Modification of HbS by MetAP2 inhibition decreases hypoxia-triggered sickling and increases oxygen binding in whole blood RBCs. Townes SCD mice were dosed intraperitoneally for 3 days with the indicated covalent MetAP2 inhibitor or vehicle control, and whole blood was collected to measure HbS modification, hypoxia-triggered RBC sickling, and oxygen affinity (p50). (A) Whole blood from SCD mice dosed with CKD-732 (10 mg/kg, daily) to achieve 50% total HbS modification was evaluated for sickling under hypoxia by SIFCA,²⁹  performed under normoxia (21% oxygen) or hypoxia (4% oxygen) for 2 to 3 hours prior to fixing and quantitating abnormal sickled cells on the Amnis ImageStream Imaging Flow Cytometer. (B) Sickling was evaluated on a PerkinElmer Operetta CLS High-Content Analysis System. Both methods confirm a decrease in hypoxia-driven sickling in whole blood samples from MetAP2 inhibitor–treated mice. Similar results were obtained in ≥3 independent studies for both methods. (C) SCD mice dosed with the indicated covalent MetAP2 inhibitors (1 mg/kg, twice daily) achieved similar high levels of HbS modification, as assessed by loss of the parent HbS peak on a Bio-Rad D-10 Hemoglobin Testing System, consistent with LC-MS analysis of lysed RBCs showing 55% total α-globin modification and 46.5% β-globin modification. (D) Oxygen affinity (p50) determined from RBC OECs in whole blood collected in EDTA. The p50 measured for MetAP2 inhibitor–treated blood samples is significantly lower, and oxygen affinity increased by 3.43 ± 0.24 torr (mean ± standard deviation) or 12.2% compared with vehicle controls. Similar results were obtained in 3 independent studies. ****P < .001, ***P < .005, 2-tailed Student t test. ns, not significant.