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Figure 1 (A) Effect of different amounts of non-activated (T–) and thrombin-activated (T+) platelets on intracellular concentration of reactive oxygen species (ROS) and superoxide in bovine aortic endothelial cells (BAECs). The BAECs were preincubated with 2',7'-dichlorofluorescein diacetate and hydroethidine at 37°C for 20 min. Different amounts of (T–) and (T+) platelets (from 0 to 4.5 x 107/ml) were added to the BAEC medium for 10 min at 37°C. Results are expressed as mean fluorescence intensity (MFI) and are the means ± SD of experiments performed in triplicate on six separate occasions. ¶p < 0.01, *p < 0.001 vs. no addition of platelets (Control). (B) Time-course of superoxide formation induced by platelets activated with different amounts of thrombin in BAECs. The BAECs were preincubated with hydroethidine for 20 min. Platelets (3 x 107/ml) activated with different doses of thrombin (from 0.03 to 1 U/l) were added to the BAEC medium for the indicated times at 37°C. The BAECs and platelets alone were used as controls. Results are expressed as MFI and are the means ± SD of experiments performed in triplicate on six separate occasions. By two-way analysis of variance there was a significant effect for the thrombin concentrations (p < 0.001) and for the times of incubation (p < 0.001) with a significant concentrations/times interaction (p < 0.001) on superoxide generation. Post-analysis of variance Tukey test for multiple comparisons: ¶p < 0.01, *p < 0.001 vs. time 0.
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