DREAM is important for platelet activation through PI3K-Iβ activity. (A-C) WT and DREAM KO platelets were pretreated (A-B) without or (C) with 0.05% dimethyl sulfoxide (DMSO) (vehicle), 50 µM LY294002 (LY), 0.1 µM wortmannin (Wort), or an isoform-specific inhibitor for PI3K-Iα (0.5 µM PIK75) or PI3K-Iβ (0.5 µM TGX221), followed by stimulation with 0.5 (for panel C), 1, or 2 µg/mL CRP. P-selectin exposure or αIIbβ3 integrin activation was analyzed by flow cytometry. The data are presented as the geometric mean fluorescence intensity (MFI) value (n = 3). (D-E) WT and DREAM KO platelets were incubated with a Ca²⁺ dye and treated with 0.5 µg/mL CRP or 100 nM A23187 in the absence of exogenous Ca²⁺ and the presence of 1 mM EGTA. Cytosolic Ca²⁺ levels were measured and quantified by area under the curve (AUC) (n = 3). (F-G) WT and DREAM KO platelets were incubated with vehicle or a PI3K inhibitor, followed by the Ca²⁺ assay as described above (n = 4). *P < .05, **P < .01, and ***P < .001 vs WT platelets after Student t test (for panels A-B and D-E) or vehicle-treated WT (white bars) or DREAM KO platelets (gray bars) after ANOVA and the Tukey test (for C and F-G). #P < .05 and ##P < .01 between 2 groups. (H-I) WT and DREAM KO platelets were pretreated with vehicle or a PI3K inhibitor as described above, followed by stimulation with 1 µg/mL CRP or 100 nM A23187. (J-K) WT and DREAM KO platelets were pretreated with vehicle or a PI3K inhibitor and then incubated (J) without or (K) with 1 mM aspirin, followed by stimulation with 0.0125 U/mL thrombin. Representative results of aggregation are presented (n = 3).