Vox, PKR activator, and GBT021601 reduced [Ca²⁺]_total levels and improved HbSS RBC occlusion by Piezo1 inhibition and enhancing residual PMCA activity under normoxic and hypoxic conditions. (A) In HbAA RBCs, Vox and PKR activator did not change the basal intracellular [Ca²⁺]_total levels (n = 5; ns, P > .05; Wilcoxon test). (B) Under normoxia, [Ca²⁺]_total levels were significantly reduced in HbSS RBCs treated with Vox (HbSS vs HbSS + Vox; n = 21), PKR activator (HbSS vs HbSS + PKR; n = 21), or GBT021601 (HbSS vs HbSS + GBT021601; n = 5), ∗∗∗P < .001, t test for all. Relatively greater reductions were observed under hypoxia (HbSS + Vox, n = 6; HbSS + PKR, n = 6; HbSS + GBT021601, n = 6; ∗∗∗P < .001; Wilcoxon test for all). (C) Piezo1 blockade with GsMTx4 reduced intracellular [Ca²⁺]_total levels under normoxia and hypoxia, but did not differ significantly with cotreatment with Vox, PKR activator, and GBT021601 (∗∗∗P < .001; n = 5; Wilcoxon test for all). (D) PMCA inhibition with vanadate increases [Ca²⁺]_total levels under both normoxia and hypoxia; cotreatment with Vox, PKR activator, and GBT021601 significantly lowered the [Ca²⁺]_total levels despite PMCA blockade (∗∗∗P < .001; n = 5; Wilcoxon test for all). (E) Similar to [Ca²⁺]_total levels, OI under GsMTx4 alone was reduced compared with untreated HbSS RBCs (∗P > .05; n = 5; Wilcoxon test), and did not differ significantly with cotreatment with Vox, PKR activator, and GBT021601 (ns, P > .05; n = 7; t test for all). (F) Vanadate increased OI under both normoxia (n = 5; ∗P < .05; Wilcoxon test) and hypoxia (n = 5; ∗∗∗P < .001; Wilcoxon test). Cotreatment with Vox, PKR activator, or GBT021601 significantly lowered the OI (n = 5; ∗P < .05, ∗∗P < .01, ∗P < .05; Wilcoxon test). Error bars are presented as mean ± SEM. Gs, Grammostola spatulata mechanotoxin 4.