Genetic inactivation of p110β catalytic subunit specifically in megakaryocytes and platelets. (A) Western blot showing class I PI3K p110 isoforms and p85 expression in PF4-Cre− p110flox/flox control (Cre−) or PF4-Cre+ p110flox/flox p110β-null (PF4-Cre+) platelet lysates. ■ represent (Cre−) platelet lysates; □, (PF4-Cre+) platelet lysates. Average results are mean ± SEM for 4 experiments (right panel). (B) p110β and p85 subunits were immunoprecipitated with specific antibodies, and the associated lipid kinase activity was assayed with phosphatidylinositol as a substrate. ■ represent (Cre−) platelet lysates; □, (PF4-Cre+) platelet lysates. Average results are mean ± SEM for 3 experiments. (C) Platelets from control or PF4-Cre/p110βflox/flox mice were stimulated under nonaggregating conditions by indicated agonists, and the radioactivity of PIP3 was determined as described in “Lipid extraction and analysis.” Results are mean ± SEM of 3 independent experiments. (D) Platelets were stimulated under aggregating conditions by thrombin, U46619, collagen, or convulxin during 7 minutes at the indicated concentration. Lysates were submitted to immunoblotting with anti–Akt-Ser(P)473 or total Akt antibodies, as indicated. Quantification by densitometric analysis of the Western blots is shown (right panels), and data are expressed as percentage of P-Akt in response to thrombin (0.5 IU/mL), U46619 (1μM), collagen (10 μg/mL), and convulxin (10nM), and are mean ± SEM of 3 independent experiments. (E) Platelets were stimulated under aggregating or nonaggregating (integrilin) conditions by thrombin (0.5 IU/mL) during different times and analyzed as in panel D. (F) Platelets were stimulated under aggregating conditions by thrombin, U46619, collagen, or convulxin in the absence or presence of TGX-221 (0.5μM) and analyzed as in panel D. (G) Platelets from p110flox/flox control (Cre−) or PF4-Cre/p110flox/flox (PF4-Cre+) mice were stimulated under nonaggregating conditions with indicated agonists, and the radioactivity of phosphatidic acid (PtdOH) was determined as described in “Lipid extraction and analysis.” Results are mean ± SEM of 3 independent experiments. Statistical analysis: **P < .01.
