PP1 or wortmannin, but not Y-27632, inhibits Rac activation during platelet spreading on collagen- or TS2/16-coated surfaces.

(A) Washed platelets were incubated without (control [C]) or with 20 μM PP1 (P), 100 nM wortmannin (W), or 20 μM Y-27632 (Y) for 10 minutes at 37°C. Then, they were seeded to BSA-coated or collagen-coated dishes and incubated at 30°C for 30 minutes. Reactions were terminated with lysis buffer B, and a small aliquot of each supernatant was used to check the equal loading of samples (lower panels). The rest of the lysate was used to precipitate active forms of Rac with PBD-bound glutathione Sepharose 4B (upper panels). The sample was then Western blotted with anti-Rac antibody. The data are representative of at least 3 experiments. (B) Washed platelets incubated without (control [C]) or with 20 μM PP1 (P), 100 nM wortmannin (W), or 20 μM Y-27632 (Y) for 10 min at 37°C. Then, they were added to BSA-coated or TS2/16-coated dishes and incubated at 30°C for 30 minutes. The procedure thereafter was the same as described for Figure 5A.