RBC-derived vesicles during storage: ultrastructure, protein composition, oxidation, and signaling components.
BACKGROUND
Red cells (RBCs) lose membrane in vivo, under certain conditions in vitro, and during the ex vivo storage of whole blood, by releasing vesicles. The vesiculation of the RBCs is a part of the storage lesion. The protein composition of the vesicles generated during storage of banked RBCs has not been studied in detail.


METHODS
Vesicles were isolated from the plasma of nonleukoreduced RBC units in citrate-phosphate-dextrose-adenine, at eight time points of the storage period and shortly afterward. The degree of vesiculation, ultrastructure, oxidation status, and protein composition of the vesicles were evaluated by means of electron microscopy and immunoblotting. RBCs and ghost membranes were investigated as controls.


RESULTS
The total protein content of the vesicle fraction and the size of the vesicles increased but their structural integrity decreased over time. The oxidation index of the vesicles released up to Day 21 of storage was greater than that of the membrane ghosts of the corresponding intact RBCs. The vesicles contain aggregated hemoglobin, band 3, and lipid raft proteins, including flotillins. They also contain Fas, FADD, procaspases 3 and 8, caspase 8 and caspase 3 cleavage products (after the 10th day), CD47 (after the 17th day), and immunoglobulin G.


CONCLUSIONS
These data indicate that the vesicles released during storage of RBCs contain lipid raft proteins and oxidized or reactive signaling components commonly associated with the senescent RBCs. Vesiculation during storage of RBCs may enable the RBC to shed altered or harmful material.
Department of Cell Biology and Biophysics, Faculty of Biology, University of Athens, Panepistimiopolis, Greece.
Anastasios G
AG
Kriebardis
Marianna H
MH
Antonelou
Konstantinos E
KE
Stamoulis
Effrosini
E
Economou-Petersen
Lukas H
LH
Margaritis
Issidora S
IS
Papassideri
D000818:Animals; D001793:Blood Preservation; D001798:Blood Proteins / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism; D022162:Cytoplasmic Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D004905:Erythrocyte Aging; D004910:Erythrocyte Membrane / Q000378:metabolism / Q000648:ultrastructure; D004912:Erythrocytes / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D006454:Hemoglobins / Q000378:metabolism; D015151:Immunoblotting; D021962:Membrane Microdomains / Q000378:metabolism / Q000648:ultrastructure; D008854:Microscopy, Electron; D010084:Oxidation-Reduction; D018384:Oxidative Stress
D001798:Blood Proteins; D006454:Hemoglobins; D020169:Caspases