New Technologies, Diagnostic Tools and Drugs Functional characterisation of Vizottin, the first factor Xa inhibitor purified from the leech Haementeria vizottoi Daniella Gorete Lourenço Oliveira 1 ; Miryam Paola Alvarez-Flores 1,2 ; Adriana Rios Lopes 1 ; Ana Marisa Chudzinski-Tavassi 1,2 1 Biochemistry and Biophysics Laboratory, Butantan Institute, São Paulo, São Paulo, Brazil; 2 Center of Applied Toxinology (CAT/CEPID), Butantan Institute, São Paulo, São Paulo, Brazil Summary The strategic position of factor Xa (FXa) in blood coagulation makes it a compelling target for the development of new anticoagulants. Blood- sucking animals have in their salivary glands mixtures of anticoagu- lants, which could be used for designing novel antithrombotic com- pounds. Herein, we describe Vizottin, the first FXa inhibitor from the salivary complex of the leech Haementeria vizottoi. Vizottin was puri- fied by gel filtration and reverse-phase chromatography, and shown to have anticoagulant effects in human plasma, prolonging the recalcifi- cation time in a dose-dependent manner (IC50 40 nM). Vizottin induced blood incoagulability in FX-deficient plasma, whereas in normal and re- constituted plasma, Vizottin doubled the prothrombin time at 160 nM. This peptide competitively inhibited human FXa (K i 2 nM) like FXa in- hibitors from other leeches, albeit via a distinct mechanism of action. At high concentrations, vizottin inhibited the amidolytic activity of factor Correspondence to: Ana Marisa Chudzinski-Tavassi, PhD Biochemistry and Biophysic Laboratory Av. Vital Brasil 1500, São Paulo SP, CEP 05503–900, Brazil Tel.: +55 11 3726 7222 ext. 2043, Fax: +55 11 3726 1505 E-mail: amchudzinski@butantan.gov.br VIIa/tissue factor (IC50 96.4 nM). Vizottin inhibited FXa in the pro- thrombinase 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 experi- ments 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 Financial support: This study was supported in part by a grant from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP 07/54257–1 and FAPESP 98/14307–9) and by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/INCT- TOX) and CAT/CEPID-FAPESP. Received: April 13, 2012 Accepted after minor revision: May 17, 2012 Prepublished online: July 10, 2012 doi:10.1160/TH12-04-0235 Thromb Haemost 2012; 108: 570–578 Introduction In the last decades, new antithrombotic agents have been intro- duced in developmental stages of clinical trials to overcome the limitations of traditional anticoagulants such as variable dose requirement and routine coagulation monitoring (1–3). Clinical studies have indicated that the most promising new anticoagulants are those that target activated factor X (FXa) and thrombin selec- tively (4). The effectiveness of FXa inhibitors as antithrombotic agents and their potentially reduced bleeding risks may offer su- perior therapeutic profiles compared to thrombin inhibitors (4). In recent decades, a complex mixture of biologically active mol- ecules has been identified in the salivary glands of haematopha- gous animals, and specific inhibitors of the blood clotting system have been particularly regarded as important tools for the study of new antithrombotic compounds (5–7). Indeed, a large number of exogenous anticoagulants that target the haemostatic system have been identified and classified based on their mechanisms of action: inhibitors of thrombin and FXa and inhibitors of the extrinsic and intrinsic tenase complex (4, 5). Among them, hirudin is a highly potent and specific inhibitor of thrombin, which was originally isolated from the leech Hirudo medicinalis (8); TAP from Ornitho- dorus moubata has more potent anticoagulant activity than hepa- rin and other direct FXa inhibitors (9, 10). In Nematoda, NAPc2 (nematode anticoagulant peptide C2) from Ancylostoma caninum is able to interact with the factor VIIa (FVIIa)/tissue factor (TF) complex through a similar mechanism of action as tissue factor pathway inhibitor (TFPI) (11). FXa is a vitamin K-dependent factor present in human blood as a two-chain glycoprotein (12). Zymogen factor X (FX) is converted to its active form, FXa, by either the extrinsic pathway complex (FVIIa/TF/cell surface/Ca 2+ ) or by the intrinsic pathway complex (FIXa/FVIIIa/cell surface/Ca 2+ ) (13, 14). FXa alone can activate prothrombin, but the rate of this reaction is not compatible with survival (15). Thus, FXa is physiologically associated with the ac- tive form of factor V (FVa, a cofactor) on the cell membrane surface in the presence of Ca 2+ to form the prothrombinase complex with a 300,000-fold greater activity than free FXa in solution, providing Thrombosis and Haemostasis 108.3/2012 570 © Schattauer 2012 For personal or educational use only. No other uses without permission. All rights reserved. Downloaded from www.thrombosis-online.com on 2012-09-12 | ID: 1000491814 | IP: 217.110.19.91