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Thromb Haemost 2001; 86(02): 604-610
DOI: 10.1055/s-0037-1616093
Review Article
Schattauer GmbH

A Sensitive Immunochemical Assay for Measuring the Concentration of the Activated Protein C-protein C Inhibitor Complex in Plasma:

Use of a Catcher Antibody Specific for the Complexed/Cleaved Form of the Inhibitor
Karin Strandberg   *
1   Department of Clinical Chemistry, University Hospital, Malmö, Lund University, Malmö, Sweden
,
Margareta Kjellberg   *
1   Department of Clinical Chemistry, University Hospital, Malmö, Lund University, Malmö, Sweden
,
Richard Knebel
2   Department of Orthopedic Surgery, University Hospital, Malmö, Lund University, Malmö, Sweden
,
Hans Lilja
1   Department of Clinical Chemistry, University Hospital, Malmö, Lund University, Malmö, Sweden
,
Johan Stenflo
1   Department of Clinical Chemistry, University Hospital, Malmö, Lund University, Malmö, Sweden
› Author Affiliations
Further Information

Publication History

Received 22 January 2001

Accepted after resubmission 30 March 2001

Publication Date:
12 December 2017 (online)

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Summary

Activated protein C (APC) is a serine proteinase that regulates blood coagulation. In plasma it is inhibited mainly by the protein C inhibitor (PCI). The plasma concentrations of APC-PCI complex is increased in hypercoagulative states such as deep venous thrombosis. Formation of the APC-PCI complex induces a drastic conformational change in PCI that exposes new epitopes (neoepitopes) on the molecule. We have devised a simple immunofluorometric sandwich assay for measurements of the concentrations of APC-PCI complex, employing as the catcher, a monoclonal antibody that has a high affinity (KD ≈ 4 × 10-11M) for a complexation-specific neoepitope that is expressed on PCI. A monoclonal antibody against protein C is employed as the tracer. The method gives a linear dose-response curve (0.06-50 πg/l), has a low detection limit (0.06 πg/l) and no crossreactivity with native PCI at physiologic plasma concentrations. We have now determined the concentration of the APC-PCI complex in healthy individuals.

Keywords

APC-PCI complex - immunochemical assay - hypercoagulability

* Both authors contributed equally to this work.


 

  • References

  • 1 Dahlbäck B, Stenflo J. The protein C anticoagulant system. The Molecular Basis of Blood Diseases. 3rd ed. Stamatoyannopoulos G, Majerus PW, Perlmutter RM, Varmus H. eds. Philadelphia, PA: W. B Saunders Co; 2001. pp. 614-56.
    Search in Google Scholar
  • 2 Esmon C, Xu J, Gu J-M, Qu D, Laszik Z, Ferrell G, Stearns-Kurosawa D, Kurosawa S, Taylor F, Esmon N. Endothelial protein C receptor. Thromb Haemost 1999; 82: 251-8.
    Thieme ConnectPubMedSearch in Google Scholar
  • 3 Bode W, Turk D, Karshikov A. The refined 1.9-Å X-ray structure of D-PhePro-Arg chloromethylketone-inhibited human α-thrombin: structure analysis, overall structure, electrostatic properties, detailed active-site geometry, and structure-function relationships. Protein Sci 1992; 1: 426-71.
    PubMedSearch in Google Scholar
  • 4 Stein PE, Carrell RW. What do dysfunctional serpins tell us about molecular mobility and disease?. Nature Struct Biol 1995; 2: 96-113.
    CrossrefPubMedSearch in Google Scholar
  • 5 Gettins PGW, Patston PA, Olson ST. Serpins: Structure, Function and Biology. Heidelberg, Germany: Springer-Verlag GmbH & Co; 1996: 33-54.
    Search in Google Scholar
  • 6 Suzuki K, Nishioka J, Hashimoto S. Protein C inhibitor. Purification from human plasma and characterization. J Biol Chem 1983; 258: 163-8.
    PubMedSearch in Google Scholar
  • 7 Suzuki K. Activated protein C inhibitor. Sem Thromb Haemost 1984; 10: 154-61.
    Thieme ConnectPubMedSearch in Google Scholar
  • 8 Heeb MJ, Griffin JH. Physiological inhibition of human activated protein C by α-1-antitrypsin. J Biol Chem 1988; 263: 11613-6.
    PubMedSearch in Google Scholar
  • 9 Pratt CW, Church FC. Heparin binding to protein C inhibitor. J Biol Chem 1992; 267: 8789-879.
    PubMedSearch in Google Scholar
  • 10 Yamada N, Wada H, Nakase T, Minamikawa K, Nagaya S, Nakamura M, Hiraoka N, Fuzioka H, Hayashi T, Suzuki K, Nakano T, Shiku H. Hemostatic abnormalities in patients with pulmonary embolism compared with that in deep vein thrombosis. Blood Coag Fibrinol 1995; 6: 627-33.
    CrossrefPubMedSearch in Google Scholar
  • 11 Tanigawa M, Wada H, Minamikawa K, Wakita Y, Nagaya S, Mori T, Tamaki S, Nishikawa H, Kakuta Y, Nakano T, Hayashi T, Suzuki K, Shiku H. Decreased protein C inhibitor after percutaneous transluminal coronary angioplasty in patients with acute myocardial infarction. Am J Hematol 1995; 49: 1-5.
    CrossrefPubMedSearch in Google Scholar
  • 12 Minamikawa K, Wada H, Wakita Y, Ohiwa M, Tanigawa M, Deguchi K, Hiraoka N, Huzioka H, Nishioka J, Hayashi T, Shirakawa S, Nakano T, Suzuki K. Increased activated protein C-protein C inhibitor complex levels in patients with pulmonary embolism. Thromb Haemost 1994; 71: 192-4.
    Thieme ConnectPubMedSearch in Google Scholar
  • 13 Espana F, Vicente V, Tabernero D, Scharrer I, Griffin JH. Determination of plasma protein C inhibitor and of two activated protein C-inhibitor complexes in normals and in patients with intravascular coagulation and thrombotic disease. Thromb Res 1990; 59: 593-608.
    CrossrefPubMedSearch in Google Scholar
  • 14 Takagi M, Wada H, Mukai K, Minamikawa K, Wakita Y, Deguchi K, Junji N, Hayashi T, Suzuki K, Shiku H. Increased activated protein C: protein C inhibitor complex and decreased protein C inhibitor levels in patients with chronic renal failure on maintenance hemodialysis. Clin Appl Thromb Haemost 1999; 5: 113-6.
    CrossrefPubMedSearch in Google Scholar
  • 15 Stratikos E, Gettins PGW. Formation of the covalent serpin-proteinase complex involves translocation of the proteinase by more than 70 Å and full insertion of the reactive center loop into β-sheet A. Proc Natl Acad Sci USA 1999; 96: 4808-13.
    CrossrefPubMedSearch in Google Scholar
  • 16 Huntington JA, Read RJ, Carrell RW. Structure of a serpin-protease complex shows inhibition by deformation. Nature 2000; 407: 923-6.
    CrossrefPubMedSearch in Google Scholar
  • 17 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; 262: 611-6.
    PubMedSearch in Google Scholar
  • 18 Laurell M, Stenflo J. Protein C inhibitor from human plasma: characterization of native and cleaved inhibitor and demonstration of inhibitor complexes with plasma kallikrein. Thromb Haemost 1989; 62: 885-91.
    Thieme ConnectPubMedSearch in Google Scholar
  • 19 Picard V, Marque P-E, Paolucci F, Aiach M, LeBonniec BF. Topology of the stable serpin-protease complex revealed by an autoantibody that fails to react with the monomeric conformers of antithrombin. J Biol Chem 1999; 274: 4586-93.
    CrossrefPubMedSearch in Google Scholar
  • 20 Nordling K, Björk I. Identification of an epitope in antithrombin appearing on insertion of the reactive-bond loop into the A β-sheet. Biochemistry 1996; 35: 10436-140.
    CrossrefPubMedSearch in Google Scholar
  • 21 Eldering E, Nuijens JH, Hack CE. Expression of functional human C1 inhibitor in COS cells. J Biol Chem 1988; 263: 11776-9.
    PubMedSearch in Google Scholar
  • 22 Björk I, Nordling K, Olson ST. Immunologic evidence for insertion of the reactive-bond loop of antithrombin into the A β-sheet of the inhibitor during trapping of target proteinases. Biochemistry 1993; 32: 6501-5.
    CrossrefPubMedSearch in Google Scholar
  • 23 Debrock S, Declerck P. Characterization of common neoantigenic epitopes generated in plasminogen activator inhibitor-1 after cleavage of the reactive center loop or after complex formation with various serine proteinases. FEBS Lett 1995; 376: 243-6.
    CrossrefPubMedSearch in Google Scholar
  • 24 Espana F, Gruber A, Heeb MJ, Hanson SR, Harker LA, Griffin JH. In vivo and in vitro complexes of activated protein C with twp inhibitors in baboons. Blood 1991; 77: 1754-60.
    PubMedSearch in Google Scholar
  • 25 Laurell M, Stenflo J, Carlson TH. Turnover of *I-protein C inhibitor and *I-α 1-antitrypsin and their complexes with activated protein C. Blood 1990; 76: 2290-5.
    PubMedSearch in Google Scholar
  • 26 Espana F, Griffin JH. Determination of functional and antigenic protein C inhibitor and its complexes with activated protein C in plasma by ELISA’s. Thromb Res 1989; 55: 671-82.
    CrossrefPubMedSearch in Google Scholar
  • 27 Strandberg K, Kjellberg M, Erb E-M, Persson U, Villoutreix BO, Mosher DF, Stenflo J. Activated protein C-protein C inhibitor complex formation: characterization of a neoepitope provides evidence for extensive insertion of the reactive center loop. Biochemistry 2000; 39: 15713-20.
    CrossrefPubMedSearch in Google Scholar
  • 28 Öhlin AK, Landes G, Bourdon P, Oppenheimer C, Wydro R, Stenflo J. β-hydroxyaspartic acid in the first epidermal growth factor-like domain of protein C. Its role in Ca2+ binding and biological activity. J Biol Chem 1988; 263: 19240-8.
    PubMedSearch in Google Scholar
  • 29 Fernlund P, Stenflo J, Roepstorff P, Thomsen J. Vitamin K and the biosynthesis of prothrombin V. γ-carboxyglutamic acids, the vitamin K-dependent structures in prothrombin. J Biol Chem 1975; 250: 6125-33.
    PubMedSearch in Google Scholar
  • 30 Hemmilä I, Dakubu S, Mukkula V-M, Siitari H, Lövgren T. Europium as a label in time-resolved immunofluorometric assays. Anal Biochem 1984; 137: 335-43.
    CrossrefPubMedSearch in Google Scholar
  • 31 Bland M. An Introduction to Medical Statistics. 2nd ed. Oxford, Great Britain: Oxford University Press Inc.; 1995. pp. 276-8.
    Search in Google Scholar
  • 32 Kisiel W. Human plasma protein C. Isolation, characterization and mechanism of activation by α-thrombin. J Clin Invest 1979; 64: 761-9.
    CrossrefPubMedSearch in Google Scholar
  • 33 Dahl OE. Mechanisms of Hypercoagulability. Thromb Haemost 1999; 82: 902-6.
    Thieme ConnectPubMedSearch in Google Scholar
  • 34 Giercksky KE, Björklid E, Prydz H, Renck H. Circulating tissue thromboplastin during hip surgery. Eur Surg Res 1979; 11: 296-300.
    CrossrefPubMedSearch in Google Scholar
  • 35 Planes A, Vochelle N, Darmon JY, Fagola M, Bellaud M, Huet Y. Risk of deep-venous thrombosis after hospital discharge in patients having undergone total hip replacement: double-blind randomised comparison of enoxaparin versus placebo. Lancet 1996; 348: 224-8.
    CrossrefPubMedSearch in Google Scholar
  • 36 Dahl OE, Gudmundsen T, Haukeland L. Late occurring clinical deep vein thrombosis in joint-operated patients. Acta Orthop Scand 2000; 71: 47-50.
    CrossrefPubMedSearch in Google Scholar
  • 37 Alcaraz A, Espana F, Sanchez Cuenza J, Zuazu I, Estelles A, Aznar J, Vicente V. Activation of the protein C pathway in acute sepsis. Thromb Res 1995; 79: 83-93.
    CrossrefPubMedSearch in Google Scholar