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.

Potential Role of Mitochondria as Modulators of Blood Platelet Activation and Reactivity in Diabetes and Effect of Metformin on Blood Platelet Bioenergetics and Platelet Activation

“…Interestingly, the decrease in oxygen consumption (corresponding to ROUTINE respiration) was already observed for 10 μM metformin [ 12 ]. Probably, the effects of such a long incubation period could drastically reduce platelet mitochondrial respiration, as observed elsewhere [ 19 ], and at the same time increase the impact of metformin on mitochondrial electron transfer system, and thus also on mitochondrial respiration. In contrary to us, no platelet activation parameters were assessed by Protti et al [ 12 ].…”

“…However, their study used a much longer incubation time. Since long-term storage adversely affects mitochondrial respiration and leads to artefactual platelet activation [ 19 ], a shorter incubation period was used in our present study. There are also several reports in the literature on the effect of metformin in vitro on other processes related to platelet functioning, such as platelet adhesion [ 21 ], platelet aggregation [ 21 , 22 ], thrombus formation [ 23 ] or thromboxane production [ 22 ].…”

Mitochondrial Respiration of Platelets: Comparison of Isolation Methods

“…Despite the lack of added Ca +2 in the apheresis solution, SSP+ PLT additive solution, residual calcium from the donor’s plasma could still be present in the PA samples, and even though we used two different systems for PA, we did not observe any significant differences between them (data not shown). These results indicated that the choice of medium for storage and the further respirometric measurements might be key considerations when using PLT as a biomarker of mitochondrial changes as suggested by Siewiera et al [ 46 ].…”

Discernment of cerebral amyloid and cognitive status using ultrasensitive blood biomarkers collected with a non-traditional anticoagulant

“…10,15 In addition, pre-analytical factors are also an important consideration when examining plasma biomarker results. 16,17 While EDTA is considered the gold standard for anti-coagulant for plasma A/T/N analyses 18 , it may interfere with other measures including functional mitochondrial outcomes 19 . In addition, anticoagulants such as acid citrate dextrose (ACD) have been shown to improve outcomes for studies focused on microparticles, such as extracellular vesicles, without interfering with downstream protein or RNA analyses.…”