Genetic Polymorphisms And Antiplatelet Effects Of Clopidogrel In Patients With Percutaneous Transluminal Angioplasty And Stenting

Percutaneous transluminal angioplasty and stenting (PTAS) is associated with an increased risk of thromboembolism as the introduction of stent into blood stream, injury to the vessel wall, and plaque disruption that lead to platelet activation during endovascular procedures. Antiplatelet therapy is often used to combat the risk of stent thrombosis and re-stenosis associated with PTAS. The inter-individual response to clopidogrel has been reported and high on-treatment platelet reactivity (HTPR) is associated with increased thromboembolic complications after neurovascular PTAS.Clopidogrel is an inactive prodrug that requires biotransformation to an active metabolite and then irreversibly inhibits the key mediator of platelet aggregation ADP P2Y12 receptor. Its pharmacokinetic and pharmacodynamic effects can be influenced by multiple factors includes age, co-morbidities, drug-drug interactions and genetic polymorphisms in clopidogrel absorption and enzymes responsible for metabolic transformation.Evidences are accumulating that the polymorphically expressed isoenzyme CYP2C19 constitutes a dominant part in conversion of clopidogrel into its active metabolite.The allele frequency of CYP2C19*2 was significantly higher in Asian populations, therefore, the prevalence of clopidogrel HTPR due to CYP2C19*2 might be particularly higher. Moreover, the CYP2C19 polymorphisms could only explain part of the interindividual variability of clopidogrel antiplatelet responsiveness. Genetic variability in a range of other receptors and key enzymes may also contribute. Recently, a common intronic G/A single nucleotide polymorphism in the platelet endothelial aggregation receptor 1 (PEAR1) locus on chromosome 1 has been linked with platelet aggregation. While there are few studies about the relationship between genetic polymorphisms and clopidogrel responses in patients after neurovascular PTAS.To address the above issues, the present study aimed to determine the impact of genetic polymorphisms on the response to clopidogrel and the prognosis for ISR occurrence in a cohort of patients after neurovascular PTAS.Part I CYP2C19*2 Gene Polymorphisms and Antiplatelet Effects of Clopidogrel in Patients with Percutaneous Transluminal Angioplasty and StentingObjective:To explore the relationships between CYP2C19*2 gene polymorphisms and high on-clopidogrel platelet reactivity in neurovascular percutaneous transluminal angioplasty and stenting patients treated with clopidogrel.Methods:Between January 2011 and March 2014, a total of 145 Chinese patients who had been treated with percutaneous transluminal angioplasty and stenting in the department of Neurology of Nanjing General Hospital of Nanjing Military Command were consecutively enrolled in this retrospective observed study. Inclusion criteria: 1)aged 18 years or older; 2) Chinese Han population; 3) diagnosed with ischemic stroke and confirmed by DSA for arterial stenosis (symptomatic stenosis> 50% or asymptomatic stenosis> 70%); 4) patients had at least one of risk factors for atherosclerosis:high blood pressure, diabetes mellitus, high cholesterol and smoking;5) prescribed dual antiplatelet therapy, which in combination with clopidogrel (75mg/d) and aspirin (100mg/d) at least six months. Exclusion criteria: 1) non-atherosclerotic arterial stenosis, such as moyamoya disease, arteritis, radiotherapy-induced arterial stenosis, muscle dysplasia and acute arterial dissection and so on.2) stroke of cardiac sources:atrial fibrillation, valvular heart disease, left ventricular mural thrombus or left ventricular myocardial infarction among six weeks;3) allergy to aspirin or clopidogrel or can not tolerate, low molecular weight heparin contraindications;4) platelet count<100 x 109/L or>450×109/L, coagulation disorders or have a bleeding tendency; 5) Class Ⅲ heart function or more severe liver and kidney function.6) took PPI, tricyclic antidepressants, antiepileptic and antipsychotics.The stents were operated by experienced interventional doctors and all stents are bare metal stents. Patients were treated with aspirin and clopidogrel (aspirin 100 mg, clopidogrel 75mg,once daily) after stenting for 6 months, and then clpidogrel 75mg/d alone. All patients undergoing neurovascular PTAS were according to the Chinese Guidelines for Endovascular Management of Ischemic Cerebrovascular Diseases. Demographic characteristics and baseline variables were obtained from NSRP and medical charts.The inhibitory effect of clopidogrel on platelet reactivity was measured by the thrombelastography (TEG) mapping assay (Haemoscope Corp., USA) in the TEG 5000. Blood samples were obtained for test while patients were on a 75mg daily clopidogrel treatment for a period of 6 months after PTAS.Clopidogrel HTPR in this study was defined as less than 30% inhibition of platelet aggregation with 2μmol/L ADP.Genomic DNA was extracted from peripheral-blood leucocytes using commercially available kits (Tiangen Biotech Co. Ltd., Beijing). Improved Multiplex Ligase Detection Reaction (iMLDR) for genotype of CYP2C19*2(681G>A) was performed. About 10% samples were randomly selected and repeated genotyped in order to ensure accuracy of results, which obtained a complete concordance.Continuous variables were tested for normal distribution with the Kolmogorov-Smirnov test and presented as mean ± standard deviation (SD) or median (inter-quartile range (IQR)). Categorical data were summarized as frequencies (percentages). For comparison among groups, the unpaired Student’s t-test was applied for normal continuous variables and abnormally distributed variables were compared using a Mann-Whitney U-test. The Chi-square test (or Fisher’s exact test when any expected cell count was< 5 for a 2x2 table) was used for categorical variables. Logistic regression analysis was used to determine the correlation between HTPR and possible risk factors.Results:General clinical data:145 patients were included in this study, the average age at baseline was 64.22±8.94 years old,66.2% of the subjects were male,65.5% with hypertension and 31% with diabetes. When compared with the nHTPR group, there was no difference between the two groups.The genotype distribution of CYP2C19*2:the minor allele frequencies (MAFs) of CYP2C19*2 was 32.8%, there were 69 patients(47.6%) carrying CYP2C19*1/*1, 57patients(39.3%) carrying CYP2C19*1/*2 and 19 patients(13.1%%) carrying CYP2C19*2/*2.Comparison of platelet inhibition rate in different genotype:the platelet inhibition rate were (58.43 ± 21.98)%, (47.79±22.93)% and (37.53±21.84)% among the individuals with the genotype of CYP2C19*1/*1> CYP2C19*1/2*、CYP2C19*2/*2, respectively.Comparison the incidence rate of HTPR in different genotype:HTPR was present in 17.4% patients of CYP2C19*1/*1,29.8% patients of CYP2C19*1/*2 and 52.6% patients of CYP2C19*2/*2. The difference of HTPR distribution among CYP2C19*1/*1 and (CYP2C19*1/*2+CYP2C19*2/*2) was significant (P=0.014).Evaluate by logistic regression analysis, the independent risk factor of HTPR was CYP2C19*2 genotype.Conclusions:The population of this study,the incidence of HTPR was 26.9%. CYP2C19*2 gene polymorphisms are associated with the occurrence of HTPR.CYP2C19*2 genotype might be relate to the increase of HTPR.PartⅡ Relationship between PEAR1 gene polymorphisms and vascular events in Patients with Percutaneous Transluminal Angioplasty and StentingObjective:To explore the relationships between PEAR1 gene polymorphisms and prognosis in neurovascular percutaneous transluminal angioplasty and stenting patients treated with clopidogrel.Methods:Between January 2011 and March 2014, a total of 145 Chinese patients who had been treated with percutaneous transluminal angioplasty and stenting in the department of Neurology of Nanjing General Hospital of Nanjing Military Command were consecutively enrolled in this retrospective observed study. Inclusion criteria: 1)aged 18 years or older; 2) Chinese Han population; diagnosed with ischemic stroke and confirmed by DSA for arterial stenosis (symptomatic stenosis≥50% or asymptomatic stenosis≥ 70%); 4) patients had at least one of risk factors for atherosclerosis:high blood pressure, diabetes mellitus, high cholesterol and smoking;5) prescribed dual antiplatelet therapy, which in combination with clopidogrel (75mg/d) and aspirin (100mg/d) at least six months. Exclusion criteria: 1) non-atherosclerotic arterial stenosis, such as moyamoya disease, arteritis, radiotherapy-induced arterial stenosis, muscle dysplasia and acute arterial dissection and so on.2) stroke of cardiac sources:atrial fibrillation, valvular heart disease, left ventricular mural thrombus or left ventricular myocardial infarction among six weeks; 3) allergy to aspirin or clopidogrel or can not tolerate, low molecular weight heparin contraindications; 4) platelet count<100×109/L or>450×109/L, coagulation disorders or have a bleeding tendency; 5) Class Ⅲ heart function or more severe liver and kidney function.6) took PPI, tricyclic antidepressants, antiepileptic and antipsychotics.The stents were operated by experienced interventional doctors and all stents are bare metal stents. Patients were treated with aspirin and clopidogrel (aspirin 100 mg, clopidogrel 75mg, once daily) after stenting for 6 months, and then clpidogrel 75mg/d alone.DNA extraction method and genotyping were agreed with the methods listed in the first chapter.After treated with PTAS, patients were followed up by neurologists of the NSRP at 1,6 and 12 months and annually thereafter via clinical visit or telephone interview. Each endpoint events was adjudicated by a physician who was blind to the genotypic results. The primary endpoint of follow-up:in-stent restenosis found by DSA (digital subtraction angiography, DSA). The secondary endpoint of the study was on-treatment ischemic events defined as composite of vascular death, and non-fatal ischemic stroke and MI. And each patient was censored once off clopidogrel. Results for each enrolled patient were recorded by observers who were blind to the results of genotypic and laboratory results.Continuous variables were tested for normal distribution with the Kolmogorov-Smirnov test and presented as mean±standard deviation (SD) or median (inter-quartile range (IQR)). Categorical data were summarized as frequencies (percentages). For comparison among groups, the unpaired Student’s t-test was applied for normal continuous variables and abnormally distributed variables were compared using a Mann-Whitney U-test. The Chi-square test (or Fisher’s exact test when any expected cell count was< 5 for a 2×2 table) was used for categorical variables. Cox regression was performed to calculate hazard ratios. Multivariable Cox regression model was applied to explore risks for primary endpoints after adjusting potential confounders. Data analysis was performed by SPSS 21.0 statistical software. P< 0.05 indicated statistical significance.Results:The minor allele frequency of rs 12041131 was 37.2%.Genotype distributions were complied with Hardy-Weinberg equilibrium. For comparing of clinical risk factors according to rs12041331 and CYP2C19*2 (rs4244285) genotypes, there were no differences between groups.According to rs12041331 genotype:the incidence of HTPR after treatment with clopidogrel differed significantly between patients with AA and (GA+GG) group (4.5% vs.30.9%, P= 0.01).During follow-up, there were 19(13.1%) patients had primary clinical endpoints. The cumulative incidence of endpoints was significantly higher in CYP2C19*2 carriers than CYP2C19*1/*1 carriers (HR=3.921,95%CI:1.300-11.824, P=0.015). As for rs12041331, there were no significant impacts on outcomes between different genotypes. As for adjustment for potential confounders, CYP2C19*2 carriers (HR=4.615,95%CI:1.507-14.132, P=0.007), diabetes mellitus (HR=2.539, 95%CI:1.010-6.383, P=0.048) and intracranial stents (HR=3.695,95%CI: 1.473-9.267, P=0.005) were independent risk factors for ISR after PTAS.Conclusion:Patients with AA genotype displayed significantly higher platelet inhibition rate than those with GG and GA genotype. The rsl2041331 gene polymorphisms are associated with the occurrence of HTPR. CYP2C19*2 gene polymorphisms, diabetes mellitus and intracranial stents might be markers for ISR for patients treated with clopidogral after PTAS.