Mitochondrial respiratory function in human platelets: Influence of sample preparation, assay buffer, and...

Circulating blood cells such as platelets represent a readily available sample type to determine mitochondrial function in humans. Here, we set out to determine the influence of sample preparation, assay buffer composition, and instrumental platform on the respiratory function of platelets isolated from human blood. Approximately 50 mL of whole blood was collected from healthy adults (n = 16) following an overnight (>12 h) fast. Platelets were immediately isolated from whole blood by centrifugation for respirometry. Respiratory function was assayed in intact and permeabilized platelets using an Oxygraph-2K (O2K) high-resolution respirometer in either RPMI or MIR05 (containing 5 mM glucose, 1 mM pyruvate, and 2 mM glutamine), or the participant's own plasma. In addition, respiratory function was determined in intact platelets using a Seahorse Extracellular Flux analyzer (XFe96) in RPMI buffer containing 1 mM pyruvate, 2 mM glutamine, and variable glucose concentrations (5, 10, and 10 mM). In assays performed in an O2K, routine and ATP-linked respiration were greater in cells assayed in RPMI compared to MIR05 (p < 0.001). However, compared to cells assayed in RPMI or MIR05, routine and ATP-linked respiration were higher in intact platelets assayed in their own plasma (p < 0.001). In digitonin-permeabilized platelets, state 3 respiration was greater when assayed in MIR05 compared to RPMI (p < 0.05). Across instrumental platforms, routine and leak respiration were lower in intact platelets assayed on an O2K versus an XFe96 (p < 0.05), whereas respiration available for ADP phosphorylation was greater in cells assayed on an O2K versus an XFe96 (p < 0.001), due to a diminished coupling response to oligomycin in cells assayed on the XFe96 (p < 0.001). Platelet respiratory function is influenced by assay buffer composition and instrumental platform. Consideration of these factors should be made by investigators planning to use platelet respiratory function as a readout of cellular energetics.