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Thromb Haemost 1988; 60(02): 334-339
DOI: 10.1055/s-0038-1647056
Original Article
Schattauer GmbH Stuttgart

Monoclonal Antibodies Against the Heparin- Dependent Protein C Inhibitor Suitable for Inhibitor Purification and Assay of Inhibitor Complexes

Martin Laurell
1   The Department of Clinical Chemistry, Malmo General Hospital, Malmo, Sweden
,
Timothy H Carlson
1   The Department of Clinical Chemistry, Malmo General Hospital, Malmo, Sweden
,
Johan Stenflo
1   The Department of Clinical Chemistry, Malmo General Hospital, Malmo, Sweden
› Author Affiliations
Further Information

Publication History

Received 18 April 1988

Accepted after revision 22 June 1988

Publication Date:
28 June 2018 (online)

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summary

Two different monoclonal antibodies against the heparin- dependent inhibitor of human activated protein C were produced, using cleaved modified inhibitor for immunization and partially purified inhibitor for screening of the hybridomas, One of the antibodies recognized free and complexed forms of the inhibitor in immunoblotting experiments. The other antibody was used to develop an assay for A PC -PCI inhibitor complexes. Using the assay the formation of complexes was studied in plasma, both in the presence and absence of heparin. The rate of complex formation was similar to that reported previously for the loss of activated protein C amidolytic activity in plasma. The same antibody was also immobilized on Sepharose and used to purify the inhibitor from fresh human plasma. The purified material appeared as two narrowly spaced bands with Mr about 57,000 in SDS-PAGE. The average yield from 1 liter of fresh plasma was 1 mg of inhibitor, The purified inhibitor formed SDS stable complexes with activated protein G and urokinase that could be identified in immunoblots using specific antibodies.

Keywords

Protein C - Protein C inhibitor
 

  • References

  • 1 Esmon CT. The regulation of natural anticoagulant pathways. Science 1987; 235: 1348-1352
    CrossrefPubMedGoogle Scholar
  • 2 Clouse LH, Comp PC. The regulation of hemostasis: the protein C system. N Engl J Med 1986; 314: 1298-1304
    CrossrefPubMedGoogle Scholar
  • 3 Dahlback B, Femlund P, Stenflo J. (eds) Inhibitors: Protein C. In: Blood Coagulation. Zwaal RFA Hemker H C . Elsevier Sci Publ. Amsterdam 1986: 285-306
    Google Scholar
  • 4 Seligsohn U, Berger A, Abend M, Rubin L, Attias D, Zivelin A, Rapaport SI. Homozygous protein. C deficiency manifested by massive venous thrombosis in the newborn. N Engl J Med 1984; 310: 559-562
    CrossrefPubMedGoogle Scholar
  • 5 Branson HE, Katz J, Marble R, Griffin JH. Inherited protein C deficiency and eoumarin-responsive chronic relapsing purpura fulminans in a newborn infant. Lancet 1983; 2: 1165-1168
    PubMedGoogle Scholar
  • 6 Griffin JH, Evatt B, Zimmerman TS, Kleiss AJ, Wideman C. Deficiency of protein C in congenital thrombotic disease. J Clin Invest 1981; 68: 1370-1373
    CrossrefPubMedGoogle Scholar
  • 7 Brockmans AW, Veltkamp JJ, Bertina RM. Congenital protein C deficiency and venous thromboembolism. A study of three Dutch families. N Engl J Med 1983; 309: 340-344
    CrossrefPubMedGoogle Scholar
  • 8 Jackson CM, Nemcrson Y. Blood coagulation. Annu Rev Biochem 1980; 49: 765-811
    CrossrefPubMedGoogle Scholar
  • 9 Kondo S, Kisiel W. Regulation of Factor Vlla activity in plasma: Evidence that Antithrombin III is the sole plasma inhibitor of human factor Vlla. Thromb Res 1987; 46: 325-335
    CrossrefPubMedGoogle Scholar
  • 10 Kiesel W, Davie EW. Protein C. Meth Enzymol 1981; 80: 320-333
    PubMedGoogle Scholar
  • 11 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
  • 12 Suzuki K, Nishioka J, Hashimoto S. Protein C inhibitor Purification from human plasma and characterization. J Biol Chem 1983; 258: 163-168
    PubMedGoogle Scholar
  • 13 Suzuki K, Nishioka J, Kusumoto H, Hashimoto S. (Tokyo) Mechanism of inhibition of activated protein C by protein C inhibitor. J Biochem 1984; 95: 187-195
    CrossrefPubMedGoogle Scholar
  • 14 Suzuki K, Deyashiki Y, Nishioka j, Jurachi 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; 262: 611-616
    PubMedGoogle Scholar
  • 15 Comp PC, Jacocks RM, Ferrell GL, Esmon CT. Activation of protein C in vivo. J Clin Invest 1982; 70: 127-134
    CrossrefPubMedGoogle Scholar
  • 16 Comp PC. Animal studies of protein c physiology. Sent Thromb Hemostas 1984; 10: 149-153
    PubMedGoogle Scholar
  • 17 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
  • 18 Marlar RA, Endres-Brooks J, Miller C. Serial studies of protein C and its plasma inhibitor in patients with disseminated intravascular coagulation. Blood 1985; 66: 59-63
    PubMedGoogle Scholar
  • 19 Francis Jr RB, Thomas W. Behaviour of protein C inhibitor in intravascular coagulation and liver disease. Thromb Haemostas 1984; 52: 71-74
    PubMedGoogle Scholar
  • 20 Laurell M, Ikeda K, Lindgren S, Stenflo J. Characterization of monoclonal antibodies against human protein C specific for the calcjum ion-induced conformation or for the activation peptide region. FEES Lett 1985; 191: 75-81
    CrossrefPubMedGoogle Scholar
  • 21 Stenflo J. A new vitamin K-dependent protein. J Biol Chem 1976; 251: 355-363
    PubMedGoogle Scholar
  • 22 Comp PC, Nixon RR, Cooper MR, Esmon CT. Familial protein S deficiency is associated with recurrent thrombosis. J Clin Invest 1984; 74: 2082-2088
    CrossrefPubMedGoogle Scholar
  • 23 Thorell JI, Johansson BG. Enzymatic iodination of polypeptides with 125I to high specific activity. BBA 1971; 251: 363-369
    CrossrefPubMedGoogle Scholar
  • 24 Borrebäck CAK, Eylar ME. Production and characterization of a monoclonal antibody against the seed lechtin of the Dolichos Biflorus plant. J Biol Chem 1981; 256: 4723-4725
    PubMedGoogle Scholar
  • 25 Burnette WN. “Western Blotting” Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrvlamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioidinated Protein A. Anal Biochem 1981; 112: 195-203
    CrossrefPubMedGoogle Scholar
  • 26 Johnsson DA, Gautch JW, Sportsman JR, Elder JH. Improved technique utilizing nonfat dry milk for analysis of proteins and nucleic acids transferred to nitrocellulose. Gene Anal Techn 1984; 1: 3-8
    CrossrefPubMedGoogle Scholar
  • 27 Blake MS, Johnston KH, Russel-Jones GJ, Gotschlich EC. A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots. Anal Biochem 1984; 136: 175-179
    CrossrefPubMedGoogle Scholar
  • 28 Suzuki K. Activated protein C inhibitor. Sem Thromb Haemostas 1984; 10: 154-161
    PubMedGoogle Scholar
  • 29 Travis J, Salvesen GS. Human plasma proteinase inhibitor. Annu Rev Biochem 1983; 52: 655-709
    CrossrefPubMedGoogle Scholar
  • 30 Stump DC, Thienpont M, Coilen D. Purification and characterization of a novel inhibitor of urokinase from human urine Quantitation and preliminary characterization in plasma. J Biol Chem 1986; 261: 12759-12766
    PubMedGoogle Scholar
  • 31 Geiger M, Binder BR, Heeb MJ, Griffin JH. Activated protein C (APC) and urokinase (UK) compete for heparin-dependent association with plasma and urinary components. Circ 1986; 74: 0II-234
    PubMedGoogle Scholar
  • 32 Heeb MJ, Espana F, Geiger M, Coilen D, Stump DC, Griffin JH. Immunologic identity of heparin-dependent plasma and urinary Protein C inhibitor and plasminogen activator inhibitor. J Biol Chem 1987; 262: 15813-15816
    PubMedGoogle Scholar
  • 33 Stief TW, Radke KP, Heimburger N. Evidence for identity of PCI and plasminogen activator inhibitor 3. Biol Chem Hoppe-Seyler 1987; 368: 1427-1433
    PubMedGoogle Scholar
  • 34 van der Meer FJM, van Tilburg NH, van der Linden IK, Briet E, Bertina RM. (Abstr) A second inhibitor for activated protein C (APC). Thromb Haemostas 1987; 58: 277
    PubMedGoogle Scholar