New sickle cell disease therapies modulate red blood cell deformability via Ca2+, ATP, and 2,3-DPG in normoxia and hypoxia

1. PKR activator and GBT021601 lower 2,3-DPG; PKR activator boosts ATP in hypoxia, enhancing sickle RBC deformability. 2. Voxelotor, GBT021601, PKR activator lower [Ca 2+ ] total by inhibiting Piezo1 and modifying clearance pathways, improving RBC deformability. Reduced deformability of red blood cells (RBCs) due to sickle hemoglobin (HbS) polymerization underlies the vaso-occlusive crises and hemolytic anemia that characterize sickle cell disease (SCD). Emerging therapies that increase hemoglobin oxygen affinity (Hb-O 2 ), voxelotor, GBT021601 (second-generation polymerization-inhibitor), and pyruvate kinase (PKR) activators have shown clinical benefits. Yet, their specific mechanistic effects on RBCs deformability under normoxic and mixed venous hypoxic conditions are not fully understood. We hypothesized that these agents differentially regulate calcium (Ca 2+ ), adenosine triphosphate (ATP), and 2,3-diphosphoglycerate (2,3-DPG), key determinants of deformability, and that hypoxia amplifies their actions. We used targeted biochemical assays to measure total intracellular calcium content ([Ca 2+ ] total ), ATP, and 2,3-DPG in sickle (HbSS) and healthy (HbAA) RBCs. Under normoxia, GBT021601 and PKR activators, but not voxelotor, reduced 2,3-DPG. Only PKR activators elevated ATP under hypoxia. All three therapies reduced [Ca 2+ ] total in HbSS RBCs by inhibiting Piezo1 activity and modulating Ca 2+ clearance pathways, including residual plasma membrane Ca 2+ -ATPase (PMCA) activity or alternative Ca 2+ efflux pathways. Decreased [Ca 2+ ] total coincided with decreases in Band-3 tyrosine phosphorylation in HbSS but not HbAA RBCs, suggesting [Ca 2+ ] total -dependent modulation of membrane-protein interactions. Mechanistically, voxelotor indirectly inhibited Ca 2+ entry; GBT021601 reduced 2,3-DPG and potentially modified Ca 2+ clearance pathways; and PKR activator paired the 2,3-DPG decrease with hypoxia-specific ATP increases to fuel PMCA activity. Our findings suggest that reduced [Ca 2+ ] total may be a common downstream mechanism by which diverse HbS-targeted and metabolic therapies restore RBC deformability, supporting the exploration of combination strategies targeting complementary pathways in Ca 2+ homeostasis and RBC deformability for SCD.