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Thromb Haemost 1993; 69(05): 422-429
DOI: 10.1055/s-0038-1651627
Original Article
Coagulation
Schattauer GmbH Stuttgart

Self-Association of Antithrombin III Relates to Multimer Formation of Thrombin-Antithrombin III Complexes

Klaus T Preissner
The Haemostasis Research Unit, Kerckhoff-Klinik, MPG, Bad Nauheim, Germany
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Publication History

Received 30 June 1992

Accepted after revision 04 January 1993

Publication Date:
25 July 2018 (online)

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Summary

During the reaction of antithrombin III (AT III) with target proteases the inhibitor serves as pseudo-substrate and undergoes profound conformational changes, becomes incorporated into a covalent stoichiometric enzyme-inhibitor complex which is, in contrast to native AT III, recognized by monoclonal antibody 4C9. In the absence of the target enzyme thrombin, incubation of AT III with 1–2 M guanidine, 0.6% deoxycholate, heating to 56° C, or buffer at pH 4 resulted in inactivation of the inhibitor with concomitant exposure of the epitope for 4C9 and formation of AT III multimers (from 3.9S to 7.1–7.4 S). Loss of activity, formation of multimers and exposure of neoepitope(s) of AT III occurred in a concerted fashion and followed second order kinetics with an activation energy of E a = 31 kcal/mol. AT III-multimerization induced by treatment with 1 M guanidine (mainly AT III-tetramers with M r of 250,000) and formation of the binary AT III-thrombin complex revealed similar self-association patterns as judged by gel electrophoresis under non-denaturing conditions. In the presence of heparin, even higher multimers of AT III-thrombin complexes were noted. Moreover, self-association products of the ternary vitronectin-thrombin-AT III complex, which is the ultimate reaction product following thrombin inhibition in the circulation, could be recognized and quantitated due to exposure of the 4C9 epitope on AT III, indicating that AT III exists in multimeric forms within binary and ternary complexes. It is proposed that the ability of these complexes to form high M r association products is at least in part mediated by the propensity of AT III to multimerize and that multimeric forms of AT III may be involved during clearance of AT III-enzyme complexes.

 

  • References

  • 1 Lane DA, Caso R. Antithrombin: Structures, genomic organization, function and inherited deficiency. Baillière Clin Haematol 1989; 2: 961-998
    PubMedGoogle Scholar
  • 2 Downing MR, Bloom JW, Mann KG. Comparison of the inhibition of thrombin by three plasma protease inhibitors. Biochemistry 1978; 17: 2649-2653
    CrossrefPubMedGoogle Scholar
  • 3 Rosenberg RD, Damus PS. The purification and mechanisms of action of human antithrombin-heparin cofactor. J Biol Chem 1973; 248: 6490-6505
    PubMedGoogle Scholar
  • 4 Jörnvall H, Fish WW, Björk I. The thrombin cleavage site in bovine antithrombin. FEBS Lett 1979; 106: 358-362
    CrossrefPubMedGoogle Scholar
  • 5 Jordan RE, Oosta GM, Gardner WT, Rosenberg RD. The kinetics of hemostatic enzyme-antithrombin interactions in the presence of low molecular weight heparin. J Biol Chem 1980; 255: 10081-10090
    PubMedGoogle Scholar
  • 6 Villanueva GB, Danishefsky I. Evidence for a heparin-induced conformational change on antithrombin III. Biochem Biophys Res Commun 1977; 74: 803-809
    CrossrefPubMedGoogle Scholar
  • 7 Nordenman B, Danielsson A, Björk I. The binding of low-affinity and high-affinity heparin to antithrombin. Fluorescence studies. Eur J Biochem 1978; 90: 1-6
    CrossrefPubMedGoogle Scholar
  • 8 Villanueva GB, Danishefsky I. Conformational changes accompanying the binding of antithrombin III to thrombin. Biochemistry 1979; 18: 810-817
    CrossrefPubMedGoogle Scholar
  • 9 Griffith MJ. Kinetics of the heparin-enhanced antithrombin III/thrombin reaction. Evidence for a template model for the mechanism of action of heparin. J Biol Chem 1982; 257: 7360-7365
    CrossrefPubMedGoogle Scholar
  • 10 Amiral J, Vissac AM, Mimilla F, Grosley B. Assay of blood activation by measurement of AT III-serine esterase complexes using a specific monoclonal antibody 4C9. Thromb Haemostas 1989; 62: 479 (Abstract)
    PubMedGoogle Scholar
  • 11 Jesty J. Dissociation of complexes and their derivatives formed during inhibition of bovine thrombin and activated factor X by antithrombin III. J Biol Chem 1979; 254S: 1044-1049
    PubMedGoogle Scholar
  • 12 Fish WW, Björk I. Release of a two-chain form of antithrombin from the antithrombin-thrombin complex. Eur J Biochem 1979; 101: 31-38
    CrossrefPubMedGoogle Scholar
  • 13 Marciniak E. Thrombin-induced proteolysis of human antithrombin III: An outstanding contribution of heparin. Br J Haematol 1981; 48: 325-336
    CrossrefPubMedGoogle Scholar
  • 14 Björk I, Fish WW. Production in vitro and properties of a modified form of bovine antithrombin, cleaved at the active site by thrombin. J Biol Chem 1982; 257: 9487-9493
    PubMedGoogle Scholar
  • 15 Villanueva GB, Allen N. Demonstration of a two-domain structure of antithrombin III during its denaturation in guanidinium chloride. J Biol Chem 1983; 258: 11010-11013
    PubMedGoogle Scholar
  • 16 Villanueva GB, Allen N. Refolding properties of antithrombin III. J Biol Chem 1983; 258: 14048-14053
    PubMedGoogle Scholar
  • 17 Fish WW, Danielsson A, Nordling K, Miller SH, Lam CF, Björk I. Denaturation behavior of antithrombin in guanidinium chloride. Irreversibility of unfolding caused by aggregation. Biochemistry 1985; 24: 1510-1517
    CrossrefPubMedGoogle Scholar
  • 18 Pepper DS, Banhegyi D, Cash JD. The different forms of antithrombin III in serum. Thromb Haemostas 1977; 38: 494-503
    PubMedGoogle Scholar
  • 19 Marciniak E, Gora-Maslak G. High molecular weight forms of antithrombin III complexes in blood. Thromb Haemostas 1983; 49: 32-36
    PubMedGoogle Scholar
  • 20 Danielsson A, Björk I. Properties of antithrombin-thrombin complex formed in the presence and in the absence of heparin. Biochem J 1983; 213: 345-353
    CrossrefPubMedGoogle Scholar
  • 21 Preissner KT, Zwicker L, Müller-Berghaus G. Formation, characterization and detection of a ternary complex between S protein, thrombin and antithrombin III in serum. Biochem J 1987; 243: 105-111
    CrossrefPubMedGoogle Scholar
  • 22 Preissner KT, Podack ER, Müller-Eberhard HJ. Self-association of the seventh component of human complement (C7): dimerization and polymerization. J Immunol 1985; 135: 452-458
    PubMedGoogle Scholar
  • 23 Podack ER, Tschopp J. Polymerization of the ninth component of complement (C9): Formation of poly(C9) with a tubular ultrastructure resembling the membrane attack complex of complement. Proc Natl Acad Sci USA 1982; 79: 574-578
    CrossrefPubMedGoogle Scholar
  • 24 Preissner KT, Podack ER, Müller-Eberhard HJ. The membrane attack complex of complement: relation of C7 to the metastable membrane binding site of the intermediate complex C5b-7. J Immunol 1985; 135: 445-451
    PubMedGoogle Scholar
  • 25 Tschopp J, Müller-Eberhard HJ, Podack ER. Formation of transmembrane tubules by spontaneous polymerization of the hydrophilic complement protein C9. Nature 1982; 298: 534-538
    CrossrefPubMedGoogle Scholar
  • 26 Miller-Andersson M, Borg H, Andersson L-O. Purification of antithrombin III by affinity chromatography. Thromb Res 1974; 5: 439-452
    CrossrefPubMedGoogle Scholar
  • 27 Miletich JP, Broze GJ, Majerus PW. Purification of human coagulation factors II, IX and X using sulfated dextran beads. Meth Enzymol 1981; 80: 221-228
    PubMedGoogle Scholar
  • 28 Lundblad RL, Uhteg LC, Vogel CN, Kingdon HS, Mann KG. Preparation and partial characterization of two forms of bovine thrombin. Biochem Biophys Res Commun 1975; 66: 482-489
    CrossrefPubMedGoogle Scholar
  • 29 Fenton II JW, Fasco MJ. Polyethylene glycol 6,000 enhancement of the clotting of fibrinogen solutions in visual and mechanical assays. Thromb Res 1974; 4: 809-817
    CrossrefPubMedGoogle Scholar
  • 30 Preissner KT, Wassmuth R, Müller-Berghaus G. Physicochemical characterization of human S-protein and its function in the blood coagulation system. Biochem J 1985; 231: 349-355
    CrossrefPubMedGoogle Scholar
  • 31 Nordenman B, Nystrom C, Björk I. The size and shape of human and bovine antithrombin III. Eur J Biochem 1977; 78: 195-203
    CrossrefPubMedGoogle Scholar
  • 32 Fenton II JW, Fasco MJ, Stackrow AB, Aronson DL, Young AM, Finlayson JS. Human thrombins. Productions, evaluation, and properties of α-thrombin. J Biol Chem 1977; 252: 3587-3598
    PubMedGoogle Scholar
  • 33 Dahlbäk B, Podack ER. Characterization of human S protein, an inhibitor of the membrane attack complex of complement. Demonstration of a free reactive thiol group. Biochemistry 1985; 24: 2368-2374
    CrossrefPubMedGoogle Scholar
  • 34 Fraker PJ, Speck JC. Protein and cell membrane iodination with a sparingly soluble chloramide, l,3,4,6-tetrachloro-3a,6a-diphenyl-glycoluril. Biochem Biophys Res Commun 1978; 80: 849-857
    CrossrefPubMedGoogle Scholar
  • 35 Preissner KT, Delvos U, Müller-Berghaus G. Binding of thrombin to thrombomodulin accelerates inhibition of the enzyme by antithrombin III. Evidence for a heparin-independent mechanism. Biochemistry 1987; 26S: 2521-2528
    PubMedGoogle Scholar
  • 36 O’Brien RD, Timpone CA, Gibson RE. The measurement of partial specific volumes and sedimentation coefficients by sucrose density centrifugation. Anal Biochem 1978; 86: 602-617
    CrossrefPubMedGoogle Scholar
  • 37 Laemmli UK. Cleavage of structure proteins during assembly of the head bacteriophage T4. Nature 1970; 227: 680-685
    CrossrefPubMedGoogle Scholar
  • 38 Pollard TD, Cooper JA. Actin and actin-binding proteins. A critical evaluation of mechanisms and functions. Annu Rev Biochem 1986; 55: 987-1035
    CrossrefPubMedGoogle Scholar
  • 39 Hantgan RR, Francis CW, Scheraga HA, Marder VJ. Fibrinogen structure and physiology. In: Hemostasis and Thrombosis. Colman RW, Hirsh J, Marder VJ, Salzman EW. (eds). Lippincott Company; Philadelphia, PA: 1987: 269-288
    Google Scholar
  • 40 Carrell RW, Evans DL. Serpins: mobile conformations in a family of proteinase inhibitors. Curr Opin Struct Biol 1992; 2: 438-446
    CrossrefPubMedGoogle Scholar
  • 41 Busby TF, Atha DH, Ingham KC. Thermal denaturation of antithrombin III. Stabilization by heparin and lyotropic anions. J Biol Chem 1981; 256: 12140-12147
    PubMedGoogle Scholar
  • 42 Carrell RW, Evans DL, Stein PE. Mobile reactive centre of serpins and the control of thrombosis. Nature 1991; 353: 576-578
    CrossrefPubMedGoogle Scholar
  • 43 Lomas DA, Evans DL, Finch JT, Carrell RW. The mechanism of Z α1-antitrypsin accumulation in the liver. Nature 1992; 357: 605-607
    CrossrefPubMedGoogle Scholar
  • 44 Lane DA, Ireland H, Olds RJ, Thein SL, Perry DL, Aiach M. Antithrombin III: a database of mutations. Thromb Haemostas 1992; 66: 657-661
    PubMedGoogle Scholar
  • 45 Tschopp J. Circular polymerization of the membranolytic ninth component of complement. Dependence on metal ions. J Biol Chem 1984; 259: 10569-10573
    PubMedGoogle Scholar
  • 46 Shifman MA, Pizzo SV. The in vivo metabolism of antithrombin III and antithrombin III complexes. J Biol Chem 1982; 257: 3243-3248
    PubMedGoogle Scholar
  • 47 Smedsröd B, Pertoft H, Custafson S, Laurent TC. Scavenger functions of the liver endothelial cell. Biochem J 1990; 266: 313-327
    CrossrefPubMedGoogle Scholar
  • 48 de Boer HC, Preissner KT, Bouma BN, de Groot PG. Binding of vitronectin-thrombin-antithrombin III complex to human endothelial cells is mediated by the heparin binding site of vitronectin. J Biol Chem 1992; 267: 2264-2268
    PubMedGoogle Scholar
  • 49 Perlmutter DH, Glover GI, Rivetna M, Schasteen CS, Fallon RJ. Identification of a serpin-enzyme complex receptor on human hepatoma cells and human monocytes. Proc Natl Acad Sci USA 1990; 87: 3753-3757
    CrossrefPubMedGoogle Scholar
  • 50 Hekman CM, Loskutoff DJ. Bovine plasminogen activator inhibitor 1: Specificity determinations and comparion of the active, latent, and guanidine-activated forms. Biochemistry 1988; 27: 2911-2918
    CrossrefPubMedGoogle Scholar
  • 51 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
  • 52 Katagiri K, Okada K, Hattori H, Yano M. Bovine endothelial cell plasminogen activator inhibitor. Purification and heat activation. Eur J Biochem 1988; 176: 81-87
    CrossrefPubMedGoogle Scholar