During thrombus formation, thrombin, which is abundantly present at sites of vascular injury, activates platelets in part via autocrine-produced ADP. We investigated the signaling pathways by which thrombin and ADP in synergy induced platelet Ca2+ elevation and procoagulant activity, and we monitored the consequences for the coagulation process. Even at high thrombin concentration, autocrine and added ADP enhanced and prolonged Ca2+ depletion from internal stores via stimulation of the P2Y12 receptors. This P2Y12-dependent effect was mediated via two distinct signaling pathways. The first is enhanced Ca2+ mobilization by the inositol 1,4,5-trisphosphate receptors due to inhibition of protein kinase A. The second pathway concerns prolonged activation of phosphoinositide 3-kinase (PI3-K) and phospholipase C. Experiments with phosphoinositide 3-kinase isoform-selective inhibitors and p110γ deficient platelets demonstrated that the phosphoinositide 3-kinase β and not the phosphoinositide 3-kinase γ isoform is responsible for the prolonged Ca2+ response and for the subsequent increases in procoagulant activity and coagulation. Taken together, these results demonstrate a dual P2Y12-dependent signaling mechanism, which increases the platelet-activating effect of thrombin by prolongation of Ca2+ elevation, thereby facilitating the coagulation process.