Functional characterisation of vizottin, the first factor Xa inhibitor purified from the leech Haementeria vizottoi.
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 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.
Biochemistry and Biophysic Laboratory, Butantan Institute, São Paulo, São Paulo, Brazil.
Daniella Gorete Lourenço
DG
Oliveira
Miryam Paola
MP
Alvarez-Flores
Adriana Rios
AR
Lopes
Ana Marisa
AM
Chudzinski-Tavassi
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D000925:Anticoagulants / Q000302:isolation & purification / Q000494:pharmacology*; D001777:Blood Coagulation / Q000187:drug effects; D001780:Blood Coagulation Tests; D020134:Catalytic Domain / Q000187:drug effects; D002850:Chromatography, Gel; D056148:Chromatography, Reverse-Phase; D002863:Chromogenic Compounds; D015195:Drug Design; D004353:Drug Evaluation, Preclinical; D004789:Enzyme Activation / Q000187:drug effects; D015942:Factor VIIa / Q000037:antagonists & inhibitors; D065427:Factor Xa Inhibitors; D005343:Fibrinolytic Agents / Q000302:isolation & purification / Q000494:pharmacology; D006801:Humans; D007865:Leeches / Q000737:chemistry*; D008074:Lipoproteins / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D012469:Salivary Glands / Q000737:chemistry; D012471:Salivary Proteins and Peptides / Q000494:pharmacology
D000590:Amino Acid Chloromethyl Ketones; D000925:Anticoagulants; D002863:Chromogenic Compounds; D065427:Factor Xa Inhibitors; D005343:Fibrinolytic Agents; C455900:Ixolaris protein, Ixodes scapularis; D008074:Lipoproteins; D012471:Salivary Proteins and Peptides; C051928:lipoprotein-associated coagulation inhibitor; C051330:glutamyl-glycyl-arginine chloromethyl ketone; D015942:Factor VIIa