Studies on the Regulation of Tissue Plasminogen Activator Activity by Physiological Templates
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Studies on the regulation of tissue plasminogen activator activity by physiological templates A thesis presented by Stella Claire Williams for the degree of Doctor of Philosophy from Imperial College London Biotherapeutics Division National Institute for Biological Standards and Control Blanche Lane, South Mimms, EN6 3QG Department of Cell and Molecular Biology Imperial College London, Exhibition Road, South Kensington, London, SW7 2AZ 1 Abstract Myocardial infarction (MI) and ischemic stroke (IS) are caused by blood clots blocking arteries and cutting off the blood supply to tissues in the heart or brain. Clots may be cleared using plasminogen activators (“clot busters”) that convert plasminogen into plasmin, which in turn digests fibrin. The major plasminogen activator in the circulation is tissue plasminogen activator (tPA) and a recombinant form Alteplase was developed for use as a thrombolytic drug. However, newer engineered thrombolytics based on tPA have often been disappointing in clinical trials for MI, and there has been little progress in treatment for IS. Some of this lack of success may be due to gaps in our understanding of the details of the action of these drugs during clot dissolution as no rational models exists to understand the quantitative relationship between fibrin binding and plasminogen activation and hence fibrin lysis. Development of such models was the goal of this project. Fibrin binding is central in the regulation of tPA activity and binding is principally via 2 tPA domains, finger (F) and kringle 2 (K2), which were the focus of attention in the design of domain variants of tPA, including fusions with jelly fish fluorescent proteins. Protease activity was investigated initially with amidolytic substrates and then with plasminogen as substrate in the presence of templates: heparin, fibrinogen and fibrin, which co-localise tPA and substrate, plasminogen, to enhance plasmin generation. Kinetics of association and dissociation and equilibrium binding studies were investigated using surface plasmon resonance technology and ELISA-style plate binding assays. The results of these experiments showed that both the strength and pattern of binding of domain variants depends on the template. Models have been developed combining intrinsic enzyme kinetic and binding data to predict plasminogen activation rates in the presence of different templates. These studies are useful for optimising the design of thrombolytic drugs to treat MI and IS. 2 Acknowledgements I wish to thank my supervisors Dr. Colin Longstaff and Dr. Craig Thelwell at The National Institute for Biological Standards and Control and Prof. Neil Fairweather and Dr Kate Brown at Imperial College for the huge amount of support, encouragement and advice they have given me throughout this project. I would also like to thank Dr Marta Silver and Colin Whitton for all their useful advice. I also thank the British Heart Foundation for the financial support extended for these studies. 3 Table of contents Abstract 2 Acknowledgements 3 Table of Contents 4 List of Figures 11 List of Tables 17 Abbreviations 19 Statement of originality 22 CHAPTER 1: INTRODUCTION 1.1 Fibrinolysis 23 1.2 Myocardial infarction and the history of thrombolytics 26 1.3 Domains of plasminogen and tPA involved in binding to fibrin and fibrinogen 27 1.4 Fibrin and Fibrinogen 29 1.4.1 The structure of fibrin and fibrinogen 29 1.4.2 Conversion of fibrinogen to fibrin by thrombin 31 1.4.3 Digestion of fibrin by plasmin 33 1.4.4 tPA and plasminogen binding sites in fibrin and fibrinogen 33 1.5 tPA and plasminogen binding to heparin 35 1.6 tPA glycosylation 38 1.7 Surface plasmon resonance – Biacore 40 4 1.7.1 The CM range of sensor chips 42 1.7.2 The SA sensor chip 43 1.8 Project aims 44 CHAPTER 2: MOLECULAR BIOLOGY, RECOMBINANT PROTEIN EXPRESSION AND PURIFICATION 2.1 Introduction 49 2.1.1 Recombinant tPA 49 2.1.2 Production of tPA in insect cells 50 2.2 Materials 56 2.2.1 Insect cell lines 56 2.2.2 Escherichia coli strains 56 2.2.3 Plasmids 57 2.2.4 Chemicals, reagents and laboratory consumables 58 2.2.5 Buffers 59 2.3 Methods 59 2.3.1 Maintenance of E. coli cultures 59 2.3.2 General methods for DNA manipulation 60 2.3.2.1 Transformation of competent -select E. coli cells 60 2.3.2.2 Plasmid isolation from E. coli 60 2.3.2.3 Restriction endonuclease digestion of plasmid DNA and agarose gel electrophoresis 60 5 2.3.2.4 Site-directed mutagenesis 61 2.3.2.5 DNA sequencing 62 2.3.3 Generating plasmid constructs for expression of itPA variants 62 2.3.3.1 Generation of pFastBac-irPA 64 2.3.3.2 Generation of pFastBac-itPA-K1P 64 2.3.3.3 Generation of pFastBac - itPA: P 64 2.3.3.4 Generation of pFastBac-itPA: F del 65 2.3.3.5 Generation of pFastBac- itPA: F del-GFP 65 2.3.3.6 Maintenance of Sf9 cultures 66 2.3.3.7 Generating recombinant bacmid DNA using DH10Bac E. coli cells 66 2.3.3.8 Transfection of Sf9 cells to generate recombinant baculovirus 67 2.3.3.9 Baculovirus titre determination 67 2.3.3.10 Baculovirus amplification 68 2.3.4 Protein expression in Sf9 cells 69 2.3.4.1 Purification of recombinant tPA variants expressed in Sf9 cells 69 2.3.4.2 Purification of itPA-GFP 70 2.3.4.3 Ion exchange chromatography 70 2.3.5 Converting itPA into tcitPA using plasmin-Sepharose 71 2.3.6 Determination of protein concentration and purity 72 2.3.6.1 Dialysis 72 2.3.6.2 Protein concentration determination using Bradford assay 72 2.3.6.3 SDS-PAGE 72 2.3.6.4 Western Blotting 73 6 2.3.7 Fluorescence measurements of itPA-GFP and itPA: F del-GFP 73 2.4 Results 75 2.4.1 Expression and purification of tPA variants 75 2.4.2 tPA-GFP and tPA: F del-GFP 80 2.4.2.1 Fluorescence measurements of itPA-GFP fusions 80 CHAPTER 3: KINETICS 3.1 Introduction 91 3.2 Materials and Methods 94 3.2.1 Materials 94 3.2.2 Methods 95 3.2.2.1 tPA amidolytic activity in solution 95 3.2.2.2 Plasminogen activation with soluble templates 96 3.2.2.3 Plasminogen activation in fibrin clots 98 3.2.3 Data collection and analysis 102 3.2.3.1 Data processing 103 3.2.3.2 Fitting data to the Michaelis-Menten equation 105 3.2.3.3 Combistats 106 3.3 Results 108 3.3.1 Amidolytic activity of tPA variants in solution 108 3.3.2 Plasminogen activation with soluble templates 112 3.3.2.1 Plasminogen activation with fibrinogen as a template 112 7 3.3.2.2 Plasminogen activation with heparin as a template 121 3.3.3 Fibrin stimulation of tPA activity 126 3.3.3.1 Measuring initial plasminogen activation rates in fibrin and the rate of fibrin lysis 126 3.3.3.2 Fibrin as a template and the effects of plasminogen concentration on the domain variants 127 3.3.3.3 Fibrin as a template and the effects of different types of fibrin on the domain variants 132 CHAPTER 4: BINDING 4.1 Introduction 141 4.2 Materials and Methods 144 4.2.1 Materials 144 4.2.2 Methods 145 4.2.2.1 Fibrinogen plates 145 4.2.2.2 Fibrin plates 147 4.2.2.3 Data Analysis 148 4.2.3 Surface Plasmon Resonance – using the Biacore system 149 4.2.3.1 Lysine immobilization 150 4.2.3.2 Fibrinogen immobilization 150 4.2.3.3 Fibrin immobilization 152 4.2.3.4 Heparin immobilization 153 8 4.2.3.5 Biacore data analysis 154 4.2.4 Solubility studies 157 4.3 Results 158 4.3.1 Binding to immobilized fibrinogen and thrombin-treated fibrinogen (fibrin) using ELISA style assays 158 4.3.1.1 Determination of fifty percent inhibitory concentration (IC50) of 6-aminohexanic acid against itPA and variants binding to fibrinogen 158 4.3.1.2 Plasminogen activator range binding to immobilized fibrinogen 162 4.3.2 Binding of itPA and variants to immobilized templates measured by Surface Plasmon Resonance –Biacore 170 4.3.2.1 Binding of itPA and variants to immobilized L-lysine on a CM5 Sensor chip 170 4.3.2.2 Interaction of itPA, variants, and plasminogen with fibrinogen immobilized indirectly via anti-fibrinogen antibodies 175 4.3.2.3 Interaction of scitPA (R275E), tcitPA, and itPA with fibrinogen immobilized directly using covalent immobilization (amine coupling) 182 4.3.2.4 Interaction of itPA and domain variants with fibrinogen 195 4.3.2.5 Interaction of itPA, variants, and plasminogen with heparin-biotin immobilized on SA chips 203 4.3.2.6 Investigating conditions for improved tPA solubility 208 9 CHAPTER 5: MODELLING 5.1 Introduction 212 5.2 Methods 215 5.3 Results 220 5.3.1 Modelling plasminogen activation by scitPA (R275E) and tcitPA with fibrinogen as a template 220 5.3.2 Modelling plasminogen activation by itPA and variants with fibrinogen as a template 223 5.3.3 Modelling plasminogen activation by itPA with heparin as a template in two buffers with different NaCl concentrations 227 CHAPTER 6 DISCUSSION 231 6.1 Future work 252 Reference List 254 10 List of figures Figure 1.1: Scheme of fibrinolysis 24 Figure 1.2: Structure of the fibrinogen molecule 30 Figure 1.3: Formation of fibrin from fibrinogen 32 Figure 1.4: Schematic diagram of normal tPA (Alteplase) 45 Figure 1.5: Schematic diagram of normal rPA (Reteplase) 45 Figure 2.1: Expression vector pFastBac1.