Key Points• Adhesion of sRBCs is synergistically regulated by hypoxia and low NO bioavailability.• P-selectin and p38 kinase pathways play a role in the synergistic adhesion of sRBCs.The molecular mechanisms by which nitric oxide (NO) bioavailability modulates the clinical expression of sickle cell disease (SCD) remain elusive. We investigated the effect of hypoxia and NO bioavailability on sickle red blood cell (sRBC) adhesion using mice deficient for endothelial NO synthase (eNOS) because their NO metabolite levels are similar to those of SCD mice but without hypoxemia. Whereas sRBC adhesion to endothelial cells in eNOS-deficient mice was synergistically upregulated at the onset of hypoxia, leukocyte adhesion was unaffected. Restoring NO metabolite levels to physiological levels markedly reduced sRBC adhesion to levels seen under normoxia. These results indicate that sRBC adherence to endothelial cells increases in response to hypoxia prior to leukocyte adherence, and that low NO bioavailability synergistically upregulates sRBC adhesion under hypoxia. Although multiple adhesion molecules mediate sRBC adhesion, we found a central role for P-selectin in sRBC adhesion. Hypoxia and low NO bioavailability upregulated P-selectin expression in endothelial cells in an additive manner through p38 kinase pathways. These results demonstrate novel cellular and signaling mechanisms that regulate sRBC adhesion under hypoxia and low NO bioavailability. Importantly, these findings point us toward new molecular targets to inhibit cell adhesion in SCD. (Blood. 2014; 123(12):1917-1926 IntroductionAlthough sickle cell disease (SCD) arises from a single mutation of b-globin, clinical severity varies significantly.1 A well-known clinical modifier is fetal hemoglobin, which is expressed at variable levels in patients and inhibits the polymerization of sickle hemoglobin. 2 The frequency of vaso-occlusive crisis was also used to evaluate disease severity of SCD patients.3 Vaso-occlusive crisis is likely triggered by multiple physiological insults such as infection and cytokine-mediated inflammation that may cause tissue hypoxia, and mediated by multistep cell adhesion mechanisms involving sickle red blood cells (sRBCs). 4 The degree of sRBC adhesion to endothelial cells was shown to correlate with SCD severity. 1 Nitric oxide (NO) bioavailability is another physiological factor capable of modulating clinical severity.5 NO bioavailability decreases during vaso-occlusive crisis, primarily because of NO scavenging by cell-free hemoglobin 6 as well as other mechanisms including arginase, 7 reactive oxygen species, 8 and NO synthases.9 NO metabolite levels vary among patients even at steady state and may inversely correlate with the frequency of vaso-occlusive crisis. 10 We showed that inhalation of low-dose NO permitted SCD mice to survive hypoxic stress. 11 The gender difference in the clinical severity of SCD may result in part from distinct levels of NO bioavailability.12 Thus, heterogeneity of the clinical expression in SCD appears ...

Breathing NO produced a rapid, protective effect to severe hypoxic stress in SAD mice. There appears to be a required loading period between NO breathing and its beneficial effect during hypoxic stress, possibly because of the total amount of NO delivered to SAD hemoglobin, blood cell components, and endothelium. NO breathing may be beneficial as a therapeutic intervention in SCD.

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.

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