Mitochondria are the major sources of ROS in hyperthermia-treated platelets.

<p>(A and B) DCFDA-loaded platelets were incubated at the indicated temperatures for 3 h (A) or 1-3 h (B). As a positive control, loaded platelets were incubated with thrombin at 37°C for 30 min. Samples were then analyzed for intracellular ROS levels by flow cytometry. Representative flow cytometric histogram is shown (A). The relative ROS levels are expressed as a percentage of platelets, which were incubated at RT for 1 h (B). Percentage of RT 1h is presented as mean ± SEM from three independent experiments. *<i>P</i><0.017 (after Bonferroni correction) as compared with RT 1 h, **<i>P</i><0.017 (after Bonferroni correction) as compared with RT 2 h, #<i>P</i><0.017 (after Bonferroni correction) as compared with RT 3h. (C–E) DCFDA-loaded platelets were pre-incubated with solvent control, DPI and apocynin (C), Mito-TEMPO and NAC (D), or L-NAME and ETYA (E) at 37 °C for 15 min, and then incubated for 3 h at 40°C or 42°C, and further analyzed by flow cytometry. The relative ROS levels are expressed as a percentage of platelets, which were pre-incubated with solvent control at 37°C for 15 min and then incubated at 40°C for 3 h. Percentage of 40°C loaded platelets pre-incubated with solvent control is presented as mean ± SEM from three independent experiments. *<i>P</i><0.025 (after Bonferroni correction) as compared with solvent control at 40°C, **<i>P</i><0.025 (after Bonferroni correction) as compared with solvent control at 42°C. Thrombin is labeled as Thr.