FIGURE 2.

Role for PKC and Akt in PAR1-mediated GSK3 phosphorylation in human platelets. A, washed human platelets were incubated with the indicated concentration of MK2206 for 10 min followed by stimulation with 0.2 unit/ml thrombin for 5 min. Platelets were extracted in NuPAGE sample buffer and immunoblotted with the indicated antibodies . B, alternatively, Akt1 was immunoprecipitated and in vitro kinase activity determined using the peptide substrate RPRAATF. In vitro kinase activity is expressed as a percentage of Akt activity in the presence of vehicle (B, mean ± S.E. (error bars), n = 3). C–H, washed human platelets were incubated with vehicle, 1 μm MK2206 (C–H), 100 nm wortmannin (WT; E and H), 10 μm U0126 (E) or 1 μm BIMIX (F–H) for 10 min before stimulation with 5 μm SFLLRN (C–E), 5 μm SFLLRN+10 μm ADP (F and G), or 100 nm PMA (H) for the indicated time period. Platelets were extracted in NuPAGE sample buffer followed by immunoblotting with the indicated antibodies (C, E, F, and H). Results (A, C, E, F, and H) are representative of at least three independent experiments. The bar graphs (D and G) depict densitometric analysis of GSK3β phosphorylation of the data shown in C and F, respectively. Data (mean ± S.E.) are expressed as the percentage of maximal GSK3 phosphorylation induced by SFLLRN (D, n = 5) and SFLLRN+ADP (G, n = 3). Star notation (D: *, p < 0.05; ***, p < 0.001) indicates a significant difference between vehicle and MK2206-treated samples at the matching time point (two-way analysis of variance with Bonferroni post test). Dagger notation (G: ††, p < 0.01) indicates a significant difference compared with 0.25-min SFLLRN+ADP treatment in the presence of vehicle, whereas double dagger notation (‡‡‡: p < 0.001) indicates a significant difference compared with 5-min SFLLRN+ADP treatment in the presence of vehicle (two-way analysis of variance with Bonferroni post test).