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

Karin Strandberg; Margareta Kjellberg; Richard Knebel; Hans Lilja; Johan Stenflo

doi:10.1055/s-0037-1616093

Thrombosis and Haemostasis, Inhaltsverzeichnis

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

Authors

Karin Strandberg^*

¹Department of Clinical Chemistry, University Hospital, Malmö, Lund University, Malmö, Sweden
Margareta Kjellberg^*

¹Department of Clinical Chemistry, University Hospital, Malmö, Lund University, Malmö, Sweden
Richard Knebel

²Department of Orthopedic Surgery, University Hospital, Malmö, Lund University, Malmö, Sweden
Hans Lilja

¹Department of Clinical Chemistry, University Hospital, Malmö, Lund University, Malmö, Sweden
Johan Stenflo

¹Department of Clinical Chemistry, University Hospital, Malmö, Lund University, Malmö, Sweden

Abstract

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 (K_D ≈ 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

Volltext

Referenzen

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.
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.
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.
4 Stein PE, Carrell RW. What do dysfunctional serpins tell us about molecular mobility and disease?. Nature Struct Biol 1995; 2: 96-113.
5 Gettins PGW, Patston PA, Olson ST. Serpins: Structure, Function and Biology. Heidelberg, Germany: Springer-Verlag GmbH & Co; 1996: 33-54.
6 Suzuki K, Nishioka J, Hashimoto S. Protein C inhibitor. Purification from human plasma and characterization. J Biol Chem 1983; 258: 163-8.
7 Suzuki K. Activated protein C inhibitor. Sem Thromb Haemost 1984; 10: 154-61.
8 Heeb MJ, Griffin JH. Physiological inhibition of human activated protein C by α-1-antitrypsin. J Biol Chem 1988; 263: 11613-6.
9 Pratt CW, Church FC. Heparin binding to protein C inhibitor. J Biol Chem 1992; 267: 8789-879.
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.
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.
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.
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.
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.
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.
16 Huntington JA, Read RJ, Carrell RW. Structure of a serpin-protease complex shows inhibition by deformation. Nature 2000; 407: 923-6.
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.
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.
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.
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.
21 Eldering E, Nuijens JH, Hack CE. Expression of functional human C1 inhibitor in COS cells. J Biol Chem 1988; 263: 11776-9.
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.
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.
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.
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.
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.
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.
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.
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.
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.
31 Bland M. An Introduction to Medical Statistics. 2nd ed. Oxford, Great Britain: Oxford University Press Inc.; 1995. pp. 276-8.
32 Kisiel W. Human plasma protein C. Isolation, characterization and mechanism of activation by α-thrombin. J Clin Invest 1979; 64: 761-9.
33 Dahl OE. Mechanisms of Hypercoagulability. Thromb Haemost 1999; 82: 902-6.
34 Giercksky KE, Björklid E, Prydz H, Renck H. Circulating tissue thromboplastin during hip surgery. Eur Surg Res 1979; 11: 296-300.
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.
36 Dahl OE, Gudmundsen T, Haukeland L. Late occurring clinical deep vein thrombosis in joint-operated patients. Acta Orthop Scand 2000; 71: 47-50.
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.