Inhibition of Thrombin-catalyzed Factor V Activation by Bothrojaracin

Véronique Arocas; Charlotte Lemaire; Marie-Christine Bouton; Annie Bezeaud; Cassian Bon; Marie-Claude Guillin; Martine Jandrot-Perrus

doi:10.1055/s-0037-1615033

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1998; 79(06): 1157-1161
DOI: 10.1055/s-0037-1615033

Rapid Communication

Schattauer GmbH

Inhibition of Thrombin-catalyzed Factor V Activation by Bothrojaracin

Véronique Arocas

¹Laboratoire de Recherche sur l’Hémostase et la Thrombose, Faculté de Médecine Xavier Bichat, Université Paris 7, Paris, France

,

Charlotte Lemaire

¹Laboratoire de Recherche sur l’Hémostase et la Thrombose, Faculté de Médecine Xavier Bichat, Université Paris 7, Paris, France

,

Marie-Christine Bouton

¹Laboratoire de Recherche sur l’Hémostase et la Thrombose, Faculté de Médecine Xavier Bichat, Université Paris 7, Paris, France

,

Annie Bezeaud

¹Laboratoire de Recherche sur l’Hémostase et la Thrombose, Faculté de Médecine Xavier Bichat, Université Paris 7, Paris, France

,

Cassian Bon

²Unité des Venins, Institut Pasteur, Paris, France

,

Marie-Claude Guillin

¹Laboratoire de Recherche sur l’Hémostase et la Thrombose, Faculté de Médecine Xavier Bichat, Université Paris 7, Paris, France

,

Martine Jandrot-Perrus

¹Laboratoire de Recherche sur l’Hémostase et la Thrombose, Faculté de Médecine Xavier Bichat, Université Paris 7, Paris, France

› Author Affiliations

Abstract

Summary

We have previously identified and characterized a potent and specific thrombin inhibitor, isolated from Bothrops jararaca, named bothrojaracin. Bothrojaracin interacts with the two positively charged recognition sites of thrombin referred to as exosite 1 and exosite 2, whereas it does not interact with the thrombin active site. Consequently, bothrojaracin inhibits thrombin-induced fibrinogen to fibrin conversion and platelet activation, without inhibition of thrombin-catalyzed cleavage of small synthetic substrates. In the present study, we show that bothrojaracin exerts an anticoagulant effect in plasma, illustrated by the prolongation of the aPTT. Using purified proteins, we observed that the anticoagulant effect of bothrojaracin was not only due to the inhibition of fibrinogen to fibrin conversion, but in addition to the inhibition of factor V activation by thrombin. Bothrojaracin decreased the rate of thrombin-catalyzed proteolysis of factor V and concurrently the generation of factor Va cofactor activity measured in a prothrombinase assay. We compared the effect of bothrojaracin with that of ligands binding specifically exosite 1 (hirudin C-terminal peptide SH54-65) or exosite 2 (heparin, prothrombin fragment 2). SH54-65 delayed thrombin catalyzed factor V activation whereas heparin or prothrombin fragment 2 did not. The thrombin derivatives β- and γ-thrombin, which are defective in their exosite 1, but present with a normally exposed exosite 2, had a reduced capacity to activate factor V, which was not further impaired by the exosite 2 ligands, bothrojaracin, heparin or prothrombin fragment 2. Altogether, our results provide further insight into the anticoagulant effect of bothrojaracin showing that it is a potent inhibitor of the feedback activation of factor V by thrombin, and thus of the up-regulation of its own production by thrombin. Inhibition of thrombin-catalyzed factor V activation by bothrojaracin is mainly mediated through the interaction of the inhibitor with thrombin exosite 1, whereas contribution of the interaction with exosite 2 does not appear to play a direct role in factor V recognition by thrombin.

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References

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