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\begin{document}

\title{Aspirin effects on platelet gene expression are associated with a
paradoxical, increase in platelet function.}



\author[1]{Rachel Myers}%
\author[1]{Thomas Ortel}%
\author[1]{Sandeep Dave}%
\author[1]{Alexander Waldrop}%
\author[1]{Geoffrey Ginsburg}%
\author[1]{Deepak Voora}%
\affil[1]{Duke University}%


\vspace{-1em}



  
  \date{May 17, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Aspirin has known effects beyond inhibiting platelet cyclooxygenase-1
(COX1) that have been incompletely characterized. Transcriptomics can
comprehensively characterize the on- and off-target effects of
medications. We used a systems pharmacogenomics approach of aspirin
exposure in volunteers coupled with serial platelet function and
purified platelet mRNA sequencing to test the hypothesis that aspirin's
effects on the platelet transcriptome are associated with platelet
function. We prospectively recruited 74 adult volunteers for a
randomized cross over study of 81- vs. 325 mg/day, each for 4 weeks.
Using mRNA sequencing of purified platelets collected before and after
each 4-week exposure, we identified 208 aspirin-responsive genes with no
evidence for dosage effects. In independent cohorts of healthy
volunteers and patients with diabetes we validated aspirin's effects on
five genes: EIF2S3, CHRNB1, EPAS1, SLC9A3R2, and HLA-DRA. Functional
characterization of the effects of aspirin on mRNA as well as platelet
ribosomal RNA demonstrated that aspirin may act as an inhibitor of
protein synthesis. Database searches for small molecules that mimicked
the effects of aspirin on platelet gene expression in vitro identified
aspirin but no other molecules that share aspirin's known mechanisms of
action. The effects of aspirin on platelet mRNA were correlated with
higher levels of platelet function both at baseline and after aspirin
exposure -- an effect that counteracts aspirin's known antiplatelet
effect. In summary, this work collectively demonstrates a
dose-independent effect of aspirin on the platelet transcriptome that
counteracts the well-known antiplatelet effects of aspirin.%
\end{abstract}%



\sloppy


\textbf{Aspirin effects on platelet gene expression are associated with
a paradoxical, increase in platelet function.}

Rachel A Myers\textsuperscript{1}, Thomas L Ortel\textsuperscript{2},
Geoffrey S Ginsburg\textsuperscript{1}, Alexander
Waldrop\textsuperscript{3}, Sandeep Dave\textsuperscript{3}, and Deepak
Voora\textsuperscript{1}

\textbf{Affiliations} : 1. Center for Applied Genomics \& Precision
Medicine, Duke University School of Medicine, Durham NC; 2. Division of
Hematology, Department of Medicine, Duke University Medical Center,
Durham NC; ~3. Center for Genomics and Computational Biology, Duke
University, Durham NC

\textbf{Financial disclosures} : None

\textbf{Funding} : This work was supported by grant funding from the
National Institutes of Health (R01HL118049).

\textbf{Keywords} : aspirin, platelet aggregometry, transcriptomics,
cardiovascular disease

\emph{Abstract}

Aspirin has known effects beyond inhibiting platelet cyclooxygenase-1
(COX1) that have been incompletely characterized. Transcriptomics can
comprehensively characterize the on- and off-target effects of
medications. We used a systems pharmacogenomics approach of aspirin
exposure in volunteers coupled with serial platelet function and
purified platelet mRNA sequencing to test the hypothesis that aspirin's
effects on the platelet transcriptome are associated with platelet
function. We prospectively recruited 74 adult volunteers for a
randomized cross over study of 81- vs. 325 mg/day, each for 4 weeks.
Using mRNA sequencing of purified platelets collected before and after
each 4-week exposure, we identified 208 aspirin-responsive genes with no
evidence for dosage effects. In independent cohorts of healthy
volunteers and patients with diabetes we validated aspirin's effects on
five genes: \emph{EIF2S3, CHRNB1, EPAS1, SLC9A3R2,} and \emph{HLA-DRA}.
Functional characterization of the effects of aspirin on mRNA as well as
platelet ribosomal RNA demonstrated that aspirin may act as an inhibitor
of protein synthesis. Database searches for small molecules that
mimicked the effects of aspirin on platelet gene expression \emph{in
vitro} identified aspirin but no other molecules that share aspirin's
known mechanisms of action. The effects of aspirin on platelet mRNA were
correlated with higher levels of platelet function both at baseline and
after aspirin exposure -- an effect that counteracts aspirin's known
antiplatelet effect. In summary, this work collectively demonstrates a
dose-independent effect of aspirin on the platelet transcriptome that
counteracts the well-known antiplatelet effects of aspirin.

\emph{Introduction}

Aspirin use is recommended by the United States Preventive Services Task
Force to prevent cardiovascular disease (CVD) in selected, high risk
patients.{[}1{]} It is well known that aspirin inhibits platelet
cyclooxygenase 1 (COX1){[}2{]}; however, aspirin has additional effects
on platelets, which are not fully explained by its effects on COX-1. For
example, 81mg/day aspirin is sufficient to fully suppress platelet
COX1{[}3{]} yet higher doses cause additional reductions in platelet
function as assessed by non-COX1 dependent platelet function assays such
as adenosine diphosphate (ADP) and collagen induced platelet
aggregation.{[}4{]} In addition, despite consistent platelet COX-1
inhibition, non-COX1 dependent measures of platelet function wane over
time{[}5{]} suggesting some adaptation of platelets to chronic aspirin
exposure. Residual platelet function measured using non-COX1 dependent
platelet function assays are risk markers for future cardiovascular
events during aspirin therapy.{[}6, 7{]} Last after decades of
uncertainty around the appropriate aspirin dose for cardiovascular risk
reduction{[}8{]} only recently has there been randomized trial data
comparing the effectiveness and safety of low- vs. high-dose aspirin.
This trial did not identify major differences in efficacy or safety
between aspirin doses; however, nearly 50\% of patients crossed over to
the alternative dose with 3 weeks of randomization. {[}9{]} Therefore,
gaps in our knowledge around one of the most prescribed medicines
worldwide remain.

Although anucleate, it is well known that platelets are a rich source of
messenger RNA (mRNA). Platelets are invested with mRNA from
megakaryocytes during thrombopoiesis in a regulated manner{[}10{]}, can
uptake transcripts from other vascular cells{[}11{]}, and are also a
major source of transcripts found in plasma extracellular vesicles
{[}12{]}. Genetic and gene expression studies have confirmed that
variable platelet mRNA levels lead to variable levels of platelet
function.{[}13-15{]} We have previously shown that a blood-based,
``aspirin response signature'' of transcripts primarily of platelet
origin correlate with platelet function on aspirin and predict future
cardiovascular events in patients taking aspirin.{[}16{]} These data
support the concept that the biologic basis for variable platelet
function lies, at least in part, in variable platelet gene expression.
We{[}17{]} and others{[}18{]} have shown that aspirin (or its stable
metabolite salicylate) can alter expression of selected genes in
megakaryocytes as well as in circulating platelets in patients exposed
to aspirin. However, these prior studies were limited in scope as they
did not systematically characterize aspirin's effects on platelet gene
expression.

Based on these knowledge gaps, we pursued the hypothesis that
variability in the platelet function response to aspirin is associated
with aspirin induced changes in the platelet transcriptome. We tested
this hypothesis using an open label, randomized crossover study of low-
vs. high-dose aspirin in human volunteers with longitudinal COX-1 direct
and indirect assays of platelet function, drug concentrations, and RNA
profiling at each time point.

\emph{Methods}

\emph{Human subject protocols}

\emph{Discovery Cohort}

We conducted a prospective, open label, randomized cross-over study to
assess the effects of antiplatelet therapy (non-enteric coated aspirin
81 mg/day, aspirin 325 mg/day, and ticagrelor 90 mg twice daily) on
platelet function and gene expression as previously described.{[}19{]}
The results of aspirin exposure are presented here. The Supplemental
Methods provides more details of inclusion/exclusion criteria. The Duke
Institutional Review Board approved the study protocol.

\emph{Validation cohorts}

Banked, platelet RNA and platelet function data from previously
described{[}20{]} cohorts of healthy volunteers or patients with
diabetes exposed to 325mg/day aspirin for 2 or 4 weeks was used for
independent validation.

\emph{Platelet function and salicylate testing}

Phlebotomy for platelet function testing was performed as previously
described with additional details in the Supplemental Methods.{[}20{]}

\emph{Platelet RNA purification}

Platelet isolation from human blood using a previously described{[}21{]}
leukocyte depletion (LD) procedure is briefly described here with
additional details in Supplemental Methods.

\emph{Platelet RNA isolation}

Platelet RNA samples from the same participant were batched together at
every stage from platelet RNA extraction through sequencing in order to
minimize introducing technical artifacts between visits.

RNA Isolation from purified platelets using a phenol-chloroform method
as described in the Supplemental Methods.

\emph{Platelet RNA quality analysis and sequencing}

Total RNA quality and quantity was assessed using the Agilent
Bioanalyzer 2100 and the Qubit fluorometer, respectively. Samples with
sufficient quantity and quality RNA went on to poly(A) mRNA capture and
construction of stranded mRNA-seq libraries from total RNA was achieved
using the commercially available KAPA Stranded mRNA-Seq Kit (catalog
\#KK8421) using the manufacturer's protocol and as described in the
Supplemental Methods.

\emph{Platelet mRNA sequencing alignment and expression quantification}

See Supplemental Methods for details regarding quantification of
platelet mRNA transcripts.

\emph{Nanostring expression profiling}

Targeted gene expression of selected transcripts identified using RNAseq
was performed in the validation cohort using Nanostring technology as
described in the Supplemental Methods.{[}22{]}

\emph{Data access}

All platelet function and RNAsequencing data used in this manuscript is
available through Gene Expression Omnibus (GSE158765)

\emph{Statistical analyses}

See Supplemental Methods for details of statistical methods used to
analyze platelet function and gene expression data.

\emph{Results}

\emph{Aspirin's effects on platelet function outcomes}

Eighty-six participants completed a baseline visit, 81 exposed to
low-dose aspirin, and 79 exposed to high-dose (Figure 1A). To assess the
extent to which platelet COX-1 was inhibited we used serum thromboxane
B2 (TXB2), and arachidonic acid (AA) induced platelet aggregation. After
4 weeks of daily dosing, both 81mg and 325mg equivalently suppressed
platelet COX-1 as assessed by (AA) induced platelet aggregation (Figure
1B) and TXB2 (Figure 1B). To assess non-COX1 dependent assays of
platelet function, we used a previously described platelet function
score (PFS, see Supplemental Methods) that aggregates nine different,
non-COX1 dependent assays of platelet function into a single score.
Higher PFS values reflect greater platelet function. After 4-weeks of
daily aspirin, both 81 and 325mg doses reduced PFS (p \textless{} 0.001)
with slightly greater reduction with 325mg vs. 81mg (p = 0.07) (Figure
1D). Therefore, daily doses of low- and high dose aspirin reduce COX-1
and non COX-1 measures of platelet function. Compared to low-dose
aspirin, high dose aspirin produces equivalent COX-1 suppression and
slightly greater non-COX 1 inhibition of platelet function.

\emph{Aspirin's effects of platelet gene expression}

After quality analysis of the platelet RNA sequencing data there were
312 platelet mRNA datasets available for analysis from 73 unique
patients representing 14333 transcripts. In general, the level of
contamination by granulocytes was low (proportion of samples
with\emph{PTPRC} /\emph{ITGA2B} count ratio [?] 0.01 =6/312) and did not
vary by treatment (likelihood ratio test P-value = 0.11). We have
previously shown that platelet gene expression levels are highly
correlated within an individual between Visits 1 and Visit 4.{[}23{]}
Therefore in all subsequent analyses, these two visits were treated
equivalently as no-drug treatment visits and no adjustment for leukocyte
contamination was performed.

To test our primary hypothesis that aspirin exposure alters the platelet
mRNA transcriptome, we used a mixed-effects regression framework to
identify transcripts that changed with any aspirin exposure compared to
no drug treatment visits. In a univariate analysis using the false
discovery rate (FDR) q-value \textless{} 0.10 to account for multiple
testing we identified 208 transcripts that were differentially expressed
(43 down regulated, 165 upregulated, Figure 2, Supplemental Data File 1)
after aspirin exposure. To test for aspirin-dose response relationships
we used two complementary approaches: 1) contrasting the effects of 81mg
vs. 325mg/day and 2) correlating gene expression with salicylate
concentrations during aspirin treatment. We found no evidence for
differences on platelet gene expression for the set of 208 gene aspirin
differentially expressed genes and higher aspirin dose or salicylate
concentrations (enrichment p-values = 0.3 and 0.9, respectively).

We used banked platelet RNA from prior research cohorts{[}19{]} of
healthy adults and patients with diabetes (n= 91 across both cohorts)
exposed to 81mg/day or 325mg/day aspirin using a custom Nanostring assay
to validate the effects of aspirin. Due to limits on the number of
transcripts available in a Nanostring assay, we prioritized the list 208
transcripts based on mean expression and robustness to various starting
points for quantifying expression, and genes that exhibited positive
correlation (r \textgreater{} 0.25) between RNAseq and Nanostring to a
subset of 51 genes to test for validation. Among these 51 genes, we
found five that independently validated in the replication cohorts
(fixed effects meta-analysis p-value for aspirin effect \textless{}
0.05) as changing in response to aspirin exposure: \emph{EIF2S3, CHRNB1,
EPAS1, SLC9A3R2,} and \emph{HLA-DRA} . (Figure 2B)

\emph{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
\textless{} 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 \textless{} 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 (\textbar{}tau\textbar{} \textgreater{} 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 \emph{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)

\emph{Effects of aspirin on platelet gene expression and platelet
function outcomes}

To test the hypothesis that aspirin's effects on platelet mRNA
correlates with aspirin's effects on non-COX1 dependent platelet
function we first summarized the overall behavior of the 51,
prioritized, differentially expressed genes identified in the Discovery
cohort into a single AES score (see Methods). By definition, the AES
score increases in response to aspirin exposure with no dosage effects
(Figure 4A). We used the aggregate AES to correlate with platelet
function scores across all cohorts (Discovery and both Validation) to
test for the relationship between the effects of aspirin on gene
expression and platelet function.

At baseline, prior to aspirin exposure, platelets with higher AES scores
can be thought of as having an endogenous ``aspirin-like'' effect in
their platelet transcriptomes. Prior to aspirin exposure, higher AES
scores correlated with higher PFS both before (effect = 0.49, 95\%
confidence interval {[}CI{]}: 0.10-0.89, random effect model p-value =
0.01, Figure 4B and 4D) with higher levels of platelet function. In
aspirin treated samples, higher AES levels reflect those participants in
whom aspirin exposure resulted in a higher AES levels (due to higher
baseline or change upon aspirin exposure). After aspirin exposure,
higher AES levels were similarly correlated with higher platelet
function (effect = 0.70, 95\% confidence interval {[}CI{]}: 0.30-1.18,
random effect model p-value = 0.002, Figure 4C and 4E). Therefore, the
biological pathways impacted by aspirin and quantified through platelet
gene expression profiling indicate that aspirin produces a paradoxical
effect that increases platelet aggregation \emph{ex vivo} . Higher
aspirin-like effects (i.e., higher AES scores) either at baseline or in
response to aspirin exposure, in aggregate, correlate with higher PFS
scores, an effect that is opposite in direction with aspirin's known
effects on inhibiting platelet COX-1 and platelet function.

\textbf{Discussion}

Aspirin is widely used to prevent cardiovascular disease and colorectal
cancers. While its role in as an antiplatelet agent is well described,
there remain large gaps in our knowledge of its diverse effects or
optimal dose beyond cardiovascular disease. In this prospective
randomized study of different aspirin doses, we aimed to comprehensively
study the effects of aspirin on platelet gene expression and function.
We tested the hypothesis that aspirin's effects on platelet gene
expression contribute to variability in platelet function in response to
aspirin exposure. Compared to daily low-dose aspirin high-dose aspirin
exhibited equivalent inhibition of platelet COX1 and slightly greater
platelet inhibition in non-COX-1 dependent measures of platelet
function. We found that aspirin exposure results in a mild perturbation
in platelet mRNA (1.5\% of transcripts affected) with no evidence for
dose effects. Using multiple, complimentary lines of evidence, we found
that aspirin exposure appears to have effects as an inhibitor of protein
synthesis in platelets and/or megakaryocytes. In terms of platelet
function, aspirin's effects on platelet gene expression appear to
counteract aspirin's platelet inhibitory effects on platelet COX-1. In
summary, using a diverse array of platelet function and molecular tools,
there appear to be effects of aspirin beyond platelet COX-1 inhibition
that are dose-independent and counteract the main antiplatelet effect of
aspirin.

Aspirin is well known to inhibit platelet COX-1{[}2{]} which is critical
for arachidonic acid-induced platelet aggregation. Additionally, aspirin
has been attributed with a wide array of additional mechanisms of action
including: NFKB/IKB inhibition{[}25{]}, AMPK activation{[}26{]}, COX2
inhibition{[}27{]}, RUNX1 pathway modulation{[}28{]}. It is unknown the
extent to which these off-target effects of aspirin contribute to its
clinical effects in preventing cardiovascular disease or if they are
relevant in typical prescribed doses (\textless{} 325 mg/day). Many of
these effects are attributed to the main metabolite of aspirin,
salicylate, which has a longer half-life than aspirin and achieves
systemic levels in the 30--100 \selectlanguage{greek}μ\selectlanguage{english}M{[}29{]} range with typical daily
dosing. Alternatively, they may arise from downstream effects of COX-1
inhibition on arachidonic acid metabolites. Aspirin and salicylates have
previously been shown to inhibit protein synthesis \emph{in vitro}
{[}30, 31{]} and \emph{in vivo} {[}31{]} in the gastrointestinal tract
of patients taking 600 mg/day. This effect appears to be mediated
through activation of AMPK and mTOR pathway{[}31{]} and acetylation of
non-COX1 targets {[}32{]}. Our findings of aspirin potentially acting as
an inhibitor of protein synthesis are in line with these previous
observations and extend the findings to platelets from humans taking
lower, therapeutic aspirin doses. Among the top genes that independently
validated as being modulated by aspirin exposure were \emph{EIF2S3}which
encodes for a core subunit of eukaryotic translation initiation factor-2
(eIF2) complex, which is essential for protein synthesis.{[}33{]} The
suppression of aspirin on 18S platelet ribosomal RNA subunit (which is
not captured in RNA sequencing) in platelets in our study further
supports this role for inhibition of protein synthesis in platelets. Our
finding that aspirin's effects in platelets is recapitulated in cellular
data suggest a potential direct role of aspirin (or its metabolite)
modifying gene expression likely in megakaryocytes. We found no
significant effect of aspirin dose on aspirin induced changes in
platelet gene expression. Therefore, at 81mg/day, any effects on
platelet gene expression appear to be saturated. Although our study was
not designed to identify the mechanism of action of the observed
effects, our analysis of the CMap data (Figure 3B) identified aspirin
but no other COX-1, COX-2, or thromboxane inhibitors as causing similar
changes in gene expression that we observed in human platelets.
Therefore, these findings raise the hypothesis of a non-canonical effect
of aspirin on protein synthesis in human megakaryocytes and/or
platelets.

The primary target of aspirin is acetylation of platelet COX-1 which is
uniformly suppressed when adherence and absorption are adequately
addressed.{[}3{]} In our studies, we show that a single 325mg dose of
non-enteric coated aspirin suppresses COX1 and that after multiple daily
doses, similar suppression is achieved with 81mg/day. However, platelet
COX-1 variably contributes to collagen, ADP, and epinephrine induced
aggregation such that in the setting of complete COX-1 inhibition there
is residual platelet aggregation using these agonists. This residual
aggregation is referred to as ``non-COX-1 dependent''{[}5{]} or ``COX-1
indirect''{[}34{]} in the literature. We have previously described an
approach to aggregate multiple measures of non-COX-1 dependent platelet
function using a platelet function score (PFS).{[}5{]} In our current
study, we found that high dose aspirin may result in further reduction
in PFS (Figure 1D) despite complete suppression of platelet COX1 (Figure
1C), a finding that is consistent with the prior ASPECT{[}4{]} trial
that found greater platelet inhibitory effects of higher aspirin dose on
non COX-1 dependent platelet function pathways. The extent to which this
greater, non-COX1 mediated platelet inhibition of high-dose aspirin on
cardiovascular events (or bleeding) is unknown but will be clarified by
the ongoing ADAPTABLE clinical trial comparing low vs. high dose aspirin
(NCT02697916). Until then, our findings suggest that there are no large
differences between 325mg/day and 81mg/day aspirin and from the
perspective of platelet function and gene expression there is no clear
advantage to the higher dosage.

To examine aspirin effects on platelet gene expression with platelet
function, we used an aspirin exposure signature which aggregates the
expression of aspirin responsive transcripts into a single ``score''
with higher scores representing a greater aspirin effect. This approach
allowed us to quantify the biological effects of aspirin using gene
expression and also assign an ``aspirin-like'' effect to all samples
used in this study before and after aspirin exposure. Using this score,
we found that the effect of aspirin on platelet gene expression appears
to attenuate the platelet inhibitory effects of aspirin on COX-1
inhibition. The overall magnitude of this effect is relatively small,
however. The average effect of aspirin on increasing the AES score was
0.50 (Figure 4A) and from the meta-analysis, this magnitude of effect
translates into 0.43 higher PFS units on aspirin (Figure 4E). The
magnitude of aspirin's effects of inhibiting platelet COX-1 is
approximately 4.0 PFS units based on the change in PFS 3 hours after a
single 325mg aspirin effect of aspirin on inhibiting platelet COX-1
(Figure 1D). Therefore, during a 4-week aspirin exposure the effects of
aspirin on platelet gene expression are relatively minor (10\% of
overall magnitude) compared to the magnitude of effect on inhibiting
platelet COX-1. The apparent time-dependent effects of aspirin on
platelet ribosomal RNA (Figure 3C) suggest that longer durations of
aspirin may be associated with larger effects on platelet gene
expression. In fact, studies of patients taking aspirin for more than 2
months showed greater changes in platelet gene expression and higher
residual, non-COX1 platelet function.{[}18{]} Therefore, future work in
patients chronically exposed to aspirin may identify a greater
contribution of aspirin's effects on platelet gene expression, high
residual platelet reactivity, and risk for cardiovascular events.

In conclusion, using a systems pharmacogenomics approach to studying the
effects of aspirin in a rigorous, human experimental model of drug
response, unbiased, genome-wide gene expression analysis, independent
validation, and correlation with \emph{ex vivo} measures of platelet
function we identify novel effects of aspirin on platelets that
counteract its canonical, pharmacological effects as a platelet
inhibitor that may be part of what has been previously described as an
adaptive response to exposure to antiplatelet therapy.{[}35{]} We extend
prior observations of this effect of aspirin to platelets from humans
exposed 81mg/day of aspirin with no evidence for dose-response. Overall,
this effect appears to attenuate the well-described inhibitory effects
of aspirin on inhibiting platelet COX-1. The extent to which these
effects on platelet gene expression impact the clinical effect of
aspirin on preventing CVD are currently not known but deserve future
consideration.

\textbf{Highlights}

\begin{itemize}
\tightlist
\item
  What is the current knowledge on the topic?
\item
  Aspirin is widely prescribed to prevent cardiovascular disease through
  inhibition of platelet COX-1. Aspirin is known to have effects beyond
  inhibiting platelet COX-1; however, characterization of non-COX1
  effects in platelets has been lacking.
\item
  What question did this study address?
\item
  This study comprehensively characterized the on- and off-target
  effects of aspirin on platelet gene expression and platelet function.
\item
  What does this study add to our knowledge?
\item
  Using an experimental protocol of human aspirin exposure and serial
  platelet transcriptomic and functional testing, we identified a
  non-canonical effect of aspirin on inhibiting protein synthesis
  pathways in platelets. This effect acts to attenuate the platelet
  inhibitory effects of aspirin on platelet COX-1
\item
  How might this change clinical pharmacology or translational science?
\item
  Compared to low-dose aspirin we found no major differences with
  high-dose aspirin with respect to platelet function or platelet
  transcriptome and thus no rationale for using high-dose aspirin.
  Patients chronically prescribed aspirin to prevent cardiovascular
  disease that have an accentuated platelet transcriptional response may
  experience smaller than expected reductions in cardiovascular risk.
\end{itemize}



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3. Grosser T, Fries S, Lawson JA, Kapoor SC, Grant GR, FitzGerald GA.
Drug Resistance and Pseudoresistance: An Unintended Consequence of
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4. Gurbel PA, Bliden KP, DiChiara J, Newcomer J, Weng W, Neerchal NK,
Gesheff T, Chaganti SK, Etherington A, Tantry US. Evaluation of
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\textbf{Figure Legends}

\textbf{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.

\textbf{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 \textless{} 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.

\textbf{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)\textbf{.} 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).

\textbf{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.\selectlanguage{english}
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