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Sample Preparation as a Critical Aspect of Blood Platelet Mitochondrial Respiration Measurements-The Impact of Platelet Activation on Mitochondrial Respiration.

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.

SUBMITTER: Siewiera K

PROVIDER: S-EPMC8430930 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Complement component 3 fragment C3a is an anaphylatoxin involved in promoting cellular responses important in immune response and host defense. Its receptor (C3a receptor, C3aR) is distributed on the plasma membrane; however, lysosomal localization in immune cells has been reported. Oxidative stress increases intracellular reactive oxygen species (ROS), and ROS activate complement signaling in immune cells and metabolic reprogramming. Here we tested oxidative stress and intracellular complement in mitochondrial dysfunction in RPE cells using high resolution live-cell imaging, and metabolism analysis in isolated mitochondria using Seahorse technology. While C3aR levels were unaffected by oxidative stress, its cell membrane levels decreased and mitochondrial (mt) localization increased. Trafficking was dependent on endocytosis, utilizing endosomal-to-mitochondrial cargo transfer. H2O2-treatment also increased C3a-mtC3aR co-localization dose-dependently. In isolated mitochondria from H2O2-treated cells C3a increased mitochondrial Ca2+ uptake, that could be inhibited by C3aR antagonism (SB290157), mitochondrial Ca2+ uniporter blocker (Ru360), and Gαi-protein inhibition (pertussis toxin, PTX); and inhibited mitochondrial repiration in an SB290157- and PTX-dependent manner. Specifically, mtC3aR activation inhibited state III ADP-driven respiration and maximal respiratory capacity. Mitochondria from control cells did not respond to C3a. Furthermore, transmitochondrial cybrid ARPE-19 cells harboring J haplogroup mitochondria that confer risk for age-related macular degeneration, showed high levels of mtC3aR and reduced ATP production upon C3a stimulation. Our findings suggest that oxidative stress increases mtC3aR, leading to altered mitochondrial calcium uptake and ATP production. These studies will have important implication in our understanding on the balance of extra- and intracellular complement signaling in controlling cellular health and dysfunction.

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Sample Preparation as a Critical Aspect of Blood Platelet Mitochondrial Respiration Measurements-The Impact of Platelet Activation on Mitochondrial Respiration.

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