CALPAIN REGULATION OF CYTOSKELETAL SIGNALING COMPLEXES IN VON-WILLEBRAND FACTOR-STIMULATED PLATELETS - DISTINCT ROLES FOR GLYCOPROTEIN IB-V-IX AND GLYCOPROTEIN IIB-IIIA (INTEGRIN ALPHA(IIB)BETA(3)) IN VON-WILLEBRAND-FACTOR-INDUCED SIGNAL-TRANSDUCTION

The adhesion of platelets to sites of vascular injury is critically de pendent on the binding of subendothelial bound von Willebrand factor ( vWf) to the platelet surface glycoprotein complexes, GP Ib-V-IX and GP IIb-IIIa (integrin alpha(IIb)beta(3)). There is growing evidence that the binding of vWf to these receptors is not only essential for stabl e platelet adhesion but is also important for the transduction of acti vation signals required for changes in platelet morphology, granule se cretion, and platelet aggregation. In this study we have investigated signaling events induced by vWf binding to GP Ib-V-IX in both spreadin g and aggregated platelets. The adhesion of platelets to vWf resulted in dramatic actin filament reorganization, as assessed by immunofluore scence with fluorescein isothiocyanate-conjugated phalloidin, and the cytoskeletal recruitment of various structural proteins (talin and int egrin alpha(IIb)beta(3)) and signaling enzymes (pp60(c-src), focal adh esion kinase (FAK), phosphatidylinositol 3-kinase (PI 3-kinase), and p rotein-tyrosine phosphatase (PTP)-1B). Time course experiments in both spreading and aggregated platelets revealed that talin, FAK, and PTP- 1B were proteolyzed after translocation to the cytoskeleton. The prote olysis of these proteins was dependent on the presence of extracellula r calcium and was specifically inhibited by pretreating platelets with the membrane-permeable calpain inhibitors calpeptin, E64d, and MDL 28 ,170, but not with the membrane-impermeable inhibitors leupeptin, E64, and calpastatin. The cytoskeletal translocation of signaling enzymes in vWf-stimulated platelets was abolished by pretreating platelets wit h an anti-GP Ib-V-IX antibody but was unaffected by blocking ligand bi nding to integrin alpha(IIb)beta(3). In contrast, calpain activation i n vWf-stimulated platelets required ligand binding to both GP Ib-V-IX and integrin alpha(IIb)beta(3). The activation of calpain in both spre ading and aggregated platelets resulted in a substantial decrease in t he level of tyrosine phosphorylation of multiple platelet proteins and was associated with a 50-80% reduction in the amount of cytoskeletal associated talin, integrin alpha(IIb)beta(3), PI 3-kinase, FAK, pp60(c -src), and PTP-1B. These studies suggest a potentially important role for calpain in regulating the formation and/or stability of cytoskelet al signaling complexes in vWf-stimulated platelets. Furthermore, they demonstrate distinct roles for GP Ib-V-IX and integrin alpha(IIb)beta( 3) in vWf-induced signal transduction.