Oxidative stress and caspase-mediated fragmentation of cytoplasmic domain of erythrocyte band 3 during blood storage.

Background. During blood bank storage, red blood cells (RBCs) undergo a number of biological and biochemical alterations collectively referred to as "storage lesions". These injuries include loss and oxidative cross-linking of band 3, the major integral protein of RBC membranes. Denaturation of hemoglobin (Hb) and damage to the amino-terminal of band 3 are recognised as the starting events for immunological recognition mechanisms and phagocytic removal of senescent or impaired RBCs from circulation. Consequently, studies focusing on the Hb-association and oxidative status of the cytoskeleton of stored RBCs intended for transfusion are of extreme interest. In this work, two storage-related fragments of band 3 were documented and biochemically characterised. Methods. Four RBC units were collected from normal volunteers and stored for 21 days under (i) standard blood bank conditions, (ii) anaerobic conditions, or (iii) in the presence of caspase 3-inhibitor. Degradation products of band 3 were followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis coupled with western blot and mass spectrometry analyses. Results. Two different degradation products of the cytoplasmic domain of the erythrocyte band 3 (CDB3) were detected in RBC membranes during storage in saline-adenine-glucose-mannitol (SAGM) preservation medium. One of these fragments showed an apparent molecular weight of 34 kDa and was demonstrated to be the product of a free-radical attack on the protein main chain, whereas another fragment of 24 kDa was the result of a caspase 3-mediated cleavage. Discussion. Although to different extent, anaerobic conditions reduced the formation of both truncated products indicating an enhanced activity of the pro-apoptotic caspase 3 enzyme following oxidative stress. Interestingly, both CDB3 fragments were tightly associated to the erythrocyte membrane supporting the involvement of Cys-201 and/or Cys-317 in clustering different band 3 monomers.