Effects of ex vivo PK activation treatment on HX RBCs. (A) RBCs of patients with HX (PIEZO1-HX, n = 6; KCNN4-HX, n = 2) were treated ex vivo with the PK activator tebapivat (2 μM) overnight, which led to an increase in PK activity as reflected by the PK:HK ratio. (B) PK thermostability showed improvements upon ex vivo treatment with tebapivat. PK thermostability after 60 minutes incubation is reported. (C-D) The effect of increased PK activity on ATP:2,3-DPG ratio and hemoglobin-oxygen affinity. (E) Effect of PK activation on intracellular calcium levels. (F-G) To determine the effect of enhanced glycolysis on RBC hydration, both the percentage of hypochromic cells (F) and the O_hyper values (G) were evaluated. (H) Along with the effect on O_hyper, the effect on EI_max was also determined. (I-J) Individual representative osmotic gradient ektacytometry curves of PIEZO1-HX (I) and KCNN4-HX (J) show that no improvement in hydration is observed in patients with PIEZO1, whereas patients with KCNN4-HX show improved O_hyper values. ∗P < .05. Square symbols indicate splenectomized patients.