Inhibition of PI3K blocks IGF-1–mediated potentiation of aggregation. (i,ii) Washed platelets were incubated with vehicle (DMSO) (A), 100 nM wortmannin (B), 20 μM LY294002 (C), 0.5 μM ZSTK474 (D), 0.5 μM PI-103 (E), 1 μM PIK-75 (F), 1 μM GTX-155 (G), 1 μM GTX-221 (H), and 1 μM IC87114 (I) for 10 minutes. Platelets were subsequently incubated in the absence (−IGF-1) or presence of 100 nM IGF-1 (+IGF-1) for 5 minutes before stimulation with 0.5 μM SFLLRN (i) or 0.7 μM SFLLRN (ii) and aggregation was recorded for a total of 5 minutes. (iii) Alternatively, platelets were incubated for 10 minutes with vehicle (DMSO) or the indicated concentration of wortmannin (B), LY294002 (C), ZSTK474 (D), PI-103 (E), PIK-75 (F), GTX-155 (G), GTX-221 (H), and IC87114 (I), before stimulation with 100 nM IGF-1 for 5 minutes. Platelets were subsequently extracted and whole-cell lysate was subjected to SDS-PAGE followed by immunoblotting with the anti-P–PKBSer473 antibody (iii). Membranes were stripped and reprobed with anti-PKBα antibody to confirm equal loading. Results are representative of 3 to 6 (i,ii) and 3 (iii) experiments.