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 alphaIIbbeta3-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 PLCbeta2, PLCbeta3a (155 kDa), and PLCgamma2 and the minor PLCs (PLCbeta1 and PLCgamma1) were located in the Triton X-100-soluble (Tx.Sol) fraction and the membrane skeleton, whereas PLCbeta3b (140 kDa) was present only in Tx.Sol fraction when examined by Western immunoblotting. Thrombin stimulation caused a rapid and transient translocation of PLCbeta3a and PLCbeta3b and a slower accumulation of PLCbeta2 and PLCgamma2 in the reorganized CSK. The translocation to CSK of both PLCbeta3a and PLCbeta3b, but not PLCbeta2, was dependent on integrin alphaIIbbeta3-mediated aggregation. Furthermore, an actin polymerization inhibitor, cytochalasin D, or a protein tyrosine kinase inhibitor, genistein, abolished the CSK association of alphaIIbbeta3, PLCbeta3a, and PLCbeta3b. In the genistein-pretreated platelets, pp60(c-)src, Gq, and protein kinase Calpha were no longer able to associate with CSK. In contrast, these agents had no or marginal inhibitory effects on the CSK association of PLCbeta2 and Gi2. The late diacylglycerol generation induced by thrombin stimulation was significantly reduced by the genistein treatment. These results suggest that the integrin alphaIIbbeta3-mediated cytoskeletal association of PLCbeta3 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.