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.

Mechanistic investigations of antitumor activity of a Rhodamine B‑oleanolic acid derivative bioconjugate

“…Cellular respiratory function was determined by the means of high resolution respirometry studies (Oxygraph-2k Oroboros Ltd.) at 37°C. To obtain a comprehensive analysis of respiratory control, a substrate-uncoupler-inhibitor titration (SUIT) protocol was followed, designed to allow the measurements of respiratory rates with both separate and convergent Complex I and II (CI+CII) electron input, as described by Petruș et al ( 28 ). The cells (1×10 6 /ml) were suspended in a mitochondrial respiration medium (MIRO5: MgCl 2 3 mM, EGTA 0.5 mM, taurine 20 mM, KH 2 PO 4 10 mM, K-lactobionate 60 mM, D-sucrose 110 mM, HEPES 20 mM, BSA 1 g/l, pH 7.1).…”

Novel Triterpenic Acid—Benzotriazole Esters Act as Pro-Apoptotic Antimelanoma Agents

“…The mitochondrial respiratory function was assessed using high respirometry studies (Oxygraph-2k Oroboros Instruments GmbH, Innsbruck, Austria) at 37 °C. To assess the mitochondrial respiratory rates of permeabilized HaCaT and A375 cells, a modified substrate uncoupler–inhibitor titration (SUIT) protocol was followed to obtain both separate and convergent Complex I and Complex II (CI + CII) electron input, as previously described by Petruș et al [ 55 ]. Prior to mitochondrial function evaluation, the cells were cultured in T75 culture flasks, treated with the tested compounds ( 1, 2, and 3 ) for 24 h, washed with PBS, trypsinized, counted, and resuspended (1 × 10 6 /mL) in mitochondrial respiration medium (MIRO5: MgCl 2 3 mM, EGTA 0.5 mM, taurine 20 mM, KH 2 PO 4 10 mM, K-lactobionate 60 mM, D-sucrose 110 mM, HEPES 20 mM, and BSA 1 g/L, pH 7.1).…”

In Vivo and Ex Vivo Mitochondrial Function in COVID-19 Patients on the Intensive Care Unit

“…Plated-rich plasma (PRP) and peripheral blood mononuclear cells (PBMCs) were isolated from the same blood tube and isolation started within 1, 2 h after the blood samples were taken. Our analysis in platelets was based on previously published methods for measurement of mitochondrial respiration in platelets [ 28 , 29 , 30 ]. Compared to the described protocols we did see the benefit of first creating a platelet pellet and resuspending it in the same plasma.…”