Inhibition of αIIbβ3 activation by Sema3A. (A) Washed platelets preincubated with the indicated concentrations of Sema3A/Fc (♦ and bold lines) or hIgG (▪ and thin lines) were activated with ADP (5 μM), PAR1-TRAP (15 μM), U46619 (2 μM), convulxin (5 ng/mL), A23187 (2.5 μM), or PMA (200 nM). Activated αIIbβ3 was detected by binding of FITC–PAC-1. Shown are representative results of 3 to 5 independent experiments. (B) Washed platelets were preincubated with Sema3A/AP (♦ and bold lines) or AP (▪ and thin lines) and activated by ADP (5 μM) or thrombin (0.5 U/mL), and then FITC–PAC-1 binding was detected. Shown are representative results of 3 independent experiments. (C) PBS-, hIgG-, or Sema3A/Fc-treated platelets were incubated with or without an αIIbβ3-activating antibody, PT25-2, and PAC1 binding was examined. Shown are representative results of 3 independent experiments.