Collagen activates platelets through a tyrosine kinase-dependent pathway, involving phospholipase Cgamma2. Functional responses such as aggregation and secretion induced by collagen are potentiated by preincubation with thrombopoietin (TPO). In this study, we show that collagen and thrombopoietin activate the phosphatidylinositol 3-kinase (PI 3-kinase) pathway and that this contributes to their respective actions. The structurally distinct inhibitors of PI 3-kinase, wortmannin, and LY294002, completely inhibit formation of phosphatidylinositol 3,4,5-trisphosphate by collagen. This leads to a substantial reduction in the formation of inositol phosphates and phosphatidic acid, 2 indices of PLC activity, and the consequent inhibition of intracellular Ca(++) [Ca(++)](i), aggregation and secretion. Potentiation of the collagen response by TPO is prevented in the presence of wortmannin and LY294002. However, when the 2 PI 3-kinase inhibitors are given after the addition of TPO but before the collagen, recovery of potentiation is observed. This suggests that potentiation is mediated through activation of PI 3-kinase. TPO stimulates aggregation of platelets from a low percentage of donors and this is also blocked by wortmannin. These results suggest that the PI 3-kinase pathway plays an important role in signaling by collagen and in the priming action of TPO.