Plasminogen Activator Inhibitor-1 Promoter 4G/5G Genotype and Plasma Levels in Relation to a History of Myocardial Infarction in Patients Characterized by Coronary Angiography
To investigate the relationship between an insertion/deletion (4G/5G) polymorphism in the promoter region of the plasminogen activator inhibitor-1 (PAI-1) gene and the phenotypes of PAI-1 levels, coronary atheroma, and a past history of coronary thrombosis, we studied 453 patients (320 men and 133 women) characterized by coronary angiography. Patients were classified as having normal vessels (n=125) or single-vessel (n=92) or multivessel (n=232) coronary disease on the basis of ≥50% stenosis. PAI-1 antigen levels were highest in patients with the 4G/4G genotype (22.5 ng/mL), with a stepwise decrease in levels as the number of 4G alleles decreased (21.5 ng/mL for 4G/5G and 15.8 ng/mL for 5G/5G, P=.02) after adjusting for age, sex, triglyceride levels, and body mass index (BMI). The association between triglyceride level and PAI-1 was genotype specific, with a steeper slope in subjects with the 4G/4G genotype (P=.004). A gene-environment interaction between BMI, PAI-1, and genotype was observed, with a steeper association in patients with the 5G/5G genotype (P=.02). The 4G/4G genotype was significantly associated with a history of myocardial infarction (P<.03; odds ratio, 2.0; 95% CI, 1.1 to 3.7). This relationship was stronger in subjects with diseased vessels (P=.006). There was no relationship between either PAI-1 genotype or levels and the presence of atheroma. Our data suggest that PAI-1 promoter polymorphism influences the development of myocardial infarction through its effect on thrombus formation in patients with preexisting coronary atheroma.
- Received December 18, 1995.
- Revision received April 4, 1996.
Plasminogen activator inhibitor-1 is the major inhibitor of fibrinolysis,1 and increased levels occur in relation to both coronary atheroma and MI. The ECAT study2 reported that PAI-1 levels are related to the presence of coronary stenosis in patients undergoing coronary angiography and that PAI-1 expression has been found to be increased in the region of atheromatous plaques.3 Hamsten et al4 demonstrated that increased PAI-1 levels predict recurrence of MI in young men. While these data indicate an association between PAI-1 level and both atheroma and thrombosis, it is unknown whether these changes represent cause, effect, both cause and effect, or a shared association with some other factor.
It has been postulated that PAI-1 responses in vivo may be mediated by changes in the rate of gene transcription and that sequence elements within the promoter region control this response.5 6 A common 4G/5G single nucleotide insertion/deletion polymorphism in the PAI-1 promoter region has been identified 675-bp upstream from the start of transcription.6 This has been related to circulating PAI-1 levels in healthy subjects, young MI patients, and patients with non–insulin-dependent diabetes, with subjects homozygous for the 4G (deletion) allele having the highest PAI-1 levels.5 6 7 8 In vitro studies have identified the differential binding of transcription-regulating proteins at the site of this polymorphism, with increased gene transcription associating with the 4G allele.5 6 This provides a potential mechanism for the increase in PAI-1 levels with the number of 4G alleles seen in population studies. If genotype contributes to the determination of PAI-1 levels, it may act as a marker for lifelong exposure to differing levels, representing a means to test the hypothesis that elevated PAI-1 levels contribute to coronary atheroma and/or thrombosis.
In this study, we examined the relationship between genotype at the PAI-1 promoter polymorphism and PAI-1 levels with the presence of atheroma as assessed by coronary angiography and thrombosis by a history of MI.
Four hundred fifty-three consecutive Caucasian European patients living in Yorkshire (320 men and 133 women) who were admitted for routine angiography for investigation of chest pain or suspected CAD were recruited from two centers, located in Leeds and Wakefield. Each subject gave informed consent, and the study was approved by the United Leeds Teaching Hospitals (NHS) Trust and Pinderfields Health Trust Research Ethics Committee.
Each patient was studied between 7:00 and 10:30 AM after an overnight fast of at least 8 hours. Free-flowing blood samples were taken from an antecubital vein using a 19-gauge butterfly needle. Blood was taken into 0.9% citrate on ice for PAI-1 assay and centrifuged at 2560g and 4°C for 30 minutes before storage at −40°C. Blood was also collected into EDTA for DNA extraction by a salt detergent method as described previously.9 Samples were collected for measurement of total cholesterol and TG levels.
Smoking history was determined, and patients were classified into two groups: current smokers, which included those who had ceased smoking in the last 10 years, and nonsmokers, which included those who had stopped smoking more than 10 years previously. BP was measured with subjects lying down and to the nearest 2 mm Hg using the Dynamap automated sphygmomanometer (model 1846 SX/P, version 086, Critikon). The diagnosis of MI was ascertained from patients' hospital records using the WHO criteria of at least 2 of 3 from the electrocardiographic finding of ST-segment elevation of 1 mm in two or more successive leads, typical chest pain longer than 20 minutes' duration, and creatinine kinase increase of more than twice the baseline value. Patients who were reported to have had a history of MI but who did not meet the WHO criteria (n=8) were excluded from the study. In a majority of cases the diagnosis was confirmed by the presence of hypokinetic and akinetic segments at angiography. Patients were recruited into the study irrespective of the time of the MI. BMI was calculated as weight in kilograms divided by the height in meters squared.
Plasma PAI-1 antigen was measured by ELISA (Imulyse, Biopool). Total cholesterol and total TG levels were measured using the Hitachi 747 automatic analyzer (Boehringer Mannheim).
PAI-1 4G/5G promoter genotype was established for each subject by allele-specific polymerase chain reaction amplification of genomic DNA as described previously.10 Each patient was classified into one of three possible genotypes: 4G/5G, 4G/4G, or 5G/5G. To validate this method of genotyping, control subjects of each genotype as shown by direct sequencing were run with each batch of samples. It was not possible to genotype 19 of the patients, and they were excluded from further analysis.
Angiography was carried out and reported by cardiologists who had no knowledge of patients' genotype or other data. Patients were classified as having normal vessels or single-vessel or multivessel disease on the basis of a ≥50% stenosis in one or more of the three major coronary arteries or their branches.
Values for PAI-1 antigen and TG were log-transformed to permit the use of parametric tests. Differences in frequencies of categorical variables (eg, genotype, extent of coronary disease) between groups were assessed by χ2 test. Differences in means of continuous variables between male and female subjects were compared by Student's t test. Differences in means of continuous variables between more than two groups were assessed by one-way ANOVA. Differences in continuous variables between groups with adjustment for other factors were assessed by factorial ANOVA. Pearson correlation coefficients were calculated to assess the relationships between the various risk factors for MI and PAI-1 levels. To investigate possible interactions between PAI-1 genotype and other factors, we compared regression slopes between genotype groups, and factorial ANOVA was used to assess the significance of any such interaction as detailed in “Results.” Statistical tests were performed using the SPSS Package for Windows, version 6.1, SPSS Inc.
The clinical and biochemical characteristics of the patients are shown in Table 1⇓. The male subjects were older, had higher BMIs and diastolic BPs, and were more likely to be smokers. There was no difference in levels of systolic BP, although adjustment was not made for an influence of BP-lowering drug therapy. Despite the fact that more women were on lipid-lowering treatments (12% versus 7%, P>.05), as a group they had higher cholesterol levels. There was a significant gender difference in the frequency of MI, with more men than women (42% versus 29% P=.02) having a history of MI; angiographically the men had a higher prevalence of stenoses affecting more than one vessel, whereas the women had a higher frequency of normal coronary angiography (P<.005).
There were 125 patients with 50% or more stenosis in no vessels and 92 and 232 with single-vessel or multivessel disease, respectively. Patients with coronary stenosis in one or more vessels were older, had a higher BMI, and higher systolic and diastolic BPs (P=.03) (Table 2⇓). A history of smoking was more frequent in subjects with one or more diseased vessels (P=.04). Cholesterol levels increased with increasing vessel involvement (P=.0009), and a history of MI was related to the number of vessels diseased (P<.0001).
There was a trend toward higher levels of PAI-1 in subjects with a history of MI (22.2 versus 18.6 ng/mL) (P=.1), but this did not reach statistical significance even when adjusted for age, sex, BMI, and TG level.
PAI-1 levels were weakly related to extent of disease (P=.06), and this association was further weakened by adjusting for TG level, BMI, age, and sex.
The frequencies of the two alleles in the whole group were 0.54 for 4G and 0.46 for 5G. The genotype frequencies were in Hardy-Weinberg equilibrium.
PAI-1 levels were highest in subjects with the 4G/4G genotype (21.4 ng/mL) and lowest in the 5G/5G group (17.2 ng/mL), with 4G/5G subjects having intermediate levels (20.3 ng/mL). When values of PAI-1 were adjusted for differences in age, sex, BMI, and TG, there was a significant difference in PAI-1 levels between genotype groups (P=.02) (Table 3⇓).
The regression slope of PAI-1 levels on TG was steeper in those with the 4G/4G and 4G/5G genotypes than in those with the 5G/5G genotype; slopes were 0.80 (95% confidence interval [CI], 0.53 to 1.1) for 4G/4G, 0.78 (95% CI, 0.53 to 1.0) for 4G/5G, and 0.30 (95% CI, –0.11 to 0.70) for 5G/5G. In contrast, the slope of PAI-1 on BMI was steeper in those with 5G/5G genotype; slopes were 0.06 (95% CI, 0.03 to 0.08) for 5G/5G, 0.04 (95% CI, 0.02 to 0.06) for 4G/5G, and 0.03 (95% CI, 0.009 to 0.05) for 4G/4G. To assess the significance of these interactions, a factorial ANOVA model was designed with PAI-1 levels as the dependent variable and the following as terms—age, sex, BMI, and TG level—and genotype as two indicator variables—4G/4G versus not 4G/4G and 5G/5G versus not 5G/5G. There were also two interaction terms: TG* (5G/5G versus not 5G/5G) and BMI* (5G/5G versus not 5G/5G). Both interaction terms remained significant with P=.004 for TG* genotype and P=.02 for BMI* genotype. TG and BMI also remained as independent and significant predictors of PAI-1 antigen levels in this model. The addition to this model of terms for interaction of TG and BMI with the other genotype indicator (4G/4G versus not 4G/4G) did not alter the outcome. In view of this paradoxical pattern of interactions, the bivariate correlation of TG and BMI was examined by Pearson's method and was r=.29, P<.0005.
The median duration (25th and 75th percentile) after MI to the time of recruitment was 16 months (range, 7 to 56 months). Only 15 patients (9%) had sustained an MI within the 3 months before recruitment, and these patients were equally distributed among the three genotype groups. Genotype frequencies differed between patients with and those without a past history of MI, with a significantly greater frequency of the 4G/4G genotype in those with a history of MI (P<.05) (Table 3⇑). In a logistic regression model, possession of the 4G/4G genotype remained significantly related to MI (P<.03; odds ratio, 2.0; 95% CI, 1.1 to 3.7), allowing for differences in age, sex, BMI, cholesterol level, and smoking history. The other significant independent factors were age (P=.04), smoking history (P=.05), BMI (P=.01), and sex (P=.0006).
In a subgroup that included only subjects with atheroma (single-vessel or multivessel disease), the increased prevalence of the 4G/4G genotype in patients with a history of MI assumed greater significance (Table 4⇓).
Likelihood χ2 ratio did not reveal any differences in genotype frequency in the different angiographic groups.
Circulating levels of PAI-1 may determine vascular risk through two separate, although related, mechanisms. Fibrin deposition is an invariable feature of atherosclerotic plaques,11 and high local levels of PAI-1 may theoretically result in increased fibrin deposition and encourage plaque formation or growth. Data showing increased circulating PAI-1 levels in subjects with coronary atheroma support this possibility.2 12 Distinct from this, acute MI is usually associated with thrombosis at the site of a ruptured atherosclerotic plaque.13 14 Because it inhibits fibrinolysis, elevated levels of PAI-1 may contribute to a prothrombotic state that increases the likelihood of coronary thrombosis and occlusion. The work of Hamsten and colleagues supports a role for elevated PAI-1 levels in coronary thrombosis.4 15 The possible influence of PAI-1 levels on atherogenesis and occlusive thrombosis should be distinguished. However, because there is some evidence that PAI-1 release is increased from atherosclerotic lesions3 and as a part of the acute phase response,16 it is possible that the elevated levels of PAI-1 seen in previous studies may result from, rather than lead to, atherogenesis or thrombosis. Furthermore, an underlying association between PAI-1 levels and coronary disease may be hidden by the confounding effect of the close correlation between levels of PAI-1 and other established cardiovascular risk factors, particularly those that occur in association with insulin resistance.17 18 19 20 21 22
The emerging evidence that circulating levels of PAI-1 relate to genotype at a common polymorphism in the promoter of the PAI-1 gene5 6 7 8 has opened the possibility of using PAI-1 genotype as a surrogate measure of pre-morbid PAI-1 levels to tease apart the cause and effect limbs of the PAI-1–coronary disease relationship. Previous studies examining the association between genotype and coronary disease have not distinguished between atherogenesis and occlusive coronary thrombosis. In this study we have examined the relationships of both PAI-1 genotype and PAI-1 levels to both coronary atheroma as visualized by angiography and to previous coronary thrombosis as assessed by WHO criteria.
PAI-1 Genotype and PAI-1 Levels
In keeping with previous studies we found an association between PAI-1 promoter (4G/5G) genotype and PAI-1 levels after adjusting for BMI and TG level, with the highest circulating levels in 4G/4G subjects, intermediate levels in 4G/5G heterozygotes, and lowest levels in 5G/5G subjects, suggesting a codominant gene effect.5 6 7 8 We have also repeated the finding of others that the relationship between fasting PAI-1 and TG levels is influenced by PAI-1 promoter genotype, with a steeper regression slope in the 4G/4G group.7 23 We also found a similar influence of genotype on the regression slope of PAI-1 on BMI, although in this case the slope was steeper in the 5G/5G group. The interactions of both TG level and BMI with genotype remained both independent and significant in a regression model. Although the interaction of TG level with genotype fits with the finding of higher levels of PAI-1 in the 4G/4G group, it is unclear exactly what the statistical interaction between BMI and the 5G/5G genotype tells us about the regulation of PAI-1. In a similar study in Pima Indians, an interaction between PAI-1 genotype and BMI was observed but with a steeper regression slope in the 4G/4G group.24 Clearly, ethnic differences or chance may account for these findings, and further studies will be required to confirm significant interaction between BMI and genotype in relationship to PAI-1 levels.
Associations With Coronary Atheroma
This study found the expected relationship between the presence of coronary stenosis with age, BMI, presence of hypertension, cigarette smoking history, and cholesterol levels. However, the relationship between PAI-1 levels and the presence of stenosis in one or more coronary arteries was weak, falling just above standard levels of significance, and there was no trend in PAI-1 levels in relation to the extent of coronary disease. In this latter respect our results are in agreement with those of the larger ECAT study.2 To the best of our knowledge this is the first study to investigate the relationship between the extent of coronary atheroma and PAI-1 promoter genotype, and we found no evidence of an association with the frequency of the potentially deleterious 4G/4G genotype, being similar in each angiographic class. This suggests that genotype and thus pre-morbid PAI-1 levels do not influence the generation of coronary atheroma. If this is the case, then the relationship between PAI-1 levels and the presence of coronary atheroma that others have shown may have resulted from the production of PAI-1 in response to or triggered by the presence of atheroma. An alternative explanation for their results is that within the patient group without significant coronary stenosis a number of patients may have had microvascular coronary disease25 and that PAI-1 genotype may influence the development of microvascular disease in the same way as macrovascular disease. However, there are no previous data to support or refute this and the majority of the patients with “clean” coronary arteries are likely to have suffered from either coronary vasospasm or noncardiac chest pain.
Associations With Coronary Thrombosis
As would be expected, a history of MI was more frequent with male sex, a history of cigarette smoking, and an increased number of stenosed coronary arteries, as well as being related to cholesterol levels, BMI, and age. Although there was only a trend to higher levels of PAI-1 in the MI group, we found a significant association between PAI-1 genotype and history of MI that remained after adjusting for the other risk factors. A single measurement of PAI-1 levels is unlikely to reflect lifelong levels because it is an acute phase reactant. Genotype, which does not change, may act as an effective marker for lifelong exposure to differing levels. This could explain why we have been able to demonstrate a relationship between genotype and a history of MI, whereas we were able to show only a trend between PAI-1 levels and a history of MI.
Our findings concur with the results of two small and restricted studies that found the 4G/4G genotype to be more frequent in young male survivors of MI6 and in non–insulin-dependent diabetes mellitus patients with a history of ischemic heart disease.10 On the other hand, our results differ from those of the large four-center ECTIM study in which no association between PAI-1 genotype and MI was found.8 The differences between our study and ECTIM are important because although they may reflect the play of chance they may also suggest the way in which PAI-1 genotype affects the processes causing coronary disease.
PAI-1 Genotype, Coronary Atheroma, and Coronary Thrombosis
In the ECTIM study, subjects who had survived MI were compared with control subjects recruited from the general population in the three French centers and Belfast.8 Although these subjects were apparently healthy, the frequency of significant, although asymptomatic, coronary disease in this control group is not known. The ECTIM study was not designed to be able to distinguish the influence of genotype on coronary thrombosis from that on atherogenesis. In contrast to this, our study examined both coronary atheroma and thrombosis, finding no relationship between PAI-1 genotype and extent of atheroma but finding an association with previous coronary thrombosis. A hypothesis whereby preexisting coronary atheroma may be necessary for the effect of PAI-1 genotype on coronary thrombosis to become evident, presumably through altered PAI-1 levels, is supported by the finding that the relationship between genotype and MI strengthened when only those patients with significant coronary artery stenosis were examined. Furthermore, this influence of PAI-1 genotype on PAI-1 levels leading to a prothrombotic state could take place at the local level. PAI-1 production is increased at and around atherosclerotic plaques,3 so the rate of both basal and stimulated PAI-1 gene transcriptions may be influenced by PAI-1 promoter genotype through molecular mechanisms being elucidated. This would account for the weak association that we found between history of MI and levels of PAI-1 in the systemic circulation.
In conclusion, our findings suggest that genotype at the PAI-1 promoter polymorphism is an independent risk factor for MI in patients being investigated for chest pain or suspected coronary disease and particularly in subjects known to have significant coronary artery stenosis. Our results indicate that this effect is mediated by involvement of the PAI-1 gene in the pathogenesis of coronary thrombosis, with the greatest risk found in those subjects possessing the 4G/4G genotype. Our findings also support the hypothesis that elevated levels of PAI-1 may predate the development of disease and indicate that PAI-1 genotype may serve as a useful marker of future risk. Prospective longitudinal studies are warranted to confirm these findings.
Selected Abbreviations and Acronyms
|CAD||=||coronary artery disease|
|CASS||=||Coronary Artery Surgery Study|
|ECAT||=||European Concerted Action on Thrombosis and Disabilities|
|ECTIM||=||Etude Cas Termoins de l'Infarctus du Myocarde|
|PAI-1||=||plasminogen activator inhibitor-1|
|WHO||=||World Health Organization|
This study was funded by the British Heart Foundation.
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McCormack LJ, Nagi DK, Stickland MH, Mansfield MW, Mohamed-Ali V, Yudkin JS, Knowler WC, Grant PJ. Promoter (4G/5G) plasminogen activator inhibitor-1 genotype in Pima Indians: relationship to plasminogen activator inhibitor-1 levels and features of the insulin resistance syndromes. Diabetologia. In press.