PLATELET TYROSINE KINASES AND FIBRINOGEN RECEPTOR ACTIVATION

Platelet adhesion and aggregation during hemostasis and thrombosis are usually limited to sites where the integrity of the vessel wall is di srupted. The high concentration of platelet agonists within these site s represents a putative control mechanism for targeting platelet activ ation. Although much has been learned about the intracellular signalin g systems controlling platelet activation, our understanding of the co nnection between signaling molecules and platelet aggregation remains limited. Tyrosine kinases are important signaling enzymes in cells and are abundant in platelets. Previous reports indicate that binding of glycoprotein IIb-IIIa (GPIIb-IIIa) to fibrinogen can induce the tyrosi ne phosphorylation of specific substrates. We show that, in turn, prot ein tyrosine kinase activity is necessary for agonist-induced activati on of GPIIb-IIIa. Genistein and the tyrphostin AG-18 are two specific tyrosine kinase inhibitors, and the former has been shown to inhibit p latelet aggregation. We use genistein and AG-18 in the present study t o demonstrate that aggregation inhibition is due to suppression of GPI Ib-IIIa activation. In contrast, genistin, an isoflavone compound rela ted to genistein, and acetylsalicylic acid do not affect the tyrosine kinase-signaling pathway, nor do they inhibit GPIIb-IIIa activation in duced by strong agonists. On identifying prominent tyrosine kinase sub strates in activated platelets, we confirm that several substrates cor respond to proteins associated with the cytoskeleton: the 85-kD subuni t of phosphatidylinositol 3-kinase, the SH3-containing and actin-assoc iating p85, pp60(Src), and pp125(FRK). Our data showing that tyrosine kinase activity is required for GPIIb-IIIa activation, together with p revious studies indicating that fibrinogen binding to its receptor res ults in tyrosine phosphorylation of platelet substrates, suggest that a dual regulatory mechanism allows for full platelet response only at sites where both pathways are activated, namely disrupted vessel walls .