Haplotype of platelet receptor P2RY12 gene is associated with residual clopidogrel on-treatment platelet reactivity

Haplotype of platelet receptor P2RY12 gene is associated with residual clopidogrel on-treatment platelet reactivity P2RY12 基因单倍体分型对急性冠脉综合征患者氯吡格雷用药后血小板反应性的影响 1 first-author †E-mail:niexy@bjmu.edu.cndrjianliu@163.comshilu@bjmu.edu.cn †E-mail:niexy@bjmu.edu.cndrjianliu@163.comshilu@bjmu.edu.cn niexy@bjmu.edu.cn 1 2 3 4 1 1 5 2 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:niexy@bjmu.edu.cndrjianliu@163.comshilu@bjmu.edu.cn †E-mail:niexy@bjmu.edu.cndrjianliu@163.comshilu@bjmu.edu.cn niexy@bjmu.edu.cn 1 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:niexy@bjmu.edu.cndrjianliu@163.comshilu@bjmu.edu.cn †E-mail:niexy@bjmu.edu.cndrjianliu@163.comshilu@bjmu.edu.cn niexy@bjmu.edu.cn School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China Department of Cardiology, Peking University People’s Hospital, Beijing 100044, China Department of Cardiology, Peking University People’s Hospital, Beijing 100044, China School of Public Health, Peking University Health Science Center, Beijing 100191, China School of Public Health, Peking University Health Science Center, Beijing 100191, China Department of Epidemiology, Fuwai Hospital, Beijing 100037, China Department of Epidemiology, Fuwai Hospital, Beijing 100037, China Department of Pharmacy, Hennepin County Medical Center, Minneapolis, Minnesota 55415, USA Department of Pharmacy, Hennepin County Medical Center, Minneapolis, Minnesota 55415, USA Objective: 2 To investigate a possible association between common variations of the P2RY12 and the residual clopidogrel on-treatment platelet reactivity after adjusting for the influence of CYP2C19 tested by thromboelastography (TEG). Methods: 2 One hundred and eighty patients with acute coronary syndrome (ACS) treated with clopidogrel and aspirin were included and platelet function was assessed by TEG. Five selected P2RY12 single nucleotide polymorphisms (SNPs; rs6798347, rs6787801, rs6801273, rs6785930, and rs2046934), which cover the common variations in the P2RY12 gene and its regulatory regions, and three CYP2C19 SNPs ( * 2, * 3, * 17) were genotyped and possible haplotypes were analyzed. Results: 2 The high on-treatment platelet reactivity (HTPR) prevalence defined by a platelet inhibition rate < 30% by TEG in adenosine diphosphate (ADP)-channel was 69 (38.33%). Six common haplotypes were inferred from four of the selected P2RY12 SNPs (denoted H 0 to H 5 ) according to the linkage disequilibrium R square (except for rs2046934). Haplotype H 1 showed a significantly lower incidence of HTPR than the reference haplotype (H 0 ) in the total study population while haplotypes H 1 and H 2 showed significantly lower incidences of HTPR than H 0 in the nonsmoker subgroup after adjusting for CYP2C19 effects and demographic characteristics. rs2046934 (T744C) did not show any significant association with HTPR. Conclusions: 2 The combination of common P2RY12 variations including regulatory regions rather than rs2046934 (T744C) that related to pharmacodynamics of clopidogrel in patients with ACS was independently associated with residual on-clopidogrel platelet reactivity. This is apart from the established association of the CYP2C19 . This association seemed more important in the subgroup defined by smoking. 目的 2 评估血小板受体基因 P2RY12 常见突变位点单倍体分型对急性冠脉综合征患者氯吡格雷用药后血小板反应性的影响。 创新点 2 首次在中国人群中对包含调控基因在内的五个常见 P2RY12 突变位点进行单倍体分析，并且校正了 CYP2C19 基因多态性的影响，发现 P2RY12 常见基因位点联合突变可降低氯吡格雷用药后血小板高反应性发生率，且作用在不吸烟人群中更明显。 方法 2 连续入选180例接受氯吡格雷药物治疗的急性冠脉综合征患者，利用血栓弹力图法检测患者用药后血小板反应性，将用药后血小板抑制率 < 30%定义为血小板高反应性（HTPR）。用多重连接酶检测反应（LDR）技术，对覆盖 P2RY12 调控基因在内的五个基因位点（rs6798347、 rs6787801、rs6801273、rs6785930和rs2046934）以及 CYP2C19 常见的三个等位基因（*2、*3和*17）进行基因分型。根据连锁不平衡系数和基因分布频率进行单倍体分析，观察在校正 CYP2C19 基因多态性影响后，不同 P2RY12 单倍体分型对氯吡格雷用药后HTPR的影响。 结论 2 将 P2RY12 基因rs6798347、rs6787801、rs6801273和rs6785930四个紧密连锁的单核苷酸多态性（SNP）分为六个常见单倍体分析（H 0 ~H 5 ，表4）。单倍体分型与氯吡格雷用药后HTPR发生率显著相关，与H 0 型相比，H 1 型HTPR发生率显著降低，而rs2046934对HTPR发生率无显著影响（表5），且在不吸烟组中单倍体分型对HTPR影响更明显（图1）。综上所述， P2RY12 基因rs6798347、rs6787801、rs6801273和rs6785930单倍体分型与氯吡格雷用药后HTPR显著相关。 P2RY12; CYP2C19; Haplotype; Single nucleotide polymorphism (SNP); Clopidogrel; Thromboelastography P2RY12 CYP2C19 单倍体分型 单核苷酸多态性 氯吡格雷 血栓弹力图 Introduction 1 Dual antiplatelet therapy with aspirin and a platelet P2Y12 adenosine diphosphate (ADP) receptor antagonist reduces recurrent adverse cardiovascular events in acute coronary syndrome (ACS) and/or following percutaneous coronary intervention (PCI) (Yusuf et al., 2001 2001 ; Beinart et al., 2005 2005 ). It is recommended for patients with ACS and/or PCI in China (Emergency Medical Branch of Chinese Medical Doctor Association et al., 2016 2016 ; Section of Interventional Cardiology of Chinese Society of Cardiology of Chinese Medical Association et al., 2016 2016 ) and worldwide (Levine et al., 2016 2016 ; SIGN, 2016 2016 ). Clopidogrel is currently one of the most commonly used platelet P2Y12 receptor inhibitors in China, although new guidelines have recommended ticagrelor as the first-line medicine in patients with ACS or following PCI. Clopidogrel is an orally administered thienopyridine prodrug, which needs to be biotransformed into its active metabolite. After oral administration, the absorption of clopidogrel is regulated by adenosine triphosphate (ATP)-binding cassette, sub-family B, member 1 (ABCB1) transporters (Mega et al., 2010a 2010a ), and the hepatic biotransformation of clopidogrel prodrug to its active metabolite is mediated by CYP450 enzymes, especially CYP2C19. After activation, the active metabolite of clopidogrel selectively and irreversibly inhibits the P2Y12 receptor located on the surface of platelets and decreases platelet aggregation. Many studies have demonstrated the close relationship between CYP2C19 genotype and high on-treatment platelet reactivity (HTPR) (Mega et al., 2010b 2010b ; Simon et al., 2011 2011 ), including some studies focusing on Chinese subjects (Chen et al., 2014 2014 ) and our previous study (Liu et al., 2015 2015 ). As well as CYP2C19 , other genetic factors may contribute to the inter-individual variability of clopidogrel, among which P2RY12 , which encodes the P2Y12 receptor on platelets, is of importance. Previous studies have implied that P2RY12 single nucleotide polymorphism (SNP) might play a role on the large variability in clopidogrel response, but the results were inconsistent and controversial (Angiolillo et al., 2007 2007 ; Zee et al., 2008 2008 ; Jana et al., 2014 2014 ; Oestreich et al., 2014 2014 ; Li et al., 2016 2016 ; Zhang et al., 2016 2016 ). Most of those studies focused on single haplotype-tagging SNPs (ht-SNPs), such as rs2046934 (i-T744C) or rs6785930, or two common haplotypes H 1 and H 2 (constituted by single nucleotide polymorphism database (dbSNP) rs10935838, rs2046934, rs5853517, and rs6809699), especially the previous studies in the Chinese population (Chen et al., 2014 2014 ; Zhang et al., 2016 2016 ). Several studies focused on rs10935838, rs5853517, and some other SNPs at the same time, but mostly in the Caucasian population (Zee et al., 2008 2008 ; Rudež et al., 2009 2009 ). rs10935838, rs2046934, rs5853517, and rs6809699 are in close linkage disequilibrium (LD) but not covering the regulatory regions of the P2RY12 gene. A comprehensive study of common variations in the P2RY12 gene that cover common haplotypes within the P2RY12 gene and its regulatory regions is lacking in the Chinese population with ACS. Moreover, most present studies focus on the P2RY12 gene separately, without adjusting for the influence of the main proven gene to affect clopidogrel on-treatment residual platelet activities—the CYP2C19 * 2 and * 3 alleles. The aim of this study was to investigate a possible association between the common genetic variations of the P2RY12 locus, including the promoter region after adjusting for the influence of CYP2C19 * 2 and * 3 alleles, and the residual on-clopidogrel platelet reactivity by thromboelastography (TEG) assay in a Chinese population with ACS. Materials and methods 1 Study population 2 Consecutive patients aged from 18 to 80 years with established ACS at Peking University Second Hospital, Beijing, China, during May 2014 to April 2015 were included in the study. The diagnosis of ACS (unstable angina or myocardial infarction) was confirmed by ischemic symptoms and electrocardiograph (ECG) changes with or without increase in creatine kinase and troponin levels plus coronary angiography if needed. The exclusion criteria included contraindications to clopidogrel, aspirin, heparin, and contrast agents as well as quantitative coronary angiography drugs, and whether there had been a major operation within the last one month and the use of a glycoprotein IIb/IIIa antagonist during hospitalization. All the eligible patients received 75 mg/d clopidogrel and 100 mg/d aspirin for at least 5 d with or without a loading dose of 300 or 600 mg clopidogrel according to institutional practice in addition to the other available medical therapy, including statins, β-blockers, calcium channel blockers, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, or antidiabetic agents. Definition 2 Smokers were defined as current or recent smokers (stopped smoking for <1 year). Hypertension, diabetes mellitus, and dyslipidemia were defined according to the World Health Organization criteria. A family history of cardiovascular disease was defined as an early history of coronary artery disease in women younger than 65 years and in men younger than 55 years among the first-degree relatives. Blood collection 2 On the 5th to 7th days after clopidogrel administration, blood samples were collected from peripheral veins with vacutainer tubes (BD Medical Systems) containing heparin or 32 g/L sodium citrate. TEG assay was conducted within 3 h after blood collection. The study protocol was approved by the Ethics Committee of Peking University, China (IRB 00001052-14087). Platelet function assay 2 Citrated whole blood (2 ml) and heparin-anticoagulated whole blood (2 ml) were drawn at least 5 d after the administration of the first clopidogrel maintenance dose. These samples were sent to the hemostasis laboratory of the hospital and TEG was performed within 3 h after blood collection according to the manufacturer’s instruction (TEG 5000, Haemoscope Corp., IL, USA) by trained technicians at the hospital. Platelet reactivity was calculated using the TEG platelet mapping system. TEG utilizes four channels to detect the effects of antiplatelet therapy activity induced by arachidonic acid or ADP. The platelet inhibition rate (PIR) in the ADP channel was recorded. A TEG PIR of less than 30% was defined as clopidogrel HTPR, while patients with non-high on-treatment platelet reactivity (nHTPR) had a PIR of at least 30% according to the instruction and previous study (Bliden et al., 2007 2007 ). Selection of SNPs 2 Based on the latest LD data of the P2RY12 locus ( P2RY12 gene with 2.5 kb flanking sequence) provided by SNP Annotation and Proxy Search (SNAP) for a population of Han Chinese in Beijing and Japanese in Tokyo (CHBJPT) ( http://www.broadinstitute http://www.broadinstitute . org/mpg/snap), we chose the five most common P2RY12 SNPs which cover common haplotypes within the P2RY12 gene and its regulatory regions to be analyzed. LD data were calculated using Haploview 4.0, based on phased genotype data from the 1000 Genomes Project (1000 Genomes Pilot 1) (Table 1 1 ). The five selected SNPs that were genotyped were rs6798347 (c.−281−3614C > t), rs6787801 (c.−217+2739T > c), rs6801273 (c.−216−4445A > g), rs6785930 (c.18C > t, which was also identified as C34T), and rs2046934 (c.742T > c, which was also identified as T744C). Linkage disequilibrium data are calculated based on phased genotype data from the 1000 Genomes Project (1000 Genomes Pilot 1), and the pairwise R 2 values of > 0.3 were selected for haplotype analysis (shown in bold numbers) 2 P2RY12 SNP 5 Pairwise R 2 rs6798347 rs6787801 rs6801273 rs2046934 rs6785930 rs6798347 rs6798347 rs6787801 0.354 rs6787801 rs6801273 0.347 0.144 rs6801273 rs2046934 0.147 0.114 0.169 rs2046934 rs6785930 0.336 0.148 0.446 0.076 rs6785930 In addition to the five selected P2RY12 genes, we also genotyped the three most common reported CYP2C19 SNPs, CYP2C19 * 2 (c.681G > A, rs4244285), CYP2C19 * 3 (c.636G > A, rs4986893) and CYP2C19 * 17 (c.806C > T, rs12248560), to adjust their effect on the platelet reactivity for the above selected SNPs. Genetic and haplotype analysis 2 After TEG testing, the used heparin-anticoagulated whole blood was collected from the hemostasis laboratory. Blood sample genomic DNA was isolated using the DNA extractor AxyPrep-96 (AXYGEN Scientific Inc., California, USA). CYP2C19 * 2 (rs4244285), CYP2C19 * 3 (rs4986893), CYP2C19 * 17 (rs12248560), and P2RY12 SNPs (rs6798347, rs6787801, rs6801273, rs2046934, and rs6785930) were identified by the ligase detection reaction (LDR)-real time polymerase chain reaction (PCR) analysis system (Perkin-Elmer Gene Amp PCR Systems 9600, Perkin-Elmer Shanghai Inc., Shanghai, China). GENESCAN™ 672 and Genemapper of the system were used to analyze the CYP2C19 alleles. In this study, 10% of the samples were randomly reanalyzed, and the results were confirmed in 98.92%. According to the LD R -square of the selected P2RY12 SNPs, SNPs with pairwise R 2 values > 0.3 were selected for haplotype analysis, which turned out to be rs6798347, rs6787801, rs6801273, and rs6785930. Statistical analysis 2 Statistical analysis was carried out using STATA for Windows, Version 12.0 (Stata Corp., Texas, USA). Demographic data were presented as means and standard deviations (SDs) for continuous variables and as counts and percentages for categorical variables. Hardy-Weinberg equilibrium analysis was conducted using a chi-square test. Multivariate logistic regression analysis was carried out to study the association between individual SNPs/haplotypes and HTPR. Homozygotes for the common allele of these SNPs were used as the reference for each comparison analysis. P2RY12 haplotype analysis was done separately in nonsmokers. Analyses were adjusted for age, sex, body mass index, smoker, diabetes, hypertension, hyperlipidemia, family history of cardiovascular disease, platelet count, clopidogrel dose regimen (with or without loading dose), prior vascular disease and diagnosis (ST-segment elevation myocardial infarction (STEMI) or non-ST-segment elevation acute coronary syndrome (NSTE-ACS)). Results were expressed as odds ratio (OR) with 95% confidence interval (95% CI). A 2-sided value of P < 0.05 was considered statistically significant. Results 1 Platelet reactivity detection and baseline characteristics 2 One hundred and eighty patients were included in this study. All were from the Chinese Han population, and 120 (66.67%) patients underwent PCI. We identified 69 (38.33%) patients with HTPR. Demographic characteristics according to different clopidogrel responses are shown in Table 2 2 . There were no significant differences among age, gender, risk factors, medications, or other general information between the two groups ( P > 0.05) except for smoking ( P =0.046). Values are expressed as mean±SD or n (%). P -values refer to comparisons between HTPR and nHTPR groups. HTPR: high on-treatment platelet reactivity; nHTPR: non-high on-treatment platelet reactivity; BMI: body mass index; DM: diabetes mellitus; FH of CVD: family history of cardiovascular disease; ACEIs/ARBs: angiotensin-converting enzyme inhibitors/angiotensin receptor blockers; CCBs: calcium channel blockers; PPIs: proton pump inhibitors; NSTE-ACS: non-ST-segment elevation acute coronary syndrome; MI: myocardial infarction; PCI: percutaneous coronary intervention Characteristics Overall ( n =180) HTPR ( n =69) nHTPR ( n =111) P -value Demographics  Age 64.09±11.08  64.91±10.47  63.59±11.46  0.434   Male 137 (76.11%)  50 (72.46%)  87 (78.38%)  0.367  Risk factors  BMI (kg/m 2 ) 25.81±3.49  25.78±3.69  25.83±3.38  0.921   Smoker 54 (30.00%)  15 (21.74%)  39 (35.14%)  0.046   DM 70 (38.89%)  28 (40.58%)  42 (37.84%)  0.714   Hypertension 122 (67.78%)  48 (69.57%)  74 (66.67%)  0.686   Dyslipideamia 78 (43.33%)  28 (40.58%)  50 (45.05%)  0.557   FH of CVD 63 (35.00%)  25 (36.23%)  38 (34.23%)  0.785  Medication  Clopidogrel loading dose 42 (23.33%)  11 (15.94%)  31 (27.93%)  0.068   β-Receptor antagonist 132 (73.33%)  46 (66.67%)  86 (77.48%)  0.113   ACEIs or ARBs 109 (60.56%)  40 (57.97%)  69 (62.16%)  0.576   CCBs 48 (26.67%)  23 (33.33%)  25 (22.52%)  0.113   Statins 165 (91.67%)  64 (92.75%)  101 (90.99%)  0.678   PPIs 28 (15.56%)  11 (15.94%)  17 (15.32%)  0.910  NSTE-ACS 124 (68.89%)  44 (63.77%)  80 (72.07%)  0.243  Prior MI 29 (16.11%)  12 (17.39%)  17 (15.32%)  0.713  Prior PCI 119 (66.11%)  47 (68.12%)  72 (64.86%)  0.654  Platelet count (10 9 L −1 ) 189.11±61.19  193.06±64.50  186.70±59.25  0.502  Haematocrit (%) 38.35±6.37  37.74±5.16  38.73±7.01  0.320  Hemoglobin (g/L) 133.50±18.52  130.40±22.43  133.50±18.52  0.319  Genotyping and haplotype results 2 Allele frequencies and genotype distribution of the genetic variations studied are shown in Table 3 3 . The distribution of all the CYP genetic variants did not deviate from Hardy-Weinberg equilibrium. Variation Non-carriers, n (%) Heterozygous carriers, n (%) Homozygous carriers, n (%) Carriers of at least one allele, n (%) Allele frequency (%) CYP2C19   * 2 (rs4244285) 91 (50.56%) 67 (37.22%) 22 (12.22%) 89 (49.44%) 30.83   * 3 (rs4986893) 162 (90.00%) 18 (10.00%) 0 (0.00%) 18 (10.00%) 5.00   * 17 (rs12248560) 179 (99.44%) 1 (0.56%) 0 (0.00%) 1 (0.56%) 0.28 P2RY12  rs6798347 94 (52.22%) 67 (37.22%) 19 (10.56%) 86 (47.78%) 29.17  rs6787801 60 (33.33%) 81 (45.00%) 39 (21.67%) 120 (66.67%) 44.17  rs6801273 71 (39.44%) 83 (46.11%) 26 (14.44%) 109 (60.56%) 37.50  rs6785930 116 (64.44%) 54 (30.00%) 10 (5.56%) 64 (35.56%) 20.56  rs2046934 122 (67.78%) 54 (30.00%) 4 (2.22%) 58 (32.33%) 17.22 3.2.1 CYP2C19 Among the 180 patients studied, 89 (49.44%) were carriers of at least one CYP2C19 * 2 loss-of-function (LOF) allele and 18 (10.00%) were carriers of at least one CYP2C19 * 3 LOF allele. For CYP2C19 * 17 gain of function (GOF) allele, only one heterozygous carrier was detected. On the basis of the distribution of genotypes, the patients were divided into extensive metabolizers (EMs) without an LOF mutation allele (LOF non-carriers), which was also the CYP2C19 * 1/ * 1 (681GG/636GG) wild type genotype, intermediate metabolizers (IMs) carrying only one LOF mutation allele ( CYP2C19 * 2 or CYP2C19 * 3) with or without the GOF mutation allele, and poor metabolizers (PMs) carrying two LOF mutation alleles ( CYP2C19 * 2 and CYP2C19 * 3), accounting for 42.78%, 42.78%, and 14.44% of all cases, respectively. 3.2.2 P2RY12 Genotype distribution and allele frequencies of the studied P2RY12 SNPs were given in Table 3 3 . According to the LD R -square of the selected P2RY12 SNPs shown in Table 1 1 , we combined the four P2RY12 ht-SNPs (rs6798347, rs6787801, rs6801273, and rs6785930) and 12 haplotype alleles were inferred, among which 6 had an allele frequency higher than 5%: (H 0 ) CTAC (7.78%), (H 1 ) tcgt (14.44%), (H 2 ) tTgt (16.11%), (H 3 ) CcgC (18.33%), (H 4 ) CcAC (21.67%), and (H 5 ) tTAC (7.22%), as shown in Table 4 4 . Finally, the defined haplotypes cover 85.56% of the total common selected DNA sequence variations in the P2RY12 locus. The last P2RY12 SNP rs2046934 was not combined as the pairwise LD R 2 values with the other four SNPs were low ( < 0.3). Haplotype alleles were coded H 1 to H 5 in the descending order of their effect on platelet inhibition rate by TEG where H 0 is the reference haplotype allele. (1)‒(4) represent rs6798347, rs6787801, rs6801273, and rs6785930, respectively, with the minor alleles in lower case Haplotype (1) (C>t) (2) (T>c) (3) (A>g) (4) (C>t) SNP allele composition Frequency (%) H 0 C T A C CTAC 7.78 H 1 t c g t tcgt 14.44 H 2 t T g t tTgt 16.11 H 3 C c g C CcgC 18.33 H 4 C c A C CcAC 21.67 H 5 t T A C tTAC 7.22 Relationship of genotypes and haplotypes with residual platelet reactivity 2 The results of multivariate logistic regression for HTPR in the entire study population are presented in Table 5 5 . After adjusting for demographic characteristics, the presence of at least one LOF allele (PMs or IMs) or P2RY12 haplotype was independently associated with HTPR in the entire study population and in nonsmokers (Fig. 1 1 ). Values are expressed as n (%). P -values refer to comparisons between HTPR and nHTPR groups by multivariate logistic regression adjusted by CYP2C19 and covariate characteristics (including smoker). HTPR: high on-treatment platelet reactivity; nHTPR: non-high on-treatment platelet reactivity; EM: extensive metabolizer; IM: intermediate metabolizer; PM: poor metabolizer. Haplotypes H 0 to H 5 were coded by the compositions of rs6798347, rs6787801, rs6801273, and rs6785930 SNP/haplotype HTPR, n (%) nHTPR, n (%) Total, n (%) P -value CYP2C19  EM 19 (24.68) 58 (75.32) 77 (42.78) Reference  IM 39 (50.65) 38 (49.35) 77 (42.78) 0.001  PM 11 (42.31) 15 (57.69) 26 (14.44) 0.091 P2RY12 (rs2046934)  Non-carriers 44 (36.07) 78 (63.93) 122 (67.78) Reference  Carriers 25 (43.10) 33 (56.90) 58 (32.33) 0.223 P2RY12 haplotype  H 0 8 (57.14) 6 (42.86) 14 (7.78) Reference  H 1 9 (34.62) 17 (65.38) 26 (14.44) 0.016  H 2 10 (34.48) 19 (65.52) 29 (16.11) 0.062  H 3 13 (39.39) 20 (60.61) 33 (18.33) 0.104  H 4 15 (38.46) 24 (61.54) 39 (21.67) 0.105  H 5 7 (53.85) 6 (46.15) 13 (7.22) 0.296 In total group smoking was also adjusted compared to nonsmoker subgroup. The subgroup of smokers was excluded from the analysis due to small sample size ( n =53) 3.3.1 CYP2C19 Carriers of at least one LOF allele (PMs and IMs) had a higher residual platelet reactivity than non-carriers (EMs), and IMs had a higher incidence of HTPR than EMs in the total study population and the nonsmoker subgroup (46 (46.46%) vs. 19 (24.68%), OR 6.00, P- value 0.000, 95% CI 2.27‒15.82; 32 (51.61%) vs. 20 (32.26%), OR 4.69, P -value 0.007, 95% CI 1.52‒14.46). PMs had 100% incidence of HTPR. 3.3.2 P2RY12 For the total study population, haplotype H 1 showed a significantly lower incidence of HTPR than the reference haplotype (H 0 ) after adjusting for demographic characteristics and smokers (9 (34.62%) vs. 8 (57.14%), OR 0.13, P -value 0.016, 95% CI 0.03–0.68; Fig. 1a 1a ). Among nonsmokers, after adjusting for demographic characteristics, haplotypes H 1 and H 2 showed a significantly lower incidence of HTPR than H 0 (8 (47.06%) vs. 7 (63.64%), OR 0.11, P -value 0.036, 95% CI 0.02–0.87; 7 (38.89%) vs. 7 (63.64%), OR 0.08, P -value 0.017, 95% CI 0.01–0.64; Fig. 1b 1b ). P2RY12 allele rs2046934 (T744C) did not show any significant association with HTPR neither in the total population nor in nonsmokers. Discussion 1 In our study, we found the possible association between the common genetic variations of the P2RY12 and residual on-clopidogrel platelet reactivity using TEG in a Chinese population with ACS after adjusting the influence of CYP2C19 * 2 and * 3 alleles. The P2RY12 SNPs we chose (rs6798347, rs6787801, rs6801273, rs6785930, and rs2046934) contained the promoter region of the P2RY12 gene, which may alter its transcriptional activity instead of changing the structure of the P2Y12 receptor as other mostly analyzed SNPs may do. In our studied population, we identified by TEG 69 (38.33%) patients with HTPR. The incidence of HTPR was similar to those of other studies on a similar population (Liu et al., 2015 2015 ; Tang et al., 2015 2015 ) and higher than those on Caucasian and African-American populations with stable coronary heart disease (Bliden et al., 2007 2007 ). The different incidences of HTPR might come from inadequate platelet inhibition in Chinese patients and because Chinese are more likely to carry the CYP2C19 LOF allele compared to Caucasian and Africans (Liu et al., 2015 2015 ). As well as the established association of the CYP2C19 * 2 and * 3 LOF alleles with HTPR, several SNPs of the P2RY12 gene have been studied. Most studies focused on rs2046934 (T744C) or rs6809699 (G52T) representing dbSNP rs10935838, rs2046934, rs5853517 and rs6809699, and rs6785930 (C34T). Some found that it can modulate platelet response to clopidogrel or affect patients’ outcomes (Fontana et al., 2003 2003 ; Zee et al., 2008 2008 ; Staritz et al., 2009 2009 ; Shalia et al., 2013 2013 ; Zoheir et al., 2013 2013 ; Li et al., 2016 2016 ), but most results were negative (Angiolillo et al., 2005 2005 ; Smith et al., 2006 2006 ; Cuisset et al., 2007 2007 ; Lev et al., 2007 2007 ; Bierend et al., 2008 2008 ; Bonello et al., 2010 2010 ; Jang et al., 2012 2012 ; Namazi et al., 2012 2012 ; Kar et al., 2013 2013 ; Kim et al., 2013 2013 ; Jana et al., 2014 2014 ). Our results show that the P2RY12 allele rs2046934 (T744C) was not significantly associated with HTPR neither in the total study population nor in nonsmokers. Instead of any single polymorphisms of the analyzed genes, we found that the coexisting of the four SNPs (rs6798347, rs6787801, rs6801273, and rs6785930) was related to on-treatment platelet reactivity tested by TEG in the total population and in the nonsmoker subgroup. Haplotype H 1 , which carried four mutation alleles of the four selected SNPs at the same time, showed a significantly lower incidence of HTPR compared with the reference wild haplotype (H 0 ). Similarly, Rudež et al. ( 2009 2009 ) composited six P2Y12 SNPs (rs6798347, rs6787801, rs9859552, rs6801273, rs9848789, and rs2046934) and found that one haplotype was associated with significantly lower residual on-clopidogrel platelet reactivity compared with the reference haplotype tested by VerifyNow and light transmission aggregometry (LTA). Rudež et al. ( 2008 2008 ) also composed five P2RY12 SNPs (rs6798347, rs10935842, rs6787801, rs6801273, and rs2046934) and found that they were associated with a higher risk of adverse cardiovascular events than the reference P2RY12 haplotype, which contains the common alleles of all the five P2RY12 SNPs. Furthermore, we analyzed the co-contribution of P2RY12 and CYP2C19 together, after adjusting for the influence of CYP2C19 LOG alleles and GOF alleles, and we found the extra coexisting impact of P2RY12 on the residual platelet reactivities besides CYP2C19 . This was partly examined in Malek et al. ( 2008 2008 )’s research with different alleles. Our study, in a similar way, found a new impact of P2RY12 genes on platelet reactivity but by a different combination of SNPs, which helps gain a better understanding of the effect of P2RY12 genes on clopidogrel on-treatment platelet reactivity. In our studied population we found that smokers had a lower percentage of HTPR (OR 0.4945, P -value 0.046, 95% CI 0.25–0.99). This might be explained by Shanker et al. ( 2006 2006 )’s study stating that smokers might have higher platelet P2Y12 activity. Similarly, Cavallari et al. ( 2007 2007 )’s study stated that rs2046934 (T744C) was associated with a higher presence of coronary artery disease, particularly in nonsmokers, which in some way demonstrated that smoking affected P2Y12’s contribution to coronary artery disease although they showed the relationship with a different P2RY12 SNP. However, we are not the first to report the association of clopidogrel on-treatment platelet reactivity with smoking (Tantcheva-Poór et al., 1999 1999 ; Bliden et al., 2008 2008 ; Gremmel et al., 2009 2009 ; Price et al., 2009 2009 ; Jeong et al., 2010 2010 ; Ueno et al., 2012 2012 ; Gurbel et al., 2013 2013 ; Peng et al., 2015 2015 ). It was early reported that smoking increases the enzymatic activity of CYP1A2, one of the predominant pathways for the first oxidative step of the pro-drug clopidogrel, through polycyclic aromatic hydrocarbons and plasma nicotine, thereby enhancing the generation of the active metabolite of clopidogrel (Tantcheva-Poór et al., 1999 1999 ). One hypothesis by Bliden et al. ( 2008 2008 )’s group was that the significant cigarette smoking might affect platelet inhibition in clopidogrel-treated patients via CYP1A2. However, a study conducted by Yousef et al. ( 2008 2008 ) showed that the elimination half-life of clopidogrel was shorter in current smokers than in nonsmokers, which confused the established hypothesis. Meanwhile, there are several studies which reported a negative association with smokers in larger observation studies (Hochholzer et al., 2011 2011 ; Sibbing et al., 2012 2012 ; Kim et al., 2016 2016 ). Kim et al. ( 2016 2016 ) deduced that hemoglobin levels might be the key driver for the observed ex vivo phenomenon of lower on-treatment platelet reactivity levels and/or an enhanced response to ADP inhibitors like clopidogrel in active smokers in most of the studies published so far. More studies are needed. Recently, the study of Patti et al. ( 2016 2016 ) found that cigarette smoking weakly influences antiplatelet effects of oral P2Y12 inhibition. The results on the association of smoking and platelet responsiveness to ADP-receptor antagonist are inconsistent and controversial within studies. There was one study of Cicko et al. ( 2010 2010 ) reporting that smoke exposure led to an up-regulation of the P2Y2R subtypes in blood neutrophils and in bronchoalveolar lavage fluid neutrophils by in vitro mouse model. This might explain the observed relationship between HTPR and smoking on ACS patients in smokers. Further studies are required to compare the different on-treatment platelet activities between nonsmokers and smokers. Conclusions 1 Our study showed that apart from the established association of the CYP2C19 * 2 and * 3 LOF alleles with HTPR, haplotypes of P2RY12 rs6798347, rs6787801, rs6801273, and rs6785930 rather than rs2046934 (T744C) that related to pharmacodynamics of clopidogrel in patients with ACS were independently associated with HTPR. This association seems to be more important in the subgroup defined by smoking. This finding suggests that P2RY12 genetic variation and common allele composition may be important in determining platelet reactivity for patients treated with clopidogrel, especially in nonsmokers. Study limitations 1 The first limitation of our study is the small sample size, especially the small sample of the population smoking. Secondly we cannot completely eliminate the possible bias by clinical risk factors despite multivariate analysis. 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P2Y12 receptor gene polymorphism and antiplatelet effect of clopidogrel in patients with coronary artery disease after coronary stenting Compliance with ethics guidelines Xiao-yan NIE, Jun-lei LI, Yong ZHANG, Yang XU, Xue-li YANG, Yu FU, Guang-kai LIANG, Yun LU, Jian LIU, and Lu-wen SHI declare that they have no conflict of interest. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008 (5). Informed consent was obtained from all patients for being included in the study. 10.1631/jzus.B1600333 Project supported by the Beijing Higher Education Young Elite Teacher Project (No. YETP0064), China 2016-07-21 2016-12-14 2016-10-20 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2017 2017 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 01 18