Metabolic Plasticity in Resting and Thrombin Activated Platelets

Abstract

Platelet thrombus formation includes several integrated processes involving aggregation, secretion of granules, release of arachidonic acid and clot retraction, but it is not clear which metabolic fuels are required to support these events. We hypothesized that there is flexibility in the fuels that can be utilized to serve the energetic and metabolic needs for resting and thrombin-dependent platelet aggregation. Using platelets from healthy human donors, we found that there was a rapid thrombin-dependent increase in oxidative phosphorylation which required both glutamine and fatty acids but not glucose. Inhibition of fatty acid oxidation or glutamine utilization could be compensated for by increased glycolytic flux. No evidence for significant mitochondrial dysfunction was found, and ATP/ADP ratios were maintained following the addition of thrombin, indicating the presence of functional and active mitochondrial oxidative phosphorylation during the early stages of aggregation. Interestingly, inhibition of fatty acid oxidation and glutaminolysis alone or in combination is not sufficient to prevent platelet aggregation, due to compensation from glycolysis, whereas inhibitors of glycolysis inhibited aggregation approximately 50%. The combined effects of inhibitors of glycolysis and oxidative phosphorylation were synergistic in the inhibition of platelet aggregation. In summary, both glycolysis and oxidative phosphorylation contribute to platelet metabolism in the resting and activated state, with fatty acid oxidation and to a smaller extent glutaminolysis contributing to the increased energy demand.

Defining the effects of storage on platelet bioenergetics: The role of increased proton leak

“…Integrating all the indices of mitochondrial function in the BHI equation, it is evident that the overall bioenergetic health of the stored platelets is significantly lower than the freshly isolated platelets suggesting that this parameter could be used as a screen of platelet bioenergetic function (Figure 2H). These data would suggest that the stored platelet would be more susceptible to oxidative stress [19]. Additionally, there were no differences in basal glycolysis, but upon inhibition of mitochondrial function, glycolytic rate was significantly enhanced in the stored compared to the freshly isolated platelet (Figure 4).…”

“…In marked contrast to other studies that have shown no changes in mitochondrial respiration, or complete loss of mitochondrial function, we demonstrated a modest decrease in individual parameters of cellular platelet bioenergetics, associated with storage. In the intact platelet the substrates for oxidative phosphorylation are supplied by the sum of the metabolic pathways capable of providing substrates for mitochondrial respiration [19]. For this reason maximal respiration in the intact platelet can be limited by the endogenous substrate supply and likely explains why the inhibition of mitochondrial electron transport at complex I and II does not result in a decrease maximal respiration.…”

“…Typically, these studies use very small sample sizes and varying protocols for platelet storage which are different from those used in transfusion medicine [8, 16, 18]. In addition, isolation of mitochondria from the platelets to analyze complex activities may lead to damage to the organelle which can be avoided by using cellular bioenergetic analysis [19]. These factors could partially explain the inconsistency of these data.…”

Modification of platelet proteins by 4-hydroxynonenal: Potential Mechanisms for inhibition of aggregation and metabolism

“…Both oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured simultaneously in the extracellular flux (XF) analyzer as described previously [20, 24]. In brief, platelets were pre-treated with 4-HNE (0–30 µM), alkyne HNE (30 µM) or 1-nonanol and nonanal (30 µM) for 1h prior to performing the assay by sequential injection of oligomycin (1 µg/ml), FCCP (0.6 µM) and antimycin A (10 µM).…”

Metabolic Reprogramming Is Required for Myofibroblast Contractility and Differentiation

“…Despite its relative inefficiency in producing ATP compared with oxidative phosphorylation (32,33), glycolysis can respond rapidly to provide both ATP and other metabolites necessary for energetically demanding processes (34 -36). Indeed, in response to increased energy demand, the stimulation of both oxidative phosphorylation and aerobic glycolysis is now emerging as a characteristic of many cells including those of the immune system and platelets (37)(38)(39)(40).…”