THROMBOSIS @Pergamon
RESEARCH 14; 697-704 Press Ltd.1979. Printed
in Great
0049-3848/79/
Britain 0415 -0697
$02.00/o
EFFECT OF PLATELET ACTIVATION ON THE PLATELET SURFACE SIALIC ACID Kenneth K. Wu' and Cecilia S.L. Ku* From the Coagulation and Thrombosis Unit of Hematology Section Department of Medicine, Rush Medical College and Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois
(Received 7.9.1978; Revised form 28.12.1978. Accepted by Editor K.M. Brinkhous)
ABSTRACT Alteration in platelet surface neuraminidase-and total releasable sialic acids in response to platelet activation was studied by treating twice-washed intact human platelets with acid soluble collagen, thrombin, adenosine diphosphate and epinephrine. At the resting state, approximately 50% of the total platelet sialic acids were removable by neuraminidase. When platelets were treated with collagen or thrombin at 37" for ten minutes, more than 80% of the total sialic acids became removable by neuraminidase. Treatment with adenosine diphosphate resulted in a mild but significanrt increase in platelet surface releasable sialic acids, while epinephrine treatment did not induce any change in sialic acids. The effect of ADP was completely abolished by 2-chloroadenosine, whereas that of collagen or thrombin was only partially affected. Acetylsalicylic acid did not inhibit the surface sialic acid increase induced by any of the three platelet activating agents. The results indicate that upon activation with ADP, collagen or thrombin, an increased amount of terminal sialic acid residues of the membrane glycoproteins become externally exposed and thus become removable by neuraminidase. We postulate that alteration of neuraminidaseremovable sialic acids is due to redistribution of the membrane glycoproteins in response to platelet activation.
INTRODUCTION There is increasing evidence that platelet surface glycoproteins play 1. To whom requests for reprints should be sent. 2. Currently a graduate student, Department of Biochemistry, Rush University 697
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.important roles in platelet function. Receptor sites for platelet activating agents on platelet membranes are probably glycoproteins (1). Glycoprotein abnormalities have been identified in patients with congenital platelet disorders, i.e., Bernard-Soulier syndrome and Glanzmann's thrombasthenia (2-3). Specific defects in the membrane glycoproteins in these two disorders tend to suggest that each glycoprotein may have a specific role in platelet adhesion and aggregation. Yet, platelet surface glycoprotein changes associated with platelet activation remain poorly understood. Phillips and Agin (4) have recently shown that treatment of platelets with thrombin is associated with a reduction of glycoprotein V, which is considered to be a receptor for thrombin. Motamed et al (5), on the other hand, have indicated that activation of platelets with adenosine diphosphate is associated with exposure of additional glycoproteins on the platelet surface. The purpose of this study is to characterize the alteration of platelet surface neuraminidase-removable sialic acids in response to activating agents such as adenosine diphosphate, collagen, thrombin and epinephrine. The inhibitory effect of Z-chloroadenosine and acetylsalicylic acid on the glycoprotein alteration was also investigated. METHODS Preparation of Washed Platelet Suspension: Twice-washed human platelet suspensions were prepared accordinq to the procedure of Rossi (6). In brief, blood was drawn from‘an antecubitai vein of'healthy volunteers-with a gauge 19 needle into acid-citrate-dextrose anticoagulant, five volumes of blood to one volume of anticoagulant. Blood was centrifuged at 220 xg for 8 minutes, platelet rich plasma was removed and centrifuged at 1000 g for 15 minutes. Supernatant was discarded and platelet pellet was resuspended and washed twice according to the procedure of Rossi (6). Platelet concentration was determined by an electronic device (Coulter ZBI, Coulter Electronics, Hialeah, Florida) and phase-contrast microscopy. Washed platelets were found to be intact (7). Measurement of Surface and Total Releasable Sialic Acid: Washed platelet suspension was divided into two equal aliquots. Aliquot A was treated with a bacterial neuraminidase (Cl. Perfringens, Sigma Chemical Co., St. Louis, MO.), which was further purified according to the method of Hatton and Regoeczi, and the caseinolytic activity of the purified preparation was negligible (8). Aliquot B was treated with sonication and acid hydrolysis (9). As neuraminidase at an optimal concentration determined experimentally for each batch of neuraminidase removed sialic acids from all externally exposed glycoproteins on the platelet surface, sialic acid content of aliquot A represented the surface releasable sialic acid, and that of aliquot B the total releasable sialic acid. The released free sialic acid was then determined by the thiobarbituric acid of Warren (10). Crystalline N-acetylneuraminic acid was used as a reference standard with each assay. Optical density readings in a Beckman spectrophotomer at 532 nm were subtraced from the readings at 549 nm and the sialic acid concentration wa: calculated according o the formula proposed by Warren (10). The result was expressed as n mole/l0 8 platelets. Platelet Aggregating and Inhibiting Agents: Acid soluble collagen from calf skin oreoared bv the method of Galluo and Seifter (11) was obtained from Siqma Chemical'Co. and the preparation was-kept at room temperature for 17 hours prior to the use for investigation. Collagen prepared in this way was capable of inducing platelet release and aggregation. Epinephrine was obtained from Parke, Davis and Co., Detroit, Mich., adenosine diphosphate (ADP) from Sigma Chemical co., thrombin (bovine) from Parke-Davis and Co., acetylsalicylic acid from Mallinkrodt Chemical, St. Louis, MO., and E-chloroadenosine from Sigma Chemical
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Co. To study the effect of platelet aggregating aqents, washed platelet suspension was incubated with an aggregating agent at 37°C for ten minutes. in the control experiment, the aggregating agent was replaced with an equal volume of saline. An attempt was made to wash the platelets after treatment with the aggregating agent. However, resuspension of platelets was often impossible because of the formation of large irreversible platelet aggregates. In certain experiments, platelets were successfully resuspended. Analysis of five such experiments showed that washing did not reduce the surface or total sialic acids. In order to obtain uniform results, all the subsequent experiments were performed without washing. To determine the inhibitory effect of acetylsalicylic acid (final concentration 1 mg/ml) or Z-chloroadenosine (5uM), washed platelet suspensions were pretreated with either agent at 37°C for ten minutes. After washing once, the platelet suspension was incubated with an aggregating agent at 37°C for ten minutes. Proper controls were always included in each experiment. Normal Donors: A group of normal volunteers were donating blood for the study. They were medical students, technologists, graduate students and housestaff. They did not take any medications or smoke cigarettes prior to the study. None of them used oral contraceptive agents. Blood samples were obtained in the morning before breakfast. Statistical Analysis: The paired student t test was used to estimate the statistical significance RESULTS The effect of epinephrine, ADP, collagen and thrombin on platelet sialic acids is shown in Figure 1. EFFECT OFPLlTPLLl W&IEWW4(I lGENTl ONPLITLLET ,I~LIC ACID CDnTEnT
FIG. 1 Effect of platelet aggregating agents on platelet surface and total releasable sialic acids. Each bar represents mean + S.E.M. The concentration of the aggregating agent was shown under each pair of bars and controls for each aggregating agent were designated as "0". Epinephrine at 5uM did not exhibit any effect on either the surface or
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total releasable sialic acid content. ADP (5uM) in the presence of fibrinogen induced a mild but significant increase in the surface neuraminidase-removable sialic acid. Total releasable sialic acids were not increased. By contrast, collagen (1 mg/ml) and thrombin (0.1 u/ml) induced a marked increase in the surface releasable sialic acid without affecting significantly the total sialic acid. The stimulatory effect of collagen and thrombin was doserelated (Figure 2).
/-
f
i Collagen
b
0:1
(mghl)
012
Thrombin(uniVml)
FIG. 2
Dose-related increment in surface-releasable sialic acids induced by collagen and thrombin. The effect reached a plateau at 1 mg/ml collagen and 0.1 u/ml thrombin. At these concentrations, over 80% of the total releasable sialic acid had become susceptible to removal with neuraminidase. The inhibitory effects of acetylsalicylic acid and E-chloroadenosine on t' aggregating agent-induced platelet surface sialic acid content are sumnarrred in Table 1. While pretreatment of washed platelets with 2-chloroadenosine completely abolished the ADP stimulatory effect, acetylsalicylic acid had no significant effect on the ADP-induced increase in platelet surface releasable sialic acid content. The collagen-induced increase in the surface releasable sialic acid was significantly inhibited by acetylsalicylic acid but was not inhibited by 2chloroadenosine. Neither acetylsalicylic acid nor 2-chloroadenosine exerted a significant effect on the surface sialic acid change induced by thrombin.
DISCUSSION In order to understand platelet membrane biochemical changes associated with platelet activation, we have studied the alteration in platelet surface releasable sialic acid content in response to stimulation with collagen, thrombin, adenosine diphosphate and epinephrine. Since terminal residues of membrane glycoproteins consist entirely of a sialic acid, >I-acetylneuraminic
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acid which is removed specifically by neuraminidase treatment (12) determination of neuraminidase-removable sialic acid from intact platelets is thus a valid parameter for estimating quantitatively the platelet surface glycoproteins. Therefore, findings in the present study can be taken to indicate
TABLE 1. EFFECTS OF 2-CHLOROADENOSINE AND ACETYLSALICYLIC ACID ON PLATELET RELEASABLE SIALIC ACIDS Agents ADP Control
No Experiments 5
Surface NANA* n mole/l0 platelets
Increase in Surface NANA-%
27 + 5
ADP (5uM)
30 + 5
CA + ADP§
26 5 2**
ASA + ADPt
30 + 4
Collagen Control
27 + 2
Collagen (lmg/ml)
47 + 4
74
CA + Collagen5
40 510
48
ASA + Collagent
38 + 2**
41
Thrombin Control
30 + 4
Thrombin (O.lu/ml)
49 f 9
63
CA + Thrombins
45 + 6
45
ASA + Thrombint
50 f 10
67
11 0 11
Abbreviations: ADP - adenosine diphosphate, CA - 2-chloroadenosine, ASA - acetylsalicylic acid, NANA - N-acetylneuraminic acid 5 Washed platelets were first treated with 2-chloroadenosine (5uM) followed by the activating agent. t Washed platelets were treated with acetylsalicylic acid (lmg/ml) followed by the activating agent. * Mean 2 S.E.M. ** Inhibitory effects were statistically significant (~~0.05) for these experiments, but insignificant for the rest.
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that upon platelet activation, increased quantities of membrane glycoproteins become externally exposed and readily removable by neuraminidase. The capability of these platelet activating agents in inducing the glycoprotein changes tends to vary. Thrombin and collagen are potent inducers, while ADP is a weak inducer. Epinephrine, on the other hand, has no inducing effect. With the exception of epinephrine, the glycoprotein-inducing potency of the platelet activating agents is comparable to their activity in inducing platelet aggregations. Increase in surface releasable sialic acids in response to platelet activation may be due to the following mechanisms: 1) synthesis of new sialic acids, 2) release of endogenous glycoproteins which become adhered to the cell surface 3) shape change with opening of the canalicular system and 4) redistribution of membrane glycoproteins. The first hypothesis concerning synthesis of sialic acids can be readily excluded because the total releasable sialic acids remained unchanged following activation. The second hypothesis, which postulates that increased surface sialic acids may be due to the release of endogenous glycoproteins (13) can be reasonably excluded because of the following findings: a) failure for acetylsalicylic acid to inhibit ADP-induced increase in sialic acids, and b). inability of epinephrine, an agent capable of inducing platelet release reaction, to cause a significant increase in surface releasable sialic acids. It should be mentioned that if an increase in surface sialic acids is due to release of glycoproteins, repeat washings should exert some influence on the sialic acid content. This is not the case in our study. Alteration in sialic acids may be due to morphological changes which increase surface area or facilitate the connection between internal membrane structures and extracellular environment. Using glutaraldehyde fixed platelets, Motamed et al (5) observed that ADP induced an increase in neuraminidase-removable sialic acid which was inhibited by adenosine. This observation led them to postulate that an increase in externally exposed glycoproteins in response to ADP activation was due to shape change. In this study, we confirm their observation. Failure for epinephrine to induce any change in surface glycoproteins provides additional support for this concept. However, shape change per se is not satisfactory in explaining the surface glycoprotein increase induced by collagen or thrombin. As the data indicate, 2-chloroadenosine has only a minor inhibitory effect on the glycoprotein increase. We postulate that, besides shape change, redistribution of membrane glycoproteins IIEY occur as a result of platelet activation by potent activating agents. Exposure Of additional glycoproteins to the external surface may provide additional receptor sites and thus may be important for completing the activating process. There is increasing evidence for enhanced platelet activity in thromboembolic disorders (14). Platelet activity has been measured by techniques based on the following principles: 1) enhanced platelet aggregability to stimulating agents in vitro (15), 2) increased circulating platelet aggregates (16), 3) reduced olatelet life soan (17) and 4) increased olatelet Droducts such as platelet factor 4 (18) and B-thromboglobulin (19). ’As our study indicates that in vitro activation of olatelets is associated with increased amounts of surface sialic acids releasable by neuraminidase, direct determination of platelet surface releasable sialic acids may be a more sensitive method for identifying activated platelets in prothrombotic states. Similarly, surface sialic acids may serve as a sensitive marker for assessing the relative effectiveness of various platelet inhibitors in controlling platelelet activity. Further investi. gations into this area should provide new insight into the chemical basis for, as well as pharmacological control of platelet hyperactivity. It should be emphasized here that several direct labeling techniques have recently been developed for the study of cell membrane glycoproteins (20-21). Utilization of
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these labeling techniques in conjunction with the sialic acid method should be quite valuable in further elucidation of the exact membrane glycoprotein changes associated with platelet activation. ACKNOWLEDGEMENTS This work was supported in part by a grant from the American Heart Association with funds contributed by the Chicago Heart Association. REFERENCES 1.
MICHAELI, 0. and ORLOFF, K.G. Molecular considerations of platelet adhesion. In: Progress in Hemostasis and Thrombosis. T.H. Spaet (ed) 3,
29,
1976.
2.
NURDEN, A.T. and CAEN, J.P. Specific roles for platelet surface glycoproteins in platelet function. Nature 255, 720, 1975.
3.
NURDEN, A.T. and CAEN, J.P. An abnormal platelet glycoprotein pattern in three cases of Glanzmann's thrombasthenia. Br. J. Haemat. 28, 253, 1974.
4.
PHILLIPS, D.R. and AGIN, P.P. Platelet plasma membrane glycoproteins. Identification of a proteolytic substrate for thrombin. Biochem. Biophys . Res. Connn. 75, 940, 1977.
5.
MOTAMED, M., MICHAL, F. and BORN, G.V.R. Increase in sialic acids removable by neuraminidase during the shape change of platelets. Biochem. J. 158, 655, 1976.
6.
ROSSI, E.C.
platelets.
The effect of albumin upon the loss of enzymes from washed J. Lab. Clin. Med. 79, 240, 1972.
7.
WU, K.K. and KU, C.S.L. Stimulation of platelet surface sialyltransferase activity by platelet aggregating agents. Thromb. Res. 13, 183, 1978.
8.
HATTDN, M.W.C. and REGOECZI, E.A. Simple method for the purification of comnercial neuraminidase preparations free from proteases. Biochem. Biophys. Acta 327, 114, 1973.
9.
AMINOFF, D. Methods for the quantitative estimate of N-acetylneuraminic acid and their application to hydrolysate of sialomucoids. Biochem. J. 81, 384, 1961.
10.
WARREN, L. The thiobarbituric acid assay of sialic acids. J. Biol. Chem. 234, 1971, 1959.
11.
GALLUP, P.M. and SEIFTER, S. Preparation and properties of soluble collagen. In: Methods of Enzymology VI, 635, 1963.
12.
MADOFF, M.A. ABBE, S. and BALDINI, M. J. Clin. Invest. 43, 870, 1964.
13.
PEERSCHKE, E.I. and ZUCKER, M.B. Shape change and the percentage of platelet sialic acid removed by neuraminidase. Fed. Proc. 37, 258A, 1978.
Sialic acid of human blood platelets
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14.
O'BRIEN, J.R. The prothrombotic state. In: Recent Advances in Blood Coagulation (edited by Poller) 2, 24, 1977.
15.
BORN, G.V.R. Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature 194, 927, 1962.
16.
WU, K.K., and HOAK, J.C. A new method for the quantitative detection of platelet aggregates in patients with arterial insufficiency. Lancet II, 924, 1974.
17.
HARKER, L.A., ROSS, R., and SCOTT, C.R. Homocystine-induced arteriosclerosis. The role of endothelial cell injury and platelet response to its genesis. J. Clin. Invest. 58, 731, 1976.
18.
HANOIN, R.I., McOONOUGH, M., and LESCH, M. Elevation of platelet factor four in acute myocardial infarction: Measurements by radioimmunoassay. J. Lab. Clin. Med. 91, 340, 1978.
19.
LUOLAM, C.A., BOLTON, A.E., MOORE, S., and CASH, J.O. New rapid method for diagnosis of deep vein thrombosis. Lancet 2, 259, 1975.
20.
GAHMBERG, C.G., and HAKOMORI, S. External labeling of cell surface galactose and glactosamine in glycolipid and glycoprotein of human erythrocytes. J. Biol. Chem 248, 4311, 1973.
21.
GAHMBERG, C.G., and ANOERSSON, L.C. Selective radioactive labeling of cell surface sialoglycoproteins by periodate-tritiated borohydride. 2. Biol. Chem.. 252, 5888, 1977.
RESEARCH 14; 697-704 Press Ltd.1979. Printed
in Great
0049-3848/79/
Britain 0415 -0697
$02.00/o
EFFECT OF PLATELET ACTIVATION ON THE PLATELET SURFACE SIALIC ACID Kenneth K. Wu' and Cecilia S.L. Ku* From the Coagulation and Thrombosis Unit of Hematology Section Department of Medicine, Rush Medical College and Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois
(Received 7.9.1978; Revised form 28.12.1978. Accepted by Editor K.M. Brinkhous)
ABSTRACT Alteration in platelet surface neuraminidase-and total releasable sialic acids in response to platelet activation was studied by treating twice-washed intact human platelets with acid soluble collagen, thrombin, adenosine diphosphate and epinephrine. At the resting state, approximately 50% of the total platelet sialic acids were removable by neuraminidase. When platelets were treated with collagen or thrombin at 37" for ten minutes, more than 80% of the total sialic acids became removable by neuraminidase. Treatment with adenosine diphosphate resulted in a mild but significanrt increase in platelet surface releasable sialic acids, while epinephrine treatment did not induce any change in sialic acids. The effect of ADP was completely abolished by 2-chloroadenosine, whereas that of collagen or thrombin was only partially affected. Acetylsalicylic acid did not inhibit the surface sialic acid increase induced by any of the three platelet activating agents. The results indicate that upon activation with ADP, collagen or thrombin, an increased amount of terminal sialic acid residues of the membrane glycoproteins become externally exposed and thus become removable by neuraminidase. We postulate that alteration of neuraminidaseremovable sialic acids is due to redistribution of the membrane glycoproteins in response to platelet activation.
INTRODUCTION There is increasing evidence that platelet surface glycoproteins play 1. To whom requests for reprints should be sent. 2. Currently a graduate student, Department of Biochemistry, Rush University 697
698
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SVRFACE
SIALIC ACID
V01.14,N0s.4/5
.important roles in platelet function. Receptor sites for platelet activating agents on platelet membranes are probably glycoproteins (1). Glycoprotein abnormalities have been identified in patients with congenital platelet disorders, i.e., Bernard-Soulier syndrome and Glanzmann's thrombasthenia (2-3). Specific defects in the membrane glycoproteins in these two disorders tend to suggest that each glycoprotein may have a specific role in platelet adhesion and aggregation. Yet, platelet surface glycoprotein changes associated with platelet activation remain poorly understood. Phillips and Agin (4) have recently shown that treatment of platelets with thrombin is associated with a reduction of glycoprotein V, which is considered to be a receptor for thrombin. Motamed et al (5), on the other hand, have indicated that activation of platelets with adenosine diphosphate is associated with exposure of additional glycoproteins on the platelet surface. The purpose of this study is to characterize the alteration of platelet surface neuraminidase-removable sialic acids in response to activating agents such as adenosine diphosphate, collagen, thrombin and epinephrine. The inhibitory effect of Z-chloroadenosine and acetylsalicylic acid on the glycoprotein alteration was also investigated. METHODS Preparation of Washed Platelet Suspension: Twice-washed human platelet suspensions were prepared accordinq to the procedure of Rossi (6). In brief, blood was drawn from‘an antecubitai vein of'healthy volunteers-with a gauge 19 needle into acid-citrate-dextrose anticoagulant, five volumes of blood to one volume of anticoagulant. Blood was centrifuged at 220 xg for 8 minutes, platelet rich plasma was removed and centrifuged at 1000 g for 15 minutes. Supernatant was discarded and platelet pellet was resuspended and washed twice according to the procedure of Rossi (6). Platelet concentration was determined by an electronic device (Coulter ZBI, Coulter Electronics, Hialeah, Florida) and phase-contrast microscopy. Washed platelets were found to be intact (7). Measurement of Surface and Total Releasable Sialic Acid: Washed platelet suspension was divided into two equal aliquots. Aliquot A was treated with a bacterial neuraminidase (Cl. Perfringens, Sigma Chemical Co., St. Louis, MO.), which was further purified according to the method of Hatton and Regoeczi, and the caseinolytic activity of the purified preparation was negligible (8). Aliquot B was treated with sonication and acid hydrolysis (9). As neuraminidase at an optimal concentration determined experimentally for each batch of neuraminidase removed sialic acids from all externally exposed glycoproteins on the platelet surface, sialic acid content of aliquot A represented the surface releasable sialic acid, and that of aliquot B the total releasable sialic acid. The released free sialic acid was then determined by the thiobarbituric acid of Warren (10). Crystalline N-acetylneuraminic acid was used as a reference standard with each assay. Optical density readings in a Beckman spectrophotomer at 532 nm were subtraced from the readings at 549 nm and the sialic acid concentration wa: calculated according o the formula proposed by Warren (10). The result was expressed as n mole/l0 8 platelets. Platelet Aggregating and Inhibiting Agents: Acid soluble collagen from calf skin oreoared bv the method of Galluo and Seifter (11) was obtained from Siqma Chemical'Co. and the preparation was-kept at room temperature for 17 hours prior to the use for investigation. Collagen prepared in this way was capable of inducing platelet release and aggregation. Epinephrine was obtained from Parke, Davis and Co., Detroit, Mich., adenosine diphosphate (ADP) from Sigma Chemical co., thrombin (bovine) from Parke-Davis and Co., acetylsalicylic acid from Mallinkrodt Chemical, St. Louis, MO., and E-chloroadenosine from Sigma Chemical
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Co. To study the effect of platelet aggregating aqents, washed platelet suspension was incubated with an aggregating agent at 37°C for ten minutes. in the control experiment, the aggregating agent was replaced with an equal volume of saline. An attempt was made to wash the platelets after treatment with the aggregating agent. However, resuspension of platelets was often impossible because of the formation of large irreversible platelet aggregates. In certain experiments, platelets were successfully resuspended. Analysis of five such experiments showed that washing did not reduce the surface or total sialic acids. In order to obtain uniform results, all the subsequent experiments were performed without washing. To determine the inhibitory effect of acetylsalicylic acid (final concentration 1 mg/ml) or Z-chloroadenosine (5uM), washed platelet suspensions were pretreated with either agent at 37°C for ten minutes. After washing once, the platelet suspension was incubated with an aggregating agent at 37°C for ten minutes. Proper controls were always included in each experiment. Normal Donors: A group of normal volunteers were donating blood for the study. They were medical students, technologists, graduate students and housestaff. They did not take any medications or smoke cigarettes prior to the study. None of them used oral contraceptive agents. Blood samples were obtained in the morning before breakfast. Statistical Analysis: The paired student t test was used to estimate the statistical significance RESULTS The effect of epinephrine, ADP, collagen and thrombin on platelet sialic acids is shown in Figure 1. EFFECT OFPLlTPLLl W&IEWW4(I lGENTl ONPLITLLET ,I~LIC ACID CDnTEnT
FIG. 1 Effect of platelet aggregating agents on platelet surface and total releasable sialic acids. Each bar represents mean + S.E.M. The concentration of the aggregating agent was shown under each pair of bars and controls for each aggregating agent were designated as "0". Epinephrine at 5uM did not exhibit any effect on either the surface or
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total releasable sialic acid content. ADP (5uM) in the presence of fibrinogen induced a mild but significant increase in the surface neuraminidase-removable sialic acid. Total releasable sialic acids were not increased. By contrast, collagen (1 mg/ml) and thrombin (0.1 u/ml) induced a marked increase in the surface releasable sialic acid without affecting significantly the total sialic acid. The stimulatory effect of collagen and thrombin was doserelated (Figure 2).
/-
f
i Collagen
b
0:1
(mghl)
012
Thrombin(uniVml)
FIG. 2
Dose-related increment in surface-releasable sialic acids induced by collagen and thrombin. The effect reached a plateau at 1 mg/ml collagen and 0.1 u/ml thrombin. At these concentrations, over 80% of the total releasable sialic acid had become susceptible to removal with neuraminidase. The inhibitory effects of acetylsalicylic acid and E-chloroadenosine on t' aggregating agent-induced platelet surface sialic acid content are sumnarrred in Table 1. While pretreatment of washed platelets with 2-chloroadenosine completely abolished the ADP stimulatory effect, acetylsalicylic acid had no significant effect on the ADP-induced increase in platelet surface releasable sialic acid content. The collagen-induced increase in the surface releasable sialic acid was significantly inhibited by acetylsalicylic acid but was not inhibited by 2chloroadenosine. Neither acetylsalicylic acid nor 2-chloroadenosine exerted a significant effect on the surface sialic acid change induced by thrombin.
DISCUSSION In order to understand platelet membrane biochemical changes associated with platelet activation, we have studied the alteration in platelet surface releasable sialic acid content in response to stimulation with collagen, thrombin, adenosine diphosphate and epinephrine. Since terminal residues of membrane glycoproteins consist entirely of a sialic acid, >I-acetylneuraminic
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acid which is removed specifically by neuraminidase treatment (12) determination of neuraminidase-removable sialic acid from intact platelets is thus a valid parameter for estimating quantitatively the platelet surface glycoproteins. Therefore, findings in the present study can be taken to indicate
TABLE 1. EFFECTS OF 2-CHLOROADENOSINE AND ACETYLSALICYLIC ACID ON PLATELET RELEASABLE SIALIC ACIDS Agents ADP Control
No Experiments 5
Surface NANA* n mole/l0 platelets
Increase in Surface NANA-%
27 + 5
ADP (5uM)
30 + 5
CA + ADP§
26 5 2**
ASA + ADPt
30 + 4
Collagen Control
27 + 2
Collagen (lmg/ml)
47 + 4
74
CA + Collagen5
40 510
48
ASA + Collagent
38 + 2**
41
Thrombin Control
30 + 4
Thrombin (O.lu/ml)
49 f 9
63
CA + Thrombins
45 + 6
45
ASA + Thrombint
50 f 10
67
11 0 11
Abbreviations: ADP - adenosine diphosphate, CA - 2-chloroadenosine, ASA - acetylsalicylic acid, NANA - N-acetylneuraminic acid 5 Washed platelets were first treated with 2-chloroadenosine (5uM) followed by the activating agent. t Washed platelets were treated with acetylsalicylic acid (lmg/ml) followed by the activating agent. * Mean 2 S.E.M. ** Inhibitory effects were statistically significant (~~0.05) for these experiments, but insignificant for the rest.
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that upon platelet activation, increased quantities of membrane glycoproteins become externally exposed and readily removable by neuraminidase. The capability of these platelet activating agents in inducing the glycoprotein changes tends to vary. Thrombin and collagen are potent inducers, while ADP is a weak inducer. Epinephrine, on the other hand, has no inducing effect. With the exception of epinephrine, the glycoprotein-inducing potency of the platelet activating agents is comparable to their activity in inducing platelet aggregations. Increase in surface releasable sialic acids in response to platelet activation may be due to the following mechanisms: 1) synthesis of new sialic acids, 2) release of endogenous glycoproteins which become adhered to the cell surface 3) shape change with opening of the canalicular system and 4) redistribution of membrane glycoproteins. The first hypothesis concerning synthesis of sialic acids can be readily excluded because the total releasable sialic acids remained unchanged following activation. The second hypothesis, which postulates that increased surface sialic acids may be due to the release of endogenous glycoproteins (13) can be reasonably excluded because of the following findings: a) failure for acetylsalicylic acid to inhibit ADP-induced increase in sialic acids, and b). inability of epinephrine, an agent capable of inducing platelet release reaction, to cause a significant increase in surface releasable sialic acids. It should be mentioned that if an increase in surface sialic acids is due to release of glycoproteins, repeat washings should exert some influence on the sialic acid content. This is not the case in our study. Alteration in sialic acids may be due to morphological changes which increase surface area or facilitate the connection between internal membrane structures and extracellular environment. Using glutaraldehyde fixed platelets, Motamed et al (5) observed that ADP induced an increase in neuraminidase-removable sialic acid which was inhibited by adenosine. This observation led them to postulate that an increase in externally exposed glycoproteins in response to ADP activation was due to shape change. In this study, we confirm their observation. Failure for epinephrine to induce any change in surface glycoproteins provides additional support for this concept. However, shape change per se is not satisfactory in explaining the surface glycoprotein increase induced by collagen or thrombin. As the data indicate, 2-chloroadenosine has only a minor inhibitory effect on the glycoprotein increase. We postulate that, besides shape change, redistribution of membrane glycoproteins IIEY occur as a result of platelet activation by potent activating agents. Exposure Of additional glycoproteins to the external surface may provide additional receptor sites and thus may be important for completing the activating process. There is increasing evidence for enhanced platelet activity in thromboembolic disorders (14). Platelet activity has been measured by techniques based on the following principles: 1) enhanced platelet aggregability to stimulating agents in vitro (15), 2) increased circulating platelet aggregates (16), 3) reduced olatelet life soan (17) and 4) increased olatelet Droducts such as platelet factor 4 (18) and B-thromboglobulin (19). ’As our study indicates that in vitro activation of olatelets is associated with increased amounts of surface sialic acids releasable by neuraminidase, direct determination of platelet surface releasable sialic acids may be a more sensitive method for identifying activated platelets in prothrombotic states. Similarly, surface sialic acids may serve as a sensitive marker for assessing the relative effectiveness of various platelet inhibitors in controlling platelelet activity. Further investi. gations into this area should provide new insight into the chemical basis for, as well as pharmacological control of platelet hyperactivity. It should be emphasized here that several direct labeling techniques have recently been developed for the study of cell membrane glycoproteins (20-21). Utilization of
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these labeling techniques in conjunction with the sialic acid method should be quite valuable in further elucidation of the exact membrane glycoprotein changes associated with platelet activation. ACKNOWLEDGEMENTS This work was supported in part by a grant from the American Heart Association with funds contributed by the Chicago Heart Association. REFERENCES 1.
MICHAELI, 0. and ORLOFF, K.G. Molecular considerations of platelet adhesion. In: Progress in Hemostasis and Thrombosis. T.H. Spaet (ed) 3,
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GALLUP, P.M. and SEIFTER, S. Preparation and properties of soluble collagen. In: Methods of Enzymology VI, 635, 1963.
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PEERSCHKE, E.I. and ZUCKER, M.B. Shape change and the percentage of platelet sialic acid removed by neuraminidase. Fed. Proc. 37, 258A, 1978.
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HARKER, L.A., ROSS, R., and SCOTT, C.R. Homocystine-induced arteriosclerosis. The role of endothelial cell injury and platelet response to its genesis. J. Clin. Invest. 58, 731, 1976.
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HANOIN, R.I., McOONOUGH, M., and LESCH, M. Elevation of platelet factor four in acute myocardial infarction: Measurements by radioimmunoassay. J. Lab. Clin. Med. 91, 340, 1978.
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LUOLAM, C.A., BOLTON, A.E., MOORE, S., and CASH, J.O. New rapid method for diagnosis of deep vein thrombosis. Lancet 2, 259, 1975.
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GAHMBERG, C.G., and HAKOMORI, S. External labeling of cell surface galactose and glactosamine in glycolipid and glycoprotein of human erythrocytes. J. Biol. Chem 248, 4311, 1973.
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GAHMBERG, C.G., and ANOERSSON, L.C. Selective radioactive labeling of cell surface sialoglycoproteins by periodate-tritiated borohydride. 2. Biol. Chem.. 252, 5888, 1977.
