High On-Treatment Platelet Reactivity: Aspirin versus Clopidogrel

Background: Antithrombotic regimen in patients on oral anticoagulation (OAC) post-percutaneous coronary intervention (PCI) is challenging. At least, one antiplatelet agent in combination with OAC is recommended after PCI for 6–12 months. Clopidogrel is used most frequently in this setting. However, data comparing P2Y12 inhibition with clopidogrel versus cyclooxygenase inhibition by acetylsalicylic acid (ASA, aspirin) is missing. It is well known that the antiplatelet effects of ASA and clopidogrel are frequently impaired (high on-treatment platelet reactivity [HTPR]). In this pilot investigation, we compared the antiplatelet effects of clopidogrel versus ASA. Methods: In this retrospective single-center database analysis, we investigated platelet reactivity by light transmission aggregometry in patients under different antiplatelet regimes. Results were presented as maximum of aggregation (MoA). HTPR to ASA and to clopidogrel were assessed. Results: 755 patients were enrolled. 677 were on ASA, 521 were on clopidogrel, and 198 had OAC. Overall mean age was 73 ± 13.4 years, and 458 (60.7%) were male. HTPR to ASA occurred in 94/677 patients (13.9%), and mean arachidonic acid-induced MoA was 14.15 ± 19.04%. HTPR to clopidogrel occurred in 241/521 patients (46.3%), and mean adenosine diphosphate-induced MoA was 50.06 ± 20.42%. HTPR to clopidogrel was significantly more frequent than HTPR to ASA; single antiplatelet therapy (SAPT)-mono ASA: 27/199 (13.6%) versus mono clopidogrel: 6/18 (33.3%); p = 0.037; SAPT with OAC-OAC with ASA: 8/35 (22.9%) versus OAC with clopidogrel: 27/60 (45%); p = 0.046. Same difference in HTPR contingency could be shown in subgroups of dual antiplatelet therapy and ASA + clopidogrel + OAC therapy. Conclusion: Impaired pharmacodynamic response to clopidogrel was more frequent as HTPR to ASA. Hence, ASA should be tested in combination with OAC post-PCI.


Introduction
Oral anticoagulation (OAC) is indicated for patients with atrial fibrillation (AF) undergoing percutaneous coronary intervention (PCI) [1]. Acetylsalicylic acid (ASA, aspirin) and clopidogrel are both established in secondary prevention after PCI. Post-procedurally, European guidelines recommend short-term antithrombotic triple therapy regimes with ASA, P2Y12 inhibitors, and OAC for less than a week. Then, therapy with ASA is stopped, and antiplatelet therapy is continued with OAC and P2Y12 inhibitors (most favorably clopidogrel) for 6-12 months [2]. Pharmacodynamic response to both agents can vary significantly. In this context, high on-treatment platelet reactivity (HTPR) is a common phenomenon and may lead to thromboembolic complications [3,4]. Previous literature indicates that incidences of HTPR might be lower for ASA as compared to clopidogrel [5,6]. As thromboembolic and bleeding events are frequent in AF patients after PCI, an optimal antithrombotic regime needs further investigation [7]. However, current data is insufficient. Studies comparing the combination of OAC and P2Y12 inhibition with clopidogrel or OAC and cyclooxygenase inhibition by ASA are completely missing. Moreover, direct comparison of ASA or clopidogrel antiplatelet effects in one cohort is sparse. Therefore, we aimed to investigate differences in the pharmacodynamic response of ASA compared to clopidogrel in patients with different antithrombotic therapy regimes. Furthermore, we investigated these effects in a subgroup of patients with OAC combined with single antiplatelet therapy (SAPT).

Study Population and Design
In this retrospective single-center database analysis, chronic coronary syndrome (CCS) patients with prior PCI from the Department of Cardiology, Pneumology, and Vascular Medicine from the University Hospital of Düsseldorf were enrolled. Study groups were defined depending on patients' antiplatelet and coagulation regimes (Fig. 1). Age under 18, antiplatelet therapy with P2Y12 inhibitors other than clopidogrel, coagulation disorders, hematological diseases, and recent acute coronary syndrome with PCI <30 days were exclusion criteria. Included patients provided informed written consent. This study protocol was reviewed and approved by the Ethics Committee of the University of Düsseldorf, approval number [4,502].

Platelet Function Testing
Blood samples were collected in citrate vacutainers (dilution 1:10). Platelet function was measured by light transmission aggregometry (LTA) in platelet-rich plasma. Platelet-rich plasma and platelet-poor plasma were generated by 10 min of whole blood centrifugation at 270 G and 1,200 G, respectively. Adenosine diphosphate (ADP) was used as an agonist to test HTPR to clopidogrel, and arachidonic acid (ARA) was used to test HTPR to ASA, respectively. Aggregation was measured as maximum of aggregation (MoA). HTPR to ASA was defined as MoA ≥20% with ARA stimulation and HTPR to clopidogrel as MoA ≥46% with ADP stimulation, as previously described and compared between groups [8,9].

Statistical Analysis
All statistics and analyses were conducted in GraphPad Prism© (GraphPad Software Inc., San Diego) and IBM SPSS© software (New York, USA). Kolmogorov-Smirnov and Shapiro-Wilks tests were used to prove normal distributions. Results and descriptive statistics are presented as mean with standard deviations (mean ± SD) for continuous variables. Binary variables are presented as absolute numbers with a percentage amount of the population in [%]). The χ 2 test was used to compare binary variables between groups; the unpaired t test was used to compare continuous variables between groups. Smoothed histograms of HTPR distribution were created in GraphPad Prism©, median with interquartile range and mean ± SD were calculated. The Fisher's exact test was used for comparison of HTPR between groups. The McNemar test was used for binomial comparison of HTPR within the same patients. p values below 0.05 were considered significant. All data generated or analyzed during this study are included in this article.

Study Population and Characteristics
We included 755 patients with antiplatelet medication in our analysis. Patients were grouped according to antiplatelet regimes at the time of platelet function measurement. Overall, 677 patients received ASA and 521 patients received clopidogrel. 199 patients received SAPT with ASA, and 18 patients had SAPT with clopidogrel. 340 patients had dual antiplatelet therapy (DAPT) with ASA and clopidogrel, and 103 had therapy with ASA, clopidogrel, and OAC. 35 patients had combined therapy with ASA and OAC, and 60 patients received clopidogrel and OAC (Fig. 1).
The mean age was 73 ± 13.4 years, and 60.7% of the patients were male. Prior myocardial infarction, prior PCI, chronic kidney disease (CKD), AF, and hypertension were frequent in this CCS cohort ( Further baseline parameters, comorbidities, comedication, and laboratory parameters were assessed and can be found in the supplements (online suppl. Tables S1-S3, available at www.karger.com/doi/10.1159/000527816).

Platelet Function Testing by LTA
Overall, mean MoA was 14.18 ± 19.09% for ARA in patients with ASA therapy and 50.06 ± 20.42% for ADP in patients with clopidogrel therapy. Patients who had monotherapy with either ASA or clopidogrel showed    (Fig. 2).

Discussion
The main results of this study were (I) that HTPR to clopidogrel showed broader variation than HTPR to ASA and (II) that incidence of HTPR was significantly higher for clopidogrel as compared to ASA throughout the whole cohort. These findings are in line with previous smaller cohort studies that reported higher incidences of HTPR to clopidogrel compared with HTPR to ASA [5, 6, 10] [11]. Clinical significance of HTPR has been demonstrated previously for clopidogrel [12,13]. However, data are contradicting for ASA, as a large prospective trial could not show an association between HTPR to aspirin and stent thrombosis but it did for clopidogrel [14]. Furthermore, HTPR to ASA is discussed to be of lower clinical significance as compared to HTPR to clopidogrel [15]. Direct comparison of HTPR to ASA versus to clopidogrel in one cohort is sparse. Moreover, this effect has not been investigated in relation to different anticoagulatory regimen. This is crucial as especially AF patients are at a high risk for ischemic events. However, this finding does not reflect the latest recommendations for post-interventional management of AF patients. Different attempts have been made to exclude ASA in cardiovascular prevention to ensure the optimal antithrombotic regime. According to current guidelines, ASA is the first medication in triple therapy with ASA + clopidogrel + OAC to be dropped after a few days, whereas P2Y12 inhibitors (mostly clopidogrel) are maintained in combination with OAC for up to 12 months after PCI [2]. These recommendations are based on large-scaled trials testing the safety of novel OAC after PCI (PIO-NEER-AF-PCI, RE-DUAL-PCI, AUGUSTUS, EN-TRUST-AF-PCI) [16][17][18][19]. In these trials, dual therapy (OAC + clopidogrel) was favored over OAC + clopidogrel + ASA due to a lower risk of bleeding. However, only the AUGUSTUS trial was designed to compare OAC + clopidogrel versus ASA + clopidogrel + OAC therapy, as it had a factorial randomization of ASA versus placebo. Again, bleeding risk was higher in the ASA + clopidogrel + OAC group. However, none of these trials was powered to detect differences in thromboembolic or major adverse cardiovascular events (MACE). Two meta-analyses showed that clopidogrel + OAC could reduce bleeding events compared to ASA + clopidogrel + OAC therapy. Interestingly, this did not lead to a decrease in mortality, as MACE (especially stent thrombosis and myocardial infarction) were higher in the clopidogrel + OAC group. In this context, the bleeding risk of clopidogrel versus ASA medication should be discussed. As our study has a retrospective design, we were not able to detect bleeding events in our study population. Thus, we could not verify if lower rates of HTPR are associated with a stronger antiplatelet effect and a higher frequency of bleeding events. The existing literature indicates that bleeding risk might be increased with ASA as compared to clopidogrel monotherapy [20]. However, an observational study comparing different anticoagulation regimes showed a lower incidence of bleeding events of ASA + OAC compared to clopidogrel + OAC in particular [21]. As there is no high quality data directly comparing ASA + OAC versus clopidogrel + OAC data remains contradicting.
Furthermore, there is a serious lack of evidence, as none of these trials investigated OAC + ASA in dual therapy. In this context, the subgroup analysis of our data could show that the antiplatelet effect of dual therapy with ASA was more reliable as compared to dual therapy with clopidogrel. If this transfers to fewer adverse clinical events, has not been investigated by now. Clinical comparison of ASA and clopidogrel in secondary prevention has been carried out before for other groups of patients but remains controversial. In this context, Vidyanti et al. [22] reported lower rates of MACE for ASA compared to clopidogrel for secondary stroke prevention. Another retrospective database analysis of stroke patients showed higher mortality rates in the clopidogrel group compared to ASA [23]. On the other hand, Park et al. [24] showed beneficial effects of clopidogrel on MACE compared to ASA for CCS patients in an observational study design. The CAPRIE trial also investigated both drugs in stroke, CCS, and peripheral artery disease patients, and showed superiority of clopidogrel compared to ASA regarding ischemic and bleeding events [25]. Thus, the superiority of one of these agents in dual therapy for AF patients after PCI remains uncertain.
In evaluation of the risk between ischemic and bleeding events, an optimal anticoagulation-antiplatelet regime is essential. Impaired pharmacodynamic response to ASA and clopidogrel has been described before [11,[26][27][28]. Mechanisms inducing HTPR to ASA are well known and precisely investigated. In this context, CKD, diabetes, and comedication with dipyrone have been described previously. Improvement of the pharmacodynamic response to ASA could be achieved if disruptive factors were taken into account [29][30][31].
In contrast, HTPR to clopidogrel is dominated by cytochrome P450 2C19 (CYP2C19)-dependent hepatic metabolism which underlies broad interindividual variability [32]. This might explain the broader distribution of the pharmacodynamic effect size as compared to ASA. In this context, poor clopidogrel metabolizers with a CYP2C19*2 loss-of-function polymorphism have been described [33]. However, Claassens et al. found evidence that genotype-guided P2Y12 medication might overcome issues of resistance to clopidogrel. In this study, genotype-guided therapy was not inferior to more reliable P2Y12 inhibitors, ticagrelor and prasugrel [34]. However, to date, genotype-guided selection of P2Y12 inhibitor is far from clinical practice. This might have contributed to broad distribution of MoA in our study.
Our finding, that HTPR to ASA is less frequent than HTPR to clopidogrel, was robust and reproducible for all subgroups -mono, dual, or triple therapy. Therefore, we hypothesize that ASA might be the preferable antiplatelet substance instead of clopidogrel in combination with OAC in AF patients.
P2Y12 inhibitors other than clopidogrel could also be a favorable alternative. Until the latest European guideline update, there was no recommendation for the use of ticagrelor or prasugrel in patients with AF after PCI. The recommendation changed, and other P2Y12 inhibitors DOI: 10.1159/000527816 could be used instead of clopidogrel [2]. In non-AF patients with PCI and stable CAD or acute coronary syndrome, the GLOBAL LEADERS trial gave evidence that ticagrelor monotherapy is non-inferior to standard DAPT (ASA + clopidogrel) [35]. But again, evidence for dual therapy with OAC in AF patients is lacking. Further large-scaled clinical trials are needed to investigate the ideal combination of an antiplatelet agent and OAC after PCI in patients with AF.

Study Limitations
This study was a retrospective, single-center database analysis. Therefore, no medical intervention and no follow-up of clinical outcomes could be conducted. We did not differentiate between different anticoagulants which could have also affected platelet function. When it comes to assessment of HTPR, available platelet function assays might have different cutoff values [36]. In this study, we chose LTA, which is the long-established gold standard to measure ASA resistance. For clopidogrel resistance, we chose the well-established cutoff of 46% MoA which was previously determined by Bonello et al. using receiveroperator characteristic curve analysis [9]. A different well-acknowledged method to define HTPR to clopidogrel is the VASP assay, which we did not include. Finally, in our study, we could only include 95 patients with either ASA or clopidogrel combined with OAC. Therefore, validity of the findings might be limited, as this represents the minority of the full study population. However, as our results were consistent across all study groups, it is likely that the choice of antiplatelet agent might be relevant in these patients.

Conclusion
Impaired pharmacodynamic response was more frequent to clopidogrel than to ASA. Hence, ASA with less risk of insufficient antiplatelet effects should be tested in combination with OAC in AF patients' post-PCI. Prevention of thrombotic complication should be set against bleeding risk in large-scale trials.

Statement of Ethics
The study conformed to the Declaration of Helsinki and was approved by the University of Düsseldorf Ethics Committee. Written informed consent was obtained from participants. This study protocol was reviewed and approved by the Ethics Committee of the University of Düsseldorf, approval number [4,502].

Conflict of Interest Statement
No conflicts to disclose.

Author Contributions
Lisa Dannenberg, René M'Pembele, and Samantha Ahlbrecht designed the study, analyzed and interpreted data, and wrote the manuscript. Philipp Mourikis, Carolin Helten, and Saif Zako collected data and wrote the manuscript. David Naguib and Kajetan Trojovsky collected data. Ragnar Huhn, Tobias Petzold, Thomas Hohlfeld, Tobias Zeus, Malte Kelm, Lisa Dannenberg, and Amin Polzin supervised the study and revised the manuscript.

Data Availability Statement
All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.