Comparison of the latency and time course of thrombin-evoked cofilin phosphorylation and actin reorganization. Platelets were rapidly mixed with thrombin at a final concentration of 0.1 U/mL or with agonist-free HBS solution (control at 0 seconds) and incubated at 37°C for various time periods (1-5 seconds) before mixing with lysis buffer (for cofilin phosphorylation) or with formaldehyde (3% in PBS; for F-actin measurement) using a quenched-flow system. For cofilin phosphorylation, proteins were separated by SDS-PAGE followed by Western blotting with either anti-phosphoSer³-cofilin antibody (Ai) or anticofilin antibody (Aii) as described in “Materials and methods.” Bands were revealed using chemiluminescence and were quantified using scanning densitometry. Positions of molecular-mass markers are shown on the right. Actin filament content was determined as described in “Materials and methods.” (B) Graph represents the quantification of cofilin phosphorylation (filled symbols) and F-actin content (open symbols). Values are mean ± SE of 15 runs made on cell preparations from 11 donors expressed as the percentage of cofilin phosphorylation or F-actin content in resting cells.