Oxidative stress and hemoglobin-c denaturation drive hemoglobin SC pathophysiology and can be ameliorated by antioxidants and hydroxyurea Free

Background: Compound heterozygous Hemoglobin SC (HbSC) disease, historically considered a mild form of sickle cell disease, is now recognized to cause substantial clinical morbidity and early mortality. HbSC red blood cells (RBC) exhibit distinct pathophysiology, characterized by cellular xerocytosis, blood hyperviscosity, and a high incidence of retinopathy. We performed the first randomized trial of hydroxyurea in HbSC disease, which showed significant reduction in vaso-occlusive pain events despite modest induction in fetal hemoglobin (HbF) levels (NEJM Evidence, Dec. 2024). Further, using our newly developed murine model of HbSC disease, we showed that hydroxyurea improves RBC deformability in HbSC mice via HbF-independent mechanisms: by reducing oxidative stress and improving membrane deformability (Blood, July 2025). These findings suggest that oxidative stress, rather than sickling and dehydration alone, may be a central driver of HbSC pathophysiology. We hypothesize that hemoglobin C (HbC) is likely more prone to oxidative damage than HbS, resulting in high hemichrome formation and hemoglobin denaturation which damage RBC membranes.

Methods: We utilized our recently characterized CRISPR-Cas9–engineered humanized HbSC mouse model, along with HbSS and HbCC littermate controls and compared the effect of oxidative stress on rheological parameters. RBC membrane deformability was assessed using shear-stress ektacytometry, sickling kinetics via oxygen-gradient ektacytometry (Oxygen-scan), and hydration via osmotic-gradient ektacytometry. Hemichrome formation, which results from clustering of band3 protein in RBC membranes with denatured hemoglobin, was assessed by Heinz body staining. Intracellular reactive oxygen species (ROS) levels were measured using DCFDA and flow cytometry. We used quercetin, a potent antioxidant, to study the redox contribution to RBC membrane damage. Quercetin (0.2 mg/kg/day) was supplemented in chow for 8 weeks. HbF levels and % F-cells were measured by HPLC and flow cytometry. Human HbSC blood samples were similarly analyzed.

Results: Heinz bodies were significantly more prevalent in murine HbCC RBC, followed by HbSC RBC, and lowest in HbSS RBC (P<.01), despite significantly higher intracellular ROS levels in HbSS RBC (P<.05), suggesting increased vulnerability of HbC to oxidant damage. Quercetin significantly reduced Heinz body formation: While control HbCC, HbSC, and HbSS mice had 71%, 56% and 48% Heinz body positive RBC, quercetin reduced these to 38%, 41% and 29%, respectively (P<.01). This change was associated with improvement in membrane deformability, which was greatest in HbCC RBC (P<0.5), and a trend toward reduced deformability in HbSC RBC. Oxygen-scan showed significant improvement in RBC elongation index max in all three groups on quercetin, and reduced point-of-sickling in HbSS and HbSC RBC. Improved RBC deformability correlated with the Heinz body burden. Quercetin did not affect hydration indices, RBC count, or hemoglobin level in either group, indicating that denatured HbC-induced membrane damage does not affect RBC volume regulation. We also analyzed Heinz body formation in 2 HbSC patients receiving hydroxyurea and 2 untreated patients. A total of 20-25 high-power fields/blood smear/patient were quantified (technical replicates). Heinz body–positive RBCs were significantly reduced in hydroxyurea-treated HbSC patients (10.1 ± 1.3%) compared to untreated patients (15.2 ± 1.3%; P = 0.003). These findings document that HbSC patients on hydroxyurea with HbF as low as 3% have reduced numbers of Heinz bodies, mirroring the findings in mice.

Conclusion: Overall, these results support a causal role for HbC-mediated oxidative membrane damage in impairing RBC rheology. Notably, hydroxyurea treatment, even with modest HbF induction, can mitigate oxidative injury and restore critical aspects of RBC membrane health, an effect similarly achieved through antioxidant supplementation in HbSC mice. Our findings highlight the central role of oxidative damage, particularly Heinz body accumulation, in the pathophysiology of HbSC disease. Quercetin treatment and hydroxyurea both reduced Heinz bodies and improved membrane mechanics, independent of hydration status and unrelated to HbF levels. These results establish oxidative injury as a third distinct and targetable contributor to HbSC pathophysiology, besides HbS and dehydration.