PTP-1B Is the Protein Tyrosine Phosphatase That Initiates Integrin αIIbβ3 Outside-In Signaling in Platelets.

Abstract

Outside-in signaling is triggered by αIIbβ3 interaction with fibrinogen or von Willebrand factor and is required for effective platelet thrombus formation on damaged vascular surfaces. It requires ligand-induced αIIbβ3 clustering, which results in the activation of several Src family tyrosine kinases that are directly associated with αIIbβ3. However, the mechanism of Src activation by αIIbβ3 is incompletely understood. Here we demonstrate that the protein tyrosine phosphatase, PTP-1B, is required for integrin activation of c-Src and for outside-in signaling in platelets. In human or mouse platelets, c-Src was constitutively-associated with αIIbβ3 through an interaction that involved the β3 cytoplasmic domain and the c-Src SH3 domain. In resting platelets, the catalytic activity of this pool of c-Src was relatively low due to auto-inhibitory constraints imposed, in part, by an intramolecular interaction between the SH2 domain and the C-terminus of c-Src. This was promoted by phosphorylation of the C-terminal tyrosine of c-Src, tyrosine 529, by the Csk kinase. When platelets became adherent to fibrinogen or bound soluble fibrinogen in response to MnCl2, PTP-1B became associated with αIIbβ3 and c-Src, as determined by co-immunoprecipitation. At the same time, Csk dissociated from the integrin complex, c-Src tyrosine 529 became dephosphorylated, and c-Src became activated, as measured by phosphorylation of activation loop tyrosine 418. In marked contrast, gene-targeted mouse platelets deficient in PTP-1B (PTP-1B−/−) exhibited no dephosphorylation of c-Src tyrosine 529 and no activation of c-Src in response to fibrinogen binding. Furthermore, unlike wild-type platelets, PTP-1B−/− platelets exhibited little or no fibrinogen-dependent tyrosine phosphorylation of c-Src substrates, and they failed to undergo cytoskeletal reorganization or to spread on fibrinogen. These abnormalities of PTP-1B−/− platelets were due to defective outside-in signaling because surface expression of αIIbβ3 was normal, and fibrinogen bound normally in response to platelet stimulation with ADP or PAR-4 receptor agonists. When Fura-2-loaded platelets were infused and visualized by real time intravital microscopy during arterial thrombus development following laser injury to the cremaster microcirculation of a living mouse, PTP-1B−/− platelets exhibited significantly reduced intracellular free Ca2+ responses and formed smaller, less stable thrombi compared to wild-type platelets. Altogether, these studies establish that PTP-1B is a necessary, positive regulator of αIIbβ3-associated c-Src during the initiation phase of outside-in signaling in platelets. Consequently, disruption of specific interactions between αIIbβ3, c-Src and PTP-1B, or abnormalities in the activation of integrin-associated c-Src or PTP-1B, may cause defects in primary hemostasis. Moreover, these interactions provide potential targets for pharmacological blockade of outside-in signaling during arterial thrombosis.