Epigenetic Regulation of <i>F2RL3</i> Associates With Myocardial Infarction and Platelet Function.

Abstract

DNA hypomethylation at the <i>F2RL3</i> (F2R like thrombin or trypsin receptor 3) locus has been associated with both smoking and atherosclerotic cardiovascular disease; whether these smoking-related associations form a pathway to disease is unknown. <i>F2RL3</i> encodes protease-activated receptor 4, a potent thrombin receptor expressed on platelets. Given the role of thrombin in platelet activation and the role of thrombus formation in myocardial infarction, alterations to this biological pathway could be important for ischemic cardiovascular disease. We conducted multiple independent experiments to assess whether DNA hypomethylation at <i>F2RL3</i> in response to smoking is associated with risk of myocardial infarction via changes to platelet reactivity. Using cohort data (N=3205), we explored the relationship between smoking, DNA hypomethylation at <i>F2RL3</i>, and myocardial infarction. We compared platelet reactivity in individuals with low versus high DNA methylation at <i>F2RL3</i> (N=41). We used an in vitro model to explore the biological response of <i>F2RL3</i> to cigarette smoke extract. Finally, a series of reporter constructs were used to investigate how differential methylation could impact <i>F2RL3</i> gene expression. Observationally, DNA methylation at <i>F2RL3</i> mediated an estimated 34% of the smoking effect on increased risk of myocardial infarction. An association between methylation group (low/high) and platelet reactivity was observed in response to PAR4 (protease-activated receptor 4) stimulation. In cells, cigarette smoke extract exposure was associated with a 4.9% to 9.3% reduction in DNA methylation at <i>F2RL3</i> and a corresponding 1.7-(95% CI, 1.2-2.4, <i>P</i>=0.04) fold increase in <i>F2RL3</i> mRNA. Results from reporter assays suggest the exon 2 region of <i>F2RL3</i> may help control gene expression. Smoking-induced epigenetic DNA hypomethylation at <i>F2RL3</i> appears to increase PAR4 expression with potential downstream consequences for platelet reactivity. Combined evidence here not only identifies <i>F2RL3</i> DNA methylation as a possible contributory pathway from smoking to cardiovascular disease risk but from any feature potentially influencing <i>F2RL3</i> regulation in a similar manner.