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Aprotinin (Trasylol) is a competitive inhibitor of activated protein C
THROMBOSIS RESEARCH 56; 751-756, 1989 0049-3848/89 $3.00 + .OO Printed in the USA. Copyright (c) 1989 Pergamon Press plc. All rights reserved.
BRIEF
COMMUNICATION
APROTININ (TRASYLOL) IS A COMPETITIVE INHIBITOR OF ACTIVATED PROTEIN C
FRANCISCO
ESPANA-, AMPARO ESTELLES", JOHN H. GRIFFIN', JUST0 AZNAR-, AND JUAN GILABERT""
Research Center* and Departments of Clinical Pathology** and Obstetrics and Gynecology+@*,Hospital La Fe, Valencia, SPAIN and Committee on Vascular Biology and Department of Molecular and Experimental Medicine, Research Institute of Scripps Clinic, La Jolla, CA, USA.*
(Received 13.9.1989; accepted in revised form 19.9.1989 by Editor H. Vinazzer)
INTRODUCTION Protein C (1,2) is a major regulatory protein of thrombus formation. Besides controlling the thrombin formation, by inactivating factors Va and it promotes fibrinolysis by inactivating the plasminogen VIIIa (3-6), activator inhibitors PAI- (7-9) and PAI- (10). APC is regulated in plasma by two major inhibitors (11). One, named protein C inhibitor (PCI) is heparindependent and has been characterized (10,12-14) and the sequence of its cDNA reported (15), and it has been shown to be identical (16) to a heparindependent urokinase inhibitor also known as PAI- (17). The second protein C inhibitor is heparin-independent and has been demonstrated to be alantitrypsin (18). Aprotinin has a broad inhibitory spectrum. It inhibits human trypsin, plasmin, plasma kallikrein, and tissue kallikreins by forming reversible stoichiometric enzyme-inhibitor complexes (19). While studying the APC inhibition by PC1 we observed that aprotinin, used to inhibit kallikrein in the study, did inhibit APC. That observation prompted us to study the interaction between APC and aprotinin by analyzing the inhibition of the APC amidolytic activity by the inhibitor.
MATERIAL AND METHODS Materials. Human protein C and APC (20) were prepared as indicated before. Aprotinin (Trasylol) was obtained from Calbiochem, La Jolla, CA. 18 U/ml aprotinin equaled to 1 pg/mL and to 150 nM. S-2366 chromogenic substrate was from Kabi, Stockholm. Bovine serum albumin (BSA) and heparin were from Sigma Chemical Co., St. Louis. Key
Words:
Protein C, Aprotinin, Trasylol 751
752
APC INHIBITION BY TRASYLOL
Vol. 56, No. 6
Amidolytic assay. To test the inhibitory effect of aprotinin on APC, purified APC (0.8 pal/L) was incubated at 37 ‘C with aprotinin (0, 0.6, 3, or 6 pal/L) in 0.05 mol/L Tris-HCl, 0.10 mol/L NaCl, and 0.1% BSA, pli 7.4, with or without the addition of heparin. Controls were performed by incubating APC and buffer containing BSA in the presence or the absence of heparin. In each case, 20 PL-aliquots were removed at time intervals and added to 180 PL of a mixture containing 1, 0.75 or 0.5 mmol/L S-2366, 0.05 mol/L Tris-HCl, 0.10 mol/L NaCl, 0.004 mol/L CaC12, pH 8.2, in the wells of microtitter plates and the rate of change in absorbance during the first three minutes was determined every 1 min using an ELISA plate reader and expressed as percent of that obtained with controls. Determination of enzyme inhibitor constants was carried out by the method reported by Dixon (21) plotting l/v versus i at three different substrate the- reaction -velocity and i is the inhibitor concentrations, where v is concentration. This gives straight lines which will cut each-other at a point the-vertical axis, at -KL, which can therefore be read off on the left of directly.
RESULTS AND DISCUSSION aprotinin concentrations resulted in timeIncubation of APC with different of its amidolytic activity. This inhibition, however, independent inhibition APC inhibition was already was dose-dependent (Figure 1). Thus, in each case after 1 ?in ? incubation with the enzyme, the extension of the completed inhibition being dependent of the inhibitor concentration used.
I Trrrllrl
I, )I
of activated protein C (APC) by trasylol. 25 /JL Figure 1. Inhibition APC (0.8 pal/L) and 25 PL trasylol were incubated at 37 ‘C for 5 removed and added to 180 NIL of a 20 PL-aliquot was min. Thereafter, 0.75 mmol/L S-2366 in the wells of microtitter plates and the APC inhibition was calculated as indicated in Methods.
To see whether the inhibitor was a competitive inhibitor, residual A three different substra determined using was amidolytic activity after 3 min incubation with the inhibitor. Figure 2 shows t concentrations, when the reciprocal of v (aA/mi modified Lineweaver & Burk plots obtained
Vol. 56, No. 6
APC INHIBITION BY TRASYLOL
was plotted versus the inhibitor concentration. The three lines cut each other at a point. This point lies at -K+. From this experiment/a value of 1.44 /JM the inhibition of APC amidolytic activity by for Ka was calculated. Thus, aprotinin is of a competitive nature. m -
Figure 2. Dixon’s plots for the inhibition of APC by trasylol. APC and trasylol were incubated as indicated in Fig.1. 20 PL-aliquots were removed and added to 180 PL of 1, 0.75 or 0.5 mmol/L S-2366 in the wells of microtitter plates and the APC inhibition was calculated as indicated in Methods.
Since APC binds to heparin and since a major plasma APC inhibitor, PCI, is heparin-dependent (10,12,14), we studied the effect of heparin on the APC inhibition by aprotinin. Figure 3 shows the results obtained. Heparin increased the affinity of the inhibitor for the enzyme in a dose dependent manner , resulting in decreased Ka values with maximum effect at about 4 u/mL.
Figure 3. Inf 1.uence of heparin on APC inhibition by trasylol. The experiment out as indicated in Fig. 2, except that APC was carried and aprot i nin were incubated in the presence of several heparin concentrat i ons . KS values were calculated as indicated in Methods.
APC INHIBITION BY TRASYLOL
754
Vol. 56, No. 6
One u/mL heparin decreased about S-fold the Ki value, whereas at 10 .u/mL heparin the inhibitor constant decreased 17-fold (Figure 3). Although the binding of aprotinin with human plasmin (K+=lnM) (22) is tighter than the binding with APC, the K+ for APC reported here, 80 to 100 nM in the presence of heparin, is in the same range that the Ki for plasma kallikrein, about 30 nM and still within the therapeutic range of aprotinin (23). Whether the APC inhibition by aprotinin is relevant in vivo, or contributes to the antifibrinolytic action of the aprotinin remains to be demonstrated. Protein C assays have been developed in which activation of protein C is achieved directly in plasma using snake venoms (24-30), and measuring the APC amidolytic and/or anticoagulant activity. In these cases, the presence of trasylol in plasma will interfere in the assays specially when, as usual, heparin is co-administered with trasylol, giving underestimation of protein C values. This could be overcome using other methods in which a previous absorption step is used (31-33).
ACKNOWLEDGEMENTS This study was supported in part by a National Institutes of Health Grant HL-31950, and by a grant No. 88/1623 from the Fondo de Investigaciones Sanitarias de la Seguridad Social, Spain.
REFERENCES A new vitamin K-dependent protein: Purification from bovine preliminary characterization. J. Biol. Chem. 251, 355-363,
1.
STENFLO, J. plasma and 1976.
2.
KISIEL, W. Human plasma protein C. Isolation, characterization and mechanism of activation by a-thrombin. J. Clin. Invest. 66, 761-769, 1979.
3.
KISIEL, W., CANFIELD, W.M., ERICSSON, L.H. and DAVIE, E.W. Anticoagulant properties of bovine plasma protein C following activation by thrombin. Biochemistry 16, 5824-5831, 1977.
4.
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. Biophys. Acta 571, 333-342, 1979.
5.
VEHAR, G. and DAVIE, E.W.Preparation and properties of bovine factor VIII. Biochemistry 2, 401-410, 1980.
6.
FULCHER, C.A., GARDINER, J.E., GRIFFIN, J.H. and ZIMMERMANN, T.S. Proteolytic inactivation of human factor VIII procoagulant protein by activated human protein C and its analogy with factor V. -Blood 63, 486-489, 1984.
7.
SAKATA, Y., CURRIDEN, S.A., LAWRENCE, D.A., GRIFFIN, J.H. and LOSKUTOFF, D.J. Activated protein C stimulates the fibrinolytic activity of cultured endothelial cells and decreases antiactivator activity. Proc. Natl. Acad. Sci. USA, g, 1121-1125, 1985.
Vol. 56, No. 6
APC INHIBITION BY TRASYLOL
755
8.
TAYLOR, F.B. and LOCKHART, M.S. A new function for activated protein C: Activated protein C prevents inhibition of plasminogen activators by releasate from mononuclear leukocytes-platelet suspensions stimulated by phorbol diester. Thromb. Res. 37_, 155-164, 1985.
9.
VAN HINSBERG, V.N.M., BERTINA, R.M., VAN WIJNGAARDEN, A., VAN TILBURG, J.H., EMEIS, J. and HAVERKATE, F. Activated protein C decreases plasminogen activator-inhibitor activity in endothelial cell-conditioned medium. Blood 65, 444-451, 1985. --
10. ESPANA, F., BERRETTINI, M. and GRIFFIN, J.H. Purification and characterization of plasma protein C inhibitor. Thromb. Res. 1989. In press. 11. HEEB, M.J., ESPANA, F. and GRIFFIN, J.H. Inhibition and complexation of activated protein C by two major inhibitors in plasma. -Blood 73, 446-454, 1989. 12. SUZUKI, K., NISHIOKA, J. and HASHIMOTO, S. Protein C inhibitor. Purification from human plasma and characterization. J. Biol. Chem. 258, 163168, 1983. 13. SUZUKI, K., NISHIOKA, J., KUSUMOTO, H. and HASHIMOTO, S. Mechanism of inhibition of activated protein C by protein C inhibitor. J. Biochem. 2, 187-195, 1984. 14. MEIJERS, J.C.M.', KANTERS, D.H.A.J., VLOOSWIJK, R.A.A., VAN ERP, H.E., HESSING, M. and BOUMA, B.N. Inactivation of human plasma kallikrein and factor_XIa by protein C inhibitor. Biochemistry -27, 4231-4237, 1988. 15. SUZUKI, K., DEYASHIKI, Y., NISHIOKA, J., KURACHI, K., AKIRA, M., YAMAMOTO, S. and HASHIMOTO, S. Characterization of a cDNA for human protein C inhibitor. J. Biol. Chem. 262, 611-616, 1987. 16. HEEB, M.J., ESPANA, F., GEIGER, M., COLLEN, D., STUMP, D. and GRIFFIN, J.H. Immunological identity of heparin-dependent plasma and urinary protein C inhibitor and plasminogen activator inhibitor-3. J. Biol. Chem. 262, 15813-15816, 1987. 17. STUMP, D.C., THIENPONT, M. and COLLEN, D. Purification and characterization of a novel inhibitor of urokinase from human urine. J. Biol. Chem. 261, 12759-12766, 1986. 18. HEEB, M.J. and GRIFFIN, J.H. Physiologic inhibition of human activated protein C by or-antitrypsin. J. Biol. Chem. 263, 11613-11616, 1988. 19. VERSTRAETE, M. Clinical application of inhibitors of fibrinolysis. Drugs 29, 236-261, 1985. 20. 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 primate model. -Blood 73, 639-642, 1989. 21. DIXON, M. The determination of enzyme inhibitor constants. Biochem. J. 3, 170-171, 1953.
756
APC INHIBITION BY TRASYLOL
Vol. 56, No. 6
22. WIMAN, B. On the reaction of plasmin or plasminstreptokinase complex aprotinin or az-antiplasmin. Thromb. Res. 5, 143,152, 1980.
with
23. PHILIPP, E. Calculations and hypothetical considerations on the inhibition
plasmin and plasma kallikrein by trasylol. In: Progress in chemical fibrinolysis and thrombolysis. Davidson et al. (Eds.) New York: Raven Press, 1978, pp. 291-295. 24. FRANCIS, R.B. and SEYFERT, U. Rapid amidolytic assay of protein C in whole
plasma using an activator from the venom of agkistrodon contortrix. Amer. J. Clin. Pathol. 87, 619-625, 1987. 25. HICKTON, C.M., FELDING, P.. IKEDA, K., MARTINSSON, G. and NILSSON, I.M. A functional assay of protein C in human plasma. Thromb. Res. 41, 501-508, 1986. 26. MARTINOLI, J.L.
and STOCKER, K. Fast functional protein C assay using protac, a novel protein C activator. Thromb. Res. 2, 253-264, 1986.
27. MCCALL, F.,
protein C in
CONKIE, J.A., WALKER, I.D. and DAVIDSON, J.F. Measurement of plasma - a fully automated assay. Thromb. Res. 5, 681-685,
1987. 28. NATHAN, I.V., PING, H. and PRADHAN, M.M.
snake venom activator. Thromb. Res. z,
Protein C functional assay using 85-91, 1987.
29. ODEGAARD, O.R.,
TRY, K. and ABILGAARD, U. Protein C: A simplified semiautomated activity assay. Thromb. Res. 42, 257-262, 1986.
30. VUKOVICH,
T.C., KNOBL, P.N.. BAUER, K., TRAGL, K.H. and DEUTSCH, E. Quantitative determination of human protein C. Evaluation of a "fast" functional assay in comparison to a "traditional" functional and an immunological assay. Thromb. Res. 49, 169-179, 1988.
31. BERTINA, R.M.,
BROEKMANS, A.W., KROMMENHOEK-VAN ES, C. and VAN WIJNGAARDEN, A. The use of a functional and immunologic assay for plasma protein C in the study of the heterogeneity of congenital protein C deficiency. Thromb. Haemostas. r, 1-5, 1984.
32. SALA, N.,
OWEN, W.G. and COLLEN, D. A functional assay of human plasma. -Blood 63, 671-675, 1984.
33. ESPANA, F., ESTELLES, A.,
protein C in
AZNAR, J. and GILABERT, J. Assay of protein C in human plasma: comparison of amidolytic, coagulation and immunochemical assays. Thromb. Res. s,, 771-782, 1986.
BRIEF
COMMUNICATION
APROTININ (TRASYLOL) IS A COMPETITIVE INHIBITOR OF ACTIVATED PROTEIN C
FRANCISCO
ESPANA-, AMPARO ESTELLES", JOHN H. GRIFFIN', JUST0 AZNAR-, AND JUAN GILABERT""
Research Center* and Departments of Clinical Pathology** and Obstetrics and Gynecology+@*,Hospital La Fe, Valencia, SPAIN and Committee on Vascular Biology and Department of Molecular and Experimental Medicine, Research Institute of Scripps Clinic, La Jolla, CA, USA.*
(Received 13.9.1989; accepted in revised form 19.9.1989 by Editor H. Vinazzer)
INTRODUCTION Protein C (1,2) is a major regulatory protein of thrombus formation. Besides controlling the thrombin formation, by inactivating factors Va and it promotes fibrinolysis by inactivating the plasminogen VIIIa (3-6), activator inhibitors PAI- (7-9) and PAI- (10). APC is regulated in plasma by two major inhibitors (11). One, named protein C inhibitor (PCI) is heparindependent and has been characterized (10,12-14) and the sequence of its cDNA reported (15), and it has been shown to be identical (16) to a heparindependent urokinase inhibitor also known as PAI- (17). The second protein C inhibitor is heparin-independent and has been demonstrated to be alantitrypsin (18). Aprotinin has a broad inhibitory spectrum. It inhibits human trypsin, plasmin, plasma kallikrein, and tissue kallikreins by forming reversible stoichiometric enzyme-inhibitor complexes (19). While studying the APC inhibition by PC1 we observed that aprotinin, used to inhibit kallikrein in the study, did inhibit APC. That observation prompted us to study the interaction between APC and aprotinin by analyzing the inhibition of the APC amidolytic activity by the inhibitor.
MATERIAL AND METHODS Materials. Human protein C and APC (20) were prepared as indicated before. Aprotinin (Trasylol) was obtained from Calbiochem, La Jolla, CA. 18 U/ml aprotinin equaled to 1 pg/mL and to 150 nM. S-2366 chromogenic substrate was from Kabi, Stockholm. Bovine serum albumin (BSA) and heparin were from Sigma Chemical Co., St. Louis. Key
Words:
Protein C, Aprotinin, Trasylol 751
752
APC INHIBITION BY TRASYLOL
Vol. 56, No. 6
Amidolytic assay. To test the inhibitory effect of aprotinin on APC, purified APC (0.8 pal/L) was incubated at 37 ‘C with aprotinin (0, 0.6, 3, or 6 pal/L) in 0.05 mol/L Tris-HCl, 0.10 mol/L NaCl, and 0.1% BSA, pli 7.4, with or without the addition of heparin. Controls were performed by incubating APC and buffer containing BSA in the presence or the absence of heparin. In each case, 20 PL-aliquots were removed at time intervals and added to 180 PL of a mixture containing 1, 0.75 or 0.5 mmol/L S-2366, 0.05 mol/L Tris-HCl, 0.10 mol/L NaCl, 0.004 mol/L CaC12, pH 8.2, in the wells of microtitter plates and the rate of change in absorbance during the first three minutes was determined every 1 min using an ELISA plate reader and expressed as percent of that obtained with controls. Determination of enzyme inhibitor constants was carried out by the method reported by Dixon (21) plotting l/v versus i at three different substrate the- reaction -velocity and i is the inhibitor concentrations, where v is concentration. This gives straight lines which will cut each-other at a point the-vertical axis, at -KL, which can therefore be read off on the left of directly.
RESULTS AND DISCUSSION aprotinin concentrations resulted in timeIncubation of APC with different of its amidolytic activity. This inhibition, however, independent inhibition APC inhibition was already was dose-dependent (Figure 1). Thus, in each case after 1 ?in ? incubation with the enzyme, the extension of the completed inhibition being dependent of the inhibitor concentration used.
I Trrrllrl
I, )I
of activated protein C (APC) by trasylol. 25 /JL Figure 1. Inhibition APC (0.8 pal/L) and 25 PL trasylol were incubated at 37 ‘C for 5 removed and added to 180 NIL of a 20 PL-aliquot was min. Thereafter, 0.75 mmol/L S-2366 in the wells of microtitter plates and the APC inhibition was calculated as indicated in Methods.
To see whether the inhibitor was a competitive inhibitor, residual A three different substra determined using was amidolytic activity after 3 min incubation with the inhibitor. Figure 2 shows t concentrations, when the reciprocal of v (aA/mi modified Lineweaver & Burk plots obtained
Vol. 56, No. 6
APC INHIBITION BY TRASYLOL
was plotted versus the inhibitor concentration. The three lines cut each other at a point. This point lies at -K+. From this experiment/a value of 1.44 /JM the inhibition of APC amidolytic activity by for Ka was calculated. Thus, aprotinin is of a competitive nature. m -
Figure 2. Dixon’s plots for the inhibition of APC by trasylol. APC and trasylol were incubated as indicated in Fig.1. 20 PL-aliquots were removed and added to 180 PL of 1, 0.75 or 0.5 mmol/L S-2366 in the wells of microtitter plates and the APC inhibition was calculated as indicated in Methods.
Since APC binds to heparin and since a major plasma APC inhibitor, PCI, is heparin-dependent (10,12,14), we studied the effect of heparin on the APC inhibition by aprotinin. Figure 3 shows the results obtained. Heparin increased the affinity of the inhibitor for the enzyme in a dose dependent manner , resulting in decreased Ka values with maximum effect at about 4 u/mL.
Figure 3. Inf 1.uence of heparin on APC inhibition by trasylol. The experiment out as indicated in Fig. 2, except that APC was carried and aprot i nin were incubated in the presence of several heparin concentrat i ons . KS values were calculated as indicated in Methods.
APC INHIBITION BY TRASYLOL
754
Vol. 56, No. 6
One u/mL heparin decreased about S-fold the Ki value, whereas at 10 .u/mL heparin the inhibitor constant decreased 17-fold (Figure 3). Although the binding of aprotinin with human plasmin (K+=lnM) (22) is tighter than the binding with APC, the K+ for APC reported here, 80 to 100 nM in the presence of heparin, is in the same range that the Ki for plasma kallikrein, about 30 nM and still within the therapeutic range of aprotinin (23). Whether the APC inhibition by aprotinin is relevant in vivo, or contributes to the antifibrinolytic action of the aprotinin remains to be demonstrated. Protein C assays have been developed in which activation of protein C is achieved directly in plasma using snake venoms (24-30), and measuring the APC amidolytic and/or anticoagulant activity. In these cases, the presence of trasylol in plasma will interfere in the assays specially when, as usual, heparin is co-administered with trasylol, giving underestimation of protein C values. This could be overcome using other methods in which a previous absorption step is used (31-33).
ACKNOWLEDGEMENTS This study was supported in part by a National Institutes of Health Grant HL-31950, and by a grant No. 88/1623 from the Fondo de Investigaciones Sanitarias de la Seguridad Social, Spain.
REFERENCES A new vitamin K-dependent protein: Purification from bovine preliminary characterization. J. Biol. Chem. 251, 355-363,
1.
STENFLO, J. plasma and 1976.
2.
KISIEL, W. Human plasma protein C. Isolation, characterization and mechanism of activation by a-thrombin. J. Clin. Invest. 66, 761-769, 1979.
3.
KISIEL, W., CANFIELD, W.M., ERICSSON, L.H. and DAVIE, E.W. Anticoagulant properties of bovine plasma protein C following activation by thrombin. Biochemistry 16, 5824-5831, 1977.
4.
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. Biophys. Acta 571, 333-342, 1979.
5.
VEHAR, G. and DAVIE, E.W.Preparation and properties of bovine factor VIII. Biochemistry 2, 401-410, 1980.
6.
FULCHER, C.A., GARDINER, J.E., GRIFFIN, J.H. and ZIMMERMANN, T.S. Proteolytic inactivation of human factor VIII procoagulant protein by activated human protein C and its analogy with factor V. -Blood 63, 486-489, 1984.
7.
SAKATA, Y., CURRIDEN, S.A., LAWRENCE, D.A., GRIFFIN, J.H. and LOSKUTOFF, D.J. Activated protein C stimulates the fibrinolytic activity of cultured endothelial cells and decreases antiactivator activity. Proc. Natl. Acad. Sci. USA, g, 1121-1125, 1985.
Vol. 56, No. 6
APC INHIBITION BY TRASYLOL
755
8.
TAYLOR, F.B. and LOCKHART, M.S. A new function for activated protein C: Activated protein C prevents inhibition of plasminogen activators by releasate from mononuclear leukocytes-platelet suspensions stimulated by phorbol diester. Thromb. Res. 37_, 155-164, 1985.
9.
VAN HINSBERG, V.N.M., BERTINA, R.M., VAN WIJNGAARDEN, A., VAN TILBURG, J.H., EMEIS, J. and HAVERKATE, F. Activated protein C decreases plasminogen activator-inhibitor activity in endothelial cell-conditioned medium. Blood 65, 444-451, 1985. --
10. ESPANA, F., BERRETTINI, M. and GRIFFIN, J.H. Purification and characterization of plasma protein C inhibitor. Thromb. Res. 1989. In press. 11. HEEB, M.J., ESPANA, F. and GRIFFIN, J.H. Inhibition and complexation of activated protein C by two major inhibitors in plasma. -Blood 73, 446-454, 1989. 12. SUZUKI, K., NISHIOKA, J. and HASHIMOTO, S. Protein C inhibitor. Purification from human plasma and characterization. J. Biol. Chem. 258, 163168, 1983. 13. SUZUKI, K., NISHIOKA, J., KUSUMOTO, H. and HASHIMOTO, S. Mechanism of inhibition of activated protein C by protein C inhibitor. J. Biochem. 2, 187-195, 1984. 14. MEIJERS, J.C.M.', KANTERS, D.H.A.J., VLOOSWIJK, R.A.A., VAN ERP, H.E., HESSING, M. and BOUMA, B.N. Inactivation of human plasma kallikrein and factor_XIa by protein C inhibitor. Biochemistry -27, 4231-4237, 1988. 15. SUZUKI, K., DEYASHIKI, Y., NISHIOKA, J., KURACHI, K., AKIRA, M., YAMAMOTO, S. and HASHIMOTO, S. Characterization of a cDNA for human protein C inhibitor. J. Biol. Chem. 262, 611-616, 1987. 16. HEEB, M.J., ESPANA, F., GEIGER, M., COLLEN, D., STUMP, D. and GRIFFIN, J.H. Immunological identity of heparin-dependent plasma and urinary protein C inhibitor and plasminogen activator inhibitor-3. J. Biol. Chem. 262, 15813-15816, 1987. 17. STUMP, D.C., THIENPONT, M. and COLLEN, D. Purification and characterization of a novel inhibitor of urokinase from human urine. J. Biol. Chem. 261, 12759-12766, 1986. 18. HEEB, M.J. and GRIFFIN, J.H. Physiologic inhibition of human activated protein C by or-antitrypsin. J. Biol. Chem. 263, 11613-11616, 1988. 19. VERSTRAETE, M. Clinical application of inhibitors of fibrinolysis. Drugs 29, 236-261, 1985. 20. 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 primate model. -Blood 73, 639-642, 1989. 21. DIXON, M. The determination of enzyme inhibitor constants. Biochem. J. 3, 170-171, 1953.
756
APC INHIBITION BY TRASYLOL
Vol. 56, No. 6
22. WIMAN, B. On the reaction of plasmin or plasminstreptokinase complex aprotinin or az-antiplasmin. Thromb. Res. 5, 143,152, 1980.
with
23. PHILIPP, E. Calculations and hypothetical considerations on the inhibition
plasmin and plasma kallikrein by trasylol. In: Progress in chemical fibrinolysis and thrombolysis. Davidson et al. (Eds.) New York: Raven Press, 1978, pp. 291-295. 24. FRANCIS, R.B. and SEYFERT, U. Rapid amidolytic assay of protein C in whole
plasma using an activator from the venom of agkistrodon contortrix. Amer. J. Clin. Pathol. 87, 619-625, 1987. 25. HICKTON, C.M., FELDING, P.. IKEDA, K., MARTINSSON, G. and NILSSON, I.M. A functional assay of protein C in human plasma. Thromb. Res. 41, 501-508, 1986. 26. MARTINOLI, J.L.
and STOCKER, K. Fast functional protein C assay using protac, a novel protein C activator. Thromb. Res. 2, 253-264, 1986.
27. MCCALL, F.,
protein C in
CONKIE, J.A., WALKER, I.D. and DAVIDSON, J.F. Measurement of plasma - a fully automated assay. Thromb. Res. 5, 681-685,
1987. 28. NATHAN, I.V., PING, H. and PRADHAN, M.M.
snake venom activator. Thromb. Res. z,
Protein C functional assay using 85-91, 1987.
29. ODEGAARD, O.R.,
TRY, K. and ABILGAARD, U. Protein C: A simplified semiautomated activity assay. Thromb. Res. 42, 257-262, 1986.
30. VUKOVICH,
T.C., KNOBL, P.N.. BAUER, K., TRAGL, K.H. and DEUTSCH, E. Quantitative determination of human protein C. Evaluation of a "fast" functional assay in comparison to a "traditional" functional and an immunological assay. Thromb. Res. 49, 169-179, 1988.
31. BERTINA, R.M.,
BROEKMANS, A.W., KROMMENHOEK-VAN ES, C. and VAN WIJNGAARDEN, A. The use of a functional and immunologic assay for plasma protein C in the study of the heterogeneity of congenital protein C deficiency. Thromb. Haemostas. r, 1-5, 1984.
32. SALA, N.,
OWEN, W.G. and COLLEN, D. A functional assay of human plasma. -Blood 63, 671-675, 1984.
33. ESPANA, F., ESTELLES, A.,
protein C in
AZNAR, J. and GILABERT, J. Assay of protein C in human plasma: comparison of amidolytic, coagulation and immunochemical assays. Thromb. Res. s,, 771-782, 1986.