Functional characterization of aspirin effects on platelet gene expression:
To characterize the functional effects of aspirin on platelet gene expression we used two complementary approaches: 1) Gene Set Enrichment Analyses (GSEA) which uses all transcripts from the mRNA sequencing experiment and 2) Connectivity Map (CMap) using a broader list (FDR < 15%) of differentially expressed genes identified through mRNA sequencing.
Using GSEA, we identified gene sets that were enriched (GSEA family wise error rate [FWER] p < 0.05) for genes that were upregulated by aspirin exposure. Many of these gene sets were related to ribosome biogenesis (Figure 3B) full list available in Supplementary File 2. Gene sets that were enriched in genes that were downregulated in response to aspirin reflected calcium/mTOR signaling, platelet aggregation, glucose metabolism, and cytoskeletal pathways (Figure 3A and Supplementary File 2). Therefore, the global effects of aspirin on the platelet transcriptome identified effects on multiple pathways including many related to protein synthesis.
CMap searches for similarities in the effect of aspirin on platelets using mRNA sequencing to the effects of 2837 small molecules on gene expression across nine cell lines. In this analysis, molecules with positive ‘tau’ scores (range -100 to +100) are interpreted as producing similar effects in cell lines as aspirin exposure in platelets and negative tau scores reflecting effects of aspirin in cells that are in the opposite direction as those found in platelets. The top molecules to emerge from this analysis (|tau| > 90) are listed in Figure 3B. In addition to identifying aspirin (tau = 94), five additional molecules not known to share mechanisms with aspirin were identified: inhibitors of protein synthesis (cephaeline and emetine), protein kinase A (H-7), protein kinase C (GSK-3-inhibitor II), and tubulin (parbendazole). In contrast, none of the 121 Cmap molecules that share known mechanisms of action with aspirin (COX1, COX2, NFKB, or TBXAS1 inhibitors or AMPK activators) produced tau scores as large as those as aspirin. (Figure 3B). Therefore, in cellular models, we validate the global effects of aspirin on gene expression and raise the hypothesis of potential non-canonical effects of aspirin, including a role in inhibiting protein synthesis.
Among the pathways identified, we chose to focus on aspirin’s effects on protein synthesis given that EIF2S3 (Eukaryotic Translation Initiation Factor 2 Subunit Gamma) validated as an aspirin-responsive gene in our human platelet data (Figure 2B), that ribosomal biosynthesis emerged from GSEA analyses (Figure 3A), and the identification of more than 1 inhibitor of protein synthesis from CMap analyses (Figure 3B). We explored the extent to which that aspirin exposure results in altered platelet ribosomal RNA (rRNA) levels which are well-known to regulate global protein synthesis.[24] Using available, banked platelet RNA samples from healthy subjects, we measured 18S rRNA levels after 4 and 8 weeks of 325 mg/day aspirin exposure using qPCR. Aspirin exposure of 325mg/day for at least 4 weeks was associated (p = 0.04) with lower platelet 18S rRNA levels with a trend towards greater effects for longer durations of aspirin exposure. (Figure 3C)