Involvement of Hic-5 in platelet activation: integrin alpha IIb beta 3-dependent tyrosine phosphorylation and association with proline-rich tyrosine kinase 2

Hic-5 and paxillin, members of the LIM protein family, have been shown to b e localized in focal adhesion and to have a role in integrin-mediated signa lling. In the present study we examined the involvement of Hic-5 in human p latelet activation: platelets express Hic-5 but not paxiliin, whereas human umbilical-vein vascular endothelial cells and MEG-01 cells express mainly paxiliin. When platelets were stimulated with thrombin, collagen or the sta ble thromboxane A(2) analogue U46619, Hic-5 was markedly tyrosine-phosphory lated, in a manner dependent on integrin alpha IIb beta3-mediated aggregati on. In addition, direct activation of protein kinase C with PMA resulted in tyrosine phosphorylation of Hic-5 only when platelets were fully aggregate d with the exogenous addition of fibrinogen. Furthermore, PMA-induced Hic-5 tyrosine phosphorylation was also observed when platelets adhered to immob ilized fibrinogen. In studies on immunoprecipitation and immunodepletion, H ic-5 seemed to associate with proline-rich tyrosine kinase 2. (Pyk2) but on ly marginally with focal adhesion kinase. When platelets were stimulated wi th thrombin, both Hic-5 and Pyk2 translocated to the cytoskeleton from the cytosol and membrane fractions in a manner dependent on alpha II beta3-medi ated aggregation. Finally, on stimulation with PMA, Hic-5: as well as Pyk2, translocated to the cell periphery, where a meshwork of actin filaments as sembled after adhesion to immobilized fibrinogen. Our results suggest that Hic-5 might be important in platelet aggregation and adhesion, in a manner dependent on alpha IIb beta3-mediated outside-in signalling, through associ ation with Pyk2.