Band 3 phosphorylation induces irreversible alterations of stored red blood cells
Authors: Slim Azouzi, Marc Romana, Nobuto Arashiki, Yuichi Takakuwa, Wassim El Nemer, Thierry Peyrard, Yves Colin, Pascal Amireault, Caroline Le Van Kim
Journal: American Journal of Hematology (2018)

Red blood cells (RBCs) transfusion is a common practice in the global patient's medical care. RBC concentrate is stored for up to 42 days after blood collection. Stored RBCs undergo abundant cellular and structural changes affecting their functional integrity and survival after transfusion and may be harmful for the blood recipient.1 Several randomized controlled trials compared transfusion of "fresher" versus "older" red cells but a correlation between the age of transfused RBCs and morbidity/mortality was not found to be conclusive.2-4 Storage lesions of RBCs leading to the progressive loss of biochemical, morphological, and mechanical properties are mostly due to irreversible changes in the membrane. These storage lesions are likely to affect RBC post-transfusion survival and increase the risks of adverse reactions in the recipients, especially in an inflammatory context.4 The underlying mechanisms leading to RBC membrane changes and vesiculation during storage are poorly known. We focused on the Band 3 anion exchanger, the most abundant protein of the RBC membrane, that plays a major role in membrane stability and deformability by linking the lipid bilayer to the skeleton via ankyrin R. Band 3 oligomerization plays a key role in the clearance of altered and old RBCs by forming senescence antigens recognized by naturally occurring autoantibodies (Nabs). Considering the absence of reliable marker of lesion storage, we aim to elucidate the molecular mechanisms leading to irreversible erythrocyte alterations during storage and to identify a relevant marker of "old" and damaged RBCs potentially harmful for patients. To investigate the oligomeric state of Band 3 in the RBC membrane, we performed the specific Eosin-5-maleimide (EMA) test used for the diagnosis of hereditary spherocytosis (Supporting Information Methods). Fluorescence intensities of EMA-labeled RBCs were measured for six blood bags at days 3, 14, 21, 28, 35, and 42 of storage (Figure 1A). We observed a progressive decrease of EMA labeling during storage with a maximum of 15% at day 42 (P < .001). This effect could be due to a decrease in Band 3 expression and/or to an increase in Band 3 oligomerization and mobility known to be due at least to Tyr phosphorylation. Then, we analyzed the phosphorylation state of Band 3 in RBC ghosts by Western blot analysis using an anti-phosphoTyr antibody. We observed an important increase in Band 3 phosphorylation between day 3 and day 42 of storage (Figure 1B, upper panel). Interestingly, we did not observe any change in the expression level of Band 3 after incubation of the same membrane with an antibody specific for the intracellular domain of Band 3 (Figure 1B, bottom panel). Similar results were obtained by flow cytometry analysis using an antibody against the extracellular domain of Band 3 (data not shown). Taken together, our findings strongly suggested that the decrease in EMA fluorescence was not due to the decrease of Band 3 expression but rather to an enhanced mobility following Band 3 phosphorylation. To further strengthen this hypothesis, we investigated the relationship between Band 3 phosphorylation and mobility by measuring the fluorescence of EMA-labeled RBCs treated with increasing concentrations of the phosphatase inhibitor O-vanadate. As expected, Band 3 tyrosine phosphorylation was induced and enhanced with increasing amounts of O-vanadate (Supporting Information Figure S1A) and this was associated with a decrease in EMA fluorescence (Supporting Information Figure S1B). Thus, it is likely that Band 3 phosphorylation during RBC storage induces its detachment from membrane skeleton, most probably by disruption from ankyrin R, increasing its mobility. To date, hyper-phosphorylated Band 3 was found to be associated with glucose-6-phosphate dehydrogenase deficiency (Ferru et al., 2011), hemoglobinopathies,5 and senescence of RBCs,6 all pathological conditions characterized by increased RBC oxidative stress and Band 3 clustering. A, EMA-labeled RBCs from six blood units sampled on days 3, 14, 21, 35, and 42. EMA fluorescence was normalized using frozen RBCs from a healthy donor, thawed one day before the experiment to avoid fluorescence variability (n = 6, mean ± SEM). B, Band 3 phosphorylation during RBC storage. Western blotting of erythrocyte membrane proteins at 3, 14, 21, 28, 35, and 42 days of storage using anti-phosphoTyr and anti-Band 3. p55 was used as loading control. Lane 1: Fresh RBCs; Lane 2: Fresh RBCs treated with 2 mM O-vanadate. C, Increase in Band 3 clustering using flow cytometry and specific antibody against the clustered form of Band 3 (n = 3). D, MPs increase during RBC storage (n = 6, mean ± SEM) To determine the impact of increased Band 3 oligomerization on its distribution and structure in the membrane, we took advantage of a unique antibody that specifically and selectively recognizes the clustered form of Band 3.7 We measured the binding capacity of this antibody to RBCs during storage by flow cytometry. A 14% increase in RBCs displaying Band 3 clusters was observed from day 3 to day 42 of storage (Figure 1C). This is of particular importance in the context of transfusion since Band 3 clustering was claimed to be the primary mechanism in the removal of RBCs from the circulation. This Band 3 alteration likely limits post-transfusion survival of RBCs. To explore the consequence of increased Band 3 phosphorylation and clustering during storage, we measured the release of microparticules (MPs), a well-known marker of irreversible lesions in the membrane in the same conditions of storage. Using flow cytometry and anti-Band 3 antibody, we found an important increase in the level of erythroid -positive MPs that all expressed Band 3 from day 28, with a 10-fold increase between day 28 and day 42 (Figure 1D). This strong elevation was associated with the increase of Band 3 phosphorylation. We hypothesized that it could represent the primary event leading to the formation of the "small cells" a sub-population of altered RBCs that accumulate during storage and are expected to be removed from the circulation in the hours following transfusion.8 To date, none of the randomized trials found a clinically significant outcome difference when comparing transfusions of red cells stored for short (1–2 weeks) or long (∼2–6 weeks) times. Interestingly, we observed that the appearance of the clustered form of Band 3 from 35 days of storage was correlated with the membrane microvesiculation. As a result, we propose that day 35 of storage represents a critical step in the red cells aging. Finally, we postulate that RBCs get "very old" from day 35 of storage and studies are needed to examine the risks associated with the transfusion of red cells stored for 35–42 days in particular in chronically transfused patients as sickle cell anemia patients. The authors thank Eliane Vera, Sirandou Tounkara, and Dominique Gien at CNRGS for providing the frozen RBC controls and EFS for providing RBC units. The authors thank Catia Pereira for help with Western blot experiments. This work was supported by the Institut National de la Santé et de la Recherche Médicale (Inserm), the Institut National de la Transfusion Sanguine and the Laboratory of Excellence GR-Ex, reference ANR-11-LABX-0051. GR-Ex is funded by the program "Investissements d'avenir" of the French National Research Agency, reference ANR-11-IDEX-0005-02. Nothing to report. SA, PA, YC, and CLVK designed the research; SA and MR performed the research; SA, YC, and CLVK analyzed the data; SA, YC, and CLVK wrote the paper; TP and WEN reviewed the manuscript with critical reading and very helpful comments; NA and YT provided the anti-Band 3 clustering antibody and useful technical advices. Additional Supporting Information may be found online in the supporting information tab for this article. Supporting Information Supporting Information Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.