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.