Exposure of platelets to LPS potentiates PI3K/Akt signalling.

<p>Washed human platelets with or without treatment with U46619 (0.25μM) in the presence or absence of LPS from <i>E</i>. <i>coli</i> O111:B4 (1μg/mL) were analysed by immunoblotting using antiphospho-Akt (Ser473) antibody. Total levels of 14-3-3 ζ were measured on each sample as a loading control (A). Human-washed platelet aggregation was performed by optical aggregometry activated with U46619 (0.25μM) in the presence or absence of LPS from <i>E</i>. <i>coli</i> O111:B4 (1μg/mL) after 3 min of incubation with LY294002 (20μM), Cal (60μM) or Akt inhibitor IV (5μM) (B). The effect of U46619 and LPS- induced fibrinogen binding and P-selectin exposure after incubation with LY294002 (20μM), Cal (60μM) or Akt inhibitor IV (5μM) were measure in PRP by flow cytometry (C and D). Washed platelets (4 x 10⁸/ml) were pre-incubated with 10μM DCFH-DA in the presence or absence of LY294002 (20μM), Cal (60μM) or Akt inhibitor IV (5μM) before being activated with U46619 (0.25μM) in the presence or absence of LPS from <i>E</i>. <i>coli</i> O111:B4 (1μg/mL) and ROS levels were analysed by flow cytometry (E). Cumulative data represent mean values ± SEM (n = 4). (Anova-Bonferroni test, * P≤ 0.05; ** P≤ 0.01; *** P≤ 0.001; Test t student ^{# #} P≤ 0.01).