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Thromb Haemost 1987; 57(02): 176-182
DOI: 10.1055/s-0038-1651089
Original Article
Schattauer GmbH Stuttgart

The Interaction of Activated Protein C and Thrombin with the Plasminogen Activator Inhibitor Released from Human Endothelial Cells

N J de Fouw
1   The Haemostasis and Thrombosis Research Unit, University Hospital Leiden, Leiden, The Netherlands
2   The TNO Gaubius Institute for Cardiovascular Research, Leiden, The Netherlands
,
V W M van Hinsbergh
2   The TNO Gaubius Institute for Cardiovascular Research, Leiden, The Netherlands
,
Y F de Jong
1   The Haemostasis and Thrombosis Research Unit, University Hospital Leiden, Leiden, The Netherlands
2   The TNO Gaubius Institute for Cardiovascular Research, Leiden, The Netherlands
,
F Haverkate
2   The TNO Gaubius Institute for Cardiovascular Research, Leiden, The Netherlands
,
R M Bertina
1   The Haemostasis and Thrombosis Research Unit, University Hospital Leiden, Leiden, The Netherlands
› Author Affiliations
Further Information

Publication History

Received 17 July 1986

Accepted after revision 08 January 1987

Publication Date:
28 June 2018 (online)

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Summary

The effects of human activated protein C (APC) and thrombin on plasminogen activator inhibitor (PAI-1) released from cultured human umbilical endothelial cells, grown in serum-free 35S-methionine containing medium, were studied in two ways: 1) measurement of PAI-1 activity with an amidolytic assay, and 2) immunoprécipitation of the medium with anti-PAI-1 IgG, antiprotein C IgG or anti-thrombin IgG followed by SDS-PAGE and autoradiography.

Addition of APC or thrombin to the endothelial cell conditioned medium results in a time and concentration dependent loss of PAI-1 activity and in the degradation of PAI-1 from 46 kD into a 42 kD product.

After incubation of the medium with APC in the presence of cells, an additional band of 95 kD was found, which could be immunoprecipitated with both anti-PAI-1 IgG and anti-protein C IgG, indicating the formation of an APC-PAI-1 complex before degradation occurs. No complex could be detected after incubation of the medium with thrombin in the presence of endothelial cells.

Blocking the active sites of APC and thrombin prevented both the formation of APC-PAI-1 complexes and the inactivation and degradation of PAI-1. After removal of the active PAI-1 from the medium, no degradation of the inactive PAI-1 by APC or thrombin could be found.

It is concluded that both APC and thrombin react with the active PAI-1, resulting in inactivation and degradation of PAI-1.

Keywords

Plasminogen activator inhibitor - Activated protein C - Thrombin - Fibrinolysis
 

  • References

  • 1 Griffin JH, Evatt B, Zimmerman TS, Kleiss AA, Wideman C. Deficiency of protein C in congenital thrombotic disease. J Clin Invest 1981; 68: 1370-1373
    CrossrefPubMedGoogle Scholar
  • 2 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
    PubMedGoogle Scholar
  • 3 Comp PC, Esmon CT. Generation of fibrinolytic activity by infusion of activated protein C into dogs. J Clin Invest 1981; 68: 1221-1228
    CrossrefPubMedGoogle Scholar
  • 4 Colucci M, Stassen JM, Collen D. Influence of protein C activation on blood coagulation and fibrinolysis in squirrel monkeys. J Clin Invest 1984; 74: 200-204
    CrossrefPubMedGoogle Scholar
  • 5 Taylor Jr F B, Lockhart MS. A new function for activated protein C: APC prevents inhibition of plasminogen activators by release from mononuclear leukocytes - platelet suspensions stimulated by phorbol-diester. Thromb Res 1985; 37: 155-164
    CrossrefPubMedGoogle Scholar
  • 6 Taylor Jr F B, Lockhart MS. Whole blood clot lysis: in vitro modulation by APC. Thromb Res 1985; 37: 639-649
    CrossrefPubMedGoogle Scholar
  • 7 De Fouw NJ, Haverkate F, Bertina RM, Koopman J, Van Wijngaarden A, Van Hinsbergh V WM. The cofactor role of protein S in the acceleration of whole blood clot lysis by activated protein C in vitro. Blood 1986; 67: 1189-1192
    PubMedGoogle Scholar
  • 8 Burdick MD, Schaub RG. Human protein C produces anticoagulation and increased fibrinolytic activity in the cat. Fed Proc 1986; 45: 1072 (Abstr 5339)
    PubMedGoogle Scholar
  • 9 Sprengers ED, Verheijen JH, Van Hinsbergh V WM, Emeis JJ. Evidence for the presence of two different fibrinolytic inhibitors in human endothelial cell conditioned medium. Biochim Biophys Acta 1984; 801: 163-170
    CrossrefPubMedGoogle Scholar
  • 10 Levin EG. Latent tissue plasminogen activator produced by human endothelial cells in culture: evidence for an enzyme-inhibitor complex. Proc Natl Acad Sci USA 1983; 80: 6804
    CrossrefPubMedGoogle Scholar
  • 11 Sprengers ED, Van Hinsbergh V WM, Jansen BG. The active and the inactive plasminogen activator inhibitor from human endothelial cell conditioned medium are immunologically and functionally related to each other. Biochim Biophys Acta. 1986 (in press)
    PubMedGoogle Scholar
  • 12 Hekman CM, Loskutoff DJ. Endothelial cells produce a latent inhibitor of plasminogen activators that can be activated by denaturants. J Biol Chem 1985; 260: 11581-11587
    PubMedGoogle Scholar
  • 13 Van Mourik JA, Lawrence DA, Loskutoff DJ. Purification of an inhibitor of plasminogen activator (antiactivator) synthesized by endothelial cells. J Biol Chem 1984; 259: 14914-14921
    PubMedGoogle Scholar
  • 14 Van Hinsbergh V WM, 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
    PubMedGoogle Scholar
  • 15 Loskutoff DJ. Effect of thrombin on the fibrinolytic activity of cultured bovine endothelial cells. J Clin Invest 1979; 64: 329-332
    CrossrefPubMedGoogle Scholar
  • 16 Levin EG, Marzec U, Anderson J, Harker LA. Thrombin stimulates t-PA release from cultured human endothelial cells. J Clin Invest 1984; 74: 1988-1995
    CrossrefPubMedGoogle Scholar
  • 17 Gelehrter TD, Sznycer-Laszuk R. Thrombin induction of plasminogen activator-inhibitor in cultured human endothelial cells. J Clin Invest 1986; 77: 165-169
    CrossrefPubMedGoogle Scholar
  • 18 Bertina RM, Broekmans AW, Krommenhoek-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
    PubMedGoogle Scholar
  • 19 Haverkate F, Brakman P. Fibrin plate assay. In Progress in Chemical Fibrinolysis and Thrombolysis. Davidson JF, Samama MM, Desnoyers PC. (eds) Raven Press; New York: 1975. 1 151-159
    Google Scholar
  • 20 Deutsch DG, Mertz ET. Plasminogen: purification from human plasma by affinity chromatography. Science 1970; 170: 1095
    CrossrefPubMedGoogle Scholar
  • 21 Verheijen JH, Chang G TG, 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
    PubMedGoogle Scholar
  • 22 Loskutoff DJ, Van Mourik JA, Erickson LA, Lawrence D. Detection of an unusually stable fibrinolytic inhibitor produced by bovine endothelial cells. Proc Natl Acad Sci USA 1983; 80: 2956-2960
    CrossrefPubMedGoogle Scholar
  • 23 Rijken DC, Wijngaards G, Welbergen J. Immunological characterization of plasminogen activator activities in human tissues and body fluids. J Lab Clin Med 1981; 97: 477-486
    PubMedGoogle Scholar
  • 24 Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins: identification by morphologic and immunologic criteria. J Clin Invest 1973; 52: 2745
    CrossrefPubMedGoogle Scholar
  • 25 Van Hinsbergh V WM, Havekes L, Emeis JJ, Van Corven E, Scheffer M. Low density lipoprotein metabolism by endothelial cells from human umbilical cord arteries and veins. Arteriosclerosis 1983; 3: 547-559
    CrossrefPubMedGoogle Scholar
  • 26 Maciag T, Cerundolo J, Ilsley S, Kelly PR, Forand R. An endothelial cell growth factor from bovine hypothalamus: identification and partial characterization. Proc Natl Acad Sci USA 1979; 76: 5674-5678
    CrossrefPubMedGoogle Scholar
  • 27 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680
    CrossrefPubMedGoogle Scholar
  • 28 Kessler SW. Rapid isolation of antigens from cells with a staphylocial protein A antibody adsorbent parameters of the interaction of antibody antigen complexes with protein A. Immunology 1976; 117: 136-142
    PubMedGoogle Scholar
  • 29 Bollen G HP M, Cohen LH, Mager WH, Klaassen AW, Planta RJ. Isolation of cloned ribosomal protein genes from the yeast Saccharomyces carlobergenosis. Gene 1981; 14: 279-287
    CrossrefPubMedGoogle Scholar
  • 30 Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979; 76: 4350-4354
    CrossrefPubMedGoogle Scholar
  • 31 Blake MS, Johnston KH, Russel-Jones GJ. A rapid sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots. Anal Biochem 1984; 136: 175-179
    CrossrefPubMedGoogle Scholar
  • 32 Levin EG. Quantitation and properties of the active and latent plasminogen activator inhibitors in cultures of human endothelial cells. Blood 1986; 67: 1309-1313
    PubMedGoogle Scholar
  • 33 Kooistra T, Sprengers ED, Van Hinsbergh V WM. Rapid inactivation of plasminogen activator inhibitor. Biochem J 1986; 239: 497-503
    CrossrefPubMedGoogle Scholar
  • 34 Sakata Y, Curriden S, Lawrence D, Griffin JH, Loskutoff DJ. Activated protein C stimulates the fibrinolytic activity of cultured endothelial cells and decrease antiactivator activity. Proc Natl Acad Sci USA 1985; 82: 1121-1125
    CrossrefPubMedGoogle Scholar
  • 35 Travis J, Salvesen GS. Human plasma proteinase inhibitors. Ann Rev Biochem 1983; 52: 655-709
    CrossrefPubMedGoogle Scholar
  • 36 Nielsen LS, Andreasen PA, Grøndahl-Hansen J, Skriver L, Danø K. Plasminogen activators catalyse conversion of inhibitor from fibrosarcoma cells to an inactive form with a lower apparent molecular mass. FEBS Lett 1986; 196: 269-273
    CrossrefPubMedGoogle Scholar
  • 37 Ny T, Sawdey M, Lawrence D, Millan JL, Loskutoff DJ. Cloning and sequence of a cDNA coding for the human β-migrating endothelial cell-type plasminogen activator inhibitor. Proc Natl Acad Sci USA 1986; 83: 6776-6780
    CrossrefPubMedGoogle Scholar
  • 38 Pannekoek H, Veerman H, Lambers H, Diergaarde P, Verwey CC, Van Zonneveld AJ, Van Mourik JA. Endothelial plasminogen activator inhibitor (PAI); a new member of the Serpin gene family. The EMBO Journal 1986; 10: 2539-2544
    PubMedGoogle Scholar
  • 39 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 Biochem 1984; 96: 455
    CrossrefPubMedGoogle Scholar
  • 40 Lockhart MS, Taylor Jr F B, Vigano D’Angelo S, D’Angelo A. Neutralization of plasminogen activator inhibitor by activated protein C: The role of protein S and phospholipids. Fibrinolysis; abstract of the eighth International Congress on Fibrinolysis. Vienna: 1986. (Abstr 38)
    Google Scholar