Convulxin Induces Platelet Activation by a Tyrosine-Kinase-Dependent Pathway and Stimulates Tyrosine Phosphorylation of Platelet Proteins, Including PLCγ2, Independently of Integrin αIIbβ3

Abstract

Convulxin (Cvx) is a well-characterized platelet aggregating glycoprotein isolated fromCrotalus durissus terrificusandC. d. cascavellavenoms. In the present report we show that Cvx induces tyrosine phosphorylation of human platelet proteins, including phospholipase C-γ 2 (PLCγ2), and also stimulates [3H]arachidonic acid ([3H]AA) mobilization, pleckstrin phosphorylation, and an increase in the cytosolic Ca2+concentration ([Ca2+]in) due to both Ca2+entry and internal 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 genistein inhibited in a dose-dependent manner platelet aggregation induced by Cvx. Both inhibitors significantly blocked to near basal levels breakdown of phosphatidylinositol 4,5-bisphosphate from [myo-2-3H]inositol-labeled platelets and the production of [3H]AA metabolites from [3H]AA-labeled platelets after challenge with Cvx. Cvx provokes an increase in [Ca2+]inin Fura-2-loaded platelets that was abolished by concentrations of staurosporine which also inhibited Cvx-induced platelet aggregation. In addition, Cvx stimulates a rapid increase in tyrosine phosphorylation of human platelets proteins with molecular masses of 40, 72/74, 78/80, 105, 120, and 145 kDa, followed by dephosphorylation. Furthermore, Cvx stimulates a rapid tyrosyl phosphorylation of a 145-kDa molecular mass protein that was identified as PLCγ2. PTP induced by Cvx was not inhibited when platelets were stimulated in the presence of indomethacin, apyrase, EDTA, or RGDS peptide. These results indicate that PTP is chronologically proximal to Cvx binding to platelets, and is independent of aggregation or fibrinogen binding to the integrin αIIbβ3. On the other hand, the dephosphorylation step is inhibited by RGDS peptide or EDTA, suggesting that integrin αIIbβ3is envolved in this step. The profile obtained with Cvx resembles that obtained in platelets adherent to an immobilized ligand, such as immobilized collagen, in which PTP is independent on integrin αIIbβ3. Thus, we suggest that Cvx is an example of a protein with adhesion molecule-like properties; i.e., it is an adhesin. In conclusion, our results show that Cvx induces multiple signaling pathways in platelets via a PTK-dependent pathway involving PLCγ2 tyrosyl phosphorylation, with the subsequent platelet responses. Cvx is unique among platelet soluble agonists because under test tube stirring conditions it induces a PTP profile independently of integrin αIIbβ3.