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Thromb Haemost 2013; 109(06): 1016-1024
DOI: 10.1160/TH12-10-0766
Blood Coagulation, Fibrinolysis and Cellular Haemostasis
Schattauer GmbH

Covalently linking heparin to antithrombin enhances prothrombinase inhibition on activated platelets

Ivan Stevic
1   Thrombosis and Atherosclerosis Research Institute, McMaster University, Hamilton, Ontario, Canada
3   Department of Pediatrics, McMaster University, Hamilton, Ontario, Canada
,
Howard H. W. Chan
1   Thrombosis and Atherosclerosis Research Institute, McMaster University, Hamilton, Ontario, Canada
2   Department of Medicine, McMaster University, Hamilton, Ontario, Canada
,
Ankush Chander
1   Thrombosis and Atherosclerosis Research Institute, McMaster University, Hamilton, Ontario, Canada
3   Department of Pediatrics, McMaster University, Hamilton, Ontario, Canada
,
Leslie R. Berry
1   Thrombosis and Atherosclerosis Research Institute, McMaster University, Hamilton, Ontario, Canada
3   Department of Pediatrics, McMaster University, Hamilton, Ontario, Canada
,
Anthony K. C. Chan
1   Thrombosis and Atherosclerosis Research Institute, McMaster University, Hamilton, Ontario, Canada
3   Department of Pediatrics, McMaster University, Hamilton, Ontario, Canada
› Author Affiliations
Further Information

Publication History

Received: 23 October 2012

Accepted after major revision: 21 February 2013

Publication Date:
22 November 2017 (online)

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Summary

Factor (F)Xa within the prothrombinase complex is protected from inhibition by unfractionated heparin (UFH), enoxaparin and fondaparinux. We have developed a covalent antithrombin-heparin complex (ATH) with enhanced anticoagulant activity. We have also demonstrated that ATH is superior at inhibiting coagulation factors when assembled on artificial surfaces. The objective of the present study is to determine the ability of ATH vs AT+UFH to inhibit FXa within the prothrombinase complex when the enzyme complex is assembled on the more native platelet system. Discontinuous inhibition assays were performed to determine final k 2-values for inhibition of FXa, FXa within the platelet-prothrombinase, or FXa within prothrombinase devoid of various components. Thrombin generation and plasma clotting was also assayed in the presence of resting/activated platelets ± inhibitors. Protection of FXa was not observed for ATH, whereas a moderate 60% protection was observed for AT+UFH. ATH inhibited platelet-prothrombinase ∼4-fold faster than AT+UFH. Relative to intact prothrombinase, rates for FXa inhibition by AT+UFH in prothrombinase complexes devoid of either prothrombin (II)/activated platelets/FVa were higher. However, inhibition by AT+UFH of prothrombinase devoid of FII yielded slightly lower rates compared to free FXa inhibition. Thrombin generation and plasma clotting was enhanced with activated platelets, while inhibition was better by ATH compared to AT+UFH, thus suggesting an overall enhanced anticoagulant activity of ATH against platelet-bound prothrombinase complexes.

Keywords

Coagulation - covalent antithrombin-heparin - heparin - platelets - prothrombinase complex
 

  • References

  • 1 Suttie JW, Jackson CM. Prothrombin structure, activation and biosynthesis. Physiol Rev 1977; 57: 1-70.
    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 Mann KG, Jenny RJ, Krishnaswamy S. Cofactor proteins in the assembly and expression of blood clotting enzyme complexes. Ann Rev Biochem 1988; 57: 915-956.
    CrossrefPubMedGoogle Scholar
  • 4 Kalafatis M, Swords NA, Rand MD. et al. Membrane-dependent reactions in blood coagulation: role of the vitamin k-dependent enzyme complexes. Biochem Biophys Acta 1994; 1227: 113-119.
    CrossrefPubMedGoogle Scholar
  • 5 Wilkens M, Krishnaswamy S. The contribution of factor Xa to exosite-dependent substrate recognition by prothrombinase. J Biol Chem 2002; 277: 9366-9374.
    CrossrefPubMedGoogle Scholar
  • 6 Davie EW, Fujikawa K, Kisiel W. The coagulation cascade: initiation, maintenance, and regulation. Biochemistry 1991; 30: 10363-10370.
    CrossrefPubMedGoogle Scholar
  • 7 Damus PS, Hicks M, Rosenberg RD. Anticoagulant action of heparin. Nature 1973; 246: 355-357.
    CrossrefPubMedGoogle Scholar
  • 8 Olds RJ, Lane DA, Mille B. et al. Antithrombin: the principal inhibitor of thrombin. Semin Thromb Hemost 1994; 20: 353-372.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 9 Jin L, Abrahams JP, Skinner R. et al. The anticoagulant activation of antithrombin by heparin. Proc Natl Acad Sci USA 1997; 94: 14683-14688.
    CrossrefPubMedGoogle Scholar
  • 10 Hatton MWC, Berry LR, Machovich R. et al. Titration of commercial heparins by reaction with NaB3WH4: chemical analysis and biological properties of the product. Analyt Biochem 1980; 106: 417-426.
    CrossrefPubMedGoogle Scholar
  • 11 Hirsh J, Anand SS, Halperin JL. et al. Mechanism of action and pharmacology of unfractionated heparin. Arterioscler Thromb Vasc Biol 2001; 21: 1094-1096.
    CrossrefPubMedGoogle Scholar
  • 12 Hoylaerts M, Owen WG, Collen D. Involvement of heparin chain length in the heparin-catalyzed inhibition of thrombin by antithrombin III. J Biol Chem 1984; 259: 5670-5677.
    PubMedGoogle Scholar
  • 13 Hirsh J, Bates SM. Clinical trials that have influenced the treatment of venous thromboembolism: A historical perspective. Ann Intern Med 2001; 134: 409-417.
    CrossrefPubMedGoogle Scholar
  • 14 Hirsh J, Warkentin TE, Shaughnessy SG. et al. Heparin and low-molecular-weight haprin: mechanism of action, pharmacokinetics, dosing, monitoring, efficacy, and safety. Chest 2001; 119: 64S-94S.
    CrossrefPubMedGoogle Scholar
  • 15 Boneu S, Caranobe C, Gabaig AM. et al. Evidence for a saturable mechanism of disappearance of standard heparin in rabbits. Thromb Res 1987; 46: 835-844.
    CrossrefPubMedGoogle Scholar
  • 16 Chong BH. Heparin-induced thrombocytopenia. J Thromb Haemost 2003; 01: 1471-1478.
    CrossrefPubMedGoogle Scholar
  • 17 Marciniak E. Factor-Xa inactivation by antithrombin. 3. Evidence for biological stabalization of factor Xa by factor V-phospholipid complex. Br J Haematol 1973; 24: 391-400.
    CrossrefPubMedGoogle Scholar
  • 18 Rezaie AR. Prothrombin protects factor Xa in the prothrombinase complex from inhibition by heparin-antithrombin complex. Blood 2001; 97: 2308-2313.
    CrossrefPubMedGoogle Scholar
  • 19 Bruffato N, Ward A, Nesheim ME. Factor Xa is highly protected from antithrombin-fondaparinux and antithrombin-enoxaparin when incorporated into the prothrombinase complex. J Thromb Haemost 2003; 01: 1258-1263.
    CrossrefPubMedGoogle Scholar
  • 20 Teitel JM, Rosenberg RD. Protection of factor Xa from neutralization by heparin-antithrombin complex. J Clin Invest 1983; 71: 1383-1391.
    CrossrefPubMedGoogle Scholar
  • 21 Schoen P, Lindhout T, Willems G, Hemker C. Antithrombin III-dependent antiprothrombinase activity of heparin and heparin fragments. J Biol Chem 1989; 264: 10002-10007.
    PubMedGoogle Scholar
  • 22 Ellis V, Scully MF, Kakkar VV. The acceleration of the inhibition of platelet prothrombinase by heparin. Biochem J 1986; 233: 161-165.
    CrossrefPubMedGoogle Scholar
  • 23 Barrowcliffe TW, Havercroft SJ, Kemball-Cook G. et al. 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
  • 24 Stevic I, Berry LR, Chan AKC. Mechanism of inhibition of the prothrombinase complex by a covalent antithrombin-heparin complex. J Biochem 2012; 152: 139-148.
    CrossrefPubMedGoogle Scholar
  • 25 Chan AKC, Berry LR, O’brodovich H. et al. Covalent antithrombin-heparin complexes with high anticoagulant activity: Intravenous, subcutaneous, and intratracheal administration. J Biol Chem 1997; 272: 22111-22117.
    CrossrefPubMedGoogle Scholar
  • 26 Parmar N, Berry LR, Post M. et al. Effect of covalent antithrombin-heparin complex on developmental mechanisms in the lung. Am J Physiol Lung Cell Mol Physiol 2009; 296: L394-L403.
    CrossrefPubMedGoogle Scholar
  • 27 Smith LJ, Mewhort-Buist TA, Berry LR, Chan AKC. An antithrombin-heparin complex increases the anticoagulant activity of fibrin clots. Res Lett Biochem 2008; 2008: 639829.
    PubMedGoogle Scholar
  • 28 Patel S, Berry LR, Chan AKC. Analysis of inhibition rate enhancement by covalent linkage of antithrombin to heparin as a potential predictor reaction mechanism. J Biochem 2007; 141: 25-35.
    PubMedGoogle Scholar
  • 29 Klement P, Berry LR, Liao P. et al. Antithrombin-heparin covalent complex reduces microemboli during cardiopulmonary bypass in a pig model. Blood 2010; 116: 5716-5723.
    CrossrefPubMedGoogle Scholar
  • 30 Chan AKC, Rak J, Berry LR. et al. Antithrombin-heparin covalent complex: A possible alternative to heparin for arterial thrombosis prevention. Circulation 2002; 106: 261-265.
    CrossrefPubMedGoogle Scholar
  • 31 Paredes N, Wang A, Berry LR. et al. Mechanisms responsible for catalysis of the inhibition of factor Xa or thrombin by antithrombin using a covalent antithrombin-heparin complex. J Biol Chem 2003; 278: 23398-23409.
    CrossrefPubMedGoogle Scholar
  • 32 Weitz JI, Hudoba M, Massel D. et al. 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
  • 33 Gaffet P, Bettache N, Bienvenue A. Phosphatidylserine exposure on the platelet plasma membrane during A23187-induced activation is independent of cytoskeleton reorganization. Eur J Cell Biol 1995; 67: 336-345.
    PubMedGoogle Scholar
  • 34 Wood J, Silveira JR, Maille NM. et al. Prothrombin activation on the activated platelet surface optimizes expression of procoagulant activity. Blood 2011; 117: 1710-1718.
    CrossrefPubMedGoogle Scholar
  • 35 Rosing J, Zwaal RFA, Tans G. Formation of meizothrombin as intermediate in factor Xa-catalyzed prothrombin activation. J Biol Chem 1986; 261: 4224-4228.
    PubMedGoogle Scholar
  • 36 Krishnaswamy S, Mann KG, Nesheim ME. The prothrombinase-catalyzed activation of prothrombin proceeds through the intermediate meizothrombin in an ordered, sequential reaction. J Biol Chem 1986; 261: 8977-8984.
    PubMedGoogle Scholar
  • 37 Lindhout T, Baruch D, Schoen P. et al. Thrombin generation and inactivation in the presence of antithrombin III and heparin. Biochemistry 1986; 25: 5962-5969.
    CrossrefPubMedGoogle Scholar
  • 38 Maccarana M, Lindahl U. Mode of interaction between platelet factor 4 and heparin. Glycobiology 1993; 03: 271-277.
    CrossrefPubMedGoogle Scholar
  • 39 Francis JL. Detection and significance of heparin-platelet factor 4 antibodies. Semin Hematol 2005; 42: S9-S14.
    PubMedGoogle Scholar
  • 40 Krauel K, Hackbarth C, Furll B. et al. Heparin-induced thrombocytopenia: in vitro studies on the interaction of dabigatran, rivaroxaban, and low-sulfated heparin, with platelet factor 4 and anti-PF4/heparin antibodies. Blood 2012; 119: 1248-1255.
    CrossrefPubMedGoogle Scholar
  • 41 Warkentin TE. Heparin-induced thrombocytopenia: diagnosis and management. Circulation 2004; 110: e454-e458.
    CrossrefPubMedGoogle Scholar