Functional characterisation of Vizottin, the first factor Xa inhibitor purified from the leech Haementeria vizottoi

Daniella Gorete Lourenço Oliveira; Miryam Paola Alvarez-Flores; Adriana Rios Lopes; Ana Marisa Chudzinski-Tavassi

doi:10.1160/TH12-04-0235

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2012; 108(03): 570-578
DOI: 10.1160/TH12-04-0235

New Technologies, Diagnostic Tools and Drugs

Schattauer GmbH

Functional characterisation of Vizottin, the first factor Xa inhibitor purified from the leech Haementeria vizottoi

Daniella Gorete Lourenço Oliveira

¹Biochemistry and Biophysics Laboratory, Butantan Institute, São Paulo, São Paulo, Brazil

,

Miryam Paola Alvarez-Flores

¹Biochemistry and Biophysics Laboratory, Butantan Institute, São Paulo, São Paulo, Brazil

²Center of Applied Toxinology (CAT/CEPID), Butantan Institute, São Paulo, São Paulo, Brazil

,

Adriana Rios Lopes

¹Biochemistry and Biophysics Laboratory, Butantan Institute, São Paulo, São Paulo, Brazil

,

Ana Marisa Chudzinski-Tavassi

¹Biochemistry and Biophysics Laboratory, Butantan Institute, São Paulo, São Paulo, Brazil

²Center of Applied Toxinology (CAT/CEPID), Butantan Institute, São Paulo, São Paulo, Brazil

› Author Affiliations

Abstract

Summary

The strategic position of factor Xa (FXa) in blood coagulation makes it a compelling target for the development of new anticoagulants. Bloodsucking animals have in their salivary glands mixtures of anticoagulants, which could be used for designing novel antithrombotic compounds. Herein, we describe Vizottin, the first FXa inhibitor from the salivary complex of the leech Haementeria vizottoi. Vizottin was purified by gel filtration and reverse-phase chromatography, and shown to have anticoagulant effects in human plasma, prolonging the recalcification time in a dose-dependent manner (IC50 40 nM). Vizottin induced blood incoagulability in FX-deficient plasma, whereas in normal and reconstituted plasma, Vizottin doubled the prothrombin time at 160 nM. This peptide competitively inhibited human FXa (K_i 2 nM) like FXa inhibitors from other leeches, albeit via a distinct mechanism of action. At high concentrations, vizottin inhibited the amidolytic activity of factor VIIa/tissue factor (IC50 96.4 nM). Vizottin inhibited FXa in the prothrombinase complex and Gla-domainless FXa. Moreover, vizottin did not interfere with FX activation induced by RVV-X, a known enzyme that requires the Gla-domain of FX for activation. Competition experiments in the presence of FXa and GGACK-FXa (active site blocked) demonstrated that the inhibition of FXa by vizottin is through binding to the active site rather than an exosite. This novel inhibitor appears to exert its inhibitory effects through direct binding to the active site of FXa in a time-dependent manner, but not involving a tight-binding model. In this context, vizottin is a promising model for designing novel anticoagulants for the treatment of thrombotic diseases.

Keywords

Leech salivary gland - Haementeria - factor Xa inhibitor - factor Xa active site - extrinsic tenase complex

Full Text

References

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