Identification of a Unique Co-operative Phosphoinositide 3-Kinase Signaling Mechanism Regulating Integrin α_IIbβ₃ Adhesive Function in Platelets^*

Abstract

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 G_i-dependent integrin α_IIbβ₃ 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 G_i-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 α_IIbβ₃ 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 α_IIbβ₃ 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 α_IIbβ₃ 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/G_i-dependent integrinα_IIbβ₃ regulation. These findings have potentially important implications for the rationale design of novel antiplatelet therapies targeting PI 3-kinase signaling pathways.