Corrigendum: Variability of extracellular vesicle release during storage of red blood cell concentrates is associated with differential membrane alterations, including loss of cholesterol-enriched domains.

[This corrects the article DOI: 10.3389/fphys.2023.1205493.].

Variability of extracellular vesicle release during storage of red blood cell concentrates is associated with differential membrane alterations, including loss of cholesterol-enriched domains.

Transfusion of red blood cell concentrates is the most common medical procedure to treat anaemia. However, their storage is associated with development of storage lesions, including the release of extracellular vesicles. These vesicles affect in vivo viability and functionality of transfused red blood cells and appear responsible for adverse post-transfusional complications. However, the biogenesis and release mechanisms are not fully understood. We here addressed this issue by comparing the kinetics and extents of extracellular vesicle release as well as red blood cell metabolic, oxidative and membrane alterations upon storage in 38 concentrates. We showed that extracellular vesicle abundance increased exponentially during storage. The 38 concentrates contained on average 7 × 1012 extracellular vesicles at 6 weeks (w) but displayed a ∼40-fold variability. These concentrates were subsequently classified into 3 cohorts based on their vesiculation rate. The variability in extracellular vesicle release was not associated with a differential red blood cell ATP content or with increased oxidative stress (in the form of reactive oxygen species, methaemoglobin and band3 integrity) but rather with red blood cell membrane modifications, i.e., cytoskeleton membrane occupancy, lateral heterogeneity in lipid domains and transversal asymmetry. Indeed, no changes were noticed in the low vesiculation group until 6w while the medium and the high vesiculation groups exhibited a decrease in spectrin membrane occupancy between 3 and 6w and an increase of sphingomyelin-enriched domain abundance from 5w and of phosphatidylserine surface exposure from 8w. Moreover, each vesiculation group showed a decrease of cholesterol-enriched domains associated with a cholesterol content increase in extracellular vesicles but at different storage time points. This observation suggested that cholesterol-enriched domains could represent a starting point for vesiculation. Altogether, our data reveal for the first time that the differential extent of extracellular vesicle release in red blood cell concentrates did not simply result from preparation method, storage conditions or technical issues but was linked to membrane alterations.

Piezo1 Regulation Involves Lipid Domains and the Cytoskeleton and Is Favored by the Stomatocyte-Discocyte-Echinocyte Transformation.

Piezo1 is a mechanosensitive ion channel required for various biological processes, but its regulation remains poorly understood. Here, we used erythrocytes to address this question since they display Piezo1 clusters, a strong and dynamic cytoskeleton and three types of submicrometric lipid domains, respectively enriched in cholesterol, GM1 ganglioside/cholesterol and sphingomyelin/cholesterol. We revealed that Piezo1 clusters were present in both the rim and the dimple erythrocyte regions. Upon Piezo1 chemical activation by Yoda1, the Piezo1 cluster proportion mainly increased in the dimple area. This increase was accompanied by Ca2+ influx and a rise in echinocytes, in GM1/cholesterol-enriched domains in the dimple and in cholesterol-enriched domains in the rim. Conversely, the effects of Piezo1 activation were abrogated upon membrane cholesterol depletion. Furthermore, upon Piezo1-independent Ca2+ influx, the above changes were not observed. In healthy donors with a high echinocyte proportion, Ca2+ influx, lipid domains and Piezo1 fluorescence were high even at resting state, whereas the cytoskeleton membrane occupancy was lower. Accordingly, upon decreases in cytoskeleton membrane occupancy and stiffness in erythrocytes from patients with hereditary spherocytosis, Piezo1 fluorescence was increased. Altogether, we showed that Piezo1 was differentially controlled by lipid domains and the cytoskeleton and was favored by the stomatocyte-discocyte-echinocyte transformation.

Aberrant Membrane Composition and Biophysical Properties Impair Erythrocyte Morphology and Functionality in Elliptocytosis.

Red blood cell (RBC) deformability is altered in inherited RBC disorders but the mechanism behind this is poorly understood. Here, we explored the molecular, biophysical, morphological, and functional consequences of α-spectrin mutations in a patient with hereditary elliptocytosis (pEl) almost exclusively expressing the Pro260 variant of SPTA1 and her mother (pElm), heterozygous for this mutation. At the molecular level, the pEI RBC proteome was globally preserved but spectrin density at cell edges was increased. Decreased phosphatidylserine vs. increased lysophosphatidylserine species, and enhanced lipid peroxidation, methemoglobin, and plasma acid sphingomyelinase (aSMase) activity were observed. At the biophysical level, although membrane transversal asymmetry was preserved, curvature at RBC edges and rigidity were increased. Lipid domains were altered for membrane:cytoskeleton anchorage, cholesterol content and response to Ca2+ exchange stimulation. At the morphological and functional levels, pEl RBCs exhibited reduced size and circularity, increased fragility and impaired membrane Ca2+ exchanges. The contribution of increased membrane curvature to the pEl phenotype was shown by mechanistic experiments in healthy RBCs upon lysophosphatidylserine membrane insertion. The role of lipid domain defects was proved by cholesterol depletion and aSMase inhibition in pEl. The data indicate that aberrant membrane content and biophysical properties alter pEl RBC morphology and functionality.

Interplay Between Plasma Membrane Lipid Alteration, Oxidative Stress and Calcium-Based Mechanism for Extracellular Vesicle Biogenesis From Erythrocytes During Blood Storage.

The shedding of extracellular vesicles (EVs) from the red blood cell (RBC) surface is observed during senescence in vivo and RBC storage in vitro. Two main models for EV shedding, respectively based on calcium rise and oxidative stress, have been proposed in the literature but the role of the plasma membrane lipid composition and properties is not understood. Using blood in K+/EDTA tubes stored for up to 4 weeks at 4°C as a relevant RBC vesiculation model, we showed here that the RBC plasma membrane lipid composition, organization in domains and biophysical properties were progressively modified during storage and contributed to the RBC vesiculation. First, the membrane content in cholesterol and linoleic acid decreased whereas lipid peroxidation and spectrin:membrane occupancy increased, all compatible with higher membrane rigidity. Second, phosphatidylserine surface exposure showed a first rapid rise due to membrane cholesterol decrease, followed by a second calcium-dependent increase. Third, lipid domains mainly enriched in GM1 or sphingomyelin strongly increased from the 1st week while those mainly enriched in cholesterol or ceramide decreased during the 1st and 4th week, respectively. Fourth, the plasmatic acid sphingomyelinase activity considerably increased upon storage following the sphingomyelin-enriched domain rise and potentially inducing the loss of ceramide-enriched domains. Fifth, in support of the shedding of cholesterol- and ceramide-enriched domains from the RBC surface, the number of cholesterol-enriched domains lost and the abundance of EVs released during the 1st week perfectly matched. Moreover, RBC-derived EVs were enriched in ceramide at the 4th week but depleted in sphingomyelin. Then, using K+/EDTA tubes supplemented with glucose to longer preserve the ATP content, we better defined the sequence of events. Altogether, we showed that EV shedding from lipid domains only represents part of the global vesiculation mechanistics, for which we propose four successive events (cholesterol domain decrease, oxidative stress, sphingomyelin/sphingomyelinase/ceramide/calcium alteration and phosphatidylserine exposure).