Participation of tyrosine phosphorylation in cytoskeletal reorganization, alpha(IIb)beta(3) integrin receptor activation, and aspirin-insensitive mechanisms of thrombin-stimulated human platelets

Background-Fibrinogen binding to the active conformation of the alpha(IIb)b eta(3) integrin receptor (glycoprotein IIb/IIIa) and cytoskeletal reorganiz ation are important events in platelet function. Tyrosine phosphorylation o f platelet proteins plays an essential role in platelet signal transduction pathways. We studied the participation of tyrosine kinases on these aspect s of platelet reactivity and their importance in cyclooxygenase (COX)-1-ind ependent mechanisms in thrombin-stimulated human platelets. Methods and Results-Using washed platelets from normal donors and tyrphosti n-A47 and aspirin as tyrosine kinase and COX-1 inhibitors, respectively, we found that tyrphostin-A47 downregulated (1) the thrombin-activated conform ational change of alpha(IIb)beta(3), (2) actin polymerization and cytoskele tal reorganization, and (3) the quantity of tyrosine-phosphorylated protein s associated with the reorganized cytoskeleton. The latter are important co mponents of multimolecular signaling complexes. Concomitantly, platelet agg regation and secretion were significantly reduced. Aspirin did not affect r eceptor activation or tyrosine phosphorylation but did decrease the initial (30-second) burst of actin polymerization, Importantly, aspirin significan tly amplified the inhibitory effect of tyrphostin-A47 on all aspects of pla telet 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 cytoskelet al assembly in thrombin-stimulated human platelets. Inhibition of these asp ects of platelet function with tyrphostin-A47 is amplified when platelets a re treated with aspirin. Therefore, tyrosine phosphorylation is a major com ponent of early signaling events and of COX-1-independent mechanisms of thr ombin-induced platelet reactivity. The study results may indicate a novel t arget for therapeutic intervention.