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

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.

1

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.

2

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.

3

Increased RBC cytoskeletal protein carbonylation induced by storage in CPDA medium. (A) A representative oxyblot analysis, i.e. immunoblot analysis of cytoskeletons from a donor stained with the anti-DNP antibody. The duration of storage is indicated in days starting from blood donation. MW of the proteins is shown in kD (right-hand side). (B) Immunoblot of the cytoskeletons probed with anti-human spectrin antibody (internal loading control). (C) The estimation of the oxidative index of the cytoskeletal samples after normalization to control values. The points in the graphs represent the average values and the error bars the standard deviation among the six tested blood donors.