G protein-coupled receptor function and signalling in atherothrombosis

G protein-coupled receptors (GPCRs), one of the largest protein families in the human genome, are the most tractable set of therapeutic targets for novel drug design. These cell surface expressed proteins translate extracellular cues which bombard the cell surface into signals which determine cellular function. GPCRs are regulated in a dynamic and complex manner, and are not static entities inserted into the plasma membrane of cells. My research examines the cell specific signals produced by individual GPCRs and the complex regulatory mechanisms controlling GPCR signalling, surface expression and intracellular sorting.  Further my research assesses the impact of such mechanisms on signalling pathways that initiate important physiological responses. In order to examine these questions we use both biochemical and imaging techniques to study receptor movement and function.

At present our research is concentrated on GPCR function in a number of cells which maintain the integrity of the cardiovascular system including platelets and smooth muscle cells. Platelets are an essential element in the pathophysiology of heart disease with platelet expressed GPCRs clinically useful pharmacological targets for antithrombotic drugs. Our research in platelets has three major strands.

Characterization of the molecular mechanisms that regulate the function of GPCR signalling in platelets.

Alterations in the responsiveness of platelet GPCRs have important consequences on platelet function with mechanisms, including desensitization, internalization and subsequent endocytic sorting regulating their function (Hardy et al., 2005; Mundell et al., 2008; Nisar et al., 2011, 2012, Cunningham et al., 2013). Our studies continue to examine the molecular mechanisms underlying the control and traffic of platelet GPCR whilst determining the consequences of such mechanisms on receptor function. A number of different techniques are employed to answer these questions ranging from measurement of cellular signalling pathways through to immunofluorescent imaging of single cells.

Genotyping and phenotyping platelet receptor function

This research seeks to identify the faulty genes that underlie bleeding in families with abnormal blood platelet function.  The study of patients with bleeding problems is a powerful approach in determining the function and regulation of important proteins in human platelets. Ultimately, this work is anticipated to identify novel platelet proteins that are targets for development of anti-thrombotics and to facilitate the wider use of antiplatelet agents in the fight against heart disease by exclusion of patients with platelet defects. This research has already identified mutations in a number of platelet GPCRs including the P2Y12 (Daly et al., 2009 and Nisar et al., 2011)  and TxA2 receptor (Mumford et al., 2010 and Gaussem et al., 2013) that change receptor function and contribute to the bleeding tendency in specific individuals.  Ongoing studies continue to identify mutations in other platelet expressed GPCRs and in the proteins that regulate receptor function.

Characterize the molecular mechanisms of currently used antithrombotics in the patient population

In collaboration with clinical colleagues in the Bristol Heart Institute and industrial collaborators we examine the interaction between and molecular mechanisms of therapies in the treatment of thrombosis, concentrating on those which inhibit platelet reactivity.  The goal of this research is to help refine the clinical use of these therapeutics by fully defining their mode of action both on their target receptor and then on their ability to impact the signalling of other platelet expressed GPCRs.

In summary this body of work aims to provide fundamental new insights into the molecular mechanisms regulating platelet receptor function leading to the development of new, more effective and safer drugs for the treatment and prevention of cardiac disease or making for a more informed use of pre-existing therapies.