Relationship Between The Platelet Glycoprotein â…¡b-â…¢a Complex Gene Polymorphism And Coronary Heart Disease

BackgroundCoronary heart disease (CHD) is multigenic disease. The rupture of atheromatous plaque and thrombosis leads to acute myocardial infarction and unstable angina. After mural thrombosis, platelet deliveries kinds of cytokines into wall of artery,encouraging cellular proliferation of contractile fiber cells. Thrombosis is controlled by variety platelet membrane receptor complex,include platelet glycoprotein(Gp) I b/ I x,Gp I a/ II a and Gp II b/ III a.The Gp II b/III a complex that is dependent on calcium ion is profibrin receptor on the surface of platelet. It controls the comitant tunnel which is dependent on profibrin of platelet aggregation. Gp IIIa polymorphism(HPA -1a/b) is 1565 T→C base substitution on exon 2 which leads Leu33(HPA-1 a, PIA1) →Pro (HPA-1 b,PIA2). Gp II b polymorphism (HPA -3a/b) is 2622 T →G base substitution which leads lle843(HPA-3a,Baka) →Ser(HPA-3b,Bakb).Previous studies showed that the group with PIA2 had susceptibility to acute coronary disease and the group had arterial sclerosis with cerebral thrombosis could be observed abnormality of Gp II blle/Ser843 gene polymorphism.Our purpose is to known the distribution of Gp IIb/ III a complex gene polymorphism in Chinese people and the relationship with CHD.Subject and Methods1. SubjectCHD(-) group:105 CHD(-) object(63 men and 42 women,mean aged 59.7 ±11.0 years) served as a control group.All subjects were consecutively collected from the hospitalized in the department of cardiology,the second affiliated hospital of medical college,Zhejiang University.All patients were confirmed have no CHD by coronary angiography.CHD(+) group:125 CHD patients(96 men and 29 women,mean aged 63.7±9.6 years) were consecutively collected from the hospitalized in the department of cardiology,the second affiliated hospital of medical college,Zhejiang University. All patients were confirmed by coronary angiography. 2. Documentation of CHD severitThe severity of coronary stenosis was determined by the number of significantly stenosed coronary arteries as follows. Angiograms were assessed by two cardiologist who were unaware that the patients were to be included in the study.Each angiogram was classified as revealing either coronary lesion with more than 50% luminal stenosis or one, two,or three major epicardial coronary arteries with more than 50% luminal obstructions.The CAD group is divided into non-myocardial infarction subgroup (n=86) and myocardial infarction subgroup (n=39) using myocardial infarction criteria defined by the World Health Organization (WHO). 3.Laboratory methods for Gp II b-IIIa complex gene polymorphism1) DNA was extracted from the peripheral blood leukocytes by standard phenol and chloroform method.2)Gp II b- III a complex genotype was determined by PCR-RFLP.The forward primer of Gp II b: 5 -CTCAAGGTAAGAGCTGGGTGG AAGAAAGA C-3 ' ;the reverse primer: 5 '-CTCACTACGAGAACGGGAT CCTGAAGCCTC- 3 ' .PCR was carried out using a 25ul reaction mix containing 10 × Reaction buffer(without MgCl₂) 2.5ul,25mM MgCl₂ 1.5ul,10mM dNTP 0.6ul,genomic DNA 100ng,Taq DNA polymerase 0.1 ul and 10pmol each primer.The PCR cycles were modified as follows:initial denaturation at 94℃ for 5 minutes followed by 28 cycles at 94 ℃ for 45 second,at 63 ℃ for 45 second and at 72℃ for 1minute,at last 72℃ for 7 minute.PCR products were then digested with Fok I at 37℃ for 16 hours and digestion product were separated on 2% agarose gels and visualized with ethidium bromide. The forward primer of Gp III a: 5 '-CTCAAGGTAAGAGCTGGGTGGAAGAAAGA C-3 ' ; the reverse primer: 5 ' -CTCACTACGAGAACGGGATCCTGAAGCCTC-3 ' .PCR was carried out using a 20ul reaction mix containing 10× Reaction buffer(without MgCl₂) 2.0ul,25mM MgCl₂ 1.2ul,10mM dNTP 0.4ul,genomic DNA 100ng,Taq DNA polymerase 0.2ul and 5pmol each primer. The PCR cycles were modified as follows:initial denaturation at 95℃ for 5 minutes followed by 30 cycles at 95℃ for 45 second,at 60 ℃ for 45 second and at 72 ℃ for 1 minute,at last 72℃ for 5 minute. PCR products were then digested with ScrFl at 37℃ for 16 hours and digestion product were separated on 2% agarose gels and visualized with ethidium bromide. 4. Statistic analysisUsing the SPSS13.0 for windows statistical package Count Data were performed by Student's t-test and measure data were performed by chi-squared test.Genotype and allele frequencies between groups were analyzed by the chi-squared test. Statistical significance was taken as p<0.05.Result1 .There is a polymorphism of Gp II b in Zhejiang Han population but not of Gp III a.The Gp II b genotype distribution of the control group and CHD group was in Hardy-Weinberg equilibrium (p>0.05).2.The distribution of genotype ab and bb in CHD group was significant more than in control group (x²=13.924, p=0.001) . The b allele frequency in CHD group is significant higher than in control group (x²=13.047, p=0.000) .3.The genotype distribution of Gp II b was no difference between single,double and triple vessel disease (x²=7.236, p=0.118) .But the allele frequency of Gp II b had significant difference, The b allele frequency in subjects with more pathological changes is higher than in subjects with less (x²=6.764, p=0.034) .4. There was a significant difference in the genotype distributionand allele frequency of Gp II b comparing patients with and without Mlwith an increased prevalence of the ab and bb type in subjects with Ml(x²=18.754, p=0.000) . The b allele frequency in objects with Ml ishigher than in subjects without Ml (x²=19.347, p=0.000) .5.There was no relationship between genotype distribution and allele frequency of Gp II b in the CHD group who are smoking (p>0.05).6.Platelet count, INR, APTT and FBG were no difference between the every genotype of Gp II b (p>0.05).Conclusions1 .There is a polymorphism of Gp II b in Zhejiang Han population.2. b allele may be a susceptibility gene of CHD.3. Gp II b gene polymorphism may be associated with the degree and type of CHD.4.Gp II b gene polymorphism may be not associated with gender,hypertension,DM and smoking.5. Gp II b gene polymorphism may be not affect the level of platelet count, INR. APTT and FBG.