- Email: [email protected]
Electrochemical features of platelet interactions
THROMBOSIS RESEARCH Printed in the United
Suppl. II, Vol. 8, Pergamon Press,
States
SECTION
1976 Inc.
V
ELECTROCHEMICAL FEATURES OF PLATELET INTERACTIONS
Geoffrey V.F. Seaman Department of Neurology University of Oregon Health Sciences Center Portland, Oregon 97201, U.S.A.
ABSTRACT Platelets participate in hemostasis and thrombus formation. The physicochemical basis at the molecular level for platelet adhesion, cohesion to form aggregates and the resultant viscous metamorphosis is not well understood. Under physiological conditions, blood platelets possess a net negative charge and decreases or changes in this charge have been implicated in both their adhesion and aggregation. The peripheral zone of the platelet contains glycoproteins and glycolipids. A variety of charged groups are present including positively charged amino groups and at least three types of negatively charged groups. The carboxyl group of terminal sialic acid residues is considered to be a major contributor to the negative charge of platelets. Removal of as little as 10 percent of the peripheral zone sialic acid by neuraminidase results in a shortened platelet lifespan _in vivo. The electrokinetic properties of blood platelets are changed by interaction with a great variety of agents. In those cases where specific adsorption occurs it is possible to study the relationship between adsorption, change in surface charge, and the process of aggregation. It should be noted that in physiological media only those charge groups within about 8 A of the electrophoretic surface of the platelet will contribute to a significant extent to the electrophoretic properties. The electrophoretic mobility of platelets has been determined for a variety of animal species and in humans for a wide spectrum of disease situations. The measurement of platelet electrophoretic mobilities before and after treatment with adenosine diphosphate and norepinephrine has been introduced as a test of platelet function. However, so many variables may influence the surface properties of blood platelets including method of preparation, time, temperature, pH, contact phenomena, shear conditions, ionic strength and composition of suspending media, conditions for mobility measurements, etc., that considerable apparent disagreement exists in the literature regarding platelet electrokinetics. 235
S EMIAN
236
INTRODUCTION Blood platelets respond readily to various stimuli by adhesion to other surfaces and by the formation of platelet aggregates (1) which are a basic component of hemostatic plugs (2) and of thrombi (3). When activated platelets release proteases and factors which increase vascular permeability (4). In addition there is evidence that endothelial damage may arise as a result of platelet action (5) which may contribute subsequently to the development of atheroma (6). A great variety of agents can influence platelet adhesion or induce platelet aggregation both -in vitro and -in vivo but the physiocochemical bases for such phenomena are not well understood. It is not surprising therefore that measurements of platelet aggregation or adhesion have not been very successful in the study of individual subjects with, or at risk from diseases involving the blood vessels. The ease with which platelets can be isolated relatively intact from blood has been both a strength and also a danger in platelet research. Properties of platelets closely related to their function in hemostasis make them more difficult to isolate and study than is apparent to the unwary investigator. The difficulties include the choice of anticoagulant (7), selection of temperature for procedures or studies (8), nature of surfaces of vessels in which experiments are conducted (8), control of other environmental factors such as pH and calcium ion concentration (9), and the conditions and composition of suspending media used in the preparation of washed platelets (10). Blood platelets under physiological conditions carry a net negative charge and decreases in this charge have been implicated in their aggregation (11). Since blood platelets because of their like charge sign would be expected to be mutually repellant it has been proposed in thrombogenesis where platelets stick to the wall of a blood vessel and attract other platelets that the platelet surface charge must be altered (12).
TABLE I Methods of Electrophoretic Analysis of Biological Cells Experimental Variable
Information Obtained
pH of suspending medium
pK values of ionogenic groups
Ionic strength of suspending medium
Distribution of ionogenic groups in the peripheral zone and role of ion redistribution
Suspending medium ion composition
Charge reversal spectra and distribution of ionogenic groups
Modification with enzymes or specific functional group reagents
Nature of surface functional groups
Treatment with specific immunological reagents or molecules with known distribution of charges
Location and distribution of surface functional groups
Suppl.
II
PLATELET
SURFACE
AND
AGGREGATION
237
ELECTROPHORETIC TECHNIQUES In order to approach the problem of the nature of electrochemical interactions between platelets or between platelets and foreign bodies or surfaces information is needed on the peripheral zone of the platelet obtained from studies in fairly simple media under conditions where no significant irreversible changes in platelet behavior have occurred, The methods of electrophoretic analysis which may be used in the elucidation of surface structure are given in Table I. The equipment and experimental procedures for the electrophoretic analysis of biological cells as well as the biological variables and physicochemical parameters which may influence the electrophoretic mobility of cells have been described by Seaman (13). Electrophoretic analysis of blood platelets has shown that in general the electrophoretic mobility of human blood platelets shows only small variations in health and is rarely abnormal in disease when examined in simple ionic media or titrated plasma (14). The pH versus mobility relationships for human blood platelets are amphoteric in nature with evidence for the presence of several types of charge groups (11,15). The ionogenic composition of the human platelet has been obtained from the following experimental evidence: (a) the marked decrease in the electrophoretic mobility (s 50%) which results from treatment with neuraminidase accompanied by the release of N-acetylneuraminic acid (11) indicating the presence of a membrane-bound sialic acid. (b) a group of pK > 9 revealed by the pH versus mobility behavior suggesting the presence of amino groups which is confirmed by (c). (c) the increase in anodic mobility ("J20%) on treatment with acetaldehyde (ll), citraconic anhydride, and 2,3-dimethyl maleic anhydride (16). (d) the decrease (Q 20%) in mobility produced by treatment with alkaline phosphatase thus implying the presence of phosphate groups (17). (e) the increase in electrophoretic mobility (Q 20%) produced by modification with 6,6'-dithiodinicotinic acid thus indicating the presence of sulfhydryl groups in the peripheral zone of the platelet (18). The remainder of the unidentified negative groups on the basis of the pH versus mobility relationship have a pK of about 4 and are most probably the free carboxyl groups of aspartyl or glutamyl residues. The ionogenic constitution of the human platelet is given in Table II. The following assumptions were made in the calculation of the parameters listed: the Gouy-Chapman equation gives a reliable estimate of electrokinetic charge density for the platelet. This assumption is valid only if no significant fraction of the peripheral zone inside the electrophoretic slip plane is available to counterions ( .g> the charge is distributed uniformly at the electrophoretic surface. the mean surface area of the platelet is 28.3 pm2.
238
TABLE II Ionogenic Constitution of the Human Blood Platelet
Groups
PK
Sialic acid carboxyl Other carboxyl Phosphate Unidentified negative Amino Sulfhydryl Total negative Total positive Total charged groups
2.6 s4.0 ? ? >9.0
Groups/platelet x 10-5
Mean separatitn of groups - A
9.4 ? 6.5 7.0 5.3 2.9 22.9 5.3 28.2
55 ? 66 64 74 99 35 73 32
The ionogenic groups of the platelet surface are part of terminal moieties which in turn are linked to surface macromolecular structures. Platelet-specific proteins appear to be arranged asymmetrically within the membrane. Lactoperoxidase-catalyzed iodination of platelet surface macromolecules have shown three major glycoproteins to be located in the peripheral zone whereas most other membrane proteins are not (20). Fifteen to twenty percent of the total platelet protein consists of actomyosin and many aspects of platelet behavior (aggregation, clot retraction, pseudopod formation and platelet release reaction) have been attributed to it (21).
INTERACTION OF PLATELETS WITH ADENOSINE DIPHOSPHATE (ADP) The addition of ADP to a suspension of washed human platelets in physiological saline produces neither aggregation nor a decrease in the electrophoretic mobility of the platelets (11). In order to study platelet-ADP interaction it is necessary to use a more physiological milieu and investigate the interaction between the platelet, suspending medium and agents added to this medium. It should be noted that for electrophoretic analysis the suspending medium must exhibit Newtonian behavior since in order to obtain meaningful electrophoretic measurements these should be independent of the electrophoretic velocity of the particle or applied electrical field strength. For example Seaman et al. (22) have examined the rate of migration of red blood cells over a range of electrical field strengths and showed that human serum was a Newtonian fluid over the shear rates tested. When ADP in low concentrations (0.2 to 1.0 uM) is added to platelet rich titrated plasma the platelets undergo an isovolumic transformation from discs to spiculated spheres and aggregate. The aggregation induced by low concentrations of ADP is reversible, dependent on the presence of calcium ions and also one or more plasma proteins including fibrinogen (23). Aggregation is inhibited by EDTA, adenosine, prostaglandin El, cyclic AMP and a variety of other substances (24). With high concentrations of ADP the release reaction occurs with liberation of endogenous ADP which induces irreversible second phase platelet aggregation (25,26). The release of intra-platelet adenosine diphosphate has been proposed as a common factor involved in the aggregation of platelets produced by a diverse range of agents. The mechanism by which ADP aggregates platelets has not been clarified (24). The addition of ADP
Suppl.
PLATELET
II
SURFACE
AND
239
AGGREGATION
to concentrations of 1 to 2 UM in platelet rich titrated plasma will produce a temporary decrease in the electrophoretic mobility of blood platelets (11) which does not occur in the presence of EDTA or absence of fibrinogen indicating that like aggregation divalent cations and fibrinogen are required for the mobility change. Hampton and Mitchell (27) while confirming the observations of Seaman and Vassar (11) for the higher concentrations of ADP also observed an increase in the electrophoretic mobility of platelets at very low concentrations of ADP ('L0.1 PM). The same workers had previously reported (28) that freshly prepared platelets in titrated plasma had a higher mobility than platelets kept for an hour at room temperature. The mobility was observed to decrease gradually during the course of an hour or so and the change could be accelerated by contact with non-siliconized glass, consequently the mobility change was termed 'the contact effect'. Hampton and Mitchell throughout their studies have used post contact platelets. We have not been able to confirm the increases in the electrophoretic mobility of platelets produced by low concentrations of ADP (see Table III). GrGttum (29) also could not confirm these observations whereas Betts et al. (30) and Larcan et al. (31) have been able to do so.
TABLE III Influence of Various Agents on the Electrophoretic Mobility of Human Blood Platelets, T = 25“C Suspending Medium Serum Citrated Plasma
Agent
0.15 M NaCl
Control mobility pm/set/v/cm
-0.88 5 0.03
Percentage change in mobility 3-6 min after addition
-1.07 t 0.04
-1.17 f 0.04
ADP
2.0 urn
ADP
0.1 urn
-1
AMP
2.0 pm
+3
+AMp2.0 mp
um
0
0
-20
0
The discrepancy between the observations may well be due to differences in experimental procedures. One difficulty with the reported increases in mobility is that they are not much beyond the limits of experimental error of the method. Precise details of the experimental procedure employed by various investigators have not been given in their publications. Such information as concentration and type of anticoagulant used, time of contact of all blood elements with plasma and container surfaces, extent of contamination of preparations with erythrocytes and leukocytes, temperature of storage of platelet rich plasma and platelet poor plasma, platelet concentration (count) in platelet rich plasma, pH of plasma, precise time sequences and durations of procedures , purity of reagents especially the ADP (i.e. how much AMP or ATP present) etc. should be provided. In a recent review Hampton and Mitchell (32) have summarized their work which has important clinical implications. They claim that the technique of platelet electrophoresis can be used diagnostically to identify individual patients with coronary artery disease with a high degree of accuracy. It therefore seems most important
SiUMAN
240
Suppl.
i-r
and worthwhile to resolve the present discrepancies by further carefully controlled experiments by investigators in several different laboratories. An important aspect of such a collaborative effort should be to establish the most appropriate environmental conditions for blood platelets consistent with obtaining electrophoretic data under conditions of electrokinetic stability, i.e., a range of environmental conditions under which platelets exhibit reversible electrophoretic behavior. The following facts should be taken into consideration:
a>Platelets
which have been exposed to cold (+4"C) tend to aggregate. All procedures or storage should be conducted at room temperature (2 20°C).
b) Platelets adhere to bare glass surfaces. Manipulations of platelet suspensions should be carried out only in siliconized glassware or plastic vessels compatible with blood platelets.
c>Platelet
suspensions should not be packed at centrifugal forces > 2500 xg since irreversible platelet aggregation tends to arise in the platelet pellet.
d) Attention should be always given to the metabolic status of platelets. In order to avoid rapid depletion of adenosine triphosphate during washing the wash fluid should contain about 5.5 mM glucose. Potassium ions should also be included in the wash medium as platelet potassium ion levels fall significantly during washing procedures.
e>Both
heparin (7) and EDTA (8) pose problems as anticoagulants for blood in platelet studies. Most studies have been conducted in titrated platelet-rich plasma (10).
PLATELET ELECTROCHEMISTRY IN DISEASE Grattum (14) investigated the electrophoretic mobility of human platelets in titrated plasma in a variety of diseases and found a slight increase only in the case of von Willebrand's disease. Zucker and Levine (33) in studies on the electrophoretic mobility of the platelet in thrombasthenia and von Willebrand's disease concluded that the values were normal. The most interesting potential application of platelet electrophoresis in disease is the measurement of platelet electrophoretic mobility before and after exposure to very low concentrations of ADP and norepinephrine (32). This approach is being used as a test of platelet function by Hampton and Mitchell (32). Low concentrations of ADP or norepinephrine (s 0.1 FM) have been reported to increase the electrophoretic mobility of normal platelets. In myocardial infarction there is an increased sensitivity to both ADP and norepinephrine as indicated by a tenfold decrease in the concentrations of these agents required to induce the maximum electrophoretic mobility increase (34). In patients with chronic ischemic heart disease or peripheral arterial disease there is an increase in sensitivity to ADP but not to norepinephrine. Further studies suggested that the increased sensitivity towards ADP in patients with arterial disease originated from lysolecithin derived from the degradation of lecithin which is present in abnormally high levels in the patient's plasma (35). Now as has been mentioned Grijttum (29) repeated the
Suppl.
II
PLATELET
SURFACE
AND
AGGREGATION
241
experiments described by Hampton and Mitchell (27,28,34) and reported that he could not confirm their results and further concluded that it seemed unlikely that methodological differences could account for the disparity. The increase in platelet mobility at very low concentrations of ADP has been confirmed by Betts et al. (30) who concluded that the mobility increase in platelets induced by ADP is dependent solely on the presence of fibrinogen. Rutty and Vine (36) have reported findings on rabbit platelets which are in accord with those described by Hampton and Mitchell for human platelets. Tomich (37) confirmed the findings of Hampton and Mitchell (38) using a field strength of 3 volts/cm and those of Grijttum (29) at 4 volts/cm. However the fact that the mobility, which is reported for platelets in the absence of aggregating agents, is not independent of applied field strength casts serious doubt on the validity of this study. The field is an interesting one with probably considerable potential for the development of useful diagnostic approaches. In the case of the studies which use low concentrations of ADP and norepinephrine further work is required to resolve some of the discrepancies which have been reported in the recent literature.
PLATELET-POLYMER
INTERACTIONS
Neutral polymers such as dextrans have been used in the prevention and therapy of thromboembolic complications especially following surgery (39). The widespread use of dextrans has led to a number of studies on the effect of dextran, if any, on platelet function. Mustard and Packham (40) in a very recent review report that dextrans in the molecular weight range 40,000 to 150,000 have no effect on -in vitro studies involving platelet adhesivein vivo ness, aggregation induced by ADP or the release reaction, whereas -4 to 6 hours after administration of dextran to human subjects, platelet adhesiveness has been found to be reduced. It is known that dextran is adsorbed to platelets (41) and produces an increase in electrophoretic mobility product (mobility corrected for the viscous effect of the suspending medium) of platelets (42,43). Other neutral polymers including ficoll and polyvinylpyrrolidone have also been reported to increase the electrophoretic mobility of cells (44). Thus the electrophoretic mobility and consequently zeta potential of a variety of cells are increased in the presence of neutral polymers. Several explanations for the increase in zeta potential of cells produced by adsorption of neutral polymers have been advanced including: a) increase in the dielectric constant of the suspending medium, b) interaction of cell surface proteins with the polymer to produce a rearrangement of the interfacial region, c) anion binding to the neutral polysaccharide, and d) an experimental artefact arising from a combination of such factors as the general effects of polymers on electrophoretic mobility measurements, non-Newtonian viscosity effects, and applicability of the electrophoresis equation to suspensions containing neutral polymers. These possibilities have been discussed by Brooks and Seaman (45) who concluded that none of these explanations for the zeta potential increase were satisfactory. A model was proposed by Brooks (46) to account for the observed increase in the zeta potential of blood cells in neutral polymer solutions. In the model the presence of adsorbed neutral polymer is predicted to increase the effective thickness of the electrical double layer as a result of excluded volume effects (counterions are not able to occupy the same volume as that occupied by dextran molecules). Thus under conditions of
constant surface charge density the surface potential will increase. Brooks (46) derived an expression for the relative zeta potential in the presence of polymer which included an adsorption term, the thickness of the polymer layer, the location of the electrophoretic plane of shear and the ionic strength of the suspending medium. He concluded that provided the plane of shear was not shifted appreciably from the cell surface the zeta potential would increase in the presence of adsorbed neutral polymer. Addition of dextrans above a certain molecular weight to suspensions of platelets in saline will produce aggregation, probably by a mechanism of polymer bridging. Since bridging by the higher molecular weight dextrans can occur at separations of more than a hundred angstroms it is not surprising that an increase in both zeta potential and aggregation is observed (47). Grattum (48) has studied the effect of the polybases, DEAE-dextran and polybrene on platelet surface charge. He found that when the mobility of the platelets had been reduced to approximately 85% of the control value aggregation ensued. These results and others which he obtained suggest that a decrease in the platelet zeta potential is essential in platelet aggregation. Zbinden et al. (49) examined the electrophoretic behavior of platelets in solutions containing poly-L-lysine, poly-L-n-ornithine and protamine sulfate. These positively charged macromolecules reduced the net negative charge of platelets, p reduced aggregation and inhibited the uptake of 5-hydroxytryptamine. Recently Massini et al. (50) reported some details on the induction of aggregation and the.release reaction in human platelets produced by polylysine. It is evident that much further work needs to be done on the interaction of polymers with blood platelets especially in regard to their effects on platelet morphology, size distribution, electrokinetic characteristics, adhesion and aggregation, nucleotide and catecholamine release and other surface changes such as platelet factor-3 availability. Techniques for the separation of cells frequently use polymers to establish density gradients and it is therefore important to establish that populations isolated by such procedures yield platelets which lack artefactual properties. Recently platelet populations have been separated by density-gradient electrophoresis. The properties of the isolated platelets are consistent with electrophoretic separation reflecting differences in platelet age, the most-rapidly migrating platelets being the youngest (51). These findings suggest the feasibility of separating platelet subpopulations by the technique of partitioning or counter current distribution in two phase aqueous polymer systems. This exquistely sensitive separation procedure has been used very successfully for the separation of other cells, particles and macromolecules (52).
ACKNOWLEDGEMENTS The studies described were supported in part by grants from the U.S. Public Health Service, HL 12787 and the Oregon Heart Association. REFERENCES 1.
MARCUS, A.J.,and ZUCRER, M.B. The Physiology of Blood Platelets: Recent Biochemical, Morphologic and Clinical Research. New York:Grune and Stratton, Inc., 1965.
Suppl.
7-1
PLATELET
SURFACE
AND
AGGREGATION
243
2.
J'$RGENSEN,L., and BORCHGREVINK, C.F. The platelet plug in normal persons. 1. The histological appearance of the plug 15, 220 minutes and 24 hours after the bleeding and its role in capillary haemostasis. Acta Path. Microbial. Stand. 57, 40, 1963.
3.
MUSTARD, J.F.
4.
MUSTARD, J.F., MOVAT, H.Z., MACMORINE, D.R.I., and SENYI, A. Release of permeability factors from the blood platelet. Proc. Sot. Exp. Biol. Med. 119, 988, 1965.
5.
MUSTARD, J.F., EVANS, G., PACKHAM, M.A., and NISHIZAWA, E.E. The platelet in intravascular immunological reactions. In: Cellular and Humoral Mechanisms in Anaphylaxis and Allergy. H.Z. Movat (Ed.). New York, S. Karger, 1969, p. 151.
6.
MUSTARD, J.F., MURPHY, E.A., ROWSELL, H.C.,and DOWNIE, H.G. and atherosclerosis. J. Atheroscler. Res. 4, 1, 1964.
7.
ZUCKER, M.B. Effect of heparin on platelet function. Haemorrh. 33, 63, 1974.
8.
BAENZIGER, N.L.,and MAJERLJS,P.W. Isolation of human platelets and platelet surface membranes. Methods in Enzymol. 2, 149, 1974.
9.
HAN, P., and ARDLIE, N.G. The influence of pH, temperature, and calcium on platelet aggregation: Maintenance of environmental vH and nlatelet function for in vztro studies in plasma stored at 37°C: Brit: J. Haematol. %,373,74.
Hemostasis and thrombosis.
Seminars Hematol. 2, 91, 1968.
Platelets
Thrombos. Diathes.
10.
MUSTARD, J.F., PERRY, D.W., ARDLIE, N.G., and PACKHAM, M.A. Preparation of suspensions of washed platelets from humans. Brit. J. Haematol. 22, 193, 1972.
11.
SEAMAN, G.V.F., and VASSAR, P.S. Changes in the electrokinetic properties of platelets during their aggregation. Arch. Biochem. Biophys. IX, 10, 1966.
12.
BORN, G.V.R. The platelet membrane and its functions. In: Metabolism and Membrane Permeability of Erythrocytes and Thrombocytes. E. Deutsch, E. Gerlach and K. Moser (Eds.). Stuttgart, Georg Thieme Verlay, 1969, p. 294.
13.
SEAMAN, G.V.F. Electrokinetic behavior of red cells. In: The Red Blood Cell Vol. II. D. MacN. Surgenor (Ed.). New York, Academic Press, 1975, p. 1135.
14.
GR@TTUM, K.A. Electrophoresis of blood platelets and erythrocytes. A study in healthy persons and patients with haematological diseases, prosthetic heart valves, virus infections and hyperlipaemia. Stand. J. Haemat. ll, 166, 1973.
15.
MEHRISHI, J.N. Molecular aspects of the mammalian cell surface. Biophys. Mol. Biol. 25, 3, 1972.
Prog.
16.
MEHRISHI, J.N. Positively charged amino groups on the surface of normaI and cancer cells. Europ. 3. Cancer. 6_, 127, 1970
17.
MEHRISHI, J.N. Phosphate groups (Receptors?) on the surface of human blood platelets. Nature. 226, 452, 1970.
18.
MEHRISHI, J.N., and GRASSETTI, D.R. Sulphydryl groups on the surface of intact Ehrlich ascites tumour cells, human blood platelet and lympho563, 1969. cytes. Nature. 3,
19.
SEAMAN, G.V.F. The surface chemistry of the erythrocyte and thrombocyte membrane. J. Supramol. Struct. Suppl. 437, 1973.
20.
PHILLIPS, D.R., and AGIN, P.P. Thrombin-induced alterations in the surface structure of the human platelet plasma membrane. Ser. Haematol. 5, 292, 1973.
21.
LtiSCHER,E.F., and BETTEX-GALLAND, M. Thrombosthenin, the contractile protein of blood platelets: new facts and new problems. Pathol. Biol. 20, suppl. 89, 1972.
22.
SEAMAN, G.V.F., KOK, D'A., and HEARD, D.H. The electrophoretic mobility of human red cells resuspended in their native serum. Clin. Sci. 23, 115, 1962.
23.
MARCUS, A.J.
Platelet function.
New Eng. J. Med. 280, 1213, 1278,
1330, 1969. 24.
WEISS, H.J.
25.
MUSTARD, J.F., and PACKHAM, M.A. Factors influencing platelet function: adhesion, release and aggregation. Pharmacol. Rev. 22, 97, 1970.
26.
HOLMSEN, H. The platelet: its membrane, physiology and biochemistry. Clin. Haematol. 1, 235, 1972.
27.
HAMPTON, J.R., and MITCHELL, J.R.A. Modification of the electrokinetic Nature. 210, 1000, response of blood platelets to aggregating agents. 1966.
28.
HAMPTON, J.R., and MITCHELL, J.R.A. Effect of glass contact on the electrophoretic mobility of human blood platelets. Nature. 209, 470, 1966.
29.
GRijTTUM,K.A. Influence of aggregating agents on electrophoretic mobility of blood platelets from healthy individuals and from patients with cardiovascular diseases. Lancet I, 1406, 1968.
30.
BETTS, J.J., BETTS, J.P., and NICHOLSON, J.T. Significance of ADP, plasma and platelet concentration in platelet electrophoretic studies. Nature. 2, 1280, 1968.
31.
LARCAN, A.J., STOLTZ, J.F., and STOLTZ, M. Modification du potentiell zeta des plaquettes humaines sous l'influence de diverses substances (ADP, A'I'P, Noradrenaline); consid&ations thgoretiques et expgrimentales concernant la stabilit.6de la suspension sanguine. Biorheolopy 5, 227, 1968.
Platelet physiology and abnormalities of platelet function. New Eng. J. Med. 293, 531, 1975.
Suppl.
II
PLATELET
SURFACE
AND
AGGREGATION
245
32.
HAMPTON, J.R., and MITCHELL, J.R.A. Platelet electrophoresis: The present position. Thromb. Diathes. Haemorrh. 3l, 204, 1974.
33.
ZUCKER, M.B., and LEVINE, R.U. Microelectrophoresis of washed and unwashed human blood platelets, with additional studies of platelets from patients with von Willebrand's disease and thrombasthenia. Thromb. Diathes. Haemorrh. I-O, 1, 1963.
34.
HAMPTON, J.R., and MITCHELL, J.R.A. Abnormalities of platelet behaviour in acute illness. Brit. Med. J. 1, 1078, 1966.
35.
BOLTON, C.H., HAMPTON, J.R., and MITCHELL, J.R.A. Nature of the transferable factor which causes abnormal behavior in vascular disease. Lancet. 2, 1101, 1967.
36.
RUTTY, D.A., and VINE, T.L. Lancet. I, 206, 1969.
37.
TOMICH, E.G. Effects of potential gradient on the electrophoretic mobility of human blood platelets in the presence of ADP or adrenaline. Biochem. Pharmacol. 2l, 3201, 1972.
38.
HAMPTON, J.R., and MITCHELL, J.R.A. Effect of aggregating agents on the electrophoretic mobility of human platelets. Brit. Med. J. 1, 1074, 1966.
39.
BYGDEMAN, S. Prevention and therapy of thromboembolic complications with dextran. Prog. Surg. 1, 114, 1969.
40.
MUSTARD, J.F., and PACKHAM, M.A. Drugs. 2, 19, 1975.
41.
ROTHMAN, S., ADELSON, E., SCHWEBEL, A., and LANGDELL, R.D. Adsorption of carbon 14 dextran to human blood platelets and human blood cells in vitro. VOX Sang. 1, 104, 1950.
42.
ROSS, S.W., and EBERT, R.V. Microelectrophoresis of blood platelets and the effcts of dextran. J. Clin. Invest. 38, 155, 1959.
43.
SEAMAN, G.V.F., HISSEN, W., LINO, L., and SWANK,,R.L. Physicochemical changes in blood arising from dextran infusions. Clin. Sci. 2, 293, 1965.
44.
POLLACK, W., HAGER, H.J., RECKEL, R., TOREN, D.A., and SINGHER, H.O. A study of the forces involved in the second stage of hemagglutination. Transfusion. 5, 158, 1965.
45.
BROOKS, D.E., and SEAMAN, G.V.F. The effect of neutral polymers on the electrokinetic potential of cells and other charged particles. I. Models for the zeta potential increase, J. Coil. Interface Sci. 43, 670, 1973.
46.
BROOKS, D.E. The effect of neutral polymers on the electrokinetic potential of cells and other charged particles. II. A model for the effect of adsorbed polymer on the diffuse double layer. J. Coil. Interface Sci. 43, 687, 1973.
Electrokinetic mobility of blood-platelets.
Platelets, thrombosis and drugs.
47.
SEAMAN, G.V.F., and BROOKS, D.E. Electrochemical aspects of platelet aggregation and adhesion. Thromb. Diathes. Haemorrh. Suppl. 42, 93, 1970.
48.
GROTTUM, K.A. Platelet surface charge and aggregation effects of polyelectrolytes. Thromb. Diathes. Haemorrh. 21, 450, 1969.
49.
ZBINDEN, G., MEHRISHI, J.N., and TOMLIN, S. Assessment of damage to human platelets after aggregation and other injuries by microscopic observation and estimation of serotonin uptake. Thromb. Diathes. Haemorrh. 23, 261, 1970
50.
MASSINI, P., METCALF, L.C., and LDSCHER, E.F. Induction of aggregation and of the release reaction in human platelets by polylysine. Haemostasis. 2, 8, 1974.
51.
CARTY, C., CRAWFORD, E.A., and GEAR, A.R.L. Separation of platelet populations by electrophoresis. Fed. Proc. 34, 289, 1975.
52.
ALBERTSSON, P.i. Partition of Cell Particles and Macromolecules. New York, Wiley (Interscience), 1971.
Suppl. II, Vol. 8, Pergamon Press,
States
SECTION
1976 Inc.
V
ELECTROCHEMICAL FEATURES OF PLATELET INTERACTIONS
Geoffrey V.F. Seaman Department of Neurology University of Oregon Health Sciences Center Portland, Oregon 97201, U.S.A.
ABSTRACT Platelets participate in hemostasis and thrombus formation. The physicochemical basis at the molecular level for platelet adhesion, cohesion to form aggregates and the resultant viscous metamorphosis is not well understood. Under physiological conditions, blood platelets possess a net negative charge and decreases or changes in this charge have been implicated in both their adhesion and aggregation. The peripheral zone of the platelet contains glycoproteins and glycolipids. A variety of charged groups are present including positively charged amino groups and at least three types of negatively charged groups. The carboxyl group of terminal sialic acid residues is considered to be a major contributor to the negative charge of platelets. Removal of as little as 10 percent of the peripheral zone sialic acid by neuraminidase results in a shortened platelet lifespan _in vivo. The electrokinetic properties of blood platelets are changed by interaction with a great variety of agents. In those cases where specific adsorption occurs it is possible to study the relationship between adsorption, change in surface charge, and the process of aggregation. It should be noted that in physiological media only those charge groups within about 8 A of the electrophoretic surface of the platelet will contribute to a significant extent to the electrophoretic properties. The electrophoretic mobility of platelets has been determined for a variety of animal species and in humans for a wide spectrum of disease situations. The measurement of platelet electrophoretic mobilities before and after treatment with adenosine diphosphate and norepinephrine has been introduced as a test of platelet function. However, so many variables may influence the surface properties of blood platelets including method of preparation, time, temperature, pH, contact phenomena, shear conditions, ionic strength and composition of suspending media, conditions for mobility measurements, etc., that considerable apparent disagreement exists in the literature regarding platelet electrokinetics. 235
S EMIAN
236
INTRODUCTION Blood platelets respond readily to various stimuli by adhesion to other surfaces and by the formation of platelet aggregates (1) which are a basic component of hemostatic plugs (2) and of thrombi (3). When activated platelets release proteases and factors which increase vascular permeability (4). In addition there is evidence that endothelial damage may arise as a result of platelet action (5) which may contribute subsequently to the development of atheroma (6). A great variety of agents can influence platelet adhesion or induce platelet aggregation both -in vitro and -in vivo but the physiocochemical bases for such phenomena are not well understood. It is not surprising therefore that measurements of platelet aggregation or adhesion have not been very successful in the study of individual subjects with, or at risk from diseases involving the blood vessels. The ease with which platelets can be isolated relatively intact from blood has been both a strength and also a danger in platelet research. Properties of platelets closely related to their function in hemostasis make them more difficult to isolate and study than is apparent to the unwary investigator. The difficulties include the choice of anticoagulant (7), selection of temperature for procedures or studies (8), nature of surfaces of vessels in which experiments are conducted (8), control of other environmental factors such as pH and calcium ion concentration (9), and the conditions and composition of suspending media used in the preparation of washed platelets (10). Blood platelets under physiological conditions carry a net negative charge and decreases in this charge have been implicated in their aggregation (11). Since blood platelets because of their like charge sign would be expected to be mutually repellant it has been proposed in thrombogenesis where platelets stick to the wall of a blood vessel and attract other platelets that the platelet surface charge must be altered (12).
TABLE I Methods of Electrophoretic Analysis of Biological Cells Experimental Variable
Information Obtained
pH of suspending medium
pK values of ionogenic groups
Ionic strength of suspending medium
Distribution of ionogenic groups in the peripheral zone and role of ion redistribution
Suspending medium ion composition
Charge reversal spectra and distribution of ionogenic groups
Modification with enzymes or specific functional group reagents
Nature of surface functional groups
Treatment with specific immunological reagents or molecules with known distribution of charges
Location and distribution of surface functional groups
Suppl.
II
PLATELET
SURFACE
AND
AGGREGATION
237
ELECTROPHORETIC TECHNIQUES In order to approach the problem of the nature of electrochemical interactions between platelets or between platelets and foreign bodies or surfaces information is needed on the peripheral zone of the platelet obtained from studies in fairly simple media under conditions where no significant irreversible changes in platelet behavior have occurred, The methods of electrophoretic analysis which may be used in the elucidation of surface structure are given in Table I. The equipment and experimental procedures for the electrophoretic analysis of biological cells as well as the biological variables and physicochemical parameters which may influence the electrophoretic mobility of cells have been described by Seaman (13). Electrophoretic analysis of blood platelets has shown that in general the electrophoretic mobility of human blood platelets shows only small variations in health and is rarely abnormal in disease when examined in simple ionic media or titrated plasma (14). The pH versus mobility relationships for human blood platelets are amphoteric in nature with evidence for the presence of several types of charge groups (11,15). The ionogenic composition of the human platelet has been obtained from the following experimental evidence: (a) the marked decrease in the electrophoretic mobility (s 50%) which results from treatment with neuraminidase accompanied by the release of N-acetylneuraminic acid (11) indicating the presence of a membrane-bound sialic acid. (b) a group of pK > 9 revealed by the pH versus mobility behavior suggesting the presence of amino groups which is confirmed by (c). (c) the increase in anodic mobility ("J20%) on treatment with acetaldehyde (ll), citraconic anhydride, and 2,3-dimethyl maleic anhydride (16). (d) the decrease (Q 20%) in mobility produced by treatment with alkaline phosphatase thus implying the presence of phosphate groups (17). (e) the increase in electrophoretic mobility (Q 20%) produced by modification with 6,6'-dithiodinicotinic acid thus indicating the presence of sulfhydryl groups in the peripheral zone of the platelet (18). The remainder of the unidentified negative groups on the basis of the pH versus mobility relationship have a pK of about 4 and are most probably the free carboxyl groups of aspartyl or glutamyl residues. The ionogenic constitution of the human platelet is given in Table II. The following assumptions were made in the calculation of the parameters listed: the Gouy-Chapman equation gives a reliable estimate of electrokinetic charge density for the platelet. This assumption is valid only if no significant fraction of the peripheral zone inside the electrophoretic slip plane is available to counterions ( .g> the charge is distributed uniformly at the electrophoretic surface. the mean surface area of the platelet is 28.3 pm2.
238
TABLE II Ionogenic Constitution of the Human Blood Platelet
Groups
PK
Sialic acid carboxyl Other carboxyl Phosphate Unidentified negative Amino Sulfhydryl Total negative Total positive Total charged groups
2.6 s4.0 ? ? >9.0
Groups/platelet x 10-5
Mean separatitn of groups - A
9.4 ? 6.5 7.0 5.3 2.9 22.9 5.3 28.2
55 ? 66 64 74 99 35 73 32
The ionogenic groups of the platelet surface are part of terminal moieties which in turn are linked to surface macromolecular structures. Platelet-specific proteins appear to be arranged asymmetrically within the membrane. Lactoperoxidase-catalyzed iodination of platelet surface macromolecules have shown three major glycoproteins to be located in the peripheral zone whereas most other membrane proteins are not (20). Fifteen to twenty percent of the total platelet protein consists of actomyosin and many aspects of platelet behavior (aggregation, clot retraction, pseudopod formation and platelet release reaction) have been attributed to it (21).
INTERACTION OF PLATELETS WITH ADENOSINE DIPHOSPHATE (ADP) The addition of ADP to a suspension of washed human platelets in physiological saline produces neither aggregation nor a decrease in the electrophoretic mobility of the platelets (11). In order to study platelet-ADP interaction it is necessary to use a more physiological milieu and investigate the interaction between the platelet, suspending medium and agents added to this medium. It should be noted that for electrophoretic analysis the suspending medium must exhibit Newtonian behavior since in order to obtain meaningful electrophoretic measurements these should be independent of the electrophoretic velocity of the particle or applied electrical field strength. For example Seaman et al. (22) have examined the rate of migration of red blood cells over a range of electrical field strengths and showed that human serum was a Newtonian fluid over the shear rates tested. When ADP in low concentrations (0.2 to 1.0 uM) is added to platelet rich titrated plasma the platelets undergo an isovolumic transformation from discs to spiculated spheres and aggregate. The aggregation induced by low concentrations of ADP is reversible, dependent on the presence of calcium ions and also one or more plasma proteins including fibrinogen (23). Aggregation is inhibited by EDTA, adenosine, prostaglandin El, cyclic AMP and a variety of other substances (24). With high concentrations of ADP the release reaction occurs with liberation of endogenous ADP which induces irreversible second phase platelet aggregation (25,26). The release of intra-platelet adenosine diphosphate has been proposed as a common factor involved in the aggregation of platelets produced by a diverse range of agents. The mechanism by which ADP aggregates platelets has not been clarified (24). The addition of ADP
Suppl.
PLATELET
II
SURFACE
AND
239
AGGREGATION
to concentrations of 1 to 2 UM in platelet rich titrated plasma will produce a temporary decrease in the electrophoretic mobility of blood platelets (11) which does not occur in the presence of EDTA or absence of fibrinogen indicating that like aggregation divalent cations and fibrinogen are required for the mobility change. Hampton and Mitchell (27) while confirming the observations of Seaman and Vassar (11) for the higher concentrations of ADP also observed an increase in the electrophoretic mobility of platelets at very low concentrations of ADP ('L0.1 PM). The same workers had previously reported (28) that freshly prepared platelets in titrated plasma had a higher mobility than platelets kept for an hour at room temperature. The mobility was observed to decrease gradually during the course of an hour or so and the change could be accelerated by contact with non-siliconized glass, consequently the mobility change was termed 'the contact effect'. Hampton and Mitchell throughout their studies have used post contact platelets. We have not been able to confirm the increases in the electrophoretic mobility of platelets produced by low concentrations of ADP (see Table III). GrGttum (29) also could not confirm these observations whereas Betts et al. (30) and Larcan et al. (31) have been able to do so.
TABLE III Influence of Various Agents on the Electrophoretic Mobility of Human Blood Platelets, T = 25“C Suspending Medium Serum Citrated Plasma
Agent
0.15 M NaCl
Control mobility pm/set/v/cm
-0.88 5 0.03
Percentage change in mobility 3-6 min after addition
-1.07 t 0.04
-1.17 f 0.04
ADP
2.0 urn
ADP
0.1 urn
-1
AMP
2.0 pm
+3
+AMp2.0 mp
um
0
0
-20
0
The discrepancy between the observations may well be due to differences in experimental procedures. One difficulty with the reported increases in mobility is that they are not much beyond the limits of experimental error of the method. Precise details of the experimental procedure employed by various investigators have not been given in their publications. Such information as concentration and type of anticoagulant used, time of contact of all blood elements with plasma and container surfaces, extent of contamination of preparations with erythrocytes and leukocytes, temperature of storage of platelet rich plasma and platelet poor plasma, platelet concentration (count) in platelet rich plasma, pH of plasma, precise time sequences and durations of procedures , purity of reagents especially the ADP (i.e. how much AMP or ATP present) etc. should be provided. In a recent review Hampton and Mitchell (32) have summarized their work which has important clinical implications. They claim that the technique of platelet electrophoresis can be used diagnostically to identify individual patients with coronary artery disease with a high degree of accuracy. It therefore seems most important
SiUMAN
240
Suppl.
i-r
and worthwhile to resolve the present discrepancies by further carefully controlled experiments by investigators in several different laboratories. An important aspect of such a collaborative effort should be to establish the most appropriate environmental conditions for blood platelets consistent with obtaining electrophoretic data under conditions of electrokinetic stability, i.e., a range of environmental conditions under which platelets exhibit reversible electrophoretic behavior. The following facts should be taken into consideration:
a>Platelets
which have been exposed to cold (+4"C) tend to aggregate. All procedures or storage should be conducted at room temperature (2 20°C).
b) Platelets adhere to bare glass surfaces. Manipulations of platelet suspensions should be carried out only in siliconized glassware or plastic vessels compatible with blood platelets.
c>Platelet
suspensions should not be packed at centrifugal forces > 2500 xg since irreversible platelet aggregation tends to arise in the platelet pellet.
d) Attention should be always given to the metabolic status of platelets. In order to avoid rapid depletion of adenosine triphosphate during washing the wash fluid should contain about 5.5 mM glucose. Potassium ions should also be included in the wash medium as platelet potassium ion levels fall significantly during washing procedures.
e>Both
heparin (7) and EDTA (8) pose problems as anticoagulants for blood in platelet studies. Most studies have been conducted in titrated platelet-rich plasma (10).
PLATELET ELECTROCHEMISTRY IN DISEASE Grattum (14) investigated the electrophoretic mobility of human platelets in titrated plasma in a variety of diseases and found a slight increase only in the case of von Willebrand's disease. Zucker and Levine (33) in studies on the electrophoretic mobility of the platelet in thrombasthenia and von Willebrand's disease concluded that the values were normal. The most interesting potential application of platelet electrophoresis in disease is the measurement of platelet electrophoretic mobility before and after exposure to very low concentrations of ADP and norepinephrine (32). This approach is being used as a test of platelet function by Hampton and Mitchell (32). Low concentrations of ADP or norepinephrine (s 0.1 FM) have been reported to increase the electrophoretic mobility of normal platelets. In myocardial infarction there is an increased sensitivity to both ADP and norepinephrine as indicated by a tenfold decrease in the concentrations of these agents required to induce the maximum electrophoretic mobility increase (34). In patients with chronic ischemic heart disease or peripheral arterial disease there is an increase in sensitivity to ADP but not to norepinephrine. Further studies suggested that the increased sensitivity towards ADP in patients with arterial disease originated from lysolecithin derived from the degradation of lecithin which is present in abnormally high levels in the patient's plasma (35). Now as has been mentioned Grijttum (29) repeated the
Suppl.
II
PLATELET
SURFACE
AND
AGGREGATION
241
experiments described by Hampton and Mitchell (27,28,34) and reported that he could not confirm their results and further concluded that it seemed unlikely that methodological differences could account for the disparity. The increase in platelet mobility at very low concentrations of ADP has been confirmed by Betts et al. (30) who concluded that the mobility increase in platelets induced by ADP is dependent solely on the presence of fibrinogen. Rutty and Vine (36) have reported findings on rabbit platelets which are in accord with those described by Hampton and Mitchell for human platelets. Tomich (37) confirmed the findings of Hampton and Mitchell (38) using a field strength of 3 volts/cm and those of Grijttum (29) at 4 volts/cm. However the fact that the mobility, which is reported for platelets in the absence of aggregating agents, is not independent of applied field strength casts serious doubt on the validity of this study. The field is an interesting one with probably considerable potential for the development of useful diagnostic approaches. In the case of the studies which use low concentrations of ADP and norepinephrine further work is required to resolve some of the discrepancies which have been reported in the recent literature.
PLATELET-POLYMER
INTERACTIONS
Neutral polymers such as dextrans have been used in the prevention and therapy of thromboembolic complications especially following surgery (39). The widespread use of dextrans has led to a number of studies on the effect of dextran, if any, on platelet function. Mustard and Packham (40) in a very recent review report that dextrans in the molecular weight range 40,000 to 150,000 have no effect on -in vitro studies involving platelet adhesivein vivo ness, aggregation induced by ADP or the release reaction, whereas -4 to 6 hours after administration of dextran to human subjects, platelet adhesiveness has been found to be reduced. It is known that dextran is adsorbed to platelets (41) and produces an increase in electrophoretic mobility product (mobility corrected for the viscous effect of the suspending medium) of platelets (42,43). Other neutral polymers including ficoll and polyvinylpyrrolidone have also been reported to increase the electrophoretic mobility of cells (44). Thus the electrophoretic mobility and consequently zeta potential of a variety of cells are increased in the presence of neutral polymers. Several explanations for the increase in zeta potential of cells produced by adsorption of neutral polymers have been advanced including: a) increase in the dielectric constant of the suspending medium, b) interaction of cell surface proteins with the polymer to produce a rearrangement of the interfacial region, c) anion binding to the neutral polysaccharide, and d) an experimental artefact arising from a combination of such factors as the general effects of polymers on electrophoretic mobility measurements, non-Newtonian viscosity effects, and applicability of the electrophoresis equation to suspensions containing neutral polymers. These possibilities have been discussed by Brooks and Seaman (45) who concluded that none of these explanations for the zeta potential increase were satisfactory. A model was proposed by Brooks (46) to account for the observed increase in the zeta potential of blood cells in neutral polymer solutions. In the model the presence of adsorbed neutral polymer is predicted to increase the effective thickness of the electrical double layer as a result of excluded volume effects (counterions are not able to occupy the same volume as that occupied by dextran molecules). Thus under conditions of
constant surface charge density the surface potential will increase. Brooks (46) derived an expression for the relative zeta potential in the presence of polymer which included an adsorption term, the thickness of the polymer layer, the location of the electrophoretic plane of shear and the ionic strength of the suspending medium. He concluded that provided the plane of shear was not shifted appreciably from the cell surface the zeta potential would increase in the presence of adsorbed neutral polymer. Addition of dextrans above a certain molecular weight to suspensions of platelets in saline will produce aggregation, probably by a mechanism of polymer bridging. Since bridging by the higher molecular weight dextrans can occur at separations of more than a hundred angstroms it is not surprising that an increase in both zeta potential and aggregation is observed (47). Grattum (48) has studied the effect of the polybases, DEAE-dextran and polybrene on platelet surface charge. He found that when the mobility of the platelets had been reduced to approximately 85% of the control value aggregation ensued. These results and others which he obtained suggest that a decrease in the platelet zeta potential is essential in platelet aggregation. Zbinden et al. (49) examined the electrophoretic behavior of platelets in solutions containing poly-L-lysine, poly-L-n-ornithine and protamine sulfate. These positively charged macromolecules reduced the net negative charge of platelets, p reduced aggregation and inhibited the uptake of 5-hydroxytryptamine. Recently Massini et al. (50) reported some details on the induction of aggregation and the.release reaction in human platelets produced by polylysine. It is evident that much further work needs to be done on the interaction of polymers with blood platelets especially in regard to their effects on platelet morphology, size distribution, electrokinetic characteristics, adhesion and aggregation, nucleotide and catecholamine release and other surface changes such as platelet factor-3 availability. Techniques for the separation of cells frequently use polymers to establish density gradients and it is therefore important to establish that populations isolated by such procedures yield platelets which lack artefactual properties. Recently platelet populations have been separated by density-gradient electrophoresis. The properties of the isolated platelets are consistent with electrophoretic separation reflecting differences in platelet age, the most-rapidly migrating platelets being the youngest (51). These findings suggest the feasibility of separating platelet subpopulations by the technique of partitioning or counter current distribution in two phase aqueous polymer systems. This exquistely sensitive separation procedure has been used very successfully for the separation of other cells, particles and macromolecules (52).
ACKNOWLEDGEMENTS The studies described were supported in part by grants from the U.S. Public Health Service, HL 12787 and the Oregon Heart Association. REFERENCES 1.
MARCUS, A.J.,and ZUCRER, M.B. The Physiology of Blood Platelets: Recent Biochemical, Morphologic and Clinical Research. New York:Grune and Stratton, Inc., 1965.
Suppl.
7-1
PLATELET
SURFACE
AND
AGGREGATION
243
2.
J'$RGENSEN,L., and BORCHGREVINK, C.F. The platelet plug in normal persons. 1. The histological appearance of the plug 15, 220 minutes and 24 hours after the bleeding and its role in capillary haemostasis. Acta Path. Microbial. Stand. 57, 40, 1963.
3.
MUSTARD, J.F.
4.
MUSTARD, J.F., MOVAT, H.Z., MACMORINE, D.R.I., and SENYI, A. Release of permeability factors from the blood platelet. Proc. Sot. Exp. Biol. Med. 119, 988, 1965.
5.
MUSTARD, J.F., EVANS, G., PACKHAM, M.A., and NISHIZAWA, E.E. The platelet in intravascular immunological reactions. In: Cellular and Humoral Mechanisms in Anaphylaxis and Allergy. H.Z. Movat (Ed.). New York, S. Karger, 1969, p. 151.
6.
MUSTARD, J.F., MURPHY, E.A., ROWSELL, H.C.,and DOWNIE, H.G. and atherosclerosis. J. Atheroscler. Res. 4, 1, 1964.
7.
ZUCKER, M.B. Effect of heparin on platelet function. Haemorrh. 33, 63, 1974.
8.
BAENZIGER, N.L.,and MAJERLJS,P.W. Isolation of human platelets and platelet surface membranes. Methods in Enzymol. 2, 149, 1974.
9.
HAN, P., and ARDLIE, N.G. The influence of pH, temperature, and calcium on platelet aggregation: Maintenance of environmental vH and nlatelet function for in vztro studies in plasma stored at 37°C: Brit: J. Haematol. %,373,74.
Hemostasis and thrombosis.
Seminars Hematol. 2, 91, 1968.
Platelets
Thrombos. Diathes.
10.
MUSTARD, J.F., PERRY, D.W., ARDLIE, N.G., and PACKHAM, M.A. Preparation of suspensions of washed platelets from humans. Brit. J. Haematol. 22, 193, 1972.
11.
SEAMAN, G.V.F., and VASSAR, P.S. Changes in the electrokinetic properties of platelets during their aggregation. Arch. Biochem. Biophys. IX, 10, 1966.
12.
BORN, G.V.R. The platelet membrane and its functions. In: Metabolism and Membrane Permeability of Erythrocytes and Thrombocytes. E. Deutsch, E. Gerlach and K. Moser (Eds.). Stuttgart, Georg Thieme Verlay, 1969, p. 294.
13.
SEAMAN, G.V.F. Electrokinetic behavior of red cells. In: The Red Blood Cell Vol. II. D. MacN. Surgenor (Ed.). New York, Academic Press, 1975, p. 1135.
14.
GR@TTUM, K.A. Electrophoresis of blood platelets and erythrocytes. A study in healthy persons and patients with haematological diseases, prosthetic heart valves, virus infections and hyperlipaemia. Stand. J. Haemat. ll, 166, 1973.
15.
MEHRISHI, J.N. Molecular aspects of the mammalian cell surface. Biophys. Mol. Biol. 25, 3, 1972.
Prog.
16.
MEHRISHI, J.N. Positively charged amino groups on the surface of normaI and cancer cells. Europ. 3. Cancer. 6_, 127, 1970
17.
MEHRISHI, J.N. Phosphate groups (Receptors?) on the surface of human blood platelets. Nature. 226, 452, 1970.
18.
MEHRISHI, J.N., and GRASSETTI, D.R. Sulphydryl groups on the surface of intact Ehrlich ascites tumour cells, human blood platelet and lympho563, 1969. cytes. Nature. 3,
19.
SEAMAN, G.V.F. The surface chemistry of the erythrocyte and thrombocyte membrane. J. Supramol. Struct. Suppl. 437, 1973.
20.
PHILLIPS, D.R., and AGIN, P.P. Thrombin-induced alterations in the surface structure of the human platelet plasma membrane. Ser. Haematol. 5, 292, 1973.
21.
LtiSCHER,E.F., and BETTEX-GALLAND, M. Thrombosthenin, the contractile protein of blood platelets: new facts and new problems. Pathol. Biol. 20, suppl. 89, 1972.
22.
SEAMAN, G.V.F., KOK, D'A., and HEARD, D.H. The electrophoretic mobility of human red cells resuspended in their native serum. Clin. Sci. 23, 115, 1962.
23.
MARCUS, A.J.
Platelet function.
New Eng. J. Med. 280, 1213, 1278,
1330, 1969. 24.
WEISS, H.J.
25.
MUSTARD, J.F., and PACKHAM, M.A. Factors influencing platelet function: adhesion, release and aggregation. Pharmacol. Rev. 22, 97, 1970.
26.
HOLMSEN, H. The platelet: its membrane, physiology and biochemistry. Clin. Haematol. 1, 235, 1972.
27.
HAMPTON, J.R., and MITCHELL, J.R.A. Modification of the electrokinetic Nature. 210, 1000, response of blood platelets to aggregating agents. 1966.
28.
HAMPTON, J.R., and MITCHELL, J.R.A. Effect of glass contact on the electrophoretic mobility of human blood platelets. Nature. 209, 470, 1966.
29.
GRijTTUM,K.A. Influence of aggregating agents on electrophoretic mobility of blood platelets from healthy individuals and from patients with cardiovascular diseases. Lancet I, 1406, 1968.
30.
BETTS, J.J., BETTS, J.P., and NICHOLSON, J.T. Significance of ADP, plasma and platelet concentration in platelet electrophoretic studies. Nature. 2, 1280, 1968.
31.
LARCAN, A.J., STOLTZ, J.F., and STOLTZ, M. Modification du potentiell zeta des plaquettes humaines sous l'influence de diverses substances (ADP, A'I'P, Noradrenaline); consid&ations thgoretiques et expgrimentales concernant la stabilit.6de la suspension sanguine. Biorheolopy 5, 227, 1968.
Platelet physiology and abnormalities of platelet function. New Eng. J. Med. 293, 531, 1975.
Suppl.
II
PLATELET
SURFACE
AND
AGGREGATION
245
32.
HAMPTON, J.R., and MITCHELL, J.R.A. Platelet electrophoresis: The present position. Thromb. Diathes. Haemorrh. 3l, 204, 1974.
33.
ZUCKER, M.B., and LEVINE, R.U. Microelectrophoresis of washed and unwashed human blood platelets, with additional studies of platelets from patients with von Willebrand's disease and thrombasthenia. Thromb. Diathes. Haemorrh. I-O, 1, 1963.
34.
HAMPTON, J.R., and MITCHELL, J.R.A. Abnormalities of platelet behaviour in acute illness. Brit. Med. J. 1, 1078, 1966.
35.
BOLTON, C.H., HAMPTON, J.R., and MITCHELL, J.R.A. Nature of the transferable factor which causes abnormal behavior in vascular disease. Lancet. 2, 1101, 1967.
36.
RUTTY, D.A., and VINE, T.L. Lancet. I, 206, 1969.
37.
TOMICH, E.G. Effects of potential gradient on the electrophoretic mobility of human blood platelets in the presence of ADP or adrenaline. Biochem. Pharmacol. 2l, 3201, 1972.
38.
HAMPTON, J.R., and MITCHELL, J.R.A. Effect of aggregating agents on the electrophoretic mobility of human platelets. Brit. Med. J. 1, 1074, 1966.
39.
BYGDEMAN, S. Prevention and therapy of thromboembolic complications with dextran. Prog. Surg. 1, 114, 1969.
40.
MUSTARD, J.F., and PACKHAM, M.A. Drugs. 2, 19, 1975.
41.
ROTHMAN, S., ADELSON, E., SCHWEBEL, A., and LANGDELL, R.D. Adsorption of carbon 14 dextran to human blood platelets and human blood cells in vitro. VOX Sang. 1, 104, 1950.
42.
ROSS, S.W., and EBERT, R.V. Microelectrophoresis of blood platelets and the effcts of dextran. J. Clin. Invest. 38, 155, 1959.
43.
SEAMAN, G.V.F., HISSEN, W., LINO, L., and SWANK,,R.L. Physicochemical changes in blood arising from dextran infusions. Clin. Sci. 2, 293, 1965.
44.
POLLACK, W., HAGER, H.J., RECKEL, R., TOREN, D.A., and SINGHER, H.O. A study of the forces involved in the second stage of hemagglutination. Transfusion. 5, 158, 1965.
45.
BROOKS, D.E., and SEAMAN, G.V.F. The effect of neutral polymers on the electrokinetic potential of cells and other charged particles. I. Models for the zeta potential increase, J. Coil. Interface Sci. 43, 670, 1973.
46.
BROOKS, D.E. The effect of neutral polymers on the electrokinetic potential of cells and other charged particles. II. A model for the effect of adsorbed polymer on the diffuse double layer. J. Coil. Interface Sci. 43, 687, 1973.
Electrokinetic mobility of blood-platelets.
Platelets, thrombosis and drugs.
47.
SEAMAN, G.V.F., and BROOKS, D.E. Electrochemical aspects of platelet aggregation and adhesion. Thromb. Diathes. Haemorrh. Suppl. 42, 93, 1970.
48.
GROTTUM, K.A. Platelet surface charge and aggregation effects of polyelectrolytes. Thromb. Diathes. Haemorrh. 21, 450, 1969.
49.
ZBINDEN, G., MEHRISHI, J.N., and TOMLIN, S. Assessment of damage to human platelets after aggregation and other injuries by microscopic observation and estimation of serotonin uptake. Thromb. Diathes. Haemorrh. 23, 261, 1970
50.
MASSINI, P., METCALF, L.C., and LDSCHER, E.F. Induction of aggregation and of the release reaction in human platelets by polylysine. Haemostasis. 2, 8, 1974.
51.
CARTY, C., CRAWFORD, E.A., and GEAR, A.R.L. Separation of platelet populations by electrophoresis. Fed. Proc. 34, 289, 1975.
52.
ALBERTSSON, P.i. Partition of Cell Particles and Macromolecules. New York, Wiley (Interscience), 1971.