Background: Previous studies of platelet metabolism use either global measurements of extracellular uptake (O2) and secretion (CO2, lactate) or metabolomics to infer changes in metabolic pathways with storage or stimuli. Neither of these directly measure the rate, or flux, of metabolites through different pathways, which is required to identify intracellular shifts in metabolism at the resolution of individual reactions.  

Aims: To generate the first metabolic flux map of resting and thrombin activated platelets based on intracellular flux measurements.

Methods: Isotope assisted metabolic flux analysis (¹³C-MFA) was used to quantify intracellular metabolism in washed human platelets. A parallel labeling approach with two isotopic carbon sources (glucose and acetate) was used to probe central metabolism. The ¹³C-labeling of intracellular metabolites was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Labeling data was analyzed with the Isotopomer Network Compartment Analysis (INCA) software and used to calculate fluxes.

Results: Resting washed human platelets demonstrate active glycolysis and primarily metabolize glucose anaerobically, producing lactate (Fig 1). A small proportion (<10%) of the glycolytic flux is shunted through the pentose phosphate pathway. Energy requirements are balanced via oxidative ATP production in the citric acid cycle from non-glucose carbon sources. Platelets activated with 1U/mL thrombin show increased in flux through all major pathways (Fig. 2). Thrombin activated platelets still maintain a glycolytic phenotype where >80% of glucose uptake contributes to lactate formation and excretion.

Resting platelet flux mapFigure 1. Resting platelet flux map

 

Thrombin activated platelet flux map. Arrow colors indicate the percent change from resting platelet fluxes normalized to the glucose uptake.
Figure 2. Thrombin activated platelet flux map. Arrow colors indicate the percent change from resting platelet fluxes.  

Conclusions: We have developed a ¹³C-MFA workflow for quantifying the intracellular metabolic flux in platelets. Resting platelets display a glycolytic phenotype and additionally oxidize non-glucose carbon sources for mitochondrial metabolism. This ¹³C-MFA approach is a new tool for probing changes in platelet metabolism at the reaction level as function of agonist and/or environmental conditions.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/13c-metabolic-flux-analysis-in-resting-and-thrombin-activated-platelets/