SI38 Characterisation of Membrane Proteins on Vesicles from Normal Red Cells and on Red Cells of Patients with Membranopathy

Background Red cell vesicles are formed either after prolonged blood storage at 4°C or during incubation in calcium-loaded medium. Unlike mature red cells, these vesicles are depleted in spectrin but carry band 3, glycophorins, acetylcholinesterase and CD47. Absorption of human anti-D by vesicles indicated the presence of Rh proteins. Study Design The aims were to identify the membrane components present on red cell vesicles, and to investigate whether red cells after vesiculation in vitro were similar to those from patients with inherited haemolytic anaemias: hereditary spherocytosis (HS) and pyropoikilocytosis (HPP). Vesicles released from fresh normal red cells in Ca2+ loaded medium were purified by filtration through 0.8 μm filters. Specific monoclonal antibodies (Mabs) were used to identify integral membrane components. The analytical techniques used were SDS-PAGE, Western blotting, confocal microscropy, and rosette formation with magnetic beads (Dynabeads) conjugated with anti-mouse immunoglobulins. Flow cytometry quantified antibody bound to red cells after vesiculation and from patients with HS and HPP. Results We confirmed depletion of spectrin and the presence of band 3, CD47 and glycophorins in vesicles released from normal red cells. Confocal microscopy demonstrated the coating of Rh(D+) vesicles with human Mab anti-D. Other membrane molecules detected on the vesicles were Duffy glycoprotein, Kell glycoprotein, glucose transporter, protein 4.2, Rh glycoprotein (RhAG), CD59, and blood group A antigen. Flow cytometry detected reductions in binding of Mabs to band 3, Rh proteins and RhAG on vesiculated red cells as well as on HS red cells. CD47 content was lower on HS than on vesiculated red cells. Greater losses of these membrane components occurred on HPP red cells with a concomitant reduction in CD59, and glycophorins A and C. Confocal microscopy of dual labelled HS and HPP red cells showed that the loss of membrane proteins was not a uniform process but ranged from strong, moderate to dim (or no) fluorescence. Conclusion Shedding a large number of membrane proteins in red cell vesicles is probably a physiological process of cell ageing. The greater loss of band 3, CD47, Rh protein and RhAG on HS and HPP red cells probably reflects an accelerated vesiculation process secondary to their underlying cytoskeletal defect.