The control of von Willebrand factor multimer size John Eshantha Pimanda A thesis submitted in fulfillment of the requirements for the degree of Doctor of Philosophy Faculty of Medicine, University of New South Wales December 2003 Acknowledgements A courageous six year old girl with congenital TTP participated in our treatment trial; I thank her and her parents for their trust. Dr. Julian Paxton, Mr. Andrew Schultz, Ramsay Health and the staff at St. John of God pathology and the children's ward at Mildura Base Hospital made the study possible. Over twenty five patients with a past diagnosis of TTP participated in a study to identify mutations in the thrombospondin-1 gene; I thank them and their physicians for their time and effort. I thank Professor Philip Hogg for supervising my work. The power of his ideas and generosity of spirit have taught me much. I have enjoyed the three years spent in his laboratory. Professor Colin Chesterman has guided my development as a haematologist and a scientist. I have benefited from the clarity of his reasoning and value his mentorship. Professor Beng Chong encouraged me to pursue a career in research at an early stage in my clinical training; Drs. Robert Lindeman and Michael Buckley gave of their time and expertise. Ms. Sue Evans, Ms. Janet Argyl, Mr. Robert Casten and Ms. Bernadette O'Reilly in the haematology department at the Prince of Wales Hospital were ever generous with their time and expertise. The staff of the blood bank accommodated my most unreasonable requests. Mr. Peter Taylor and the staff in the molecular genetics department provided valuable technical advice and practical help. Professor Michael Berndt, Monash University and Dr. Emanuel Favaloro, ICPMR Westmead were gracious with time, materials and ideas. My colleagues at the Centre for Vascular Research were a joy to work with. Dr Xing-mai Jiang and Ms. Akiko Maekawa introduced me to the fundamentals of molecular biology and protein expression. Drs. Angelina Lay, Mark Raftery and Troels Wind furthered my understanding in protein chemistry. Ms. Lisa Sun and Ms. Lakmini Weerakoon helped with material preparation and Mr. Geoff Kershaw (at the Royal Prince Alfred Hospital) and Mr. Tim Ganderton with running VWF multimer gels. Dr. Susan Maastricht and Ms. Christine Sutter facilitated the animal work. The National Health and Medical Research Council of Australia provided financial support for which I am most grateful. My parents in law, Rita and Hector Welgampola, with their presence have been a major asset to the completion of this thesis. My parents, Frank and Sriyani Pimanda continue to contribute selflessly to my happiness as they always have. I dedicate this thesis to my wife, Miriam ii Publications arising from this thesis Refereed journal articles published Pimanda JE, Maekawa A, Wind T, Paxton J, Chesterman CN, Hogg PJ. Congenital Thrombotic Thrombocytopenic Purpura in association with a mutation in the second CUB domain of ADAMTS13. Blood 2003 (prepublished online September 25) Pimanda JE, Annis DS, Raftery M, Mosher DF, Chesterman CN, Hogg PJ. The von Willebrand factor-reducing activity of thrombospondin-1 is located in the calcium binding/C-terminal sequence and requires a free thiol at position 974. Blood. 2002 Oct 15; 100(8):2832-8. Pimanda JE, Chesterman CN, Hogg PJ. A Perspective on the Measurement of ADAMTS13 in Thrombotic Thrombocytopaenic Purpura. European Journal of Haematology. 2003 Apr; 70 (4):257-62. Pimanda J, Hogg P. Control ofvon Willebrand factor multimer size and implications for disease. Blood Rev. 2002 Sep; 16 (3):185-92 Pimanda JE, Lowe HC, Hogg PJ, Chesterman CN, Kachigian LM. Novel and emerging therapies in cardiology and haematology. Current Drug Targets­ Cardiovascular & Haematological Disorders, 2003; 3 (2):101-123 iii Refereed journal articles submitted Pimanda JE, Ganderton T, Lawler J, Kershaw G, Maekawa A, Chesterman CN, Hogg PJ. Role ofthrombospondin 1 in control ofvon Willebrand Factor multimer size in mice. Referenced abstracts Pimanda JE, Xie LJ, Chesterman CN, Hogg PJ. Control ofvon Willebrand Factor Multimer Size. Blood 2001 Nov 16, 98(11): 160a. Pimanda JE, Annis DS, Raftery M, Mosher DF, Chesterman CN and Hogg PJ. The von Willebrand Factor Reducing Activity ofThrombospondin-1 Centres around a Free Thiol at Position 974. Blood2002 Nov 16, 100(11): 980a. Pimanda JE, Kershaw G, Lawler J, Chesterman CN, Hogg PJ. The control of von Willebrand Factor multimer size by Thrombospondin-1. Journal of Thrombosis and Haemostasis 2003 (in press). Presented abstracts Xie LJ, Pimanda JE, Chesterman CN, Hogg PJ. The control ofvon Willebrand factor multimer size by thrombospondin-1. Haematology Society of Australia and New Zealand/ Australasian Society of Blood Transfusion/ Australasian Society of Thrombosis and Haemostasis Joint Annual meeting.(HSANZ/ ASBT / ASTH) Brisbane. September 2001. iv Pimanda JE, Annis DS, Raftery M, Mosher DF, Chesterman CN and Hogg PJ. Localization of the von Willebrand factor reducing activity of thrombospondin-1. The Australian Society of Medical Research Scientific Meeting. Sydney. June 2002. Pimanda JE, Annis DS, Raftery M, Mosher DF, Chesterman CN and Hogg PJ. The von Willebrand Factor Reducing Activity ofThrombospondin-1 is located in the Calcium-binding/C-globular Domain and Requires a Free Thiol at Position 974. HSANZ/ASBT/ASTH Joint Annual Meeting. Adelaide. September 2002. Pimanda JE, Kershaw G, Lawler J, Chesterman CN, Hogg PJ. The control of vWF multimer size by thrombospondin-1. A study of Plasma and platelet VWF multimer size in thrombospondin-1 null mice. The Royal College of Pathologists of Australasia­ Pathology Update 2003. Sydney. March 2003. Pimanda JE, Ganderton T, Kershaw G, Lawler J, Chesterman CN, Hogg PJ. Role of thrombospondin-1 in the control of VWF multimer size in mice. The Australian Vascular Biology Society and Australian Atherosclerosis Society-annual scientific meeting. Ballarat, Victoria, September 2003. Pimanda JE, Ganderton,T, Kershaw G, Lawler J, Chesterman CN, Hogg PJ. Role of thrombospondin-1 in the control ofVWF multimer size in mice. HSANZ/ASBT/ASTH­ joint annual scientific meeting, Christchurch, New Zealand, October 2003 V Table of contents page List of abbreviations 1 Abstract 4 Preface 6 Chapter 1: Literature review 8 Introduction 9 1.1 von Willebrand factor 10 1.1.1 Synthesis 10 1.1.2 Storage and secretion 12 1.2 Control of plasma VWF multimer size-by proteolytic cleavage 13 1.2.1 ADAMTS13-a novel VWF cleaving protease 15 1.2.1.1 Tracing the ADAMTS13 gene 15 1.2.1.2 Phylogeny of ADAMTS13 17 1.2.1.3 The ADAMTS13 proenzyme 17 1.2.1.4 The domain structure of ADAMTS13 and relevance to function 19 1.2.1.5 The Tyr1605 and Met1606 cleavage site resides within the A2 domain of VWF 21 1.2.1.6 Glycosylation of ADAMTS13 23 1.2.1.7 Measurement of ADAMTS13 activity in plasma 24 1.3 Control of plasma VWF multimer size -by a VWF reductase 29 1.3.1 The thrombospondin gene family 35 1.3.1.1 Evolution of the thrombospondin gene family 37 1.3.1.2 The structure ofTSP-1 and relevance to function 38 1.3.1.3 The structure and function relationship between TSP-1 and VWF 44 vi 1.3.1.4 TSP-1 as an enzyme inhibitor 45 1. 3 .1. 5 The phenotype of the TSP-1 null mouse 46 1.4 Control of platelet VWF multimer size 47 1.5 VWF multimer size and disease 50 1.5.1 Deficiency of high molecular weight VWF multimers and bleeding 50 1.5.2 Persistence of high molecular weight VWF multimers and thrombosis 52 1.5.2.1 The thrombotic microangiopathies 52 1.5.2.2 VWF: atherosclerosis and arterial thrombosis 56 1.5.2.3 Pre-eclampsia, eclampsia and the HELLP syndrome 58 Chapter 2: The VWF reducing activity ofthrombospondin-1 60 2.1 Summary 61 2.2 Introduction 62 2.3 Materials and methods 64 2.4 Results 69 2.5 Discussion 81 Chapter 3: Role ofthrombospondin-1 in the control of VWF multimer size in mice 85 3.1 Summary 86 3.2 Introduction 87 3.3 Materials and methods 90 3.4 Results 97 3.5 Discussion 112 vii Chapter 4: Role ofthrombospondin-1 in TTP 117 5.1 Summary 118 5.2 Introduction 119 5.3 Patients and methods 120 5.4 Results and discussion 125 Chapter 5: TTP in association with a mutation in the second CUB domain of ADAMTS13 135 4.1 Summary 136 4.2 Introduction 136 4.3 Materials and methods 138 4.4 Results 141 4.5 Discussion 146 Chapter 6: Summary and future directions 150 References 157 viii List of abbreviations and acronyms used AEBSF 4-(2-aminoethyl) benzensulfonyl fluoride ADAM ~ gisintegrin-like ~d metalloprotease ADAMTS ~ .disintegrin-like ~nd metalloprotease with thrombo.s_pondin type 1 motif BSA bovine serum albumin CBA collagen binding activity CK cysteine knot COMP cartilage oligomeric matrix protein COS-7 cell line (monkey, African green, Kidney) fibroblast like CP123 procollagen and properdin (type 1) repeats 1-3 CPA cone and plate (let) analyzer CUB £Omplement components Cl r/Cls, sea yrchin epidermal growth factor and .b_one morphogenetic protein DDAVP 1-desamino-8-D-arginine vasopressin DTNB 5, 5 '-dithiobis (2-nitrobenzoic acid) E3CaG third type 2, type 3 and C-terminal globular domain EDTA ethylenediaminetetraacetic acid EGF epidermal growth factor ELISA enzyme linked immunosorbant assay ER endoplasmic reticulum FFP fresh frozen plasma 1 GSH reduced glutathione HBS HEPES buffered saline HELLP haemolysis, elevated liver function and low proteins HEPES (N-[2-Hydroxyethyl] piperazine-N' -[2-ethanesulfonic acid]) HIV human immunodeficiency virus HMWM high molecular weight multimers HUS haemolytic uremic syndrome HUVEC human umbilical vein endothelial cell