Bothropstoxin-II (Bthtx-II), an Asp-49 phospholipase A(2) (D-PLA(2)) isolated from Bothrops jararacussu snake venom is able to induce platelet aggregation in a concentration-dependent manner. This effect was not due to the release of ADP from platelets since the aggregation was not suppressed by ADP scavenger systems. PMSF and PPACK were unable to inhibit Bthtx-II-induced platelet aggregation. Thus, a thrombin-like proaggregating activity of Bthtx-II can be excluded as its mechanism of action. On the other hand, indomethacin at low concentrations inhibited more markedly the ATP-release reaction than the aggregation induced by Bthtx-II, indicating that generation of cyclooxigenase products is not the most important event for the platelet aggregation reaction. It was also found that staurosporine and genistein suppressed both platelet aggregation and ATP-release reactions, but not the platelet shape-change induced by Bthtx-II. Substances that either directly activates adenylyl cyclase enzyme (forskolin and PGE(1)) or cell-permeant increasing agents (dibutyril-cAMP) inhibited in a concentration-dependent fashion, the platelet aggregation effects induced by the protein. It is concluded that Bthtx-II induces platelet aggregation and secretion through multiple signal transduction pathways.