Relative Efficacy of Active Site-Blocked Factors IXa, Xa in Models of Rabbit Venous and Arterio-Venous Thrombosis

Summary

In order to investigate the respective roles of prothrombinase and intrinsic tenase (IXa/VIIIa) in venous thrombosis, we compared the antithrombotic efficacy of inhibitors of these two coagulation complexes. The agents tested were dansyl-Glu-Gly-Arg chloromethyl ketone-inactivated bovine factor IXa (IXai) and Glu-Gly-Arg chloromethyl ketone- inactivated human factor Xa (Xai). In vitro formation of active complexes (prothrombinase or tenase) was inhibited by Xai and IXai resulting in IC₅₀ values of 3 nM and 5 nM, respectively. Antithrombotic activity was measured by inhibition of clot accretion on cotton threads placed in the abdominal vena cava of anesthetized rabbits. Intravenous bolus dosing followed by infusion of Xai during the experimental protocol resulted in a dose dependent reduction of clot weight, a dosage of 16.0 μg/kg + 0.28 μg/kg/min being sufficient to produce a 96% inhibition of thrombosis. A much higher dose of IXai (1.0 mg/kg + 17.3 |ig/kg/min) resulted in a 39% reduction of clot weight. In a rabbit arterio-venous shunt model mimicking arterial thrombosis, the relative efficacy of the two agents was found to be more comparable. The doses required for optimum antithrombotic activity were 128.0 μg/kg + 2.2 μg/kg/min for Xai and 1.0 mg/kg+ 17.3 μg/kg/min for IXai. We conclude that, in this study, prothrombinase rather than tenase inhibition was more effective in reducing venous thrombosis and that these effects can be achieved without disruption of extravascular hemostasis.

• References

• 1 Davie EW, Fujikawa K, Kisiel W. The coagulation cascade: Initiation, maintenance and regulation. Biochemistry 1991; 30: 10363-10370
• 2 Lynch JJ, Sitko GR, Lehman ED, Vlasuk GP. Primary prevention of coronary arterial thrombosis with the factor Xa inhibitor rTAP in a canine electrolytic injury model. Thromb Haemost 1995; 74: 640-645
• 3 Ragosta M, Gimple LW, Gertz SD, Dunwiddie CT, Vlasuk GP, Haber HL, Powers ER, Roberts WC, Sarembock IJ. Specific factor Xa inhibition reduces restenosis after balloon angioplasty of atherosclerotic femoral arteries in rabbits. Circulation 1994; 89: 1262-1271
• 4 Hara T, Yokoyama A, Tanabe K, Ishihara H, Iwamoto M. DX-9065a, an orally active, specific inhibitor of factor Xa, inhibits thrombosis without affecting bleeding time in rats. Thromb Haemost 1995; 74: 635-639
• 5 Hollenbach S, Sinha U, Lin PH, Needham K, Frey L, Hancock TE, Wong A, Wolf DL. A comparative study of prothrombinase and thrombin inhibitors in a novel rabbit model of non-occlusive deep vein thrombosis. Thromb Haemost 1994; 71: 357-362
• 6 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: 1760-1765
• 7 Wolf DL, Sinha U, Hancock TE, Lin PH, Messier TL, Esmon CT, Church WR. Design of constructs for the expression of biologically active recombinant human factors X and Xa. J Biol Chem 1991; 266: 13726-13730
• 8 Sinha U, Hancock TE, Nzerem JJ, Lin PH, Tomlinson JE, Wolf DL. Effect of gamma carboxylation on prothrombinase inhibitory activity of catalytically inactive factor Xa. Thromb Res 1994; 75: 427-436
• 9 Benet LZ, Mitchell JR, Sheiner LB. Pharmacokinetics: The Dynamics of Drug Absorption, Distribution, and Elimination. In: The Pharmacological Basis of Therapeutics. Gilman AG, Rail TW, Nies AS, Taylor P. eds Pergamon Press; New York, NY: 1990. pp 22-26
• 10 Knabb RM, Kettner CA, Timmermans PB, Reilly TM. In vivo characterization of a new synthetic thrombin inhibitor. Thromb Haemost 1992; 67: 56-59
• 11 Nesheim ME, Kettner C, Shaw E, Mann KG. Cofactor dependence of factor Xa incorporation into the prothrombinase complex. J Biol Chem 1981; 256: 6537-6540
• 12 Sinha U, Hollenbach S, Wolf DL. Macromolecular complex assembly of prothrombinase is a central process of thrombosis. Ann NY Acad Sci 1994; 714: 32-40
• 13 Benedict CR, Ryan J, Todd J, Kuwabara K, Tijburg P, Cartright 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. Blood 1993; 81: 2059-2066
• 14 Hoffman M, Monroe DM, Oliver JA, Roberts HR. Factors IXa and Xa play distinct roles in tissue factor-dependent initiation of coagulation. Blood 1995; 86: 1794-1801
• 15 Kirchhofer D, Tschopp TB, Baumgartner HR. Active site-blocked factors Vila and IXa differentially inhibit fibrin formation in a human ex vivo thrombosis model. Arterioscler Thromb Vase Biol 1995; 15: 1098-1106
• 16 Cadroy Y, Hanson SR, Kelly AB, Marzec UM, Evatt BL, Kunicki TJ, Montgomery RR, Harker LA. Relative antithrombotic effects of monoclonal antibodies targeting different platelet glycoprotein-adhesive molecule interactions in non-human primates. Blood 1994; 83: 3218-3224
• 17 Hanson SR, Harker LA. Interruption of acute platelet-dependent thrombosis by the synthetic antithrombin D-phenylalanyl-L-prolyl-L-arginyl chloromethyl ketone. Proc Natl Acad Sci USA 1988; 85: 3184-3188