Red blood cell nitric oxide synthase activation is increased in patients with sickle cell hemoglobin <scp>C</scp> disease
Authors: Anaïs Mozar, Marijke Grau, Yann Lamarre, Linda Weyel, Frank Suhr, Bianca Collins, Marie‐Dominique Hardy‐Dessources, Marc Romana, Nathalie Lemonne, Maryse Etienne‐Julan, Wilhelm Bloch, Philippe Connes
Journal: American Journal of Hematology (2014)

To the Editor: Red blood cells (RBC) are able to produce nitric oxide (NO) via an endothelial-like NO synthase (eNOS) termed RBC-NOS 1. In healthy subjects it was shown that the NO produced by RBC-NOS directly modulates RBC deformability through S-nitrosylation of the cytoskeletal proteins, α- and β-spectrins 2 and seems to play a role in vascular physiology as well 3. Grau et al 4 recently reported that RBC-NOS was highly activated in patients with sickle cell anemia (SCA) compared to healthy individuals. While many studies have focused their attention on the pathophysiology and the RBC rheological abnormalities of SCA, very few have been devoted to sickle cell-hemoglobin C disease (SC). We set out to assess RBC-NOS content, its activation signaling pathway, NO concentration, S-nitrosylation of RBC cytoskeleton proteins, and RBC deformability in this population and in healthy individuals (AA). Twenty subjects (12 SC and 8 AA) gave written informed consent to participate in this study, which was approved by the Regional Ethics Committee (DIRC Sud/Ouest Bordeaux/DOM, registration number: 2009-A00211-56/project SAPOTILLE/HYPOSNAD). Parameters including basic hematological parameters, RBC deformability, immunohistochemical staining of total RBC-NOS, RBC-NOS phosphorylation sites serine 1177, serine 116 and threonine 495, nitrotyrosine and phospho-Akt-Ser473, plasma and RBC nitrite levels, and S-nitrosylation of α- and β- spectrins were determined as previously described 4. The results showed decreased hemoglobin concentration, hematocrit, and mean cell volume and increased mean corpuscular hemoglobin concentration in SC compared to AA. In SC, level of activated Akt (phosphorylation at Ser473) was significantly higher than in AA. Accordingly, RBC-NOS activation, reflected by phosphorylation of its Ser1177 residue, was also significantly increased in SC. Phosphorylation of RBC-NOS-Ser116 and RBC-NOS-Thr495—two inhibitory phosphorylation sites—was barely detectable and not different between the two groups. Total RBC-NOS protein content did not reveal any significant difference between AA and SC. RBC nitrite concentration, reflecting RBC-NOS dependent NO production, was significantly higher in SC compared to AA while no significant difference in RBC nitrotyrosine and plasma nitrite levels were observed between the two groups. Higher S-nitrosylation of α-spectrin and β-spectrin was found in SC compared to AA, indicating increased binding of NO produced by RBC-NOS to these proteins in SC. RBC deformability was significantly decreased in SC compared to AA (Table 1). Higher Akt and RBC-NOS activation, RBC nitrite content, and S-nitrosylation of cytoskeletal proteins in SC strongly support higher ability of RBC from SC patients to produce NO than AA RBC, as it has been recently demonstrated in SCA patients, too 4. However, in contrast to SCA patients, RBC nitrotyrosine level was not greater in SC than in AA, suggesting no major difference in the RBC oxidative/nitrositative stress between these two groups. This finding is in line with recent findings by Hierso et al. 5 who reported similar RBC glutathione (GSH) level in SC and AA, and only a very mild elevation of RBC reactive oxygen species content in SC. Grau et al. 2 demonstrated that increased S-nitrosylation of α- and β-spectrins obtained by RBC-NOS activation improved RBC deformability in healthy individuals. The present study shows that SC RBC exhibit higher levels of S-nitrosylated α- and β-spectrins as a consequence of higher RBC-NOS activation, but RBC deformability was still decreased in comparison with AA. The reduction of RBC deformability in SC is caused by the presence of both hemoglobin C and hemoglobin S in RBCs, which results in severe cellular dehydration, a reduction of the mean cell volume and a rise in the mean corpuscular hemoglobin concentration. These changes are at the origin of the moderate RBC deformability reduction in SC individuals 6. NO has been assumed to modulate oxygen affinity of sickle cell erythrocytes which would attenuate sickling, and thereby, could partly limit further reduction of RBC deformability in sickle cell patients but further studies are needed to test this hypothesis in SC. We suspect that addition of extracellular NO or enhancement of RBC NO production could positively influence the rheology of SC RBCs and might reduce the risks for several complications 6. AM, MG, YL, FS, MDHD, MR, WB and PC designed the research. AM, MG, YL, LW, FS, BC, NL and PC performed the experiments. AM, MG, FS, MDHD, MR, NL, WB and PC analyzed and interpreted the data. AM, MG, YL, LW, FS, BC, MDHD, MR, NL, MEJ, WB and PC wrote the article. The authors would like to express their sincere gratitude to all the patients who accepted to participate in the study. This study was supported in part by the German Research Foundation. A.M. is funded by the Region Guadeloupe. Anaïs Mozar,1,2 Marijke Grau,3,4 Yann Lamarre,1,2 Linda Weyel,3 Frank Suhr,3,4 Bianca Collins,3 Marie-Dominique Hardy-Dessources,1,2 Marc Romana,1,2 Nathalie Lemonne,5 Maryse Etienne-Julan,1,2,5 Wilhelm Bloch,3,4 and Philippe Connes1,2,6* 1 UMR Inserm U1134, Université des Antilles et de la Guyane, Pointe-à-Pitre, Guadeloupe, France 2 Laboratoire d'Excellence du Globule Rouge (LABEX GR-Ex), PRES Sorbonne, Paris, France 3 Department of Molecular and Cellular Sport Medicine, Institute of Cardiovascular Research and Sport Medicine, German Sport University Cologne, Cologne, Germany 4 The German Research Center of Elite Sport, German Sport University Cologne, Cologne, Germany 5 Unité Transversale de la Drépanocytose, Centre Hospitalier Universitaire de Pointe-à-Pitre, Pointe-à-Pitre, Guadeloupe, France;6 Institut Universitaire de France (IUF), Paris, France