Activated Protein C Increases Fibrin Clot Lysis by Neutralization of Plasminogen Activator Inhibitor No Evidence for a Cofactor Role of Protein S

N J de Fouw; Y F de Jong; F Haverkate; R M Bertina

doi:10.1055/s-0038-1647055

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1988; 60(02): 328-333
DOI: 10.1055/s-0038-1647055

Original Article

Schattauer GmbH Stuttgart

Activated Protein C Increases Fibrin Clot Lysis by Neutralization of Plasminogen Activator Inhibitor No Evidence for a Cofactor Role of Protein S

N J de Fouw

¹The Gaubius Institute TNO, University Hospital, Leiden, The Netherlands

²The Haemostasis and Thrombosis Research Unit, University Hospital, Leiden, The Netherlands

,

Y F de Jong

¹The Gaubius Institute TNO, University Hospital, Leiden, The Netherlands

²The Haemostasis and Thrombosis Research Unit, University Hospital, Leiden, The Netherlands

,

F Haverkate

¹The Gaubius Institute TNO, University Hospital, Leiden, The Netherlands

,

R M Bertina

²The Haemostasis and Thrombosis Research Unit, University Hospital, Leiden, The Netherlands

› Author Affiliations

Abstract

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summary

The effect of purified human activated protein G (APC) on fibrinolysis was studied using a clot iysis system consisting of purified glu-plasminogen, tissue-type plasminogen activator, plasminogen activator inhibitor (released from endothelial cells or blood platelets), fibrinogen, ¹²⁵T-fibrinogen and thrombin. All proteins were of human origin.

In this system APC could increase fibrinolysis in a dose dependent way, without affecting fibrin formation or fibrin crosslinking. However, this profibrinolytic effect of APC could only be observed when plasminogen activator inhibitor (PAI-l) was present. The effect of APC was completely quenched by pretreatment of APC with anti-protein C IgG or di-isopropylfluorophosphate. Addition of the cofactors of APC:protein S, Ca²⁺-ions and phospholipid-alone or in combination did not enhance the profibrinolytic effect of APC. These observations indicate that human APC can accelerate in vitro clot lysis by the inactivation of PAI-1 activity. However, the neutralization of PAI-1 by APC is independent of the presence or absence of protein S, phospholipid and Ca²⁺-ions.

Keywords

Activated protein C - Plasminogen activator inhibitor-1 - Protein S - Fibrinolysis

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References

References
1 Esmon CT, Esmon NL. Protein C activation. Semin Thromb Hemostas 10: 122-130
2 Fulcher CA, Gardiner JE, Griffin JH, Zimmerman TS. Proteolytic inactivation of human factor VIII procoagulant protein by activated human protein C and its analogy with factor V. Blood 1984; 53: 486-489
3 Odegaard B, Mann K. Proteolysis of Factor Va by Factor Xa and activated protein C. J Biol Chem 1987; 262: 11233-11238
4 Marlar RA, Kleiss AJ, Griffin JH. Mechanism of action of human activated protein C, a thrombin-dependent anticoagulant enzyme. Blood 1982; 59: 1067-1072
5 Comp PC, Esmon GT. Generation of fibrinolytic activity by infusion of activated protein C into dogs. J Clin Invest 1981; 68: 1221-1228
6 Coined M, Stassen JM, Collen DC. Influence of protein C activation on blood coagulation aud fibiiuolysis in squirrel monkeys. J Clin Invest 1984; 74: 200-204
7 Taylor FB, Lockhart MS. Whole blood clot lysis: in vitro modulation by activated piolein C. Tliionib Res 1984; 37: 639-649
8 De Fouw NJ, Ilaverkate F, Bertina RM, Koopman J, Van Wijngaarden A, Van Hinsbergh VWM. The eofactor role of protein S in the acceleration of whole blood clot lysis by activated protein C in vitro. Blood 1986; 67: 1189-1192
9 Burdick MD, Schaub RG. Human protein C produces antieoagulation and increased fibiiuolylic activity the cat. Thromb Res 1987; 45: 413-419
10 Suzuki K, Nishioka J, Matsuda M, Murayama H, Hashimoto S. Protein S is essential for the activated protein C catalyzed inactivation of platelet associated factor Va. J Biocliem 1984; 96: 455-460
11 Dahlback B. Purification of human vitamin K-dependent protein S is and its limited proteolysis by thrombin. Biochem J 1983; 209: 837-846
12 Walker FJ. Protein S and the regulation of APC. Semin Thromb Hemostas 1984; 10: 131-138
13 Marlar RA, Kleiss AJ, Griffin JH. Human protein C: inactivation of factor V and VIII in plasma by the activated molecule. Ann NY Acad Sci 1981; 370: 303-310
14 Van Hinsbergh VM, Bertina RM, Van Wijngaarden A, Van Tilburg NH, Emeis JJ, Haverkate F. Activated protein C decreases plasminogen activator inhibitor activity in endothelial cell conditioned medium. Blood 1985; 65: 444-451
15 Taylor FB, Lockhart MS. A new function of activated protein C: activated protein C prevents inhibition of plasminogen activators by releasate from mononuclear leukocytes-platelet suspensions stimulated by phorhol diester. Thromb Res 1985; 37: 155-164
16 Sakata Y, Curriden S, Lawrence D, Griffin JH, Loskutoff DJ. Activated protein C stimulates the fibrinolytic activity of cultured endothelial cells and decreases antiactivator activity. Proc Natl Acad Sci USA 1985; 82: 1121-1125
17 Taylor Jr FB, Lockhart MS. A new function for activated protein C: APC prevents inhibition of plasminogen activators by release from mononuclear leukocytes-platelets suspensions stimulated by phorhol- diester. Thromb Res 1985; 37: 155-164
18 Sakata Y, Loskutoff DJ, Giadson CL, Hekman CM, Griffin JH. Mechanism of protein C-dependent clot lysis: role of plasminogen activator inhibitor. Blood 1986; 68: 1218-1223
19 D’Angelo A, Lockhart MS, D’Angelo SV, Taylor FB. Protein S is a cofactor for activated protein C neutralization of an inhibitor of plasminogen activation. released from platelets Blood 1987; 69: 231-237
20 De Fouw NJ, Van Hinsbergh VWM, De Jong YF, Haverkate F, Bertina RM. The interaction of activated rprotein C and thrombin with the plasminogen activator inhibitor released from human endothelial cells. Thromb Haemostas 1987; 57: 176-182
21 De Fouw NJ, Bertina RM, Haverkate F. (Abstr) Stimulation of fibrinolysis by activated protein C (APC). Thromb Haemostas 1987; 58: 48
22 Deutsch DG, Mertz ET. Plasminogen: purification from human plasma by affinity chromatography. Science 1970; 170: 1095-1096
23 Kluft C, Van Wezel AL, Van der Velden CAM, Emeis JJ, Verheijen JH, Wijngaards G. (eds) Large scale production of extrinsic (tissue type) plasminogen activator from human melanoma cells In: Advances in Biotechnological Processes Mizrahi A Van Wezel A L Alan R Liss. New York 1983; 2: 097-110
24 Rijken DC, Collen D. Purification and characterization of the plasminogen activator secreted by human melanoma cells in culture. J Biol Chem 1981; 256: 7035-7041
25 Verheijen JH, Mullaart E, Chang GTG, Kluft C, Wijngaards G. A simple, sensitive spectrophotometric assay for extrinsic (tissue-type) plasminogen activator applicable to measurements in plasma. Thromb Haemostas 1982; 48: 266-269
26 Bertina RM, Broekmans AW, Krotnmenhoek-van EsC, 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 1984; 51: 1-5
27 Bertina RM, Broekmans AW, Van der Linden IK, Mertens K. Protein C deficiency in a Dutch family with thrombotic disease. Thromb Haemostas 1982; 48: 1-5
28 Bertina RM, Van Wijngaarden A, Reinalda-Poot J, Poort SR, Bom VJJ. Determination of plasma protein S the protein cofactor of activated protein C. Thromb Haemostas 1985; 53: 268-272
29 Verheijen JH, Chang GTG, Kluft C. Evidence for the occurrence of a fast-acting inhibitor for tissue-type plasminogen activator in human plasma. Thromb Haemostas 1984; 51: 392-395
30 Sprengers ED, Akkerman JWN, Jansen BG. Blood platelet plasminogen activator inhibitor: two different pools of endothelial cell type plasminogen activator inhibitor in human blood. Thromb JIaemostas 1986; 55: 325-329
31 Folch L, Lees M, Sloane SGH. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 1957; 226: 497-509
32 Botteher CJF, Van Gent CM, Pries C. A rapid and sensitive submicro phosphorus determination. Anal Chim Acta 1961; 24: 203-204
33 Van den Besselaar AMHP, Ram IE, Alderkamp GHJ, Bertina RM. The role of factor IX in tissue thromboplastin induced coagulation. Thromb Haemostas 1982; 48: 54-58
34 Mertens K, Cupers R, Van Wijngaarden A, Bertina RM. Binding of human blood coagulation Factors IXa and X to phospholipid membranes. Biochem J 1984; 223: 599-605
35 De Fouw NJ, Haverkate F, Bertina RM. (ed) In: Protein C and Related Proteins Biochemical and Clinical Aspects Bertina R M Churchill Livingstone. Edinburgh 1988: 71-90
36 Erickson LA, Heckman CM, Loskutoff DJ. The primary plasminogen-activator inhibitors in endothelial cells platelets, serum and plasma are immunologically related. Proe Natl Acad Sci USA 1985; 82: 8710-8714
37 Erickson LA, Ginsberg MH, Loskutoff DJ. Detection and partial characterization of an inhibitor of plasminogen activator in human platelets. J Clin Invest 1984; 74: 1465-1472