Mechanisms of red cell turnover (Eryptosis)

AbstractTitle: Mechanisms of Red Cell Turnover (Eryptosis).Author: Kris Patrick JeremyA thesis submitted in partial fulfilment of the requirements of the University of the West of England, Bristol for the degree of Doctor of Philosophy.When CD47 is ligated with a specific ligand, phosphatidylserine exposure is observed on the surface of the erythrocyte in a process known as eryptosis, which is similar to apoptosis.The aim of this study was to identify new proteins within the CD47 receptor mediated phosphatidylserine pathway and to elucidate the functions of any newly identified proteins. Previous research had suggested a link between protein 4.1R and CD47. Therefore, diseased protein 4.1R deficient erythrocytes were used as a molecular tool to help explore the role of protein 4.1R in the CD47 pathway. To achieve this aim, flow cytometry to measure phosphatidylserine exposure, 1D and 2D protein immunoblotting, 1D and 2D electrophoresis, RS100 ProteinChips coupled to SELDI-TOF, Q-TOF and MALDI-TOF, calcium influx, 2D serine/threonine phosphoblots and protein kinase A and protein kinase C inhibition were employed.Results showed increased phosphatidylserine exposure in 4.1R deficient erythrocytes and protein immunoblots highlighted a novel interaction between CD44 and protein 4.1R. RS100 ProteinChip experiments also confirmed an interaction between CD44 and protein 4.1R. Phosphatidylserine exposure in normal erythrocytes was increased when protein kinase A was inhibited. Conversely phosphatidylserine exposure was decreased when protein kinase C was inhibited. In 4.1R deficient erythrocytes, protein kinase A inhibition further increased phosphatidylserine exposure and protein kinase C inhibition further decreased phosphatidylserine exposure. Phosphorylation of protein 4.1R and calcium influx was also observed in response to CD47 ligation.The study concludes that protein 4.1R, protein kinase A and protein kinase C have a direct effect on phosphatidylserine exposure and can therefore be assumed to be a part of the CD47 pathway. Calcium influx and phosphorylation of protein 4.1R also play a critical role in the CD47 pathway. The novel interaction between CD44 and protein 4.1R and the role that CD44 might play in the CD47 pathway is worthy of further research