Alteration of α-spectrin ubiquitination after hemorrhagic shock

Background RBC deformability after trauma and hemorrhagic shock (T/HS) leads to the microcirculatory dysfunction and clinical manifestations of organ failure. However, the cellular mechanism of this phenomenon remains unknown. Spectrins are important for the shape and physical properties of erythrocytes, such as deformability and resistance to mechanical stress. Previous studies have shown that erythrocyte α-spectrin is ubiquitinated. Studies of sickled cells and aged erythrocytes, 2 conditions known to have decreased RBC deformability, have shown decreased α-spectrin ubiquitination, which may contribute to the inability of these cells to change shape. It was hypothesized that decreased α-spectrin ubiquitination could participate in the mechanism(s) whereby T/HS erythrocytes become less deformable. Methods The level of α-spectrin ubiquitination in erythrocytes isolated from T/HS rats was determined and compared with erythrocytes from control sham-shocked (T/SS) animals. After T/SS (n = 4) or T/HS (n = 7), α- and β-spectrin subunits were isolated using a low ionic–strength buffer at 37°C for 30 minutes. The relative amount of ubiquitinated α-spectrin was evaluated by Western blotting using a monoclonal antibody to ubiquitin. Results The relative level of α-spectrin ubiquitination (normalized to total α-spectrin in the same preparation) was found to be significantly decreased after T/HS (.319 ± .03) compared with T/SS control erythrocytes (.485 ± .06, P < .05). To evaluate the content and relative amounts of the other membrane proteins, the profiles of T/HS and T/SS erythrocytes were compared by sodium dodecyl sulphate–polyacrylamide gel electrophoresis. This did not reveal any significant quantitative differences between T/SS and T/HS spectrin or other membrane proteins. Conclusions The finding of decreased α-spectrin ubiquitination after T/HS suggests that this mechanism could contribute to increased rigidity of the erythrocyte membrane.