Effects of Pyruvate Kinase Activators on Red Blood Cell Rheology, Sickling and Senescence in Sickle Cell Disease

Introduction: Pyruvate Kinase Activators (PKA) are a new class of pharmacological agents that enhance pyruvate kinase (PKR) activity and glycolytic flux in red blood cells (RBC), resulting in increased intracellular adenosine triphosphate (ATP) and decreased 2,3-diphosphoglycerate (2,3-DPG) contents. In patients with sickle cell disease (SCD), decreased PKR activity and thermostability result in decreased ATP/2,3-DPG ratio, which promotes HbS polymerization and sickling. Previous ex-vivo studies have shown that AG-348 (mitapivat) could represent a therapeutic option for those patients by restoring PK activity and thermostability and by increasing RBC deformability and the ATP/2,3-DPG ratio, thus reducing the propensity of RBC to sickle under deoxygenation (1). Recently, a new PKA molecule has been developed (AG-946) but its effects on RBC deformability and sickling have not been fully studied (2). Moreover, the effects of the two molecules in a context of enhanced oxidative stress - a situation frequently encountered in SCD - have not been investigated.

Materials and Methods: Blood samples from non-transfused adults and children with SCD (3 to 53 years-old) were collected in EDTA tubes. HbF levels ranged from 1.3 to 47.1%. Washed RBC were resuspended in phosphate buffer saline after buffy-coat removal and incubated for 4 hours at 37°C, with or without 0.4 mM of cumene hydroperoxide (CH), a molecule that causes a considerable oxidative stress to the cells.

Three different incubation conditions (AG-348 at 50 µM, AG-946 at 5 µM and a vehicle control) were systematically compared for the following ex-vivo experiments on RBC: (i) determination of the partial pressure of oxygen at which the hemoglobin is 50% saturated with oxygen (p50) with the Hemox® device; (ii) RBC deformability at normoxia (EImax and SS_1/2 parameters), in hypoxia (EImin) and the partial pressure of oxygen at which sickling starts during deoxygenation (PoS; Point of Sickling) by ektacytometry; (iii) analyses of RBC senescence markers (i.e. Phosphatidylserine externalization, reactive oxygen species, intra-cellular calcium (Ca²⁺) and membrane CD47) by flow cytometry. One-way ANOVA with the Newman-Keuls method was performed for multiple comparisons and the significant level was set at 0.05.

Results: A significant decrease of the p50 and PoS values was observed for both AG-molecules, with and without CH, compared to the control condition (Fig. 1-A). Without CH, both AG-molecules decreased SS_1/2 (i.e., the shear stress for half-maximal deformation of RBCs) and AG-946 increased EImin and EImax. With CH, the two molecules still decreased the PoS compared to the control condition and a trend through an increase of EImin was observed with the AG-946 molecule. A slight increase of intra-cellular Ca²⁺ with the two AG-molecules (trend for AG-946) was observed in absence of oxidative stress (Fig. 2). The other RBC senescence markers were not modified, regardless of the incubation condition.

Conclusion: Our results show that both PKAs increase the oxygen affinity and reduce the sickling propensity of RBCs from patients with SCD in vitro, in the presence or absence of enhanced oxidative stress. The observed small increase of intracellular Ca²⁺ upon drug treatment may be a result of increased overall cellular metabolic activity, as a consequence of PKR activation. Interestingly, the increased intracellular Ca²⁺ is not accompanied by enhanced RBC senescence. Overall, these data support testing PKA in prospective clinical trials for the reduction of vaso-occlusive crisis in patients with SCD.

Bibliography: (1): Decreased activity and stability of pyruvate kinase in sickle cell disease: a novel target for mitapivat therapy. Rab MAE, Bos J, van Oirschot BA, van Straaten S, Kosinski PA, Chubukov V, Kim H, Mangus H, Schutgens REG, Pasterkamp G, Dang L, Kung C, van Beers EJ, van Wijk R.Blood. 2021 PMID: 33690814.

(2): Pharmacodynamic effects of AG-946, a highly potent next-generation activator of pyruvate kinase, in ex vivo treatment of red blood cells from sickle cell disease patients. Rab, MAE, Van Dijk MJ, Van Oirschot BA, Gerrits J Kosinski PA, Kung C, Jans JJM, Van Beers EJ, Dang L and Van Wijk R. 2021 American Society of Hematology Congress, Abstract 113.

Joly:Agios Pharmaceuticals: Research Funding. Nader:Global Blood Therapeutics: Consultancy, Research Funding. Connes:Global Blood Therapeutics: Consultancy, Research Funding; Agios Pharmaceuticals: Research Funding.