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Thromb Haemost 1989; 62(03): 885-891
DOI: 10.1055/s-0038-1651022
Original Article
Schattauer GmbH Stuttgart

Protein C Inhibitor from Human Plasma: Characterization of Native and Cleaved Inhibitor and Demonstration of Inhibitor Complexes with Plasma Kallikrein

Martin Laurell
The Department of Clinical Chemistry, Malmö General Hospital Malmö, Sweden
,
Johan Stenflo
The Department of Clinical Chemistry, Malmö General Hospital Malmö, Sweden
› Author Affiliations
Further Information

Publication History

Received 17 March 1989

Accepted after revision 20 July 1989

Publication Date:
30 June 2018 (online)

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Summary

Protein C inhibitor (PCI) was purified from human plasma using immunoaffinity chromatography and heparin Sepharose chromatography, a method that allowed the purification of active and inactive inhibitor. PCI purified from outdated plasma was inactive and either in complex with plasma kallikrein or proteo-lytically degraded. Sequence analysis of cleaved PCI and of complexes between PCI and activated protein C or urokinase identified the previously recognized inhibitor cleavage site Arg354-Ser355. Two additional cleavage sites were observed in the modified inhibitor i.e. Arg357-Leu358 and Arg362-Leu363 which probably represent secondary cleavage of the inhibitor. Furthermore the sequence analysis of the inhibitor, whether purified from fresh or outdated plasma, revealed that it was microheterogeneous in the NH2-terminus as a result of cleavage by a trypsin like enzyme(s).

Keywords

Protein C - Protein C inhibitor - Plasma kallikrein - Urokinase - Heparin
 

  • References

  • 1 Walker FJ, Sexton PW, Esmon CT. The inhibition of blood coagulation by activated Protein C through the selective inactivation of activated factor V. Biochim Biophys Acta 1979; 571: 333-342
    CrossrefPubMedGoogle Scholar
  • 2 Dahlbäck B, Femlund P, Stenflo J. Inhibitors: protein C. Elsevier. Amsterdam 1986; 285-306
    PubMedGoogle Scholar
  • 3 Esmon CT. The regulation of natural anticoagulant pathways. Science 1987; 235: 1348-1352
    CrossrefPubMedGoogle Scholar
  • 4 Marlar RA, Griffin JH. Deficiency of protein C inhibitor in combined factor V/VIII deficiency disease. J Clin Invest 1980; 66: 1186-1189
    CrossrefPubMedGoogle Scholar
  • 5 Suzuki K, Nishioka J, Hashimoto S. Protein C inhibitor. Purification from human plasma and characterization. J Biol Chem 1983; 258: 163-168
    PubMedGoogle Scholar
  • 6 Suzuki K, Deyashiki Y, Nishioka J, Kurachi K, Akira M, Yamamoto S, Hashimoto S. Characterization of a cDNA for human protein C inhibitor. A new member of the plasma serine protease inhibitor superfamily. J Biol Chem 1987; 2262: 611-616
    PubMedGoogle Scholar
  • 7 Van der Meer FJ M, Van Tillburg NH, Van der Linden IK, Briet E, Bertina RM. A second inhibitor for activated protein C (APC). Thromb Haemostas 1987; 58: 277 (Abstr).
    PubMedGoogle Scholar
  • 8 Heeb MJ, Griffin JH. Physiologic inhibiton of human activated protein C by alpha 1-antitrypsin. J Biol Chem 1988; 263: 11613-11616
    PubMedGoogle Scholar
  • 9 Suzuki K, Nishioka J, Kusumoto H, Hashimoto S. Mechanism of inhibiton of activated protein C by protein C inhibitor. J Biochem (Tokyo) 1984; 95: 187-195
    CrossrefPubMedGoogle Scholar
  • 10 Travis J, Salvesen GS. Human plasma proteinase inhibitor. Annu Rev Biochem 1983; 52: 655-709
    CrossrefPubMedGoogle Scholar
  • 11 Carell R, Travis J. Alfa 1-antitrypsin and the serpins: variation and countervariation. TIBS 1985; 10: 20-24
    PubMedGoogle Scholar
  • 12 Kazama Y, Niwa M, Yamagishi R, Takahashi K, Sakuragawa N, Takeheiko K. Specificity of sulfated polysaccharides to accelerate the inhibition of activated protein C inhibitor. Thromb Res 1987; 48: 179-185
    CrossrefPubMedGoogle Scholar
  • 13 Silverberg M, Diehl SV. The activation of the contact system of human plasma by polysaccharide sulfates. Anal NY Acad Sci 1987; 516: 269-279
    PubMedGoogle Scholar
  • 14 Stump DC, Thienpont M, Collen D. Urokinase related proteins in human urine. J Biol Chem 1986; 261: 1267-1273
    PubMedGoogle Scholar
  • 15 Stump DC, Thienpont M, Collen D. Purification and characterization of a novel inhibitor of urokinase from human urine. Quantitation and preliminary characterization in plasma. J Biol Chem 1986; 261: 1259-1266
    PubMedGoogle Scholar
  • 16 Heeb MJ, Espana F, Geiger M, Collen D, Stump DC, Griffin JH. Immunologic identity of heparin-dependent plasma and urinary Protein C inhibitor and plasminogen activator inhibitor-3. J Biol Chem 1987; 262: 15813-15816
    PubMedGoogle Scholar
  • 17 Stief TW, Radke K-P, Heimburger N. Evidence for identity of PCI and plasminogen activator inhibitor 3. Biol Chem Hoppe-Seyler 1987; 368: 1427-1433
    PubMedGoogle Scholar
  • 18 Laurell M, Carlson T, Stenflo J. Monoclonal antibodies against the heparin dependant protein C inhibitor ; suitable for inhibitor purification and assay of inhibitor complexes. Thromb Haemostas 1988; 60: 334-339
    PubMedGoogle Scholar
  • 19 Comp PC, Nixon RR, Cooper MR, Esmon CT. Familial protein S deficiency is associated with recurrent thrombosis. J Clin Invest 1984; 674: 2082-2088
    PubMedGoogle Scholar
  • 20 Laurell M, Ikeda K, Lindgren S, Stenflo J. Characterization of monoclonal antibodies against human protein C specific for the calcium ion-induced conformation or for the activation peptide region. FEBS Lett 1985; 191: 75-81
    CrossrefPubMedGoogle Scholar
  • 21 Kiesel W, Davie EW. protein C. Meth Enzymol 1981; 80: 320-333
    PubMedGoogle Scholar
  • 22 Angeles-Cano E, Sultan Y. A solid phase fibrin immunoassay for the specific detection of monoclonal antibodies against different epitopic determinants of tissue plasminogen activators. J Immunol Meth 1984; 69: 115-127
    CrossrefPubMedGoogle Scholar
  • 23 Johansson BG. Agarose screen electrophoresis. In: Electrophoretic and electro-immunochemical analysis of plasma proteins. Laurell CB. (ed) (; 12429). Universitätsförlaget Oslo; 1972: 07-19
    Google Scholar
  • 24 Olafsson I, Gudmunsson G, Abrahamson M, Jensson O, Grubb A. The aminoterminal portion of cerebrospinal fluid cystatin C in hereditary cystatin C amyloid angiopathy is not truncated. Direct sequence analysis from agarose electropherograms. Accepted for publication in: Scand. J Clin Invest 1989
    Google Scholar
  • 25 Maizel Jr JV. Methods. Virology 1971: 179-246
    Google Scholar
  • 26 Matsudaira P. Sequence from picomole quantities of proteins electro-blotted onto polyvinylidene difluoride membranes. J Biol Chem 1987; 262: 10035-10038
    PubMedGoogle Scholar
  • 27 Chung DW, Fujikawa K, McMullen BA, Davie EW. Human plasma prekallikrein, a zymogen to a serine protease that contains four tandem repeats. Biochemistry 1986; 25: 2410-2417
    CrossrefPubMedGoogle Scholar
  • 28 Carell RW, Boswell DR, Brennan SO, Owen MC. Active site of antitrypsin; homologous site in antithrombin. Biochem Biophys Res Commun 1980; 93: 399-402
    CrossrefPubMedGoogle Scholar
  • 29 Radke KP, Stief TW, Heimburger N. A new and simple isolation procedure for human protein C inhibitor. Evidence for a second inhibitor for activated protein C present in human plasma. Biol Chem Hoppe-Seyler 1988; 369: 965-974
    PubMedGoogle Scholar
  • 30 Schapira M. Major inhibitors of the contact phase coagulation factors. Semin Thromb Hemostas 1987; 13: 69-78
    Article in Thieme ConnectPubMedGoogle Scholar
  • 31 Meijers JC M, Kanters DH A J, Vloswijk RA A, van Erp HE, Hessing M, Bouma BB. Inactivation of human plasma kallikrein and Factor XI a by protein C inhibitor. Biochemistry 1988; 27: 4231-4237
    CrossrefPubMedGoogle Scholar
  • 32 Kluft C, Dooijewaard G, Emesis JJ. Role of contact system in fibrinolysis. Sem Thromb Hemostas 1987; 13: 50-68
    Article in Thieme ConnectPubMedGoogle Scholar
  • 33 Geiger M, Heeb MJ, Binder BR, Griffin JH. Competition of activated protein C (APC) and urokinase (UK) for a heparin-dependent inhibitor. FASEB J 1988; 2: 2263-2267
    CrossrefPubMedGoogle Scholar
  • 34 Carrell RW, Christey PB, Boswell DR. Serpins: antithrombin and other inhibitors of coagulation and fibrinolysis. Evidence from amino acid sequence Leuven University Press; Leuven: 1987: 01-15
    Google Scholar
  • 35 Potempa J, Shieh B-H, Travis J. Alpha-2-antiplasmin: A serpin with two separate but overlapping reactive sites. Science 1988; 241: 699-700
    CrossrefPubMedGoogle Scholar
  • 36 Salvesen GS, Catanese JJ, Kress LF, Travis J. Primary structure of the reactive site of human Cl-inhibitor. J Biol Chem 1985; 260: 2432-2436
    PubMedGoogle Scholar
  • 37 Morii M, Odani S, Koide T, Ikenaka T. Human alphax-antitrypsin. Characterization and N-and C-terminal sequences. J Biochem 1978; 83: 269-277
    CrossrefPubMedGoogle Scholar