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Platelet and Erythrocyte Functional Defects Caused by Mutations of Transcription Factor GFI1B

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Platelet and Erythrocyte Functional Defects Caused by Mutations of Transcription Factor GFI1B Platelet and erythrocyte functional defects caused by mutations of transcription factor GFI1B Lucinda Beutler A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Faculty of Medicine University of Sydney December 2018 I praise you because…… I am fearfully and wonderfully made; Your works are wonderful - I know that full well. Psalm 139:14 (NIV) TABLE OF CONTENTS STATEMENT OF ORIGINALITY ...................................................................................................... vi ACKNOWLEDGEMENTS .............................................................................................................. vii ABSTRACT ................................................................................................................................... ix PUBLICATIONS AND PRESENTATIONS ........................................................................................... x LIST OF FIGURES .......................................................................................................................... xi LIST OF TABLES........................................................................................................................... xv ABBREVIATIONS ........................................................................................................................ xvii 1. Literature review ...................................................................................................................... 2 1.1 Introduction ....................................................................................................................... 2 1.2 Haemostasis and bleeding disorders ................................................................................... 2 1.2.1 Haemostasis................................................................................................................. 2 1.2.2 Bleeding disorders ........................................................................................................ 5 1.3 Platelets ............................................................................................................................. 5 1.3.1 Platelet structure ......................................................................................................... 7 1.3.2 Platelet adhesion ......................................................................................................... 8 1.3.3 Platelet activation ........................................................................................................ 8 1.3.4 Platelet aggregation, platelet plug, and fibrinolysis ....................................................... 9 1.4 Overview of haematopoiesis, megakaryopoiesis and thrombopoiesis, and erythropoiesis .... 9 1.4.1 Haematopoiesis ........................................................................................................... 9 1.4.2 Megakaryopoiesis and thrombopoiesis ....................................................................... 11 1.4.3 Erythropoiesis ............................................................................................................ 12 1.5 Regulation of haematopoiesis ........................................................................................... 13 1.5.1 Haematopoietic growth factors .................................................................................. 13 1.5.2 Haematopoietic transcription factors .......................................................................... 15 1.5.3 Overview of transcription factors in erythro-megakaryopoiesis ................................... 17 1.6 Transcription factor defects in erythropoietic and megakaryopoietic disorders .................. 23 1.6.1 GATA1 ....................................................................................................................... 23 1.6.2 RUNX1 ....................................................................................................................... 25 1.6.3 FLI1 ............................................................................................................................ 26 1.6.4 KLF1 ........................................................................................................................... 28 1.6.5 GFI1B ......................................................................................................................... 29 1.7 Investigation into a familial bleeding disorder due to GFI1B mutation .............................. 31 1.7.1 Background ................................................................................................................ 31 1.7.2 Laboratory investigations ........................................................................................... 32 i 1.7.3 Genetic testing ........................................................................................................... 32 1.8 Hypotheses and aims ...................................................................................................... 34 1.8.1 Hypothesis ................................................................................................................. 34 1.8.2. Aims ......................................................................................................................... 34 2. General materials and methods .............................................................................................. 36 2.1 K562 cell culture ............................................................................................................... 36 2.2 Mouse models .................................................................................................................. 36 2.2.1 Mouse lines ............................................................................................................... 36 2.2.2 DNA extraction and PCR genotyping ........................................................................... 37 2.2.4 Mouse surgery ........................................................................................................... 42 2.2.5 Mouse blood and tissue collection .............................................................................. 43 2.2.6 Pregnancy trial ........................................................................................................... 46 2.2.7 Mouse tail bleeding times .......................................................................................... 47 2.2.8 Mouse thrombopoietin (TPO) ..................................................................................... 47 2.2.9 Cytospin ..................................................................................................................... 47 2.2.10 Flow cytometry – mouse tissue ................................................................................. 48 2.2.11 Histology .................................................................................................................. 50 2.2.12 Immunohistochemistry............................................................................................. 51 2.2.13 Transmission electron microscopy (TEM) .................................................................. 53 2.2.14 Cell culture of megakaryocytic progenitor cells .......................................................... 54 2.2.15 BSA gradient preparation of megakaryocytes from GFI1B TG and control mice ........... 55 2.2.16 Nested PCR for DNA sequencing of cDNA from extracted RNA of megakaryocytes from GFI1B TG and control mice .................................................................................................. 56 2.2.17 Transfection of FLAG vector in CHO cells for use as positive control for immunofluorescence (IF) microscopy and Western blot (WB) for GFI1B TG and control mice 57 2.2.18 Immunofluorescence microscopy of megakaryocytes from GFI1B TG and control mice .......................................................................................................................................... 58 2.2.19 Western blot of FLAG-tagged GFI1B (nuclear) protein in GFI1B TG and control mice ... 60 2.2.20 Data analysis ............................................................................................................ 62 3. Effect of GFI1Bc.880-881insc on erythropoiesis in an erythroleukaemic cell culture model ............... 64 3.1 Introduction ..................................................................................................................... 64 3.1.1 Background ................................................................................................................ 64 3.1.2 Red cell membrane and cytoskeletal abnormalities ..................................................... 65 3.1.3 Experimental design: cell model system for studying the effect of GFI1Bc.880-881insC on erythropoiesis .................................................................................................................... 66 3.1.4 K562 erythroleukaemic cell line in human disease research ......................................... 67 3.2 Establishment of cell culture ............................................................................................. 68 ii 3.2.1 Transfection ............................................................................................................... 68 3.2.2 Cell culture ................................................................................................................. 68 3.3 Cell growth and proliferation ...........................................................................................
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