Studies on the regulation of tissue plasminogen activator activity by physiological templates

275 Pages·2012·2.89 MB·English

by  Stella Williams

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

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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.
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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.

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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
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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
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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
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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
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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
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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 (IC ) of
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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

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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

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