Agonist-dependent changes in [GTP]rac1, [GTP]cdc42, and cytoskeletal IQGAP2.

(A-B) GFP (5 × 10⁷ platelets/lane; 100 μg starting protein) were preincubated with GST-PBD and stimulated with 10 nM α-thrombin, 10 μg/mL collagen, or 10 μM ADP for various time points, followed by lysis and immunoblot analysis for the presence of GTP[rac1] (upper panel) or GTP[cdc42] (middle panel). Data are presented as percentage of [GTP]-bound GTPase determined by densitometric comparison with 100 μg platelet lysates loaded with saturating concentrations (100 μM) of GTPγS or GDPβS (lanes marked accordingly). Nonspecific binding by GST-PBD was adjusted for by subtracting the “background” GDPβS component. Note the rapid increase of cytosolic GTP[rac1] with a concomitant decrease in [GTP]cdc42, demonstrable only in α-thrombin-stimulated platelets. (C) Triton-X insoluble cytoskeletons from agonist-stimulated platelet (8.5 × 10⁶ platelets/lane corresponding to 17 μg total protein) were immunoblotted for the presence of IQGAP2, and data were reported as the percentage of total platelet IQGAP2 determined by densitometric comparison to platelet control lysates. F-actin contents were essentially as outlined in Figure 4. All results are representative of one complete set of experiments, repeated on 2 occasions with 2 healthy volunteers.