Abstract
Blood platelets are considered as promising candidates as easily-accessible biomarkers of mitochondrial functioning. However, their high sensitivity to various stimulus types may potentially affect mitochondrial respiration and lead to artefactual outcomes. Therefore, it is crucial to identify the factors associated with platelet preparation that may lead to changes in mitochondrial respiration. A combination of flow cytometry and advanced respirometry was used to examine the effect of blood anticoagulants, the media used to suspend isolated platelets, respiration buffers, storage time and ADP stimulation on platelet activation and platelet mitochondria respiration. Our results clearly show that all the mentioned factors can affect platelet mitochondrial respiration. Briefly, (i) the use of EDTA as anticoagulant led to a significant increase in the dissipative component of respiration (LEAK), (ii) the use of plasma for the suspension of isolated platelets with MiR05 as a respiration buffer allows high electron transfer capacity and low platelet activation, and (iii) ADP stimulation increases physiological coupling respiration (ROUTINE). Significant associations were observed between platelet activation markers and mitochondrial respiration at different preparation steps; however, the fact that these relationships were not always apparent suggests that the method of platelet preparation may have a greater impact on mitochondrial respiration than the platelet activation itself.
Highlights
There has been growing interest in blood platelets as mitochondria-related biomarkers of pathological changes in various diseases, including Alzheimer’s disease [1], sickle cell disease [2], sepsis [3], asthma [4] and diabetes [5]
To gain a greater understanding of the potential of platelets as biomarkers of mitochondrial functioning, the present study assesses whether the manner of the preparation of samples for respirometry measurements may lead to artefactual changes in platelet mitochondrial respiration; it examines whether these artefactual changes are related to platelet activation and how much the results obtained with the use of some compared methods/variants differ from each other when used for the same biological material originating from a single subject
In the case of ethylenediaminetetraacetic acid (EDTA), the platelets were further activated during centrifugation of blood to platelet-rich plasma (PRP), which was not observed in the case of other anticoagulants (Figure 1B)
Summary
There has been growing interest in blood platelets as mitochondria-related biomarkers of pathological changes in various diseases, including Alzheimer’s disease [1], sickle cell disease [2], sepsis [3], asthma [4] and diabetes [5]. Since the activation of platelets depends on the energy produced in the process of oxidative phosphorylation [13,14,15], changes in activation may lead to alterations in mitochondria respiration, such as an increase in routine cell respiration related to elevated energy demands or a decrease in mitochondrial respiration related to the loss of mitochondria in the process of microparticle release. To gain a greater understanding of the potential of platelets as biomarkers of mitochondrial functioning, the present study assesses whether the manner of the preparation of samples for respirometry measurements may lead to artefactual changes in platelet mitochondrial respiration; it examines whether these artefactual changes are related to platelet activation and how much the results obtained with the use of some compared methods/variants differ from each other when used for the same biological material originating from a single subject
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