Characterisation, development and application of a clinical model of thrombosis and fibrinolysis
Lucking, Andrew John
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The demonstration of antithrombotic efficacy in man is challenging. Most techniques evaluate specific plasma or cellular components under static conditions in vitro. In contrast, in vivo thrombus initiation and growth occur in whole blood, under conditions of continuous flow and in the presence of vascular injury. An in vivo model for use in clinical studies presents significant safety issues and does not currently exist. The Badimon chamber is an ex vivo model of thrombosis that is suitable for use in clinical studies and has previously been used to assess novel antithrombotic regimens. Although well-established, previous characterisation studies were performed in a porcine system and using methodology that has since been superceded. In addition, it has a number of disadvantages that limit its broader applicability and has not previously been used to assess fibrinolysis. Having established The Badimon Chamber within my own institution, I developed the methodology and performed careful validation and characterisation studies with a particular emphasis on reproducibility. These developments allowed more efficient data analysis and the accurate addition of compounds to the extracorporeal circuit, both of which broaden the applicability of the technique. In subsequent studies, using a series of double-blind randomised controlled crossover studies in healthy volunteer cohorts, I utilised the updated methodology to address questions in separate but overlapping areas of cardiovascular medicine. The dynamic regulation of intravascular thrombus formation by the endogenous fibrinolytic system is central to the pathogenesis of acute atherosclerotic events, particularly within the coronary circulation. Previous work within our institution has provided novel insights into the role of endogenous fibrinolysis. Despite a growing body of evidence, a key limitation of studies to date is that the effects of acute endogenous t-PA release on in situ thrombus formation have not been demonstrated. Is endogenous endothelial t-PA released under agonist stimulation functionally active and able to enhance fibrinolysis of in situ thrombus? Firstly, I demonstrated that the addition of exogenous t-PA into the extracorporeal circuit of The Badimon Chamber results in a dose dependent increase in plasma D-dimer associated with a dose dependent reduction in thrombus formation, consistent with enhanced fibrinolysis. Having validated the model, I proceeded to investigate whether freshly released endogenous t-PA would have similar effects to exogenous t-PA. By combining intraarterial infusion of bradykinin into the human forearm in order to stimulate acute release of endogenous t-PA with an assessment of thrombus formation in the Badimon Chamber, I demonstrated that endogenous t-PA released acutely from the human vascular endothelium enhances fibrinolysis and limits in situ thrombus formation. These data validate the forearm model as a relevant model with which to assess acute fibrinolytic capacity, confirm the functional significance of t-PA released during agonist stimulation and suggest that further studies to explore its therapeutic manipulation are warranted. I went on to evaluate a promising small molecule PAI-1 inhibitor, PAI-749, using assessments of ex vivo thrombosis complimented by extensive in vitro studies. Interestingly, in contrast to the promising results seen with this compound in preclinical models, we were unable to demonstrate efficacy in any of the clinical models used, highlighting the potential pitfalls of relying solely on in vitro and preclinical models during early compound development. In the final phase of this work, I used the chamber to explore the prothrombotic effects of exposure to air pollution. A plethora of observational data exist to suggest that acute exposure to particulate air pollution can trigger vascular events including myocardial infarction although the underlying mechanisms are only partly understood. Using a unique human exposure facility, we demonstrated that inhalation of diesel exhaust causes platelet activation and enhances thrombus formation. These data provide a plausible mechanism linking exposure to particulate air pollution with acute cardiovascular events including myocardial infarction. Furthermore, in a separate study we were able to demonstrate that reducing the particulate component of the exposure using a commercially available particle trap prevents the detrimental effects on ex vivo thrombosis and endothelial function. These data support calls for the application of particle traps to diesel-powered vehicles in order to limit a range of adverse cardiovascular effects that result from exposure to traffic-derived air pollution.