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

Kriebardis AG, Antonelou MH, Stamoulis KE, Economou-Petersen E, Margaritis LH, Papassideri IS - J. Cell. Mol. Med. (2007 Jan-Feb)

Bottom Line: A proportion of the ghost-bound Hb consists of non-reducible crosslinkings of probably oxidized(denatured Hb or hemichromes.The oxidative index of the cytoskeletal proteins was found increased, signalizing oxidative modifications in spectrin and possibly other cytoskeletal proteins.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Biophysics, Faculty of Biology, University of Athens, Panepistimiopolis, Athens, Greece.

ABSTRACT
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.

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Immunoblotting analysis of the cytoskeletons extracted from the ghost membranes of RBCs stored in CPDA. (A) Western blot analysis of a representative cytoskeleton preparation performed with anti-human Hb and cytoskeletal proteins-specific antibodies. The cytoskeletons under storage accumulate Hb, non-reducible Hb oligomers and high MW aggregates.The duration of storage is indicated in days starting from blood donation. MW of the proteins is shown in kDa (right-hand side). (B) Densitometry analysis of the relative proportion of Hb in the cytoskeletons. The points in the graphs represent the average values and the error bars the standard deviation among the six blood donors tested, after normalization to control values.
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fig02: Immunoblotting analysis of the cytoskeletons extracted from the ghost membranes of RBCs stored in CPDA. (A) Western blot analysis of a representative cytoskeleton preparation performed with anti-human Hb and cytoskeletal proteins-specific antibodies. The cytoskeletons under storage accumulate Hb, non-reducible Hb oligomers and high MW aggregates.The duration of storage is indicated in days starting from blood donation. MW of the proteins is shown in kDa (right-hand side). (B) Densitometry analysis of the relative proportion of Hb in the cytoskeletons. The points in the graphs represent the average values and the error bars the standard deviation among the six blood donors tested, after normalization to control values.

Mentions: To determine if the membrane-bound denatured Hb was associated with the membrane skeletons as well, we achieved a subsequent immunoblotting analysis of the Triton insoluble cytoskeletons after the fractionation of the stored RBC membrane samples with Triton X-100. The present findings showed, for the first time, that the same storage effect of Hb concerns the cytoskeletons of the stored cells as well, since a progressive accumulation of Hb, cross-linked multimers and high MW (< 260 kD) aggregates to them in amounts proportionate to the age of storage was revealed (Fig. 2). Of note, the significant increase in the Hb content of the cytoskeletons extracted from stored RBC ghosts was independent of their protein band 3 content (data not shown). In control membrane preparations (including the RBCs stored for 0 and 2 days in CPDA), the cytoskeletons do not contain detectable amounts of globin. After the extraction with Triton, the globin follows its physical linker, that is, the cytoplasmic site of band 3 [16], in the Triton-soluble fraction of the membrane (data not shown). The cytoskeletons of the six examined RBC units that had been stored even for long periods in CPDA did not exhibit any sign of severe proteolysis or fragmentation, as confirmed by immunoblotting analysis and probing for spectrin, actin and 4.1R proteins (Fig. 2).


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

Kriebardis AG, Antonelou MH, Stamoulis KE, Economou-Petersen E, Margaritis LH, Papassideri IS - J. Cell. Mol. Med. (2007 Jan-Feb)

Immunoblotting analysis of the cytoskeletons extracted from the ghost membranes of RBCs stored in CPDA. (A) Western blot analysis of a representative cytoskeleton preparation performed with anti-human Hb and cytoskeletal proteins-specific antibodies. The cytoskeletons under storage accumulate Hb, non-reducible Hb oligomers and high MW aggregates.The duration of storage is indicated in days starting from blood donation. MW of the proteins is shown in kDa (right-hand side). (B) Densitometry analysis of the relative proportion of Hb in the cytoskeletons. The points in the graphs represent the average values and the error bars the standard deviation among the six blood donors tested, after normalization to control values.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4401228&req=5

fig02: Immunoblotting analysis of the cytoskeletons extracted from the ghost membranes of RBCs stored in CPDA. (A) Western blot analysis of a representative cytoskeleton preparation performed with anti-human Hb and cytoskeletal proteins-specific antibodies. The cytoskeletons under storage accumulate Hb, non-reducible Hb oligomers and high MW aggregates.The duration of storage is indicated in days starting from blood donation. MW of the proteins is shown in kDa (right-hand side). (B) Densitometry analysis of the relative proportion of Hb in the cytoskeletons. The points in the graphs represent the average values and the error bars the standard deviation among the six blood donors tested, after normalization to control values.
Mentions: To determine if the membrane-bound denatured Hb was associated with the membrane skeletons as well, we achieved a subsequent immunoblotting analysis of the Triton insoluble cytoskeletons after the fractionation of the stored RBC membrane samples with Triton X-100. The present findings showed, for the first time, that the same storage effect of Hb concerns the cytoskeletons of the stored cells as well, since a progressive accumulation of Hb, cross-linked multimers and high MW (< 260 kD) aggregates to them in amounts proportionate to the age of storage was revealed (Fig. 2). Of note, the significant increase in the Hb content of the cytoskeletons extracted from stored RBC ghosts was independent of their protein band 3 content (data not shown). In control membrane preparations (including the RBCs stored for 0 and 2 days in CPDA), the cytoskeletons do not contain detectable amounts of globin. After the extraction with Triton, the globin follows its physical linker, that is, the cytoplasmic site of band 3 [16], in the Triton-soluble fraction of the membrane (data not shown). The cytoskeletons of the six examined RBC units that had been stored even for long periods in CPDA did not exhibit any sign of severe proteolysis or fragmentation, as confirmed by immunoblotting analysis and probing for spectrin, actin and 4.1R proteins (Fig. 2).

Bottom Line: A proportion of the ghost-bound Hb consists of non-reducible crosslinkings of probably oxidized(denatured Hb or hemichromes.The oxidative index of the cytoskeletal proteins was found increased, signalizing oxidative modifications in spectrin and possibly other cytoskeletal proteins.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Biophysics, Faculty of Biology, University of Athens, Panepistimiopolis, Athens, Greece.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus