Biological and Genetic Determinants of Red Blood Cell Glycolysis

Abstract Glycolysis is a central metabolic pathway in health and disease, 1 an essential one in mature red blood cells (RBCs), which rely on the Embden-Meyerhof-Parnas pathway, i.e., glycolysis, as the sole source of energy generation. 2 During aging in vivo and in vitro under blood bank conditions, 3 the adenosine triphosphate (ATP) generated via glycolysis in mitochondria-devoid mature RBCs is essential for the modulation of oxygen kinetics, 4 deformability 2 and, ultimately, intra- and extra-vascular hemolysis. 3 Here we draw upon a cohort of 13,029 volunteers from the Recipient Epidemiology and Donor Evaluation Study (REDS) to identify biological and genetic traits associated with heterogeneity in glycolytic phenotypes. Age, sex and ethnicity influenced glycolysis, with additional effects due to genetic polymorphisms in regions coding for rate-limiting enzymes of glycolysis, 1 phosphofructokinase 1 (PFKP) and hexokinase 1 (HK1), and for the ADP-ribosyl cyclase CD38. The gene-metabolite associations were validated by testing glycolysis in fresh and stored RBCs from 350 Diversity Outbred mice. 5 These findings were further corroborated via multi-omics analyses of 1,929 samples at storage day 10, 23 and 42 in 643 RBC units from donors who were selected amongst the index cohort based on their extreme hemolytic propensity. Finally, we show that glycolysis, ATP levels, breakdown and deamination into hypoxanthine are associated with increased hemolytic propensity and vesiculation ex vivo , as well as with extravascular hemolysis in vivo, both in healthy autologous transfusion recipients from the Donor Iron Deficiency Study and in 4,700 heterologous critically ill patients receiving blood products from the REDS donors. This work advances our understanding of genetic and non-genetic factors that regulate glycolysis in mature RBCs, a simplified model of mammalian cell metabolism, 6 a cell type where dysregulated glycolysis holds broader implications in hemolytic disorders, offering potential avenues for novel therapeutic interventions and transfusion strategies. Graphical abstract