Vesiculation of Red Blood Cells in the Blood Bank: A Multi-Omics Approach towards Identification of Causes and Consequences

Abstract

Microvesicle generation is an integral part of the aging process of red blood cells in vivo and in vitro. Extensive vesiculation impairs function and survival of red blood cells after transfusion, and microvesicles contribute to transfusion reactions. The triggers and mechanisms of microvesicle generation are largely unknown. In this study, we combined morphological, immunochemical, proteomic, lipidomic, and metabolomic analyses to obtain an integrated understanding of the mechanisms underlying microvesicle generation during the storage of red blood cell concentrates. Our data indicate that changes in membrane organization, triggered by altered protein conformation, constitute the main mechanism of vesiculation, and precede changes in lipid organization. The resulting selective accumulation of membrane components in microvesicles is accompanied by the recruitment of plasma proteins involved in inflammation and coagulation. Our data may serve as a basis for further dissection of the fundamental mechanisms of red blood cell aging and vesiculation, for identifying the cause-effect relationship between blood bank storage and transfusion complications, and for assessing the role of microvesicles in pathologies affecting red blood cells.

Storage conditions determine the characteristics of red blood cell derived extracellular vesicles

“…According to Pistorius et al 54 we assigned the band at 1152 cm −1 to the C–O–C stretching of glucose; they selected the same band to characterize the glucose level of RBC concentrates using IR spectroscopy. Indeed, the latest proteomics results on REV samples performed by Bosman and coworkers 22 corroborate the presence of proteins related to the energy metabolism of RBCs. It is interesting to note, that for the rest of the REVs the glucose-related band is suppressed and the band at 1125 cm −1 , corresponding to lactate is increasing 54 .…”

“…It seems plausible that during storage the vesicles are enriched not only in hemoglobin but also in red blood cell membrane specific glycoproteins such as band 3 or glycophorin A-D. Recent proteomics on REVs isolated at different storage times from blood banks shows that the enrichment of band 3 protein in the vesicles achieved a maximum after 3 weeks of storage 22 . Regarding our experiment we speculate that in PBS-stored REVs hemoglobin and band 3 might also be present at a higher percentage than in SAGM-related REVs.…”

“…Alterations in band 3 aggregation/degradation related to RBC aging were also revealed 25 . All these findings suggest that the breakdown of connections between the cytoskeleton and the lipid bilayer provided by band 3—ankyrin interaction might be a crucial step in the vesiculation process 1 , 19 , 22 . Prudent et al reported that flotillin can associate with band 3 in stored RBCs, further confirming the involvement of band 3 complexes in microvesiculation 26 .…”

“…Controversially, recent findings based on proteomics 22 and phospholipidomics 23 have suggested that the main mechanism of storage-induced RBC vesiculation is linked to the membrane protein band 3 rather than to rafts. Band 3, the major protein component of the RBC membrane, is considered to play key roles in membrane reorganization during RBC storage (band 3 clustering model) 5 , 22 . Indeed, band 3 and actin were detected in REVs but other integral membrane and cytoskeletal proteins, such as spectrin and ankyrin were not found 24 .…”

Nanoscale Changes on RBC Membrane Induced by Storage and Ionizing Radiation: A Mini-Review

“…Among the changes induced by blood bank storing, RBC's vesiculation is probably the result of spontaneous curvature, compression, and altered membrane protein conformation (Li and Lykotrafitis, 2015). Disturbances in the interaction between the cytoskeleton and the lipid bilayer may lead to microparticle detachment (Leal et al, 2018;Freitas Leal et al, 2020). This cooperative phenomenon affects the structure and conformation of spectrin and actin proteins, located in the cytoskeleton, the two domain structures of band 3, one of them present in the cytosol and the other in the lipid bilayer and the ankyrin-binding protein (Kim et al, 2015;Lux, 2016;Dinarelli et al, 2018).…”