Effect Of Age and Gender On Human Platelet mRNA and Micro-RNA Levels

The well-known inter-individual variation in human platelet reactivity is heritable, but there is limited understanding of the responsible genetic mechanisms. Prior work supports an effect of age, gender, race, body mass index (BMI) and other demographic variables on platelet function, but there is little or no data addressing whether these variables are associated with platelet gene expression. Quantitative and qualitative variants of different classes of platelet RNAs are important windows into megakaryocyte and platelet gene expression, and emerging evidence indicates the utility of specific transcripts as disease biomarkers, as vectors for systemically transmitting genetic information in microparticles, as mediators of pharmacogenetic effects, and as potential therapies. However, to date the small sample sizes of platelet transcriptomic studies have not permitted testing for associations between demographic variables and platelet RNA levels. The Platelet RNA And eXpression-1 (PRAX1) study was designed to characterize mRNAs and microRNAs (miRNAs) in highly purified platelets from a group of healthy, non-diabetic subjects. 163 participants (84 whites and 70 blacks) were recruited, and after exclusion due to use of anti-platelet medication (defined as non-responsiveness to arachidonic acid-induced platelet aggregation) or abnormal hematological parameters, 154 subjects were included for RNA profiling and analyses. Leukocyte-depleted platelet RNA was profiled using the Human Gene 1.0 ST Array (Affymetrix) for mRNA and the nCounter (Nanostring) for miRNA. After normalization, mRNAs and miRNAs were defined as commonly expressed if they were above background in over 65% of subjects. This yielded 5813 common mRNAs and 181 common miRNAs. These profiles were validated using a separate cohort with similar demographic characteristics (n=19) and by plotting rank correlations of mRNAs (r = 0.57; p = 2.3x10^-311) and miRNAs (r = 0.69; p = 7.3x10^-21). We identified 130 mRNAs and 15 miRNAs that were differentially expressed (DE) by age (Q<0.05). These 130 mRNAs were enriched for putative binding sites for these 15 miRNAs (p<0.001). We identified a network of DE miRNAs targeting DE mRNAs, in which the miRNA and mRNA were significantly and inversely correlated by age. Mitochondrial mRNAs were also inversely correlated with age. Second, we identified 54 mRNAs and 9 miRNAs DE by gender (Q<0.05). As expected, the Y-chromosome genes, EIF1AY, TMSB4Y, UTY and DDX3Y were expressed more highly in males (p = 1.22x10^-82, p = 9.28x10^-70, p = 2.89x10^-68 and p = 7.45x10^-58, respectively). A network of miRNAs and mRNAs, both DE by gender, was identified in which the miRNAs were predicted to target the mRNAs. Lastly, a single miRNA but no mRNAs were DE by BMI. In summary, levels of platelet mRNAs and miRNAs are strongly associated with age and gender, but for the most part, not with BMI. The inverse relationship between these two DE RNA classes suggests miRNAs may regulate mRNA levels between genders and upon aging. Future association studies between platelet RNAs and either ex vivo platelet function or in vivo platelet-mediated hemostasis and thrombosis must account for age and gender.