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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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Globin oligomerization/crosslinking events on the membranes of RBCs stored in CPDA. (A) Western blot analysis of the ghosts of a representative donor performed with anti-human Hb polyclonal antibody.The duration of storage is indicated in days starting from blood donation. (B) Western blot analysis of ghosts against human actin (internal control). MW of the proteins is shown in kDa (right-hand side). (C) Densitometry analysis performed on ECL-developed films, regarding the relative proportion of Hb (monomers and oligomers) in the ghosts. The points in the graphs represent the average values among the six tested blood donors, after normalization to control values. Error bars demonstrate the standard deviation between the six donors.
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fig01: Globin oligomerization/crosslinking events on the membranes of RBCs stored in CPDA. (A) Western blot analysis of the ghosts of a representative donor performed with anti-human Hb polyclonal antibody.The duration of storage is indicated in days starting from blood donation. (B) Western blot analysis of ghosts against human actin (internal control). MW of the proteins is shown in kDa (right-hand side). (C) Densitometry analysis performed on ECL-developed films, regarding the relative proportion of Hb (monomers and oligomers) in the ghosts. The points in the graphs represent the average values among the six tested blood donors, after normalization to control values. Error bars demonstrate the standard deviation between the six donors.

Mentions: To determine the nature of the Hb chains interacting with the membranes of stored RBCs and to estimate the proportion of the total membrane-associated Hb that is specifically bound to the cytoskeleton, the non-fractionated ghosts from stored RBCs were probed for Hb by immunoblotting techniques. As expected, the Hb was increasingly associated with the membrane in proportion to the duration of storage (Fig. 1A and C). Our study clearly demonstrated that a proportion of the membrane-bound total Hb consists of non-reducible crosslinkings of probably oxidized/denatured chains or hemichromes. Clearly viewed high molecular weight (MW) Hb-immunopositive bands representing these pathologic multimers were evident after 4 days of storage and were more prominent after a storage period of 22 days (Fig. 1A). Interestingly, the quantity of cross-linked forms of globin were found to increase dramatically as the cells approach the end of the storage period in comparison to the also increased amount of the globin monomers, demonstrating that the defective Hb-membrane association was strongly affected by the prolonged storage. The membrane-bound Hb aggregates are not obviously stabilized solely by disulfide bonds, but rather by non-reducible linkages (such as amides or free radical-generated adducts, e.g. bityro-sine), since they are partly resistant to reduction with beta mercaptoethanol. Despite the fact that the globin multimers were usually present in membranes enriched in globin monomers, in the globin-loaded control ghosts found occasionally, they were always undetectable (see control ghosts in Fig. 1A). On the contrary, stored RBC membrane ghosts with fewer globin monomers in comparison to some controls exhibited aberrant globin-assigned bands (see day 17 preparation in Fig. 1A).


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)

Globin oligomerization/crosslinking events on the membranes of RBCs stored in CPDA. (A) Western blot analysis of the ghosts of a representative donor performed with anti-human Hb polyclonal antibody.The duration of storage is indicated in days starting from blood donation. (B) Western blot analysis of ghosts against human actin (internal control). MW of the proteins is shown in kDa (right-hand side). (C) Densitometry analysis performed on ECL-developed films, regarding the relative proportion of Hb (monomers and oligomers) in the ghosts. The points in the graphs represent the average values among the six tested blood donors, after normalization to control values. Error bars demonstrate the standard deviation between the six donors.
© Copyright Policy
Related In: Results  -  Collection

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

fig01: Globin oligomerization/crosslinking events on the membranes of RBCs stored in CPDA. (A) Western blot analysis of the ghosts of a representative donor performed with anti-human Hb polyclonal antibody.The duration of storage is indicated in days starting from blood donation. (B) Western blot analysis of ghosts against human actin (internal control). MW of the proteins is shown in kDa (right-hand side). (C) Densitometry analysis performed on ECL-developed films, regarding the relative proportion of Hb (monomers and oligomers) in the ghosts. The points in the graphs represent the average values among the six tested blood donors, after normalization to control values. Error bars demonstrate the standard deviation between the six donors.
Mentions: To determine the nature of the Hb chains interacting with the membranes of stored RBCs and to estimate the proportion of the total membrane-associated Hb that is specifically bound to the cytoskeleton, the non-fractionated ghosts from stored RBCs were probed for Hb by immunoblotting techniques. As expected, the Hb was increasingly associated with the membrane in proportion to the duration of storage (Fig. 1A and C). Our study clearly demonstrated that a proportion of the membrane-bound total Hb consists of non-reducible crosslinkings of probably oxidized/denatured chains or hemichromes. Clearly viewed high molecular weight (MW) Hb-immunopositive bands representing these pathologic multimers were evident after 4 days of storage and were more prominent after a storage period of 22 days (Fig. 1A). Interestingly, the quantity of cross-linked forms of globin were found to increase dramatically as the cells approach the end of the storage period in comparison to the also increased amount of the globin monomers, demonstrating that the defective Hb-membrane association was strongly affected by the prolonged storage. The membrane-bound Hb aggregates are not obviously stabilized solely by disulfide bonds, but rather by non-reducible linkages (such as amides or free radical-generated adducts, e.g. bityro-sine), since they are partly resistant to reduction with beta mercaptoethanol. Despite the fact that the globin multimers were usually present in membranes enriched in globin monomers, in the globin-loaded control ghosts found occasionally, they were always undetectable (see control ghosts in Fig. 1A). On the contrary, stored RBC membrane ghosts with fewer globin monomers in comparison to some controls exhibited aberrant globin-assigned bands (see day 17 preparation in Fig. 1A).

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