The Correlation Analysis Of Venous Thromboembolism And Acute Myocardial Infarction With The Levels And Polymorphisms Of Coagulation And Anticoagulation Factors

The development of thrombotic disorders in humans is one of the most common causes of morbidity and mortality in the world.Thrombotic diseases can be classified into venous thromboembolism and arterial thromboembolism.Venous thromboembolism(VTE)results in two major clinical manifestations:deep venous thrombosis(DVT)and pulmonary embolism (PE).Arterial thrombosis includes coronary artery disease(CAD),ischemic stroke(IS)and peripheral arterial occlusion(PAO).Acute thrombosis at the site of a ruptured,lipid-rich atherosclerotic plaque is understood as a pivotal event in the transition from stable or subclinical atherosclerotic disease to acute myocardial infarction(MI)in the development of CAD.Over the past 20 years,several hematologic disorders have been identified as related to the risks of thrombotic disorders,these disorders include deficiency of anticoagulants and excessiveness of procoagulant factors.Recent investigations have shown that deficiencies of protein C,protein S and antithrombin;high clotting activities of coagulation factors FⅡ(FⅡ:C),FⅤ(FⅤ:C), FⅧ(FⅧ:C)and FⅨ(FⅨ:C);high plasma levels of fibrinogen and D-dimer(D-D); hyperhomocysteinemia(HHc)and activated protein C resistance(APCR)might be associated with an increased risk for thrombotic disorders,but the data is still a matter of debate.The occurrence of thrombotic disorder is a culmination of environmental and genetic risk factors,but approximately half of all thrombotic events occur in patients without environmental risk factors.Thus,in the current era of elucidation of the human genome,investigators have focused on the molecular genetics of thrombosis and thrombotic disorders to improve their understanding of the pathobiology of thrombotic disorders,and a range of gene polymorphisms contributing to disease risks have been identified.Such as the polymorphisms -148C/T and -455G/A in the promoter region of fibrinogen B(FGB)beta gene,the polymorphisms of FⅡG20210A and 19911A/G,the polymorphisms of FⅤ(FⅤLeiden,FⅤCambridge, FⅤHong Kong,FⅤAsp79His and FⅤI359T),the -1476A/T and -1654C/T of PC gene promoter polymorphisms and methylenetetrahydrofolate reductase(MTHFR)gene polymorphisms (1298A/C,1793G/A and 1317T/C).Nevertheless,the distribution of these polymorphisms are different in the different area and different ethnic populations,the relation between these polymorphisms and the risk of thrombotic disease are still controversial.Finally,there are no reports about relationship of FⅡ19911A/G,MTHFR1793G/A and 1317T/C polymorphisms with the VTE or MI in China. The present study was designed to study plasma concentration levels of fibrinogen and D-D; activies of plasma FⅡ:C,FⅤ:C,FⅧ:C,FⅨ:C,protein C and antithrombin;the prevalences of APCR in 95 patients with VTE,95 patients with acute myocardial infarction(AMI)and 95 normal subjects and their clinical significance.Meanwhile,through determination of the genotype prevalences and allele frequencies of these polymorphisms in the above population,a linkage of the polymorphisms of these genes with the risk of thrombosis was identifed.Finally, the prevalence of combined gene polymorphisms in patients with VTE and AMI,and the associated risks,were determined.In this study,-148C/T and -455G/A of fibrinogen B(FGB)beta gene,the polymorphisms of FⅡ19911 A/G,FⅤAsp79His and FⅤI359T,the -1476A/T and -1654C/T PC gene promoter polymorphisms,and the MTHFR gene polymorphisms of A1298C,T1317C and G1793A were assayed by matrix-assisted laser desorption ionization / time-of-flight mass spectrometry (MALDI-TOF)technique.FⅤLeiden,FⅤCambridge,FⅤHongKong and prothrombin G20210A mutation were analyzed by specific polymerase chain reactions and restriction enzyme analysis.The activated protein C resistance(APCR)was evaluated by means of APC-APFT. The levels of AT and PC were detected by a chromogenic substrate,levels of fibrinogen were determined by full-automatical biochemic apparatus and D-Dimer were determined using immuneoturbidimetric determination,the activities of FⅡ,FⅤ,FⅧand FⅨwere measured by one-step clotting assays and SYSMEX CA-7000 fully automated coagulation analysis.The results showed that the levels of fibrinogen and D-Dimer,activities of FⅡ,FⅤ,FⅧ, FⅨ,PC and AT in patients with VTE were 4.49±2.41g/L,0.68±0.49 mg/L,100.88%±23.67 %,108.03%±28.29%,109.56%±35.06%,90.61%±18.00%,98.77%±24.63%and 102.53%±22.53%respectively.The levels of fibrinogen and D-Dimer,the activies of FⅡ,FⅤand FⅧwere remarkably higher in patient with VTE than those in healthy group(P＜0.05), and the activities of FIX,PC and AT were of no significant difference between VTE group and healthy group(P＞0.05).The mean levels of fibrinogen(4.00±0.83g/L vs 3.26±0.83 g/L,P＜0.001),D-Dimer(0.36±0.41 mg/L vs 0.18±0.12 mg/L,P=0.020),FⅤ:C(92.97%±32.25%vs 81.33%±28.15%,P=0.034)and FⅧ:C(106.05%±32.14%vs 109.56%±35.06%,P=0.000)were significantly higher in AMI group than that in controls,while,no significant difference of FⅡ:C(98.02%±22.40%vs 93.24%±26.08%,P＞0.050),FⅨ:C (89.77%±21.75%vs 84.73%±32.82%,P=0.4433),PC:A(94.70%±25.52%vs 108.78 %±37.23%,P=0.077)and AT:A(102.52%±13.39%vs 105.85%±23.68%,P=0.481) between AMI patients and the control group.The genotype frequencies of -148CC,CT and TT in the control group were 0.720,0.258 and 0.022 respectively;the genotype frequencies of-148CC,CT and TT in the VTE group were 0.598,0.315 and 0.087 respectively;the genotype frequencies of -148CC,CT and TT in AMI group were 0.710,0.247 and 0.043 respectively;the -455G/A genotype frequencies were the same as that of-148C/T in VTE,AMI and control group.The frequencies of rare alleles -148T and -455A were 0.151 and 0.151 in the control group respectively,the common allele frequencies were 0.849 for -148C and 0.849 for -455G.The relationship between -455G and -148C was completely concordant.There were no statistically significant difference of genotype between VTE and control groups(P＞0.05);whereas clear difference were observed for the allele frequencies between -148C/T and -455G/A(P=0.023).Neither genotype frequencies of -148CC,CT and TT nor allele frequencies of -148C and -148T were found to be of significant difference between AMI group and control group.The respective influences of CC,CT and TT genotypes on the fibrinogen plasma concentration were then analyzed in controls,VTE and AMI patients.In controls,-148CT+ -148TT was associated with higher fibrinogen concentrations[3.58±0.84 g/L(CT+TT)vs 3.14±0.80 g/L(CC)],One Way ANOV analysis showed that fibrinogen concentrations were of significant difference(P=0.021)between the two groups,while General Factorial ANOV analysis disclosed no significant difference(P=0.293).In VTE,no difference was found in the fibrinogen concentrations between the -148CT+-148TT group and CC group(4.94±2.66 g/L vs 4.69±2.21 g/L,P=0.644).In AMI,General Factorial ANOV analysis found the -148CT + -148TT genotype was also associated with significantly increased plasma fibrinogen levels(4.51±1.31 g/L vs 3.80±1.21 g/L,P＜0.050).There were significant difference in plasma fibrinogen levels between -148CT + -148TT genotype and CC genotype(4.40±2.00 g/L vs 3.83±1.59 g/L,General Factorial ANOV analysis,P＜0.05)in analyses of all subjects.FⅡG20210A mutation was not found in three groups.The distribution frequencies of FⅡA19911G allele were significantly different between VTE group and normal group(P=0.017); but there was no difference of genotype distribution or allele frequencies between AMI cases and control subjects(P＞0.050).There were also no difference of genotype distributions or allele frequencies between the AG+AA genotype and GG genotype in all analysed subjects(97.55%±26.62%vs 96.01%±21.60%,P=0.632)or in control subjects(94.69%±23.85%vs 92.13%±28.36%,P=0.644),VTE cases(102.60%±20.56%vs 101.12%±24.86%,P= 0.805)or AMI cases(98.82%±21.66%vs 96.69%±23.86%,P=0.663).No FⅤLeiden,FⅤCambridge,FⅤHong Kong,FⅤAsp79His and FⅤI359T mutations were found in all subjects.The incidence rate of APC resistance was 20.0%(13 of 65 cases)in patients with VTE and 5.0%(3 of 60 cases)in normal controls(P=0.012),but there were no difference of APC resistance frequencies between AMI and control(P=0.804). The genotype distributions or allele frequencies of PC- 1654C/T were similar between VTE cases and control subjects(P＞0.050),it was so between AMI cases and control subjects(P＞0.050),while there was significant difference of-1476TT genotype distributions(11.0%vs 1.1%,P=0.005)and T allele frequencies(25.3%vs 16.3%,P=0.030)in -1476A/T between VTE and control subjects,and there were no difference of that between AMI and control.Three (CC/TT,CT/AT and TT/AA)of all possible haplotypes of -1654 C/T and -1476A/T PC gene promoter polymorphisms in control subjects were observed with frequencies of 0.011,0.209 and 0.308 respectively.The distribution of CC/TT haplotypes was significantly different between VTE and control subjects(11.2%vs 1.1%,P＜0.050),but the distributions of other haplotypes were of no difference in the two groups.The distributions of AA/TT,AT/CT and TT /CC haplotypes were also of no difference between AMI and control groups.The genotypes of PC-1654C/T were not associated with circulating PC concentration in group of control subjects[101.91%±27.91%(CC),116.29%±80.92%(CT),108.78%±27.91%(TT);P=0.718],VTE cases[101.67%±24.54%(CC),95.48%±25.20%(CT), 101.09%±25.09%(TT);P=0.539]or AMI cases[105.65%±25.07%(CC),101.65%±26.05%(CT),91.35%±18.61%(TT);P=0.124];and it was the same with -1476A/T [Control:116.42%±73.74%(AA)vs 103.65%±24.17%(AT+TT),P=0.367;VTE: 100.33%±26.13%(AA)vs 95.35%±22.93%(AT+TT),P=0.354;MI:97.74%±24.29 %(AA)vs 103.69%±27.09%(AT + TT),P=0.312].There was no difference of circulating PC concentrations among the different haplotypes in control group[108.86%±29.01%(AA/TT),104.55%±23.03%(AT/CT+TT/CC),118.17%±84.66%(others);P =0.664],VTE[101.09%±25.09%(AA/TT),88.23%±20.75%(AT/CT+TT/CC), 100.99%±25.83%(others);P=0.210]and AMI[91.10%±19.61%(AA/TT),101.89%±20.44%(AT/CT and TT/CC),102.63%±27.87%(others);P=0.352].No 1317 T/C mutation was found in three groups.The prevalences of 1298 AA,1298 AC and 1298CC for MTHFR 1298 A/C were 73.3%,24.4%and 2.2%in controls respectively.The GG,GA and AA genotype distributions or allele frequencies of 1793 G/A were 85.0%,8.0%, 1.0%and 4.3%respectively.Three of all possible haplotypes(AA/GG,AC/GA and CC/AA) of 1298 A/C and 1793 G/A polymorphisms in the control subjects were observed with frequencies of 0.733,0.100 and 0.011 respectively.There were no difference of genotype distributions or allele frequencies in both 1298 A/C and 1793 G/A polymorphisms between VTE cases and control subjects.There was significant difference of AC+CC genotype(P=0.027) distribution or C allele frequency(P=0.013)in 1298 A/C polymorphisms between AMI cases and control subjects,and it was so with the GA genotype(P=0.012)in 1793 G/A polymorphisms. Two or more gene polymorphisms were detected in some of VTE patients,AMI cases and controls.Because the number of patients with VTE or AMI affected by combined polymorphisms was rather small,the attributed risks of VTE or AMI was estimated by only a X~2 (R×C)model.Only some of joint occurrence of above polymorphisms might increase the occurrence rate of VTE or AMI.Conclusions:1.There were high levels of fibrinogen,D-Dimer,FⅤ:C,FⅧ:C and FⅨ:C in VTE and AMI.This proved the importance of combined functions of several coagulant factors in causing VTE and AMI,and further confirmed that "improved functions of coagulant factors" may be more common risk factors for VTE and AMI.2.APCR was detected in both VTE and AMI,but APCR was only associated with VTE, this confirmed that APCR and FⅤLeinden are two independent risk factors leading VTE.3.There was a complete linkage disequilibrium between -148C/T and -455G/A in this study. The carriage of the T allele for the C148T mutation in theβ-fibrinogen promoter gene might be associated with an increased risk of VTE,but not for AMI.The -148C/T polymorphism of theβ-fibrinogen gene are physiologically relevant mutations with a significant impact on the plasma fibrinogen concentration,but the fibrinogen levels are strongly influenced by age,sex and environmental factors.4.The prothrombin 20210G/A polymorphism was not found in this study.Carriage of the 19911G allele might increase the risk of VTE,but not for AMI.There was no linkage between FⅡ19911A/G and circulating FⅡ:C levels among the groups of control,VTE and AMI.5.FⅤLeiden,FⅤCambridge,FⅤHong Kong,FⅤAsp79His and FⅤI359T mutations were not found in all subjects.APCR was found in three groups,APCR was a risk factor for VTE,but not for AMI.APCR may not be associated with mutations of FⅤLeiden,FⅤCambridge, FⅤHong Kong,FⅤAsp79His and FⅤI359T polymorphisms,other factors needed to be studied in APCR.This further confirmed that APCR and factor V Leiden mutations were independent risk factors of VTE.6.There was incompact linkage between PC-1654C/T and -1476A/T polymorphisms. -1476A/T and -1654C/T were not associated with plasma PC activities.PC-1654C/T polymorphism was not associated with VTE,while -1476A/T polymorphism was associated with VTE.PC -1476A/T and -1654C/T polymorphisms were not associated with AMI.7.No 1317 T/C polymorphism was found in MTHFR gene.There was compact linkage disequilibrium between 1298 A/C and 1793 G/A polymorphisms.The genetic polymorphisms of MTHFR 1298A/C and 1793G/A may be related to the development of AMI,but not VTE. 8.The presence of two or more of the above polymorphisms had positive cooperativity in the development of AMI and VTE,but the number of patients with VTE or AMI affected by combined polymorphisms was rather small.In the future study,selecting appropriate gene polymorphisms is very important.