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Activation of SCD RBC adhesion by oxidative stress
known to undergo skeletal and membrane remodeling during maturation.33,34
Several studies have investigated the dynamics and rhe- ology of SS RBC under flow conditions using microfluidic devices. Alapan et al. assessed sickle RBC adhesion in fibronectin-coated microfluidic chips and observed signif- icantly greater numbers of adhered non-deformable than deformable RBC.35 Our study extends these findings by comparing the adhesion of deformable and non- deformable RBC within both the young and mature pop- ulations. As a matter of fact, fibronectin is the substrate of integrin α4β1 that is expressed only in very young reticu- locytes, restricting the analysis to a very small subpopula- tion of RBC, while the laminin receptor Lu/BCAM is expressed on RBC at all the maturation stages.
We show that oxidation can activate RBC adhesion to laminin by inducing post-translational modifications of Lu/BCAM that modify its distribution at the cell surface generating aggregates with high binding potential to laminin. This mechanism targets and abolishes Lu/BCAM cis-interaction with GPC at the cell surface32 and seems to be specific for Lu/BCAM and to target GPC primarily, as another IgSF member, i.e., LW/ICAM-4, was not impacted by in vitro oxidation and did not show altered distribution at the surface of SS RBCs. Abnormal RBC adhesion to laminin was reported in several pathologies15 with two triggering mechanisms including Lu/BCAM phosphoryla- tion17,24,27 and Lu/BCAM dissociation from the spectrin- based skeleton.25,26,36 Here, the oxidation-driven mecha- nism seems to be at the origin of increased adhesion of HD RBC in the absence of Lu/BCAM phosphorylation and seems in line with the high adhesion levels of HD RBC in the absence of responsiveness to cAMP inducers.28 Moreover, although abnormal actin oxidation has been reported in irreversibly sickled cells affecting cytoskeletal dynamics,37 in our study oxidation and the subsequent loss of interaction with GPC do not alter Lu/BCAM bind- ing to the skeleton as we did not see a difference in Lu/BCAM Triton extractability between in vitro oxidized and non-oxidized AA RBC (data not shown). This is sup- ported by the unchanged mobility of Lu/BCAM at the sur- face of neuraminidase-treated RBC as measured by fluo- rescence recovery after photobleaching assay.32 As a mat- ter of fact, oxidation may impact the interactions of mem- brane proteins with the skeleton as it was reported for Band 338-41 but here Lu/BCAM activation seems to be trig- gered by modifications at the extracellular rather than the intracellular side.
During erythrocyte lifespan, sialic acid levels gradually
decrease.42-44 The sialic acid levels were lower on HD than on LD RBC for all blood samples, corroborating the find- ing that sialoglycoproteins are enriched in membranes of young reticulocytes45 and indicating an accelerated aging- like phenotype between the two stages despite the very short lifespan of RBC in SCD.46,47 This suggests increased damage of the RBC surface in SCD, targeting the glycoca- lyx, probably mediated by high levels of oxidative stress effectors in the plasma including free hemoglobin.48
Our study extends our recent findings on the novel mechanism activating Lu/BCAM-mediated RBC adhesion and suggests that this mechanism could be triggered by oxidative stress activating the adhesion of dense sickle RBC in the absence of Lu/BCAM phosphorylation. It would be interesting to determine the impact of anti-oxi- dant drugs on this specific mechanism and to evaluate their potential of reducing or attenuating RBC adhesion in SCD.
Disclosures
No conflicts of interests to disclose.
Contributions
MALI and SDL conducted experiments, acquired and ana- lyzed data and wrote the manuscript; VB provided blood samples and discussed data; SC, SEH, AF, MD and SA conducted experiments, analyzed data and edited the manuscript; CLVK and FRL discussed data and edited the manuscript; OF, BLP, TK and RvB conducted experiments, discussed data and edited the manuscript; WEN designed research, analyzed data and wrote the manuscript.
Acknowledgments
We thank Mr Mickaël Marin, Mr Harvey Nagy and Dr Jean-Philippe Semblat for technical support.
Funding
The work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), the Institut National de la Transfusion Sanguine, the Laboratory of Excellence GR-Ex, reference ANR-11-LABX-0051, and the Laboratory of Excellence LaSIPS (ANR-10-LABX-0040-Lasips). The labex GR-Ex is funded by the IdEx program “Investissements d’avenir” of the French National Research Agency, reference ANR-18-IDEX-0001. MALI and SEH were funded by the Ministère de l’Enseignement Supérieur et de la Recherche (Ecole Doctorale BioSPC); they received financial support from: Club du Globule Rouge et du Fer and Société Française d’Hématologie.
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