Mark Wefers Bettink

Chapter 7 132 of the platelets for 15 minutes at 150 × g at room temperature (21 – 24 °C). 1.0 mL of this PRP was collected. Previously published methods for measurement of mitochondrial respiration in platelets were followed.(18, 19, 24) Compared to the described protocols we did not see the benefit of first creating a platelet pellet and resuspending it in the same plasma. Therefore, we altered the platelet isolation protocol used in these studies. Hereby creating a PRP instead of a near protein free plasma and platelet pellet and 1 ml of PRP was transferred to a separate tube. The residue (hematocrit plus PRP) was centrifuged a second time for 6 minutes at 4,000 × g at room temperature (21 – 24 °C) and all plasma was collected. For the determination of free mitochondrial DNA in plasma as marker of mitochondrial damage(25), 200 µL of plasma was transferred to a 1.5 mL sterile tube (Sterile Safe-Lock™, Eppendorf ® , Hamburg, Germany) and snap frozen in liquid nitrogen and stored at -80°C. High-Resolution Respirometry (HRR) Measurement of oxygen consumption in intact thrombocytes’ mitochondria was performed in a high-resolution oxygraph (Oxygraph-2k, Oroboros Instruments, Innsbruck, Austria). Volume calibration was performed at the start of each week. Air calibration was performed on the air-saturated plasma of the patient at the start of each experiment. The oxygen solubility factor was set to 0.89 for plasma, the stirrer speed was set to 750 rpm and chamber temperature was set to 37℃. . Data was recorded with DatLab 5.2 software (Oroboros Instruments, Innsbruck, Austria) with sampling rate set to 2 seconds. The 2 mL glass chamber was filled with 1.6 mL of the air-saturated collected plasma of the patient, after which the oxygen calibration was performed. Previous published data suggested that a concentration of 100-200 × 10 6 platelets per milliliter in the oxygraph chamber yields superior results in respiration measures.(26) 0.5 mL of PRP was added to the plasma of both chambers. If necessary, reoxygenation was performed until an adequate oxygen concentration was reached. After 10 – 15 minutes of stabilization, the unstimulated respiration state (state 4o) was reached. Oligomycin (4 µL, 5 mM) – an ATP synthase inhibitor – was added to induce an ADP phosphorylation independent respiration state in 15 – 30 minutes. The oxidative phosphorylation uncoupler carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) (20 mM) was repeatedly added in volumes of 1 µL, until no further increase in respiration was detected in two successive additions. 1 µL of rotenone and 2 µL of antimycin A were added, resulting in a remaining oxygen respiration primarily