Platelet DREAM plays a critical role in PI3K activation through GPVI-, integrin-, or A23187-mediated signaling. (A) WT and DREAM KO platelets were stimulated by 0.5 µg/mL CRP for 1, 2, and 5 minutes under stirring conditions (1000 rpm) in an aggregometer. An equal amount of cell lysate protein (50 µg) was immunoblotted, followed by densitometry (arbitrary unit [AU], n = 5). (B-D) WT and DREAM KO platelets were stimulated with 0.5 µg/mL CRP for 2 minutes under stirring conditions. The lysates were immunoprecipitated with control IgG or antibodies against (B) LAT, (C) DREAM, or (D) total PI3K p85 (t-PI3K p85) followed by immunoblotting and densitometry (n = 4). (E-F) Human platelets were stimulated with 0.5 µg/mL CRP for 2 minutes under stirring conditions, followed by coimmunoprecipitation as shown in panels C and D. (G-H) WT and DREAM KO platelets were stimulated by 10 µM ADP for 1, 2, or 5 minutes in the (G) absence or (H) presence of 30 µg/mL FG under stirring conditions (n = 4). (I-J) WT and DREAM KO platelets were pretreated (I) without or (J) with 20 µg/mL eptifibatide following stimulation with 100 nM A23187 for 1, 2, or 5 minutes with stirring. An equal amount of lysate protein was immunoblotted, followed by densitometry (n = 4). *P < .05 and **P < .01 vs WT platelets at each time point (or resting platelets for panels C-F) after the Student t test.