PB2299 AN INVESTIGATION INTO THE PRESENCE OF COMPLEMENT PROTEINS ON ERYTHROCYTE VESICLES IN SICKLE CELL DISEASE

Background: A single amino acid substitution in the globin gene segment is the underlying genetic defect in sickle cell disease (SCD) creating a haemoglobin molecule which polymerizes on deoxygenation. Recent insights into the pathophysiology of the disease have pointed to the roles inflammation and hypercoagulability play in disease manifestation and severity caused, in part, by increased phosphatidylserine exposure and extracellular vesiculation by sickle red cells. Current works have identified some of these vesicles as those released from reticulocytes in the process of maturation to the red blood cell. These vesicles are termed ‘inside‐out’ autophagic vesicles as they are the result of the fusion of endocytosed parts of the cell membrane with autophagic endosomes and are positive for intracellular glycophorin A (GPA) epitope using BRIC163 antibody. Further analysis has shown that some of these vesicles are also positive for extracellular GPA epitope using BRIC256 antibody raising the possibility that the vesicle membrane may have become permeabilized. One possible permeabilizing mechanism is the membrane attack complex of the complement system. Aims: The aim of this project was to investigate the presence of complement proteins on vesicles isolated from plasma or attached to erythrocytes using flow cytometry and confocal microscopy Methods: Vesicles were isolated from SCD plasma (n = 2) and healthy control (n = 1) using 2 different techniques: membrane‐based isolation technique and fluorescent‐activated cell sorter (FACS) and studied with flow cytometry using BRIC163, BRIC256, C3, C4, C3c and C3d antibodies. Cultured reticulocyte (n = 2), sickle red cells (n = 3) and healthy control red cells (n = 2) were analyzed with confocal microscopy using Annexin‐V, BRIC163, C3, C4, C3c and C3d antibodies. Results: Results show the presence of complement proteins on vesicles in both SCD plasma and healthy control in levels as high as 100% for BRIC163 vesicles in healthy control. Confocal analyses reveal this deposition is also present on vesicles prior to release and is associated with PS exposure. Summary/Conclusion: Complement proteins are present on vesicles in plasma and erythrocytes and may suggest activation and formation of the MAC complex. Further work is required to determine the involvement of these proteins in vesicle formation and release.