The relationship between ADAMTS13 genotype and phenotype in congenital thrombotic thrombocytopenic purpura and characterisation of ADAMTS13 mutants Mary Imy Underwood Thesis submitted to University College London for the degree of Doctor of Philosophy University College London 2015 1 I, Mary Imy Underwood confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. …………………………………………………. 2 Abstract Congenital thrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy, usually involving ADAMTS13 gene defects. ADAMTS13 processes the multimeric plasma glycoprotein Von Willebrand factor making it less reactive to platelets. Patients differ in terms of disease severity and evidence suggests a relationship between ADAMTS13 genotype and disease phenotype. Over 140 mutations have been identified in patients but only ~30% of these has been expressed in vitro. The aim of this thesis was to study certain ADAMTS13 mutations identified in a homozygous form in congenital TTP patients to assess in vitro their effect on the secretion and activity of ADAMTS13 and to assess their contribution to disease phenotype. ADAMTS13 mutants (p.R102H, p.I143T and p.Y570C) and wild type (WT) were expressed in HEK293T cells. The p.R102H mutation partially affected the secretion of ADAMTS13 and reduced the catalytic efficiency of the mutant but not to the extent predicted based upon levels measured in patient plasma. Expressing this mutant with three ADAMTS13 polymorphisms (p.Q448E, p.P618A and p.A900V) which were also identified in the patient with this mutation further reduced the secretion and activity of ADAMTS13. When these three polymorphisms were expressed separately in WT ADAMTS13, the p.P618A polymorphism reduced the secretion and subsequently the activity of ADAMTS13 suggesting that this polymorphism in particular was responsible for the reduction observed. These results highlight the importance of ADAMTS13 polymorphisms. The p.I143T and p.Y570C mutations severely affected ADAMTS13 secretion. Immunofluorescence studies showed localisation of these mutants within the ER but less extensive localisation within the cis Golgi compared to WT ADAMTS13. The p.I143T mutant was characterised further and was shown to be degraded by the cell proteasome. Addition of a chemical chaperone (betaine) appeared to rescue the secretion defect caused by the p.I143T mutation. This may have future therapeutic implications for the treatment of some congenital TTP patients. 3 Acknowledgements I would like to thank my supervisors Dr. Ian Mackie, Professor Samuel Machin and Professor Flora Peyvandi for their help and support throughout the last four years. I also would like to thank all staff at the Haemostasis Research Unit in UCL and the Angelo Bianchi Bonomi Hemophilia and Thrombosis Centre, Milan, in particular Dr. Isabella Garagiola. I would also like to thank Dr. Raimondo De Cristofaro, Dr. Stefano Lancelloti, Mr George Chennel and Mr Tomas Adejumo for their technical support. Finally I would like to thank my family for their support throughout. 4 Contents Abstract ...................................................................................................................... 3 Acknowledgements .................................................................................................... 4 Chapter 1 Introduction ........................................................................................ 21 1.1 Von Willebrand Factor ............................................................................ 21 1.1.1 VWF gene, synthesis, secretion ......................................................... 21 1.1.2 VWF function .................................................................................... 22 1.1.3 ADAMTS13 regulates the size of VWF ............................................ 25 1.2 The ADAMTS13 gene, synthesis and secretion ....................................... 27 1.2.1 Gene ................................................................................................... 27 1.2.2 Synthesis ............................................................................................ 28 1.2.3 Secretion ............................................................................................ 28 1.3 The ADAMTS13 protein......................................................................... 29 1.3.1 The ADAMTS family ........................................................................ 29 1.3.2 ADAMTS13 ....................................................................................... 30 1.3.3 Propeptide .......................................................................................... 31 1.3.4 Metalloprotease domain ..................................................................... 31 1.3.5 Disintegrin-like domain ..................................................................... 32 1.3.6 TSP-1 domain .................................................................................... 33 1.3.7 Cysteine-rich domain ......................................................................... 34 1.3.8 Spacer domain .................................................................................... 34 1.3.9 TSP-1:2→TSP-1:8, CUB-1 and CUB-2 domains ............................. 35 1.4 ADAMTS13 cleavage of VWF ............................................................... 37 1.4.1 Factors affecting cleavage of VWF by ADAMTS13 ........................ 39 1.4.2 Disulphide bond reduction of VWF by ADAMTS13 ........................ 41 1.4.3 VWF and ADAMTS13 cleavage ....................................................... 42 1.5 Thrombotic thrombocytopenic purpura................................................... 42 5 1.5.1 Signs and symptoms .......................................................................... 42 1.5.2 Incidence ............................................................................................ 43 1.5.3 Pathophysiology ................................................................................. 44 1.5.4 Triggers:infection ............................................................................... 44 1.5.5 Triggers: Pregancy ............................................................................. 45 1.5.6 Treatment ........................................................................................... 45 1.6 Congenital TTP ....................................................................................... 46 1.6.1 ADAMTS13 mutations ....................................................................... 47 1.6.2 ADAMTS13 polymorphisms .............................................................. 47 1.6.3 Mouse models of congenital TTP ...................................................... 48 1.6.4 Residual ADAMTS13 activity ........................................................... 50 1.7 Acquired TTP .......................................................................................... 54 1.7.1 Baboon models of acquired TTP ....................................................... 55 1.8 New therapeutic options for TTP ............................................................ 55 1.9 ADAMTS13 deficiency in the setting of other diseases ......................... 59 1.10 ADAMTS13 genetic variation in diseases other than TTP ...................... 60 1.11 Aims of thesis .......................................................................................... 60 Chapter 2 Materials and methods ....................................................................... 61 2.1 In silico analysis of ADAMTS13 missense mutations ............................. 61 2.2 Creation of ADAMTS13 missense mutations .......................................... 62 2.2.1 Sequencing ADAMTS13 cDNA ......................................................... 62 2.2.2 Site directed mutagenesis ................................................................... 62 2.2.3 DpnI digestion and agarose gel electrophoresis................................. 63 2.2.4 Transformation ................................................................................... 63 2.2.5 Mini preparation................................................................................. 64 2.2.6 Sequencing of construct to confirm presence of mutation ................. 64 2.2.7 Large scale plasmid preparation ........................................................ 65 6 2.3 Transient transfection of ADAMTS13 variants ...................................... 66 2.3.1 HEK 293T cells.................................................................................. 66 2.3.2 Transient transfection......................................................................... 66 2.3.3 Normalisation after transient transfection .......................................... 67 2.3.4 Concentrating media .......................................................................... 68 2.4 Creation of stable lines ............................................................................ 68 2.5 Western blotting ...................................................................................... 69 2.6 ADAMTS13 antigen ELISA ................................................................... 70 2.6.1 In-house ADAMTS13 antigen ELISA............................................... 70 2.6.2 Commercial ADAMTS13 antigen ELISA ......................................... 71 2.7 ADAMTS13 activity assay: FRETS-VWF73 ........................................