Assessment of mitochondrial respiration in human platelets

Abstract

It has been long recognized that the impairment of platelet mitochondrial function occurs in a broad spectrum of diseases. Accordingly, the assessment of platelet respiratory dys/function has emerged as a putative approach allowing the characterization of the early impairment of human bioenergetic profile in several chronic pathologies. The aim of this study was to standardize the methodology for platelet isolation from peripheral blood and the measurement of mitochondrial oxygen consumption by means of high-resolution respirometry, respectively. The platelet isolation protocol consisted of two consecutive centrifugations of the whole blood collected from adult healthy females (n = 10) yielding a platelet-rich plasma sample. Respiration was measured at 370C using the Oxygraph-2k (Oroboros Instruments, Austria) according to a classic substrate-uncoupler-inhibitor-titration protocol. Platelets permeabilized with digitonin were allowed to respire in the presence of complex I (glutamate and malate) and complex II (succinate) substrates. We obtained a respiratory control ratio of 2.77 ± 3.65 that indicates an accurate coupling efficiency of oxidative phosphorylation. The in vitro measurement of platelet respiration is a reliable method to evaluate the bioenergetic profile in humans. The standardized technique will be further used to assess the occurrence of mitochondrial dysfunction in peripheral blood in the setting of various chronic non-communicable diseases.