Glycoprotein (GP) VI is a critical platelet collagen receptor. PI3-kinase (PI3-K) plays an important role in GPVI-mediated platelet activation, yet the major PI3-K isoforms involved in this process have not been identified. In addition, stimulation of GPVI results in the activation of Akt, a downstream effector of PI3-K. Thus, we investigated the contribution of PI3-K isoforms to GPVI-mediated platelet activation and Akt activation. A protein kinase C inhibitor GF 109203X or a P2Y12 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 PI 3-K inhibitor LY294002 and PI3-Kbeta inhibitor TGX-221 indicating an essential role of PI3-Kbeta in Akt activation directly downstream of GPVI. Moreover, GPVI-mediated platelet aggregation, secretion, and intracellular Ca(2+) mobilization were significantly inhibited by TGX-221, and less strongly inhibited by PI3-Kalpha inhibitor PIK75, but were not affected by PI3-Kgamma inhibitor AS252424 and PI3-Kdelta inhibitor IC87114. Consistently, GPVI-induced integrin alphaIIbbeta3 activation of PI3-Kgamma-/- and PI3-Kdelta-/- platelets also showed no significant difference compared to wild-type platelets. These results demonstrate that GPVI-induced Akt activation in platelets is dependent in part on Gi stimulation through P2Y12 receptor activation by secreted ADP. In addition, a significant portion of GPVI-dependent, ADP-independent Akt activation also exists, and PI 3-Kbeta plays an essential role in GPVI-mediated platelet aggregation and Akt activation.