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Energy metabolism in RBCs is characterized by O2-responsive variations in flux through the Embden Meyerhof pathway (EMP) or the hexose monophosphate pathway (HMP). Therefore, the generation of ATP, NADH, and 2,3-DPG (EMP) or NADPH (HMP) shift with RBC O2content because of competition between deoxyhemoglobin and key EMP enzymes for binding to the cytoplasmic domain of the Band 3 membrane protein (cdB3). Enzyme inactivation by cdB3 sequestration in oxygenated RBCs favors HMP flux and NADPH generation (maximizing glutathione-based antioxidant systems). We tested the hypothesis that sickle hemoglobin disrupts cdB3-based regulatory protein complex assembly, creating vulnerability to oxidative stress. In RBCs from patients with sickle cell anemia, we demonstrate in the present study constrained HMP flux, NADPH, and glutathione recycling and reduced resilience to oxidative stress manifested by membrane protein oxidation and membrane fragility. Using a novel, inverted membrane-on-bead model, we illustrate abnormal (O2-dependent) association of sickle hemoglobin to RBC membrane that interferes with sequestration/inactivation of the EMP enzyme GAPDH. This finding was confirmed by immunofluorescent imaging during RBC O2loading/unloading. Moreover, selective inhibition of inappropriately dispersed GAPDH rescues antioxidant capacity. Such disturbance of cdB3-based linkage between O2gradients and RBC metabolism suggests a novel mechanism by which hypoxia may influence the sickle cell anemia phenotype.

BACKGROUND:In sepsis, many red blood cell (RBC) defects are described: ↑ oxygen (O2) affinity, ↓ deformability, ↑ aggregation & adhesion, and dysregulated RBC-based vasoregulation. We suggest these comprise a unique class of organ (RBC) failure, which we term sepsis-induced RBC dysfunction (SiRD). We propose SiRD arises from RBC metabolic failure and depowered antioxidant systems - resulting in impaired RBC physiology that disables O2transport. We employed a murine cecal ligation and puncture (CLP) sepsis model to characterize SiRD features and explore mechanisms of SiRD initiation.