PAK activation is required for platelet lamellipodia and actin-rich adhesion formation in response to thrombin. A: purified human platelets spread on a surface of thrombin, stained for PAK, Rac, and actin and visualized by fluorescence deconvolution microscopy. Scale bar = 2 μm. B: representative DIC images of human platelets treated with vehicle (DMSO), increasing concentrations of IPA-3 or 10 μM PIR 3.5 on a surface of thrombin. Scale bar = 10 μm. C: super-resolution analysis of platelet focal adhesion formation of vehicle and 10 μM IPA-3 treated platelets on a surface of thrombin, visualized by SR-SIM of actin and vinculin staining. Scale bar = 2 μm. D: purified human platelets were spread on a surface of thrombin. After 30 min, unbound platelets were removed and activated platelets were treated with vehicle (0.1% DMSO), 10 μM IPA-3, or 10 μM PIR 3.5 for an additional 15 min. Platelet spreading and lamellipodial withdrawal were evaluated by DIC microscopy. Scale bar = 10 μm.