Kinetics of activation of p38, ERK, and MLC in platelets spreading on fibrinogen. Washed platelets at a concentration of 4 × 107/mL were incubated with 500 μM aspirin and 50 μM P2Y12 inhibitor, 2MeSAMP for 5 minutes at 37°C. Platelets were added to polystyrene dishes coated with 100 μg/mL fibrinogen to induce adhesion and spreading at 37°C for indicated times or kept in suspension. Dishes were rinsed to remove nonadherent platelets, and platelets were solubilized as described in “Immunoblot detection of p38, ERK, and MLC phosphorylation in platelets.” Activation of p38, ERK, and MLC were analyzed by Western blot analysis using phospho-specific antibodies against phosphorylated Thr180/Tyr182 (p38), Thr-202/Tyr204 (ERK), and Thr18/Ser19 (MLC) (Cell Signaling Technologies). Monoclonal antibody against tubulin was used to verify equal loading.
