No abstract

Studies with inhibitors have implicated protein kinase C (PKC) in the adhesive functions of integrin ␣ IIb ␤ 3 in platelets, but the responsible PKC isoforms and mechanisms are unknown. ␣ IIb ␤ 3 interacts directly with tyrosine kinases c-Src and Syk. Therefore, we asked whether ␣ IIb ␤ 3 might also interact with PKC. Of the several PKC isoforms expressed in platelets, only PKC␤ co-immunoprecipitated with ␣ IIb ␤ 3 in response to the interaction of platelets with soluble or immobilized fibrinogen. PKC␤ recruitment to ␣ IIb ␤ 3 was accompanied by a 9-fold increase in PKC activity in ␣ IIb ␤ 3 immunoprecipitates. RACK1, an intracellular adapter for activated PKC␤, also co-immunoprecipitated with ␣ IIb ␤ 3 , but in this case, the interaction was constitutive. Broad spectrum PKC inhibitors blocked both PKC␤ recruitment to ␣ IIb ␤ 3 and the spread of platelets on fibrinogen. Similarly, mouse platelets that are genetically deficient in PKC␤ spread poorly on fibrinogen, despite normal agonist-induced fibrinogen binding. In a Chinese hamster ovary cell model system, adhesion to fibrinogen caused green fluorescent protein-PKC␤I to associate with ␣ IIb ␤ 3 and to co-localize with it at lamellipodial edges. These responses, as well as Chinese hamster ovary cell migration on fibrinogen, were blocked by the deletion of the ␤ 3 cytoplasmic tail or by co-expression of a RACK1 mutant incapable of binding to ␤ 3 . These studies demonstrate that the interaction of ␣ IIb ␤ 3 with activated PKC␤ is regulated by integrin occupancy and can be mediated by RACK1 and that the interaction is required for platelet spreading triggered through ␣ IIb ␤ 3 . Furthermore, the studies extend the concept of ␣ IIb ␤ 3 as a scaffold for multiple protein kinases that regulate the platelet actin cytoskeleton.

Outside-in integrin αIIbβ3 signaling is required for normal platelet thrombus formation and is triggered by c-Src activation through an unknown mechanism. In this study, we demonstrate an essential role for protein–tyrosine phosphatase (PTP)–1B in this process. In resting platelets, c-Src forms a complex with αIIbβ3 and Csk, which phosphorylates c-Src tyrosine 529 to maintain c-Src autoinhibition. Fibrinogen binding to αIIbβ3 triggers PTP-1B recruitment to the αIIbβ3–c-Src–Csk complex in a manner that is dependent on c-Src and specific tyrosine (tyrosine 152 and 153) and proline (proline 309 and 310) residues in PTP-1B. Studies of PTP-1B–deficient mouse platelets indicate that PTP-1B is required for fibrinogen-dependent Csk dissociation from αIIbβ3, dephosphorylation of c-Src tyrosine 529, and c-Src activation. Furthermore, PTP-1B–deficient platelets are defective in outside-in αIIbβ3 signaling in vitro as manifested by poor spreading on fibrinogen and decreased clot retraction, and they exhibit ineffective Ca2+ signaling and thrombus formation in vivo. Thus, PTP-1B is an essential positive regulator of the initiation of outside-in αIIbβ3 signaling in platelets.