Mitochondrial Respiration of Platelets: Comparison of Isolation Methods
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...
| Main Authors: | , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2021-12-01
|
| Series: | Biomedicines |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2227-9059/9/12/1859 |
| _version_ | 1797506550271574016 |
|---|---|
| author | Andrea Vernerova Luiz Felipe Garcia-Souza Ondrej Soucek Milan Kostal Vit Rehacek Lenka Kujovska Krcmova Erich Gnaiger Ondrej Sobotka |
| author_facet | Andrea Vernerova Luiz Felipe Garcia-Souza Ondrej Soucek Milan Kostal Vit Rehacek Lenka Kujovska Krcmova Erich Gnaiger Ondrej Sobotka |
| author_sort | Andrea Vernerova |
| collection | DOAJ |
| description | 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 platelets with phosphate buffer containing 10 mmol·L<sup>−1</sup> ethylene glycol-bis (2-aminoethyl ether)-<i>N</i>,<i>N</i>,<i>N</i>′,<i>N</i>′-tetra-acetic acid, suggesting that several components, particularly Ca<sup>2+</sup> 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. |
| first_indexed | 2024-03-10T04:34:09Z |
| format | Article |
| id | doaj.art-d4f34fef9825464b942a10477dc3c5c9 |
| institution | Directory Open Access Journal |
| issn | 2227-9059 |
| language | English |
| last_indexed | 2024-03-10T04:34:09Z |
| publishDate | 2021-12-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biomedicines |
| spelling | doaj.art-d4f34fef9825464b942a10477dc3c5c92023-11-23T03:56:42ZengMDPI AGBiomedicines2227-90592021-12-01912185910.3390/biomedicines9121859Mitochondrial Respiration of Platelets: Comparison of Isolation MethodsAndrea Vernerova0Luiz Felipe Garcia-Souza1Ondrej Soucek2Milan Kostal3Vit Rehacek4Lenka Kujovska Krcmova5Erich Gnaiger6Ondrej Sobotka7Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Kralove, Charles University, Akademika Heyrovskeho 1203/8, 500 05 Hradec Kralove, Czech RepublicOroboros Instruments GmbH, Schoepfstrasse 18, A-6020 Innsbruck, AustriaDepartment of Clinical Immunology and Allergology, University Hospital Hradec Kralove, Sokolska 581, 500 05 Hradec Kralove, Czech Republic4th Department of Internal Medicine—Hematology, University Hospital Hradec Kralove, Sokolska 581, 500 05 Hradec Kralove, Czech RepublicTransfusion Department, University Hospital Hradec Kralove, Sokolska 581, 500 05 Hradec Kralove, Czech RepublicDepartment of Analytical Chemistry, Faculty of Pharmacy in Hradec Kralove, Charles University, Akademika Heyrovskeho 1203/8, 500 05 Hradec Kralove, Czech RepublicOroboros Instruments GmbH, Schoepfstrasse 18, A-6020 Innsbruck, Austria3rd Department of Internal Medicine—Metabolic Care and Gerontology, University Hospital Hradec Kralove, Sokolska 581, 500 05 Hradec Kralove, Czech RepublicMultiple 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 platelets with phosphate buffer containing 10 mmol·L<sup>−1</sup> ethylene glycol-bis (2-aminoethyl ether)-<i>N</i>,<i>N</i>,<i>N</i>′,<i>N</i>′-tetra-acetic acid, suggesting that several components, particularly Ca<sup>2+</sup> 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.https://www.mdpi.com/2227-9059/9/12/1859mitochondriahuman plateletsthrombocytesdensity gradient centrifugationplatelet apheresisflow cytometry |
| spellingShingle | Andrea Vernerova Luiz Felipe Garcia-Souza Ondrej Soucek Milan Kostal Vit Rehacek Lenka Kujovska Krcmova Erich Gnaiger Ondrej Sobotka Mitochondrial Respiration of Platelets: Comparison of Isolation Methods Biomedicines mitochondria human platelets thrombocytes density gradient centrifugation platelet apheresis flow cytometry |
| title | Mitochondrial Respiration of Platelets: Comparison of Isolation Methods |
| title_full | Mitochondrial Respiration of Platelets: Comparison of Isolation Methods |
| title_fullStr | Mitochondrial Respiration of Platelets: Comparison of Isolation Methods |
| title_full_unstemmed | Mitochondrial Respiration of Platelets: Comparison of Isolation Methods |
| title_short | Mitochondrial Respiration of Platelets: Comparison of Isolation Methods |
| title_sort | mitochondrial respiration of platelets comparison of isolation methods |
| topic | mitochondria human platelets thrombocytes density gradient centrifugation platelet apheresis flow cytometry |
| url | https://www.mdpi.com/2227-9059/9/12/1859 |
| work_keys_str_mv | AT andreavernerova mitochondrialrespirationofplateletscomparisonofisolationmethods AT luizfelipegarciasouza mitochondrialrespirationofplateletscomparisonofisolationmethods AT ondrejsoucek mitochondrialrespirationofplateletscomparisonofisolationmethods AT milankostal mitochondrialrespirationofplateletscomparisonofisolationmethods AT vitrehacek mitochondrialrespirationofplateletscomparisonofisolationmethods AT lenkakujovskakrcmova mitochondrialrespirationofplateletscomparisonofisolationmethods AT erichgnaiger mitochondrialrespirationofplateletscomparisonofisolationmethods AT ondrejsobotka mitochondrialrespirationofplateletscomparisonofisolationmethods |