Interactions of heparin and a covalently-linked antithrombin-heparin complex with components of the fibrinolytic system

Summary

Unfractionated heparin (UFH) is used as an adjunct during thrombolytic therapy. However, its use is associated with limitations, such as the inability to inhibit surface bound coagulation factors. We have developed a covalent conjugate of antithrombin (AT) and heparin (ATH) with superior anticoagulant properties compared with UFH. Advantages of ATH include enhanced inhibition of surface-bound coagulation enzymes and the ability to reduce the overall size and mass of clots in vivo. The interactions of UFH or ATH with the components of the fibrinolytic pathway are not well understood. Our study utilised discontinuous second order rate constant (k₂ ) assays to compare the rates of inhibition of free and fibrin-associated plasmin by AT+UFH vs ATH. Additionally, we evaluated the effects of AT+UFH and ATH on plasmin generation in the presence of fibrin. The k₂ values for inhibition of plasmin were 5.74 ± 0.28 x 10⁶ M^-1 min^-1 and 6.39 ± 0.59 x 10⁶ M^-1 min^-1 for AT+UFH and ATH, respectively. In the presence of fibrin, the k₂ values decreased to 1.45 ± 0.10 x 10⁶ M^-1 min¹ and 3.07 ± 0.19 x 10⁶ M^-1 min^-1 for AT+UFH and ATH, respectively. Therefore, protection of plasmin by fibrin was observed for both inhibitors; however, ATH demonstrated superior inhibition of fibrin-associated plasmin. Rates of plasmin generation were also decreased by both inhibitors, with ATH causing the greatest reduction (approx. 38-fold). Nonetheless, rates of plasmin inhibition were 2–3 orders of magnitude lower than for thrombin, and in a plasma-based clot lysis assay ATH significantly inhibited clot formation but had little impact on clot lysis. Cumulatively, these data may indicate that, relative to coagulant enzymes, the fibrinolytic system is spared from inhibition by both AT+UFH and ATH, limiting reduction in fibrinolytic potential during anticoagulant therapy.

Keywords

Fibrinolysis - plasmin generation - antithrombin - heparin - antithrombin-heparin covalent complex

• References

• 1 Lucas MA. et al. The binding of human plasminogen to fibrin and fibrinogen. J Biol Chem 1983; 258: 4249-4256.
• 2 de Vries C. et al. Identification of the domains of tissue-type plasminogen activator involved in the augmented binding to fibrin after limited digestion with plasmin. J Biol Chem 1989; 264: 12604-12610.
• 3 Rijken DC. et al. Fibrinolytic properties of one-chain and two-chain human extrinsic (tissue-type) plasminogen activator. J Biol Chem 1982; 257: 2920-2925.
• 4 Hoylaerts M. et al. Kinetics of the activation of plasminogen by human tissue plasminogen activator. J Biol Chem 1982; 257: 2912-2919.
• 5 Fredenburgh JC, Nesheim ME. Lys-plasminogen is a significant intermediate in the activation of Glu-plasminogen during fibrinolysis in vitro. J Biol Chem 1992; 267: 26150-26156.
• 6 Rosenberg RD, Damus PS. The purification and mechanism of action of human antithrombin-heparin cofactor. J Biol Chem 1973; 248: 6490-6505.
• 7 O’Gara PT. et al. 2013 ACCF/AHA Guideline for the management of ST-elevation myocardial infarction: a report of the American College of Cardiology Foundation/American Heart Association Task Force on practice guidelines. Circulation 2013; 127: e362-e425.
• 8 Eisenberg PR. Role of heparin in coronary thrombolysis. Chest 1992; 101: 131S-139S.
• 9 Liang JF. et al. The potential mechanism for the effect of heparin on tissue plasminogen activator-mediated plasminogen activation. Thromb Res 2000; 97: 349-358.
• 10 Edelberg JM, Pizzo SV. Kinetic analysis of the effects of heparin and lipoproteins on tissue plasminogen activator mediated plasminogen activation. Biochemistry 1990; 29: 5906-5911.
• 11 Soeda S. et al. Localization of the binding sites of porcine tissue-type plasminogen activator and plasminogen to heparin. Biochim Biophys Acta 1987; 916: 279-287.
• 12 Young TN. et al. Ionic modulation of the effects of heparin on plasminogen activation by tissue plasminogen activator: the effects of ionic strength, divalent cations, and chloride. Arch Biochem Biophys 1992; 296: 530-538.
• 13 Rijken D. et al. Interaction of plasminogen activators and plasminogen with heparin: effect of ionic strength. Thromb Haemost 1993; 70: 867-872.
• 14 Andrade-Gordon P, Strickland S. Interaction of heparin with plasminogen activators and plasminogen: effects on the activation of plasminogen. Biochemistry 1986; 25: 4033-4040.
• 15 Paques E-P. et al. Study on the mechanism of action of heparin and related substances on the fibrinolytic system: relationship between plasminogen activators and heparin. Thromb Res 1986; 42: 797-807.
• 16 Weitz JI. et al. Standard and low molecular weight heparin have no effect on tissue plasminogen activator induced plasma clot lysis or fibrinogenolysis. Thromb Haemost 1991; 65: 541-544.
• 17 Estes JW. Clinical pharmacokinetics of heparin. Clin Pharmacokinet 1980; 5: 204-220.
• 18 Young E. et al. Heparin binding to plasma proteins, an important mechanism for heparin resistance. Thromb Haemost 1992; 67: 639-643.
• 19 Weitz JI. et al. Clot-bound IIa is protected from inhibition by heparin-anti-thrombin III but is susceptible to inactivation by antithrombin III independent inhibitors. J Clin Invest 1990; 86: 385-391.
• 20 Weitz JI. et al. Thrombin binds to soluble fibrin degradation products where it is protected from inhibition by heparin-antithrombin but susceptible to inactivation by antithrombin-independent inhibitors. Circulation 1998; 97: 544-552.
• 21 Walker FJ, Esmon CT. The effects of phospholipid and factor Va on the inhibition of factor Xa by antithrombin III. Biochem Biophys Res Commun 1979; 90: 641-647.
• 22 Chan A. et al. Covalent antithrombin-heparin complexes with high anticoagulant activity. Intravenous, subcutaneous and intratracheal administration. J Biol Chem 1997; 272: 22111-22117.
• 23 Berry LR. et al. Inhibition of fibrin-bound thrombin by a covalent antithrombin-heparin complex. J Biochem 2002; 132: 167-176.
• 24 Stevic I. et al. Mechanism of inhibition of the prothrombinase complex by a covalent antithrombin-heparin complex. J Biochem 2012; 152: 139-148.
• 25 Stevic I. et al. Covalently linking heparin to antithrombin enhances prothrombinase inhibition on activated platelets. Thromb Haemost 2013; 109: 1016-1024.
• 26 Stevic I. et al. Inhibition of the prothrombinase complex on red blood cells by heparin and covalent antithrombin-heparin complex. J Biochem 2013; 153: 103-110.
• 27 Chan AKC. et al. A novel antithrombin-heparin covalent complex: antithrombotic and bleeding studies in rabbits. Blood Coagul Fibrinolysis 1998; 9: 587-595.
• 28 Paredes N. 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.
• 29 Patel S. et al. Analysis of inhibition rate enhancement by covalent linkage of antithrombin to heparin as a potential predictor of reaction mechanism. J Biochem 2007; 141: 25-35.
• 30 Ewart MR. et al. The proteolytic action of arvin on human fibrinogen. Biochem J 1970; 118: 603-609.
• 31 Schneider M, Nesheim M. A study of the protection of plasmin from antiplasmin inhibition within an intact fibrin clot during the course of clot lysis. J Biol Chem 2004; 279: 13333-13339.
• 32 Kim PY. et al. A high affinity interaction of plasminogen with fibrin is not essential for efficient activation by tissue-type plasminogen activator. J Biol Chem 2012; 287: 4652-4661.
• 33 Jordan RE. et al. The kinetics of haemostatic enzyme-antithrombin interactions in the presence of low molecular weight heparin. J Biol Chem 1980; 255: 10081-10090.
• 34 Anonick PK. et al. Regulation of plasmin, miniplasmin, and streptokinase-plasmin complex by α2-antiplasmin, α2-macroglobulin, and antithrombin III in the presence of heparin. Thromb Res 1990; 59: 449-462.
• 35 Berry L. et al. Investigation of the anticoagulation mechanisms of a covalent antithrombin-heparin complex. J Biol Chem 1998; 273: 34730-34736.
• 36 Collen D, Wiman B. Fast-acting plasmin inhibitor in human plasma. Blood 1978; 51: 563-569.
• 37 Wiman B, Collen D. On the kinetics of the reaction between human antiplasmin and plasmin. Eur J Biochem 1978; 84: 573-578.
• 38 Wiman B. et al. On the kinetics of the reaction between human antiplasmin and a low-molecular-weight form of plasmin. Eur J Biochem 1978; 87: 143-146.
• 39 Wiman B, Collen D. On the mechanism of the reaction between human alpha 2-antiplasmin and plasmin. J Biol Chem 1979; 254: 9291-9297.