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Effects of Resistin on Platelet Function and Its Receptor, Adenylyl Cyclase Associated Protein 1

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Effects of Resistin on Platelet Function and Its Receptor, Adenylyl Cyclase Associated Protein 1 Effects of resistin on platelet function and its receptor, adenylyl cyclase associated protein 1 Pooja Laxmikant Joshi PhD in Medical Sciences The University of Hull and the University of York Hull York Medical School March 2017 ABSTRACT Resistin is an adipokine which is found elevated in patients suffering from metabolic syndrome, affecting a large proportion of the population. It is characterised by obesity, insulin resistance (IR), type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD), which are the biggest contributors to morbidity and mortality in the modern world. Platelets play a major role in CVD, where their activation leads to formation of thrombi, which when uncontrolled, leads to the pathophysiological effects of CVD. The role of resistin in CVD progression and its mechanism of action on its target cells still remains debatable and surrounded by controversies. In a bid to assess this, the present study aims at illustrating the effect of resistin on platelet activation by deriving conclusions from platelet functional assays. This thesis makes novel observations that resistin blunts thrombin-mediated platelet aggregation and secretion and seemingly affects the platelet actin cytoskeleton. It also finds that resistin activates downstream effectors of phosphoinositide 3 kinase (PI3K) and mitogen activated protein kinase (MAPK) pathways in platelets. Adenylyl cyclase associated protein 1 (CAP1), a cytoskeletal protein, was recently identified as a receptor for resistin. When CAP1 was investigated in platelets, interesting observations were made, where CAP1 temporally translocated from the membrane fraction in response to thrombin mediated activation. Furthermore, in an attempt to improve our knowledge of platelet biology and to delineate future avenues for exploration, an observational proteomics investigation of cytoskeletal proteins and the change in abundance 2 upon thrombin activation was carried out, further highlighting their importance in platelet regulation. Additionally the subcellular localisation of specific cytoskeletal proteins (IQGAP1, Arp2/3, coronin1a, profilin, cofilin, villin, l-plastin, myosin IIa, and tropomyosin) that are either known or hypothesised to interact with CAP1 was also conducted. Further investigations with proteins IQGAP1 and Arp2/3 revealed that these proteins did not interact with CAP1. 3 TABLE OF CONTENTS ABSTRACT ..................................................................................................................... 2 TABLE OF CONTENTS .................................................................................................. 4 TABLE OF FIGURES .................................................................................................... 10 LIST OF TABLES .......................................................................................................... 13 LIST OF ABBREVIATIONS .......................................................................................... 14 DEDICATIONS ............................................................................................................. 21 ACKNOWLEDGEMENTS ............................................................................................. 22 AUTHOR’S DECLARATION ......................................................................................... 24 CHAPTER 1 INTRODUCTION ..................................................................................... 25 1.1 Platelets in metabolic syndrome: role of adipokines ............................................... 25 1.2 Adipokines .............................................................................................................. 31 1.2.1 Leptin ................................................................................................................ 33 1.2.2 Adiponectin ....................................................................................................... 35 1.2.3 Other true adipokines ....................................................................................... 36 1.2.4 Other adipokines ............................................................................................... 37 1.3 Resistin ................................................................................................................... 39 1.3.1 Resistin structure .............................................................................................. 40 1.3.2 Resistin gene expression .................................................................................. 43 1.3.3 Mechanism of action of resistin in endothelial and immune cell lines ............... 44 1.3.4 Resistin function ............................................................................................... 49 1.3.4.1 Resistin in MetS and associated CVD progression .................................... 49 1.3.4.2 Resistin in inflammation: Effects on human monocytes and endothelial cells ...................................................................................................... 50 1.3.4.3 Function of resistin in adipokine regulation in diabetic mouse models ....... 51 1.3.4.4 Function of resistin in glucose metabolism in rodent disease models and human endothelial cell lines ............................................................................ 52 1.3.4.5 Upregulation of resistin in cancerous cells: Role in cell proliferation .......... 53 1.4 Platelets .................................................................................................................. 54 4 1.4.1 Platelet morphology .......................................................................................... 54 1.4.2 Platelet plug formation ...................................................................................... 59 1.4.2.1 Platelet adhesion: vWF/GP1b complex mediated platelet activation .......... 63 1.4.2.2 Platelet adhesion: Collagen/GPVI mediated platelet activation .................. 63 1.4.2.3 GPCR mediated platelet activation ............................................................. 64 1.4.2.4 Amplification of platelet activation: Granule secretion ................................ 70 1.4.2.5 Integrin αIIbβ3 activation as a central event in platelet activation ................. 71 1.4.2.6 Regulation of platelet activation .................................................................. 73 1.5 The platelet cytoskeleton ........................................................................................ 77 1.5.1 Cytoskeleton of the resting platelet ................................................................... 79 1.5.2 Actin rearrangement in stimulated platelets: A CAP1 perspective .................... 82 1.5.3 Cytoskeleton of the activated platelet: Formation of actin structures ................ 85 1.5.2.1 Filopodia formation in platelets ................................................................... 87 1.5.2.2 Lamellipodia formation in platelets ............................................................. 87 1.5.2.3 Actin nodules in platelets ............................................................................ 88 1.5.2.4 Stress fibres in platelets ............................................................................. 88 1.5.3 Brief overview of signalling related to actin cytoskeletal reorganisation ............ 90 1.6 Adenylyl cyclase-associated protein 1 (CAP1) ........................................................ 95 1.6.1 CAP1 structure ................................................................................................. 95 1.6.2 CAP1 function ................................................................................................. 101 1.6.2.1 Adenylyl cyclase activity ........................................................................... 101 1.6.2.2 Role of CAP1 in actin regulation ............................................................... 102 AIMS AND OBJECTIVES ........................................................................................... 109 CHAPTER 2 MATERIALS AND METHODS ............................................................... 111 2.1 Preparation of human blood platelets ................................................................ 111 2.1.1 Isolation of human blood platelets ............................................................... 111 2.1.2 Determination of platelet count .................................................................... 112 2.2 Turbidimetric analysis of platelet aggregation .................................................... 112 2.3 Measurement of platelet dense granule secretion ............................................. 112 2.4 Platelet visualisation .......................................................................................... 113 2.4.1 Spreading on fibrinogen or collagen ............................................................ 113 5 2.4.2 Platelet stimulation in suspension ............................................................... 114 2.4.3 Immunostaining platelets ............................................................................. 115 2.4.4 Imaging platelets ......................................................................................... 115 2.4.5 Analysis of imaged platelets ........................................................................ 116 2.5 Preparation of whole cell lysate ......................................................................... 117 2.6 BCA (Bicinchoninic acid) Protein determination assay .....................................
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