Isolation and characterization of Bothrops jararaca snake venom proteins that induce thrombocytopenia

ANA TEREZA AZEVEDO SACHETTO; MARÍA ELISA PEICHOTO; JAQUELINE GOMES ROSA; RICARDO JOSÉ SOARES TORQUATO; APARECIDA SADAE TANAKA; MARCELO LARAMI SANTORO

São Pedro

Congreso; XV Congress of the Brazilian Society of Toxinology; 2019

Brazilian Society of Toxinology

Bothrops jararaca snake venom (BjV) induces thrombocytopenia in human patients and experimental models. Although various proteins that activate or inhibit platelets in vitro and ex vivo have been isolated and characterized from BjV, it is not known which ones account for the development of thrombocytopenia in vivo. In previous studies, we showed that BjV incubated with catalytic inhibitors of metalloproteinases or serine proteinases, and/or anti-botrocetin antibodies, still evoked platelet consumption in rats and mice. Thus, we initiated the isolation, identification and characterization of BjV toxins responsible for inducing thrombocytopenia in vivo, using ultrafiltration and liquid chromatography, and in vivo and ex vivo testing of fractions. All procedures involving the use of animals or human donors were approved by the Institutional Animal Care and Use Committee from Instituto Butantan (CEUAIB 5937060618) and Plataforma Brasil (CAAE: 51368615.5.0000.0065), respectively. By ultrafiltration, we observed that only venom components passing 50-kDa molecular weight cutoff membranes caused thrombocytopenia in mice, and this activity was completely independent of the hemorrhagic or coagulant activity of BjV. Ultrafiltered proteins were further separated by hydrophobic interaction and ion exchange chromatography, and two 30-kDa proteins (called E6 and D4) that induced thrombocytopenia in mice were isolated. Surprisingly, E6 and D4 did not induce platelet aggregation in platelet rich plasma or whole blood from humans or mice. Currently, experiments are being carried out to understand their mechanisms of action and their primary structure. These findings reinforce previous results that thrombocytopenia occurs simultaneously to hemorrhage and coagulation during envenomation, but they are independent phenomena. Such results are important to understand the pathophysiology of B. jararaca envenomation, particularly of hemostatic disorders that may complicate the clinical picture.