Platelet Metabolic Profiling Reveals Glycolytic and 1-Carbon Metabolites Are Essential for GP VI-Stimulated Human Platelets-Brief Report

Abstract

Background: Evolving evidence suggests that besides signaling pathways, platelet activation involves a complex interplay between metabolic pathways to support thrombus growth. Selective targeting of metabolic checkpoints may inhibit platelet activation and provide a novel antiplatelet strategy. We, therefore, examined global metabolic changes that occur during the transition of human platelets from resting to an activated state to identify metabolites and associated pathways that contribute to platelet activation.

Methods: We performed metabolic profiling of resting and convulxin-stimulated human platelet samples. The differential levels, pathway analysis, and PCA (principal component analysis) were performed using Metaboanalyst. Metascape was used for metabolite network construction.

Results: Of the 401 metabolites identified, 202 metabolites were significantly upregulated, and 2 metabolites were downregulated in activated platelets. Of all the metabolites, lipids scored highly and constituted ≈50% of the identification. During activation, aerobic glycolysis supports energy demand and provides glycolytic intermediates required by metabolic pathways. Consistent with this, an important category of metabolites was carbohydrates, particularly the glycolysis intermediates that were significantly upregulated compared with resting platelets. We found that lysophospholipids such as 1-palmitoyl-GPA (glycero-3-phosphatidic acid), 1-stearoyl-GPS (glycero-3-phosphoserine), 1-palmitoyl-GPI (glycerophosphoinositol), 1-stearoyl-GPI, and 1-oleoyl-GPI were upregulated in activated platelets. We speculated that platelet activation could be linked to 1-carbon metabolism, a set of biochemical pathways that involve the transfer and use of 1-carbon units from amino acids, for cellular processes, including nucleotide and lysophospholipid synthesis. In alignment, based on pathway enrichment and network-based prioritization, the metabolites from amino acid metabolism, including serine, glutamate, and branched-chain amino acid pathway were upregulated in activated platelets, which might be supplemented by the high levels of glycolytic intermediates.

Conclusions: Metabolic analysis of resting and activated platelets revealed that glycolysis and 1-carbon metabolism are necessary to support platelet activation.

Keywords: 1-carbon metabolism; glycolysis; glycoprotein VI; platelet activation; platelet metabolism.

Figure 1:. Changes in the metabolic profiles of activated human platelets.

A) Principal Component Analysis of the normalized metabolites from resting and activated human platelets depicting clear separation between the two groups. B) Heatmaps of hierarchically clustered metabolites (with increasing or decreasing trend) in resting (red color) and convulxin-stimulated (green color) human platelets. Columns represent individual samples, and rows represent metabolites. C) Heatmap of Top 10 metabolites with highest fold change values between resting and convulxin-stimulated platelets. B and C) Normalized metabolite intensities are displayed using a color scale ranging from red to blue, with red representing higher intensities and blue representing lower intensities. Di - Dviiii) The mechanistic view and levels of the top 10 identified metabolites and their interaction with other metabolites. Values are mean ± SEM, n=5.

Figure 2:. Pathway and network analysis of metabolites reveals backbone metabolites and collective mechanistic links.

A) The metabolite backbone network. The backbone network is derived from a master network constructed using metascape for significant metabolites identified. B) The figure provides the pathway for one-carbon metabolism. C, D, E, F, G, and H) The bar charts represent the metabolite levels between resting and convulxin-stimulated platelets. Values are mean ± SEM, n=5.