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Regulation of Blood Platelet Function by Nitric Oxide

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Regulation of Blood Platelet Function by Nitric Oxide THE UNIVERSITY OF HULL REGULATION OF BLOOD PLATELET FUNCTION BY NITRIC OXIDE JONATHAN D WAKE SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY POST GRADUATE MEDICAL INSTITUTE AND HULL YORK MEDICAL SCHOOL 2013 I Abstract Upon vascular injury, platelets instantly adhere to the exposed extracellular matrix resulting in platelet activation and aggregation to form a haemostatic plug. This self- amplifying mechanism requires a tight control to prevent uncontrolled platelet aggregate formation that could occlude the vessel. Endothelial-derived nitric oxide (NO) and prostacyclin (PGI 2) are strong negative regulators that modulate platelet adhesion, activation, aggregation, secretion and shape change. In this study the effects of NO on Ca2+ dependent and independent pathways of activation were investigated. The data produced during the course of this study reveals new insights into the mechanisms by which NO regulates platelet responses via the activation of the AGC family of Ser/Thr protein kinases. NO inhibited platelet shape change in a concentration dependent manner. Platelet shape change is driven by phosphorylation of myosin light chain (MLC) and the experimental data shows that NO blocked this critical phosphorylation event. Phospho-MLC generated in response to platelet agonists occurs through a Ca2+ dependent and RhoA kinase (ROCK)- dependent mechanisms and NO differentially inhibits both pathways. Activation of the ROCK pathway via RhoA leads to the phosphorylation MLC phosphatase Threonine 696/853 , which inhibits enzyme activity. Experimental evidence in this thesis indicates that NO, acting through cGMP and protein kinase G, prevents this inhibitory phosphorylation of MLCP by at least two mechanisms, (i) inhibiting the ROCK pathway that phosphorylates MLCP, and (ii) directly phosphorylating MLCP at an independent site, Serine 695 . These original observations hint at a novel mechanism for platelet regulation by the NO-cGMP-signalling pathway. II Acknowledgments Many of life’s challenges lay ahead, though now they all seem just that little more achievable due to this thesis and the people that I have had the honour to know and to work with. Privileged to be supervised by Prof. K. Naseem and Prof. S. Atkin, without whose aid and direction I would surely have foundered, and indeed thankful to meet such great friends and personalities throughout my time. To meet characters such as Dr Ahmed Aburima, Dr Simba Magwenzi, Zaher Raslan, Katie Wraith, Sreemoti Banerjee and of course Benjamin Spurgeon for the encouragement and advice they have given, and many others to whom I owe a great deal. Though most crucially, to my family, for their support, inspiration and devotion throughout the years. III Table of content Abstract II Acknowledgment III Table of content IV Table of figures XI Abbreviations XIV CHAPTER 1 INTRODUCTION XVIII 1.1 Overview 1 1.2 Platelet formation 1 1.3 Blood platelets 3 1.4 Platelet Structure 4 1.5 Platelet activation and thrombus formation 7 1.6 Platelet adhesion to vessel wall 7 1.7 Extension of the platelet plug 11 1.7.1 Platelet activation by soluble agonists 12 1.8 Platelet Aggregation 16 1.9 Perpetuation 17 1.10 Platelet Pro-coagulant Activity 18 1.10.1 Calcium signalling in platelets 22 1.10.2 Store-Operated Calcium Entry 24 1.10.3 Role of STIM 24 1.10.4 Role of ORAI 25 1.10.5 Role of TRPC 25 1.11 Shape change 28 1.12 Platelet Secretion 31 IV 1.12.1 Role of Adenosine Diphosphate 32 1.12.2 Role of Thromboxane A2 33 1.12.3 Role of Thrombin in Platelet Activation 33 1.13 Role of Myosin IIA 36 1.13.1 Role of Myosin Light Chain in Platelet Shape Change 37 1.13.2 Regulation of Myosin in blood platelets 37 1.13.3 Role of RhoA/Rho-Kinase 38 1.13.4 Inhibition of Myosin Phosphatase via Rho-Kinase mediated phosphorylation 39 1.14 Regulation of platelet functions 42 1.15 Regulation of platelet activation and aggregation 45 1.15.1 Nitric Oxide 45 1.15.2 Nitric oxide synthesis 46 1.15.3 S-Nitrosothiols 47 1.16 Role of nitric oxide dependent guanylyl cyclase 48 1.17 Mechanisms of cGMP regulation of platelet function 49 1.18 AGC Protein Kinases 53 1.18.1 PKA 53 1.18.2 PKG 54 1.18.3 PKC 56 1.19 Regulation of cyclic nucleotide levels 56 1.20 Aims of the study 59 CHAPTER 2 METHODS 60 2. Methods 61 V 2.1 Methods for the study of platelet function 61 2.1.1 Isolation and preparation of human platelets 61 2.1.2 Calculation of platelet count 62 2.1.3 Determination of protein content 62 2.1.4 Analysis of platelet function via light transmission aggregometry 62 2.2 Analysis of protein phosphorylation 63 2.2.1 SDS-PAGE 63 2.2.2 Sample preparation for SDS-PAGE 64 2.2.3 Methods for SDS-PAGE 65 2.2.4 Western immunoblotting 65 2.2.5 Stripping and reprobing of membranes 66 2.3 Calcium Assay 67 2.3.1 Platelet preparation 67 2.3.2 Measurement of cytosolic calcium 67 2.4 SKOV-3 analysis 68 2.4.1 Cell culture 68 2.4.2 RT-PCR 68 2.4.3 Antibodies 68 2.4.4 Immunostaining 69 2.4.5 Small interference RNA transfection 69 2.4.6 Cell proliferation assay 70 2.5 Statistical analysis 70 VI CHAPTER 3 ROLE OF CALCIUM STORES AND SIGNALLING PROTEINS 71 3. Introduction 72 3.1 Store-operated calcium entry 72 3.2 Results 73 3.2 Role of calcium stores and signalling proteins 74 3.2.1 Investigation into calcium signalling proteins 74 3.2.1.1 Confirmation of TRPC 74 3.2.1.2 Confirmation of Stim and Orai 77 3.2.1.3 SKOV3 cell proliferation was inhibited by store-operated channel blockers 79 3.2.1.4 Role of TRPC channels in cell proliferation 81 3.3.1 Role of calcium pathway in platelets 84 3.3.2 Influence of NO on calcium mediated MLC phosphorylation 86 3.4 Discussion 88 CHAPTER 4 REGULATION OF PLATELET FUNCTION BY NITRIC OXIDE (NO) 91 4. Introduction 92 4.1 Regulation of platelet function by nitric oxide 92 4.2 Results 94 4.2 The regulation of platelet function by nitric oxide 94 4.2.1 Agonist induced platelet aggregation 94 VII 4.2.2 Influence of Nitric Oxide (NO) on platelet aggregation 97 4.2.2.1 Nitric Oxide inhibits platelet aggregation in a dose dependent manner 97 4.2.2.2 Nitric Oxide inhibits platelet aggregation in a time dependent manner 99 4.2.2.3 Nitric Oxide inhibits platelet aggregation via cGMP pathway 99 4.2.3 Nitric Oxide induced phosphorylation of Vasodilator Stimulated Phosphoprotein (VASP) 102 4.2.3.1 NO induces a concentration and time dependent increase in VASP at serine 239 102 4.2.4 Inhibition of PAR 1 and 4 induced platelet aggregation via Nitric Oxide 105 4.2.4.1 Inhibition of Protease Activated Receptors PARs) 1 and 4 via a dose and time dependent manner 105 5.3 Discussion 110 CHAPTER 5 REGULATION OF MYOSIN LIGHT CHAIN PHOSPHORYLATION BY NITRIC OXIDE 116 5. Introduction 117 5.1 Regulation of myosin light chain phosphorylation by nitric oxide 117 5.2 Results 119 5.2 Regulation of Myosin Light Chain phosphorylation by NO 119 5.2.1 Platelet shape change 119 5.2.2 Thrombin induces a dose and time dependent MLC VIII phosphorylation in platelets 122 5.2.3 Thrombin induces MLC phosphorylation via biphasic mechanism 123 5.2.4 Reversible phosphorylation via the action of NO 126 5.2.5 Dissection of the pathways regulating NO mediated regulation of MLC phosphorylation 129 5.2.6 NO modulation of the RhoA-mediated pathway 132 5.3 Discussion 135 CHAPTER 6 THE REGULATION OF MYOSIN LIGHT CHAIN PHOSPHATASE (MLCP) 139 6. Introduction 140 6.1 The regulation of myosin light chain phosphatase 140 6.2 Results 141 6.2 The regulation of myosin light chain phosphatase 141 6.2.1 Rho-Kinase dependent inhibition of MLCP 141 6.2.2 Agonist triggered phosphorylation of MYPT1 residues 142 6.2.3 Influence of NO on platelet MLCP phosphorylation 143 6.2.4 Reversible phosphorylation of MLCP upon influence of NO 146 6.2.5 Action of NO mediating MLC phosphorylation via MLCP 148 6.3 Discussion 150 CHAPTER 7 GENERAL DISCUSSION 154 7. General Discussion 155 IX 7.1.1 First key finding 157 7.1.2 Second key finding 157 7.1.3 Third key finding 158 7.1.4 Fourth key finding 159 8. Further Work 161 Appendix I 165 Appendix II 177 Appendix III 178 Appendix IV 179 Appendix V 179 References 181 X Table of figures Figure 1.1 Platelet adhesion and aggregation on the ECM 10 Figure.1.2 Overview of platelet activation 15 Figure.1.3 Intrinsic and extrinsic systems of the coagulation cascade 21 Figure.1.4 Platelet calcium 23 Figure.1.5 Store-operated calcium entry (SOCE) in platelets 27 Figure.1.6 Platelet activation and subsequent shape change 30 Figure.1.7 Various stages of platelet activation 35 Figure.1.8 Ca 2+ sensitization and Ca 2+ dependent pathways 41 Figure.1.9 Model for platelet-endothelial interactions 44 Figure.1.10 Schematic diagram of cAMP/cGMP signalling network in platelets 52 Figure 1.11 Regulation and known effector sites of cyclic nucleotides in platelets 58 Figure.3.1 Expression of TRPC channels in SKOV3 cells 76 Figure.3.2 Expression of Orai and Stim channels in SKOV3 cells 78 Figure.3.3.
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