Identification of a Unique Co-operative Phosphoinositide 3-Kinase Signaling Mechanism Regulating Integrin αIIbβ3 Adhesive Function in Platelets

Phosphoinositide (PI) 3-kinases play an important role in regulating the adhesive function of a variety of cell types through affinity modulation of integrins. Two type I PI 3-kinase isoforms (p110β and p110γ) have been implicated in Gi-dependent integrin αIIbβ3 regulation in platelets, however, the mechanisms by which they coordinate their signaling function remains unknown. By employing isoform-selective PI 3-kinase inhibitors and knock-out mouse models we have identified a unique mechanism of PI 3-kinase signaling co-operativity in platelets. We demonstrate that p110β is primarily responsible for Gi-dependent phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2) production in ADP-stimulated platelets and is linked to the activation of Rap1b and AKT. In contrast, defective integrin αIIbβ3 activation in p110γ-/- platelets was not associated with alterations in the levels of PI(3,4)P2 or active Rap1b/AKT. Analysis of the effects of active site pharmacological inhibitors confirmed that p110γ principally regulated integrin αIIbβ3 activation through a non-catalytic signaling mechanism. Inhibition of the kinase function of PI 3-kinases, combined with deletion of p110γ, led to a major reduction in integrin αIIbβ3 activation, resulting in a profound defect in platelet aggregation, hemostatic plug formation, and arterial thrombosis. These studies demonstrate a kinase-independent signaling function for p110γ in platelets. Moreover, they demonstrate that the combined catalytic and non-catalytic signaling function of p110β and p110γ is critical for P2Y12/Gi-dependent integrinαIIbβ3 regulation. These findings have potentially important implications for the rationale design of novel antiplatelet therapies targeting PI 3-kinase signaling pathways.