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There is an unmet need for thrombopoietic agents that can increase platelet counts in patients with thrombocytopenia. The development of recombinant versions of the main thrombopoietic hormone, thrombopoietin (Tpo), were terminated due to the unwanted formation of antibodies to endogenous thrombopoietin. Eltrombopag is an orally bioavailable, non-peptide, small molecule agonist of the thrombopoietin receptor that increases platelet counts in humans, by driving differentiation and proliferation of megakaryocytes. The development of eltrombopag as a treatment for thrombocytopenia involved iterative experiments in a series of in vitro and in vivo preclinical studies in which eltrombopag was shown to activate the Tpo receptor leading to signal transduction via the JAK-ST AT pathway in megakaryocyte cell lines, drive megakaryopoiesis in primary human bone marrow cells and increase platelet counts after oral administration to chimpanzees. The target exposure for increasing platelet counts was 30-90~g.h/ml, while the no observed adverse effect level was 164~g.h/ml. When administered to healthy subjects for 10 days, eltrombopag increased platelet counts in a dose dependent manner at doses of 30, 50 and 75mg, where exposures exceeded the predicted 30~g.h/ml. A Phase II study in adult subjects with chronic ITP showed that doses of 50mg and 75mg of eltrombopag increased platelet counts in up to 80% of subjects, compared to only 11 % of subjects that received placebo. Thorough characterization of the pharmacology of eltrombopag during preclinical and early clinical development allowed for data-driven dose selection decisions and provided a strong foundation for successful Phase III clinical studies and regulatory approval of eltrombopag as a much needed treatment for thrombocytopenic patients. Jenkins ABSTRACT Cardiovascular Medicine

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Platelets play a pivotal role in both normal hemostasis and pathological bleeding and importantly also contribute to the development of atherothrombosis. Even though platelet function tests traditionally are utilised mainly for the diagnosis and management of patients presenting with bleeding problems rather than thrombosis, new and improved platelet function tests are now increasingly used to monitor anti-platelet therapy in patients and to identify patients at risk of arterial disease. Based on light transmission traditional aggregometry, this thesis reports data from a new model of platelet aggregation using a modified 96-well plate format. This method allows examination of many agonists at a range of concentrations at the same time. Thus, more information can be collated about different aspects of platelet function and smaller assay volumes can be used while still obtaining reliable results. To further utilise this method, agonist combinations were used in the 96-well plate approach that resemble the actions of machines such as the PFA-100, which uses combined agonists within a cartridge, but at much lower cost. Platelet cyclooxygenase has been widely studied; however, the functions of platelet 12-lipoxygenase and NADPH oxidase in platelets are still generally not understood. Data presented here demonstrate that both pathways are partly essential in platelet activation following exposure to stimulatory agonists. To further explore the relationship between dietary intake and the risk of atherothrombosis, an in vivo study was performed to observe the antiplatelet effects following from consumption of dark chocolate in baseline hypertensive patients. Based on findings in this thesis, it can be 4 concluded that this new method of evaluating platelet aggregation and adhesion in a 96-well plate format is very useful, and that new observations into influences on platelets of pathways other than cyclooxygenase may be beneficial in the development of new antiplatelet drugs.

This project sets out to create a reference laboratory service capable of detecting platelet function disorders using the latest techniques and based on the most current research. Platelet function disorders are difficult to diagnose due to differing phenotypic presentation and multiple causative agents. Platelet research has moved rapidly over the past decade and has included new reagents, analysers and techniques in the way platelet function disorders are diagnosed. A forward-looking diagnostic laboratory needs translate this ongoing research into routine laboratory practice, whilst ensuring that techniques used in the laboratory comply with the current guidelines and performed in a standardised and scientifically rigorous way. Literature searches were used to develop a pre-analytical questionnaire and this has been adopted. It has proved to be an important tool for standardisation of the pre-analytical procedure which is now in use at other diagnostic centres. New light transmission aggregation equipment and agonists have been introduced, standardised and reference ranges generated, driven by evidence based practice. Reagent comparison studies have been undertaken to asses’ cost-effectiveness of the assays in the laboratory. Platelet nucleotide reference ranges have been generated and are in use. The flow cytometric analysis of glycoproteins has been brought ‘in-house’, has been standardised, and is now being offered as a routine assay to specialist haematology clinical staff, improving the service the laboratory offers. This work has enabled an extended range of assays available to the laboratory and now has capacity for specialist testing of inherited platelet disorders. This together with expert clinical staff creates the scientific and technical environment required for the establishment of a specialist regional referral centre. The reputation of the laboratory has been additionally enhanced, through presentations and collaborations with manufacturers, other healthcare scientists and professional bodies. All these improvements based on strong scientific research and rigorous application have enabled the patient to undergo a thorough investigation with the minimum of inconvenience and enabled the health care provider to utilise resources more effectively.