ON THE ROLE OF THE PLATELET MEMBRANE SKELETON IN MEDIATING SIGNAL-TRANSDUCTION - ASSOCIATION OF GP-IIB-IIIA, PP60(C-SRC), PP62(C-YES), AND THE P21(RAS) GTPASE-ACTIVATING PROTEIN WITH THE MEMBRANE SKELETON

The platelet plasma membrane is lined with a membrane skeleton compose d of short actin filaments, actin-binding protein, spectrin, vinculin, and other unidentified proteins. It is connected to the outside of th e cell through association with the cytoplasmic domains of transmembra ne receptors. In detergent-lysed platelets, cytoplasmic actin filament s are sedimented by centrifugation at 15,600 X g, but the sedimentatio n of membrane skeleton fragments requires higher g-forces (100,000 X g ). In the present study, we show that the major platelet integrin, gly coprotein (GP) IIb-IIIa, sediments from detergent-lysed platelets at 1 00,000 x g together with fragments of the membrane skeleton that conta in the cytoskeletal proteins spectrin, vinculin, and talin. In additio n, this cell fraction contained the tyrosine kinases pp60c-src and pp6 2c-yes and the p21ras GTPase-activating protein (GAP). After thrombin- induced platelet aggregation mediated by fibrinogen binding to GP IIb- IIIa on adjacent platelets, we detected a redistribution of spectrin, talin, vinculin, pp60c-src, and pp62c-yes to the fraction that sedimen ts at 15,600 X g. The redistribution of these proteins from the high-s peed detergent-insoluble fraction to the low-speed fraction correlated with the extent of aggregation and was not detected in aggregation-de fective thrombasthenic platelets (which lack the GP IIb-IIIa complex). In addition, many of the proteins phosphorylated on tyrosine in activ ated platelets were present in detergent-insoluble fractions. These re sults are consistent with the possibilities that 1) GP IIb-IIIa, pp60c -src, pp62c-yes, and GAP associate with a membrane skeleton fraction t hat contains spectrin, vinculin, and talin, 2) the association of GP I Ib-IIIa with adhesive ligand in a platelet aggregate causes components of the membrane skeleton to undergo altered association with cytoplas mic actin filaments, and 3) many of the proteins phosphorylated on tyr osine residues in activated platelets are components of the cytoskelet on. The results imply that the membrane skeleton may play an important role in binding signaling molecules at sites of integrin-cytoskeleton interactions and in mediating signal transduction events in platelets . Further, GP IIb-IIIa-induced redistribution of components of the mem brane skeleton and associated signaling molecules may represent an imp ortant step in regulating integrin-induced motile events in platelets.