Platelet Microrna-mRNA Co-Expression Profiles Correlate with Platelet Reactivity

There is extreme inter-individual variation in platelet reactivity, which likely impacts the variation in both risk and clinical outcome of ischemic vascular disease since platelet hyperreactivity has prospectively been shown to be a risk for recurrent coronary syndromes. Although heritability strongly influences the inter-individual variation in platelet reactivity, there is a lack of understanding of the molecular and genetic mechanisms responsible for this variability. To understand some of these mechanisms, we have previously performed mRNA microarray analysis on platelets of subjects with differing levels of platelet reactivity. We showed a differentially expressed (DE) transcript (VAMP8) was associated with platelet reactivity. Intriguingly, we identified a possible role for microRNA (miRNA)-96 in the regulation of VAMP8 mRNA and protein expression. MiRNAs regulate numerous aspects of normal cell physiology and cause disease by altering protein expression, and recent data demonstrate a role for miRNAs in both normal and diseased human megakaryocytopoiesis. Although others and we have observed miRNAs in platelets, their biology is largely unexplored.

To test whether platelet miRNA levels were associated with platelet reactivity in 19 healthy subjects. Because we had previously obtained platelet mRNA profile data on these 19 subjects, we also had a unique opportunity to test for relationships between differentially expressed miRNAs and target DE mRNAs.

MiRNA microarray analysis was performed on leukocyte depleted platelets from 19 healthy subjects with marked variability in platelet responsiveness. Bioinformatics approaches were used analyze the miRNA data in platelets. Subsequently transfection experiments in cell lines to assess miRNA knockdown of target gene products and reporter gene assays were used for functional assessment of miRNA binding to 3’UTRs of the target genes.

We found that human platelets express 284 miRNAs, some at very high levels. Unsupervised hierarchical clustering of miRNA profiles resulted in two groups of subjects that appeared to cluster by platelet aggregation phenotypes. Seventy-four miRNAs were differentially expressed between subjects grouped according to platelet aggregation to epinephrine, a subset of which predicted the platelet reactivity response. We profiled miRNAs from HEL and Meg-01 cells and found a strong correlation between normal human platelets and both HEL cells and Meg-01 cells. Using whole-genome mRNA expression data on these same 19 subjects, we computationally generated a high-priority list of miRNA-mRNA pairs where the differentially expressed platelet miRNAs had binding sites in 3’UTRs of differentially expressed mRNAs, and the levels were negatively correlated. From this list, three miRNA-mRNA pairs (miR-200b:PRKAR2B, miR-495:KLHL5 and miR-107:CLOCK) were selected, and all three miRNAs knocked down the protein expression of the target mRNA. Co-transfection experiments using reporter gene constructs engineered to contain the candidate mRNA 3’UTR and corresponding miRNA demonstrated that the miRNA of interest directly targeted the 3’UTR of the candidate mRNA.

Results from this study demonstrated (1) platelet miRNAs are able to repress expression of platelet proteins; (2) platelet miRNA profiles are associated with and may predict platelet reactivity and (3) bioinformatic approaches can successfully identify functional miRNAs in platelets. Our findings suggest that selected platelet miRNAs may have potential as biomarkers for vascular thrombosis. It will be important to consider the repertoire and levels of miRNAs when attempting to elucidate the molecular mechanisms responsible for inter-individual variation in platelet reactivity.

No relevant conflicts of interest to declare.