Effects of Duvernoy’s gland secretion of Philodryas patagoniensis from the north-east of Argentina on blood coagulation

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

Philodryas patagoniensis is a colubrid snake spread by all South America. It is considered as not venomous although it has a Duvernoy’s gland secretion with toxicity such as to cause severe lesions, even the death (DL50 2.93 μg/g, similar to that of Bothrops species), in snake bitten patients. We have recently demonstrated that this secretion exhibits high hemorrhagic activity (MHD = 0.035 μg), containing enzymes that are able to degrade the vascular wall. In this work we aim to determine if the secretion, besides to cause hemorrhage, affects the blood coagulation. For this reason, procoagulant and coagulant activities were evaluated on plasma and fibrinogen, respectively. The delay in the thrombin clotting time of fibrinogen previously incubated with the secretion was also determined. Specific hydrolysis of fibrinogen and fibrin pre-incubated with the secretion at different time intervals was shown by electrophoresis on polyacrylamide gel. To determine the structural characteristics of the enzymes degrading fibrinogen and fibrin, secretion was incubated with equal volumes of 45 mM Na2EDTA or 40 mM Benzamidine before the incubation with fibrinogen or fibrin, respectively. Duvernoy’s gland secretion of P. patagoniensis did not clot plasma or fibrinogen; therefore it has neither thrombin-like activity nor procoagulant enzymes. On the contrary, it exhibited a potent fibrinogenolytic activity degrading the Aα-chain faster than the Bβ-chain, whereas γ-chain was resistant, being stable after 1 hour of hydrolysis. This latter corresponded with a strong delay in the clotting time of fibrinogen (4 mg/ml) pre-incubated with the secretion by thrombin, being 9.53 μg the amount of protein that increased the thrombin clotting time from 20 to 60 sec. The secretion also hydrolyzed fibrin degrading the α-chain. Inhibition studies with Na2EDTA and Benzamidine showed that metalloproteinases and serinoproteinases are the main enzymes responsible for the proteolytic activity on fibrinogen and fibrin. All these results demonstrate that Duvernoy’s gland secretion of P. patagoniensis possesses enzymes able to hydrolyze plasma components playing a relevant role in the blood coagulation. These hydrolyzing activities and those acting on the wall of blood vessels let the secretion exhibit a high hemorrhagic activity, which may result in permanent sequelae or even cause the death of the victims bitten by this colubrid snake.