Effects of pyruvate kinase activators on red blood cell properties in sickle cell disease.

Pyruvate kinase activators (PKA) are a new class of pharmacological agents that enhance red blood cell pyruvate kinase (PK) activity and glycolytic flux resulting in increased intracellular adenosine triphosphate (ATP) and decreased 2,3-diphosphoglycerate (2,3-DPG).1, 2 In patients with sickle cell disease (SCD), decreased PK activity and thermostability result in increased 2,3-DPG3 and decreased ATP/2,3-DPG ratio, promoting the polymerization of haemoglobin S (HbS) and RBC sickling. Previous ex-vivo studies demonstrated that mitapivat (AG-348) could represent a therapeutic option for patients with SCD by restoring PK activity and thermostability. Incubation of RBCs from patients with SCD with mitapivat has been shown to decrease 2,3-DPG content which increased haemoglobin-oxygen affinity and RBC deformability and reduced RBC sickling. The increase in cellular ATP levels improved RBC integrity and cellular hydration.4 A recent phase 1 and phase 2 open-label study in patients with SCD confirmed that mitapivat decreased 2,3-DPG, increased the haemoglobin-oxygen affinity and decreased sickling tendency.5, 6 The authors also reported decreased haemolysis and increased haemoglobin concentration, which was sustained at 4 weeks after stopping mitapivat, suggesting an improvement in RBC survival. However, no study has investigated the effects of PKA on RBC senescence. Moreover, another PKA, AG946, has been recently developed, yet it is unknown whether this molecule also improves the properties of RBCs in SCD. The present study investigated the ex-vivo effects of mitapivat and AG946 on the properties of RBCs in patients with SCD. In addition, the effects of the two PKA were also tested in the presence of cumene hydroperoxide to mimic elevated cellular oxidative stress. Blood samples (EDTA) from non-transfused adults and children with SCD (HbSS genotype) were collected and centrifuged (1000 g, 10 min at 20°C) to remove the plasma and buffy coat. RBCs were washed in PBS 1x and then resuspended in PBS (haematocrit of 20%). RBCs were then incubated for 4 h at 37°C with mitapivat (50 μM), AG946 (5 μM) or the vehicle. Several parameters reflecting the haemoglobin-oxygen affinity and RBC properties were measured. The Hemox-Analyzer (TCS, Medical Products Division) has been used to determine the oxyhemoglobin dissociation curve (ODC) and p50 values.7 RBC deformability and sickling tendency were determined by oxygen gradient ektacytometry (Oxygenscan, LORRCA; Mechatronics).8, 9 Oxygenscan method allows the determination of the maximal RBC deformability (i.e., in normoxia; EImax), the minimum RBC deformability (i.e., in hypoxia; EImin) and the Point of Sickling (PoS), which corresponds to the pO2 at which RBCs start to sickle. Several RBC senescence markers were measured by flow cytometry (BD Accuri C6)10: percentage of RBCs with externalized phosphatidylserine (Annexin V-FITC; 1:200 dilution; Beckman Coulter), intracellular reactive oxygen species (2′,7′–dichlorofluorescin diacetate; Sigma-Aldrich), intra-cellular calcium (Ca2+; Fluo3/AM probe; Biotium) and membrane CD47 (anti-CD47-PE; 1:33 dilution; Miltenyi Biotec) levels. For the second set of experiments, RBCs were incubated with PKAs for 4 h, and then, cumene hydroperoxide (0.4 mM) was added to the RBCs suspension for 15 min (37°C) to mimic a situation of increased oxidative stress. The same parameters were measured. The study was conducted in accordance with the guidelines set by the Declaration of Helsinki and was approved by the Regional Ethics Committees (L14-127). For the experiments performed without cumene hydroperoxide, 23 patients were included for haemoglobin-oxygen affinity and RBC properties analyses (26 ± 13 years; HbF = 12.5 ± 7.9%; HbS = 82.0 ± 10.1%, Hb = 8.8 ± 1.3 g/dL, reticulocyte count = 230 ± 116 109/L, 7 under hydroxyurea) and 17 others were included for the RBC senescence markers measurements (23 ± 11 years; HbF = 15.0 ± 12.0%; HbS = 79.0 ± 13.0%, Hb = 9.1 ± 1.8 g/dL, 195 ± 109 109/L, 13 under hydroxyurea). Both mitapivat and AG946 decreased the p50 (Figure 1A) and PoS (Figure 1B) compared to vehicle. AG946 increased EImin (Figure 1C) and EImax (Figure 1D). RBC Ca2+ was slightly increased with mitapivat compared to the vehicle condition (Figure 1E). No effect of the two PKA was observed for the other RBC senescence markers (Figure 1F–H). Nevertheless, further studies are needed to test the in-vivo effects of these two PKA on RBC senescence as Kalfa et al11 recently reported a decrease in externalized PS in SCD patients receiving etavopivat. For the experiments with cumene hydroperoxide, 13 patients were included for haemoglobin-oxygen affinity and RBC properties analyses (25 ± 11 years; HbF = 13.2 ± 9.9%; HbS = 83.0 ± 9.7%, Hb = 8.8 ± 1.2 g/dL, 241 ± 71 109/L, 5 under hydroxyurea) and 11 others were included for the RBC senescence markers measurements (25 ± 10 years; HbF = 16.0 ± 6.3%; HbS = 80.3 ± 6.3%, Hb = 8.5 ± 1.6 g/dL, 155 ± 79 109/L, 9 under hydroxyurea). We first evaluated the effects of cumene hydroperoxide alone on RBCs from 8 SCD patients independently of PKA effects (Figure 2A–H). Incubation for 15 min at 37°C with cumene hydroperoxide caused a decrease of p50 (Figure 2A), which may partly be explained by the formation of methemoglobin.12 Methemoglobin has been shown to affect the equilibrium between oxygen and haemoglobin, as does carbon monoxide.12 In addition, cumene hydroperoxide decreased EImax (Figure 2D), and increased the percentage of RBC with externalized PS (Figure 2E) and RBC ROS (Figure 2G) level. Then, the effects of cumene hydroperoxide were tested on PKA-treated RBCs. Both PKAs decreased p50 (Figure 2I) and PoS (Figure 2J) values. AG946 tended to increase EImin (Figure 2K). The experiments on RBC senescence markers were performed only with AG946 and showed a slight trend towards an increase of RBC Ca2+ (Figure 2M) but no effect on the other parameters (Figure 2N–P). Our results show that AG946, like mitapivat, increases the haemoglobin-oxygen affinity and reduces RBC sickling in patients with SCD ex-vivo. This effect was also observed with both molecules in the presence of increased oxidative stress. There was a non-significant increase of intra-cellular Ca2+ with AG946, which could be due to the higher PK activity driven by the PKA molecules being calcium-dependent enzymes.13 However, it is worth noting that the increase of intracellular Ca2+ had no effect on RBC senescence. Overall, these data support the continued evaluation of PKA in prospective clinical trials in patients with SCD to reduce vaso-occlusive crisis frequency and/or to decrease the severity of anaemia. Recent results obtained in healthy individuals (phase 1 clinical trial) showed a favourable safety profile of AG946.14 Philippe Joly, Elie Nader, Céline Renoux and Philippe Connes designed the study. Philippe Joly, Elie Nader, Flora Ketels, Camille Boisson and Romain Carin performed the experiments. Philippe Joly, Elie Nader, Flora Ketels and Philippe Connes analysed and interpreted the data. Alexandra Gauthier, Solene Poutrel and Yves Bertrand included the patients. Philippe Connes wrote the manuscript. Philippe Joly, Elie Nader, Flora Ketels, Camille Boisson, Romain Carin, Céline Renoux, Alexandra Gauthier, Solene Poutrel and Yves Bertrand reviewed and corrected the manuscript. All authors approved the final version. This research has been selected for oral communication at the 64th American Society of Hematology (ASH) Annual Meeting and Exposition (December 2022). Agios Pharmaceuticals: Research Funding. P.C. is consultant for Global Blood Therapeutics (GBT). The remaining authors declare no competing financial interests. The study was conducted in accordance with the guidelines set by the Declaration of Helsinki and was approved by the Regional Ethics Committees (L14-127). Data are available upon reasonable request to the corresponding author.