326 - The Role of Cytoskeletal S-Nitrosation in Red Blood Cell Deformability

Abstract

The deformability of red blood cells (RBC) is an important characteristic of RBC functionality, and contributes to define blood rheological properties and blood viscosity. The ability of RBCs to change their shape mainly depends on the flexibility of the interactions between cytoskeletal proteins, in particular of the structure of the main cysteine rich protein spectrin. In this study we aimed to analyze the effects of S-nitrosation of spectrin on changes in RBC deformability. We found that treatment with S-nitrosocystein (SNOC) leads to S-nitrosation of purified spectrin as assessed by chemiluminescence detection (CLD). Similarly, SNOC-induced nitrosation of cytoskeletal proteins of RBC ghosts and RBC were measured by CLD and the biotin switch assay. Western blotting following the biotin switch assay confirmed that spectrin was one of the nitrosated cytoskeletal proteins in RBC ghosts and RBCs. However, treatment with SNOC or DEA/NO did not affect deformability of RBCs as assessed by ektacytometry measured in a LORRCA system, nor blood viscosity measured in a low shear rotational viscometer under the same shear rate conditions. Instead, we found that changes in redox state of RBC by treatment with high concentrations of tert-Butyl hydroperoxide decreased deformability and increased blood viscosity. Our results demonstrate that incubation of RBC, RBC ghosts and purified spectrin with a nitrosothiol result in significantly increased S-nitrosation of RBC cytoskeletal spectrin. However, nitrosation did not improve RBC deformability or change blood viscosity. Hence, in our setting change in redox state/oxidative modification rather than nitrosation reactions of cytoskeletal proteins like spectrin have an impact on RBC deformability and blood viscosity.