Platelets are the main orchestrators of processes such as wound healing and tissue regeneration post vascular injury. They not only mediate haemostasis, but also promote immune cell recruitment to the site of vascular injury. Platelets are also key promoters of angiogenesis, and they support the formation of novel ‘scar’ tissue. However, inappropriate platelet activation can be responsible for pathological thrombosis, most commonly in the coronary vasculature. Platelet non-reactivity is tightly regulated by several surface receptors. The P2Y₁₂ receptor is one such platelet receptor that is stimulated by ADP released from activated platelets to further potentiate platelet reactivity. The P2Y₁₂ receptor is now an established therapeutic target, with antagonism of this receptor used in the treatment of atherothrombosis. It has recently been established that this receptor exhibits a high level of agonist-independent activation (constitutive activity). Ticagrelor, a widely used therapeutic platelet inhibitor that blocks ADP-dependent P2Y₁₂ receptor stimulation, was recently shown to act as an inverse agonist at the P2Y₁₂ receptor, reducing its basal constitutive activity. Interestingly, although marketed as a reversible drug, ticagrelor was recently shown to be irreversible throughout the life span of platelets. The present study aims to characterize several molecular modulators of the P2Y₁₂ receptor constitutive activity and to probe why ticagrelor may be irreversible in platelets. Using a BRET-based approach, this current study discovered that Zn2+ acts as a non-selective positive regulator of the P2Y₁₂ receptor activity. Tetherin (BST-2), a molecular regulator of membrane microdomains, acts as a negative regulator at the receptor. Studies probing the mode of action of ticagrelor have found that neither cholesterol, nor P2Y₁₂ downstream signalling affect the ability of this drug to act in an irreversible manner. Mutant P2Y₁₂ receptor studies could also not decipher the irreversible nature of ticagrelor post-washout, however it discovered key residues in the receptor structure which may be important in regulation of the constitutive activity of the receptor and the inverse agonism of ticagrelor. Further structural studies are required to understand the molecular pharmacology, i.e., binding of ticagrelor at the receptor. The findings of this study will contribute to the understanding of platelet hyperreactivity and the mode of action of ticagrelor, which would ultimately improve clinical outcomes in thrombotic patients.
Defining the molecular pharmacology of ticagrelor
Dimofte, T. (Author). 6 Dec 2022
Student thesis: Master's Thesis › Master of Science by Research (MScR)