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Characterization of Cyclic Adenosine Monophosphate/Protein Kinase a Signalling Networks in Blood Platelets. Zaher Raslan a Thesi

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Characterization of Cyclic Adenosine Monophosphate/Protein Kinase a Signalling Networks in Blood Platelets. Zaher Raslan a Thesi Characterization of Cyclic Adenosine Monophosphate/Protein Kinase A Signalling Networks in Blood Platelets. Zaher Raslan A thesis submitted for the degree of Doctor of Philosophy in Medical Sciences The University of Hull and the University of York Hull York Medical School (HYMS) September 2012 III Abstract Platelet activation is a critical physiological event, whose main role is to prevent excessive blood loss and repair vessel wall injuries. However, platelet activation must be controlled to prevent unwanted and exaggerated responses leading to the occlusion of the blood vessel. The endothelial-derived inhibitors prostacyclin (PGI2) and nitric oxide (NO) are known to play a critical role in the control of platelet activity, although the mechanism underlying their actions remains unclear beyond the triggering of cyclic nucleotides signaling pathways. The aim of this study was to improve our understanding of platelet regulation by cAMP signaling networks. We observed differences in cAMP signaling depending on the agonists used. Using phosphorylation of PKA substrates as a marker of PKA activity, it was observed that PKA substrates were phosphorylated and dephosphorylated at different time points in a unique temporal pattern. Consistent with this observation we found that individual PKA isoforms, PKA I and II, were localized in distinct subcellular compartments, with PKA I being identified as a lipid raft protein. Our experimental data suggest that the localization of PKA I to lipid rafts is mediated by interaction with A-kinase anchoring proteins (AKAPs). Additionally, PKA signaling events were reversed when potential PKA type I interactions with AKAPs were disrupted with competitive peptides. Using this approach we found that the redistribution of PKA I to lipid rafts facilitated the phosphorylation of GPIbβ and the inhibition of von- Willebrand factor-mediated aggregation. Our data also demonstrated for the first time that the chemical disruption of lipid rafts increased platelet sensitivity to PGI2, through increased cAMP production and IV PKA activity. The mechanism by which this occurs may involve sequestering a population of adenylyl cyclase 5/6 to a location remote from Gαs. In conclusion, data presented in this thesis suggest differential roles of PKA subtypes in the regulation of platelet activity. This involves, at least in part, the localisation of PKA I into specific subcellular compartments through an interaction with AKAPs. The potential presence of PKAII-AKAP interactions and the identification of specific AKAPs will be the main aim of future work. V Publications Oral Presentation The role of A-Kinase Anchoring Proteins in mediating the inhibitory effect of Protein Kinase A in blood platelets. 12th UK Platelet Meeting, Nottingham, UK (2010). (Winner of best oral presentation award). Poster presentations Raslan, Z., Aburima, A.A., Magwenzi, S.G., Tasken, K. & Naseem, K.M. Targeting of Type I Protein Kinase A to lipid rafts is required for platelet inhibition by the cAMP- signaling pathway. 4th British Pharmacological Society (BPS) Focussed Meeting on Cell Signalling. Leicester, UK (2012). Raslan, Z. & Naseem, K.M. The role of lipid rafts in the compartmentalisation of cAMP/PKA signalling in blood platelets. 13th UK Platelet Meeting. Cardiff, UK (2011). Raslan, Z. & Naseem, K.M. The role of lipid rafts in the compartmentalisation of cAMP/PKA signalling in blood platelets. Northern Cardiovascular Research Group (NCRG) meeting, Hull, UK (2011). VI Table of Contents Abstract .............................................................................................................. III Publications ......................................................................................................... V Table of Contents ............................................................................................... VI Abbreviations .................................................................................................... XV List of tables ..................................................................................................... XIX List of Figures .................................................................................................... XX Acknowledgment ............................................................................................ XXVI Author’s declaration ..................................................................................... XXVIII Chapter 1: General Introduction .......................................................................... 1 1.1 Introduction ............................................................................................... 1 1.2 Platelet formation ...................................................................................... 2 1.3 Platelet structure ....................................................................................... 3 1.3.1 Platelet membrane .............................................................................. 5 1.3.2 Platelet cytoskeleton ........................................................................... 5 1.3.3 Platelet organelles. .............................................................................. 6 1.4 Platelet activation ...................................................................................... 8 1.4.1 Platelet adhesion to the vessel wall ................................................... 10 1.4.2 Platelet signal transduction ................................................................ 14 1.4.3 Amplification of platelet activation .................................................... 20 VII 1.4.4 Platelet aggregation ........................................................................... 22 1.5 Platelet regulation by the endothelium .................................................... 25 1.5.1 The regulation of platelet function by nitric oxide .............................. 25 1.5.2 The regulation of platelet function by prostacyclin ............................. 27 1.6 Platelet inhibition by cAMP/PKA signalling pathway ................................. 35 1.6.1 cAMP synthesis by adenylyl cyclase in blood platelets ........................ 38 1.6.2 cAMP degradation by phosphodiesterases in blood platelets ............. 42 1.6.3 Protein kinase A in platelets ............................................................... 44 1.6.4 Protein kinase A structure and activation ........................................... 47 1.6.5 Protein kinase A substrates in blood platelets .................................... 51 1.7 Compartmentalisation of cAMP/PKA signalling in platelets and other cell types ............................................................................................................. 58 1.8 A-Kinase Anchoring Proteins ..................................................................... 62 1.9 Aims of this study ..................................................................................... 68 Chapter 2: Materials and methods ..................................................................... 70 2.1 Materials .................................................................................................. 70 2.2 Methodologies used in the preparation of human blood platelets. ........... 70 2.2.1 Procurement of human blood: ........................................................... 70 2.2.2 Isolation of platelets from whole blood by lowering of blood pH. ....... 71 2.2.3 Quantification of platelet numbers. ................................................... 72 VIII 2.3 Assessment of platelet functions .............................................................. 74 2.3.1 Platelet aggregation ........................................................................... 74 2.3.2 Measurement of platelet dense granule secretion .............................. 77 2.4 Methodologies used in the analysis of platelet proteins ............................ 78 2.4.1. Immunoprecipitation ........................................................................ 78 2.4.2 Sodium dodecyl sulphate -polyacrylamide gel electrophoresis (SDS- PAGE) ......................................................................................................... 81 2.4.3 Immunoblotting ................................................................................. 85 2.5 Isolation of platelet lipid rafts ................................................................... 89 2.5.1 Lipid raft isolation by sucrose ultracentrifugation. .............................. 90 2.6 Determination of cAMP levels ...................................................................... 92 2.6.1 Sample preparation for cAMP EIA ...................................................... 94 2.7 Determination of cholesterol concentrations ............................................ 95 2.8 Investigation of potential AKAP-PKA interactions in platelets. .................. 96 2.8.1 R-I Anchoring Disruptor (RIAD) synthesis and determination of loading conditions .................................................................................................. 96 2.8.2 RIAD-Arg11 conjugation with fluorescein ............................................ 98 2.9 Data representation and statistical analysis .............................................. 99 2.10 Supplementary methods ...................................................................... 100 2.10.1 Visualization of RIAD uptake by adhered platelets .......................... 100 IX 2.10.2 Platelet aggregation under flow
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