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)