CONVULXIN INDUCES PLATELET ACTIVATION BY A TYROSINE-KINASE-DEPENDENT PATHWAY AND STIMULATES TYROSINE PHOSPHORYLATION OF PLATELET PROTEINS, INCLUDING PLC-GAMMA-2, INDEPENDENTLY OF INTEGRIN ALPHA(IIB)BETA(3)

(1)Convulxin (Cvx) is a well characterized platelet aggregating glycop rotein isolated from Crotalus durissus terrificus and C. d cascavella venoms. In the present report we show that Cvx induces tyrosine phosph orylation of human platelet proteins, including phospholipase C-gamma 2 (PLC gamma 2), and also stimulates [H-3]arachidonic acid ([H-3]AA) m obilization, pleckstrin phosphorylation, and an increase in the cytoso lic Ca2+ concentration ([Ca2+](in)) due to both Ca2+ entry and interna l Ca2+ mobilization. Staurosporine, a potent protein kinase inhibitor, and genistein, a specific inhibitor of protein tyrosine kinases (PTK) , were used to evaluate the role of protein tyrosine phosphorylation ( PTP) in the signal transduction evoked by Cvx. Staurosporine and genis tein inhibited in a dose-dependent manner platelet aggregation induced by Cvx. Both inhibitors significantly blocked to near basal levels br eakdown of phosphatidylinositol 4,5-bisphosphate from [myo-2-H-3]inosi tol-labeled platelets and the production of [H-3]AA metabolites from [ H-3]AA-labeled platelets after challenge with Cvx. Cvx provokes an inc rease in [Ca2+](in), in Fura-loaded platelets that was abolished by co ncentrations of staurosporine which also inhibited Cvx-induced platele t aggregation. In addition, Cvx stimulates a rapid increase in tyrosin e phosphorylation of human platelets proteins with molecular masses Of 40, 72/74, 78/80, 105, 120, and 145 kDa, followed by dephosphorylatio n. Furthermore, Cvx stimulates a rapid tyrosyl phosphorylation of a 14 5-kDa molecular mass protein that was identified as PLC gamma 2. PTP i nduced by Cvx was not inhibited when platelets were stimulated in the presence of indomethacin, apyrase, EDTA, or RODS peptide. These result s indicate that PTP is chronologically proximal to Cvx binding to plat elets, and is independent of aggregation or fibrinogen binding to the integrin alpha(IIb)beta(3). On the other hand, the dephosphorylation s tep is inhibited by RGDS peptide or EDTA, suggesting that integrin alp ha(IIb)beta(3) is envolved in this step. The profile obtained with Cvx resembles that obtained in platelets adherent to an immobilized ligan d, such as immobilized collagen, in which PTP is independent on integr in alpha(IIb)beta(3). Thus, we suggest that Cvx is an example of a pro tein with adhesion molecule-like properties; i.e., it is an adhesin. I n conclusion, our results show that Cvx induces multiple signaling pat hways in platelets via a PTK-dependent pathway involving PLC gamma 2 t yrosyl phosphorylation, with the subsequent platelet responses. Cvx is unique among platelet soluble agonists because under test tube stirri ng conditions it induces a PTP profile independently of integrin alpha (IIb)beta(3). (C) 1998 Academic Press.