Fig 2. Mitochondrial assay of platelets exposed to thrombin.

Platelets were plated on XF96 plates in MAS buffer, and first a basal rate of oxygen consumption was measured, followed by injection of thrombin (0.5 U/ml). This was followed by injection of (A) saponin (60 μg/ml), pyruvate (5 mM), malate (2.5 mM) and ADP (1 mM) for complex I substrates (D) or saponin (60 μg/ml), succinate (10 mM) and ADP (1 mM) for complex II substrates. Then oligomycin (1 μg/ml) and antimycin A (10 μM) were injected sequentially. From this different parameters of respiration were calculated—(B, E) state 3 (substrate sensitive—oligomycin sensitive OCR) and state 4 (oligomycin sensitive—AA sensitive OCR). (C, F) Respiratory control ratio (RCR) was calculated using the formulae—state3/state4. Data expressed as mean±SEM from one representative donor, n = 3–5 replicates per sample. *p<0.01, different from control.