Frontiers in Hematology · Published 2026-06-29 · DOI 10.3389/frhem.2026.1766692
Apoorva Jagadish, Youwei Chen, Hongmei Zhu, Weijia Mai, Shein-Chung Chow, Zhong Li, Grace M. Lee, Marilyn J. Telen, Tim J. McMahon
BackgroundSickle cell disease (SCD) represents a significant global health burden and is characterized by hemolytic anemia, vaso-occlusive crises, end-organ damage, and early mortality. Red blood cells (RBCs) depend on glycolysis due to the virtual absence of mitochondria. RBC glycolysis generates adenosine triphosphate (ATP) and regulates levels of 2,3-diphosphoglycerate (2,3-DPG). Intracellular ATP is essential for numerous RBC functions, such as maintaining ion homeostasis, cell membrane integrity, redox protection, and RBC deformability. Sickle (SS) RBCs have reduced intracellular ATP compared to normal RBCs. RBCs also export ATP, which can modulate blood flow in response to hypoxia, inhibit intercellular adhesion, and prevent excess capillary permeability. We sought primarily to investigate a novel approach to augment ATP export from SS vs. healthy RBCs. Pyruvate kinase (PK) catalyzes the final step of glycolysis, generating RBC intracellular ATP. PK activators (PKAs), such as mitapivat (AG-348), are now approved in PK deficiency and are being studied in SCD patients. By increasing PKR (RBC PK) activity, they increase intracellular ATP while decreasing upstream 2,3-DPG. Our secondary aim was to compare the effects of a PKR activator (PKRA) on ATP export by RBCs in SCD vs. healthy (HC) individuals.MethodsWhole blood (WB) from SCD patients and healthy adults (IRB-approved) was incubated with AG-348 (10 µM) or vehicle (DMSO) at 4 °C for 18–24 h. RBCs were isolated, washed, and exposed to normoxic (21% O2) or hypoxic (1% O2) conditions. Supernatant and intracellular ATP was measured by luciferase assay, and hemoglobin was quantified to assess hemolysis. For adhesion assays, treated or control WB was perfused through laminin-coated microfluidic channels under increasing shear stress (0.3–10 dyn/cm²). Images were obtained after each shear increment, and adhesion was quantified as the percentage of cells remaining using ImageJ.ResultsWe obtained WB from pediatric SCD patients. PKRA treatment ex vivo raised intra-RBC ATP in HC blood, consistent with prior reports, but did not increase intra-RBC ATP significantly in SCD blood. Exported ATP was increased after HC RBC exposure to hypoxia (94.3 ± 24.3 nM (mean ± SEM) vs. 79.7 ± 20.6 nM in normoxia). ATP export from HC RBCs increased after treatment with the PKRA AG-348 in normoxia (80.3 ± 22.3 nM vs. 109.0 ± 30.2 nM) and hypoxia. ATP exported by SCD RBCs increased in hypoxia vs. normoxia. Exported ATP did not change significantly in SCD RBCs treated with the PKRA AG-348 (79.7 ± 20.6 nM vs. 77.1 ± 19.9 nM). Post-assay lysis levels were 1% or lower under all conditions. These low levels of lysis in ATP export assays did not differ as a function of PKRA treatment or between HC and SCD RBCs. As compared to the effects of vehicle alone, SS RBC adhesion to laminin after WB exposure to AG-348 was decreased at the physiologically relevant shear stresses of 0.3, 1, and 3 dyne/cm2.ConclusionsTo our knowledge, our data show for the first time that ATP export from HC RBCs increases after treatment with a PKRA, which also increased HC intra-RBC ATP, confirming prior reports. In contrast, exposure of SCD RBCs to PKRA under identical conditions did not significantly increase intra-RBC or exported ATP in SCD in normoxia or hypoxia. ATP export from untreated SS RBCs rose in hypoxia. Ongoing work addresses the determinants of cellular and exported ATP in HC and SCD RBCs (basal and treated) and the basis of the antiadhesive effect of this PKRA on SS RBCs.
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Jagadish, A., Chen, Y., Zhu, H., et al. (2026). Influence of the pyruvate kinase activator mitapivat (AG-348) on RBC ATP content and export and RBC adhesivity in blood from healthy subjects and sickle cell disease patients. Frontiers in Hematology. https://doi.org/10.3389/frhem.2026.1766692