Post-P2Y₁₂-receptor signalling mechanisms and platelet responses to clopidogrel

Anti-aggregatory agents such as clopidogrel limit platelet responses to adenosine diphosphate (ADP) by blocking the P2Y₁₂ receptor and have pivotal roles in the prevention of coronary artery stent thrombosis. However anti-aggregatory responses to such agents vary between individuals and this may predispose to stent thrombosis: the bases for this variablility are incompletely understood. Loss of function CYP2C19 genotypes, impeding clopidogrel bioactivation, have received considerable attention in the literature. However, this alone does not account for the increased rates of lack of response to clopidogrel commonly seen in diseases such as diabetes, obesity and acute coronary syndromes. Furthermore, assessment of aggregation on treatment, while clinically useful, does not precisely measure response to anti-aggregatory therapy, and standardisation of drug dosing may lead to relative underdosing in obese individuals. P2Y12 receptor activation by ADP is linked via Giα protein, to inhibition of adenylate cyclase and thus suppression of cyclic AMP formation. This results in nett pro-aggregatory effects. Hence clopidogrel acts in part by reversing this cascade. A number of prostanoids including prostacyclin and prostaglandin E₁ function as physiological activators of adenylate cyclase and have been shown to potentiate the effects of P2Y₁₂ inhibitors. Furthermore the integrity of the prostanoid/adenylate cyclase signalling pathway is impaired in both cardiovascular disease states and diseases predisposing to cardiovascular disease such as obesity and diabetes. Cyclic AMP formation contributes, together with that of cyclic GMP, to phosphorylation of vasomotor stimulated phosphoprotein (VASP) an effector pathway for ADP effects and their inhibition. The principal hypothesis tested in this thesis was that adenylate cyclase signalling integrity predicted 7 day response to clopidogrel. This was tested by measuring response to clopidogrel (both regarding changes in aggregation and in VASP phosphorylation) in both normal subjects and patients with known ischaemic heart disease. Integrity of prostanoid/adenylate cyclase signalling was measured via pre-clopidogrel platelet response to PGE₁. Putative determinants of clopidogrel response, including genotype and cGMP formation, were evaluated via univariate followed by multivariate analyses, and it was found that PGE₁ response (but not genotype) strongly predicted clopidogrel response. In related analyses, it was also shown that (i) weight adjusted clopidogrel dosing could be utilised to show that variable BMI does not markedly influence response to clopidogrel and that (ii) presence of symptomatic cardiac disease was not a significant cause of variability in response. Finally, exploratory analyses were undertaken to evaluate:- (i) the role of insulin resistance (ii) the putative involvement of the phosphoinositide 3-kinase (PI3- kinase) pathway and (iii) the matricellular protein thrombospondin-1 (TSP-1) as further modulators of clopidogrel effect. Overall the data demonstrate marked inter-individual heterogeneity of responsiveness to clopidogrel, which is engendered at least in part, by variable post-receptor signal transduction.Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2016