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
1   Laboratoire de Recherche sur l’Hémostase et la Thrombose, Faculté de Médecine Xavier Bichat, Université Paris 7, Paris, France
,
Charlotte Lemaire
1   Laboratoire de Recherche sur l’Hémostase et la Thrombose, Faculté de Médecine Xavier Bichat, Université Paris 7, Paris, France
,
Marie-Christine Bouton
1   Laboratoire de Recherche sur l’Hémostase et la Thrombose, Faculté de Médecine Xavier Bichat, Université Paris 7, Paris, France
,
Annie Bezeaud
1   Laboratoire de Recherche sur l’Hémostase et la Thrombose, Faculté de Médecine Xavier Bichat, Université Paris 7, Paris, France
,
Cassian Bon
2   Unité des Venins, Institut Pasteur, Paris, France
,
Marie-Claude Guillin
1   Laboratoire de Recherche sur l’Hémostase et la Thrombose, Faculté de Médecine Xavier Bichat, Université Paris 7, Paris, France
,
Martine Jandrot-Perrus
1   Laboratoire de Recherche sur l’Hémostase et la Thrombose, Faculté de Médecine Xavier Bichat, Université Paris 7, Paris, France
› Author Affiliations
This work was supported by grants from the European Community (CI1* CT940073), the Ministère de l’Education Nationale, de l’Enseignement Superieur et de la Recherche (ACC-SV9) and Université Paris 7.
Further Information

Publication History

Received 24 October 1997

Accepted after resubmission 05 February 1998

Publication Date:
07 December 2017 (online)

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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