Effect of AG-348 on calpain activity, ATP production, and Ca²⁺ efflux. (A-B) PTP1B in HbAA vs HbSS RBCs. The levels of membrane-associated intact PTP1B in RBCs from 6 pairs of healthy controls (AA1-6) and HbSS patients (SS1-6) were analyzed by western blotting and quantified by densitometry. The relative levels of PTP1B to actin in AA1-6 RBCs were 0.42, 0.84, 0.81, 0.63, 0.39, and 0.75, respectively, with an average of 0.64; and in SS1-6 RBCs were 0.28, 0.63, 0.37, 0.21, 0.51, and 0.20, respectively, with an average of 0.37. ∗P ˂ .05. (C) Effect of AG-348 on calpain activity. HbSS RBCs were treated with DMSO (Con) or different concentrations (1, 3, 10, and 30 μM) of AG-348 in DMSO overnight, and the activity of calpain was measured using Calpain Activity Assay Kit. Mean reductions in calpain activity of RBCs treated with 1-, 3-, 10-, and 30-μM AG-348 were 9%, 15%, 24%, and 26%, respectively. ∗P ˂ .05. (D-E) Effect of AG-348 on ATP production. HbSS RBCs were treated with DMSO (Con) or 30-μM AG-348 in DMSO for different times (2, 4, 6, and 8 hours) or different concentrations (1, 3, 10, and 30 μM) of AG-348 in DMSO for 8 hours; ATP level was analyzed using CellTiter-Glo 2.0 Cell Viability Assay kit. The mean increase in ATP levels of RBCs treated with 30-μM AG-348 for 2, 4, 6, and 8 hours were 2%, 5%, 11%, and 21%, respectively, and with 1-, 3-, 10-, and 30-μM AG-348 for 8 hours were 2%, 5%, 14%, and 26%, respectively. Three biological replicates of HbSS donors were used in the study here. ∗P ˂ .05. (F-H) Effect of AG-348 on Ca²⁺ efflux. (F) Ca²⁺ efflux kinetics were measured using extracellular Fluo-8. Symbols represent mean ± standard error of the mean (SEM) from triplicate measurements from single HbAA and HbSS donors (dark blue and red circles, respectively). Notice the slower efflux with the SS donor. (G) Mean ± SEM efflux halftimes from 4 donors, measured similar to panel F. The slower efflux observed from SS erythrocytes did not reach statistical significance. (H) Mean ± SEM efflux halftimes for indicated cells after a 6-hour treatment with 30-μM AG-348 or matched DMSO control (blue and red bars, respectively, for each genotype). RBCs from 4 pairs of ethnic-matched healthy controls (HbAA) and HbSS patients were used in each assay, and the results represent a mean from the 4 separate assays.