Voxelotor and Red Blood Cell Pyruvate Kinase Activator Enhance Sickle Erythrocyte Deformability By Calcium Efflux

Introduction: Reduced red blood cell (RBC) deformability due to the polymerization of sickle hemoglobin (HbS) is central to the pathophysiology of sickle cell disease (SCD) [1]. Novel therapies such as voxelotor and RBC pyruvate kinase (PKR) activators increase hemoglobin's oxygen (O₂) affinity, thereby theoretically reducing HbS polymerization and hemolysis [2, 3]. However, the mechanisms by which these therapies affect RBC deformability are not yet fully understood. We hypothesize that voxelotor and PKR activators modify intracellular processes critical for maintaining membrane integrity, thus protecting against the loss of RBC deformability.

Methods: We used the OcclusionChip [4-6] to investigate the effect of voxelotor (S8540) and PKR activator (HY-19702) on the RBC deformability, rigidity, and overall occlusion capacity in both SCD (HbSS) and healthy (HbAA) blood samples under normoxic and hypoxic conditions. Blood samples were centrifuged at 500 g for 5 min. Plasma and buffy coats were removed. Isolated, washed RBCs were re-suspended in PBS+ Glucose (5mM, PBS+G) at 20% hematocrit and mixed with voxelotor to a final concentration of 600 µM (in DMSO) (HbSS+vox or HbAA+vox) or PKR activator at 10 mM (in DMSO) (HbSS+ PKR or HbAA+ PKR) and incubated at 37 ^°C for 6 hours. For controls, HbSS- or HbAA-containing RBCs at 20% hematocrit were mixed with PBS+G containing 0.5% v/v DMSO and incubated at 37 ^°C for 6 hours. We then simultaneously measured intracellular levels of 2,3-DPG, ATP, Ca²⁺I, and the degree of phosphorylation of the membrane protein Band 3 in all samples under normoxia. Some RBCs were pretreated with the Piezo 1 inhibitor Grammostola Spatulata mechanotoxin 4 (GsMTx4) (P1205) before subjecting them to PKR and voxelotor treatment.

Results and Discussion: We found that occlusion indices (OIs) for HbAA samples under normoxia were significantly lower, i.e., more normal, than OI values for HbSS samples (p = 0.001). When HbSS samples were treated with either voxelotor or PKR activators, OI values decreased, by approximately 38.63% for HbSS+vox, and 39.40 % for HbSS+PKR, when compared to HbSS vehicle control (p = 0.001 for both). ATP levels of untreated HbSS RBCs did not differ relative to HbSS+vox or to HbSS+PKR treated RBCs; (p= 0.47, p= 0.12, respectively). 2,3 DPG levels from PKR-treated but not Voxelotor-treated HbSS RBCs were reduced compared to the untreated HbSS RBCs (p= 0.001 and p= 0.18, respectively). Ca²⁺ levels of untreated HbAA RBCs did not differ from Voxelotor- or PKR-exposed HbAA RBCs (p= 0.67, p = 0.85, respectively). However, intracellular Ca²⁺ levels in HbSS+vox or HbSS+PKR treated RBCs were significantly reduced compared with control HbSS RBCs (p = 0.01, p = 0.05, respectively), but this effect was lost following Piezo1 inhibition by GsMTx4 (p=0.65, p=0.34, respectively). Band 3 phosphorylation levels for untreated HbAA did not differ from treated HbAA+ vox or HbAA+ PKR cells (p = 0.59 and p = 0.37 respectively, However, Band 3 tyrosine phosphorylation levels in HbSS+ vox or HbSS+ PKR treated RBCs were significantly reduced compared with the control vehicle HbSS RBCs (p = 0.001, p = 0.0015).

Conclusion: Based on our findings, PKR or voxelotor acted similarly on HbSS RBCs in vitro, by increasing RBC deformability in HbSS in vitro, whilst ATP levels were unchanged relative to controls. 2,3-DPG levels in HbSS RBCs were considerably reduced by PKR activator treatment but not voxelotor. Intracellular Ca²⁺ levels that were high in HbSS, but not HbAA, RBCs, were lowered by PKR activators or Vox, unless the cells were pre-treted with a Piezo1 blocking agent. Ca²⁺ efflux is associated with decreased Band 3 tyrosine phosphorylation for both voxelotor and PKR activator treatments in HbSS RBCs, but not in HbAA RBCs. These findings suggest that Ca²⁺ efflux is a common pathway through which voxeletor and PKR activators affect Band 3 phosphorylation and RBC deformability; these pathways can be manipulated pharmacologically and understanding this may be relevant to multi-drug therapies.

References

1. Kato, G.J., et al., Nat Rev Dis Primers, 2018. 4: p. 18010.

2. Schroeder, P., et al., J Pharmacol Exp Ther, 2022. 380(3): p. 210-219.

3. Sekyonda, Z., et al., Blood, 2023. 142: p. 1114.

4. Man, Y., et al., Frontiers in Physiology, 2022. 13: p. 954106.

5. Man, Y., et al., Microcirculation, 2021. 28(2): p. e12662.

6. Man, Y., et al., Lab Chip, 2020. 20(12): p. 2086-2099.