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

Abstract

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).

Microvesicle Formation Induced by Oxidative Stress in Human Erythrocytes

“…Two major mechanisms are described for EV formation including an increase in intracellular calcium concentration and oxidative stress (OS)-induced Hb oxidation [ 16 ]. Ca 2+ -activated protease calpain is involved in vesiculation by binding and degrading band 3, band 4.1, and ankyrin, which lead to alteration of cell deformability [ 9 , 16 , 17 , 18 , 19 ]. OS triggers membrane and Hb oxidation, clustering, and disruption of band 3-cytoskeleton anchorage, and all these alterations lead to EV formation [ 7 , 17 , 20 , 21 , 22 , 23 ].…”

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

“…Before experiments, blood was diluted 10-fold in glucose- and HEPES-containing medium (Invitrogen, Carlsbad, CA, USA). RBCs were then collected, washed twice by centrifugation at 200× g for 2 min and resuspended, as in [ 13 ]. The number of RBCs used for experiments were as follows: (i) imaging, 12.5 × 10 6 ; (ii) phosphatidylserine (PS) surface exposure by flow cytometry, 0.5 × 10 6 ; (iii) cholesterol assay, 37.5 × 10 6 ; (iv) lipidomics, 250 × 10 6 ; (v) Ca 2+ and ROS contents, 15 × 10 6 ; (vi) intracellular ATP, 5 × 10 6 ; and (vii) hemoglobin release, 12.5 × 10 6 .…”

“…GM1-enriched domain abundance increases upon PIEZO1 activation while sphingomyelin-enriched domain abundance increases with Ca 2+ efflux [ 10 , 12 ]. A fourth type of domain, mainly enriched in ceramide, has been recently identified at the inner leaflet [ 13 ].…”

“…None of the patient’s relatives were consulted for symptoms nor were they diagnosed with elliptocytosis, but her mother (mother of pEl, or pElm) nevertheless appeared mildly affected and her RBCs were used for comparison. Blood from healthy donors upon storage at 4 °C, known to be associated with RBC vesiculation, biochemical and morphology changes [ 13 , 20 ], was also used as internal control.…”

Microvesicle Formation Induced by Oxidative Stress in Human Erythrocytes

“…Two major mechanisms are described for EV formation including increase of intracellular calcium concentration and oxidative stress (OS)-induced Hb oxidation [16]. Ca 2+ -activated protease calpain is involved in vesiculation by binding and degrading band 3, band 4.1 and ankyrin which lead to alteration of cell deformability [9,[16][17][18][19]. OS triggers membrane and Hb oxidation, clustering and disruption of band 3 -cytoskeleton anchorage, and all these alterations lead to EV formation [17,[20][21][22][23][24].…”