Analysis of platelet activation in PI3Kγ^KD and PI3Kβ^KD mice. ADP (A), U46619 (B), or convulxin (C) induced platelet aggregation (i), Rap1b activation (ii), and Akt phosphorylation (iii) in wild-type (WT), PI3Kγ^KD (γKD), and PI3Kβ^KD (βKD) mice. (i) Platelet aggregation was stimulated by 1, 5, or 10 μM ADP (in the presence of 200 μg/mL fibrinogen) or U46619, or with 50 and 100 ng/mL convulxin (CVX). The percentage of platelet aggregation was determined 5 minutes after addition of the agonist. Aggregation of PI3Kγ^KD was also measured upon preincubation with 0.5 μM TGX221 (a gift from Dr Peter R. Shepherd, University of Auckland, Auckland, New Zealand) for 5 minutes. For each agonist, maximal aggregation induced by the highest dose of agonist was considered as 100%. In the case of CVX stimulation, the dose of 100 ng/mL was sufficient to trigger maximal platelet aggregation (light transmission higher than 95%) under our experimental conditions. Results are the mean ± SD of 3 (A-B) or 6 (C) different experiments (where not visible, SD bar is masked by the mark). Traces are as follows: ●, wild-type platelets; ■, PI3Kγ^KD platelets; □, PI3Kγ^KD platelets treated with TGX221; ♦, PI3Kβ^KD platelets. Analysis with t test performed at each dose of agonists revealed that aggregation of both PI3Kβ^KD and PI3Kγ^KD platelets stimulated with ADP or U46619 was always significantly different from that of wild-type platelets (P < .01). Upon stimulation with CVX, PI3Kβ^KD platelets showed significantly reduced aggregation relative to wild-type platelets at both concentrations tested, whereas aggregation of PI3Kγ^KD platelets was significantly reduced relative to that of wild-type platelets only at 100 ng/mL (P < .05). (ii) Analysis of Rap1b activation was performed upon platelet stimulation with 1 or 5 μM ADP or U46619 or with 50 ng/mL CVX for 60 seconds. When indicated, stimulation of platelets was also performed upon preincubation with 0.5 μM TGX221 (+ TGX) for 5 minutes, while Rap1b activation induced by CVX was also analyzed in the presence of 2 U/mL apyrase (APY). Active Rap1b was precipitated with GST-tagged RalGDS-RBD and visualized by immunoblotting with a specific antibody. Identical aliquots of total cell lysates from each sample were also tested for the total Rap1b expression by immunoblotting. Quantitative analysis was performed by densitometric scanning of the immunoblots. For ADP and U46619, variable results were obtained, and thus histograms summarizing the quantitative analysis of 3 different experiments for wild-type (■), PI3Kγ^KD (□), and PI3Kβ^KD () platelets are reported. Statistically significant differences in Rap1b activation in the PI3Kγ^KD or PI3Kβ^KD versus wild-type platelets (t test, P < .05) are indicated by *. For CVX-treated platelets, a representative immunoblot of 3 identical experiments is reported, as either a normal or a completely inhibited activation of Rap1b was constantly observed. (iii) Wild-type, PI3Kγ^KD, and PI3Kβ^KD platelets were stimulated with 5 μM ADP, 5 μM U46619, or 50 ng/mL CVX for the indicated times. When indicated, platelets were preincubated with 0.5 μM TGX221 for 5 minutes before stimulation. Proteins from identical aliquots of whole-cell lysates were separated by SDS-PAGE on a 10% acrylamide gel, transferred to polyvinylidene difluoride, and probed with anti–phospho-Akt (Ser473) antibody (top panels). Membranes were stripped and then reprobed with anti-Akt antibody (bottom panels). Immunoblots representative of 3 different experiments giving identical clearcut results are reported.