Cross-neutralization of the coagulant activity of Crotalus durissus terrificus venom from the Northeast of Argentina by bivalent bothropic antivenom

RODRIGUEZ JUAN PABLO; GAY CLAUDIA CAROLINA; FUSCO LUCIANO; GAUNA MARIA DEL CARMEN; ACOSTA OFELIA; LEIVA LAURA

Mar del Plata, Buenos Aires

Congreso; XLII Reunión Anual de la Sociedad Argentina de Farmacología Experimental; 2010

Snake venoms, particularly those belonging to Viperidae, often induce disorder of the blood coagulation system, and this represents a serious complication in snake bite. Crotalus durissus terrificus (C.d.t.) venom may cause complete and long lasting incoagulability of blood in patients, due to the hypofrinogenemia developed after the bite. Beside the substitution therapy with plasma or plasma derived, the use of antivenoms is the only effective treatment to this disturbance of hemostasis. Owing to the therapeutics implications in this medically relevant intoxication, the aim of this work was to investigate the neutralization of C.d.t. venom coagulant activity by bivalent antivenom raised in horses against the species mainly responsible for bothropic envenomations from the northeast of Argentina: Bothrops alternatus (B.a.) and Bothrops diporus (B.d.). Horse hyperimmune antisera were obtained by immunization with different amount of B.a. or B.d. venom per animal mixed with Freund’s adjuvant. The antibody levels in the sera were monitored by ELISA and its specificity was tested by immunoblotting. Antibodies were purified by ion exchange chromatography in a FPLC System. Crotalic thrombin-like activity neutralization was test incubating different amount of venom with antivenom for 30 min at 37°C and the clotting time measured in a Fibrintimer Winner Lab® Coagulometer. According to demonstrate that crotalic thrombine-like enzymes (TLEs) from C.d.t. venom are neutralized by the antivenom produced, a TLE were purified from the whole venom and similar neutralization tests were developed. Our experiments confirmed that the antivenom obtained with venoms from the northeast of Argentina neutralize thrombin-like activities of C.d.t. venom but, higher amount of antivenom is required compeered to bothropic venoms neutralization. These results provide crucial information for a rational and effective treatment of snake bite and are also useful for the development of new antivenoms formulations.