Cleavage of fibrinogen and fibrin by a hemorrhagin isolated from the venom of Bothrops alternatus from the north-east region of Argentina

GAY, C.; MARUÑAK, S.; M. E. PEICHOTO; LEIVA, L. C.; ACOSTA, O.

Angra dos Reis (Brasil)

Congreso; VIII Congreso de la Sociedad Brasileña de Toxinologia. VIII Symposium of the Pan American Section of the International Society on Toxinology; 2004

Sociedad Brasileña de Toxinología

The majority of snakebites in the north-east region of Argentina are caused by Bothrops alternatus. The hemorrhage induced by the venom of this snake represents a serious problem because of its local and systemic effects. Others authors have isolated a hemorrhagin metalloproteinase/disintegrin, called alternagin, which is a potent inhibitor of the collagen binding to alpha2beta1 integrin  In this work, a hemorrhagin, probably alternagin, was isolated and its effects on fibrinogen and fibrin were studied. The enzyme was isolated by DEAE-Cellulose chromatography and gel filtration on Sephadex G-75. The hemorrhagic activity was evaluated by the skin method of Kondo. The capacity of hydrolysing fibrinogen and fibrin was shown by electrophoresis on polyacrilamide gel. To determine the structural characteristics of the enzyme, the previous assays were carried out in presence of inhibitor, preincubating the hemorrhagin isolated with equal volumes of 45 mM Na2EDTA or 40 mM Benzamidine for 1 hr at room temperature. The molecular weight of the isolated enzyme was of 55 kDa. It exhibited a high hemorrhagic activity with a minimal hemorrhagic dose of 1.9 µg, almost two fold minor than the whole venom (3.6 µg). The enzyme hydrolysed fibrinogen and fibrin. It rapidly digested the Aá-chain of fibrinogen, followed by Bâ-chain degradation and leaving the ã-chain unaffected. It also degraded the á-chain of fibrin. fibrinogenolytic, fibrinolytic and hemorrhagic activity were inhibited by EDTA (metalloproteinases inhibitor), whereas they were not inhibited by benzamidine (serinoproteinases inhibitor). These results demonstrate that the Bothrops alternatus venom from north-east region of Argentina possesses a hemorrhagic metalloprotease, probably alternagin, which is able to degrade fibrinogen and fibrin. These hydrolytic activities could contribute to the hemorrhagic effect of the venom, acting synergically with thrombin-like enzymes that this latter possesses, which is evidenced by a local and systemic profuse bleeding of the victim bitten.