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Thromb Haemost 1995; 73(04): 648-653
DOI: 10.1055/s-0038-1653835
Original Articles
Coagulation
Schattauer GmbH Stuttgart

Inhibition of Prothrombinase by Antithrombin-Heparin at a Macroscopic Surface

Han Speijer
The Department of Biochemistry, Cardiovascular Research Institute Maastricht, University of Limburg, Maastricht, The Netherlands
,
Didier Billy
*   Supported by Program Grant 900-526-192 from the Dutch Organization for Scientific Research (NWO)
The Department of Biochemistry, Cardiovascular Research Institute Maastricht, University of Limburg, Maastricht, The Netherlands
,
George Willems
The Department of Biochemistry, Cardiovascular Research Institute Maastricht, University of Limburg, Maastricht, The Netherlands
,
H Coenraad Hemker
The Department of Biochemistry, Cardiovascular Research Institute Maastricht, University of Limburg, Maastricht, The Netherlands
,
Theo Lindhout
The Department of Biochemistry, Cardiovascular Research Institute Maastricht, University of Limburg, Maastricht, The Netherlands
› Author Affiliations
Further Information

Publication History

Received 14 November 1994

Accepted after revision 13 January 1995

Publication Date:
09 July 2018 (online)

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Summary

The antithrombin-dependent inhibition of prothrombinase, assembled at a macroscopic surface, was studied under flow conditions utilizing a tubular flow reactor that consists of a phospholipid-coated glass capillary. Prothrombinase activity was determined from steady-state rates of thrombin production upon perfusion with prothrombin and from factor Va-associated factor Xa activity present in the flow reactor. The prothrombinase density was maintained at a low level (0.03 fmol/cm2) to assure that the rate of thrombin production reflected the amount of prothrombinase present in the capillary. Perfusion of the flow reactor with antithrombin resulted in an exponential decrease of prothrombinase activity in time. The second order rate constant (8.5 × 104 M-1min-1) is comparable with the rate of inactivation of free factor Xa. Inhibition was much faster when antithrombin was complexed with heparin. The second order rate constants of inhibition decreased with decreasing heparin chain length: 9.6 × 107, 4.5 × 107 and 0.39 × 107 M-1min-1 for unffactionated heparin, low molecular weight heparin and synthetic pentasaccharide heparin, respectively. In the presence of prothrombin (0.2 μM), however, the heparin-dependent rate of inhibition of prothrombinase was about 50-fold lower. The heparin-independent inhibition of prothrombinase by antithrombin (4 μM) in the presence of prothrombin (0.2 μM) was virtually negligible. At a 70-fold higher surface density of prothrombinase (2 fmol/cm2) prothrombinase activity was much faster inactivated. The rate of thrombin production, however, was not affected. In conclusion, at low prothrombinase densities, prothrombin efficiently protects prothrombinase from inhibition. At high densities, prothrombinase is much less protected but the higher rate of prothrombinase inactivation has no consequences for the thrombin production because of the transport-limited regime.

 

  • References

  • 1 Hemker HC, Esnouf MP, Hemker PW, Swart AC W, MacFarlane RG. Formation of prothrombin converting activity. Nature 1967; 215: 248-251
    CrossrefPubMedGoogle Scholar
  • 2 Nesheim ME, Taswell JB, Mann KG. The contribution of bovine factor V and factor Va to the activity of prothrombinase. J Biol Chem 1979; 254: 10952-10962
    PubMedGoogle Scholar
  • 3 Rosing J, Tans G, Govers-Riemslag JW P, Zwaal RF, Hemker HC. The role of phospholipids and factor Va in the prothrombinase complex. J Biol Chem 1980; 255: 274-283
    PubMedGoogle Scholar
  • 4 Rosing J, van Rijn JL M L, Bevers EM, van Dieijen G, Comfurius P, Zwaal RF A. The role of activated human platelets in prothrombin and factor X activation. Blood 1990; 65: 319-332
    PubMedGoogle Scholar
  • 5 Mann KG, Nesheim ME, Church WR, Haley P, Krishnaswamy S. Surface- dependent reactions of the vitamin K-dependent enzyme complexes. Blood 1990; 76: 1-16
    CrossrefPubMedGoogle Scholar
  • 6 Zwaal RF A, Comfurius P, Bevers EM. Platelet procoagulant activity and microvesicle formation - its putative role in hemostasis and thrombosis. Biochim Biophys Acta 1992; 1180: 1-8
    CrossrefPubMedGoogle Scholar
  • 7 Esmon CT. The roles of protein C and thrombomodulin in the regulation of blood coagulation. J Biol Chem 1989; 264: 4743-4746
    PubMedGoogle Scholar
  • 8 Egeberg O. Inhereted antithrombin deficiency causing thrombophilia. Thromb Diath Haemorrh 1965; 13: 516-530
    PubMedGoogle Scholar
  • 9 Abildgaard U. Binding of thrombin to antithrombin III. Scand J Clin Lab 1969; 24: 23-27
    CrossrefPubMedGoogle Scholar
  • 10 Rosenberg RD, Damus PS. The purification and mechanism of action of human antithrombin-heparin cofactor. J Biol Chem 1973; 248: 6490-6505
    PubMedGoogle Scholar
  • 11 Danielsson A, Raub E, Lindahl U, Björk I. Role of ternary complexes, in which heparin binds both antithrombin and proteinase, in the acceleration of the reactions between antithrombin and thrombin or factor X a. J Biol Chem 1986; 261: 15467-15473
    CrossrefPubMedGoogle Scholar
  • 12 Olson ST. Transient kinetics of heparin-catalyzed protease inactivation by antithrombin III. Linkage of protease-inhibitor-heparin interactions in the reaction with thrombin J Biol Chem 1988; 263: 1698-1708
    PubMedGoogle Scholar
  • 13 Craig PA, Olson ST, Shore JD. Transient kinetics of heparin-catalyzed protease inactivation by antithrombin III. Characterization of assembly, product formation, and heparin dissociation steps in the factor Xa reaction J Biol Chem 1989; 264: 5452-5461
    PubMedGoogle Scholar
  • 14 Hogg PJ, Jackson CM. Fibrin monomer protects thrombin from inactivation by heparin-antithrombin III: Implications for heparin efficacy. Proc Natl AcadSci USA 1989; 86: 3619-3623
    CrossrefPubMedGoogle Scholar
  • 15 Weitz JI, Hudoba M, Massel D, Maraganora JM, Hirsh J. Clot-bound thrombin is protected from inhibition by heparin-antithrombin III but is susceptible to inactivation by antithrombin III-independent inhibitors. J Clin Invest 1990; 86: 385-391
    CrossrefPubMedGoogle Scholar
  • 16 Eisenberg PR, Siegel JE, Abendschein DR, Miletich JP. Importance of factor Xa in determining the procoagulant activity of whole-blood clots. J Clin Invest 1993; 91: 1877-1883
    CrossrefPubMedGoogle Scholar
  • 17 Marciniak E. Factor-Xa Inactivation by antithrombin III: Evidence for biological stabilization of factor Xa by factor V-phospholipid complex. Br J Haematol 1973; 24: 391-400
    CrossrefPubMedGoogle Scholar
  • 18 Miletich JP, Jackson CM, Majerus PW. Properties of the factor Xa binding site on human platelets. J Biol Chem 1978; 253: 6908-6916
    PubMedGoogle Scholar
  • 19 Teitel JM, Rosenberg RD. Protection of factor Xa from neutralization by the heparin-antithrombin complex. J Clin Invest 1983; 71: 1383-1391
    CrossrefPubMedGoogle Scholar
  • 20 Lindhout T, Baruch D, Schoen P, Franssen J, Hemker HC. Thrombin generation and inactivation in the presence of antithrombin III and heparin. Biochemistry 1986; 25: 5962-5969
    CrossrefPubMedGoogle Scholar
  • 21 Ellis V, Scully MF, Kakkar VV. The acceleration of the inhibition of platelet prothrombinase complex by heparin. Biochem J 1986; 233: 161-165
    CrossrefPubMedGoogle Scholar
  • 22 Barrowcliffe TW, Havercroft SJ, Kemball-Cook G, Lindahl U. The effect of Ca2+, phospholipid, and factor V on the anti-(factor Xa) activity of heparin and its high affinity oligosaccharides. Biochem J 1987; 243: 31-37
    CrossrefPubMedGoogle Scholar
  • 23 Schoen P, Lindhout T, Willems GM, Hemker HC. Antithrombin III dependent anti-prothrombinase activity of heparins of various molecular weight. J Biol Chem 1989; 264: 10002-10007
    PubMedGoogle Scholar
  • 24 Schoen P, Lindhout T. Flow and the inhibition of prothrombinase by antithrombin III and heparin. Blood 1991; 78: 118-124
    PubMedGoogle Scholar
  • 25 Billy D, Speijer H, Willems G, Hemker HC, Lindhout T. Prothrombin activation by prothrombinase in a tubular flow reactor. A kinetic study J Biol Chem 1995; 270: 1029-1034
    CrossrefPubMedGoogle Scholar
  • 26 Schoen P, Wielders S, Petitou M, Lindhout T. The effect of sulfation on the anticoagulant and antithrombin III-binding properties of a heparin fraction with low affinity for antithrombin III. Thromb Res 1990; 57: 415-423
    CrossrefPubMedGoogle Scholar
  • 27 Lindhout T, Govers-Riemslag JW P, van deWaart, Hemker HC, Rosing J. Factor Va-factor Xa interaction. Effects of phospholipid vesicles of varying composition Biochemistry 1982; 21: 5494-5502
    CrossrefPubMedGoogle Scholar
  • 28 Levich VG. Physiochemical Hydrodynamics. Englewood Cliffs, NJ: Prentice-Hall; 1962. p 75
    Google Scholar
  • 29 Atha DH, Lormeau JC, Petitou M, Rosenberg RD, Choay J. Contribution of monosaccharide residues in heparin binding to anti thrombin III. Biochemistry 1985; 24: 6723-6729
    CrossrefPubMedGoogle Scholar
  • 30 Olson ST, Björk I, Sheffer R, Craig PA, Shore JD, Choay J. Role of the antithrombin-binding pentasaccharide in heparin acceleration of antithrombin-proteinase reactions. J Biol Chem 1992; 267: 12528-12538
    PubMedGoogle Scholar
  • 31 Schoen P, Lindhout T, Hemker HC. Ratios of anti-factor Xa to antithrombin activities of heparins as determined in recalcified human plasma. Brit J Haematol 1992; 81: 255-262
    CrossrefPubMedGoogle Scholar