| Clopidogrel is a kind of antiplatelet drug that becomes the primary therapeutic drug for patients with CAHD who undergo percutaneous coronary intervention(PCI) or those with ACS. Clopidogrel coule reduces the incidence of stent thrombosis efffectively, significantly improves the prognosis of coronary heart disease(CHD), and is thus one of the most important drugs for antiplatelet therapy after PCI. Recent studies show that the reactions to antiplatelet drugs are dependent on individuals. Some patients, especially those who received coronary stenting due to adverse cardiovascular events,still have to face the recurred adverse cardiovascular events, even if they are taking adequate antiplatelet drugs such as clopidogrel for a long term.Clopidogrel resistance(CR)is related to many factors, such as individual differences, interactions between drugs, polymorphisms of platelet genes,basic platelet reactivity, platelet sizes, etc. However, the molecular mechanism is still unknown.Clopidogrel is activated through two~step oxidation reaction by CYP450 in the liver. Thereinto, 45% and 21% of clopidogrel are mediated by CYP2C19 protein in each step. Thus, CYP2C19 gene is very important in the transformation of clopidogrel. The studies reveal that CYP2C19 gene is polymorphism, and the variants *2/*3 are associated with the lack of metabolic function(referred as the loss of functional genes). Some recent studies have shown that the levels of clopidogrel active metabolite and platelet inhibition are lower in patients with CYP2C19*2 or *3 loss~functional genotype, leading to an increased risk in recurrence of cardiovascular events.Moreover, mutation frequency of CYP2C19 loss of functional gene is higher in Asian compared wit other ethnic groups. Additionally, many studies prove that high levels of micro RNA(mi RNA or mi R) expression exists in platelets,and plays important roles in the pathology of diseases involving platelets.However, there are few studies on the effect of mi RNA in CR. Thus, this study firstly investigated the relationship between CYP2C19 gene polymorphisms and CR in the patients of coronary atherosclerotic heart disease(CAHD) who accept percutaneous coronary intervention(PCI) as well as the relationship with long~term prognosis of patients after PCI. In addition,this study also investigated whether the level of mi RNAs in platelets are related to CR in patients after PCI. Furthermore, this study also investigated the effects of maintenance dose of Clopidogrel, Tongxinluo capsule combined with dual antiplatelet therapy and Ticagrelor on patients with CYP2C19 gene polymorphisms after PCI to provide better therapeutic for patients.Part 1 Influence of CYP2C19 gene polymorphisms on clopidogrelresistance in the patients accepting PCIObjective : To explore the association between CYP2C19 gene polymorphisms and clopidogrel resistance in patients of coronary atherosclerotic heart disease(CAHD) who has underwent PCI.Methods : This study included 312 patients of CAHD who have underwent PCI from June 2012 to December 2013 in the People's Hospital of Hebei Province. Using the Taqman~realtime PCR technology to detect the CYP2C19 genotypes in all the patients. TEG was used to dected the level of platelate aggregation rate and MA~ADP. Meanwhile, flow cytometry was applied to detect the level of VASP, s CD40 and P~selectin. SPSS13.0 software was conducted to analysis the relationship between the polymorphisms of CYP2C19 gene and clopidogrel resistance.Results:1 203 males and 109 females were included in this study, and the mean age was(58.67±10.34) years, from 39 to 78 years.2 There were 135 patients carrying CYP2C19*1/*1, 124 patients CYP2C19*1/*2,22 patients CYP 2C19*1/*3, 23 patients CYP2C19*2/*2, 6patents CYP 2C19*2/*3 and 2 patients CYP 2C19*3/*3The allele frequency of CYP2C19*1, *2, *3 were 66.7%, 28.2% and 5.1%.Based on the distribution of genotype, patients were divided into normal genotype group(CYP2C19*1/*1)including 135 patients and lost~functional genotypes(CYP2C19*1/*2?*1/*3?*2/*2?*2/*3?*3/*3)including 177patients?3 Compared with normal genotype group, the platelet inhibition rate was lower(P<0.001) and MA~ADP was higher(P<0.001) of the lost~functional genotype group.4 Compared with normal genotype group, the level of VASP was lower(P<0.001) in the lost~functional genotype group; while, the level of s CD40 and P~selectin were much higher(P<0.001) in the lost~functional genotype group.5 Patients were divided into clopidogrel resistance group(68 patients)and non~clopidogrel resistance group(244 patients), based on the platelet inhibition rate. The proportion of clopidogrel resistance was 21.8%. While, in each genotype(*1/*1 ? *1/*2 ? *1/*3 ? *2/*2 ? *2/*3 and *3/*3), the proportion of clopidogrel resistance were 16.3%, 25.0%, 27.3%, 26.1%,33.3% and 50.0%, respectively. Additionally the proportion of clopidogrel resistance was 83.7% in the lost~functional genotype group, and it was significantly higher compared with normal genotype group(?2=4.221,P=0.040).Conclusions:The polymorphism of CYP2C19 gene influences platelet inhibition rate, MA~ADP and the level of VASP, s CD40 and P~selectin.Part 2 Association between CYP2C19 polymorphism and cardiovascularevents in patients with Coronary Artery Disease after PCIObjective:To analyze the association of CYP2C19(*2 and *3) gene polymorphism and long~term prognosis of patients after PCI.Methods : From June 2012 to December 2013, 312 CAD patientsaccepting PCI in the People's Hospital of Hebei Province were enrolled in this study. Only 275 patients accomplished the follow~up. Using Taqman~realtime PCR method to detect the genotype of CYP2C19(*2 and *3) in patients. Patients were divided into 3 groups, including Extensive Metabolizer(EM); Intermediary Metabolism(IM) and Poor Metabolism(PM). Through telephone, outpatient service and readmission records, the follow~up was carried out. The endpoint of this study is Major Adverse Cardiac events in 1year after PCI. Among three groups, the incidence of MACE was compared and the relationship between CYP2C19 gene polymorphisms as well as the MACE.Results:1 The distribution of CYP2C19 are as follows: CYP2C19*1/*1(122patients), CYP2C19*1/*2(112 patients), CYP2C19*1/*3(18 patients),CYP2C19*2/*2(19 patients), and CYP2C19*2/*3(4 patients). The allele frequency of CYP2C19*1 was 68.0%, CYP2C19*2/*2 was 28.0% and CYP2C19*3was 4.0%. 275 patients were divided into three groups: Extensive metabolizers(122 patients),Intermediate metabolizers(130 patients), Poor metabolizers(23 patients).2 The incidence of non~fatal MI in Extensive metabolizers, Intermediate metabolizers and Poor metabolizers were 0.8% ? 1.5% and 21.7%(P<0.001), respectively. The incidence of MACE as well as the combined MACE was much higher in patients with CYP2C19*2/*3 lose of function allele.The polymorphisms of CYP2C19*2/*3 genotype was independently correlated with MACE(OR=9.373,95%CI:1.756~50.044;P=0.009).Conclusion:The polymorphism of CYP2C19(*2 and *3)is related to clinical outcomes of coronary heart disease patients receiving PCI.Part 3 Association between the level of micro RNA~26a expressed inplatelets participates and clopidogrel resistance in CHD patientsObjective : This study is to evaluate the relationship between platelet micro RNA(mi RNA)~26a expression and clopidogrel resistance in patientsafter coronary stenting.Methods : From January 2013 to January 2014, 43 patients with chronary heart disease underwent PCI in our hospital. Meanwhile, 20 healthy volunteers without history of cardiovascular disease were enrolled as the control group. Flow cytometry was used to measure the phosphorylation levels of VASP, as well as PRI. Low response to clopidogrel was defined as PRI ?50% on day 7 after drug intake. The protein expression of VASP was evaluated by Western blotting. Quantitative real~time polymerase chain reaction was performed to determine the levels of m RNA and mi RNAs.Bioinformatics tools were employed to predict that mi R~26a, mi R~199, and mi R~23a might target VASP.Results:Activity of platelets in patients with low clopidogrel response is higher than that in healthy subjects. VASP gene transcription is maintained at relatively low levels in healthy subjects and patients with high clopidogrel response, but kept at relatively high levels in patients with low clopidogrel response. Expression of platelet mi RNA~26a, but not mi RNA~199 and mi RNA~23a, is related to high platelet reactivity. Serum mi RNA~26a,mi RNA~199 and mi RNA~23a are not involved in clopidogrel resistance.Conclusions:Platelet mi RNA~26a plays important roles in clopidogrel resistance. Combination of mi RNA and VASP PRI tests will be helpful in the early diagnosis and prediction of clopidogrel resistance.Part 4 Effects of dual dose of Clopidogrel,Tongxinluo capsule combinedwith antiplatelet therapy and Ticagrelor on patients after PCIObjective:To investigate the Influence of dual dose of Clopidogrel,Tongxinluo capsule combined with antiplatelet therapy and Ticagrelor on patients with CYP2C19 gene polymorphisms after PCI.Methods:During the period of June 2012 to October 2014, 458 CAD patients in the People's Hospital of Hebei Province were enrolled in this study.Using Taqman~realtime PCR method to detect the polymorphisms of CYP2C19*2 gene in selected patients. Additionally TEG was applied to detect the level of paltelate inhibition rate. The patients with CYP2C19*1*2 orCYP2C19*2*2 were enrolled, and were divided into four groups:(1) regular dose of Clopidogrel group;(2)double dose of Clopidogrel group,(3)Tongxinluo capsule combined with regular antiplatelet therapy group and(4)Ticagrelor group. The Major Adverse Cardiac events(MACE) was considered as the endpoint of the study after PCI in 1 year. The PAG and the incidence of MACE within 1 year among four groups was compared.Results:1 After 1 month of taking drugs, the platelet inhibition rate was Ticagrelor group > double dose of Clopidogrel group > Tongxinluo capsule combined with antiplatelet therapy group > regular dose of Clopidogrel group.2 After 3 month of taking drugs, the platelet inhibition rate was Ticagrelor group > Tongxinluo capsule combined with antiplatelet therapy group > double dose of Clopidogrel group =regular dose of Clopidogrel group.3 After 6 month of taking drugs, the platelet inhibition rate was Ticagrelor group > regular dose of Clopidogrel group >Tongxinluo capsule combined with antiplatelet therapy group =double dose of Clopidogrel group.4 After 12 month of taking drugs, the platelet inhibition rate was Ticagrelor group > Tongxinluo capsule combined with antiplatelet therapy group =double dose of Clopidogrel group =regular dose of Clopidogrel group.5 The total incidence of MACE was much lower in Ticagrelor group,double dose of Clopidogrel group and Tongxinluo capsule combined with dual antiplatelet therapy group compared with regular dose of Clopidogrel group(all P<0.05).6 The incidence of hemorrhage in Ticagrelor group was much higher than other three groups.Conclusions:1 The effects of double dose of Clopidogrel group, Tongxinluo capsule combined with antiplatelet therapy group and Ticagrelor group in inhibiting platelate aggregation was better than regular dose of Clopidogrel group, and Ticagrelor was the best.2 The total incidence of MACE was much lower in double dose ofClopidogrel group, Tongxinluo capsule combined with antiplatelet therapy group and Ticagrelor group. While the incidence of hemorrhage in Ticagrelor group was higher. |
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