Inhibition of PIEZO1 prevents echinocytosis and hemolysis in HX001 RBCs. (A) Representative images of RBCs from a HD before and after centrifugation. (B-C) Representative images of the RBCs from HX001 with various concentrations of GsMTx-4 (B) and Benz (C) before and after centrifugation. (D-E) Effect of GsMTx-4 (D) and Benz (E) on spin-induced RBC echinocytosis (see “Methods” for details). (F) Representative photos of 2 μM Yoda1(Y1)-induced hemolysis in HX RBCs with and without 5 μM GsMTx-4. 0 Yoda1 (left) and H₂O (right) serve as negative and positive controls, respectively. (G) Representative images of 2 μM Yoda1-induced hemolysis with and without 20 μM Benz at different time points as indicated. (H) Time course of 2 μM Yoda1(Y1)-induced with and without 5 μM spider toxin GsMTx-4 in HX001 RBCs. Results were normalized to the water-induced fully lysed group. (I) Time course of 2 μM Yoda1 (Y1)-induced hemolysis with and without 20 μM Benz in HX001 RBCs. Results were normalized to the water-induced fully lysed group. (J) Yoda1-induced RBCs hemolysis percentage at 120 minutes with and without GsMTx-4 and Benz. Unpaired 2-sided Student t test, ∗∗∗∗P < .0001, n = 3 to 6 repeats for each condition. (K) Partially inhibiting PIEZO1 can effectively disrupt PIEZO1-TMEM16F coupling in HX RBCs and prevent HX-associated complications.