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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. https://doi.org/10.26124/mitofit:2021-00062021-12-08 published in Biomedicines

» MitoFit Preprints 2021.6.

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Mitochondrial respiration of platelets: comparison of isolation methods

MitoFit Preprints (2021-09-16) 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


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O2k-Protocols: Isolation of blood cells for HRR.
2020-10-22Bioblast pdf  »Versions

Support

Template NextGen-O2k.jpg
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).