Mitochondrial Respiration of Platelets: Comparison of Isolation Methods (original) (raw)
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Assessment of Mitochondrial Respiration in Human Platelets
Revista de Chimie, 2017
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...
Mitochondrion, 2013
Studying whole cell preparations with intact mitochondria and respiratory complexes has a clear benefit compared to isolated or disrupted mitochondria due to the dynamic interplay between mitochondria and other cellular compartments. Platelet mitochondria have a potential to serve as a source of human viable mitochondria when studying mitochondrial physiology and pathogenic mechanisms, as well as for the diagnostics of mitochondrial diseases. The objective of the present study was to perform a detailed evaluation of platelet mitochondrial respiration using high-resolution respirometry. Further, we aimed to explore the limits of sample size and the impact of storage as well as to establish a wide range of reference data from different pediatric and adult cohorts. Our results indicate that platelet mitochondria are well suited for ex-vivo analysis with the need for minute sample amounts and excellent reproducibility and stability.
The mitochondrial membrane potential in human platelets: a sensitive parameter for platelet quality
Transfusion, 2005
BACKGROUND: Deterioration of platelet (PLT) quality during storage is accompanied by an increase in lactate production, indicating a decrease in mitochondrial function. In this study, the optimal conditions under which the fluorescent dye JC-1 can be used to detect changes in mitochondrial function in PLTs were established. STUDY DESIGN AND METHODS: PLTs were incubated at 37 ∞ C in synthetic medium under various conditions of JC-1 loading. In the presence of a high membrane potential, this dye accumulates in the mitochondria with a concomitant increase in red fluorescence. After JC-1 loading, the ratio of red (FL2) to green (FL1) fluorescence was determined by flow cytometry. RESULTS: The FL2-to-FL1 ratio of PLTs (3 ¥ 10 7 /mL, loaded with 0.5 m mol/L JC-1) amounted to about 5 in 1day-old PLTs. At higher dye concentrations, the FL2-to-FL1 ratio was significantly lower, suggesting uncoupling by the dye itself. Plasma concentrations above 3 percent significantly affected the JC-1 signal. The FL2-to-FL1 ratio showed a dose-dependent decrease to an uncoupler of oxidative phosphorylation or to inhibition of the respiratory chain. JC-1-loaded PLTs showed a clear decrease in FL2to-FL1 ratio after prolonged storage or upon ultraviolet (UV) illumination. Only after UV treatment did changes in JC-1 signal correlate with changes in CD62P expression. CONCLUSION: The FL2-to-F1 ratio of PLTs loaded with JC-1 is a reliable and sensitive indicator of the mitochondrial membrane potential, provided that the proper experimental conditions have been applied.
Cells, 2021
Human blood cells may offer a minimally invasive strategy to study systemic alterations of mitochondrial function. Here we tested the reliability of a protocol designed to study mitochondrial respiratory control in human platelets (PLTs) in field studies, using high-resolution respirometry (HRR). Several factors may trigger PLT aggregation during the assay, altering the homogeneity of the cell suspension and distorting the number of cells added to the two chambers (A, B) of the Oroboros Oxygraph-2k (O2k). Thus, inter-chamber variability (∆ab) was calculated by normalizing oxygen consumption to chamber volume (JO2) or to a specific respiratory control state (flux control ratio, FCR) as a reliable parameter of experimental quality. The method’s reliability was tested by comparing the ∆ab of laboratory-performed experiments (LAB, N = 9) to those of an ultramarathon field study (three sampling time-points: before competition (PRE, N = 7), immediately after (POST, N = 10) and 24 h after ...
Redox Biology, 2014
The assessment of metabolic function in cells isolated from human blood for treatment and diagnosis of disease is a new and important area of translational research. It is now becoming clear that a broad range of pathologies which present clinically with symptoms predominantly in one organ, such as the brain or kidney, also modulate mitochondrial energetics in platelets and leukocytes allowing these cells to serve as "the canary in the coal mine" for bioenergetic dysfunction. This opens up the possibility that circulating platelets and leukocytes can sense metabolic stress in patients and serve as biomarkers of mitochondrial dysfunction in human pathologies such as diabetes, neurodegeneration and cardiovascular disease. In this overview we will describe how the utilization of glycolysis and oxidative phosphorylation differs in platelets and leukocytes and discuss how they can be used in patient populations. Since it is clear that the metabolic programs between leukocytes and platelets are fundamentally distinct the measurement of mitochondrial function in distinct cell populations is necessary for translational research.
Platelet aerobic metabolism: new perspectives
Journal of Unexplored Medical Data, 2019
Although the role of platelets in hemostasis and thrombotic disorders as well as their contribution to inflammation are known, recent studies support the notion that much remains to be learned about platelet bioenergetics. Recent data suggest that platelets possess extra-mitochondrial oxidative phosphorylation (OXPHOS), which could represent one of sources of the chemical energy necessary for the prompt platelets activation. However, the extra-mitochondrial OXPHOS can play both beneficial and pathological roles, since the OXPHOS is the principal responsible of oxidative stress generation. For this reason, several authors evaluated the effects of polyphenols and other antioxidants on the modulation of the platelets oxidative stress production. In conclusion, we believe that a better understanding of platelet oxidative metabolism would allow a deeper knowledge of their physiology and the designing novel treatments targeting the role of platelets in many human diseases.
Laboratory Investigation, 2013
Peripheral blood mononuclear cells and platelets have long been recognized as having the potential to act as sensitive markers for mitochondrial dysfunction in a broad range of pathological conditions. However, the bioenergetic function of these cells has not been examined from the same donors, yet this is important for the selection of cell types for translational studies. Here, we demonstrate the measurement of cellular bioenergetics in isolated human monocytes, lymphocytes, and platelets, including the oxidative burst from neutrophils and monocytes from individual donors. With the exception of neutrophils, all cell types tested exhibited oxygen consumption that could be ascribed to oxidative phosphorylation with each having a distinct bioenergetic profile and distribution of respiratory chain proteins. In marked contrast, neutrophils were essentially unresponsive to mitochondrial respiratory inhibitors indicating that they have a minimal requirement for oxidative phosphorylation. In monocytes and neutrophils, we demonstrate the stimulation of the oxidative burst using phorbol 12-myristate 13-acetate and its validation in normal human subjects. Taken together, these data suggest that selection of cell type from blood cells is critical for assessing bioenergetic dysfunction and redox biology in translational research.
Transfusion, 2012
BACKGROUND: Intracellular adenosine triphosphate (ATP) levels decline significantly during storage of platelet (PLT) products, in part due to PLT degranulation. However, metabolic ATP stores also become depleted during storage through an unclear mechanism. Since both anaerobic glycolysis and oxidative phosphorylation are important for PLT ATP production, it is possible that the reduction in metabolic ATP reflects impaired oxidative phosphorylation. To assess this, we evaluated the kinetic activity and protein expression of cytochrome C oxidase (CcOX) in stored apheresis PLTs. STUDY DESIGN AND METHODS: Apheresis PLTs were collected and stored with agitation at 22 Ϯ 2°C for 7 days. In vitro measurements of PLT metabolic state, function, and activation were performed on Days 0, 2, 4, and 7 of storage. Total PLT ATP content, steady-state CcOX kinetic activity, and protein immunoblotting for CcOX Subunits I and IV were also performed using isolated PLT mitochondria from simultaneously collected samples. RESULTS: Intra-PLT ATP and steady-state PLT CcOX activity declined significantly and in a progressive manner throughout storage while steady-state levels of CcOX I and IV protein remained unchanged. Timedependent decline in CcOX activity correlated with progressive ATP depletion over time. CONCLUSION: During storage of apheresis PLTs for 7 days, the parallel decline in CcOX function and intra-PLT ATP suggests development of an acquired impairment in PLT oxidative phosphorylation associated with perturbed ATP homeostasis in stored PLTs. ABBREVIATIONS: CcOX = cytochrome C oxidase; MPV = mean platelet volume; sCD40L = soluble CD40 ligand. Steady-state levels of PLT CcOX Subunits I ( ) and IV ( ) were determined. A representative Western blot is depicted; the housekeeping mitochondrial protein, porin, was used to assess for adequate protein loading. Day 0 measurements served as baseline control and were arbitrarily set to equal a relative density of 1. n = 10, ANOVA p values were nonsignificant.
Transfusion, 2010
BACKGROUND: The gas permeability of platelet (PLT) storage bags influences the retention of in vitro PLT parameters during storage. The aim of this study was to evaluate mitochondrial function of PLTs stored in first- and second-generation bags with different gas permeabilities.STUDY DESIGN AND METHODS: Identical whole blood–derived PLT concentrates were stored in second-generation CLX (Pall Corp.) and first-generation PL146 (Baxter Healthcare Corp.) bags (n = 12). PLTs were assayed for standard in vitro PLT assays as well as for mitochondrial membrane potential (MMP), accumulation of reactive oxygen species, Annexin V binding, mitochondrial mass, and activity of mitochondrial reduction power on Days 1, 4, 5, 6, and 7. Results were analyzed by paired t test and by multiple regression analysis.RESULTS: With PLTs stored in PL146 bags that underwent large pH declines, there was greater superoxide production, greater peroxide accumulation, and greater mitochondrial membrane depolarization. Superoxide anion generation was correlated with higher levels of carbon dioxide (p = 0.0001) and lower oxygen levels (p = 0.0064; multiple regression R2 = 0.9204). Changes in MMP were correlated with higher levels of carbon dioxide (p = 0.0288) and PLT activation (p = 0.0178; multiple regression R2 = 0.9511).CONCLUSION: Prolonged periods of elevated carbon dioxide levels, potentially coupled with other factors, is associated with PLT mitochondria dysfunction and poor pH control during storage.
Blood Advances, 2019
Historically, metabolic studies in platelets have primarily investigated events occurring during ex vivo platelet storage, less so the consequences of metabolic alterations on platelet homeostasis and physiologic function. The importance of such studies is emphasized by the recent finding of platelet mitochondrial dysfunction in type 2 diabetes, sickle cell disease, and sepsis. 1-7 These early investigations used radiometric methods with labeled glucose to study glycolysis and oxidative phosphorylation in platelets. 8,9 Recently, these techniques have been supplanted by more sophisticated investigative tools that allow more rapid analysis of small volume samples, including high-resolution respirometry 10,11 and Seahorse extracellular flux analysis. 12-14 Glycolysis and oxidative phosphorylation, as well as glutaminolysis and fatty acid b oxidation, all function to support the metabolic demand in platelets. 10,14-16 The metabolic flexibility of the platelet has been emphasized by investigations demonstrating that activated platelets exhibit a glycolytic phenotype while preserving mitochondrial function and can easily switch between glucose and fatty acid catabolism to support activation. 12 The recently published study by A. K. Chauhan's group further advances our understanding of the understudied field of platelet metabolism. 13 In this paper, the authors demonstrate that dichloroacetate, an inhibitor of pyruvate dehydrogenase kinases, alters platelet metabolism and function. 13 Finding salutary antiaggregatory and antithrombotic effects of dichloroacetate, the authors propose targeting of the platelet metabolic response as a novel antithrombotic approach. However, there are few points of clarification we would like to add, which we believe will be of great benefit for the platelet and mitochondria research community in their exploration of this new therapeutic avenue. 13