Background— This study was designed to determine whether (1) P2Y 12 antagonism synergizes with other antithrombotics and (2) anticoagulants (thrombin inhibitors) affect the antithrombotic activity elicited by P2Y 12 antagonism. Methods and Results— Thrombosis was achieved by perfusion of human and murine blood through type III collagen–coated capillaries at arterial shear rate. CT50547, a direct-acting P2Y 12 antagonist, inhibited thrombosis in PPACK- but not heparin-anticoagulated human blood. In contrast, CT50547 inhibited thrombosis in aspirin-treated individuals independently of the anticoagulant. Thrombin and TXA 2 also synergized with P2Y 12 in the absence of anticoagulation, because combined treatment of aspirin or C921-78 (a factor Xa inhibitor) with CT50547 or 2-MeSAMP (a P2Y 12 antagonist) inhibited the thrombotic process, whereas all treatments failed to inhibit thrombosis when used individually. Synergism was also observed ex vivo when P2Y 12 -deficient (P2Y 12−/− ) mice were administered aspirin or coagulation inhibitors (C921-78 and bivalirudin). Finally, using intravital microscopy, we found that both C921-78 and bivalirudin abrogated the thrombotic process in P2Y 12+/− mice, whereas each showed only partial efficacy in P2Y 12+/+ animals. Conclusions— Our study indicates that (1) thrombin inhibitors and aspirin have a demonstrable synergy of antithrombotic activity with P2Y 12 antagonism and (2) the in vitro analysis of the antithrombotic activity of P2Y 12 antagonists is affected by the anticoagulant used for blood collection. This suggests that the antithrombotic potential of P2Y 12 antagonists in vitro may be overestimated in anticoagulated samples of blood and best achieved in vivo by the inclusion of aspirin and/or a thrombin inhibitor.

2000-04-01·European Journal of Pharmacology3区 · 医学

Antithrombotic and hemostatic capacity of factor Xa versus thrombin inhibitors in models of venous and arteriovenous thrombosis

3区 · 医学

Article

作者: Paul Ku ; Stanley J Hollenbach ; John Malinowski ; Pei Hua Lin ; Charles K Marlowe ; Robert M Scarborough ; Paul W Wong ; Bing Yan Zhu ; Uma Sinha

Thrombin plays a central role in venous and arterial thrombosis. We utilized two different rabbit models of in vivo thrombosis to investigate the effect of inhibitors of thrombin generation and thrombin activity. The agents tested were specific inhibitors of factor Xa (fXa) [N2-[(phenylmethyl)sulfonyl]-D-arginyl-N-[(1S)-4-[(aminoiminomethyl++ +)a mino]-1-(2-thiazolylcarbonyl)butyl]-glycinamide (C921-78)] and thrombin [D-phenylalanyl-N-[4-[(aminoiminomethyl)amino]-1-(chloroacetyl)but yl]-L-prolinamide (PPACK)], as well as drugs that affect both thrombin and fXa, unfractionated and low molecular weight (enoxaparin) heparin. The agents administered as constant intravenous infusion were evaluated for antithrombotic efficacy in anesthetized rabbits. All four agents were capable of dose dependent inhibition of thrombosis in venous and arteriovenous thrombosis models. However, due to the more aggressive nature of thrombotic stimulation in the arteriovenous shunt model, complete cessation of thrombus growth was not achieved for any of the agents at the doses tested. Comparison between the agents focused on the differences in extension of coagulation parameters (activated partial thromboplastin time, prothrombin time, thrombin clotting time), changes in hematological parameters, and extension of rabbit cuticle bleeding time at doses required to produce maximum inhibition in the thrombosis models. In the venous thrombosis model at the maximally effective dose, C921-78 had minimal extension of ex vivo clotting parameters, while enoxaparin and unfractionated heparin demonstrated a two to sevenfold increase in activated partial thromboplastin times, and PPACK had a threefold extension of thrombin clotting times. In addition, unlike the other three agents, which exhibited no significant changes in hematological parameters, PPACK demonstrated dose dependent thrombocytopenia. A standardized cuticle bleeding time was used as a measure of perturbation of hemostasis. The agents were evaluated for significant increases in bleeding time at doses up to eight times that needed to completely inhibit venous thrombus formation. Unfractionated heparin displayed a significant bleeding time effect at the dose required to inhibit venous thrombosis (100 u/kg+2 u/kg/min). Enoxaparin and PPACK caused significant bleeding time extensions at four times the fully efficacious venous dose (800 u/kg+8 u/kg/min and 30 microg/kg/min). By contrast, C921-78 did not significantly increase bleeding time even at eight times the maximally effective dose (240 microg/kg+7.2 microg/kg/min). Our results demonstrate that specific inhibition of fXa can be utilized to derive potent antithrombotic activity without disrupting extravascular hemostasis.

1999-11-01·Biochemistry3区 · 生物学

Inhibition of Factor Xa by a Peptidyl-α-ketothiazole Involves Two Steps. Evidence for a Stabilizing Conformational Change

3区 · 生物学

Article

作者: Wong, Paul W. ; Sinha, Uma ; Betz, Andreas

Recently, peptidylketothiazoles have been shown to be potent inhibitors of proteases, but the details of the interaction have not yet been studied. In the work presented here, the interaction of factor Xa, a coagulation protease, with the transition state inhibitor BnSO(2)-D-Arg-Gly-Arg-ketothiazole (C921-78) is characterized. C921-78 is a tight and selective inhibitor of the coagulation protease factor Xa (K(d) = 14 pM). The hydrolytic activity of factor Xa was inhibited by C921-78 in a time-dependent manner. The rate-limiting step of the bimolecular combination of inhibitor and enzyme was competitive with the substrate. Conversely, the inhibitor could be displaced from the active site of the enzyme after exposure of the preformed complex to an excess of substrate or to the active site inhibitor dansyl-Glu-Gly-Arg-chloromethyl ketone (DEGR-CMK) in a slow reaction. The formation of the C921-78-factor Xa complex resulted in a 60% increase in the magnitude of the fluorescence emission spectrum. Rapid mixing of the enzyme and inhibitor produces a monophasic fluorescence increase, compatible with spectral transition in a single step. The rate constant for this reaction increased hyperbolically with the concentration of C921-78, but the amplitude remained constant. These results are consistent with the initial formation of an enzyme-inhibitor complex (EI), followed by a unimolecular conversion of EI to EI linked to a spectral transition. The rate constants of the isomerization provide an estimate of 300000-fold stabilization. Thus, the inhibition of factor Xa by C921-78 follows a mechanism similar to that described classically for slow tight binding inhibitors. However, the two steps of the reaction cannot be kinetically separated by the rapid equilibrium assumption, and therefore, the formation of EI is partially rate-limiting, too. The driving energy for the unusually fast isomerization step may result from the highly favorable interactions of the inhibitor in the primary binding site.

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