Influence of Genetic and Epigenetic Factors of P2Y12 Receptor on the Safety and Efficacy of Antiplatelet Drugs

Purpose P2Y12 receptor inhibitors are drugs that decrease the risk of stent thrombosis and lower the long-term risk of non-stent-related myocardial infarction and stroke. They inhibit the binding of adenosine diphosphate (ADP) to the P2Y12 receptor and effectively reduce platelet reactivity. However, considerable variability in the pharmacodynamics response contributes to a failure of antiplatelet therapy; this phenomenon is especially notorious for older drugs, such as clopidogrel. Some genetic polymorphisms associated with these drugs’ metabolic pathway, especially in the CYP2C19 gene, can significantly decrease antiplatelet efficacy. There are few reports on the variability stemming from the target of this drug class that is the P2Y12 receptor itself. Results and conclusion This review summarizes the results of research that focus on the influence of P2Y12 genetic polymorphisms on the pharmacodynamics and the efficacy of P2Y12 inhibitors. We found that the conclusions of the studies are unequivocal, and despite several strong candidates, such as G52T (rs6809699) or T744C (rs2046934), they may not be independent predictors of the inadequate response to the drug. Most probably, P2Y12 genetic polymorphisms contribute to the effect exerted by other gene variants (such as CYP2C19*2/*3/*17), drug interactions, or patient habits, such as smoking. Also, epigenetic modifications, such as methylation or miRNA levels, may play a role in the efficacy of antiplatelet treatment.


Introduction
Combined with aspirin, P2Y 12 receptor inhibitors are used in dual antiplatelet therapy (DAPT).This treatment aims to prevent thrombotic events after percutaneous coronary intervention (PCI) or myocardial infarction (MI) and stroke [1].The basis of the therapeutic action of this drug class is the inhibition of adenosine diphosphate (ADP) binding to the P2Y 12 receptor.Two platelet receptors, P2Y 1 and P2Y 12 , react to ADP in physiological conditions and induce platelet aggregation [2].The introduction of P2Y 12 inhibitors attenuates ADP interaction with its platelet receptor and effectively reduces platelet reactivity (Fig. 1).
Besides the most widely used clopidogrel, newer P2Y 12 inhibitors gain interest, and their use increases (Table 1).For example, Chest Pain-Myocardial Infarction Registry in the United States noted that ticagrelor use in patients with ST-segment-elevation myocardial infarction (STEMI) increased from 18.0 to 44.0% [3].Last year, selatogrel (ACT-246475), a novel subcutaneous P2Y 12 inhibitor, completed phase 2 studies [4,5].The declining use of clopidogrel stems from a sizeable observed variability in the therapeutic response.The "clopidogrel resistance" phenomenon can affect as much as 16-50% of the population [6], leading to the failure of antiplatelet therapy.Significant contributors to this resistance are genetic polymorphisms of CYP450 enzymes, especially CYP2C19 [7].The presence of loss-of-function CYP2C19 alleles increases the risk of major adverse cardiovascular events (MACE) during DAPT with clopidogrel [8][9][10][11].However, some authors claim that alterations in the P2Y 12 gene could also play a role in thrombotic events during antiplatelet therapy (Tables 2  and 3) [12,13].As these polymorphisms may affect the receptor's functionality, its potential influence may translate to other P2Y 12 inhibitors.Therefore, this narrative review focuses on presenting available data on the influence of P2Y 12 genetic polymorphism on the efficacy and safety of currently used antiplatelet drugs.

H1/H2 Haplotype and Related Alleles
In 2003, Fontana et al. [13] identified that some healthy volunteers had significantly greater ADP-induced maximal platelet aggregation (MPA).The authors performed genotyping and found that four of the studied polymorphisms (G52T (rs6809699), i-C139T (rs10935838), i-T744C (rs2046934), and i-ins801A (rs5853517); Table 2) were in linkage disequilibrium.When a subject carried G variant in G52T, C in i-C139T, T in i-T744C, and lacked insertion in 801A, he carried H1 haplotype.Otherwise, an H2 haplotype was present.P2Y12 genotyping revealed that greater MPA and a more significant reduction in cAMP concentration correlated with the presence of H2 haplotype.increased ADP-induced MPA and increase platelet reacƟvity (H2 haplotype) -lower ADP-induced MPA (rs6787801) -more frequent occurrence of resistance (T744C, G52T, C34T, H2 haplotype) -increased risk of CAD (T744C) -increased risk of neurological events (C34T) -higher risk of coronary artery aneurism in Kawasaki disease (rs7637803) Fig. Further investigations showed that the H2 haplotype could be related to thrombotic-related diseases.For example, a case-control study showed that patients with peripheral arterial disease are more likely to be H2 carriers (30% in cases vs. 21% in healthy controls) [12].Likewise, a recent China-based study revealed that 26.3% of subjects with cerebral infarction carried H2 haplotype, while this variant occurred less frequently (16.7%) in healthy controls [64].The most widely used allele indicative of the H1/H2 haplotype is the intronic variant T744C.Several case-control studies conducted on patients with acute coronary syndrome (ACS) or coronary artery disease observed that the mutant

H1/H2 Haplotype Alleles and Clopidogrel
Two meta-analyses investigated the association of P2Y 12 single nucleotide polymorphisms (SNPs) linked with H1/ H2 haplotypes and the efficacy of clopidogrel.The first one focused on the risk of clopidogrel resistance, defined either as lower than 10% platelet inhibition or ADP-induced platelet aggregation greater than 70% [65].The authors found that under the dominant model, only G52T increased the odds ratio of clopidogrel resistance (OR = 1.45, 95% CI: 1.14-1.85,p = 0.003 for G52T).The other investigated SNP, T744C, did not influence the odds of inadequate response to clopidogrel.The second meta-analysis, by Zhao et al. [66], showed a significant association of T744C and ischemic events in the Han Chinese population but not in the Caucasian population.Moreover, the significance was observed only under a recessive model (CC vs. CT + TT, OR = 3.32, 95% CI: 1.62-6.82,p = 0.001).In contrast, Liu et al. [41] found that in the case of patients with ischemic stroke, the presence of the C allele was associated with a lower incidence of clopidogrel resistance (OR = 0.407, 95% CI: 0.191-0.867,p = 0.018).Another study on the population of Asian patients with symptomatic extracranial or intracranial stenosis showed a significant adverse influence of the T allele.When a patient carried the T allele, there were significantly greater odds of transient ischemic attack, ischemic stroke, MI, or vascular-related mortality during a 1-year follow-up (OR: 2.01, 95% CI: 1.10-3.67,p = 0.041) [42].Despite these reports, a number of studies failed to notice any influence of T744C on the prevalence of HTPR or adverse events [32, 34, 39, 43-50, 58, 67, 68].It is also possible that the effect of T744C on platelet function and the efficacy of clopidogrel treatment may depend on the coexistence of other factors, such as compliance, drug interactions, and comorbidities [51,69,70].For example, a study on 222 ACS patients showed that the concomitance of a mutant allele of T744C and polymorphic alleles of CYP2B6 (*9 and *1B) were associated with high platelet reactivity and poor response to clopidogrel [71].
Another SNP associated with H1/H2 haplotype, G52T, can negatively impact PCI outcomes in elderly patients.In a recent study that included 811 patients aged ≥ 75 years of age, TT homozygotes had a higher risk of bleeding during a 1-year follow-up compared with GG and GT genotypes (adjusted HR: 3.87, 95% CI: 1.41-10.68,p = 0.009) [72].
Although T744C was not one of the investigated variants, the T allele in G52T is associated with the H2 haplotype.Therefore, the observation of Cha et al. could translate to the combined effect of this haplotype on the efficacy of DAPT with clopidogrel (75 mg) and aspirin (100 mg).

H1/H2 Haplotype Alleles and Other P2Y 12 Inhibitors
Available resources regarding the influence of H1/H2 on the efficacy of other P2Y 12 inhibitors are less abundant than for clopidogrel.One study investigated the role of two alleles comprising H1/H2 haplotypes (T744C and G52T) on ticagrelor's ex vivo antiplatelet effect [33].The carriers of the T744C and G52T mutant alleles had significantly lower baseline platelet aggregation.However, there were no statistically significant differences in peak and late aggregation or inhibition of platelet aggregation at low (15 μM) and high (50 μM) ticagrelor concentrations.
In a cangrelor-focused study, Bouman et al. [36] investigated ex vivo peak and late platelet aggregation at low and high drug concentrations.The carriers of the T744C CC genotype exhibited higher peak platelet aggregation and lower peak inhibition of platelet aggregation at low (0.05 µM) cangrelor concentrations but the differences were not statistically significant.In contrast, Oestreich et al. [37] reported a significant recessive effect of H2 haplotype, represented by the T744C variant, on platelet reactivity stimulated by thrombin receptor-activating peptide (TRAP).The response to TRAP was markedly reduced (25-42%) in H2/H2 carriers whose isolated platelets were subjected to cangrelor.

C34T and P2Y 12 Inhibitors
Two of the meta-analyses cited above investigated the influence of C34T (rs6785930) on the efficacy of clopidogrel therapy.Both found a significant relationship between the presence of the T allele and the risk of insufficient response to the drug.In the first analysis, the presence of the T allele increased over two-fold the risk of clopidogrel resistance (OR = 2.30, 95% CI: 1.50-3.51,p = 0.0001) [65].In the other study, the odds ratio of ischemic events was 1.7 (95% CI: 1.22-2.36,p = 0.002) in carriers of the T allele, but the effect was significant in Han Chinese population only [66].Also, this polymorphism was not associated with an increased risk of bleeding events.
Lack of the influence of C34T on clopidogrel therapy was reported for European populations [34,44].Ulehlova et al. [44] did not observe significant differences in the frequencies of C34T alleles between clopidogrel-resistant patients with AMI and the entire investigated group.This polymorphism also seems not to influence the endothelial function or arterial wall properties [73].However, an interesting observation was reported for smokers [74].Carriers of mutant C34T allele with coronary artery disease (CAD) undergoing PCI who also smoked had a higher risk of reaching the primary endpoint (death from cardiovascular causes, nonfatal MI, revascularization, stroke, recurrent cardiac ischemia, or transient ischemic attack) than the CC homozygotes (HR 2.23, 95% CI: 1.05-6.01,p = 0.04).
The influence of the C34T allele on the efficacy of ticagrelor may also be insignificant.Although there are no clinical reports, the ex vivo study showed that there might be a difference in platelet aggregation and the inhibition caused by ticagrelor [33].The CT heterozygotes had significantly lower platelet aggregation and greater inhibition of platelet aggregation at the highest studied ticagrelor concentrations than TT homozygotes.

Other P2Y12 Haplotypes and Alleles
Besides H1/H2, some reports distinguish other haplotypes that may influence platelet reactivity.Nie et al. [32] investigated SNPs in the promoter and regulatory region of the P2Y 12 gene.The authors inferred six commonly occurring haplotypes of four SNPs-rs6798347 (C > t), rs6787801 (T > c), rs6801273 (A > g), and rs6785930 (C > t).Among the haplotypes labeled H0-H5, haplotype H1 (tcgt) had a lower incidence of high-on-treatment platelet reactivity (HTPR) in all of the 180 patients with ACS compared with the reference H0 (CTAC) haplotype (adjusted OR 0.13, 95% CI: 0.03-0.68,p = 0.016).The influence was significant after adjusting for other covariates-patients' demographics and CYP2C19 LoF alleles.
In a study with patients with ischemic stroke who underwent a stenting procedure, two P2Y 12 SNPs, rs6787801 and rs6798347, comprised three haplotypes [42].The patients were on DAPT with clopidogrel and aspirin and were monitored for the occurrence of ischemic events for 12 months after the procedure.None of the distinguished haplotypes was associated with a greater risk of subsequent vascular events.
Li et al. [33] distinguished six common haplotypes (A-F) from nine investigated P2Y 12 SNPs.One of the haplotypes ('D', TAA TAG CCT) included, among others, the minor C allele of T744C, the major C allele of C34T, and the minor T allele of G52T.The carriers of this haplotype exhibited significantly lower baseline platelet aggregation than the most common haplotype.However, it did not translate to significantly lower aggregation or lower inhibition of platelet reactivity caused by ticagrelor.Hence, the authors concluded that the investigated P2Y 12 haplotypes do not influence the antiplatelet effect of the drug.
Bouman et al. [36] distinguished six common haplotypes (A-F) from five SNPs in the P2Y 12 gene.The studies SNPs were rs6798347, rs6787801, rs9859552, rs6801273, and T744C (rs2046934).The authors found that the C haplotype, comprising the minor T744C C allele and the major variants of the other SNPs, had the most significant influence on cangrelor-induced platelet aggregation.Both peak and late ex vivo platelet aggregation were greater than the reference haplotype under low and high cangrelor concentrations.At the same time, the C haplotype had lower inhibition of platelet aggregation, but the effect was most pronounced 360 s after stimulation with ADP.
Another study showed that the minor G of rs3732759 (A > G) allele occurred more frequently in patients with cardiovascular diseases treated with clopidogrel than in healthy controls (42.7% vs. 26.7%,OR = 1.43, 95% CI: 1.05-1.93,p = 0.021) [35].This observation was also confirmed for the frequency of clopidogrel resistance, as 54.2% of the resistant and only 28.6% of sensitive patients carried GG genotype (p = 0.017).Decreased sensitivity to clopidogrel translated to the occurrence of MACE.The patients with MACE were more likely to carry the rs3732759 GG genotype.The authors paired three analyzed SNPs into four common haplotypes in the same paper.It allowed capturing the interplay between the SNPs.Although the rs3732759 GG negatively influenced clopidogrel therapy, the only haplotype associated with an increased risk of CHD was TCA (rs7428575 T, rs2046934 C, and rs3732759 A; OR: 1.57, 95% CI: 1.14-2.17,p = 0.005).
Recently, Rath et al. [34] investigated patients with 103 ischemic stroke or transient ischemic attack and investigated the influence of selected SNPs on the platelet reactivity and the degree of aggregation inhibition during clopidogrel therapy.The minor T allele (rs9859552) carriers appeared to have greater platelet reactivity values; the effect was not statistically significant.

Epigenetic Studies on P2Y12
Epigenetic studies are a new direction to identify the mechanism of variable response to antiplatelet therapy.Several reports suggest that methylations of promoters or other epigenetic changes contribute to the altered expression of P2Y 12 and could be responsible for the epigenetic mechanisms of clopidogrel resistance.Su et al. [75] reported that DNA methylation levels of two cytosine-phosphate-guanine (CpG) dinucleotides on P2Y 12 promoter (CpG1 and CpG2) were related to platelet activity measured by the VerifyNow P2Y 12 assay in patients with CAD.Lower methylation of two CpGs indicated the poorer clopidogrel response in alcoholabusing status.Moreover, lower methylation levels of CpG1 correlated with higher platelet activity in the active smokers and patients with albumin concentrations ≤ 35 g/L.In the study by Li et al. [76], the impact of the P2Y 12 promoter DNA methylation on the recurrence of ischemic events was evaluated in patients with cerebrovascular disease.Among sixteen tested CpG dinucleotides, three CpG sites (CpG11 and CpG12 + 13) showed lower methylation levels, which correlated with ADP inhibition rate and ADP-induced platelet-fibrin cloth strength measured by thromboelastography.The lower methylation status of CpG11 and CpG12 + 13 was also associated with an increased risk of clinical events, including vascular-related mortality, ischemic stroke, transient ischemic attack, or MI.
Evidence suggests that platelet-related miRNAs could play a pivotal role as biomarkers of antiplatelet therapy efficacy because they act as modulators of P2Y 12 expression.miRNAs are small non-coding sequences of nucleotides that bind to mRNA sites, block transcription, and, in consequence, cause a decrease in protein production [77].Landry et al. [78] reported that the P2Y 12 receptor is a target of miRNA-223, and complexes of Argonaute 2 protein with miRNA-223 may be involved in regulating P2Y 12 receptor expression in platelets.These results seem to align with the study by Chyrchel et al. [79], who reported that miRNA-223 expression in plasma was elevated in patients with ACS exhibiting increased platelet inhibition in response to DAPT with aspirin plus clopidogrel, prasugrel, or ticagrelor.The effect was stronger for newer P2Y 12 antagonists.Similarly, an association between decreased platelet miRNA-223 levels and high on-treatment platelet reactivity in patients treated with clopidogrel was confirmed in other studies [80,81].Alteration in platelet aggregation due to reduced P2Y 12 expression caused by miRNA-126 inhibition was confirmed in patients with ACS [82].In another study on that group of patients, the statistically significant connection between miRNA-29 and miRNA-34 expression levels and P2Y 12R gene polymorphism (A > G, rs3732759) was reported [83].Syam et al. [84] noticed a significant association of high miRNA-26a platelet expression but not DNA methylation of the P2Y 12 gene on platelet reactivity in patients with acute STEMI undergoing clopidogrel therapy.The role of an elevated miRNA-92a as a biomarker of an increased risk of ACS was confirmed in coronary heart disease patients with type 2 diabetes mellitus [85].However, as Kramer et al. [86] claimed, further studies are needed to elaborate a standardized protocol for miRNA sample handling to minimize preanalytical variability and the kinetics of platelet miRNA release in response to platelet activation.

Summary
With an inadequate response to P2Y12 inhibitors, thrombotic events may occur, leading to treatment failure.As shown in Table 3, some authors claim that alterations in the P2Y 12 gene could also play a role in thrombotic events during antiplatelet therapy.This review outlines that the H2 haplotype could be most strongly related to thrombotic-related diseases and possibly influences platelet reactivity.However, several studies showed that only G52T, one of the polymorphisms comprising H1/ H2 haplotypes, increased the odds of clopidogrel resistance, simultaneously exposing the lack of influence of T744C polymorphism.The coexistence of other factors should be considered regarding platelet function and the efficacy of clopidogrel treatment.The non-genetic factors include the origin of study population or smoking status.While for the newer antiplatelet drugs, the reports are scarce and inconclusive, and some clopidogrel co-factors are well described.For example, the two-step activation process involves several CYP450 enzymes, among which CYP2C19 is regarded as the most influential [7,87].The carriers of CYP2C19 alleles are at the greater risk of MACE, MI, or stent thrombosis.The association is so pronounced that some clinicians postulate guided antiplatelet therapy that includes an option of switching from clopidogrel to newer, CYP2C19-independent alternatives [88].As CYP2C19 is crucial in the second step of the activation, its inhibitors may also decrease clopidogrel's efficacy.For example, in a recent meta-analysis, it was shown that concomitant intake of proton pump inhibitors increases the risk of MACE by 63% in CYP2C19 loss-offunction allele carriers (95% CI: 1.31-2.03,p < 0.0001) [89].These findings indicate that studies on P2Y 12 polymorphisms in patients treated with clopidogrel should account for the CYP2C19 genotype.
Recent evidence indicates that epigenetic factors such as P2Y 12 DNA methylation and miRNAs play a role in the observed variability of platelet reactivity and may serve as biomarkers of response to P2Y 12 inhibitors.Lower DNA methylation of the P2Y 12 gene promoter increases the risk of resistance to antiplatelet therapy, while decreased miRNA levels are connected to high on-treatment platelet reactivity and ischemic events in patients treated with P2Y 12 inhibitors.Due to a broad range of platelet miR-NAs, further studies are needed on a reliable miRNAbased biomarker to assess the in vivo platelet activation and response to P2Y 12 inhibitors.

Conclusions
The current review underlines the multifactorial causes of the observed response variability to P2Y 12 receptor inhibitors.Most of the reported P2Y 12 genetic polymorphisms (e.g., T allele in G52T) may be responsible for changes in the structure of the P2Y12 receptor, therefore decreasing the efficacy of antiplatelet therapy.However, the impact of some factors (e.g., T744C, C34T, smoking) on antiplatelet therapy is not yet fully determined, and their coexistence may lead to resistance to antiplatelet therapy.

Table 2
Genetic polymorphisms in the P2Y12 gene sequence with possible influence on the efficacy of antithrombotic drugs.Data taken from the dbSNP database (ncbi.nlm.nih.gov/ snp/).Global allele fre-quencies (AF) taken from the Ensembl database, 1000 Genomes Project Phase 3 (ensem bl.org).The alleles leading to a possible function alteration are in bold CVD cardiovascular disease, HTPR high on-treatment platelet reactivity