Apyrase does not inhibit platelet spreading or PAK activation in platelets on collagen- or TS2/16-coated surfaces.
(A) Washed platelets were incubated without or with 1 U/mL or 3 U/mL apyrase for 10 minutes at 37°C. Then, they were seeded on surfaces coated with 50 μg/mL collagen or 10 μg/mL anti–integrin β1 (TS2/16) for 30 minutes at 30°C. After unbound platelets were removed, the platelets were fixed, then incubated with TRITC-conjugated phalloidin for actin fiber staining. The extent of platelet spreading on collagen-coated surfaces was tentatively evaluated as described in the legend for Figure 2. After evaluating at least more than 25 platelets, the total points for 10 platelets were calculated as the platelet spreading index. The data represent the mean ± SD of the total points. (B) Washed platelets were incubated without or with 3 U/mL apyrase for 10 minutes at 37°C. Then they were added to BSA- or collagen-coated dishes and incubated at 30°C for 10 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.
