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Characterisation of Cellular Fibrinogen Phosphorylation and Its Functional Implications in Clot Formation

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Characterisation of Cellular Fibrinogen Phosphorylation and Its Functional Implications in Clot Formation Characterisation of Cellular Fibrinogen Phosphorylation and its Functional Implications in Clot Formation Esther J. Cooke Submitted in accordance with the requirements for the Degree of Doctor of Philosophy The University of Leeds School of Medicine February 2015 The candidate confirms that the work submitted is her own, except where work which has formed part of jointly authored publications has been included. The contribution of the candidate and the other authors to this work has been explicitly indicated below. The candidate confirms that appropriate credit has been given within the thesis where reference has been made to the work of others. The work in Chapters 2 and 3 of the thesis has appeared in publication as follows: Smith, K.A., Pease, R.J., Avery, C.A., Brown, J.M., Adamson, P.J., Cooke, E.J., Neergaard-Petersen, S., Cordell, P.A., Ariëns, R.A., Fishwick, C.W., Philippou, H., Grant, P.J. 2013. The activation peptide cleft exposed by thrombin cleavage of FXIII- A(2) contains a recognition site for the fibrinogen α chain. Blood, 121 (11), 2117-2126. The candidate was responsible for the expression and purification of recombinant fibrinogen. The other authors contributed to study design, laboratory work, data analysis and writing of the manuscript. This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. © 2015 The University of Leeds and Esther J. Cooke The right of Esther J. Cooke to be identified as Author of this work has been asserted by her in accordance with the Copyright, Designs and Patents Act 1988. ii Acknowledgements I would first of all like to thank my supervisors Professor Peter Grant and Dr Kerrie Smith for the opportunity to undertake this PhD project, and for all the support and guidance you have given me over the last four years. In addition to your scientific ideas and contributions, you have advised and challenged me, and helped me to develop confidence and rigour in conducting independent research. I would particularly like to thank Kerrie for all that you have taught me, your patience and your encouragements. Secondly, I wish to thank my fellow Grant group members. I owe a huge thank you to Paul for your ideas, knowledge and generosity; you have given up a great deal of time to help me and it is much appreciated. Richard, thank you for your input and the many words of wisdom, and Jane, your technical support has been invaluable. I would also like to thank Cora, May, Kingsley and Kathryn for your help and suggestions. You have all made my time here enjoyable and I will miss the eventful excursions. There are many other people whom I have had the pleasure of working with in LIGHT. I would like to thank Ced for your CHO expertise and willingness to help me over the years. Thanks go to Katie, Emma, Amy, Fladia, Jess, Romana, Matt, Katy, Natalie, Fraser, Marco, Helen, Lewis, Carla, Nathan and Pooja for your advice and company in the lab, your friendship, and for Monday afternoon cake provision. I am also grateful to Gareth Howell for his assistance with the confocal microscope and image analysis. Last but not least, I would like to thank my friends and family for your prayers and support, for all the fun times I’ve had in Leeds, for keeping me sane when experiments failed for the nth time and for looking out for me. I am especially thankful to my parents, who have believed in me, provided for me in many ways, and been a real encouragement. I could not have achieved this without your support, thank you. iii Abstract Fibrinogen is a vital component of coagulation; cleavage of fibrinogen yields fibrin monomers that polymerise to form a network of fibres, constituting the blood clot. Human fibrinogen is secreted from hepatocytes in its phosphorylated form, with 20-25 % of circulating fibrinogen phosphorylated exclusively at A chain Ser3 and Ser345. Phosphorylation of fibrinogen is elevated in acute phase conditions, venous thrombosis and ovarian cancer, but little is known about the regulation and effects of this modification. The aims of this PhD project were to characterise the cellular mechanism and functional role of fibrinogen phosphorylation in vivo. Human hepatoma cells were incubated in the presence and absence of IL-6 and the phosphate content of secreted fibrinogen was analysed by western blotting. Interleukin-6 caused a 3.1-fold increase in fibrinogen phosphorylation, demonstrating for the first time that the up-regulation of this modification in acute phase conditions is regulated at the cellular level. Using real-time PCR, IL-6 was found to significantly enhance (6.0-fold) the expression of Golgi casein kinase Fam20A, whose recognition sequence matches the Ser3 and Ser345 phosphorylation sites. Expression of other potential fibrinogen kinases, including CK2, Fam20B and Fam20C, were unchanged. This finding suggests that Fam20A plays an important role in the hepatocellular response to acute phase conditions and may phosphorylate fibrinogen in vivo. Chromatographic enrichment of phosphorylated human plasma fibrinogen was conducted for functional analyses. Binding and activity assays found no effect of fibrinogen phosphorylation on FXIII cross-linking of fibrin α and γ chains, plasmin(ogen) binding to fibrinogen, or α2-antiplasmin incorporation. Analysis by SDS-PAGE revealed a small decrease in the rate of fibrinogen degradation by plasmin with increasing phosphorylation, indicating a possible role in protection from fibrinolysis. Scanning electron microscopy and turbidimetric assays revealed thinner fibres and more extensive branching in clots with a higher phosphate content, which typically represents a pro-thrombotic structure. This work highlights the importance of fibrinogen phosphorylation in maintaining the balance between clot formation and lysis. Investigations have shown that increased intracellular kinase activity leads to elevated fibrinogen phosphorylation in acute phase conditions. The observed alterations to clot phenotype with elevated fibrinogen iv phosphorylation suggest this modification may help to stem bleeding following trauma. Furthermore, it may have important implications in the development of thrombosis, which would make it a valuable target for therapeutic intervention in associated pathologies. v Table of Contents Acknowledgements ................................................................................................... iii Abstract ...................................................................................................................... iv Table of Contents ....................................................................................................... vi List of Figures .......................................................................................................... xiv List of Tables ........................................................................................................... xvii List of Abbreviations ............................................................................................. xviii Chapter 1 ..................................................................................................................... 1 Introduction ................................................................................................................... 1 1.1. Haemostasis and coagulation ............................................................................ 1 1.1.1. The coagulation cascade ........................................................................... 2 1.1.1.1. Tissue factor pathway ......................................................................... 2 1.1.1.2. Contact activation pathway ................................................................. 2 1.1.1.3. Common pathway ............................................................................... 3 1.1.2. The cell-based model of haemostasis ........................................................ 4 1.1.3. Anticoagulant pathways ............................................................................. 5 1.1.4. The fibrinolytic system ................................................................................ 6 1.1.4.1. Plasminogen and its activators ........................................................... 7 1.1.4.2. Plasmin digestion of fibrin(ogen) ......................................................... 7 1.1.4.3. Fibrinolysis inhibitors .......................................................................... 9 1.2. Fibrin(ogen) ..................................................................................................... 10 1.2.1. Genetic regulation of fibrinogen ............................................................... 10 1.2.1.1. Splice variants and polymorphisms .................................................. 10 1.2.1.2. Dysfibrinogenemias .......................................................................... 11 1.2.2. Fibrinogen biosynthesis ........................................................................... 12 1.2.3. Fibrinogen structure ................................................................................. 15 1.2.3.1. Disulphide bonds .............................................................................. 16 vi 1.2.3.2. Crystal Structures ............................................................................. 17 1.2.4. Functions of fibrinogen in clot formation ................................................... 17 1.2.4.1. Fibrin polymerisation
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