Abstract
Acute coronary syndrome (ACS) arises from a reduction in blood flow to the heart, leading to severe cardiovascular events, including myocardial infarction. Atherosclerotic plaque formation drives arterial occlusions seen in ACS, and is linked to dysregulated platelet response in the vasculature. Thus, using anti-platelet therapies, and more specifically targeting the platelet P2Y12 receptor(P2Y12R), is the gold-standard clinical treatment for ACS patients. Recent studies have demonstrated that P2Y12R exhibits constitutive activity (CA), and hence anti-P2Y12R blockers, such as ticagrelor, have been re-defined as inverse agonists. Following this discovery, our understanding of P2Y12R
activity remains ill-defined. Therefore, this thesis explores both the molecular structure of P2Y12R, and the influence of the membrane environment upon receptor activation. Focusing on in vitro assessment of receptor activation using bioluminescence resonance energy transfer (BRET) assays, this work explores the effects of missense mutations in key residues identified in P2Y12R on ligand-dependent activity and CA. This work revealed that transfection level-dependent
increases in P2Y12R surface expression increase CA. Among the different mutant receptor constructs there was a significant increase in agonist-dependent activity at the Y105A and D294N mutants was observed, whilst the patient-derived R265P mutation demonstrated a global reduction in ligand-dependent activity. Further, Y209, a key residue within the NPxxY motif of P2Y12R, was identified as
being important for CA. Study examining the relationship between the membrane microenvironment and P2Y12R activity was also undertaken. Changes in membrane cholesterol significantly affected P2Y12R activity with cholesterol-dependent increases in P2Y12R CA were observed using BRET. In platelets meanwhile there was a global reduction in platelet aggregation response upon changing cholesterol levels. This thesis therefore presents an in vitro characterisation of ligand-dependent and independent activity in key P2Y12R mutations, and begins to explore the effects of manipulating the cholesterol
plasma membrane environment on receptor activity, and global platelet function. Exploring two factors affecting the molecular mechanisms of P2Y12R helps to further our understanding of P2Y12R and subsequently improves our understanding of anti-P2Y12R therapies in the treatment of ACS.
| Date of Award | 5 Dec 2023 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Stuart J Mundell (Supervisor) & Thomas W Johnson (Supervisor) |