MARCELO LARAMI SANTORO
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- Bothrops jararaca Venom Metalloproteinases Are Essential for Coagulopathy and Increase Plasma Tissue Factor Levels during Envenomation(2014) YAMASHITA, Karine M.; ALVES, Andre F.; BARBARO, Katia C.; SANTORO, Marcelo L.Background/Aims: Bleeding tendency, coagulopathy and platelet disorders are recurrent manifestations in snakebites occurring worldwide. We reasoned that by damaging tissues and/or activating cells at the site of the bite and systemically, snake venom toxins might release or decrypt tissue factor (TF), resulting in activation of blood coagulation and aggravation of the bleeding tendency. Thus, we addressed (a) whether TF and protein disulfide isomerase (PDI), an oxireductase involved in TF encryption/decryption, were altered in experimental snake envenomation; (b) the involvement and significance of snake venom metalloproteinases (SVMP) and serine proteinases (SVSP) to hemostatic disturbances. Methods/Principal Findings: Crude Bothrops jararaca venom (BjV) was preincubated with Na-2-EDTA or AEBSF, which are inhibitors of SVMP and SVSP, respectively, and injected subcutaneously or intravenously into rats to analyze the contribution of local lesion to the development of hemostatic disturbances. Samples of blood, lung and skin were collected and analyzed at 3 and 6 h. Platelet counts were markedly diminished in rats, and neither Na-2-EDTA nor AEBSF could effectively abrogate this fall. However, Na-2-EDTA markedly reduced plasma fibrinogen consumption and hemorrhage at the site of BjV inoculation. Na-2-EDTA also abolished the marked elevation in TF levels in plasma at 3 and 6 h, by both administration routes. Moreover, increased TF activity was also noticed in lung and skin tissue samples at 6 h. However, factor VII levels did not decrease over time. PDI expression in skin was normal at 3 h, and downregulated at 6 h in all groups treated with BjV. Conclusions: SVMP induce coagulopathy, hemorrhage and increased TF levels in plasma, but neither SVMP nor SVSP are directly involved in thrombocytopenia. High levels of TF in plasma and TF decryption occur during snake envenomation, like true disseminated intravascular coagulation syndrome, and might be implicated in engendering bleeding manifestations in severely-envenomed patients.
- Bothrops jararaca envenomation: Pathogenesis of hemostatic disturbances and intravascular hemolysis(2015) SENISE, Luana V.; YAMASHITA, Karine M.; SANTORO, Marcelo L.To attain fully functional biological activity, vitamin-K dependent coagulation factors (VKDCF) are -carboxylated prior to secretion from liver. Warfarin impairs the -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 24h, 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.