Figure Legends
Figure 1 Aspirin exposure study design and effects on platelet
function. Panel A: Sequence of drug exposures and washout periods for
volunteers enrolled in the discovery cohort. Panel B: Serum thromboxane
B2 (TXB2, y-axis) concentrations (ng/mL) at baseline and after 4 weeks
of 81 md/day and 325mg/day aspirin exposure. Panel C: Maximum light
transmittance aggregometry percent aggregation (y-axis) induced by
arachidonic acid (AA) before and after 3-hours or 4-weeks of daily
aspirin exposure (81- and 325 mg/day). Panel D: An aggregate platelet
function score (PFS, y-axis, see Methods) of non-COX1 dependent platelet
function measures measured before and 3-hours and 4-weeks after daily
aspirin exposure. Box-whisker plots indicate median (bold line),
interquartile range (box), and outliers. Values connecting boxes
indicate p-values for comparison between exposures.
Figure 2 Discovery and validation of aspirin responsive
transcripts. Panel A: “Volcano” plot of genes detected by messenger
RNA sequencing and tested for association with aspirin exposure. Each
point represents a gene of interest, its fold change with aspirin
exposure (log2, x-axis), and p-value (-log10, y-axis). Colored points
represent those that were significant after adjustment for multiple
comparisons (false discovery rate < 0.1). Panel B: “Forest”
plots of meta-analysis for each independently validated aspirin
responsive gene (n = 5). For each gene the treatment effect (TE) and its
standard error (seTE) is plotted for each of the two Validation cohorts
(1 and 2). Results of fixed and random effects meta-analysis for the log
fold change (FC) of aspirin exposure for each gene and 95% confidence
intervals (CI) are plotted.
Figure 3 Functional characterization of aspirin’s effects on
platelet gene expression. Panel A: Selected, top pathways to emerge
from Gene Set Enrichment Analysis (GSEA, see Methods). For each
selected pathway, its rank as well as direction of enrichment is listed.
See Supplementary File 2 for full GSEA results. Panel B: Connectivity
Map analyses (see Methods). A density plot of summary scores (tau,
x-axis) for different sets of small molecules in the Connectivity Map
database: All small molecules (n = 2837) or subsets based on their
mechanisms of action: “Cyclooxygenase inhibitors” (n = 67),
“Thromboxane synthase inhibitors” (n = 13), “NFKB inhibitors” (n =
35), and “AMPK activators” (n = 4). The table lists those small
molecules with the highest tau summary scores, their individual scores,
and attributed mechanisms of action. Panel C: Fold change in platelet
18S ribosomal RNA (rRNA, y-axis) before (Visit 1) or after 4- or 8-weeks
of 325mg/day aspirin therapy (Visits 2 and 3, respectively).
Figure 4 Aspirin’s effect on platelet gene expression and
association with non-COX1 dependent platelet function measures. Panel
A: Effect of aspirin on an aggregate Aspirin Exposure Signature (AES,
see Methods) that aggregates the effects of aspirin on the top
aspirin-responsive genes into a single score. Values indicate p-value
for comparisons between exposures. Panel B and C: The AES (x-axis) in
each of the cohorts (Discovery, Validation 1, and Validation 2) is
plotted against a previously described platelet function scores (PFS,
y-axis) which is an aggregate a panel of individual non-COX1 dependent
platelet function assays. Points represent individual patients, straight
line represents best fit linear regression, shaded region represents
95% confidence intervals. Platelet function and AES scores were
measured at baseline (Panel B) as well as after 4 weeks of aspirin
exposure (Panel C). Panels D and E: Forest plots of effects (TE) and
their standard errors (seTE) from linear regression models of AES vs.
PFS in each cohort as well as results from fixed- and random-effects
meta-analyses across cohorts. Results from baseline samples (Panel D)
and on-aspirin samples (Panel E) are plotted.
Acknowledgements: None
Author Contributions:
Wrote Manuscript (RM, DV), Designed Research (DV, GSG, TLO, SSD),
Performed Research (RM, DV, AW), Analyzed Data (RM, AW).
Supplementary File names and contents:
Supplementary File 1 – “AspirinDifferentialExpression.xlsx” –
Provides the results of the effects of aspirin (either dose) on platelet
gene expression for all genes that passed quality control
(AspirinDifferentialExpression tab). Column header definitions the
‘DataDictionary’ tab.
Supplementary File 2 – “AspirinGSEA.xlsx” – Provides the full gene
set enrichment analysis (GSEA) results for gene sets that were enriched
for genes associated with aspirin exposure (‘AspirinEnrichment’ tab) or
no drug exposure (‘NoDrugEnrichment’ tab). The ‘DataDictionary’ tab
provides column definitions.