Poster 231 - The action of snake venom metalloproteases on blood coagulation and endothelial cells

Poster 231. Congresso da Sociedade Brasileira de Toxinologia, 8., Symposium of the Pan American Section of the International Society on Toxinology, 8., 2004, Angra dos Reis, Brasil. Abstracts... J. Venom. Anim. Toxins incl.Trop. Dis., 2004, 10, 3, p.590.

The Action Of Snake Venom Metalloproteases On Blood Coagulation And Endothelial Cells

From the Brazilian snake Bothropsinsularis which is restricted to Queimada Grande Island (São Paulo coast), and from the Bothropsbrazili which is found in the Amazon Forest, three Metalloproteases group-I (SVMPs-I), named P141 (B. insularis), B151 and RQ2 (B. brazili) were previously purified in our laboratory. The objective of this work is to compare the activity of these SVMPs on the blood coagulation and in the endothelial cells viability. The fibrino(geno)lytic activity was observed by SDS-PAGE and by fibrin plates; the prothrombin activation as well as the Factor X activation were measured indirectly using chromogenic subtracts (respectively S-2238 and S-2765), the action on washed platelets was determined using collagen and thrombin as agonists. The apoptosis induction and viability of human umbilical vein endothelial cells (HUVECs) were determined in presence of these proteins by immunofluorescence cell staining with acrinidine orange and ethidium bromide. All the enzymes were able to degrade fibrin, fibrinogen (a and b-chains) and to convert prothrombin in thrombin, moreover, the P141 also activated Factor X. The proteins B151 and P141 inhibited platelet aggregation induced by collagen, in a dose-dependent manner, whereas RQ2 did not present this effect. The P141 and B151 caused a slight inhibition on the platelet aggregation induced by thrombin (35 %) that could be related to the interference on GPIb as reveled by flow citometry assays. GPIIb and P-selectin (basal or induced by thrombin) were not affected by all proteins. In HUVECs, B151 and RQ2 induced monolayer cells detach and cells clusters, and the percentage of apoptotic cells induced by B151 and RQ2 were greater than control samples. In conclusion, despite similar structures these enzymes differs potentially in its activities suggesting the role of HXXXHXXXXXHZ domain as well as others sequences presents in their structure.