Effect of PK activators on Tyr-p-bd3. (A) In vivo effect of AG-348 on Tyr-p-bd3, PTP1B, and ankyrin-1. The levels of Tyr-p-bd3, intact PTP1B, and membrane-associated ankyrin-1 were quantified by western blotting in frozen blood samples from 15 patients (Tyr-p-bd3 and PTP1B) or 11 patients (ankyrin-1) treated twice daily with 5-, 20-, 50-, and 100-mg AG-348. ∗P ˂ .05; ∗∗P ˂ .01; ∗∗∗P ˂ .001. (B-E) Ex vivo effect of AG-348 on Tyr-p-bd3. HbSS RBCs were treated with DMSO (Con) or different concentrations (1, 3, 10, and 30 μM) of AG-348 in DMSO for 4 hours (B-C) or 30-μM AG-348 for different times (1, 2, 4, and 6 hours) (D-E). The level of Tyr-p-bd3 was analyzed by western blotting and quantified by densitometry analysis. The mean reduction on Tyr-p-bd3 of RBC treated with 1-, 3-, 10-, and 30-μM AG-348 were 1%, 22%, 35%, and 50%, respectively, and with 30-μM AG-348 for 2, 4, and 6 hours were 6%, 28%, and 59%, respectively. ∗P ˂ .05; ∗∗P ˂ .01. (F-I) Ex vivo effect of AG-946 on Tyr-p-bd3. HbSS RBCs were treated with DMSO (Con) or different concentrations (1, 3, 10, and 30 μM) of AG-946 in DMSO for 4 hours (F-G) or 30-μM AG-946 for different times (1, 2, 4, and 6 hours) and the level of Tyr-p-bd3 analyzed by western blotting and quantified by densitometry analysis (H-I). The mean reduction on Tyr-p-bd3 of RBC treated with 1-, 3-, 10-, and 30-μM AG-946 were 3%, 24%, 41%, and 56%, respectively, and with 30-μM AG-946 for 4 and 6 hours were 29% and 49%, respectively. ∗P ˂ .05; ∗∗ P ˂ .01. Three biological replicates (blood samples from 3 different HbSS donors) were used in all ex vivo assays, except in panels H and I, which had 4 biological replicates. ∗P ˂ .05; ∗∗P ˂ .01.