Royal College of Surgeons in Ireland e-publications@RCSI PhD theses Theses and Dissertations 11-1-2015 Characterization of a protein: protein interaction between RNF181 and the platelet integrin, αIIbβ3 Seamus Allen Royal College of Surgeons in Ireland, [email protected] Citation Allen S. Characterization of a protein: protein interaction between RNF181 and the platelet integrin, αIIbβ3.[PhD Thesis]. Dublin: Royal College of Surgeons in Ireland; 2015. This Thesis is brought to you for free and open access by the Theses and Dissertations at e-publications@RCSI. It has been accepted for inclusion in PhD theses by an authorized administrator of e-publications@RCSI. For more information, please contact [email protected]. — Use Licence — Creative Commons Licence: This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 License. This thesis is available at e-publications@RCSI: http://epubs.rcsi.ie/phdtheses/179 Characterization of a protein: protein interaction between RNF181 and the platelet integrin, αIIbβ3 Seamus T Allen BSc Platelet Biology Group, Department of Molecular and Cellular Therapeutics RCSI A Thesis submitted to the School of Postgraduate Studies, Faculty of Medicine and Health Science, Royal College of Surgeons in Ireland, in fulfillment of the degree of Doctor of Philosophy Supervisor: Professor Niamh Moran July 2015 Thesis Declaration I declare that this thesis, which I submit to RCSI for examination in consideration of the award of a higher degree of Doctor of Philosophy, is my own personal effort. Where any of the content presented is the result of input or data from a related collaborative research programme, this is duly acknowledged in the text such that it is possible to ascertain how much of the work is my own. I have not already obtained a degree in RCSI or elsewhere on the basis of this work. Furthermore, I took reasonable care to ensure that the work is original, and, to the best of my knowledge, does not breach copyright law, and has not been taken from other sources except where such work has been cited and acknowledged within the text. Signed _____________________________________________________ Student Number ______________________________________________ Date _______________________________________________________ 2 Table of contents Abbreviations ...................................................................................................... 7 Figures ................................................................................................................ 9 Tables ................................................................................................................ 12 Summary ........................................................................................................... 14 Acknowledgments ............................................................................................ 15 Chapter 1 ........................................................................................................... 18 General Introduction ........................................................................................ 18 1.1: Platelets in haemostasis ..................................................................................................... 19 1.2: Integrins ................................................................................................................................ 19 1.2.1: Integrin Extracellular domains ........................................................................................ 22 1.2.2: The integrin transmembrane domains .......................................................................... 23 1.2.3: The integrin cytoplasmic domains ................................................................................. 24 1.3: Integrin αIIbβ3 in platelet function ....................................................................................... 26 1.4: Structure and function of the integrin αIIb transmembrane and cytoplasmic tail domains ........................................................................................................................................ 27 1.5: Proteins binding to the integrin αIIb cytoplasmic tails ..................................................... 29 1.5.1: RNF181 ............................................................................................................................. 30 1.5.2: Calcium and integrin binding protein 1 (CIB1) ............................................................. 32 1.5.3: Chloride channel regulatory protein (ICln) ................................................................... 34 1.5.4: Protein phosphatase type 1 (PP1) and protein phosphatase type 2A (PP2A) ....... 34 1.5.5: Ancient ubiquitous protein 1 (AUP1) ............................................................................. 35 1.5.6: Triosephosphate isomerase (TIM) ................................................................................ 36 1.6: Platelets in cardiovascular disease .................................................................................. 36 1.6.1: Anti – platelet drugs ......................................................................................................... 37 1.6.2: Antagonists of the platelet integrin ................................................................................ 38 1.7: Potential for new anti-platelet drugs ................................................................................. 40 1.7.1: Extracellular domain inhibitors ....................................................................................... 40 1.7.2: Intracellular inhibitors ....................................................................................................... 41 Chapter 2 ........................................................................................................... 44 Materials and Methods ..................................................................................... 44 2.1: Materials ............................................................................................................................... 45 3 2.2: Methods ................................................................................................................................ 47 2.2.1: Preparation of washed platelets .................................................................................... 47 2.2.2: Platelet aggregation ......................................................................................................... 47 2.2.3: Cell adhesion assay for confocal microscopy .............................................................. 48 2.2.4: Platelet lysate generation ................................................................................................ 49 2.2.5: Tissue culture of CHO cells ............................................................................................ 50 2.2.6: Generation of CHO cell lysate ........................................................................................ 50 2.2.7: Determining the expression levels of αIIbβ3 in CHO cells ........................................... 51 2.2.8: Bradford assay.................................................................................................................. 51 2.2.9: Gel electrophoresis .......................................................................................................... 52 2.2.9.1: SDS-PAGE .................................................................................................................... 52 2.2.9.2: Sample preparation for SDS-PAGE ........................................................................... 53 2.2.10: Western blot .................................................................................................................... 53 2.2.11: Coomassie blue staining ............................................................................................... 54 2.2.12: Immunoprecipitation (IP) ............................................................................................... 55 2.2.12.1: Co-immunoprecipitation of RNF181 with integrin αIIbβ3 ........................................ 55 2.2.13: RNA interference............................................................................................................ 56 2.2.13.1: siRNA design ............................................................................................................... 56 2.2.13.2: siRNA delivery ............................................................................................................. 57 2.2.14: Recombinant poly His-tagged RNF181 protein expression in E.coli ..................... 58 2.2.14.1: Purification of poly His-tagged RNF181 protein ..................................................... 59 2.2.15: Integrin αIIbβ3 preparation .............................................................................................. 60 2.2.16: PepSpot peptide arrays on cellulose .......................................................................... 61 2.2.17: Pull down of native and purified integrin IIb3 with RNF181 derived peptides.... 63 2.2.18: Isothermal titration calorimetry (ITC)........................................................................... 63 2.2.19: Microscale Thermophoresis (MST) ............................................................................. 64 Chapter 3 ..........................................................................................................