Nitric oxide reduces sickle hemoglobin polymerization: Potential role of nitric oxide-induced charge alteration in depolymerization

Abstract

We previously demonstrated that inhaling nitric oxide (NO) increases the oxygen affinity of sickle red blood cells (RBCs) in patients with sickle cell disease (SCD). Our recent studies found that NO lowered the P50 values of sickle hemoglobin (HbS) hemolysates but did not increase methemoglobin (metHb) levels, supporting the role of NO, but not metHb, in the oxygen affinity of HbS. Here we examine the mechanism by which NO increases HbS oxygen affinity. Because anti-sickling agents increase sickle RBC oxygen affinity, we first determined whether NO exhibits anti-sickling properties. The viscosity of HbS hemolysates, measured by falling ball assays, increased upon deoxygenation; NO treatment reduced the increment. Multiphoton microscopic analyses showed smaller HbS polymers in deoxygenated sickle RBCs and HbS hemolysates exposed to NO. These results suggest that NO inhibits HbS polymer formation and has anti-sickling properties. Furthermore, we found that HbS treated with NO exhibits an isoelectric point similar to that of HbA, suggesting that NO alters the electric charge of HbS. NO–HbS adducts had the same elution time as HbA upon high performance liquid chromatography analysis. This study demonstrates that NO may disrupt HbS polymers by abolishing the excess positive charge of HbS, resulting in increased oxygen affinity.

A systematic review of known mechanisms of hydroxyurea-induced fetal hemoglobin for treatment of sickle cell disease

“…Recently, a rabbit erythrocyte model was used to demonstrate that HU regulates the production of NO by the enzyme endothelial nitric oxide synthase (eNOS) [114], and further showing that the released NO induces blood flow-induced ATP, which is a known mediator of vasodilation through endothelial cell purinergic receptor binding [115–118] and subsequent release of endothelial NO [119–121]. The improved blood flow results from the NO-induced increase in oxygen affinity of sickle erythrocytes in SCD patients because NO exhibits anti-sickling properties and inhibits HbS polymerization [85,122]. NO may be released by endothelial cells and/or erythrocytes, establishing a feedback loop with flow-derived ATP release from erythrocytes and provide rationale for some of the immediate reported benefits of HU treatment (improved blood flow and attenuated sickling events in SCD patients) [123].…”

Imaging flow cytometry for automated detection of hypoxia‐induced erythrocyte shape change in sickle cell disease

“…After the incubation, a fixative solution is added and the number of sickled cells is manually counted under a light microscope. In several preclinical and early phase pharmacologic trials in SCD, this assay has been used as an outcome variable to predict clinical effect . Although such a sickling assay is useful, it has several disadvantages: there are questions about its sensitivity and variability and it lacks automated methods to quantitate the percentage of sickled cells from large samples.…”

Visualizing red blood cell sickling and the effects of inhibition of sphingosine kinase 1 using soft X-ray tomography

“…The morphology of sickled RBCs has been studied extensively using light microscopy (Christoph et al, 2005;Coletta et al, 1988;Horiuchi et al, 1988;Pierre, 2002;van Beers et al, 2014;Zhang et al, 2014) and scanning electron microscopy (SEM; Horiuchi et al, 1988;Kaul and Xue, 1991;Pierre, 2002) to gather information on cell morphology. In fact, preclinical trials and treatment-testing experiments routinely use lightmicroscopy-derived percentages of sickled cells as predictors of outcome (Abdulmalik et al, 2005;Chang et al, 1983a,b;Ikuta et al, 2011;Zhang et al, 2014). However, both light microscopy and SEM provide only two-dimensional data and, in the case of light microscopy, at relatively low resolution.…”