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Laboratory Investigation of Platelet Function in Patients with Mild Bleeding Disorders

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Laboratory Investigation of Platelet Function in Patients with Mild Bleeding Disorders LABORATORY INVESTIGATION OF PLATELET FUNCTION IN PATIENTS WITH MILD BLEEDING DISORDERS by Rashid Hafidh Rashid Al Ghaithi A thesis submitted to the University of Birmingham for the degree of DOCTOR OF PHILOSOPHY Institute of Inflammation and Ageing College of Medical and Dental Sciences University of Birmingham January 2018 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Abstract ABSTRACT Platelets play a crucial role in haemostasis by preventing bleeding at sites of vascular injury. Inherited or acquired platelet defects can impair haemostasis resulting in bleeding symptoms of varying severity ranging from mild to excessive which can be life threatening. Diagnosis of mild platelet-based bleeding disorders is challenging due to the absence of a gold standard technique and their variable bleeding symptoms and bleeding phenotypes observed in healthy individual as well as other haemostatic disorders. Furthermore their bleeding symptoms often only manifest after haemostatic challenge. The work in this thesis built on the previous studies in the genotyping and platelet phenotyping project allowing further characterization of inherited platelet function defects in individuals with mild bleeding disorders. The bleeding symptoms of patients were evaluated using the bleeding assessment tool, and its likelihood in diagnosing platelet function defects was assessed and recorded. Platelet aggregation and secretion in samples from 206 patients were investigated during the course of this thesis and were categorised on the basis of the observed defects. Surprisingly, in over a half of these patients, an ex vivo platelet function defect was not found. The genetic investigation of selected cases using whole exome sequencing identified mutations in number of genes previously known to be critical in platelet biology. However, subsequent functional studies are required to further understand their contribution to the platelet defect observed in these patients. This thesis also focused on evaluation of three other platelet techniques by comparison with lumi-aggregometry to assess their overall potential in detecting platelet function defects. Further studies are still needed to further assess the potential of these techniques before they can be applied in routine clinical diagnosis. i Publications arising from this work PUBLICATIONS ARISING FROM THIS WORK i) Published manuscripts a. Al Ghaithi R, Drake S, Watson SP, Morgan NV and Harrison P Comparison of multiple electrode aggregometry with lumi-aggregometry for the diagnosis of patients with mild bleeding disorders. Journal of Thrombosis and Haemostasis 2017 15(10):2045-2052. b. Jun Mori, Zoltan Nagy, Giada Di Nunzio, Christopher W. Smith, Mitchel J. Geer, Rashid Al Ghaithi et al. Maintenance of platelet homeostasis by the kinase-phosphatase pair Csk- CD148 in mice. Submitted ii) Published abstracts a. R. Al Ghaithi, J. Mori, L. Hardy, H. Philippou, E. Hethershaw, YA. Senis, NV. Morgan and P. Harrison. Evaluation of the Total Thrombus-formation System (T-TAS): Application to Human and Mouse Blood Analysis. Accepted for poster presentation at International Society on Thrombosis and Haemostasis 2017 Congress (Berlin, Germany). (abstract reference PB 2230) b. R. Al Ghaithi, J. Mori, L. Hardy, H. Philippou, E. Hethershaw, NV. Morgan, YA. Senis and P. Harrison. Evaluation of the Total Thrombus-formation System (T-TAS): Application to Human and Mouse Blood Analysis. Accepted for oral presentation at A Joint Meeting of BSHT / AiP / UK Platelet Group 2016 (Leeds, UK). c. Al Ghaithi R, Drake S, Watson SP, Morgan NV and Harrison P Comparison of multiple electrode aggregometry with lumi-aggregometry for the diagnosis of patients with mild bleeding disorders (abstract published in the platelet journal ) ii Dedication DEDICATION I wish to dedicate this work firstly to my parents who brought me up in the best possible manner. Then to my family, my beloved wife Nasra Al Jahdhami whose sacrifice and her unconditional love have greatly contributed to the success of this work, and to my three lovely children, Hafidh, Muthla and Maher who always stand proud for their father’s achievements iii Acknowledgements Acknowledgements In the name of Allah, Most gracious, Most merciful. Praised being to Him who granted me with good health and gave me the strength to complete this work. Deepest thanks must go to Dr Paul Harrison for giving me the opportunity to study and work on this research under his supervision and for his continuous encouragment, advice and support for me throughout my research. Special thanks for Professor Steve Watson to allow me to be part of the Birmingham platelet group and to work in his laboratory and also for his supervision, advice and support. Special thanks also go to Dr Neil Morgan whom I worked very closely in the GAPP project and his advice and guidance for the genetic study. I am grateful to the Government of Sultanate of Oman who funded this research and financially supported me thought out my study. Special thanks go to the Ministry of Health in Oman especially to Dr Qasem Al Salmi and Dr Muhanna Al Musalhi to nominate, motivate and supported me for this study. Special thanks also go to the Oman cultural attaché in London for facilitating all my academic and financial issues. Special thanks go to people who worked with me in the GAPP project, Dr Sarah Fletcher, Mrs Sian Drake, Dr Ben Johnson and Miss Annabel Maclachlan for their help and support. Thanks also to Dr Ban Dawood, Dr Marie Lordkipanidze and Dr Gillian Lowe for their previous contributions and for setting up the GAPP project. Thanks to Professor Yotis Senis and his team (platelet signalling group) for collaboration and help with the animal models. Thanks to Dr Alexander Brill for assessing all of my annual reports and providing me with feedback and advice. Thanks to Dr Steve Thomas and Dr Natalie Poulter for teaching me platelet spreading and analysis of platelet under the electron microscopy. Special thanks also to Vikki Harrison, Stephanie Watson, Beata Grygielska and Gayle Halford for all of their help and support. I thank my office mates and members of the Watson (platelet) group for all the wonderful time I spent throughout my studies. Thanks to everyone in the research laboratory at the QEH for their help and support. I am very grateful to all of my Omani friends and family members here in UK and back home for their encouragement and support. Thank you very much. iv Contents LIST OF ABBREVIATIONS ACMG - American College of Medical Genetics and Genomics ADP - Adenosine diphosphate ARC - Arthrogryposis-renal dysfunction-cholestasis ATP - Adenosine triphosphate AUC - Area under the curve BAT - Bleeding Assessment Tool BSS - Bernard-Soulier syndrome BT - Bleeding time CHS - Chediak-Higashi syndrome CLEC-2 - C-type lectin-like receptor-2 COX1 - Cyclooxygenase 1 CPA - Cone and platelet analyser CRP - Collagen-related peptide DNA - Deoxyribonucleic acid GAPP - Genotyping and Phenotyping of Platelets GT - Glanzmann thrombasthenia GTP - Guanosine-5’- triphosphate GP - Glycoprotein GPCRs - G protein–coupled receptors GDP - Guanosine diphosphate GPS - Grey platelet syndrome HMW - High molecular weight HPO - Human Phenotype Ontology HPS - Hermansky-Pudlak syndrome Ig - Immunoglobulin IPA - Impedance platelet aggregometry IPF - Immature platelet fraction ISTH - International Society on Thrombosis and Haemostasis ITAM - Immunotyrosine-based activation motif ITP - Immune thrombocytopenic purpura JAMs - Junctional adhesion molecules KO - Knock out Lumi-LTA - Lumi-aggregometry LTA - Light transmission aggregometry MAF - Minor allele frequency MEA - Multiple Electrode Aggregometry MFI - Median fluorescence intensity MKs - Megakaryocyte MPV - Mean platelet volume NGS - Next generation sequencing v Contents NO - Nitric oxide NPV - Negative predictive value OCA - Oculocutaneous albinism OCS - Open canalicular system OT - Occlusion time PCR - Polymerase Chain Reaction PFD - Platelet function defects PFT - Platelet function testing PGI2 - Prostacyclin PI3K - Phosphoinositide 3-kinases PLCβ - Phospholipase C-β POC - Point of care PPP - Platelet poor plasma PPV - Positive predictive value PRP - Platelet rich plasma PS - Phosphatidylserine PT-VWD - Platelet-type von Willebrand disease PTF - Platelet thrombus formation QPD - Quebec platelet disorder ROC - Receiver-operator curve ROTEM - Rotational thromboelastometry RPFT - Remote platelet function test SIFT - Sorting Intolerant from Tolerant SLE - Systemic lupus erythematosus SLMA - Signalling lymphocyte activation molecules SNV - Single nucleotide variant SPD - Storage pool disease T-TAS - Total Thrombus-formation System TEG - Thromboelastography TPO - Thrombopoietin TRAP - Thrombin receptor activating peptide TTF - Total thrombus-formation TxA2 - Thromboxane A2 uPA - Urokinase plasminogen activator VWD - von Willebrand disease VWF - von Willebrand factor WES - Whole exome sequencing WGS - Whole-genome sequencing WT - Wild type WTF - White thrombus
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