Red blood cell aging markers during storage in citrate-phosphate-dextrose-saline-adenine-glucose-mannitol.

BACKGROUND: It has been suggested that red blood cell (RBC) senescence is accelerated under blood bank conditions, although neither protein profile of RBC aging nor the impact of additive solutions on it have been studied in detail. STUDY DESIGN AND METHODS: RBCs and vesicles derived from RBCs in both citrate-phosphate-dextrose (CPD)-saline-adenine-glucose-mannitol (SAGM) and citrate-phosphate-dextrose-adenine (CPDA) were evaluated for the expression of cell senescence markers (vesiculation, protein aggregation, degradation, activation, oxidation, and topology) through immunoblotting technique and immunofluorescence or immunoelectron microscopy study. RESULTS: A group of cellular stress proteins exhibited storage time- and storage medium-related changes in their membrane association and exocytosis. The extent, the rate, and the expression of protein oxidation, Fas oligomerization, caspase activation, and protein modifications in Band 3, hemoglobin, and immunoglobulin G were less conspicuous and/or exhibited significant time retardation under storage in CPD-SAGM, compared to the CPDA storage. There was evidence for the localization of activated caspases near to the membrane of both cells and vesicles. CONCLUSIONS: We provide circumstantial evidence for a lower protein oxidative damage in CPD-SAGM-stored RBCs compared to the CPDA-stored cells. The different expression patterns of the senescence markers in the RBCs seem to be accordingly related to the oxidative stress management of the cells. We suggest that the storage of RBCs in CPD-SAGM might be more alike the in vivo RBC aging process, compared to storage in CPDA, since it is characterized by a slower stimulation of the recognition signaling pathways that are already known to trigger the erythrophagocytosis of senescent RBCs.

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. STUDY DESIGN AND 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. CONCLUSION: 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.

Storage-dependent remodeling of the red blood cell membrane is associated with increased immunoglobulin G binding, lipid raft rearrangement, and caspase activation.

BACKGROUND: The elucidation of the storage lesion is important for the improvement of red blood cell (RBC) storage. Ex vivo storage is also a model system for studying cell-signaling events in the senescence and programmed cell death of RBCs. The membrane hosts critical steps in these mechanisms and undergoes widespread remodeling over the storage period. STUDY DESIGN AND METHODS: Fresh and CPDA-stored RBCs from 21 blood donors were evaluated as whole cells, membrane ghosts, and cytoskeletons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, immunofluorescence microscopy, and in situ assays. Band 3 content, immunoglobulin G (IgG) content, specific protein movement to and from the membrane, and caspase system activation were measured. RESULTS: During storage, Band 3 protein was aggregated and its content decreased as did the content of several lipid raft-related proteins. IgG binding to the membrane increased. Sorcin and synexin moved from the cytosol to the membrane, stomatin and flotillins left the membrane, the Fas protein was oligomerized, and caspase was activated. CONCLUSION: The remodeling of the RBC membrane during storage includes loss and oxidative cross-linking of Band 3 as well as IgG binding. This process occurs with lipid raft development and loss and is probably driven by caspase activation. Oxidative injury appears to be an important driver of RBC aging during storage.

Progressive oxidation of cytoskeletal proteins and accumulation of denatured hemoglobin in stored red cells.

Red blood cell (RBC) membrane proteins undergo progressive pathological alterations during storage. In conditions of increased cellular stress, the cytoskeleton also sustains certain modifications. The hemoglobin (Hb) content and oxidative status of the RBC cytoskeletons as a function of the storage period remain unclear. The possible Hb content and oxidative alterations occurring in the cytoskeletons in the course of storage were monitored in six units, by means of electrophoresis, immunoblotting and protein carbonylation assays. A proportion of the ghost-bound Hb consists of non-reducible crosslinkings of probably oxidized(denatured Hb or hemichromes. The defective Hb-membrane association was strongly affected by the prolonged storage. A progressive accumulation of Hb monomers, multimers and high molecular weight aggregates to corresponding cytoskeletons were also evident. The oxidative index of the cytoskeletal proteins was found increased, signalizing oxidative modifications in spectrin and possibly other cytoskeletal proteins. The reported data corroborate the evidence for oxidative damage in membrane proteins with emphasis to the cytoskeletal components. They partially address the pathophysiological mechanisms underlying the RBC storage lesion, add some new insight in the field of RBC storage as a hemoglobin- and cytoskeleton-associated pathology and suggest the possible use of antioxidants in the units intended for transfusion.

Membrane protein carbonylation in non-leukodepleted CPDA-preserved red blood cells.

Transfusion of allogeneic blood products is associated with adverse reactions and complications. Some of the negative effects of RBC transfusion are associated with the storage lesion. The importance of RBC oxidative damage in the storage lesion is not well documented. We monitored the storage-induced membrane protein oxidation in CPDA-preserved non-leukodepleted RBCs units from five blood donors in the course of the storage period, as assessed by protein carbonylation levels estimation. Carbonylated protein content was determined following 2,4-dinitrophenylhydrazine derivatization and SDS-polyacrylamide gel electrophoresis coupled with Western blotting. Immunoblotting with dinitrophenol-specific antibody revealed increased RBC membrane protein carbonyls with prolonged storage in CPDA units. This finding supports the idea of oxidation as a part of the storage lesion.

A large GLC1C Greek family with a myocilin T377M mutation: inheritance and phenotypic variability.

PURPOSE: POAG is a complex disease; therefore, families in which a glaucoma gene has been mapped may carry additional POAG genes. The goal of this study was to determine whether mutations in the myocilin (MYOC) gene on chromosome 1 are present in two POAG families, which have previously been mapped to the GLC1C locus on chromosome 3. METHODS: The three exons of MYOC were screened by denaturing (d)HPLC. Samples with heteroduplex peaks were sequenced. Clinical findings were compared with genotype status in all available family members over the age of 20 years. RESULTS: A T377M coding sequence change in MYOC was identified in family members of the Greek GLC1C family but not in the Oregon GLC1C family. Individuals carrying both the MYOC T377M variant and the GLC1C haplotype were more severely affected at an earlier age than individuals with just one of the POAG genes, suggesting that these two genes interact or that both contribute to the POAG phenotype in a cumulative way.