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

  • Blood Adv. 2024 Nov 12;8(21):5653-5662. doi: 10.1182/bloodadvances.2024013504.
Kang Le  1 ,  Xunde Wang  1 ,  Jonathan Chu  2 ,  Maureen Lundt  1 ,  Yuan Yee Lee  1 ,  Anna Conrey  1 ,  Ingrid Frey  1 ,  Silvia Giannini  3 ,  Penelope A Kosinski  3 ,  John M Hausman  4  5 ,  Philip S Low  4  5 ,  Neal Jeffries  6 ,  Sanjay A Desai  2 ,  Swee Lay Thein  1
Affiliations
  • 1. Laboratory of Sickle Cell Genetics and Pathophysiology, Sickle Cell Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD.
  • 2. Apicomplexan Molecular Physiology Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD.
  • 3. Agios Pharmaceuticals, Inc, Cambridge, MA.
  • 4. Department of Chemistry, Purdue University, West Lafayette, IN.
  • 5. Institute for Drug Discovery, Purdue University, West Lafayette, IN.
  • 6. Office of Biostatistics Research, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD.
PMID: 39265169 DOI: 10.1182/bloodadvances.2024013504
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 activity, and increased Ca2+ efflux. Altogether, our data indicate that ATP increase is a key mechanism underlying the increase in Hemoglobin levels upon PK activation in SCD. This trial was registered at www.clinicaltrials.gov as #NCT04000165.

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