Platelet activation by either immobilized or solution-phase heparin requires PI3K. (A) Inhibition of platelet spreading on heparin. Washed human platelets at a concentration of 2.5 × 10⁸/mL were preincubated for 3 minutes with 3μM wortmannin (right panels), or its vehicle control dimethyl sulfoxide (DMSO; left panels) before being added to chamber slides that had been coated with either fibrinogen (Fg; 100 μg/mL) or UFH (10 units/mL) under the same conditions described in Figure 1. Platelets were allowed to spread for 45 minutes at 37°C before being photographed. Note that platelet spreading on immobilized heparin was markedly attenuated in the presence wortmannin. (B) Inhibition of heparin-induced platelet potentiation by wortmannin. SDS-PAGE/immunoblot analysis of washed human platelets (2.5 × 10⁸/mL) that had been pretreated with 3μM wortmannin or its DMSO vehicle control for 3 minutes before addition of 1 U/mL of unfractionated, pharmaceutical-grade heparin (Hep). Note that the strong activation of Akt and GSK3-β induced by exposure to heparin in solution is eliminated by wortmannin.