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.
1Convulxin (Cvx) is a well-characterized platelet aggregating glycoprotein isolated from Crotalus durissus terrificus and C. d. cascavella venoms. In the present report we show that Cvx induces tyrosine phosphorylation of human platelet proteins, including phospholipase C-gamma 2 (PLC gamma 2), 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+]in in 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 PLC gamma 2. 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 alpha IIb beta 3. On the other hand, the dephosphorylation step is inhibited by RGDS peptide or EDTA, suggesting that integrin alpha IIb beta 3 is 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 alpha IIb beta 3. 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 PLC gamma 2 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 alpha IIb beta 3.
Francischetti
I M
IM
Department of Medical Biochemistry, Federal University of Rio de Janeiro, Cidade Universitária, Ilha do Fundão, Brazil.
Ghazaleh
F A
FA
Reis
R A
RA
Carlini
C R
CR
Guimarães
J A
JA
D000818:Animals; D003435:Crotalid Venoms / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D037181:Lectins, C-Type; D051966:Phospholipase C gamma; D010766:Phosphorylation; D010974:Platelet Aggregation / Q000187:drug effects*; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011817:Rabbits; D015398:Signal Transduction; D012996:Solutions; D019311:Staurosporine / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
D003435:Crotalid Venoms; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D037181:Lectins, C-Type; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D012996:Solutions; C028890:convulxin; D014443:Tyrosine; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D019311:Staurosporine