Participation of tyrosine phosphorylation in cytoskeletal reorganization, αIIbβ3 integrin receptor activation, and aspirin-insensitive mechanisms of thrombin-stimulated human platelets

Background-Fibrinogen binding to the active conformation of the alpha(IIb)beta(3) integrin receptor (glycoprotein IIb/IIIa) and cytoskeletal reorganization are important events in platelet function. Tyrosine phosphorylation of platelet proteins plays an essential role in platelet signal transduction pathways. We studied the participation of tyrosine kinases on these aspects of platelet reactivity and their importance in cyclooxygenase (COX)-1-independent mechanisms in thrombin-stimulated human platelets. Methods and Results-Using washed platelets from normal donors and tyrphostin-A47 and aspirin as tyrosine kinase and COX-1 inhibitors, respectively, we found that tyrphostin-A47 downregulated (1) the thrombin-activated conformational change of alpha(IIb)beta(3), (2) actin polymerization and cytoskeletal reorganization, and (3) the quantity of tyrosine-phosphorylated proteins associated with the reorganized cytoskeleton. The latter are important components of multimolecular signaling complexes. Concomitantly, platelet aggregation and secretion were significantly reduced. Aspirin did not affect receptor activation or tyrosine phosphorylation but did decrease the initial (30-second) burst of actin polymerization, Importantly, aspirin significantly amplified the inhibitory effect of tyrphostin-A47 on all aspects of platelet reactivity that we evaluated. Conclusions-Tyrosine protein phosphorylation is a regulatory control system of the inside-out mechanism of alpha(IIb)beta(3) activation and cytoskeletal assembly in thrombin-stimulated human platelets. Inhibition of these aspects of platelet function with tyrphostin-A47 is amplified when platelets are treated with aspirin. Therefore, tyrosine phosphorylation is a major component of early signaling events and of COX-1-independent mechanisms of thrombin-induced platelet reactivity. The study results may indicate a novel target for therapeutic intervention.