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 (< 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 μM[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 in vitro [30,
31] and 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 EIF2S3which 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 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.