Effects of Sema3A on F-actin contents, cofilin phosphorylation, and Rac1 activation. (A) Sema3A/Fc-(gray bars) or hIgG-treated (black bars) platelets were activated by 30 μM PAR1-TRAP or 0.5 U/mL thrombin at 37°C for 30 seconds without stirring. After fixation, F-actin was stained with bodipy-phallacidin. Specific phallacidin binding was obtained by subtraction of FL1 fluorescence with a 300-fold more excess of unlabeled phallacidin from FL1 fluorescence without unlabeled phallacidin, and fold increase against fluorescence of no agonist sample was calculated. Data represent mean and SE of 3 independent experiments. *P < .05. (B) Sema3A/Fc- or hIgG-treated platelets were activated with 30 μM PAR1-TRAP for the indicated time at 37°C without stirring. Then, cells were lysed and SDS-PAGE was performed. Phospho-cofilin was detected by anti–phospho-cofilin–specific antibody. After stripping, total cofilin was detected by anticofilin antibody. Optical density of the bands was measured by NIH Image software, and relative increase against phospho-cofilin in IgG-treated platelets without thrombin was calculated. Mean and SE of 3 independent experiments was plotted in bottom panel. *P < .05; **P < .01. (C) Sema3A/Fc- or hIgG-treated platelets were activated with 30 μM PAR1-TRAP for the indicated time at 37°C without stirring. GTP-form of Rac1 was precipitated by incubation with GST-PAK1-PBD and glutathione beads. After SDS-PAGE electrophoresis, Rac1 was detected by a Rac1-specific antibody. Total Rac was detected by electrophoresis of total lysates on an SDS-PAGE gel followed by detection with the same antibody. Optical density of the bands was measured by NIH Image software, and relative increase against GTP-Rac in IgG-treated platelets without thrombin was calculated. Sema3A/Fc is indicated by ▪ and bold lines; hIgG, by ♦ and thin lines. Mean and SE of 3 independent experiments was plotted in lower panel. *P < .05.