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 (p110 beta and p110 gamma) have been implicated in G(i)-dependent integrin alpha(IIb)beta(3) 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 p110 beta is primarily responsible for G(i)-dependent phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)) production in ADP-stimulated platelets and is linked to the activation of Rap1b and AKT. In contrast, defective integrin alpha(IIb)beta(3) activation in p110 gamma(-/-) platelets was not associated with alterations in the levels of PI(3,4)P(2) or active Rap1b/AKT. Analysis of the effects of active site pharmacological inhibitors confirmed that p110 gamma principally regulated integrin alpha(IIb)beta(3) activation through a non-catalytic signaling mechanism. Inhibition of the kinase function of PI 3-kinases, combined with deletion of p110 gamma, led to a major reduction in integrin alpha(IIb)beta(3) activation, resulting in a profound defect in platelet aggregation, hemostatic plug formation, and arterial thrombosis. These studies demonstrate a kinase-independent signaling function for p110 gamma in platelets. Moreover, they demonstrate that the combined catalytic and non-catalytic signaling function of p110 beta and p110 gamma is critical for P2Y(12)/G(i)-dependent integrin alpha(IIb)beta(3) regulation. These findings have potentially important implications for the rationale design of novel antiplatelet therapies targeting PI 3-kinase signaling pathways.
Highlights
The phosphoinositide (PI)2 3-kinases are a well defined family of lipid kinases that participate in a broad range of signaling
Isoform-selective p110␥ inhibitors are currently being evaluated in a broad range of inflammatory disorders [13]. p110␦ appears to have an important role in adaptive immunity, in T- and B-cell, and mast cell signaling [14] with evidence that inhibition of p110␦ reduces allergic responses [15]
Our studies indicate that p110 predominately regulates Gi-dependent integrin ␣IIb3 activation through a classical lipid kinase-dependent mechanism, involving Rap1 and AKT, whereas p110␥ appears to regulate integrin ␣IIb3 principally through a non-catalytic signaling mechanism
Summary
The phosphoinositide (PI)2 3-kinases are a well defined family of lipid kinases that participate in a broad range of signaling. To investigate the relative contribution of type I PI 3-kinase isoforms to ADP-dependent integrin ␣IIb3 regulation, fibrinogen binding studies were performed on p110␥Ϫ/Ϫ and p110␦Ϫ/Ϫ platelets and on wild type platelets treated with the isoform-selective pharmacological inhibitors against p110 (TGX221) and p110␣ (PIK-75).
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