The late and sustained activation of PI 3-kinase is required for PtdIns(3,4)P₂ accumulation and irreversible aggregation induced by TRAP in the presence of ADP. Platelet aggregation was measured as described in Materials and Methods. Various concentrations of the 2 unrelated PI 3-kinase inhibitors, wortmannin (A) or LY294002 (B), were added 2 minutes after stimulation with 40 μmol/L TRAP, as indicated by the arrow. Data are representative of 3 independent experiments with very similar results. (C) [³²P]-labeled human platelets (7.5 × 10⁸/mL) were stimulated with 40 μmol/L TRAP. After 2 minutes, 50 nmol/L wortmannin (•), 25 μmol/L LY 294002 (○), or 0.1% Me₂SO (▪) was added. The reactions were stopped at different times by addition of chloroform/methanol and the level of [³²P]PtdIns(3,4)P₂ was measured by HPLC as indicated in Materials and Methods.