Figure 2
From: Long-term treated HIV infection is associated with platelet mitochondrial dysfunction
Platelet energy phenotype determined by the Seahorse extracellular flux analyzer and mitochondrial membrane potential and ROS production. Samples were measured with five replicates, 1 × 107 washed platelets per well. Five age-and sex-matched donors per group. (A) Real-time mitochondrial respiration depicted as oxygen consumption rate (OCR) at 18 timepoints. Four agonists or inhibitors were added in the following order: (1) Thrombin Receptor Activator Peptide-6 (TRAP-6; 50 µM) or medium causing platelet activation; (2) Oligomycin which inhibits cellular ATP production; (3) Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP), an uncoupling agent causing maximum oxygen consumption through complex IV, and (4) Antimycin A which inhibits all mitochondrial respiration (CRP-XL can be found in Supplemental Fig. 9). (B) Energymap showing the change in mitochondrial respiration (as change in OCR) and glycolysis (extracellular acidification rate (ECAR) from basal conditions after ex vivo stimulation with platelet agonists. ((A)) Depicts the real-time ECAR after (1) medium or TRAP-6 (2) oligomycine, (3) FCCP and (4) antimycin A. Samples were measured with five replicates, 1 × 107 washed platelets per well. Five age-and sex-matched donors per group. (D) Basal OCR as mean of first three measurements. (E) Membrane potential was determined using Tetramethylrhodamine ethyl ester (TMRE) staining with the uncoupler FCCP disrupting mitochondrial membrane potential as a negative control for every sample. Data depicted as delta geometric mean fluorescence intensity (MFI). (F) Mitochondrial ROS production with MitoSox staining in MFI. Data are depicted as mean ± standard error of the mean (SEM) and analyzed using the unpaired student’s T-test.
