Differential translocation of phospholipase C isozymes to integrin-mediated cytoskeletal complexes in thrombin stimulated human platelets

To investigate a role of phospholipase C (PLC) isozymes in the integrin alpha(IIb)beta(3)-mediated signaling, their location was examined in thrombin-activated human platelets, revealing different regulation of their translocation to the cytoskeleton (CSK). In resting platelets, the major PLCs such as PLC beta 2, PLC beta 3a (155 kDa), and PLC gamma 2 and the minor PLCs (PLC beta 1 and PLC gamma 1) were located in the Triton X-100-soluble (Tx . Sol) fraction and the membrane skeleton, whereas PLC beta 3b (140 kDa) was present only in Tx . Sol fraction when examined by Western immunoblotting, Thrombin stimulation caused a rapid and transient translocation of PLC beta 3a and PLC beta 3b and a slower accumulation of PLC beta 2 and PLC gamma 2 in the reorganized CSK. The translocation to CSK of both PLC beta 3a and PLC beta 3b, but not PLC beta 2, was dependent on integrin alpha(IIb)beta(3)-mediated aggregation, Furthermore, an actin polymerization inhibitor, genistein, abolished the CSK association of alpha(IIb)beta(3), PLC beta 3a, and PLC beta 3b. In the genistein pretreated platelets, pp60(c-src), G(q), and protein kinase C alpha were no longer able to associate with CSK, In contrast, these agents had no or marginal inhibitory effects on the CSK association of PLC beta 2 and G(i2). The late diacylglycerol generation induced by thrombin stimulation was significantly reduced by the genistein treatment, These results suggest that the inte grin alpha(IIb)beta 3-mediated cytoskeletal association of PLC beta 3 is regulated by protein tyrosine kinase and also that the activation of the relocated PLC may play a role in the late platelet-to-platelet aggregation in thrombin-stimulated human platelets.