Role of Phosphoinositide 3-Kinase β in Glycoprotein VI-mediated Akt Activation in Platelets

Glycoprotein (GP) VI is a critical platelet collagen receptor. Phosphoinositide 3-kinase (PI3K) plays an important role in GPVI-mediated platelet activation, yet the major PI3K isoforms involved in this process have not been identified. In addition, stimulation of GPVI results in the activation of Akt, a downstream effector of PI3K. Thus, we investigated the contribution of PI3K isoforms to GPVI-mediated platelet activation and Akt activation. A protein kinase C inhibitor GF 109203X or a P2Y(12) receptor antagonist AR-C69931MX partly reduced GPVI-induced Akt phosphorylation. Platelets from mice dosed with clopidogrel also showed partial Akt phosphorylation, indicating that GPVI-mediated Akt phosphorylation is regulated by both secretion-dependent and -independent pathways. In addition, GPVI-induced Akt phosphorylation in the presence of ADP antagonists was completely inhibited by PI3K inhibitor LY294002 and PI3K beta inhibitor TGX-221 indicating an essential role of PI3K beta in Akt activation directly downstream of GPVI. Moreover, GPVI-mediated platelet aggregation, secretion, and intracellular Ca2+ mobilization were significantly inhibited by TGX-221, and less strongly inhibited by PI3K alpha inhibitor PIK75, but were not affected by PI3K gamma inhibitor AS252424 and PI3K delta inhibitor IC87114. Consistently, GPVI-induced integrin alpha(IIb)beta(3) activation of PI3K gamma(-/-) and PI3K delta(-/-) platelets also showed no significant difference compared with wild-type platelets. These results demonstrate that GPVI-induced Akt activation in platelets is dependent in part on G(1) stimulation through P2Y12 receptor activation by secreted ADP. In addition, a significant portion of GPVI-dependent, ADP-independent Akt activation also exists, and PI3K beta plays an essential role in GPVI-mediated platelet aggregation and Akt activation.