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Inhibitory effect of activated protein C on platelet aggregation induced by the prothrombin-converting reaction
THROMBOSIS RESEARCH 67; 705-710, 1992 00493848/92 $5.00 + .OOPrinted in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.
INHIBITORY EFFECT OF ACTIVATED PROTEIN C ON PLATELET AGGREGATION INDUCED BY THE PROTHROMBIN-CONVERTING REACTION
Keiji Komoriyal, Kyoko Inoue l, Katsushi Takahashil and Koji Suzuki2 1Teijin Institute for Bio-medical Research 11, Hino, Tokyo 191, and 2Department of Molecular Biologv on Genetic Disease, Mie University School of Medicine, Tsu-city, Mie 514, Japan.
(Received 2951992;
ABSTRACT
accepted in revised form 23.7.1992 by Editor S. Iwanaga)
The present study was undertaken to elucidate the effect on platelet aggregation of the prothrombin-converting reaction on platelets with or without activated protein C (APC). A reaetion mixture of washed platelets from human individuals, Factor Xa and prothrombin markedly induced platelet aggregation: maximum aggregation rates, 31.3-92.5%, and times to reach to maximum aggregation, ll.6 to 20.1 min. This aggregation was of APC with 50% inhibition inhibited by the addition concentration (IC5o) value of 14.4 U/ml. APC also inhibited thrombin generation in the reaction mixture in a dosedependent manner with IC56 value of 0.96 U/ml. However, APC did not inhibit the thrombin (0.1 CU/ml)-induced platelet aggregation at concentrations of up to 30 U/ml. These findings suggest that APC has no direct inhibitory effect on platelet aggregation and that APC inhibits platelet aggregation through inhibition of thrombin generation.
INTRODUCTION Activated protein C (APC) exerts anticoagulant effects due to the through its selective proteolytic inhibition of thrombin generation inactivating of coagulation factors Va and VIIIa (1-5). APC also neutralizes plasminogen activator inhibitor-1 and exerts fibrinolytic actions in vitro (6). In in vivo experiments, APC inhibits platelet-dependent thrombus formation in primates and rats (7-9). These properties of APC wil1 be useful for the Key words: activated protein C, platelet aggregation,
705
thrombin
PROTEIN C AND PLATELETS
706
Vol. 67, No. 6
treatment of thrombosis (10). It is considered that platelet aggregation plays an important role in the initiation of thrombus formation. However, the effect of APC on platelet aggregation is poorly understood. In this study we report that APC inhibits platelet aggregation through inhibition of thrombin generation. MATERIALS
AND METHODS
Materials. Reagents used were as follows: bovine serum albumin (Nakarai Tesque Inc., Kyoto), human factor Xa (Enzyme Research Laboratories Inc., South Bend. IN), thrombin (Mochida Pharmaceutical Co., Ltd., Tokyo), BocVal-Pro-Arg-MCA (Peptide Institute, Inc., Osaka), prothrombin and phenylmethylsulfonyl fluoride (Sigma Chemical, St. Louis, MO). adenosine 5’-diphosphate sodium salt (Yamasa Shoyu Co., Ltd., Chiba), collagen (Hormon-Chemie, Munchen), and platelin plus activator (Organon Teknika, Tokyo). Purity of prothrombin, thrombin and factor Xa are at least >95% as determined using SDS-PAGE gels developed with silver stain. Human protein C was purified from fresh frozen plasma by APC -* immunoaffinity chromatography using monoclonal antibody to protein C. APC was prepared by activation of protein C with human thrombin, and purified by cation exchange chromatography. APC preparation (Lot No. 032) containing 671 U APC, 25 mg human serum albumin, 6 mg sodium citrate, 5 mg aminoacetic acid and 7 mg NaCl, was kindly supplied by the ChemoSero-Therapeutic Research Institute, Kumamoto. APC which prolonged activated partial thromboplastin time (APIT) of human plasma twice in comparison with the clotting time without APC was referred to as one unit (IJ). APC preparation was dissolved in distilled water, gel-filtrated through a PD-10 column (Pharmacia, Uppsala, Sweden), and diluted with HEPESTyrode solution, pH 7.4, consisting of 137 mM NaCl, 2.68 mM KCl, 0.42 mM NaH2P04, 1.7 mM MgC12, ll.9 mM NaHC03, 5.6 mM glucose, 10 mM HEPES and 0.35% bovine serum albumin. The control was added the same concentration of additives. Prenaration of nlatelets. Acid citrate-treated human blood was obtained from male healthy volunteers and platelet-rich plasma (PRP) was obtained by centrifugation at 1,000 r.p.m. for 10 min at 20°C. Residual blood was centrifuged at 3,000 r.p.m. for 10 min for preparation of platelet poor plasma (PPP). To obtain washed platelets, PRP was passed through a Sepharose 2B column and the platelets were finally suspended to 108 cells/ml in HEPES-Tyrode solution. Incubation of nlatelets. factor Xa and m-othrombin. Ten microliters of washed platelets and 10 ~1 of 20 mM Tris-HCl buffer (pH 7.5) were incubated with or without 10 ~1 of APC (1.5, 5, 15, 50 and 150 U/ml) for 5 min at 37’C, and then mixed with 10 ~1 of factor Xa (3 pg/ml) and 10 ~1 of prothrombin (65 or 21.7 pg/ml). After 3 min incubation, 10 ~1 of 0.2 M EDTA was added to stop the reaction, and the reaction mixture was used for platelet aggregation and determination of thrombin activity. Platelet
aggregation.
To washed platelets (285 ~1) was added 7.5 ~1 of 60
*
77.5 NT NT NT NT NT
0
NT 91 .o 91.7 87.9 46.3 31.3 69.6k12.8
Control
NT 93.8 79.5 87.1 41.7 27.9 67.5k10.8
APC 3
Volunteers A B C D E F MeankSEM NT, nof tesfed.
0.0 34.1 23.1 13.0 16.8 1.6 3.3
Control 35.0 13.9 10.5 10.5 1.7 52
Control 45.5 18.0 10.7 11.8 1.7 4.6
1 .o 29.8 8.9 2.5 2.7 1.1 3.9
TABLE
2
Control 54.0 19.6 9.9 16.1 2.3 4.7
3.0 15.2 5.3 1.7 2.1 0.9 3.5
APC Inhibit. % 72.3 73.0 82.8 86.8 44.0 24.5 63.9+10.0
Thrombin activitv KlJ/ml) Addition (Wml) Control 61.8 15.2 12.3 24.1 2.5 5.5
10.0 10.0 6.5 1.1 1.2 0.7 3.4
APC
30.0 6.4 2.1 1.2 09 0.9 2.3
APC Inhibit. % 86.4 07.3 83.3 97.2 45.5 71.1 79.9Ik8.9
Inhibit. % -3.5 94.5 92.7 53.3 78.2 56.5 62.O-tl4.9
Control 46.8 16.5 7.2 31.2 1.7 7.8
77.9 5.0 6.5 41.7 9.8 13.8 25.8411.8
APC 30
reaction
Inhibit. % 83.8 57.2 81.1 94.9 70.9 38.7 71.1 k8.3
reaction
75.3 91.5 89.6 89.2 45.0 31.7 70.4-elO.5
Control
by the prothrombin-converting
induced by the prothrombin-converting
Inhibit. % 34.6 50.6 76.6 77.3 51.7 16.2 51.2k9.7
APC
Inhibit. % -23.6 29.5 64.8 2.4 12.7 1.9 14.6k12.3
APC
0.3 43.3 9.8 3.7 10.3 1.4 5.1
generation
NT, not tested.
-3.1 13.3 0.9 9.9 10.9 6.41k3.2
Inhibit. %
Effect of APC on thrombin
‘Mean value was obtained from five volunteers.
A B C D E F MeankSEM
Volunteers
induced
Addition (WmI) APC Control 10 Inhibit. % Maximum platelet aggregation (%) NT NT NT NT 85.3 28.0 67.2 92.5 69.2 25.2 48.8 32.1 34.2 33.8 17.1 49.4 65.1 k14.2 36.6k11.3 44.Ok9.2
Effect of APC on platelet aggregation
TABLE 1
708
PROTEIN C AND PLATELETS
Vol. 67, No. 6
mM CaC12. After 30 sec incubation, 7.5 ~1 of the reaction mixture described Platelet aggregation was determined at 37’C while above was added. stirring at 1,000 r.p.m. in an aggregometer (CHRONO-LOG, Baxter The aggregation rate was calculated against the Healthcare Co., Tokyo). transmittance of platelet suspension and a HEPES-Tyrode solution, which was designated as 0 and 100%. respectively. Determination of thrombin activitv. The activity of thrombin generated was measured using Bot-Val-Pro-At-g-MCA. Thirty microliters of the reaction mixture was incubated with 1 ml of 0.1 mM Boe-Val-Pro-Arg-MCA for 10 min at 37’C. The reaction was terminated by the addition of 0.1 ml of 20 mM phenylmethylsulfonyl fluoride. The activity of thrombin was measured as the fluorescente of 7-amino-4-methylcoumarin (AMC) liberated from the substrate using a fluorescente spectrophotometer (F-3000, Hitachi, Ltd., Tokyo), and was expressed as the amount of thrombin. Platelet aggregation bv various stimuli. PRP (3-4x108 cells/ml) was stimulated with, 5 PM 5’-adenosine diphosphate disodium salt (ADP) or 10 pg/ml of collagen. The aggregation rate was calculated against the transmittance of PRP and PPP, which was designated as 0 and lOO%, respectively. RESULTS Platelet aggregation. The reaction mixture incubated with platelets, factor Xa and prothrombin induced platelet aggregation. Maximum aggregation rates were shown from 31.3 to 92.5%. and aggregation reached a maximum ll.6 to 20.0 min after the addition. As shown in Table 1, APC inhibited this aggregation in a concentration-dependent manner, at concentrations of 10 to 30 U/ml. At 30 U/ml, the inhibition was observed in five out of six volunteers at 53.3-94.5%. The IC50 value was 14.4 U/ml. However, APC showed no obvious effect on the time to attain the maximum aggregation rate. Platelet aggregation bv various stimuli. APC showed no inhibition of platelet aggregation which was induced by 0.1 CU/ml of thrombin, 5 PM ADP or 10 pg/ml of collagen at concentrations of up to 30 U/ml (data not shown). These results mean the process of platelet aggregation is not affected by APC. Thrombin activitv. To clarify the mode of action of APC, thrombin activity of the reaction mixture was measured. As shown in Table 2, APC inhibited thrombin generation in a concentration-dependent marmer, at 14.6-79.9%, at concentrations of 0.3-30 U/ml. The IC!50value was 0.96 U/ml. DISCUSSION APC exerts anticoagulant effects due to the inhibition of thrombin generation through inactivation of factors Va and VIIIa (l-5), but its effect on platelet aggregation is poorly understood. We expected APC to inhibit
Vol. 67, No. 6
PROTEIN C AND PLATELETS
709
platelet aggre ation of PRP stimulated with tissue factor. When human PRP (0.75- 1.0x10 $ celIs/ ~1) was incubated with APC for 2 min and then stimulated with tissue factor (0.56 mg/50 ~1: Simplastin, Organon Teknika, Tokyo), platelets from 8 out of 13 volunteers were aggregated; maximum aggregation rate, 67.6f2.60/ o and time of attainment of maximum aggregation rate, 10.4k1.3 min. APC only showed a slight inhibition of the maximum aggregation rate and a slight prolongation of the time of attainment of maximum aggregation rate at concentrations of up to 30 U/ml (data not shown). Factor V comprising about 20% in whole blood, is contained in the agranule of human platelets, is released from the granule by stimuli such as thrombin, collagen, epinephrine, and ADP (11, 12). and is converted into factor Va by thrombin (13). The factor Va serves as a receptor for factor Xa. APC inactivates the platelet-associated factor Va concomitantly with a decrease in the binding of factor Xa to the platelets (14, 15). APC inhibits prothrombin conversion when APC is incubated with factor Va bound to platelets, factor Xa and prothrombin (16). We next investigated the effect of this prothrombin converting reaction on platelet aggregation, and the effect of APC on that reaction. The reaction mixture incubated with platelets, factor Xa and prothrombin, caused platelet aggregation. APC inhibited’ the platelet aggregation in five out of six volunteers at concentrations of 10-30 U/ml, with IC50 value of 14.4 U/ml. APC also inhibited thrombin generation in al1 volunteers at a concentration of 0.3 U/ml or more, with IC50 value of 0.96 U/ml. Since the platelet aggregation induced by thrombin, ADP or collagen was not influenced by APC, it is thought that APC inhibits platelet aggregation through its inhibitory effect of thrombin generation. However. there is a discrepancy in these IC50 values. In a certain case, APC markedly inhibited thrombin generation, but not platelet aggregation (Volunteer A in Table 1 and 2). This discrepancy may be accounted for as a critical concentration in the thrombin-induced platelet aggregation as reported by Thomas (17). APC has been demonstrated to inhibit arterial thrombosis in primates and rats (7-9) and show the therapeutic effect on the patients with disseminated intravascular coagulation (10). These properties of APC may be explained by the inhibition of platelet aggregation through its inhibition of thrombin generation. REFERENCES 1. Kisiel, W., Canfield, W.M., Ericsson, L.H. and Davie, E.W. properties of bovine plasma protein C following activation Biochemistry 16, 5824-583 1, 1977.
Anticoagulant by thrombin.
2. Walker, F.J., Sexton, P.W. and Esmon, C.T. The inhibition of blood coagulation by activated protein C through the selective inactivation of activated Factor V. Biochim. Bioplys. Acta 571, 333-342. 1979. 3. Marlar, R.A., Kleiss, A.J. and Griffin, J.H. Mechanism of action of human activated protein C, a thrombin-dependent anticoagulant enzyme. Bloed 59, 1067-1072, 1982.
PROTEIN C AND PLATELETS
710
Vol. 67, No. 6
B. Inactivation of 4. Suzuki, K., Stenflo, J.. Dahlback. B. and Teodorsson, human coagulation factor V by activated protein C. J. Biel. Chem. 258. 1914- 1920. 1983. 5. Vehar, G.A. and Davie, E.W. Preparation and properties of bovine factor Biochemislry 19, 401-409, 1980. VIII (Antihemophilic factor). 6. Sakata, Activated endothelial U.S.A. 82,
Y.. Curriden, S., Lawrence, D., Griffin. J.H. and Loskutoff, D.J. protein C stimulates the fibrinolytic activity of cultured cells and decreases antiactivator activity. Proc. Natl. Acad. Sci. 1121-1125, 1985.
7. Gruber, A., Griffin, J.H., Harker, L.A. and Hanson, S.R. Inhibition of platelet-dependent thrombus formation by human activated protein C in a 1989. primate model. Bloed 73, 639-642, 8. Gruber, A., Hanson, S.R., Kelly, A.B., Yan, B.S., Bang, N., Griffin, J.H. and Harker, L.A. Inhibition of thrombus formation by activated recombinant protein C in a primate model of arterial thrombosis. Circulation 82, 578585. 1990. 9. Araki, H.. Nishi, K., Ishihara. N. and Okajima, K. Inhibitory effects of activated protein C and heparin on thrombotic arterial occlusion in rat mesenteric arteries. Thrombosis Res. 62. 209-216, 199 1. 10. Okajima, K., Imamura, H., Koga, S., Inoue, M., Takatsuki, K. and Aoki, N. Treatment of patients with disseminated intravascular coagulation by protein C. Am. J. Hematol. 33, 277-278, 1990. 11. Chesney, C.H., Pifer, D., and Colman, R.W. Subcellular locarization and secretion of factor V from human platelets. Proc. Natl. Acad. Sci. U.S.A. 78, 5180-5184, 1981. 12. Tracy, P.B., Eide, L.L., Bowie, E.J.W., and Mann, K.G. Radioimmunoassay for factor V in human plasma and platelets Bloed 60, 59-63, 1982. 13. Miletich, J.P., Jackson, C.M. and Majerus, P.W. Properties of the factor Xa binding site on human platelets. J. Bio!. Chem. 253, 6908-6916, 1978. 14. Comp, P.C. and Esmon, C.T. Activated protein C inhibits prothrombin-converting activity. Bloed 54, 1272- 128 1, 1979.
platelet
15. Dahlback, B. and Stenflo, J. Inhibitory effect of activated protein C on activation of prothrombin by platelet-bound factor X(a). Eur. J. Biochem. 107, 331-335, 1980. 16. Suzuki, K., Nishioka, J., Matsuda, M., Murayama, H. and Hashimoto, Protein S is essential for the activated protein C-catalyzed inactivation platelet-associated factor Va. J. Biochem. 96, 455-460, 1984. 17. Thomas, by thrombin.
D.P. Effect of catecholamines Nature 215. 298-299, 1967.
on platelet aggregation
S. of
caused
INHIBITORY EFFECT OF ACTIVATED PROTEIN C ON PLATELET AGGREGATION INDUCED BY THE PROTHROMBIN-CONVERTING REACTION
Keiji Komoriyal, Kyoko Inoue l, Katsushi Takahashil and Koji Suzuki2 1Teijin Institute for Bio-medical Research 11, Hino, Tokyo 191, and 2Department of Molecular Biologv on Genetic Disease, Mie University School of Medicine, Tsu-city, Mie 514, Japan.
(Received 2951992;
ABSTRACT
accepted in revised form 23.7.1992 by Editor S. Iwanaga)
The present study was undertaken to elucidate the effect on platelet aggregation of the prothrombin-converting reaction on platelets with or without activated protein C (APC). A reaetion mixture of washed platelets from human individuals, Factor Xa and prothrombin markedly induced platelet aggregation: maximum aggregation rates, 31.3-92.5%, and times to reach to maximum aggregation, ll.6 to 20.1 min. This aggregation was of APC with 50% inhibition inhibited by the addition concentration (IC5o) value of 14.4 U/ml. APC also inhibited thrombin generation in the reaction mixture in a dosedependent manner with IC56 value of 0.96 U/ml. However, APC did not inhibit the thrombin (0.1 CU/ml)-induced platelet aggregation at concentrations of up to 30 U/ml. These findings suggest that APC has no direct inhibitory effect on platelet aggregation and that APC inhibits platelet aggregation through inhibition of thrombin generation.
INTRODUCTION Activated protein C (APC) exerts anticoagulant effects due to the through its selective proteolytic inhibition of thrombin generation inactivating of coagulation factors Va and VIIIa (1-5). APC also neutralizes plasminogen activator inhibitor-1 and exerts fibrinolytic actions in vitro (6). In in vivo experiments, APC inhibits platelet-dependent thrombus formation in primates and rats (7-9). These properties of APC wil1 be useful for the Key words: activated protein C, platelet aggregation,
705
thrombin
PROTEIN C AND PLATELETS
706
Vol. 67, No. 6
treatment of thrombosis (10). It is considered that platelet aggregation plays an important role in the initiation of thrombus formation. However, the effect of APC on platelet aggregation is poorly understood. In this study we report that APC inhibits platelet aggregation through inhibition of thrombin generation. MATERIALS
AND METHODS
Materials. Reagents used were as follows: bovine serum albumin (Nakarai Tesque Inc., Kyoto), human factor Xa (Enzyme Research Laboratories Inc., South Bend. IN), thrombin (Mochida Pharmaceutical Co., Ltd., Tokyo), BocVal-Pro-Arg-MCA (Peptide Institute, Inc., Osaka), prothrombin and phenylmethylsulfonyl fluoride (Sigma Chemical, St. Louis, MO). adenosine 5’-diphosphate sodium salt (Yamasa Shoyu Co., Ltd., Chiba), collagen (Hormon-Chemie, Munchen), and platelin plus activator (Organon Teknika, Tokyo). Purity of prothrombin, thrombin and factor Xa are at least >95% as determined using SDS-PAGE gels developed with silver stain. Human protein C was purified from fresh frozen plasma by APC -* immunoaffinity chromatography using monoclonal antibody to protein C. APC was prepared by activation of protein C with human thrombin, and purified by cation exchange chromatography. APC preparation (Lot No. 032) containing 671 U APC, 25 mg human serum albumin, 6 mg sodium citrate, 5 mg aminoacetic acid and 7 mg NaCl, was kindly supplied by the ChemoSero-Therapeutic Research Institute, Kumamoto. APC which prolonged activated partial thromboplastin time (APIT) of human plasma twice in comparison with the clotting time without APC was referred to as one unit (IJ). APC preparation was dissolved in distilled water, gel-filtrated through a PD-10 column (Pharmacia, Uppsala, Sweden), and diluted with HEPESTyrode solution, pH 7.4, consisting of 137 mM NaCl, 2.68 mM KCl, 0.42 mM NaH2P04, 1.7 mM MgC12, ll.9 mM NaHC03, 5.6 mM glucose, 10 mM HEPES and 0.35% bovine serum albumin. The control was added the same concentration of additives. Prenaration of nlatelets. Acid citrate-treated human blood was obtained from male healthy volunteers and platelet-rich plasma (PRP) was obtained by centrifugation at 1,000 r.p.m. for 10 min at 20°C. Residual blood was centrifuged at 3,000 r.p.m. for 10 min for preparation of platelet poor plasma (PPP). To obtain washed platelets, PRP was passed through a Sepharose 2B column and the platelets were finally suspended to 108 cells/ml in HEPES-Tyrode solution. Incubation of nlatelets. factor Xa and m-othrombin. Ten microliters of washed platelets and 10 ~1 of 20 mM Tris-HCl buffer (pH 7.5) were incubated with or without 10 ~1 of APC (1.5, 5, 15, 50 and 150 U/ml) for 5 min at 37’C, and then mixed with 10 ~1 of factor Xa (3 pg/ml) and 10 ~1 of prothrombin (65 or 21.7 pg/ml). After 3 min incubation, 10 ~1 of 0.2 M EDTA was added to stop the reaction, and the reaction mixture was used for platelet aggregation and determination of thrombin activity. Platelet
aggregation.
To washed platelets (285 ~1) was added 7.5 ~1 of 60
*
77.5 NT NT NT NT NT
0
NT 91 .o 91.7 87.9 46.3 31.3 69.6k12.8
Control
NT 93.8 79.5 87.1 41.7 27.9 67.5k10.8
APC 3
Volunteers A B C D E F MeankSEM NT, nof tesfed.
0.0 34.1 23.1 13.0 16.8 1.6 3.3
Control 35.0 13.9 10.5 10.5 1.7 52
Control 45.5 18.0 10.7 11.8 1.7 4.6
1 .o 29.8 8.9 2.5 2.7 1.1 3.9
TABLE
2
Control 54.0 19.6 9.9 16.1 2.3 4.7
3.0 15.2 5.3 1.7 2.1 0.9 3.5
APC Inhibit. % 72.3 73.0 82.8 86.8 44.0 24.5 63.9+10.0
Thrombin activitv KlJ/ml) Addition (Wml) Control 61.8 15.2 12.3 24.1 2.5 5.5
10.0 10.0 6.5 1.1 1.2 0.7 3.4
APC
30.0 6.4 2.1 1.2 09 0.9 2.3
APC Inhibit. % 86.4 07.3 83.3 97.2 45.5 71.1 79.9Ik8.9
Inhibit. % -3.5 94.5 92.7 53.3 78.2 56.5 62.O-tl4.9
Control 46.8 16.5 7.2 31.2 1.7 7.8
77.9 5.0 6.5 41.7 9.8 13.8 25.8411.8
APC 30
reaction
Inhibit. % 83.8 57.2 81.1 94.9 70.9 38.7 71.1 k8.3
reaction
75.3 91.5 89.6 89.2 45.0 31.7 70.4-elO.5
Control
by the prothrombin-converting
induced by the prothrombin-converting
Inhibit. % 34.6 50.6 76.6 77.3 51.7 16.2 51.2k9.7
APC
Inhibit. % -23.6 29.5 64.8 2.4 12.7 1.9 14.6k12.3
APC
0.3 43.3 9.8 3.7 10.3 1.4 5.1
generation
NT, not tested.
-3.1 13.3 0.9 9.9 10.9 6.41k3.2
Inhibit. %
Effect of APC on thrombin
‘Mean value was obtained from five volunteers.
A B C D E F MeankSEM
Volunteers
induced
Addition (WmI) APC Control 10 Inhibit. % Maximum platelet aggregation (%) NT NT NT NT 85.3 28.0 67.2 92.5 69.2 25.2 48.8 32.1 34.2 33.8 17.1 49.4 65.1 k14.2 36.6k11.3 44.Ok9.2
Effect of APC on platelet aggregation
TABLE 1
708
PROTEIN C AND PLATELETS
Vol. 67, No. 6
mM CaC12. After 30 sec incubation, 7.5 ~1 of the reaction mixture described Platelet aggregation was determined at 37’C while above was added. stirring at 1,000 r.p.m. in an aggregometer (CHRONO-LOG, Baxter The aggregation rate was calculated against the Healthcare Co., Tokyo). transmittance of platelet suspension and a HEPES-Tyrode solution, which was designated as 0 and 100%. respectively. Determination of thrombin activitv. The activity of thrombin generated was measured using Bot-Val-Pro-At-g-MCA. Thirty microliters of the reaction mixture was incubated with 1 ml of 0.1 mM Boe-Val-Pro-Arg-MCA for 10 min at 37’C. The reaction was terminated by the addition of 0.1 ml of 20 mM phenylmethylsulfonyl fluoride. The activity of thrombin was measured as the fluorescente of 7-amino-4-methylcoumarin (AMC) liberated from the substrate using a fluorescente spectrophotometer (F-3000, Hitachi, Ltd., Tokyo), and was expressed as the amount of thrombin. Platelet aggregation bv various stimuli. PRP (3-4x108 cells/ml) was stimulated with, 5 PM 5’-adenosine diphosphate disodium salt (ADP) or 10 pg/ml of collagen. The aggregation rate was calculated against the transmittance of PRP and PPP, which was designated as 0 and lOO%, respectively. RESULTS Platelet aggregation. The reaction mixture incubated with platelets, factor Xa and prothrombin induced platelet aggregation. Maximum aggregation rates were shown from 31.3 to 92.5%. and aggregation reached a maximum ll.6 to 20.0 min after the addition. As shown in Table 1, APC inhibited this aggregation in a concentration-dependent manner, at concentrations of 10 to 30 U/ml. At 30 U/ml, the inhibition was observed in five out of six volunteers at 53.3-94.5%. The IC50 value was 14.4 U/ml. However, APC showed no obvious effect on the time to attain the maximum aggregation rate. Platelet aggregation bv various stimuli. APC showed no inhibition of platelet aggregation which was induced by 0.1 CU/ml of thrombin, 5 PM ADP or 10 pg/ml of collagen at concentrations of up to 30 U/ml (data not shown). These results mean the process of platelet aggregation is not affected by APC. Thrombin activitv. To clarify the mode of action of APC, thrombin activity of the reaction mixture was measured. As shown in Table 2, APC inhibited thrombin generation in a concentration-dependent marmer, at 14.6-79.9%, at concentrations of 0.3-30 U/ml. The IC!50value was 0.96 U/ml. DISCUSSION APC exerts anticoagulant effects due to the inhibition of thrombin generation through inactivation of factors Va and VIIIa (l-5), but its effect on platelet aggregation is poorly understood. We expected APC to inhibit
Vol. 67, No. 6
PROTEIN C AND PLATELETS
709
platelet aggre ation of PRP stimulated with tissue factor. When human PRP (0.75- 1.0x10 $ celIs/ ~1) was incubated with APC for 2 min and then stimulated with tissue factor (0.56 mg/50 ~1: Simplastin, Organon Teknika, Tokyo), platelets from 8 out of 13 volunteers were aggregated; maximum aggregation rate, 67.6f2.60/ o and time of attainment of maximum aggregation rate, 10.4k1.3 min. APC only showed a slight inhibition of the maximum aggregation rate and a slight prolongation of the time of attainment of maximum aggregation rate at concentrations of up to 30 U/ml (data not shown). Factor V comprising about 20% in whole blood, is contained in the agranule of human platelets, is released from the granule by stimuli such as thrombin, collagen, epinephrine, and ADP (11, 12). and is converted into factor Va by thrombin (13). The factor Va serves as a receptor for factor Xa. APC inactivates the platelet-associated factor Va concomitantly with a decrease in the binding of factor Xa to the platelets (14, 15). APC inhibits prothrombin conversion when APC is incubated with factor Va bound to platelets, factor Xa and prothrombin (16). We next investigated the effect of this prothrombin converting reaction on platelet aggregation, and the effect of APC on that reaction. The reaction mixture incubated with platelets, factor Xa and prothrombin, caused platelet aggregation. APC inhibited’ the platelet aggregation in five out of six volunteers at concentrations of 10-30 U/ml, with IC50 value of 14.4 U/ml. APC also inhibited thrombin generation in al1 volunteers at a concentration of 0.3 U/ml or more, with IC50 value of 0.96 U/ml. Since the platelet aggregation induced by thrombin, ADP or collagen was not influenced by APC, it is thought that APC inhibits platelet aggregation through its inhibitory effect of thrombin generation. However. there is a discrepancy in these IC50 values. In a certain case, APC markedly inhibited thrombin generation, but not platelet aggregation (Volunteer A in Table 1 and 2). This discrepancy may be accounted for as a critical concentration in the thrombin-induced platelet aggregation as reported by Thomas (17). APC has been demonstrated to inhibit arterial thrombosis in primates and rats (7-9) and show the therapeutic effect on the patients with disseminated intravascular coagulation (10). These properties of APC may be explained by the inhibition of platelet aggregation through its inhibition of thrombin generation. REFERENCES 1. Kisiel, W., Canfield, W.M., Ericsson, L.H. and Davie, E.W. properties of bovine plasma protein C following activation Biochemistry 16, 5824-583 1, 1977.
Anticoagulant by thrombin.
2. Walker, F.J., Sexton, P.W. and Esmon, C.T. The inhibition of blood coagulation by activated protein C through the selective inactivation of activated Factor V. Biochim. Bioplys. Acta 571, 333-342. 1979. 3. Marlar, R.A., Kleiss, A.J. and Griffin, J.H. Mechanism of action of human activated protein C, a thrombin-dependent anticoagulant enzyme. Bloed 59, 1067-1072, 1982.
PROTEIN C AND PLATELETS
710
Vol. 67, No. 6
B. Inactivation of 4. Suzuki, K., Stenflo, J.. Dahlback. B. and Teodorsson, human coagulation factor V by activated protein C. J. Biel. Chem. 258. 1914- 1920. 1983. 5. Vehar, G.A. and Davie, E.W. Preparation and properties of bovine factor Biochemislry 19, 401-409, 1980. VIII (Antihemophilic factor). 6. Sakata, Activated endothelial U.S.A. 82,
Y.. Curriden, S., Lawrence, D., Griffin. J.H. and Loskutoff, D.J. protein C stimulates the fibrinolytic activity of cultured cells and decreases antiactivator activity. Proc. Natl. Acad. Sci. 1121-1125, 1985.
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