A Comparative Study of Prothrombinase and Thrombin Inhibitors in a Novel Rabbit Model of Non-Occlusive Deep Vein Thrombosis

Summary

A quantitative and non-occlusive deep vein thrombosis model was developed in rabbits. We used this model to test the antithrombotic activity of the prothrombinase complex inhibitors factor rXai and its chemical analog glutamyl-glycyl-arginyl chloromethyl ketone inactivated human factor Xa (EGR-Xai), along with the thrombin inhibitors D-phenylalanyl-prolyl-arginyl chloromethyl ketone (PPACK) and heparin. Dose dependent effects of the inhibitors during constant infusion were monitored. Measurements included thrombus weights, hemostatic parameters and both cuticle and ear bleeding times. In this model, factor rXai and EGR-Xai had comparable in-vivo efficacy, and showed 80%-93% inhibition at plasma levels of 6.5 nM (rXai) and 8 nM (EGR-Xai). Effects on ex-vivo clotting times varied among the inhibitors. At 80-100% thrombus inhibition, factor rXai and EGR-Xai had no statistically significant effect, while PPACK extended thrombin clotting time (TCT) times 2.3-fold, and heparin prolonged both activated partial thromboplastin time (APTT), prothrombin time (PT) and TCT ex-vivo clotting times 6.9-, 1.2-, and 7-fold respectively. At these dosages, cuticle and ear bleeding times were prolonged for all inhibitors and showed increases of 177%-389% (cuticle) and 45%-129% (ear). Our results demonstrate that direct inhibition of prothrombinase complex assembly is effective in arresting venous thrombosis.

• References

• 1 Hirsh J, Salzman EW, Marder VJ, Colman RW. Pathogenesis of thrombosis. In: Hemostasis and Thrombosis. Colman RW, Hirsh J, Marder VJ, Salzman EW. (eds) JB Lippincott; Philadelphia: 1987. pp 1063-72
• 2 Mann KG, Nesheim ME, Curch WR, Haley P, Krishnaswamy S. Surface-dependent reactions of the vitamin K-dependent enzyme complexes. Blood 1990; 76: 1-16
• 3 Nesheim ME, Kettner C, Shaw E, Mann KG. Co-factor dependence of factor Xa incorporation into the prothrombinase complex. J Biol Chem 1981; 256: 6537-40
• 4 Skogen WF, Esmon CT, Cox AC. Comparison of coagulation factor Xa and des-(l-44) factor Xa in the assembly of prothrombinase. J Biol Chem 1984; 259: 2306-10
• 5 Sinha U, Hancock T, Lin PH, Hollenbach S, Wolf D. Expression, purification and characterization of inactive human coagulation factor Xa (Asn^332:Ala⁴¹⁹). Prot Exp and Purif 1992; 3: 518-24
• 6 Hanson SR, Harker LA. Interruption of acute platelet-dependent thrombosis by the synthetic antithrombin D-phenylananyl-L-prolyl-L-arginyl chloromethyl ketone. Proc Natl Acad Sci USA 1988; 85: 3184-8
• 7 Stein B, Fuster V, Halperin JL, Chesebro JH. Antithrombotic therapy in cardiac disease: An emerging approach based on pathogenesis and disk. Circulation 1989; 80: 1501-13
• 8 Wolf DL, Sinha U, Hancock IE, Lin PH, Messier TL, Esmon CT, Church WR. Design of constructs for the expression of biologically active recombinant human factors X and Xa. Kinetic analysis of the expressed proteins. J Biol Chem 1991; 266: 13726-30
• 9 Di Scipio RG, Hermodson MA, Davie EW. Activation of human factor X (Stuart factor) by a protease fioiu Russell’s viper venom. Biochemistry 1977; 16: 5253-60
• 10 Kirlc RE. Experimental design: procedures for the behavioral sciences. 2nd ed. (Belmont) Brooks/Cole; CA: 1982. pp 106-9
• 11 Colien D, Stassen JM, Verstraete M. Thrombolysis with human extrinsic (tissue-type) plasminogen activator in rabbits with experimental jugular vein thrombosis. Effect of molecular form and dose of activator, age of the thrombus, and route of administration. J Clin Invest 1983; 71: 368-76
• 12 Haskel JE, Torn ST, Day KC, Palmier M, Wun TC, Sobel BE, Abend-schein DR. Prevention of arterial reocclusion after thrombolysis with recombinant lipoprotein-associated coagulation inhibitor. Circulation 1991; 84: 821-7
• 13 Hirsh J. Rationale for development of low-molecular-weight heparins and their clinical potential in the prevention of postoperative venous thrombosis. Am JSurg 1991; 161: 512-8
• 14 Waxman L, Smith DE, Arcuri KE, Vlasuk GP. Tick anticoagulant peptide (TAP) is a novel inhibitor of blood coagulation factor Xa. Science 1990; 248: 593-6
• 15 Benedict CR, Ryan J, Wolitzky B, Ramos R, Gerlach M, Tijburg P, Stern D. Active site-blocked factor IXa prevents intravascular thrombus formation in the coronary vasculature without inhibiting extravascular coagulation in a canine thrombosis model. J Clin Invest 1991; 88: 1760-5
• 16 Benedict CR, Ryan J, Todd J, Kuwabra K, Tijburg P, Cartwright J, Stern D. Active site-blocked factor Xa prevents thrombus formation in the coronary vasculature in parallel with inhibition of extravascular coagulation in a canine thrombosis model..
• 17 Girard TJ, MacPhail LA, Likert KM, Novotny WF, Miletich JP, Broze Jr GJ. Inhibition of factor Vlla-tissue factor coagulation activity by a hybrid protein. Science 1990; 248: 1421-4