Fig. 2

Effect of methylene blue and NV118 on coupled respiration in rotenone-intoxicated human platelets. a Representative traces of mitochondrial respiration in human platelets with complex I inhibition induced by rotenone (2 μM). Mitochondrial respiration due to coupled phosphorylation of rotenone-intoxicated platelets treated with vehicle (rotenone, dark gray trace), methylene blue (20 μM, dark blue trace) or the cell-permeable succinate prodrug NV118 (250 μM, dark green trace), here referred to as coupled respiration, was evaluated by subsequent addition of the ATP-synthase inhibitor oligomycin (1 μg/ml) to block the phosphorylation pathway, and calculated as the difference in respiration before and after the inhibition of the ATP-synthase. The protocol was continued by adding the complex III inhibitor antimycin A (1 μg/ml) followed by the complex IV inhibitor sodium azide (10 mM). Control experiments were performed without the addition of oligomycin to account for background drift of oxygen consumption in the presence of the bypass agents (light blue trace, light green trace). b Quantification of coupled respiration of rotenone-intoxicated platelets treated with vehicle (rotenone, black circle), methylene blue (20 μM, dark blue square), or the cell-permeable succinate prodrug NV118 (250 μM, dark green triangle) was calculated as the difference in respiration before and after inhibition of the ATP-synthase. c The coupled respiration, non-coupled respiration, and non-mitochondrial respiration was evaluated in rotenone-intoxicated platelets treated with vehicle, methylene blue, or NV118. Non-mitochondrial respiration was defined as respiration remaining after addition of sodium azide, which all other respiratory values were corrected for. Data are expressed as individual scatter plot and mean ± SD (b) or mean (c). Rotenone: n = 21, NV118 and methylene blue: n = 7. One-way ANOVA with Dunnet post hoc test was used for comparison of the drug’s effect on coupled respiration. ***p < 0.001. CII–V: complex II to V, CII–IV: complex II to IV, CIV: complex IV