Molecular mechanisms of platelet G protein-coupled receptor gene variants

• Main Author: Norman, Jane Eleanor
• Published: University of Bristol 2015
• Subjects: 616.1
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.687283

G protein-coupled receptors (GPCRs) are critical mediators of platelet responses to regulatory agonists and are essential drug targets. This project aimed to identify informative platelet GPCR gene variants and to characterise the mechanism of loss of receptor function for selected variants. Variants were sought in 2400 cardiac surgery patients in which preoperative platelet function test results were used to select subgroups with GPCR dysfunction potentially explained by loss of function P2Y12 receptor, thromboxane A2 receptor and protease-activated receptor 1 gene variants. This approach did not identify variants likely to affect GPCR function. Re-sequencing the protease-activated receptor 4 (PAR4) gene yielded seven different missense variants. After evaluation using computational prediction and homology modelling, the predicted tyrosine 157 to cysteine (Y157C) substitution was demonstrated to reduce PAR4 reactivity and was studied further. Compared to controls, Y157C platelets showed reduced functional responses to PAR4 activating peptide and a greater reduction in responses to a-thrombin in the presence of a PAR1 antagonist, vorapaxar, together consistent with a PAR4 defect. Y157C platelets, showed similar total PAR4 expression levels to controls but reduced surface expression, accounting at least in part for the reduced of PAR4 reactivity. HEK293 cells transfected with a PAR4 Y157C expression construct also showed reduced PAR4 surface expression and functional responses. PAR4 Y157C co-localised with an ER marker in the cell cytoplasm and had an expression pattern consistent with reduced N-glycosylation. Mutagenesis of the putative hydrogen bond partner for the substituted Y157 residue caused a similar phenotype. These findings suggest the Y157C substitution results in receptor mis-trafficking due to a disruption of an intra-molecular hydrogen bond. This first reported characterisation of a variant affecting PAR4 demonstrates that rare variants in the PAR4 gene are a potential source of inter-individual variation in the platelet haemostatic response and the effect of anti-platelet drugs that target the PAR system.