Role of Antioxidants and Polyphenols in Thrombosis and Haemostatic Function

236 Pages·2014·3.29 MB·English

by  Abishek Bommannan

Role of Antioxidants and Polyphenols in
Thrombosis and Haemostatic Function

Abishek Bommannan Santhakumar
(B.Tech, SRM University; MSc, RMIT University)

School of Medical Science
Heart Foundation Research Centre
Griffith University

Submitted in fulfilment of the requirements of the degree of
Doctor of Philosophy

August 2014
STATEMENT OF ORIGINALITY

This work has not previously been submitted for a degree or diploma in any
university. To the best of my knowledge and belief, the thesis contains no material
previously published or written by another person except where due reference is made
in the thesis itself.

Name Abishek Bommannan, Santhakumar

Signature ____________________

Date ____________________

2
Table of Contents
ACKNOWLEDGEMENTS ................................................................... 14
1. Review of Literature ............................................................... 20
1.1 Introduction.............................................................................................. 22

1.2 Oxidative stress, antioxidants and antiplatelet therapy ....................... 24

1.3 Oxidative stress induced thrombosis and disease ................................. 26

1.4 Platelet activation and induced oxidative stress ................................... 31

1.5 Antioxidant rich functional foods .......................................................... 33

1.5.1 Polyphenols ............................................................................................ 37

1.5.2 Relationship between structure and antioxidant activity ....................... 41

1.6 Polyphenols and platelet function .......................................................... 42

1.7 Antiplatelet therapy ................................................................................. 51

1.8 Conclusion ................................................................................................ 54

1.9 Aims and Hypothesis ............................................................................... 55

2. Materials and Methods ........................................................... 58

2.1 Research ethics ......................................................................................... 59

2.2 Volunteer recruitment and Randomisation ........................................... 59

2.3 Evaluation of Food Diaries – FoodWorks ............................................. 61

2.4 Sample collection - Blood ........................................................................ 61

2.5 Sample processing - Blood ...................................................................... 63

2.6 Sample collection and Processing - Urine .............................................. 64

2.7 Full Blood Examination .......................................................................... 64

2.8 Platelet aggregation assay ....................................................................... 65

2.9 Platelet surface marker expression – Flowcytometry .......................... 67

2.10 Coagulation assay .................................................................................... 71

2.11 Biochemical assays ................................................................................... 71

2.12 Beverage analysis, plasma and urinalysis .............................................. 72

3. Taurine and Caffeine .............................................................. 73

3.1 Introduction.............................................................................................. 75

3.2 Materials and Methods ........................................................................... 78

3.2.1 Volunteers............................................................................................... 78

3.2.2 Study design ........................................................................................... 79

3.2.3 Blood collection ..................................................................................... 79

3.2.4 Full blood examination (FBE) ............................................................... 79

3.2.5 Taurine and Caffeine working solution .................................................. 79

3
3.2.6 Platelet aggregation assay ...................................................................... 81
3.2.7 Coagulation assay .................................................................................. 81

3.2.8 Lipid profile and C-reactive protein (CRP) assays ................................ 82

3.2.9 Statistical analysis .................................................................................. 82

3.3 Results ....................................................................................................... 82

3.4 Discussion ................................................................................................. 86

3.5 Conclusion ................................................................................................ 89

4. Hippuric acid – an active metabolite of polyphenols ........... 91

4.1 Introduction.............................................................................................. 93

4.2 Methods and Materials ........................................................................... 96

4.2.1 Subject screening and recruitment ......................................................... 96

4.2.2 Hippuric acid .......................................................................................... 96

4.2.3 Activation dependant platelet surface-marker expression ..................... 96

4.2.4 Statistical analysis .................................................................................. 97

4.3 Results ....................................................................................................... 97

4.4 Discussion ............................................................................................... 100

5. Polyphenols and Thrombosis ............................................... 104

5.1 Introduction............................................................................................ 106

5.2 Materials and methods .......................................................................... 108

5.2.1 Study participants and experimental design ........................................ 108

5.2.2 Study beverages ................................................................................... 111

5.2.3 Dietary intake monitoring .................................................................... 112

5.2.4 Blood and urine sample collection ....................................................... 112

5.2.5 Full blood examination and Biochemical assays ................................. 112

5.2.6 Platelet aggregation assay .................................................................... 113

5.2.7 Activation-dependent platelet surface marker expression ................... 113

5.2.8 Coagulation assay ................................................................................ 113

5.2.9 Statistical analysis ................................................................................ 113

5.3 Results ..................................................................................................... 114

5.4 Discussion ............................................................................................... 126

5.5 Conclusion .............................................................................................. 133

6. Polyphenols and Thrombosis under Oxidative stress ........ 135

6.1 Introduction............................................................................................ 137

6.2 Materials and methods .......................................................................... 139

6.2.1 Study participants and experimental design ........................................ 139

6.2.2 Blood Sampling ................................................................................... 143

4
6.2.3 Induced oxidative stress – Exercise tests ............................................. 143
6.2.4 Incremental exercise test ...................................................................... 143

6.2.5 Familiarisation trial .............................................................................. 144

6.2.6 Constant-load exercise tests ................................................................. 144

6.2.7 Study Beverages ................................................................................... 145

6.2.8 Platelet aggregation assay .................................................................... 145

6.2.9 Activation-dependant platelet surface-marker expression ................... 145

6.2.10 Coagulation assay ................................................................................ 145

6.2.11 Dietary intake monitoring .................................................................... 145

6.2.12 Statistical analysis ................................................................................ 146

6.3 Results ..................................................................................................... 146

6.3.1 Exercise induced oxidative stress ........................................................ 146

6.3.2 Ex-vivo analysis ................................................................................... 147

6.4 Discussion ............................................................................................... 154

6.5 Conclusion .............................................................................................. 159

7. Polyphenols and Gene Expression ....................................... 161

7.1 Introduction............................................................................................ 162

7.2 Materials and Methods ......................................................................... 163

7.2.1 Study design and sample collection ..................................................... 163

7.2.2 RNA isolation and cDNA synthesis ..................................................... 164

7.2.3 Primers ................................................................................................. 164

7.2.4 Real-Time PCR .................................................................................... 165

7.2.5 Statistical analysis ................................................................................ 166

7.3 Results and Discussion .......................................................................... 167

8. Summary and Future directions .......................................... 172

9. References .............................................................................. 178

10. Appendices ............................................................................. 202

10.1 Appendix 1 .............................................................................................. 203

10.2 Appendix 2 .............................................................................................. 205

10.3 Appendix 3 .............................................................................................. 207

10.4 Appendix 4 .............................................................................................. 212

10.5 Appendix 5 .............................................................................................. 218

10.6 Appendix 6 .............................................................................................. 223

10.7 Appendix 7 .............................................................................................. 234

10.8 Appendix 8 .............................................................................................. 235

10.9 Appendix 9 .............................................................................................. 236

5
LIST OF TABLES

Table 1. Health benefits of antioxidant rich functional foods or its components ............. 35
Table 2. Effect of different dietary phenolic compounds on platelet function ................. 48
Table 3. Blood tube types used in sample analysis .......................................................... 63
Table 4. Master Mix preparation for Antibody and Isotype control ................................. 69
Table 5. The effect of dose and time response of caffeine on platelet aggregation
induced by ADP & collagen .............................................................................................. 80
Table 6. Baseline parameters of full blood examination, platelet aggregation and
coagulation assays ............................................................................................................. 83
Table 7. Baseline parameters of study participants .......................................................... 98
Table 8. Baseline biochemical and haematological parameters of study participants ..... 109
Table 9. Composition of the three study beverages by analysis ....................................... 115
Table 10. Descriptive values of blood cell counts and biochemical parameters in 20
participants at baseline (pre-treatment) and end of 4 week intervention (post-
treatment) .......................................................................................................................... 116
Table 11. Mean values of total vitamins, caffeine and alcohol calculated from dietary
intake of 20 subjects over the course of three separate supplementation treatments ........ 119
Table 12. Baseline characteristics of study participants ................................................... 140
Table 13. Average heart rate values attained during the constant-load exercise trials ..... 147
Table 14. Mean total vitamins, caffeine and alcohol levels during QGPJ and PBO
supplementation period ..................................................................................................... 148
6
Table 15. Changes to haematological parameters before and after exercise induced
oxidative stress .................................................................................................................. 150
Table 16. Real-Time PCR primers ................................................................................... 165
Table 17. Analysis of mean dietary levels of total vitamins, caffeine and alcohol
during the 4-week supplementation period ....................................................................... 168
Table 18. Anthropometric data of participants before and after supplementation ........... 169

7
LIST OF FIGURES
Figure 1. Platelet activation in type 2 diabetes mellitus and metabolic syndrome .......... 30
Figure 2. Role of platelets in thrombosis ......................................................................... 32
Figure 3. Mechanism of inhibition of platelet aggregation/activation by
polyphenols/other antioxidants and anti-platelet drugs .................................................... 47
Figure 4. The potential impact of synergistic effect of antioxidant rich polyphenols
and current antiplatelet therapy in reducing thrombotic risks .......................................... 54
Figure 5. Step by step protocol followed in the evaluation of activation-dependant
platelet surface marker expression by flowcytometry ...................................................... 70
Figure 6. The effect of taurine, caffeine and taurine combined with caffeine (T+C) on
platelet aggregation induced by ADP ............................................................................... 84
Figure 7. The effect of taurine, caffeine and taurine combined with caffeine (T+C) on
platelet aggregation induced by collagen .......................................................................... 84
Figure 8. The effect of taurine (500 µM), caffeine (700 µM) and taurine combined
with caffeine (T+C) on prothrombin clotting time ........................................................... 85
Figure 9. The potential mechanism of absorption, metabolism and elimination of
anthocyanins into hippuric acid ........................................................................................ 95
Figure 10. The effect of varying concentrations of hippuric acid on P-selectin/CD62P
surface marker expression ................................................................................................. 99
Figure 11. The effect of different concentrations of hippuric acid on PAC-1 activation
dependant surface marker expression ............................................................................... 100
Figure 12. Double-blind, randomized, placebo controlled, dietary intervention trial ...... 111
8
Figure 13. Effect of 4-week supplementation of QGPJ, PJ and PBO on platelet
aggregation induced by platelet stimulants ....................................................................... 120
Figure 14. Activation-dependent platelet surface marker P-selectin/CD62P expression
before and after QGPJ, PJ and PBO supplementation ...................................................... 121
Figure 15. Effect of QGPJ, PJ, and PBO supplementation (4-weeks) on activated
partial thromboplastin clotting time and fibrinogen concentration ................................... 123
Figure 16. Plasma malondialdehyde (MDA) levels pre and post supplementation ......... 124
Figure 17. Hippuric acid concentrations in plasma and urine pre and post
supplementation ................................................................................................................ 125
Figure 18. Study design.................................................................................................... 142
Figure 19. Platelet aggregation at rest and post-exercise, before (QGP )and after 4-
PRE
weeks of Queen-Garnet plum juice supplementation (QGP ) ..................................... 149
POST
Figure 20. Platelet surface marker expression at rest and post exercise, before
(QGP ) and after 4weeks of Queen-Garnet plum juice supplementation (QGP ) ... 151
PRE POST
Figure 21. Effect of QGPJ supplementation on coagulation parameters ......................... 153
Figure 22. SELP gene expression before (QGP ) and after (QGP ) 28 day QGPJ
PRE POST
supplementation ................................................................................................................ 168
Figure 23. Summary of the studies conducted ................................................................. 177

9
LIST OF ABBREVIATIONS
ADP Adenosine diphosphate
AGE Advance glycation end-products
AM Aroniamelanocarpa
ANOVA Analysis of variance
AOX Antioxidant
APC Allophycocyanin
AP-1 Activator protein-1
aPTT Activated partial thromboplastin time
BMI Body mass index
BP Blood pressure
C3GE Cyanidin-3-glucoside equivalents
CABG Coronary artery bypass grafting
cAMP Cyclic adenosine monophosphate
cDNA complementary DNA
CHD Coronary heart disease
COX-1 Cyclooxygenase-1
CRP C-reactive protein
C Cycle threshold
T
CVD Cardiovascular disease
EC Endothelial cells
EDTA Ethylene diamine tetra-acetic acid
EGCG Epigallocatechin-3-gallate
eNOS Endothelial nitric oxide synthase
10