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The Expression of Prion Protein in the Vasculature

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The Expression of Prion Protein in the Vasculature The Expression of Prion Protein in the Vasculature Richard David Starke BSc (Hons) A thesis submitted to the University of London for the degree of Doctor of Philosophy University College London, 2003 ProQuest Number: U643764 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest U643764 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract The aim of my thesis was to study the expression of prion protein (PrP^) in the vasculature. The function of PrP^ is unknown but it is thought to refold into a pathogenic form termed PrP^^ which damages neurones in the brain causing transmissible spongiform encephalopathies. I studied the expression of PrP^ in CD34 positive stem cells, reticulocytes, erythrocytes and megakaryocytes. These cells were found to be positive for PrP^ with a general trend for increased expression during megakaryocyte maturation and a decrease in expression during erythropoiesis. Since megakaryocytes were positive for PrP^ I hypothesised that they were the source of platelet PrP^. I studied the expression, location and function of PrP^ in platelets. PrP^ was found in platelet alpha-granules and was transported to the cell surface upon cellular activation. Crosslinking PrP^ on the platelet surface was found to be associated with signal transduction phosphorylation events, but there was no apparent increase in platelet function. I hypothesised that the origin of blood plasma PrP^ was the vascular endothelium. Microvascular and macrovascular endothelial cells were found to express and release soluble PrP^ in a constitutive manner with no increase in expression upon cellular activation. Measurement of plasma PrP^ levels in patients with varying blood cell counts demonstrated no association between platelet, erythrocyte or leucocyte counts and the PrP^ concentration. Patients with a variety of chronic vascular diseases had a normal PrP^ concentration in their blood plasma. However there was a higher level of PrP^ in plasma from patients with renal failure that was not removed by renal dialysis. In summary I have provided a detailed analysis of the expression of PrP^ in several cells of the vasculature for the first time. Although its function is unclear PrP^ must be of significant importance for such a large and widespread distribution. Table of Contents The Expression of Prion Protein in the Vasculature...................................................... 1 Abstract.................................................................................................................................. 2 Table of Contents................................................................................................................. 3 List of Tables.......................................................................................................................10 List of Figures......................................................................................................................11 Abbreviations......................................................................................................................16 Publications..........................................................................................................................20 Acknowledgements............................................................................................................. 21 1. General Introduction................................................................................................22 1.1 Prion Protein........................................................................................................ 22 1.1.1 Introduction....................................................................................................22 1.1.2 Prion protein hypothesis............................................................................... 23 1.1.3 Protein X .........................................................................................................25 1.1.4 Model of PrP^^ formation............................................................................26 1.1.5 Mutations in the prion gene and hereditary CJD....................................27 1.1.6 Structure......................................................................................................... 29 1.1.7 Cell biology of PrP^...................................................................................... 30 1.1.8 Hypothesised functions................................................................................. 40 1.2 Transmissible Spongiform Encephalopathy...................................................42 1.2.1 Animal TSEs...................................................................................................42 1.2.2 BSE epidemic.................................................................................................. 43 1.2.3 Species variation.............................................................................................44 1.2.4 Human TSEs...................................................................................................44 1.2.5 Prion disease and Haematology................................................................. 50 1.2.6 Route of infection......................................................................................... 58 1.3 CD34+Stem Cells................................................................................................. 61 1.4 Megakaryocytes..................................................................................................... 64 1.4.1 Development..................................................................................................64 1.4.2 Structure........................................................................................................ 66 1.4.3 Granules......................................................................................................... 66 1.4.4 In vitro culture of megakaryocytes............................................................68 1.4.5 Platelet production....................................................................................... 71 1.5 Platelets...................................................................................................................73 1.5.1 Structure........................................................................................................ 73 1.5.2 Granules......................................................................................................... 75 1.5.3 Activation....................................................................................................... 75 1.5.4 Platelet adhesion and aggregation............................................................. 76 1.5.5 Shear................................................................................................................78 1.5.6 Thrombin....................................................................................................... 79 1.5.7 Collagen.......................................................................................................... 80 1.5.8 Intracellular signalling................................................................................ 80 1.6 Endothelial Cells................................................................................................... 83 1.6.1 Introduction..................................................................................................... 83 1.6.2 Structure........................................................................................................ 83 1.6.3 Function......................................................................................................... 85 1.6.4 Granules......................................................................................................... 91 1.6.5 Activation....................................................................................................... 91 1.6.6 Endothelial cell culture................................................................................ 92 1.7 Erythrocytes.......................................................................................................... 93 1.8 Aims of Thesis ....................................................................................................... 95 General Methods....................................................................................................... 97 2.1 Collection and separation of blood samples.....................................................97 2.1.1 Platelet rich plasma (FRF).......................................................................... 97 2.1.2 Platelet poor plasma (FFF).......................................................................... 97 2.1.3 Pure platelets................................................................................................. 97 2.1.4 Buffy coat........................................................................................................98 2.2 Molecular biology.................................................................................................98
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