Fig 3. Exposure of platelets to LPS potentiates PI3K/Akt signalling.

Washed human platelets with or without treatment with U46619 (0.25μM) in the presence or absence of LPS from E. coli 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 E. coli 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 E. coli 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).