A Large Cluster of Micrornas At 14q32 Defines an RNA Expression Module That Accounts for Racial Differences in Protease Activated Receptor 4-Mediated Platelet Reactivity

Abstract 380

Myocardial infarction and stroke typically result from an occlusive platelet thrombus formed at the site of atherosclerotic plaques. There is reproducible variation in platelet reactivity among different individuals – a variation that likely contributes to thrombosis risk. The inter-individual variation in platelet reactivity is highly heritable, and this heritability is greater in African Americans (AAs) than European Americans (EAs), but there is limited understanding of the genetic mechanisms responsible for this variability. The Platelet RNA And eXpression-1 (PRAX1) study was designed to identify novel genes and miRNAs responsible for inter-individual variation in platelet reactivity. We assessed platelet aggregation in 70 AA and 84 EA healthy subjects and profiled miRNA and mRNA from highly purified blood platelets. The richness of these data provided unique opportunities to establish miRNA-mRNA-physiology relationships that led to a number of novel and unexpected results.

Although 607 miRNAs were detected above background, some were virtually absent in some samples. Using highly stringent criteria based on a biphasic distribution of expression in the 154 samples, we defined 178 platelet miRNAs present in at least 65% of subjects. Statistical analysis identified miRNAs that were differentially expressed (DE) by age, gender and race. A miRNA-miRNA correlation matrix uncovered a cluster of 52 miRNAs located at 14q32 that were highly co-regulated and that were expressed at higher levels in platelets from EAs than AAs. Unexpectedly, platelet aggregation in response to PAR4-AP was significantly higher in AAs than in EAs, whereas no racial difference was observed for the platelet aggregation response to arachidonic acid, ADP or PAR1-AP. Having established that 14q32 miRNAs were DE by race and that PAR4-mediated platelet reactivity was different by race, we sought to determine whether there were mRNAs that linked these two discoveries. Using a simple linear regression with an FDR q<.25, we found 119 mRNAs that were positively correlated with PAR4 reactivity. Notably, the gene encoding PAR4 (F2RL3) was not DE by race. A substantially higher proportion of 14q32 miRNAs were represented among the miRNAs enriched for targets among PAR4-positively-correlated mRNAs than would be expected by chance (p = 0.0092, OR=3.71). In other words, the racially-differentially expressed miRNAs located on 14q32 target mRNAs that are positively associated with PAR4 reactivity. A conditional correlation analysis showed that the significant association between PAR4 reactivity and race vanished after accounting for RNA expression, indicating that these two traits are highly collinear. We were especially intrigued with the gene, PCTP, which encodes phosphatidylcholine (PC) transfer protein (TP), because of the well-established importance of fatty acids, phospholipids and PC in platelet reactivity and because PC-TP catalyzes the transfer of PC between membranes. PCTP was DE by race (q value=10⁻¹⁹) and by PAR4 reactivity (p=10⁻⁶), and PC-TP protein was higher in AAs than EAs. PCTP is predicted to be targeted by 14q32 miRNA, hsa-miR-376c, and we validated that PC-TP mRNA and protein were knocked down by hsa-miR-376c.

In summary, we have discovered racial differences in PAR4-mediated platelet activation and uncovered a genetic regulatory module in which racial differences in expression of miRNAs located at 14q32 and their target mRNAs account for this racial difference in platelet function. High levels of 14q32 miRNAs in EAs specifically target mRNAs that positively regulate PAR4 activity, resulting in lower sensitivity to PAR4-mediated activation. The inverse is true of AAs. Compared to EAs, AAs have a 2-fold increase in the incidence of CHD and a lower long-term survival. In addition, it is unknown whether the racial difference in platelet reactivity impacts the benefits and risks of anti-platelet therapies. Because novel inhibitors of PAR1 and 4 are currently in clinical development, it is critical to know whether dosing of such agents should be adjusted by race to maximize benefit and avoid toxicity. Lastly, since PAR4 is expressed in tissues other than platelets (e.g., heart, brain, liver), if 14q32 miRNA targets are DE by race in non-platelet tissues, it is possible that the consequent altered gene expression could contribute to non-thrombotic diseases known to show racial differences.