Biological and Genetic Determinants of Glycolysis: Phosphofructokinase Isoforms Boost Energy Status of Stored Red Blood Cells and Transfusion Outcomes

Abstract

Glycolysis is a central metabolic pathway in health and disease,1an 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.2During aging in vivo and in vitro under blood bank conditions,3the adenosine triphosphate (ATP) generated via glycolysis in mitochondria-devoid mature RBCs is essential for the modulation of oxygen kinetics,4deformability2and, ultimately, intra- and extra-vascular hemolysis.3Here 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,1phosphofructokinase 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.5These 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 vesiculationex 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,6a cell type where dysregulated glycolysis holds broader implications in hemolytic disorders, offering potential avenues for novel therapeutic interventions and transfusion strategies.Graphical abstract

RBC-GEM: a Knowledge Base for Systems Biology of Human Red Blood Cell Metabolism

“…RBCs are well known for their near exclusive reliance on glycolysis for the generation of ATP, the pentose phosphate pathway for NADPH regeneration, and the purine salvage pathway for maintaining adequate nucleotide concentrations. Many of these rate-limiting enzymes are pH sensitive, including hexokinase ( HK1 ), phosphofructokinase isozymes ( PFKL , PFKM , PFKP )[102], and G6PD . The product of glycolysis in RBCs is lactate [31], which exists in equilibrium with lactic acid at physiological pH.…”

“…At low oxygen saturation, the bound glycolytic enzymes are outcompeted by deoxyhemoglobin and released. The release of glycolytic enzymes to the cytosol corresponds to an increase in the activity of these rate-limiting enzymes the glycolytic pathway[102,106,107], consequently forming an intricate feedback loop in which oxygen transport and delivery is finely regulated through RBC metabolic demands [108].…”

“…Therefore, we included catecholamines such as epinephrine, norepinephrine, and dopamine along with subsequent oxidation and glutathione conjugation reactions[215]. We also included various post translational modifications and repair reactions, including deglycation by fructose 3-kinase ( FN3K )[102,216] and related protein ( FN3KRP )[217], methylation by various methyltransferases including PCMT1 [185], glycosylation by residual activity of blood group proteins[218,219], signaling through phosphorylation and dephosphorylation[28,220,221], protein degradation through ubiquitin-mediated proteolysis[83,222], and alkylation via transglutaminase ( TGM2 )[223]. The multitude of post-translational modifications (PTMs) and protein repair reactions included in RBC-GEM are therefore indicative of the importance of PTMs for RBC metabolic reprogramming, adaptivity, and timing of senescence in a cell devoid of standard protein synthesis and degradation.…”

Modulation of the allosteric and vasoregulatory arms of erythrocytic oxygen transport

“…ATP (and other indices of glycolytic activity such as lactate and hypoxanthine) in turn associated significantly with hemolytic propensity ex vivo and after RBC transfusion in critically ill patients. PKLR (encoding PK in the liver and RBCs) was among the genes for which metabolite quantitative trait loci associated significantly with glycolysis, suggesting a druggable target for potential intervention ( Nemkov et al, 2024 ).…”

“…Large studies of the degree of post-transfusion recovery of RBCs in recipients have revealed substantial variability from donor to donor, and high reproducibility of post-transfusion recovery (PTR) of RBCs from a given donor, consistent with the importance of heritable traits governing this essential measure of transfusion effectiveness. The variability in PTR is clearly linked to variable donor genetics and metabolomes ( Nemkov et al, 2024 ).…”