Figure 1.
PK activity, thermostability, and cellular metabolism is compromised in RBCs from patients with SCD and correlates with markers of clinical severity. (A) PK/HK ratio, as a means to evaluate PK activity in the presence of high number of reticulocytes, is decreased in RBCs from patients with SCD (red, N = 22) compared with healthy controls (Ctr; gray, N = 10), indicating a deficiency of PK. (B-C) The deficiency of PK is accompanied by a decrease in PK thermostability in RBCs of patients with SCD (N = 22) compared with controls (N = 10) (B), as well as a decrease in ATP/2,3-DPG ratio in patients with SCD (N = 14) compared with healthy controls (N = 10) (C). (D) PK thermostability correlates with PoS (pO2 at which RBCs start to sickle) in patients with SCD (N = 22), a novel potential biomarker of clinical severity determined by oxygen gradient ektacytometry.20 (E-F) Ex vivo treatment of purified SCD RBCs with mitapivat increases PK activity and ATP levels. PK activation and ATP response (%) were assessed from purified RBCs of patients with SCD (green, N = 7) and 14 healthy controls (Ctr; purple, N = 14) after 24 hours’ incubation in the presence (light green and light purple) or absence (dark green and dark purple) of 10 μM mitapivat. An increase in PK activity is seen in patients with SCD (mean increase, 129%; range, 113% to 148%) and controls (mean increase, 144%; range, 111% to 188%) (E). This increase is accompanied by an increase in ATP response in patients with SCD (mean increase, 131%; range, 123% to 139%) and controls (mean increase, 133%; range, 101% to 155%) (F). (G-L) Ex vivo treatment of buffy coat–depleted whole blood with mitapivat restores PK thermostability, increases ATP/2,3-DPG ratio, decreases p50 and PoS, and increases membrane health. PK thermostability (% residual PK activity), after 22 hours’ incubation in the presence of 50 μM (light green) and 100 μM (dark blue) mitapivat, is restored to 79% (100 μM) compared with vehicle controls (32%; dimethyl sulfoxide [DMSO] control; light gray, N = 11) (G), causing a 25% increase in ATP/2,3-DPG ratio (N = 11) (H) and a 5% decrease in p50 (N = 11) (I). (J) Representative oxygen gradient ektacytometry curve shows improved PoS and EImax after incubation in the presence of 100 μM mitapivat (dark blue) compared with vehicle control (light gray). (K-L) PoS and EImax of 9 patients with SCD improve with 9% and 2%, respectively, after 22 hours’ incubation in the presence of 100 μM (dark blue) mitapivat compared with vehicle controls (light gray). Error bars represent standard deviation. ****P < .0001; ***P < .001; **P < .01.

PK activity, thermostability, and cellular metabolism is compromised in RBCs from patients with SCD and correlates with markers of clinical severity. (A) PK/HK ratio, as a means to evaluate PK activity in the presence of high number of reticulocytes, is decreased in RBCs from patients with SCD (red, N = 22) compared with healthy controls (Ctr; gray, N = 10), indicating a deficiency of PK. (B-C) The deficiency of PK is accompanied by a decrease in PK thermostability in RBCs of patients with SCD (N = 22) compared with controls (N = 10) (B), as well as a decrease in ATP/2,3-DPG ratio in patients with SCD (N = 14) compared with healthy controls (N = 10) (C). (D) PK thermostability correlates with PoS (pO2 at which RBCs start to sickle) in patients with SCD (N = 22), a novel potential biomarker of clinical severity determined by oxygen gradient ektacytometry.20  (E-F) Ex vivo treatment of purified SCD RBCs with mitapivat increases PK activity and ATP levels. PK activation and ATP response (%) were assessed from purified RBCs of patients with SCD (green, N = 7) and 14 healthy controls (Ctr; purple, N = 14) after 24 hours’ incubation in the presence (light green and light purple) or absence (dark green and dark purple) of 10 μM mitapivat. An increase in PK activity is seen in patients with SCD (mean increase, 129%; range, 113% to 148%) and controls (mean increase, 144%; range, 111% to 188%) (E). This increase is accompanied by an increase in ATP response in patients with SCD (mean increase, 131%; range, 123% to 139%) and controls (mean increase, 133%; range, 101% to 155%) (F). (G-L) Ex vivo treatment of buffy coat–depleted whole blood with mitapivat restores PK thermostability, increases ATP/2,3-DPG ratio, decreases p50 and PoS, and increases membrane health. PK thermostability (% residual PK activity), after 22 hours’ incubation in the presence of 50 μM (light green) and 100 μM (dark blue) mitapivat, is restored to 79% (100 μM) compared with vehicle controls (32%; dimethyl sulfoxide [DMSO] control; light gray, N = 11) (G), causing a 25% increase in ATP/2,3-DPG ratio (N = 11) (H) and a 5% decrease in p50 (N = 11) (I). (J) Representative oxygen gradient ektacytometry curve shows improved PoS and EImax after incubation in the presence of 100 μM mitapivat (dark blue) compared with vehicle control (light gray). (K-L) PoS and EImax of 9 patients with SCD improve with 9% and 2%, respectively, after 22 hours’ incubation in the presence of 100 μM (dark blue) mitapivat compared with vehicle controls (light gray). Error bars represent standard deviation. ****P < .0001; ***P < .001; **P < .01.

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