Effect of Btk inhibition on the latency and time course of thrombin-evoked cofilin phosphorylation. Human platelets were preincubated for 10 minutes in the presence of 10 μM LFM-A13 (○) or the vehicle (▪) and then rapidly mixed with thrombin (0.1 U/mL) or with agonist-free HBS solution (control at 0 seconds) and incubated at 37°C for various time periods before mixing with lysis buffer using a quenched-flow system. Proteins were separated by SDS-PAGE followed by Western blotting with either anti-phosphoSer³-cofilin antibody (Ai) or anticofilin antibody (Aii) as described in “Material and methods.” C indicates control; T, LFM-A13-treated cells. Bands were revealed using chemiluminescence and were quantified using scanning densitometry. Positions of molecular-mass markers are shown on the right. (B) Graph represents the quantification of cofilin phosphorylation. Values are mean ± SE of 6 runs made on cell preparations from 6 donors expressed as the percentage of cofilin phosphorylation in resting cells.