FIGURE 2.

Effects of agonists on Rac1 activation in platelets both in suspension and on a fibrinogen surface. Equal numbers of washed human (A) or murine (B) platelets (5 × 10⁸/ml) were stimulated with thrombin (thr; 1 unit/ml) or ADP (10μM) in the absence or presence of ADP scavenger apyrase (2 units/ml) for 60 s before lysis. Subsequently platelet lysates were incubated with agarose beads coupled with GST-PBD fusion proteins to bind GTP-bound forms of Rac1. Total lysates or material binding to the GST-PBD beads were solubilized, separated by SDS-PAGE, and detected by immunoblotting with an anti-Rac1 monoclonal antibody. In separate experiments, washed human (C) and murine (D) platelets (5 × 10⁸/ml) were added to BSA- or fibrinogen (FG)-coated dishes in the absence or presence of thrombin (1 unit/ml) and incubated for 10 min. Dishes coated with fibrinogen were washed twice to remove nonadherent cells. Platelets adherent to fibrinogen or in suspension over BSA were lysed, and Rac activity was determined as described above. Each panel depicts one experiment representative of three independent experiments.