PP1 or wortmannin inhibits PAK activation during platelet spreading on surfaces coated with collagen or TS2/16.
(A) Washed platelets were incubated without (cont) or with 20 μM PP1 (PP1), 100 nM wortmannin (wo) for the indicated durations at 37°C. Then, they were added to BSA- or collagen-coated dishes and incubated at 30°C for 30 minutes. After removing unbound platelets, reactions were terminated with a lysis buffer. After the protein concentrations were adjusted, PAK proteins were isolated by immunoprecipitation with anti-PAK antibody. The immunoprecipitates were used for in vitro kinase assays with myelin basic protein as the exogenous substrate. The data are representative of at least 3 experiments. (B) Washed platelets were incubated without (cont) or with 20 μM PP1 (PP1), 100 nM wortmannin (wo) for 10 minutes at 37°C. The platelets were then added to BSA- or F(ab)′2 fragments of TS2/16-coated dishes and incubated at 30°C for 30 minutes. The procedures thereafter were the same as described for A. (C) Activation of PAK was quantified with Quantity One image analyzing software.
