Characterization of CD109 by Joseph Yao Abotsi Prosper A thesis submitted in conformity with the requirements Doctor of Philosophy Graduate Department of Medical Biophysics University of Toronto. © Copyright by Joseph Yao Abotsi Prosper 2011 Characterization of CD109 Joseph Yao Abotsi Prosper Doctor of Philosophy 2011 Department of Medical Biophysics, University of Toronto ABSTRACT: CD109 is a 170kD glycosylphosphatidylinositol-anchored protein expressed on subsets of fetal and adult CD34+ haematopoietic stem cells, endothelial cells, activated T cells, and activated platelets. Cloning of the CD109 cDNA by our group identified the molecule as a novel member of the 2M/C3/C4/C5 family of thioester containing proteins. Curiously, CD109 bears features of both the 2M and complement branches of the gene family. Additionally CD109 carries the antigenic determinant of the Gov alloantigen system, which has been implicated in a subset of immune mediated platelet destruction syndromes. In this thesis, the status of CD109 in the evolution and phylogeny of the A2M family has been clarified. First, I elucidated the evolutionary relationships of CD109, and of the other eight human A2M/C3/C4/C5 proteins, using sequence analysis and a detailed comparison of the organization of the corresponding loci. Extension of this analysis to compare CD109 to related sequences extending back to placazoans, defined CD109 as a member of a distinct and archaic branch of the A2M phylogenetic tree. Second, in conjunction with collaborators, the molecular basis of the Gov alloantigen system was identified as an allele specific A2108C; Y703S polymorphism. Utilizing cDNA and genomic sequence we then developed methods to accurately and precisely genotype the Gov system. Finally, the expression kinetics of platelet CD109 was elucidated, in order to obtain basic information regarding its expression and ii subcellular localization, and to resolve discrepancies in reported platelet CD109 expression. Quantitative flow cytometry demonstrated that CD109 was expressed on the surface of activated platelets at very low levels in most healthy volunteers. In resting platelets, CD109 was localized to the OCS and intracellular storage granules. CD109 displayed differential agonist induced expression in comparison to GPIIb/IIIa epitope unmasking, and surface expression of CD62P and CD63. CD109 was rapidly expressed on the cell surface in response to low doses of both strong and weak agonists. This early expression is likely the result of CD109’s proximity to the plasma membrane in resting platelets. As such, CD109 is positioned to participate at early stages of primary haemostasis. iii ACKNOWLEDGEMENTS: For those of you who work at, or have visited the Princess Margaret Hospital you may have noticed the inspirational whiteboard on the main floor adjacent to the phlebotomy clinic. Today’s message - “Its people not things that are life’s most valued treasures” - is the perfect framework as I reflect on my PhD. I would like to thank my parents for all the support and sacrifices they have made for me (and my siblings). My siblings, I am fortunate to have such wonderful people I can call family, thank-you for being you. I would like to thank Dr. Andre Schuh, for taking me on as a student in his lab. Thank- you, for the guidance, support, and sharing of your scientific and clinical knowledge. I would also like to thank-you (on behalf of my family as well) for your compassion during my father’s illness, I am eternally grateful. This thesis manuscript is the culmination of years of work, with its associated crests and nadirs. There have been a number of people that have been instrumental in helping me along the journey. I would like to take this opportunity to thank a few of them. Wendy, for her technical and intellectual input, and of course our diverse topics of discussion. The evergreen Xiang-Fu, I can only dream of isolating proteins with your yield. Thank-you to all my student colleagues of the Schuh lab, past and present: Denise, Sandra, Abhijit, Martin, Quang, Iris, and Kaustav. Special thanks to Andrea, Boyko, Rachel, Smita, and Joanne. I have been blessed to have some great people in my life. Your friendship has and will continue to be invaluable to me. Comfort, Ricki, Ivy, Isabel, Kwesi, Bright, Stephen, Imtiaz, Joanne K., Jose, Abhijit, Ryan, Sam, Mandy, Kristina, Joanne S., Neil C., Ockie, Rajal, Teresa, Hyun, Neal, Lan, Sandeep, Rich, Dennis, Fernando, Bart, Jon N., Nosh, Sian, Graeme, Christine, Ron, Monica, and Reza, words cannot express the gratitude I have for you all. My soccer team- mates thanks for the good times on and off the pitch. If I have missed anyone I meant no slight. J. iv TABLE OF CONTENTS: pg. ABSTRACT ii ACKNOWLEDGEMENTS iv TABLE OF CONTENTS v LIST OF TABLES viii LIST OF FIGURES Ix ABBREVIATIONS x Chapter 1: GENERAL INTRODUCTION 1 1.1 PREFACE 1 1.2 CD109 6 1.2.1 Historical description of CD109 6 1.2.2 Cloning of CD109 6 1.2.3 CD109 is a member of the 2macroglobulin/C3, C4, C5 family 7 1.2.4 Functional attributes of 2macroglobulin 7 1.2.5 Complement C3, C4, and C5 8 1.2.6 CD109 shares features of both Complement C3 and 2M 11 1.3 PLATELET PHYSIOLOGY 12 1.3.1 Platelet surface receptors 13 1.3.1.1 GPIb-IX-V 13 1.3.1.2 GPIIb/IIIa 15 1.3.1.3 Collagen receptors 17 1.3.1.4 Protease activated receptors 18 1.3.1.5 ADP receptor 22 1.3.1.6 Thromboxane receptor 23 1.3.1.7 Epinephrine receptor, 2A-adrenoreceptor 24 1.3.1.8 Serotonin receptors 26 1.3.1.9 Human platelet antigens 27 1.3.2 Platelet ultrastructure 28 1.3.2.1 Open canalicular system (OCS) 28 1.3.2.2 Dense tubular system (DTS) 30 1.3.2.3 Dense granules (-granules) 30 1.3.2.4 Alpha granules (-granules) 31 1.3.2.5 Lysosomes 32 1.3.3 Granule biogenesis 32 1.3.4 Platelet degranulation 33 1.3.4.1 Platelet release reaction 34 1.3.4.2 Heterogeneity and differential release of granules 38 Chapter 2: CD109 GENOMIC STRUCTURE AND PHYLOGENETIC ANALYSIS 52 v 2.1 INTRODUCTION 52 2.2 OBJECTIVE AND AIMS 56 2.3 EXPERIMENTAL PROCEDURES 57 2.3.1 Chromosomal localization of CD109 57 2.3.2 Genomic structure of CD109 58 2.3.3 Sequence analysis 59 2.4 RESULTS 61 2.4.1 Chromosomal Mapping of the CD109 locus 61 2.4.2 Genomic Structure of CD109 61 2.4.3 The Human A2M Family 62 2.4.4 Conservation of Genomic Structure in the Human A2M Family 63 2.4.5 Conservation of Genomic Structure of Mammalian and Avian CD109 64 2.4.6 Phylogenetic Position of CD109 65 2.5 DISCUSSION 81 Chapter 3: MOLECULAR DETERMINATION OF GOV ALLOANTIGEN DETERMINANTS 91 3.1 INTRODUCTION 91 3.2 OBJECTIVE AND AIMS 94 3.3 EXPERIMENTAL PROCEDURES 95 3.3.1 Donor panels 95 3.3.2 Gov phenotyping 95 3.3.3 cDNA synthesis and analysis of CD109 sequence 95 3.3.4 DNA sequence analysis of introns flanking Gova/b polymorphism-bearing CD109 exon 97 3.3.5 PCR-SSP analysis of Gova/b alleles 97 3.3.6 PCR-RFLP analysis of Gova/b alleles 97 3.3.7 Allele-specific real-time PCR analysis 98 3.4 RESULTS 99 3.5 DISCUSSION 106 CHAPTER 4 : QUANTIFICATION, SUBCELLULAR LOCALIZATION, AND EXPRESSION OF PLATELET CD109 111 4.1 INTRODUCTION 111 4.2 OBJECTIVES AND AIMS 113 4.3 EXPERIMENTAL PROCEDURES 114 4.3.1 Materials 114 4.3.2 Blood collection 114 4.3.3 Platelet preparation 115 4.3.4 Flow cytometric determination of epitope binding sites 115 4.3.5 Flow cytometric analysis of agonist induced responses 116 4.3.6 Flow cytometric determination of intracellular pools of CD109 116 4.3.7 Subcellular fractionation of platelet lysates 117 4.3.8 Immunoprecipitation 118 4.3.9 Immunofluorescence microscopic analysis of platelets 118 4.3.10 Immunoelectron microscopic analysis of platelets 119 4.3.11 Statistical Analysis 120 4.4 RESULTS 121 4.4.1 Quantification of CD109 levels on PAR1-AP stimulated platelets 121 vi 4.4.2 Cell surface CD109 is upregulated on suboptimally stimulated platelets 121 4.4.3 CD109 activation kinetics 122 4.4.4 Flow cytometric analysis reveals a subcellular pool of CD109 123 4.4.5 CD109 codistributes with granular and membrane fractions in sucrose density gradients 124 4.4.6 Subcellular localization of CD109 by immunofluorescence microscopy 125 4.4.7 Immunoelectron microscopic detection of platelet CD109 125 4.5 DISCUSSION 143 CHAPTER 5: SUMMARY AND FUTURE DIRECTIONS 150 5.1 Summary 150 5.2 Future Studies 151 REFERENCES 159 vii LIST OF TABLES: Table pg. 1 -1 Platelet granule contents 50 1-2 Platelet granule membrane proteins 51 2 -1 Oligonucleotides used for the determination of the intron/exon structure of human CD109 68 2-2 Intron/exon structure of CD109 69 2-3 Conservation of intron phase among human A2M family genes 70 2-4 Conservation of intron phase among mammalian and avian CD109 71 2-5 Conservation of intron phase among human CD109, C. elegans, and Drosophila TEP 72 viii LIST OF FIGURES: Figure pg. 1 - 1 CD109 cDNA schematic 39 1 - 2 CD109 primary structure 40 1 - 3 2M thioester reaction mechanism 41 1 - 4 C3 thioester reaction mechanism 42 1 - 5 Classical and lectin complement pathways 43 1 - 6 Alternative complement pathway 44 1 - 7 Overview of haemostasis 45 1- 8 Adhesion and activation of platelets 46 1 - 9 Platelet agonist receptors 47 1 - 10 Major input signals regulating calcium in platelets 48 1 - 11 Role of SNAREs in -granule secretion 49 2 - 1 The genomic organization of CD109 73 2 - 2 Phylogenetic relationship of human A2M family proteins 74 2 - 3 Conservation of genomic structure among human A2M family members 75 2 - 4 Conservation of genomic structure among CD109 orthologues 76 2- 5 A cross-phyla A2M family phylogenetic tree 77 Chapter 2 figure legends 79