. The degradation of serotonin and accumulation of lipid peroxides in platelets, the interference with melanosome formation in melanocytes and dense bodies in thrombocytes, and retention of large amounts of lipofuchsin and ceroid in macrophages may all be related to a failure of regulation in the same catabolic system. b. Storage Pool Disease The second congenital disorder of platelet function to be associated with an ultrastructural defect is very similar to the HPS 56'57. Patients with platelet storage pool deficiency (SPD) do not have albinism nor do they accumulate ceroid-like pigment in macrophages of the bone marrow or reticuloendothelial system. In at least one reported family the disorder appears to be inherited as an autosomal dominant. The bleeding symptoms of patients with SPD resemble those observed in the HPS and the platelet defect is similar, if not identical (Figs. 11A, 11B). SPD platelets are very deficient in the non-metabolic storage pool of adenine nucleotides. ADP, a major constituent of dense bodies, is considerably more reduced than ATP, a minor component. Serotonin is also reduced, but the degree of deficiency is variable and

Figs. l l A , I1B. Storage pool disease. Platelets from patients with storage pool disease (SPD) are indistinguishable from those of patients with HPS. Dense bodies are virtually absent from platelets obtained from patients with either condition. Mags. l l A x 27,500; l l B × 16,500.

usually less than observed in HPS platelets. Weiss has noted that serotonin levels in SPD platelets were reduced in proportion to the reduction in platelet A T P 53. The ability of SPD platelets to absorb 14C serotonin may be more compromised than observed in HPS cells. The SPD platelets take up serotonin initially at the same rate as normal cells but are quickly saturated. Serotonin accumulated by normal platelets is retained but the absorbed 5-HT in SPD platelets is discharged at a steady rate in the form of metabolites. When SPD platelets prelabeled with 14C serotonin are stimulated by aggregating agents, they release the ~4C-5-HT

more slowly than normal cells, suggesting that the release reaction may be defective in SPD 5s. WmLiS and WEISS have found that SPD platelets are deficient in their ability to synthesize intermediates of prostaglandin biosynthesis 59. After stimulation by collagen SPD platelets produced less than 20% of the PGE2 and PGF20t synthesized by normal cells. Since serotonin and A D P may act as cofactors for PG synthase, 6°'61 WILLIS and WEISS suggested that the defect in PG synthesis might be closely linked to the storage pool deficiency in platelet SPD. The ultrastructural defect in SPD platelets appears essentially identical to that observed in H P S 62. Morphological characteristics are similar to normal platelets except for the profound reduction in the number of dense bodies. The decrease in dense bodies correlates with the deficiency in serotonin and adenine nucleotides, the impaired response of the cells to aggregating agents and the clinical symptoms of the patients. Thus, SPD is the second disorder in which impaired platelet function can be directly associated with an ultrastructural defect in the cells as'19. However, the normal pigmentation of individuals with this disorder and the absence of an unusual accumulation of ceroid or lipofuchsin in macrophages suggests that the cause is basically different than that responsible for platelet storage pool deficiency in HPS. c. Other storage pool diseases In his review of abnormalities in platelet function due to defects in the release reaction WEiss pointed out the probable existence of heterogeneity in platelet storage pool disease 63. In addition to the two disorders, HPS and SPD, discussed above, a deficiency of serotonin and non-metabolic adenine nucleotides has been reported in patients with Wiskott-Aldrich syndrome 64, the TAR (thrombocytopenia, absent radii) syndrome 6s, the Chediak-Higashi s y n d r o m e 66-69 and in acquired conditions such as leukemia 7° and patients with circulating antiplatelet antibody 71, Unfortunately, electron micrographs of the defective platelets have not accompanied most reports of storage pool deficiency in these inherited and acquired syndromes. As a result, it has been difficult to determine whether the low levels of serotonin and adenine nucleotides found in platelets from patients with the various disorders is associated with decreased numbers or abnormal dense 119

bodies. Recently we have carried out investigations in patients with one of these syndromes, chronic myelogenous leukemia. 1. Chronic Myelogenous Leukemia Several investigators have reported that platelets from patients with leukemia, particularly chronic myelogenous leukemia (CML), respond abnormally to aggregating agents and may be storage pool deficient7z-74. This finding is of interest because platelets appear to be one of the cell lines involved in CML as shown by

the presence of the Philadelphia chromosome in megakaryocytes from the patients 75. We have recently studied eight patients with classic CML, seven of whom were Philadelphia chromosome positive 76. Although the reaction varied from time to time, the response of the platelets from seven patients to epinephrine was abnormal on at least one occasion and collagen induced aggregation was defective in five individuals. Measurement of serotonin and adenine nucleotides in platelets of five patients manifesting abnormal function revealed marked deficiencies of 5-HT and ADP in all of them. In addition, the number of dense bodies in their platelets was significantly reduced (Figs. 12A,12B). Thus, a true storage pool deficiency exists in a significant percentage of patients with CML. Its origin and relationship to other features of CML is not yet known. d. Possible storage pool disorders The classic storage pool deficiencies, HPS and SPD, and other similar conditions, such as CML, have been grouped together because platelets from affected patients were reported to be deficient in serotonin, adenine nucleotides and the dense bodies in which these products are stored. In addition, there are other inherited disorders in which the storage pool products are reduced, but the content of dense bodies appears to be normal. Also, some patients have been found whose platelets contain normal levels of 5-HT and adenine nucleotides, but are deficient in products found in other storage organelles. 1. Chediak-Higashi syndrome

Figs. 12A,12B. Acquired storage pool disease. The platelets in the two examples are from a child with chronic myelogenous leukemia and acquired platelet storage pool disease. The cell in 12A has an increased content of channels of the dense tubular system (DTS), but most of the platelets appear normal. Dense bodies (DB) were greatly reduced in the childs cells. Mags. 12A x 21,000. 12B × 15,000.

120

The Chediak-Higashi syndrome (CHS) is a rare, autosomally inherited disorder characterized clinically by photophobia, nystagmus, pseudoalbinism, marked susceptibility to infection, hepatosplenomegaly, lymphadenopathy, and early death, due frequently to development of a lymphoreticular malignancy77-81. Laboratory diagnosis is based on the presence of giant organelles in nearly all leukocytes on Wrightstained peripheral blood smears. The massive granules have been found in neutrophils, eosinophils, lymphocytes and monocytes from blood and in their bone marrow precursors. Despite thrombocytopenia which develops during the accelerated phase of CHS and an

early report describing two patients with markedly decreased platelet serotonin a2, blood platelets have not been considered a major problem in this disease. Recently, however, several studies have shown that platelets express the genetic fault of the disorder 66-69. Platelets from patients with CHS are biochemically, physiologically and functionally abnormal. The defect has been related to a profound deficiency in the storage pool of adenine nucleotides and serotonin, though the numbers of dense bodies which store these substances was found to be normal 69 (Figs. 13A,13B). Elevated levels of cyclic 3'5-adenosine monophosphate (cAMP) were noted in platelets from one infant with CHS. However, the level of cAMP was cor-

Figs, 13A,13B. Chediak-Higashi syndrome (CHS). Platelets from our patient with CHS are essentially normal. They contain circumferential bands of microtubules and dense bodies ( I' ). Rare platelets contain giant granules (GG) which are markedly larger than normal sized organelles (G). Mags. 13Ax27,500; 13Bx50,000.

rected to normal by treatment with ascorbate without apparent improvement in platelet function. Thus, the platelet appears to be involved in the expression of CHS along with other blood cells containing giant cytoplasmic granules. However, it has recently been suggested that the basic defect leading to functional abnormalities of CHS blood cells is not due to the presence of giant organelles but to a defect in cyclic nucleotide metabolism which prevents the cells from polymerizing microtubules 83-86. The defect in microtubule assembly was observed in leukocytes from animal models of CHS in which levels of cyclic quanosine 3'5'-monophosphate (cGMP) were low and cAMP normal, and in human CHS neutrophils in which cGMP was normal, but levels of cAMP elevated. Recently we have evaluated the morphology of platelets from a patient with clinical and laboratory features characteristic of c n s 87. Giant granules of a type never seen in normal platelets or in other platelet disorders were found in his platelets in a ratio of about 1 per 1000 cells in thin sections. The number, structure and organization of microtubules in the patients discoid platelets were comparable to normal cells. Exposure of the CHS platelets to collagen resulted in their movement toward the cell center where they encircled centrally clumped granules, an entirely normal response. When the CHS platelets were exposed to low temperature microtubules disappeared from the cells. On rewarming the chilled cells to 37 °C, they recovered their discoid shape and circumferential bands of microtubules as rapidly as normal cells. It is difficult to draw inferences from studies on a single patient or on a cell type that was not examined in previous studies describing the basic defect in microtubule assembly in CHS. However, since CHS platelets are functionally defective, share the cyclic nucleotide abnormality of leukocytes, originate from the same stem cell as granulocytes and contain giant granules characteristic of the disorder, it seemed reasonable to examine CHS platelet microtubules to determine if the proposed defect in microtubule assembly was a generalized feature of the disease. Results of our studies on this patient's platelets are inconsistent with the concept that inability to assemble microtubules is the fundamental abnormality 121

responsible for functional defects in CHS cells. 2) Decreased or absent granules a. Gray platelet syndrome The Gray platelet syndrome (GPS) is an extremely rare disorder, and only one case of this condition has been reported in the literature ss. As a result, its mode of inheritence is unknown. The propositus was evaluated for thrombocytopenia as a child and found to have large, agranular platelets which appeared gray or blue-gray on Wright stained blood smears. Splenectomy corrected the thrombocytopenia and the child remained well throughout adolesence. However, well over half of his platelets retained the characteristic features observed prior to splenectomy.

Figs. 14A,14B. Gray platelet syndrome. Platelets from the two patients with GPS are very large and virtually devoid of granules (G). Dense bodies, mitoehondria (M) and peroxisomes (P) are present in normal numbers. The dense tubular system (DTS) is very prominent in some cells as shown in 14B. Mags.x 14A x 19,000; 14Bx 39,000. 122

We have studied this patient on several occasions. Stirred platelet samples respond less well than normal cells to aggregating agents. Levels of platelet serotonin and adenine nucleotides are within normal limits. Examination of his cells in the electron microscope revealed wide variations in platelet size, internal organization and content of organelles (Figs. 14A, 14B). About ~ of his platelets are quite similar to normal cells while 2 of the cells range from slightly abnormal to bizarre. Some of the cells in thin section are giant bags of cytoplasm without any organelles. Others are similarly large but the cytoplasm is largely replaced by vacuoles. In many, the number of mitochondria present is greater than all other organelles combined. A few granules are present and the frequency of dense bodies is comparable to normal cells. Gray plate!ets absorb, retain and secrete 14C serotonin normally. Thus, despite the profound abnormality in granule formation and abberations in cytoplasmic organization, Gray platelets appear to be capable of fairly normal function. We have noted that the patient may have mild bleeding symptoms when his platelet count is between 100-150,000/mm 3. Therefore, it is possible that abnormal function of Gray platelets is modified by the population of relatively normal cells when his count is normal. Attempts to identify truly separate populations of platelets by Coulter sizing has not been successful. He appears to have a single population of platelets manifesting a broad continuum in size from normal to extremely large. Recently, we have found a second patient with GPS. The new patient, a young girl, also has Goldenhar's syndrome. Other patients with Goldenhar's syndrome were not found to have GPS and our original patient does not have any evidence of another inherited disorder. Platelets from the second patient appear identical to those of the first young man. In collaboration with Dr. David Phillips we have studied platelet proteins from both patients ag. Thrombin sensitive protein is profoundly deficient in their cells and is not secreted during the release reaction stimulated by aggregating agents, though 14C serotonin is extruded normally. The patients represent the first two cases in which a platelet granule associated protein deficiency has been defined.

b. Defects in the process of granule labilization The process of platelet granule labilization is poorly understood at present. One agent, phorbol myristate acetate, has been shown to selectively initiate labilization of granules 23. No specific defects of this function have been described as yet. Agents which elevate platelet cyclic AMP levels will inhibit granule labilization, but appear somewhat less effective in accomplishing the inhibition than in inhibiting contraction. Known effects of cyclic AMP on calcium raise the possibility that a localized flux of calcium might be critical. Haslam has studied phosphorylation of platelet proteins by aggregating agents and raised the possibility that one of the roles of such phosphorylation might be granule labilization 9°. Recently, SHULMAN, using inhibitors such as suramin has suggested that anions such as C1- or O H - might be important in labilization of platelet granules 9~. 4. Defects in platelet contraction The primary response of platelets to many aggregating agents including collagen, thrombin, serotonin, vasopressin and arachidonic acid is contractile in nature 92'93. Reactions of platelets to the secreted product, A D P 96, and to endogenously synthesized thromboxane A2 are also contractile 97"98, raising the possibility that the effects of the first group of aggregating agents are mediated by either A D P or thromboxane A2. Studies with inhibitors suggest that the effects of arachidonic acid and of low concentrations of collagen on internal platelet contraction are mediated almost exclusively by thromboxane A299. The primary effects of high concentrations of collagen, thrombin, serotonin and vasopressin are independent of secreted ADP or thromboxane A2 synthesis 99"~°°. However, the secondary or autocatalytic responses to all of these agents and to epinephrine are dependent on both secretion of A D P and thromboxane A2 production 98. Studies with the calcium ionophore A23187 have shown that internal platelet contraction is critically dependent on raising the cytoplasmic calcium level 1°1'1°2. The effect of calcium appears to activate the contraction of platelet actin and myosin, since platelet actomyosin has been demonstrated to show calcium sensitivity as it does in other muscle systems ~°3. Studies using

2-deoxy-D-glucose and antimycin A have shown that internal contraction is also dependent on the generation of metabolic energy (ATP) 98. Thus, a defect in platelet contraction can be at the level of the receptor, transmission of the signal from the receptor to initiate calcium flux, a defect in platelet contractile proteins, or a defect in metabolism of ATP. a) Defects in receptors As yet, no inherited defect in platelet receptors has been definitely identified. Certain receptors, such as that for epinephrine, can be specifically blocked by drugs like phentolamine 1°4. Several receptors including these for epinephrine, ADP, and serotonin can be specifically blocked by pretreatment of the platelets with the same agent. This also occurs in some circumstances in vivo, as in the carcinoid syndrome, where exposure to serotonin results in platelets which respond weakly or not at all to serotonin 1°5. Recently studies in children with acute lymphocytic leukemia being treated with Lasparaginase have provided evidence for a defect either in the collagen receptor or at a very early step in the platelet interaction with collagen 1°6. b) Defects in transmission of the signal to initiate calcium flux Thrombin, ADP, serotonin, vasopressin and collagen at high concentrations appear to act through their receptors to directly initiate a flux of calcium 94. The source of calcium and the nature of this initial calcium flux is not clear, although some evidence suggests that A D P releases calcium from a store on the inside of the external plasma membrane 99. The nature of this calcium flux is probably different with A D P than with thrombin, since, in the absence of prostaglandin synthesis, A D P produces only a limited degree of contraction and secretion, whereas thrombin can generate a forceful contraction and secretion. A secondary effect of A D P and thrombin is to stimulate synthesis of thromboxane A2. The thromboxane A2 can then augment the degree of contraction and secretion probably by releasing additional calcium from the dense tubular system 98. Synthesis of thromboxane A2 is probably initiated by activation of phospholipase A2 releasing arachidonic acid from the platelet phospholipids and making it available to the cyclo-oxygenase and thromboxane synthetase enzymes which convert the 123

arachidonic acid to thromboxane A2 a°7. Since the phospholipase A2 enzyme can be calcium activated 9s'1°~-~1°, the possibility exists that the initial calcium flux produced by ADP and thrombin produces internal contraction and phospholipase A2 activation. In response to low concentrations of collagen and during the second wave of epinephrine aggregation an internal platelet contraction occurs which is almost completely dependent on thromboxane A2 production 99. If this effect of epinephrine and collagen is also due to calcium activation of the phospholipase A2, then these two agents must be able to trigger a selective flux of calcium which activates phospholipase A2 but does not initiate internal contraction. From the foregoing discussion it can be seen that the transmission of the signal from the receptor to produce the calcium flux necessary for contraction is a fairly complicated process and may vary from one aggregating agent to another. Defects in transmission of the signal to produce calcium flux can be produced by agents such as local anesthetics or similar compounds including TMB-6 and TMB-8 which appear to act by inhibiting intracellular calcium flux ~1~-~3. Agents which act by elevating intracellular cyclic AMP also inhibit calcium dependent processes by promoting transfer of calcium from the cytoplasm to the storage site in the dense tubular system 1°1'114. Both these classes of compounds will inhibit not only internal contraction, but also phospholipase A2 activation, presumably acting to inhibit the calcium flux necessary for the two steps. The only known inherited defect in internal contraction is due to defective signal transmission. 1) Cyclo-oxygenase Deficiency MALMSTEN et al. and DECHEVANN~et al. have both described patients with defective cyclooxygenase activity las'116. The platelets will aggregate with PGG2 but not with arachidonic acid. Addition of arachidonic acid to the platelets showed production of HETE, the lipoxygenase product, but no production of H H T or thromboxane B2, the products of the cyclo-oxygenase enzyme. No ultrastructural studies were done on the original patients but other cases with the same defect appear to have normal platelet morphology. Ultrastructural 124

studies have been carried out on the effects of aspirin and indomethacin which inhibits the cyclo-oxygenase enzyme and no morphologic defect has been observed 1°2. Aspirin or indomethacin largely prevented internal contraction with epinephrine, collagen, or arachidonic acid but had little effect on the internal contraction produced by ADP or thrombin. c) Defects in platelet contractile proteins Platelets possess actin, myosin, tropomyosin and troponin 117. Platelet actomyosin shows calcium sensitivity as does the actomyosin of other muscle cells. To date no inherited abnormalities of these contractile proteins have been demonstrated. d) Defects in platelet metabolism 1) Wiskott-Aldrich syndrome It has been particularly difficult to obtain accurate information on the biochemistry, function and morphology of platelets from patients with the Wiskott-Aldrich syndrome (WAS) because of their profound thrombocytopenia l~s'Hg. In the past, we were able to obtain adequate specimens of peripheral blood and bone marrow from three patients for electron microscopy and aggregometry. Platelets from these patients were ½ to 2 the size of normal cells ls'19. However, the small platelets appeared to contain normal numbers of granules, mitochondria and electron dense bodies (Figs. 15A, 15B). Megakaryocytes from the patients were similar in all respects to the large cells in normal marrow. Functionally, the platelets appeared to respond to aggregating agents and latex particles when carefully concentrated. However, they never reacted as well as normal platelets. On the basis of our studies we concluded that bleeding problems in Wiskott-Aldrich syndrome were most likely due to the thrombocytopenia and reduction in cell size leading to a very decreased circulating platelet mass. The deficiencies in levels of serotonin and adenine nucleotides reported by others 12°'~21 and confirmed in our patients did not correspond with a marked decrease in the numbers of platelet dense bodies. It occurred to us that the low levels of adenine nucleotides identified in WAS platelets might be due to a defect in the generation of ATP, rather than to an inability to form storage organelles. Because of the difficulties in carrying out studies on the small, thrombocytopenic

Figs. 15A,15B. Wiskott-Aldrichsyndrome(WAS). Platelets from patients with the WAS are, on the average,½to za normal size. Althoughthe number of dense bodies may be reduced in some patients they appear to be quite normal in others ( ~"). Mags.x 15A 17,500; 15B× 36,000. children with the disease, we elected to pursue the investigation on women who are known carriers of the WAS gene. Evaluation of platelets from carrier mothers revealed that they were normal in number and size and responded in a completely normal manner to all aggregating agents on the platelet aggregometer. This seemed to rule out a metabolic defect in the WAS carrier platelets. However, it was also possible that the genetic fault in carrier platelets was latent, and that levels of ATP sufficient for normal function could be generated by their cells. Measurements of their platelet levels of ATP, ADP and AMP confirmed this suspicion.

It seemed to us, however, that it might be possible to bring out a latent defect in adenine nucleotide metabolism 122. ATP formation in platelets is dependent on two metabolic pathways, enzymatic glycolysis and oxidative phosphorylation. By choosing specific inhibitors it is possible to block either pathway separately. Antimycin A, which blocks oxidative phosphorylation, had no more effect on epinephrine induced aggregation of WAS carrier platelets than on normal cells. An inhibitor of glycolysis, 2-deoxy-D-glucose, however, at concentrations which had no effect on the biphasic response of normal platelets to epinephrine, regularly blocked the second wave of aggregation in samples of WAS platelets. The result has been successfully repeated in 18 unrelated carriers of the WAS. Our findings suggest that WAS carrier platelets lack an enzyme critical to ATP generation via the Kreb's cycle and oxidative phosphorylation pathway, and thus are unusually sensitive to inhibition of ATP generation via glycolysis. Results of this study have provided a new test for detection of the carrier state in WAS and focused attention on the specific pathway in which the enzymatic defect may exist 122. Furthermore, the findings may explain why the patients with WAS were considered to have storage pool disease 120. Adequate levels of ATP are required for secretion. Even though the patients may have normal numbers of dense bodies, the low levels of metabolic ATP would be inadequate to drive the systems necessary for their extrusion from activated cells. Thus, WAS may not represent a variety of storage pool deficiency disease like HPS and SPD, but an abnormality of secretion related to a defect in ATP synthesis.

C. Abnormalities o[ platelet [unction associated with hyperactive platelets Evaluation of patients with hyperactive platelets resulting in a variety of thrombotic disorders is still in an early stage 4. Classification of the disorders employing the same system used for hypoactive platelets should be possible in the near future. At present it is only possible to mention several disease states where reasonable evidence links hyperactive platelets to a thrombotic tendency in the disorder. CAaVALnO and associates have evaluated platelet function in 125

patients with type II hyperbetalipoproteinemia 123. Families with this disorder have a high frequency of thrombotic complications. Their platelets were found to be m o r e sensitive to a variety of aggregating agents than cells from control individuals. CARVALHO et al. have recently suggested that the abnormal sensitivity m a y be due to an increased production and prolonged half life of t h r o m b o x a n e A2. COLWELL and his associates have carried out detailed studies of platelet function in patients with diabetes melitus TM. Diabetic platelets were found to be hyperactive c o m p a r e d to control cells and produced increased amounts of prostaglandins in response to aggregating agents, The hyperactive state was inhibited by aspirin. T h e findings of these investigators suggest that diabetic platelets m a y be hypersensitive due to a b n o r m a l arachidonic acid metabolism. H o w ever, the hypersensitivity of diabetic platelets may result from the altered general metabolic state of the patient rather than an intrinsic abnormality of the cells. W e have studied one child with systemic lupus erythemtosis and pulmonary hypertension. This boy had a markedly reduced platelet survival and was found to be depositing chromium labelled platelets in his lungs. Treatm e n t of the child with aspirin and prednisone restored platelet survival to normal, p r e v e n t e d deposition of platelets in the lungs and improved the pulmonary hypertension. These studies implicate hyperactive platelets as a factor in the disease of this child. H o w e v e r , the precise nature of the platelet abnormality was not defined. Patients with this type of plateletrelated thrombotic disease m a y be far m o r e c o m m o n than we have realized in the past. Studies of HoAI¢ et al. on patients with recurrent venous t h r o m b o e m b o l i s m show that some patients have an increased n u m b e r of circulating platelet aggregates I25. Again, hyperactive platelets are implicated, although the precise abnormality is not yet defined. T h e few examples described above indicate that information is accumulating rapidly relating hypersensitive platelets to a variety of thrombotic states. In the near future it should be possible to place these conditions into a logical sequence which will facilitate understanding and treatment.

126

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