Quantitation of GTP-rac from high-speed supernatant prepared from resting platelets or platelets stimulated with 25 μmol/L TRAP in the presence or absence of PI-3-kinase inhibitor wortmannin or 1 U/mL thrombin.

(A) Graph quantifying the increase in GTP-rac contained in the soluble fraction of lysates prepared from resting (rest) or TRAP-activated cells. The graph is an average of a minimum of 4 experiments (mean ± SD). Resting cells contained from 2% to 10% of the total rac. Activation increases the amount of GTP-rac by 6-fold to 30% to 40% of the total rac protein in the high-speed supernatant. GTP-rac formation after PAR-1 ligation is not affected by wortmannin. (inset) Representative anti-rac immunoblots of rac collected from high-speed supernatant of resting or TRAP activated platelets. GTP-bound rac was collected with the GBD-binding domain of PAK1 bound to Sepharose beads and is visualized in immunoblots (top) with an anti-rac mouse monoclonal antibody and a HRP-goat antimouse antibody. Platelets were incubated with 100 nmol/L wortmannin for 15 minutes before stimulation with TRAP (bottom). The amount GTP-rac collected and the kinetics of GTP-charging in the presence of 100 nmol/L wortmannin are similar to that in high-speed supernatant from cells activated without inhibitors. (B) Quantitation of the content of GTP-rac in high-speed supernatant from resting platelets and platelets activated using 1U/mL thrombin. (inset) In a representative experiment, the relative amount of GTP-rac collected using the PAK1-bead complexes.