Ex vivo activation of pyruvate kinase improves red blood cell metabolism and hydration in hereditary spherocytosis

• The activity of the key glycolytic enzyme PK is relatively decreased in HS RBCs. • Ex vivo treatment with PK activators leads to increased levels of adenosine triphosphate levels and an improved hydration state. Hereditary spherocytosis (HS) is a hereditary hemolytic anemia with limited treatment options. A relative decrease in the activity of pyruvate kinase (PK), an essential glycolytic enzyme in red blood cells (RBCs), has been described in patients with HS. Owing to their dependence on glycolysis, PK activation therapy could therefore potentially improve red cell health in HS. This study aimed to evaluate PK activity in HS RBCs and to investigate the effect of 2 PK activators (mitapivat and tebapivat). Blood samples from 18 patients with non-transfusion-dependent HS (splenectomized and nonsplenectomized) were analyzed. Our results confirmed impaired glycolysis in HS at baseline, indicated by a relatively decreased PK activity (PK-to-hexokinase ratio, 7.6 in HS vs 11.4 in controls). PK activity (increase >50%) and adenosine triphosphate levels (increase >44%) improved upon ex vivo treatment with mitapivat and tebapivat. Metabolomics showed various metabolic alterations upon treatment. Furthermore, the hydration state of the cells improved (O hyper increase >2.1%). No improvements were found in deformability, intracellular calcium, and cellular adhesion to laminin. When comparing splenectomized patients with nonsplenectomized patients, we found that PK thermostability in nonsplenectomized patients was decreased more than in splenectomized patients. After this, PK activator therapy improved PK thermostability to a greater extent in RBCs from splenectomized patients, which seems to relate to the degree of reticulocytosis. Overall, we demonstrated that PK activation improves the metabolic and cellular properties of HS RBCs ex vivo, supporting the rationale for further evaluation of PK activation in HS.