Neutrophil elastase (NE) upregulates the fibrinogen binding activity of the platelet integrin αIIbβ3 through proteolysis of the αIIb subunit. This cleavage allows a strong potentiation of platelet aggregation induced by low concentrations of cathepsin G (CG), another neutrophil serine proteinase. During this activation process, we observed a strong fibrinogen binding and aggregation-dependent phosphatidylinositol 3,4-bis-phosphate (PtdIns(3,4)P2) accumulation. PtdIns(3,4)P2 has been suggested to play a role in the stabilization of platelet aggregation, possibly through the control of a maintained αIIbβ3 integrin activation. Here we show that inhibition of phosphoinositide 3-kinase (PI 3-K) by very low concentrations of wortmannin or LY294002 transformed the irreversible platelet aggregation induced by a combination of NE and low concentrations of CG into a reversible aggregation. However, although inhibition of PI 3-K was very efficient in inducing platelet disaggregation, it did not modify the level of αIIbβ3 activation as assessed by binding of an activation-dependent antibody. These results indicate that PI 3-K activity can control the irreversibility of platelet aggregation even under conditions where αIIbβ3 integrin remains activated.