RESUMO
To attain fully functional biological activity, vitamin-K dependent coagulation factors (VKDCF) are γ-carboxylated prior to secretion from liver. Warfarin impairs the γ-carboxylation, and consequently their physiological function. Bothrops jararaca snake venom (BjV) contains several activators of blood coagulation, especially procoagulant enzymes (prothrombin and factor X activators) and thrombin-like enzymes. In order to clarify the relative contribution of prothrombin and factor X activators to the hemostatic disturbances occurring during experimental B. jararaca envenomation, warfarin was used to deplete VKDCF, prior to BjV administration. Male Wistar rats were pretreated with saline (Sal) or warfarin (War) and inoculated subsequently with BjV or saline, thus forming four groups: Sal + Sal (negative control), Sal + BjV (positive control), War + Sal (warfarinization control), and War + BjV. Three hours after inoculation, prothrombin and factor X levels fell 40% and 50%, respectively; levels of both factors decreased more than 97% in the War + Sal and War + BjV groups. Platelet counts dropped 93% and 76% in Sal + BjV and War + BjV, respectively, and plasma fibrinogen levels decreased 86% exclusively in Sal + BjV. After 6 and 24 h, platelet counts and fibrinogen levels increased progressively. A dramatic augmentation in plasma hemoglobin levels and the presence of schizocytes and microcytes in the Sal + BjV group indicated the development of intravascular hemolysis, which was prevented by warfarin pretreatment. Our findings show that intravascular thrombin generation has the foremost role in the pathogenesis of coagulopathy and intravascular hemolysis, but not in the development of thrombocytopenia, in B. jararaca envenomation in rats; in addition, fibrinogenases (metalloproteinases) may contribute to coagulopathy more than thrombin-like enzymes.
Assuntos
Hemólise , Hemostasia/efeitos dos fármacos , Mordeduras de Serpentes , Venenos de Serpentes/química , Animais , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/metabolismo , Bothrops , Eritrócitos/metabolismo , Fator X/química , Fibrinogênio/química , Hemostáticos/química , Masculino , Contagem de Plaquetas , Protrombina/química , Ratos , Ratos Wistar , Trombina/química , Trombocitopenia/sangue , Varfarina/químicaRESUMO
Snake venom metalloproteinases (SVMPs) are major components in most viperid venoms that induce disturbances in the hemostatic system and tissues of animals envenomated by snakes. These disturbances are involved in human pathology of snake bites and appear to be essential for the capture and digestion of snake's prey and avoidance of predators. SVMPs are a versatile family of venom toxins acting on different hemostatic targets which are present in venoms in distinct structural forms. However, the reason why a large number of different SVMPs are expressed in some venoms is still unclear. In this study, we evaluated the interference of five isolated SVMPs in blood coagulation of humans, birds and small rodents. P-III class SVMPs (fractions Ic, IIb and IIc) possess gelatinolytic and hemorrhagic activities, and, of these, two also show fibrinolytic activity. P-I class SVMPs (fractions IVa and IVb) are only fibrinolytic. P-III class SVMPs reduced clotting time of human plasma. Fraction IIc was characterized as prothrombin activator and fraction Ic as factor X activator. In the absence of Ca2+, a firm clot was observed in chicken blood samples with fractions Ic, IIb and partially with fraction IIc. In contrast, without Ca2+, only fraction IIc was able to induce a firm clot in rat blood. In conclusion, functionally distinct forms of SVMPs were found in B. neuwiedi venom that affect distinct mechanisms in the coagulation system of humans, birds and small rodents. Distinct SVMPs appear to be more specialized to rat or chicken blood, strengthening the current hypothesis that toxin diversity enhances the possibilities of the snakes for hunting different prey or evading different predators. This functional diversity also impacts the complexity of human envenoming since different hemostatic mechanisms will be targeted by SVMPs accounting for the complexity of the response of humans to venoms.
Assuntos
Venenos de Crotalídeos/enzimologia , Hemostáticos/química , Metaloproteases/química , Proteínas de Répteis/química , Adaptação Biológica , Sequência de Aminoácidos , Animais , Bothrops , Galinhas , Venenos de Crotalídeos/isolamento & purificação , Venenos de Crotalídeos/farmacologia , Fator X/química , Feminino , Hemostáticos/isolamento & purificação , Hemostáticos/farmacologia , Humanos , Masculino , Metaloproteases/isolamento & purificação , Metaloproteases/farmacologia , Camundongos , Proteólise , Protrombina/química , Ratos , Proteínas de Répteis/isolamento & purificação , Proteínas de Répteis/farmacologia , Mordeduras de SerpentesRESUMO
Ixolaris is a two-Kunitz tick salivary gland tissue factor pathway inhibitor (TFPI). In contrast to human TFPI, Ixolaris specifically binds to factor Xa (FXa) heparin-binding exosite (HBE). In addition, Ixolaris interacts with zymogen FX. In the present work we characterized the interaction of Ixolaris with human FX quantitatively, and identified a precursor state of the heparin-binding exosite (proexosite, HBPE) as the Ixolaris-binding site on the zymogen. Gel-filtration chromatography demonstrated 1:1 complex formation between fluorescein-labeled Ixolaris and FX. Isothermal titration calorimetry confirmed that the binding of Ixolaris to FX occurs at stoichiometric concentrations in a reaction which is characteristically exothermic, with a favorable enthalpy (DeltaH) of -10.78 kcal/mol. ELISA and plasmon resonance experiments also indicate that Ixolaris binds to plasma FX and FXa, or to recombinant Gla domain-containing FX/FXa with comparable affinities ( approximately 1 nM). Using a series of mutants on the HBPE, we identified the most important amino acids involved in zymogen/Ixolaris interaction-Arg-93 >>> Arg-165 > or = Lys-169 > Lys-236 > Arg-125-which was identical to that observed for FXa/Ixolaris interaction. Remarkably, Ixolaris strongly inhibited FX activation by factor IXa in the presence but not in the absence of factor VIIIa, suggesting a specific interference in the cofactor activity. Further, solid phase assays demonstrated that Ixolaris inhibits FX interaction with immobilized FVIIIa. Altogether, Ixolaris is the first inhibitor characterized to date that specifically binds to FX HBPE. Ixolaris may be a useful tool to study the physiological role of the FX HBPE and to evaluate this domain as a target for anticoagulant drugs.