J. BIOMED. MATER. RES.
VOL. 10, PP. 429-443 (1976)
Adhesion of Platelets to Artificial Surfaces: Effect of Red Cells J. L. BRASH, J. M. BROPHY, and I. A. FEUERSTEIN, Departments of Chemical Engineering and Pathology, AlcMaster University, Ham ilton , Ontario, Canada
Summary Adhesion of platelets to several polymer- and protein-coated glass surfaces has been studied in vitro. The apparatus consists of a cylindrical probe rotating in a test tube containing the platelet medium and allows close control of fluid shear and mass transport. Suspensions of washed pig platelets constitute the basic platelet medium, and can be modified by adding back red cells and plasma proteins. Adhesion is measured via 50-labeling of platelets. In the absence of red cells, identical low levels of adhesion were seen on all surfaces and saturation was reached within 2 min. I n the presence of red cells, adhesion was greater. Saturation on all surfaces except fibrinogen and collagen again occurred within 2 min. The adhesion levels on polymer surfaces and glass were indistinguishable, while those 011 albumin were lower and those on fibrinogen were higher. Collagen was the most reactive surface. It did not equilibrate within 15 min, and kinetic data indicated a platelet diffusivity strongly dependent on hematocrit. These effects were attributed to rotational and translational motion of the red cells causing increased diffusion and surface-platelet collision energy.
INTRODUCTION It is widely recognized that platelet adhesion is important in hemostasis, intravascular thrombosis, and thrombus formation on vascular implant materials. A considerable amount of literature now exists on platelet adhesion, the emphasis of particular investigators being in three general areas: 1) adhesiveness of platelets with a view to clinical diagnosis of diseases, such as von Willebrand’s disease and predisposition to t h r o m b ~ s i s ; l - ~2) modification of adhesion by various drugs in model studies for the treatment of t h r o m b ~ s i s ; ~ - ~ and 3) evaluation of materials for use as vascular implants by using platelet adhesion as a measure of thrombogenic 429 @ 1976 by John Wiley & Sons, Inc.
430
BRASH, BROPHY, AND FEUERSTEIN
There are many important factors in the study of platelet adhesion, all of which must be taken into account if valid results are to be obtained. Some of these factors are: 1) the necessity t o measure adhesion as distinct from aggregation; 2 ) close control of the platelet medium, e.g., whether to use plasma, whole blood, or suspensions of washed platelets, which anticoagulant to use, and what should be the protein content of the medium; 3) details of the test system such as definition of flow and transport conditions, and avoidance of transfer to the test surface of protein films a t the air-liquid interface; and 4) adequate purification and characterization of test surfaces. Previous investigations have been concerned with some but not all of these factors, and the particular factors recognized in a given study have tended to depend on the motivational emphasis as outlined above. I n the work reported here, an attempt has been made t o study the adhesion of platelets to a variety of surfaces including reconstituted collagen, glass, and various polymeric materials. While the primary motivation is t o investigate platelet adhesion in relation to surface thrombogenesis, the results have a bearing on intravascular thrombosis as well. Most importantly, cognizance has been taken in the experimental design of the various important factors indicated above.
EXPERIMENTAL Materials Segmented polyurethanes were of two types, a hydrophobic material based on poly(oxypropy1ene) and a hydrophilic material based on poly(oxyethy1ene). Methods of synthesis have been described previously. lo The polymers were dissolved in dimethylformamide and applied to Delrin specimen tubes by solution coating. Polystyrene was obtained from Monsanto Canada Ltd., and coated on Delrin from solution in tetrahydrofuran. Sulfonated polystyrene was prepared by copolymerization of styrene and sodium styrene sulfonate as described previously. lo The copolymer used in this work contained 10 mole-% of the sulfonate monomer, and was coated on Delrin from solution in dimethyl sulfoxide. Human albumin was from Rehringswerke (West Germany) and was 1 0 0 ~ oimmunochemically pure. Fibrinogen was from Kabi
EFFECT OF RED CELLS ON PLATELET ADHESION
431
(Stockholm, Sweden) with a clottability of 95%. Fibrinogen and albumin were applied to glass specimen tubes by immersion in solutions a t a concentration of 100 mg/100 ml for 2 hr. Under those conditions, surface concentrations corresponding to close-packed monolayers are obtained." Coating of glass specimen tubes with reconstituted collagen fibers was carried out using the procedure of Cazenave et Glass surfaces for adhesion experiments were washed successively in 1 N HCI, 1 N IIOH, and distilled water.
Platelet Preparations The basic platelet medium consisted of pig platelets, twice washed and suspended in Tyrode solution. The preparation has been described p r e v i o ~ s l y . Important ~ ~ ~ ~ ~ features in the context of the present work are: 1) coagulation factors and anticoagulants are absent, thus allowing study of adhesion independent of these complicating and little understood factors; 2 ) the protein composition is known and can be varied; 3) washed erythrocytes can be added back t o the suspensions a t various concentrations (These are prepared by washing three times in Tyrode-ACD solution after separation from the plasma.); 4) the platelets are labeled using 51Cr-sodi~m chromate, and adhesion is quantitated by radioisotope counting; 5) apyrase is added to stabilize the suspensions and to degrade adventitious ADP, thus allowing adhesion t o be observed independent of ADP-induced aggregation. A typical suspension contained 3 pg/ml apyrase, 0.35 g/100 ml albumin, and the platelet concentration was 750,0OO/p1. The calcium and magnesium concentrations were 2 mM and 1 mM, respectively, in the fluid portion of the suspension. In some experiments, fibrinogen (0.30 g/100 ml) was substituted for albumin; in others, protein was omitted entirely. Experiments were also carried out in a suspending medium consisting of the original pig plasma anticoagulated with sodium citrate. Each suspension was checked before use for normal response to ADP in the aggregometer.
Apparatus The apparatus, shown in Figure I, is essentially a rotating probe device consisting of two rods on which can be mounted test specimens,
432
BRASH, BIZOPHY, AXl) FEUEKSTEIN
PROBE
STAND
SIDE VIEW
I
I
-TACHOMETER TOP VIEW
Fig. 1 . Schematic of rotational apparatus for study of platelet adhesion. For simplicity, the right-side probe arid left-side test tube are riot shown.
5 cm in length, of outside diameter 0.7 cm (glass or Delrin). The specimens can be rotated a t speeds between 50 and 200 rpm within two removable test tubes of 2.3 em internal diameter. With this arrangement, duplicate specimens can be run simultancously. Alternatively, one side of the apparatus can be used for a control and the other for the test specimen. The fluid dynamic characteristics of the device are discussed in detail elsewhere.14 It is pertinent to note, however, that the flow is laminar with circular streamlines. Mass transport to the probe surface is by diffusion alone with no convective component, and the probe surface is uniformly accessible. At the speeds used, centrifugal forces on platelets are not greater than gravitational forces.
EFFECT OF RED CELLS ON PLATELET ADHESION
433
Procedure I n preliminary work, a few experiments were performed a t 37°C and showed adhesion levels indistinguishable from those a t room temperature. As a matter of convenience, therefore, the data reported were obtained a t room temperature (22°C). I n a typical experimental run, the specimens mounted on the probes are immersed in Tyrode solution for 2 min. After draining off the bulk of the fluid, a firmly adherent layer, 15-25 pm in average thickness (as measured using 51Cr-labeled Tyrode solution), remains. It is believed that the layer of aqueous fluid on the test surface is equivalent t o the lead fluid that is generally employed in systems where internal surfaces are being studied, thus eliminating the transfer of the protein film a t the air-fluid interface t o the specimen surface. The test tubes, each containing about 60 ml of test fluid, are secured t o the apparatus and the probes positioned in the test tubes. The D.C. motor is started and the speed adjusted by a rheostat on a D.C. power supply. All experiments reported here were run a t 100 rpm corresponding t o a shear rate of 19 sec-'. After rotating for the desired time, the probes are removed and shaken to remove loosely adherent fluid. Rinsing is completed by rotating in Tyrode solution at 100 rpm for 30 sec. The specimens are then placed in vials for counting in a Beckman Biogamma solid scintillation gamma counter. Platelet surface density is estimated by comparison of the radioactivity of the specimen with that of an aliquot of test fluid. Representative scanning electron micrographs of all surfaces were obtained and showed platelets adhering singly. Normal shape was also retained except in the case of glass where pseudopods were usually observed. A typical micrograph for collagen is shown in Figure 2. Visual counting of platelets in the electron microscope agreed well with counts obtained by the labeling method. After each experiment, the fluid was centrifuged and the supernatant radioactivity and hemoglobin content (when red cells were present) determined. The radioactivity gave an indication of platelet lysis and was typically on the order of 2y0 of total radioactivity in the suspension, indicating negligible lysis. Hemoglobin content was used as an indication of hemolysis. Only trace quantities
434
BRASH, BROPHY, A M ) FEUERSTEIN
Fig. 2. Scanning electron micrograph of a collagen-coated glass rod which was exposed to platelet-red cell suspension (hematocrit, 10%) for 30 min. Platelet concentration 750,00O/rl. Collagen fibers and adhering platelets are readily visible.
( < 1 mg/100 ml) were usually detected, indicating no significant hemolysis as would be anticipated from the mild fluid shear conditions.
RESULTS AND DISCUSSION Kinetic data between 0 and 15 min were obtained for all systems. However, only for collagen and fibrinogen surfaces in the presence of red cells was there a marked time-dependence of adhesion. Therefore, the bulk of the data are presented in tabular form and the values of adhesion averaged over the time range studied are given (Tables 1-111). Typical kinetic data are shown for a few polymer surfaces (Fig. 3 ) and for collagen (Fig. 4). The dependence of adhesion on shear rate between 0 and 30 sec-' was minimal and all data reported are for a shear rate of 19 sec-1.
3.3 f 1 . 3 2.3 f 0.6
6.6 f 2 . 0 -
Polystyrene
-
2.6 f 0 . 3
Albumin on glass
2.4 f O . 3
Fibrinogen on glass
f
1.6 1.0 f 0.3
5.9
-
-~
Collagen on glass
a Conditions: shear rate, 19 sec-l, exposure time, 2-15 min; platelet concn, 750,00O/pl. (Data are platelets per 1000 pm2 f S.11.) h Data for glam in plain Tyrode and Tgrode containing 0.3 g/100 ml fibrinogen were 3.6 f 1.7 and 2.7 f 0.5, respectively, averaged over the time period 2-15 min.
3.7 f 0 . 9 2.0 f 0.3
1.3
2.0 f 0 . 3
f
4.3
Plasma
Glassh
Hydrophobic Hydrophilic polyurethane polyurethane
Tgrode albumin
~~
TABLE I Adhesion of Platelets in the Absence of Red Cells.
436
BRASH, BROPHY, AND FEUERSTEIN
Table I shows results in the absence of red cells. It can be seen that adhesion is of the order of 4 ~latelets/1000pm2 with similar values on all surfaces. Marginally lower values are seen for albumin and fibrinogen, and adhesion in plasma may be less than in Tyrode albumin, but the differences are not highly significant. Interestingly, it appears, from a set of data on glass, that adhesion in a protein-free medium (plain Tyrode) is not different from that in media containing plasma proteins. The surface concentrations all reach saturation within 2 min. Observation of differences in kinetics among the surfaces, if such exist, would presumably require different experimental conditions, perhaps shorter times and lower platelet concentration. Coverage in terms of available area is relatively sparse. It can be estimated that a close-packed layer of single platelets would contain about 250 platelets/1000 pm2. Under these experimental conditions, it is thus difficult t o conceive of the interaction proceeding to the stage of surface localized aggregation. The platelets do not appear to spread or to deploy pseudopods. The interaction is thus probably of a nonspecific van der Waals type which, in agreement Ixith observation, would not be expected to vary greatly from surface to surface. Experiinents in the absence of red cells have been reported previously by Jenkins et al.4 and Lyman et aL6 The latter found adhcsion on collagen of 1 platelet/1000 pmZand the formrr reported values for albumin of about 2 and, for fibrinogen, 10 platelets/1000 pm2. Although the present data are quantitatively similar, they do not show significant differences among any of these surfaces. It should be pointed out that in the experiments of Jenkins et al., film transfer from the fluid-air intrrface can occur both in surface preparation and in the adhesion test. It may be that fibrinogen transferred in this way is more reactive than material adsorbed from solution. Table I1 shows adhesion data a t 20yohematocrit. I n thc presence of red cells, it is seen that adhesion patterns are in some respects the sarnr and in others greatly altered. Adhesion to the polymer surfacrs is again not time-drpendent, arid no significant difference is seen among the various materials. Also, adhesion in plasma is somcwhat lower than in Tyrodr albumin. Adhesion t o albumin follows a similar course that shoxx s a slightly lower saturation level. I n contrast to the data in Table I, adhesion to collagen and fibrinogen
5 . 3 f 0.8
___ _______________
-
-
__.____ ~-
-
(J
8
Collagen
38.4 & 3 . 2 __
-
63.2 f 6 . 8
on glassc
(Data are platelets per 1000
-
-
32.0 f 4 . 6
14.3 f 1 . 6
5.1 i 0 . 4
6 . 7 f 1.7
Fibrinogen on glass
Albumiri or1 glass
Sulfonated polystyrene
Conditions: shear rate, 19 sec-'; exposure time, 15 sec-15 min; platelet coricn, 750,0OO/pl. pm2 f S.11.) b Value for glass in Tyrode-fibrinogen was 5.9 f 1.4. Data obtained a t 15% hematocrit arid 10 miri. Protein-free.
~ _ _ _ _ _ _ _ _ ~ ~ ~ _ .
5.6 f 0 . 6
5.5 f 1 . 1
Plasma
19.8 f 3.0
2.2
30.2 f 2 . 8
Plain Tyroded 31.9
6 . 4 f 1.0
8 . 7 f 1.4
7.2 f 1.0
Polystyrene
Hydrophobic polyurethane
Tyrode albumin
Glassb
TABLE I1 Adhesion of Platelets in the Presence of Red Cells (Hematocrit 20%)*
z
0
r
0
BRASH, BROPHY, AND FEUERSTEIX
438
is greater than on the other surfaces, and collagen is the most reactive. Levels of adhesion to the various polymer surfaces, of the order of 8 per 1000Mm2,are significantly greater than the red-cell-free experiments, a fact which is also clear from Figure 3 . Again, however, coverage of the available area falls well short of close-packing in all systems. The higher adhesion on fibrinogen and somewhat lower adhesion on albumin surfaces is in agreement with the results of .Jenkins et al.* and Packham et al.,15although as indicated above, red cells were not present in their experiments. Mason et a1.16 have also reported higher adhesion levels on fibrinogen compared to several other plasma proteins, with values between 0.2 and 2 platelets/1000 pm2 in a static whole-blood system. Table I1 also shows that adhesion in a protein-free medium is significantly greater than in any of the protein-containing media. Also, polystyrene shows lower adhesion under those conditions than any of thc other surfaces. It is tempting to suggest that lower
0
5
10
15
TIME (MIN)
Fig. 3. Adhesion of platelets to various artificial surfaces: effect of red cell conhydrophobic polyurethane. centration. (A)Glass; ( 0)polystyrene;).(
EFFECT OF RED CELLS OX PLATELET ADHESION
439
values in the presence of protein may be due to protein adsorption. However, such adsorption occurs independent of the presence of red cells; it is therefore difficult to see why similar differences in adhesion with and without protein are not seen in the absence of red cells (Table I). It may be speculated that the true explanation lies in the increased energy of platelet-surface interaction in the presence of red cells due to their radial and translational motions (see discussion below), such energy increases being more effective with the “bare” surface than with the adsorbed protein surface. Concerning this point, it may be noted that platelet adhesion in blood is believed t o take place on a layer of adsorbed protein rather than on the material surface itself. While this is certainly a reasonable hypothesis, the present results show that adhesion to the “bare” surface can also occur and does so to a greater extent. Both fibrinogen and collagen show a strong time-dependence and the kinetics of adhseion to collagen are shown in Figure 4 a t 25y0 hematocrit. (Data a t 0% are also shown for contrast.) Collagen is the most reactive surface investigated, not in the sense of showing 80r
>
60
-
OK Hematocrit i7
Y 0
5
10
15
TIME IMIN)
Fig. 4. Kinetics of platelet adhesion to collagen-coated glass at 0 and 25% hematocrit.
BRASH, BROPHY, AN11 FEUERSTEIN
440
faster adhesion, but because it shows a higher capacity for adhesion. The reaction of platelets with collagen is a fundamental step in hemostasis and thrombosis, and although the precise mechanism is a t present a matter of debate,17j1sit is probably reasonable to assume that specific chemical reactions are involved, as opposed to the general physical interactions that probably take place with the other surfaces. Collagen adhesion data can be fitted to a kinetic modrl in which diffusion and reaction rates a t the surface are considered equal. Such treatment yields values of platelet diffusion coefficient and a reaction rate constant. This approach is the subject of another p ~ b l i c a t i o n , where '~ it is shown that these parameters, particularly diffusivity, are strongly dependent on hematocrit and less so on shear rate. Table I11 shows data a t 457, hematocrit, some of which is also plotted in Figure 3. The level of adhesion to polymer surfaces is of the order of 18 platelets/1000 pm2 and is thus significantly higher than a t 20y0 hematocrit. Again, the various surfaces reach indistinguishable equilibria within 2 min, and kinetics are not accessible under these conditions. Adhesion in plasma is less than in Tyrodr albumin, the difference a t 45'3, hematocrit being more significant than a t 0 or 20%. The principal finding of this study is that red cells increase the levels of platelet adhesion. Red cell influence on adhesion has been noted previously, particularly in the early developement of adhesiveness tests19,20The effect has generally been attributed to ADP released from the red cells,20but since such tests probably evaluate aggregation rather than adhesion, a n ADI' effect is t o be expected. TABLE 111 Adhesion of Platelets in the Presence of Bed Cells (Hematocrit 457,)s
Tyrode albumin Plasma
Glass
Hydrophobic polyurethane
Polystyrene
19.1 f 3 . 2
16.7 f 2.8
18.6 f 1.8
7 . 2 f 1.4
8.2 f 1.0
7.3 f 0.7
a Conditions: shear rate, 19 sec-'; exposure time, 5-15 min; platelet concn, 750,00O/pl. (Data are platelets per 1000 pm2 f S.D.)
EFFECT OF REI) CELLS O N PLATELET ADHESION
441
Nonrthelrss, the possibility that red cell-induced augmentation of adhesion is due to ADI’ must be givrn consideration. With resprct t o effects related t o ADP-induced aggrrgation in thc fluid, i t can be argued that these are not operative since apyrasc was prrsent, hemolysis was minimal, and platclrt aggregates were not observed on the surface or in the medium. The possibility remains that the red cells could, on contact with the surface, deposit some “sticky” substance that would result in increasrd adhesion. To explore this possibility, experiments were performed in which either glass or collagen was exposrd first to a red cell suspension without platelets, then to a platelet suspension either with or without red cells. The results, shown in Table IV, were obtained in plasma as TABLE I V Effect of Preexposure to Red Cell Suspension (45% Hematocrit) on Platelet Adhesionarh Hematocrit of platelet suspension 0% ~
______
-
Hematocrit of platelet suspension 45% - -
-
__
With Without preexposure preexposure
With preexposure
_ _ _
Without preexposure
Glass
1.6 f 0 . 5
1.0 f 0 . 2
4.9 f 0 . 5
3.6 f 0 . 2
Collagen-coatedglass
0.8 f 0 . 5
1.0 f 0 . 3
25.1 f 2.1
25.3 f 1.0
a Conditions: shear rate, 19 sec-1; exposure time, 4 min; platelet coiicn, 400,00O/pl. b Data obtained in citrated plasma. Values given as means & 95y0 confidence interval.
susprnding fluid. In no case was thrrc a significant diffrrence, at a confidence level of 5% or less, between adhesion following exposure t o red cells and adhesion without such prior exposure. The most striking difference is, as before, between adhesion in the presencr and in the absence of red cells. These results suggest that deposition of an adhesive substance by red cell-surface contact does not occur. Anothrr possible explanation of red cell-augmented adhesion may reside in the rotational motion of the rcd cells and their fluctuating motions perpendicular to fluid streamlines as described by Goldsmith.21s22 Such motions have brcn found by several groups to
442
BRASH, BROPHY, AN11 FEUERSTEIN
increase platelet d i f f u ~ i o n . It ~ ~seems ~ ~ ~possible that the increase in platelet momentum induced by red cell motions could result in platelet,-surface collisions of higher cnergy, and allow thc occupation of surface sites of higher activation energy. It must be emphasized that there is no direct evidence for this explanation and i t should b r regarded simply as a working hypothesis. With respect to adhesion on different polymer surfaces, the results of this work are in agreement with those of Friedman et a1.8 and in conflict with those of Lyman et al.' The latter invcstigation was carried out in a flowing whole-blood system and indicated that adhesion levels increased with critical surface tension in a series of polymer surfaces. The values obtained for 1 min exposure ranged from 0.1 to 1 per 1000 ,mi2. A recent study24of flow patterns in the test cell of Lyman et al. indicated very low shear rates of the order of 1 sec-1; therefore, the motion-induced effccts of red cells may be much less under those conditions. Another important difference from the present work and from that of Friedman et al. is that the coagulation system was intact. I n view of the known variations of and inasmuch as in viuo thrombogenesis of the various surfaces, the present study examines platelet adhesion independent of aggregation and coagulation and shows no variations among surfaces, it may reasonably be concluded that early platelet adhesion per se is not a strong determinant of eventual thrombus formation. The combined effects of platelets and coagulation, manifest as adhesion and surface-localized aggregation of platelets, may well be the critical factor. The aut,hors wish to thank Mr. ill. J. Kiilczycki for experimental assistance and Ijr. Itathbone and Ijr. J-P. Cazenave for helpful discussions. This study was supported by grants from the Medical Research Council of Canada ( I X 103 and I)G 104).
References 1. E. W. Salzman, J . Lub. Clin. M e d . , 62, 724 (1963). 2. J. N. George, Blood, 40, 862 (1972). 3. It. G . Mason arid J. M. (:ilkey, Thronb. Diath. Haemorrh., 25, 21 (1971). 4. C. S. P. Jenkins, 31. A. Packham, M. A . Guccione, and J . F. Mustard, J . Lab. Clin. M e d . , 81, 280 (1973). 5. J. P. Cazenave, M. A. Packham, ill. A. Guccione, and J. F. Mustard, J . Lab. Clin. Med., 83, 797 (1974). 6. B. Lyman, L. Itosenberg, and S. Karpatkiri, J. Clin. Invest., 50, 1854 (1971).
E F F E C T OF R E D CELLS ON PLATELET ADHESIONS
443
7. I). J. Lyman, K. G. Klein, J. L. Brash, and B. K. Fritxinger, Il'hromb. Diath. Haemorrh., 23, 120 (1970). 8. L. I. Friedman, H. Liem, E. F. Grabowski, E. F. Leonard, and C. W. McCord, Trans. Amer. SOC.Artif. I n t . Organs, 16, 63 (1970). 9. E. W. Salzman, E. W. Merrill, A. Binder, C. F. W. Wolf, T. P. Ashford, and G. W. Austen, J . Biomed. Muter. Res., 3, 69 (1969). 10. J. L. Brash, B. K . Fritzinger, and B. H. Loo, Development of Materials for Heart Assist Devices, Contract P H 43-64-84, Annual Report to the National Heart and Lung Institute, April, 1972. 11. J. L. Brash and V. J. Ilavidson, unpublished observation. 12. J. P. Caaenave, M. A . Packham and J. F. Mustard, J . Lab. Clin. Med., 82, 978 (1973). 13. J. F. Mustard, 1). W. Perry, N. G. Ardlie, and M. A. Packham, Brit. J . Haenaatol., 22, 193 (1972). 14. I. A. Feuerstein, J. Brophy, and J . L. Brash, Trans. Amer. SOC.Artif. Int. Organs, 21,427 (1975). 15. M. A. Packham, G. Evans, M. F. Gljmn, and J. F. Mustard, J . Lab. Clin. Med., 73, 686 (1969). 16. K. G. Mason, R. W. Shermer, W. H. Zucker, and K. M. Brinkhous, Erythrocytes, l'hrombocytes, Leukocytes, 2nd Internat. S y m p . 1972, E. Gerlach, Ed., Georg Thieme, Stuttgart, 1973, pp. 263-266. 17. A. J. Barber and G. A. Jamieson, Biochim. Biophys. Acta., 252, 533 (1971). 18. 11. Puett, B. K. Wasserman, J. D. Ford, and L. W. Cunningham, J. Clin. Invest., 52, 2495 (1973). 19. A. J. Hellem, Scand. J . Clin. Lab. Invest., 12 Suppl., 51 (1960). 20. A. Gaarder, J. Jonsen, S. Laland, A. Hellem, and P. A. Owren, Nature, 192, 531 (1961). 21. H. L. Goldsmith and S. G. Mason in Rheology, Vol. IV, F. R. Eirich, Ed., Academic Press, New York, 1967. 22. H. L. Goldsmith, Fed. Proc., 30, 1578 (1971). 23. V. T. Turrito, A. M. Benis, and E . F. Leonard, I n d . Eng. Chem. Fundam., 11, 216 (1972). 24. R. Day, I. A. Feuerstein, and J. L. Brash, to be published. 25. V. L. Gott and A. Furuse, Fed. Proc., 30, 1679 (1971).
Received July 23, 1975 Revised September 19, 1975
VOL. 10, PP. 429-443 (1976)
Adhesion of Platelets to Artificial Surfaces: Effect of Red Cells J. L. BRASH, J. M. BROPHY, and I. A. FEUERSTEIN, Departments of Chemical Engineering and Pathology, AlcMaster University, Ham ilton , Ontario, Canada
Summary Adhesion of platelets to several polymer- and protein-coated glass surfaces has been studied in vitro. The apparatus consists of a cylindrical probe rotating in a test tube containing the platelet medium and allows close control of fluid shear and mass transport. Suspensions of washed pig platelets constitute the basic platelet medium, and can be modified by adding back red cells and plasma proteins. Adhesion is measured via 50-labeling of platelets. In the absence of red cells, identical low levels of adhesion were seen on all surfaces and saturation was reached within 2 min. I n the presence of red cells, adhesion was greater. Saturation on all surfaces except fibrinogen and collagen again occurred within 2 min. The adhesion levels on polymer surfaces and glass were indistinguishable, while those 011 albumin were lower and those on fibrinogen were higher. Collagen was the most reactive surface. It did not equilibrate within 15 min, and kinetic data indicated a platelet diffusivity strongly dependent on hematocrit. These effects were attributed to rotational and translational motion of the red cells causing increased diffusion and surface-platelet collision energy.
INTRODUCTION It is widely recognized that platelet adhesion is important in hemostasis, intravascular thrombosis, and thrombus formation on vascular implant materials. A considerable amount of literature now exists on platelet adhesion, the emphasis of particular investigators being in three general areas: 1) adhesiveness of platelets with a view to clinical diagnosis of diseases, such as von Willebrand’s disease and predisposition to t h r o m b ~ s i s ; l - ~2) modification of adhesion by various drugs in model studies for the treatment of t h r o m b ~ s i s ; ~ - ~ and 3) evaluation of materials for use as vascular implants by using platelet adhesion as a measure of thrombogenic 429 @ 1976 by John Wiley & Sons, Inc.
430
BRASH, BROPHY, AND FEUERSTEIN
There are many important factors in the study of platelet adhesion, all of which must be taken into account if valid results are to be obtained. Some of these factors are: 1) the necessity t o measure adhesion as distinct from aggregation; 2 ) close control of the platelet medium, e.g., whether to use plasma, whole blood, or suspensions of washed platelets, which anticoagulant to use, and what should be the protein content of the medium; 3) details of the test system such as definition of flow and transport conditions, and avoidance of transfer to the test surface of protein films a t the air-liquid interface; and 4) adequate purification and characterization of test surfaces. Previous investigations have been concerned with some but not all of these factors, and the particular factors recognized in a given study have tended to depend on the motivational emphasis as outlined above. I n the work reported here, an attempt has been made t o study the adhesion of platelets to a variety of surfaces including reconstituted collagen, glass, and various polymeric materials. While the primary motivation is t o investigate platelet adhesion in relation to surface thrombogenesis, the results have a bearing on intravascular thrombosis as well. Most importantly, cognizance has been taken in the experimental design of the various important factors indicated above.
EXPERIMENTAL Materials Segmented polyurethanes were of two types, a hydrophobic material based on poly(oxypropy1ene) and a hydrophilic material based on poly(oxyethy1ene). Methods of synthesis have been described previously. lo The polymers were dissolved in dimethylformamide and applied to Delrin specimen tubes by solution coating. Polystyrene was obtained from Monsanto Canada Ltd., and coated on Delrin from solution in tetrahydrofuran. Sulfonated polystyrene was prepared by copolymerization of styrene and sodium styrene sulfonate as described previously. lo The copolymer used in this work contained 10 mole-% of the sulfonate monomer, and was coated on Delrin from solution in dimethyl sulfoxide. Human albumin was from Rehringswerke (West Germany) and was 1 0 0 ~ oimmunochemically pure. Fibrinogen was from Kabi
EFFECT OF RED CELLS ON PLATELET ADHESION
431
(Stockholm, Sweden) with a clottability of 95%. Fibrinogen and albumin were applied to glass specimen tubes by immersion in solutions a t a concentration of 100 mg/100 ml for 2 hr. Under those conditions, surface concentrations corresponding to close-packed monolayers are obtained." Coating of glass specimen tubes with reconstituted collagen fibers was carried out using the procedure of Cazenave et Glass surfaces for adhesion experiments were washed successively in 1 N HCI, 1 N IIOH, and distilled water.
Platelet Preparations The basic platelet medium consisted of pig platelets, twice washed and suspended in Tyrode solution. The preparation has been described p r e v i o ~ s l y . Important ~ ~ ~ ~ ~ features in the context of the present work are: 1) coagulation factors and anticoagulants are absent, thus allowing study of adhesion independent of these complicating and little understood factors; 2 ) the protein composition is known and can be varied; 3) washed erythrocytes can be added back t o the suspensions a t various concentrations (These are prepared by washing three times in Tyrode-ACD solution after separation from the plasma.); 4) the platelets are labeled using 51Cr-sodi~m chromate, and adhesion is quantitated by radioisotope counting; 5) apyrase is added to stabilize the suspensions and to degrade adventitious ADP, thus allowing adhesion t o be observed independent of ADP-induced aggregation. A typical suspension contained 3 pg/ml apyrase, 0.35 g/100 ml albumin, and the platelet concentration was 750,0OO/p1. The calcium and magnesium concentrations were 2 mM and 1 mM, respectively, in the fluid portion of the suspension. In some experiments, fibrinogen (0.30 g/100 ml) was substituted for albumin; in others, protein was omitted entirely. Experiments were also carried out in a suspending medium consisting of the original pig plasma anticoagulated with sodium citrate. Each suspension was checked before use for normal response to ADP in the aggregometer.
Apparatus The apparatus, shown in Figure I, is essentially a rotating probe device consisting of two rods on which can be mounted test specimens,
432
BRASH, BIZOPHY, AXl) FEUEKSTEIN
PROBE
STAND
SIDE VIEW
I
I
-TACHOMETER TOP VIEW
Fig. 1 . Schematic of rotational apparatus for study of platelet adhesion. For simplicity, the right-side probe arid left-side test tube are riot shown.
5 cm in length, of outside diameter 0.7 cm (glass or Delrin). The specimens can be rotated a t speeds between 50 and 200 rpm within two removable test tubes of 2.3 em internal diameter. With this arrangement, duplicate specimens can be run simultancously. Alternatively, one side of the apparatus can be used for a control and the other for the test specimen. The fluid dynamic characteristics of the device are discussed in detail elsewhere.14 It is pertinent to note, however, that the flow is laminar with circular streamlines. Mass transport to the probe surface is by diffusion alone with no convective component, and the probe surface is uniformly accessible. At the speeds used, centrifugal forces on platelets are not greater than gravitational forces.
EFFECT OF RED CELLS ON PLATELET ADHESION
433
Procedure I n preliminary work, a few experiments were performed a t 37°C and showed adhesion levels indistinguishable from those a t room temperature. As a matter of convenience, therefore, the data reported were obtained a t room temperature (22°C). I n a typical experimental run, the specimens mounted on the probes are immersed in Tyrode solution for 2 min. After draining off the bulk of the fluid, a firmly adherent layer, 15-25 pm in average thickness (as measured using 51Cr-labeled Tyrode solution), remains. It is believed that the layer of aqueous fluid on the test surface is equivalent t o the lead fluid that is generally employed in systems where internal surfaces are being studied, thus eliminating the transfer of the protein film a t the air-fluid interface t o the specimen surface. The test tubes, each containing about 60 ml of test fluid, are secured t o the apparatus and the probes positioned in the test tubes. The D.C. motor is started and the speed adjusted by a rheostat on a D.C. power supply. All experiments reported here were run a t 100 rpm corresponding t o a shear rate of 19 sec-'. After rotating for the desired time, the probes are removed and shaken to remove loosely adherent fluid. Rinsing is completed by rotating in Tyrode solution at 100 rpm for 30 sec. The specimens are then placed in vials for counting in a Beckman Biogamma solid scintillation gamma counter. Platelet surface density is estimated by comparison of the radioactivity of the specimen with that of an aliquot of test fluid. Representative scanning electron micrographs of all surfaces were obtained and showed platelets adhering singly. Normal shape was also retained except in the case of glass where pseudopods were usually observed. A typical micrograph for collagen is shown in Figure 2. Visual counting of platelets in the electron microscope agreed well with counts obtained by the labeling method. After each experiment, the fluid was centrifuged and the supernatant radioactivity and hemoglobin content (when red cells were present) determined. The radioactivity gave an indication of platelet lysis and was typically on the order of 2y0 of total radioactivity in the suspension, indicating negligible lysis. Hemoglobin content was used as an indication of hemolysis. Only trace quantities
434
BRASH, BROPHY, A M ) FEUERSTEIN
Fig. 2. Scanning electron micrograph of a collagen-coated glass rod which was exposed to platelet-red cell suspension (hematocrit, 10%) for 30 min. Platelet concentration 750,00O/rl. Collagen fibers and adhering platelets are readily visible.
( < 1 mg/100 ml) were usually detected, indicating no significant hemolysis as would be anticipated from the mild fluid shear conditions.
RESULTS AND DISCUSSION Kinetic data between 0 and 15 min were obtained for all systems. However, only for collagen and fibrinogen surfaces in the presence of red cells was there a marked time-dependence of adhesion. Therefore, the bulk of the data are presented in tabular form and the values of adhesion averaged over the time range studied are given (Tables 1-111). Typical kinetic data are shown for a few polymer surfaces (Fig. 3 ) and for collagen (Fig. 4). The dependence of adhesion on shear rate between 0 and 30 sec-' was minimal and all data reported are for a shear rate of 19 sec-1.
3.3 f 1 . 3 2.3 f 0.6
6.6 f 2 . 0 -
Polystyrene
-
2.6 f 0 . 3
Albumin on glass
2.4 f O . 3
Fibrinogen on glass
f
1.6 1.0 f 0.3
5.9
-
-~
Collagen on glass
a Conditions: shear rate, 19 sec-l, exposure time, 2-15 min; platelet concn, 750,00O/pl. (Data are platelets per 1000 pm2 f S.11.) h Data for glam in plain Tyrode and Tgrode containing 0.3 g/100 ml fibrinogen were 3.6 f 1.7 and 2.7 f 0.5, respectively, averaged over the time period 2-15 min.
3.7 f 0 . 9 2.0 f 0.3
1.3
2.0 f 0 . 3
f
4.3
Plasma
Glassh
Hydrophobic Hydrophilic polyurethane polyurethane
Tgrode albumin
~~
TABLE I Adhesion of Platelets in the Absence of Red Cells.
436
BRASH, BROPHY, AND FEUERSTEIN
Table I shows results in the absence of red cells. It can be seen that adhesion is of the order of 4 ~latelets/1000pm2 with similar values on all surfaces. Marginally lower values are seen for albumin and fibrinogen, and adhesion in plasma may be less than in Tyrode albumin, but the differences are not highly significant. Interestingly, it appears, from a set of data on glass, that adhesion in a protein-free medium (plain Tyrode) is not different from that in media containing plasma proteins. The surface concentrations all reach saturation within 2 min. Observation of differences in kinetics among the surfaces, if such exist, would presumably require different experimental conditions, perhaps shorter times and lower platelet concentration. Coverage in terms of available area is relatively sparse. It can be estimated that a close-packed layer of single platelets would contain about 250 platelets/1000 pm2. Under these experimental conditions, it is thus difficult t o conceive of the interaction proceeding to the stage of surface localized aggregation. The platelets do not appear to spread or to deploy pseudopods. The interaction is thus probably of a nonspecific van der Waals type which, in agreement Ixith observation, would not be expected to vary greatly from surface to surface. Experiinents in the absence of red cells have been reported previously by Jenkins et al.4 and Lyman et aL6 The latter found adhcsion on collagen of 1 platelet/1000 pmZand the formrr reported values for albumin of about 2 and, for fibrinogen, 10 platelets/1000 pm2. Although the present data are quantitatively similar, they do not show significant differences among any of these surfaces. It should be pointed out that in the experiments of Jenkins et al., film transfer from the fluid-air intrrface can occur both in surface preparation and in the adhesion test. It may be that fibrinogen transferred in this way is more reactive than material adsorbed from solution. Table I1 shows adhesion data a t 20yohematocrit. I n thc presence of red cells, it is seen that adhesion patterns are in some respects the sarnr and in others greatly altered. Adhesion to the polymer surfacrs is again not time-drpendent, arid no significant difference is seen among the various materials. Also, adhesion in plasma is somcwhat lower than in Tyrodr albumin. Adhesion t o albumin follows a similar course that shoxx s a slightly lower saturation level. I n contrast to the data in Table I, adhesion to collagen and fibrinogen
5 . 3 f 0.8
___ _______________
-
-
__.____ ~-
-
(J
8
Collagen
38.4 & 3 . 2 __
-
63.2 f 6 . 8
on glassc
(Data are platelets per 1000
-
-
32.0 f 4 . 6
14.3 f 1 . 6
5.1 i 0 . 4
6 . 7 f 1.7
Fibrinogen on glass
Albumiri or1 glass
Sulfonated polystyrene
Conditions: shear rate, 19 sec-'; exposure time, 15 sec-15 min; platelet coricn, 750,0OO/pl. pm2 f S.11.) b Value for glass in Tyrode-fibrinogen was 5.9 f 1.4. Data obtained a t 15% hematocrit arid 10 miri. Protein-free.
~ _ _ _ _ _ _ _ _ ~ ~ ~ _ .
5.6 f 0 . 6
5.5 f 1 . 1
Plasma
19.8 f 3.0
2.2
30.2 f 2 . 8
Plain Tyroded 31.9
6 . 4 f 1.0
8 . 7 f 1.4
7.2 f 1.0
Polystyrene
Hydrophobic polyurethane
Tyrode albumin
Glassb
TABLE I1 Adhesion of Platelets in the Presence of Red Cells (Hematocrit 20%)*
z
0
r
0
BRASH, BROPHY, AND FEUERSTEIX
438
is greater than on the other surfaces, and collagen is the most reactive. Levels of adhesion to the various polymer surfaces, of the order of 8 per 1000Mm2,are significantly greater than the red-cell-free experiments, a fact which is also clear from Figure 3 . Again, however, coverage of the available area falls well short of close-packing in all systems. The higher adhesion on fibrinogen and somewhat lower adhesion on albumin surfaces is in agreement with the results of .Jenkins et al.* and Packham et al.,15although as indicated above, red cells were not present in their experiments. Mason et a1.16 have also reported higher adhesion levels on fibrinogen compared to several other plasma proteins, with values between 0.2 and 2 platelets/1000 pm2 in a static whole-blood system. Table I1 also shows that adhesion in a protein-free medium is significantly greater than in any of the protein-containing media. Also, polystyrene shows lower adhesion under those conditions than any of thc other surfaces. It is tempting to suggest that lower
0
5
10
15
TIME (MIN)
Fig. 3. Adhesion of platelets to various artificial surfaces: effect of red cell conhydrophobic polyurethane. centration. (A)Glass; ( 0)polystyrene;).(
EFFECT OF RED CELLS OX PLATELET ADHESION
439
values in the presence of protein may be due to protein adsorption. However, such adsorption occurs independent of the presence of red cells; it is therefore difficult to see why similar differences in adhesion with and without protein are not seen in the absence of red cells (Table I). It may be speculated that the true explanation lies in the increased energy of platelet-surface interaction in the presence of red cells due to their radial and translational motions (see discussion below), such energy increases being more effective with the “bare” surface than with the adsorbed protein surface. Concerning this point, it may be noted that platelet adhesion in blood is believed t o take place on a layer of adsorbed protein rather than on the material surface itself. While this is certainly a reasonable hypothesis, the present results show that adhesion to the “bare” surface can also occur and does so to a greater extent. Both fibrinogen and collagen show a strong time-dependence and the kinetics of adhseion to collagen are shown in Figure 4 a t 25y0 hematocrit. (Data a t 0% are also shown for contrast.) Collagen is the most reactive surface investigated, not in the sense of showing 80r
>
60
-
OK Hematocrit i7
Y 0
5
10
15
TIME IMIN)
Fig. 4. Kinetics of platelet adhesion to collagen-coated glass at 0 and 25% hematocrit.
BRASH, BROPHY, AN11 FEUERSTEIN
440
faster adhesion, but because it shows a higher capacity for adhesion. The reaction of platelets with collagen is a fundamental step in hemostasis and thrombosis, and although the precise mechanism is a t present a matter of debate,17j1sit is probably reasonable to assume that specific chemical reactions are involved, as opposed to the general physical interactions that probably take place with the other surfaces. Collagen adhesion data can be fitted to a kinetic modrl in which diffusion and reaction rates a t the surface are considered equal. Such treatment yields values of platelet diffusion coefficient and a reaction rate constant. This approach is the subject of another p ~ b l i c a t i o n , where '~ it is shown that these parameters, particularly diffusivity, are strongly dependent on hematocrit and less so on shear rate. Table I11 shows data a t 457, hematocrit, some of which is also plotted in Figure 3. The level of adhesion to polymer surfaces is of the order of 18 platelets/1000 pm2 and is thus significantly higher than a t 20y0 hematocrit. Again, the various surfaces reach indistinguishable equilibria within 2 min, and kinetics are not accessible under these conditions. Adhesion in plasma is less than in Tyrodr albumin, the difference a t 45'3, hematocrit being more significant than a t 0 or 20%. The principal finding of this study is that red cells increase the levels of platelet adhesion. Red cell influence on adhesion has been noted previously, particularly in the early developement of adhesiveness tests19,20The effect has generally been attributed to ADP released from the red cells,20but since such tests probably evaluate aggregation rather than adhesion, a n ADI' effect is t o be expected. TABLE 111 Adhesion of Platelets in the Presence of Bed Cells (Hematocrit 457,)s
Tyrode albumin Plasma
Glass
Hydrophobic polyurethane
Polystyrene
19.1 f 3 . 2
16.7 f 2.8
18.6 f 1.8
7 . 2 f 1.4
8.2 f 1.0
7.3 f 0.7
a Conditions: shear rate, 19 sec-'; exposure time, 5-15 min; platelet concn, 750,00O/pl. (Data are platelets per 1000 pm2 f S.D.)
EFFECT OF REI) CELLS O N PLATELET ADHESION
441
Nonrthelrss, the possibility that red cell-induced augmentation of adhesion is due to ADI’ must be givrn consideration. With resprct t o effects related t o ADP-induced aggrrgation in thc fluid, i t can be argued that these are not operative since apyrasc was prrsent, hemolysis was minimal, and platclrt aggregates were not observed on the surface or in the medium. The possibility remains that the red cells could, on contact with the surface, deposit some “sticky” substance that would result in increasrd adhesion. To explore this possibility, experiments were performed in which either glass or collagen was exposrd first to a red cell suspension without platelets, then to a platelet suspension either with or without red cells. The results, shown in Table IV, were obtained in plasma as TABLE I V Effect of Preexposure to Red Cell Suspension (45% Hematocrit) on Platelet Adhesionarh Hematocrit of platelet suspension 0% ~
______
-
Hematocrit of platelet suspension 45% - -
-
__
With Without preexposure preexposure
With preexposure
_ _ _
Without preexposure
Glass
1.6 f 0 . 5
1.0 f 0 . 2
4.9 f 0 . 5
3.6 f 0 . 2
Collagen-coatedglass
0.8 f 0 . 5
1.0 f 0 . 3
25.1 f 2.1
25.3 f 1.0
a Conditions: shear rate, 19 sec-1; exposure time, 4 min; platelet coiicn, 400,00O/pl. b Data obtained in citrated plasma. Values given as means & 95y0 confidence interval.
susprnding fluid. In no case was thrrc a significant diffrrence, at a confidence level of 5% or less, between adhesion following exposure t o red cells and adhesion without such prior exposure. The most striking difference is, as before, between adhesion in the presencr and in the absence of red cells. These results suggest that deposition of an adhesive substance by red cell-surface contact does not occur. Anothrr possible explanation of red cell-augmented adhesion may reside in the rotational motion of the rcd cells and their fluctuating motions perpendicular to fluid streamlines as described by Goldsmith.21s22 Such motions have brcn found by several groups to
442
BRASH, BROPHY, AN11 FEUERSTEIN
increase platelet d i f f u ~ i o n . It ~ ~seems ~ ~ ~possible that the increase in platelet momentum induced by red cell motions could result in platelet,-surface collisions of higher cnergy, and allow thc occupation of surface sites of higher activation energy. It must be emphasized that there is no direct evidence for this explanation and i t should b r regarded simply as a working hypothesis. With respect to adhesion on different polymer surfaces, the results of this work are in agreement with those of Friedman et a1.8 and in conflict with those of Lyman et al.' The latter invcstigation was carried out in a flowing whole-blood system and indicated that adhesion levels increased with critical surface tension in a series of polymer surfaces. The values obtained for 1 min exposure ranged from 0.1 to 1 per 1000 ,mi2. A recent study24of flow patterns in the test cell of Lyman et al. indicated very low shear rates of the order of 1 sec-1; therefore, the motion-induced effccts of red cells may be much less under those conditions. Another important difference from the present work and from that of Friedman et al. is that the coagulation system was intact. I n view of the known variations of and inasmuch as in viuo thrombogenesis of the various surfaces, the present study examines platelet adhesion independent of aggregation and coagulation and shows no variations among surfaces, it may reasonably be concluded that early platelet adhesion per se is not a strong determinant of eventual thrombus formation. The combined effects of platelets and coagulation, manifest as adhesion and surface-localized aggregation of platelets, may well be the critical factor. The aut,hors wish to thank Mr. ill. J. Kiilczycki for experimental assistance and Ijr. Itathbone and Ijr. J-P. Cazenave for helpful discussions. This study was supported by grants from the Medical Research Council of Canada ( I X 103 and I)G 104).
References 1. E. W. Salzman, J . Lub. Clin. M e d . , 62, 724 (1963). 2. J. N. George, Blood, 40, 862 (1972). 3. It. G . Mason arid J. M. (:ilkey, Thronb. Diath. Haemorrh., 25, 21 (1971). 4. C. S. P. Jenkins, 31. A. Packham, M. A . Guccione, and J . F. Mustard, J . Lab. Clin. M e d . , 81, 280 (1973). 5. J. P. Cazenave, M. A. Packham, ill. A. Guccione, and J. F. Mustard, J . Lab. Clin. Med., 83, 797 (1974). 6. B. Lyman, L. Itosenberg, and S. Karpatkiri, J. Clin. Invest., 50, 1854 (1971).
E F F E C T OF R E D CELLS ON PLATELET ADHESIONS
443
7. I). J. Lyman, K. G. Klein, J. L. Brash, and B. K. Fritxinger, Il'hromb. Diath. Haemorrh., 23, 120 (1970). 8. L. I. Friedman, H. Liem, E. F. Grabowski, E. F. Leonard, and C. W. McCord, Trans. Amer. SOC.Artif. I n t . Organs, 16, 63 (1970). 9. E. W. Salzman, E. W. Merrill, A. Binder, C. F. W. Wolf, T. P. Ashford, and G. W. Austen, J . Biomed. Muter. Res., 3, 69 (1969). 10. J. L. Brash, B. K . Fritzinger, and B. H. Loo, Development of Materials for Heart Assist Devices, Contract P H 43-64-84, Annual Report to the National Heart and Lung Institute, April, 1972. 11. J. L. Brash and V. J. Ilavidson, unpublished observation. 12. J. P. Caaenave, M. A . Packham and J. F. Mustard, J . Lab. Clin. Med., 82, 978 (1973). 13. J. F. Mustard, 1). W. Perry, N. G. Ardlie, and M. A. Packham, Brit. J . Haenaatol., 22, 193 (1972). 14. I. A. Feuerstein, J. Brophy, and J . L. Brash, Trans. Amer. SOC.Artif. Int. Organs, 21,427 (1975). 15. M. A. Packham, G. Evans, M. F. Gljmn, and J. F. Mustard, J . Lab. Clin. Med., 73, 686 (1969). 16. K. G. Mason, R. W. Shermer, W. H. Zucker, and K. M. Brinkhous, Erythrocytes, l'hrombocytes, Leukocytes, 2nd Internat. S y m p . 1972, E. Gerlach, Ed., Georg Thieme, Stuttgart, 1973, pp. 263-266. 17. A. J. Barber and G. A. Jamieson, Biochim. Biophys. Acta., 252, 533 (1971). 18. 11. Puett, B. K. Wasserman, J. D. Ford, and L. W. Cunningham, J. Clin. Invest., 52, 2495 (1973). 19. A. J. Hellem, Scand. J . Clin. Lab. Invest., 12 Suppl., 51 (1960). 20. A. Gaarder, J. Jonsen, S. Laland, A. Hellem, and P. A. Owren, Nature, 192, 531 (1961). 21. H. L. Goldsmith and S. G. Mason in Rheology, Vol. IV, F. R. Eirich, Ed., Academic Press, New York, 1967. 22. H. L. Goldsmith, Fed. Proc., 30, 1578 (1971). 23. V. T. Turrito, A. M. Benis, and E . F. Leonard, I n d . Eng. Chem. Fundam., 11, 216 (1972). 24. R. Day, I. A. Feuerstein, and J. L. Brash, to be published. 25. V. L. Gott and A. Furuse, Fed. Proc., 30, 1679 (1971).
Received July 23, 1975 Revised September 19, 1975
