464 - Characterization of Microparticles and Exosomes from Red Blood Cells During Storage: Implications for Transfusion Toxicity

Abstract

Red blood cell (RBC) storage for up to 42 days is a mainstay therapeutic in transfusion medicine. During storage several biochemical changes and metabolic alterations occur in RBCs that associate with increased morbidity and mortality in transfusion recipients. These changes include formation of smaller microvesicles comprising microparticles (MPs) and exosomes (Exo). Importantly, depending on the size and composition, these vesicles may elicit circulatory dysfunction and inflammation. In addition, our recent data has identified free heme and cell free hemoglobin as independent mediators of pro-inflammatory and oxidant activity associated with stored RBC transfusions. In this study we determined the relative formation of MPs and Exo from leukoreduced RBCs stored for 7-10d or 35-52d and assessed their composition with hemoglobin, heme and antioxidants (glutathione and Peroxiredoxin-2, catalase, superoxide dismutase, and Thioredoxin). Both MPs and Exo increased with storage, with the former being ∼10-fold higher in total number than the latter. The total glutathione content normalized to oxyHb (pro-oxidant) was significantly decreased by 2.5 and 6 fold respectively in MPs and Exo compared to intact RBC. Interestingly, high molecular weight antioxidant proteins, Peroxiredoixin-2, Cu/Zn SOD and Thioredoxin were detected in both MPs and Exo, but present as higher MWt complexes (under reducing condition) indicative of oxidatively-cross linked proteins. Moreover, these complexes were more abundant in Exo compared to MPs normalized to both protein and OxyHb. Furthermore, addition of H 2 O 2 failed to alter Prx-2 complex MWt indicating that the Prx-2 system is unable to metabolize and protect MPs or Exo against H 2 O 2 induced stress. These data suggest that MP’s and Exo harbor more oxidatively modified proteins and mediators, and contain lower antioxidants. We speculate that transfusion with these microvesicles will lead to pro-oxidant stress and contribute to inflammatory stress associated with transfused with older stored RBCs.