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.