Activating pyruvate kinase improves red blood cell integrity by reducing band 3 tyrosine phosphorylation

作者：Kang Le, Xunde Wang, Jonathan M. Chu, Maureen Lundt, Yuan Yee Lee, Anna Conrey, Ingrid Frey, Silvia Giannini, Penelope A. Kosinski, John M. Hausman, Philip S. Low, Neal Jeffries, Sanjay A. Desai, Swee Lay Thein · 发表于：Blood Advances · 年份：2024 · DOI：10.1182/bloodadvances.2024013504 · 被引用次数：16 · 研究领域：Erythrocyte Function and Pathophysiology、Hemoglobinopathies and Related Disorders、Calcium signaling and nucleotide metabolism

ABSTRACT: In a phase 1 study (NCT04000165), we established proof of concept for activating pyruvate kinase (PK) in sickle cell disease (SCD) as a viable antisickling therapy. AG-348 (mitapivat), a PK activator, increased adenosine triphosphate (ATP) and decreased 2,3-diphosphoglycerate levels while patients were on treatment, in line with the mechanism of the drug. We noted that the increased hemoglobin (Hb) persisted for 4 weeks after stopping AG-348 until the end of study (EOS). Here, we investigated the pathways modulated by activating PK that may contribute to the improved red blood cell (RBC) survival after AG-348 cessation. We evaluated frozen whole blood samples taken at multiple time points from patients in the phase 1 study, from which RBC ghosts were isolated and analyzed by western blotting for tyrosine phosphorylation of band 3 (Tyr-p-bd3), ankyrin-1, and intact (active) protein tyrosine phosphatase 1B (PTP1B) levels. We observed a significant dose-dependent decrease in mean Tyr-p-bd3 from baseline in the patients, accompanied by an increase in the levels of membrane-associated ankyrin-1 and intact PTP1B, all of which returned to near baseline by EOS. Because PTP1B is cleaved (inactivated) by intracellular Ca2+-dependent calpain, we next measured the effect of AG-348 on ATP production and calpain activity and the plasma membrane Ca2+ ATPase pump-mediated efflux kinetics in HbAA and HbSS erythrocytes. AG-348 treatment increased ATP levels, decreased calpain activit...