PIEZO1 inhibitor GsMTx-4 breaks PIEZO1-TMEM16F coupling and prevents excessive PS exposure in HX RBCs. (A) GsMTx-4 dose-dependently inhibits Yoda1–induced Ca²⁺ influx in HX001 RBCs. Error bars represent ± SEM from 3 replicates. (B-C) Representative flow cytometry data of GsMTx-4 inhibition on Yoda1 (2 μM)-induced PS exposure in HX001 (B) and HX002 (C) RBCs. CF488-AnV was used as a fluorescence PS marker. Each concentration of GsMTx4 was repeated in 3 independent experiments. (D) Dose-response curves of GsMTx-4 inhibition on Ca²⁺ increase (solid circle) and PS exposure (open square) in the RBCs from HX001 (black) and HX002 (red) patients. All signals were normalized to 0 GsMTx-4 condition and fitted with the Hill equation (see “Methods”). (E) IC₅₀ of GsMTx-4 on Yoda1-induced Ca²⁺ influx and PS exposure in HX001 and HX002 RBCs. Unpaired 2-sided Student t test. ∗∗P < .01, n = 3 to 4 repeats.