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Vernerova 2021 MitoFit PLT

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Vernerova 2021 MitoFit PLT

Publications in the MiPMap
Vernerova A, Garcia-Souza LF, Soucek O, Kostal M, Rehacek V, Krcmova LK, Gnaiger E, Sobotka O (2021) Mitochondrial respiration of platelets: comparison of isolation methods. published in Biomedicines

» MitoFit Preprints 2021.06.

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Mitochondrial respiration of platelets: comparison of isolation methods

MitoFit Prep 2021.6. (2021) MitoFit Prep

Abstract: Journal publication 2021-12-08 in Biomedicines

Graphical abstract

Multiple non-aggregatory functions of human platelets (PLT) are widely acknowledged, yet their functional examination is limited mainly due to a lack of standardized isolation and analytic methods. Platelet apheresis (PA) is an established clinical method for PLT isolation aiming at the treatment of bleeding diathesis in severe thrombocytopenia. On the other hand, density gradient centrifugation (DC) is an isolation method applied in research for the analysis of the mitochondrial metabolic profile of oxidative phosphorylation (OXPHOS) in PLT obtained from small samples of human blood.

We studied PLT obtained from 29 healthy donors by high-resolution respirometry for comparison of PA and DC isolates. ROUTINE respiration and electron transfer capacity of living PLT isolated by PA were significantly higher than in the DC group, whereas plasma membrane permeabilization resulted in a 57 % decrease of succinate oxidation in PA compared to DC. These differences were eliminated after washing the PA cells with phosphate buffer containing 10 mmol·L-1 EGTA, suggesting that several components, particularly Ca2+ and fuel substrates, were carried over into the respiratory assay from the serum in PA. A simple washing step was sufficient to enable functional mitochondrial analysis in subsamples obtained from PA.

The combination of the standard clinical PA isolation procedure with PLT quality control and routine mitochondrial OXPHOS diagnostics meets an acute clinical demand in biomedical research of patients suffering from thrombocytopenia and metabolic diseases.

Keywords: mitochondria mt, human platelets PLT, thrombocytes, platelet apheresis PA, oxidative phosphorylation OXPHOS, high-resolution respirometry HRR, density gradient centrifugation DC, flow cytometry Bioblast editor: Gnaiger E

ORCID: ORCID.png Vernerova A, ORCID.png Garcia-Souza LF, ORCID.png Soucek O, ORCID.png Kostal M, ORCID.png Rehacek V, ORCID.png Krcmova LK, ORCID.png Gnaiger E, ORCID.png Sobotka O

Data availability

Original files are available Open Access at Zenodo repository: 10.5281/zenodo.5482452


Braganza A, Corey CG, Santanasto AJ, Distefano G, Coen PM, Glynn NW, Nouraie SM, Goodpaster BH, Newman AB, Shiva S (2019) Platelet bioenergetics correlate with muscle energetics and are altered in older adults. JCI Insight 5:128248.2019PMID: 31120438 Open Access
Brand MD, Nicholls DG (2011) Assessing mitochondrial dysfunction in cells. Biochem J 435: 297-312.2011
Bynum JA, Adam Meledeo M, Getz TM, Rodriguez AC, Aden JK, Cap AP, Pidcoke HF (2016) Bioenergetic profiling of platelet mitochondria during storage: 4°C storage extends platelet mitochondrial function and viability. Transfusion 56 Suppl 1:S76-84.2016PMID: 27001365
Chacko BK, Kramer PA, Ravi S, Johnson MS, Hardy RW, Ballinger SW, Darley-Usmar VM (2013) Methods for defining distinct bioenergetic profiles in platelets, lymphocytes, monocytes, and neutrophils, and the oxidative burst from human blood. Lab Invest 93:690-700.2013PMID: 23528848 Open Access
Doerrier C, Garcia-Souza LF, Krumschnabel G, Wohlfarter Y, Mészáros AT, Gnaiger E (2018) High-Resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibers from small biopsies of muscle, and isolated mitochondria. Mol Biol 1782:31-70. PMID: 29850993 »O2k-brief
Ferguson MA, Sutton RM, Karlsson M, Sjövall F, Becker LB, Berg RA, Margulies SS, Kilbaugh TJ (2016) Increased platelet mitochondrial respiration after cardiac arrest and resuscitation as a potential peripheral biosignature of cerebral bioenergetic dysfunction. J Bioenerg Biomembr 48:269-79.2016PMID: 27020568
Fišar Z, Jirák R, Zvěřová M, Setnička V, Habartová L, Hroudová J, Vaníčková Z, Raboch J (2019) Plasma amyloid beta levels and platelet mitochondrial respiration in patients with Alzheimer's disease. Clin Biochem 72:71-80.2019PMID: 30954436
Gnaiger E (2020) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 5th ed. Commun 2020.2. Open Access pdf published online 2020-12-30

Gnaiger E et al ― MitoEAGLE Task Group (2020) Mitochondrial physiology. Commun 2020.1. Open Access pdf published online 2020-05-20

Gnaiger E, Steinlechner-Maran R, Méndez G, Eberl T, Margreiter R (1995) Control of mitochondrial and cellular respiration by oxygen. Bioenerg Biomembr 27:583-96. PMID: 8746845
Bioblast pdf
Gvozdjakova A, Sumbalova Z, Kucharska J, Chladekova A, Rausova Z, Vancova O, Komlosi M, Ulicna O, Mojto V (2019) Platelet mitochondrial bioenergetic analysis in patients with nephropathies and non-communicable diseases: a new method. Bratisl Lek Listy 120:630-35.2019PMID: 31475544 Open Access
Gvozdjáková A, Sumbalová Z, Kucharská J, Komlósi M, Rausová Z, Vančová O, Számošová M, Mojto V (2020) Platelet mitochondrial respiration, endogenous coenzyme Q10 and oxidative stress in patients with chronic kidney disease. Diagnostics (Basel) 10:176.2020PMID: 32210203 Open Access »O2k-brief
Hoppel F, Calabria E, Pesta DH, Kantner-Rumplmair W, Gnaiger E, Burtscher M (2021) Effects of ultramarathon running on mitochondrial function of platelets and oxidative stress parameters: a pilot study. Physiol 12:632664. PMID: 33679442 Open Access »O2k-brief
Hsu CC, Tsai HH, Fu TC, Wang JS (2019) Exercise training enhances platelet mitochondrial bioenergetics in stroke patients: a randomized controlled trial. J Clin Med 8:E2186.2019PMID: 31835774 Open Access
Kline JA, Puskarich MA, Pike JW, Zagorski J, Alves NJ (2020) Inhaled nitric oxide to control platelet hyper-reactivity in patients with acute submassive pulmonary embolism. Nitric Oxide 96:20-8.2020PMID: 31940502
McCann MR, McHugh CE, Kirby M, Jennaro TS, Jones AE, Stringer KA, Puskarich MA (2020) A multivariate metabolomics method for estimating platelet mitochondrial oxygen consumption rates in patients with sepsis. Metabolites 10:E139.2020PMID: 32252461 Open Access
Perales Villarroel JP, Figueredo R, Guan Y, Tomaiuolo M, Karamercan MA, Welsh J, Selak MA, Becker LB, Sims C (2013) Increased platelet storage time is associated with mitochondrial dysfunction and impaired platelet function. J Surg Res 184:422-9.2013PMID: 23830370 Open Access
Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibers from small biopsies of human muscle. Methods Mol Biol 810:25-58. 22057559
Bioblast pdf
Puskarich MA, Kline JA, Watts JA, Shirey K, Hosler J, Jones AE (2016) Early alterations in platelet mitochondrial function are associated with survival and organ failure in patients with septic shock. J Crit Care 31:63-7.2016PMID: 26511963
Renner K, Geiselhöringer AL, Fante M, Bruss C, Färber S, Schönhammer G, Peter K, Singer K, Andreesen R, Hoffmann P, Oefner P, Herr W, Kreutz M (2015) Metabolic plasticity of human T cells: Preserved cytokine production under glucose deprivation or mitochondrial restriction, but 2-deoxy-glucose affects effector functions. Eur J Immunol 45:2504-16.2015PMID: 26114249
Schmidl C, Renner K, Peter K, Eder R, Lassmann T, Balwierz PJ, Itoh M, Nagao-Sato S, Kawaji H, Carninci P, Suzuki H, Hayashizaki Y, Andreesen R, Hume DA, Hoffmann P, Forrest AR, Kreutz MP, Edinger M, Rehli M (2014) Transcription and enhancer profiling in human monocyte subsets. Blood 123:e90-9.2014PMID: 24671955
Siewiera K, Kassassir H, Talar M, Wieteska L, Watala C (2016) Higher mitochondrial potential and elevated mitochondrial respiration are associated with excessive activation of blood platelets in diabetic rats. Life Sci 148:293-304.2016PMID: 26872978
Sjoevall F, Morota S, Hansson Magnus J, Friberg H, Gnaiger E, Elmer E (2010) Temporal increase of platelet mitochondrial respiration is negatively associated with clinical outcome in patients with sepsis. Crit Care 14:R214.2010PMID: 21106065 Open Access
Sjövall F, Morota S, Asander Frostner E, Hansson Magnus J, Elmer E (2014) Cytokine and nitric oxide levels in patients with sepsis - temporal evolvement and relation to platelet mitochondrial respiratory function. PLoS One 9:e97673.2014PMID: 24828117 Open Access
Stadlmann S, Renner K, Pollheimer J, Moser PL, Zeimet AG, Offner FA, Gnaiger E (2006) Preserved coupling of oxidative phosphorylation but decreased mitochondrial respiratory capacity in IL-1ß treated human peritoneal mesothelial cells. Cell Biochem Biophys 44:179-86.2006PMID: 16456220
Bioblast pdf
Stadlmann S, Rieger G, Amberger A, Kuznetsov AV, Margreiter R, Gnaiger E (2002) H2O2-mediated oxidative stress versus cold ischemia-reperfusion: mitochondrial respiratory defects in cultured human endothelial cells. Transplantation 74:1800-3.2002PMID: 12499903
Wang L, Wu Q, Fan Z, Xie R, Wang Z, Lu Y (2017) Platelet mitochondrial dysfunction and the correlation with human diseases. Biochem Soc Trans 45:1213-23.2017PMID: 29054925
O2k-Protocols: Isolation of blood cells for HRR.
2020-10-22Bioblast pdf  »Versions


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The study was supported by Project SVV 260 548, the University Hospital in Hradec Kralove MH CZ - DRO (UHHK, 00179906), and COST Action CA15203 MitoEAGLE. The O2k respirometers were provided by Oroboros Instruments (Innsbruck, Austria). This work was partially funded by the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 859770, NextGen-O2k project (EG).