The primary inhibitor of fibrinolysis is PAI-1, and a 4/5-guanine-tract (4G/5G) polymorphism was identified in the promoter of the PAI-1 gene, located 675 base pairs upstream from the start of transcription (17). The PAI-1 4G allele is associated with elevated PAI-1 levels in plasma (frequency of PAI-1 4G-allele in whites 0.52) (17-19), particularly among subjects with relatively high plasma levels of triglycerides, insulin, or glucose (20,21). In vitro studies indicate that the 4G-allele is unable to bind a repressor and is associated with increased transcription of the PAI-1 gene (17,18). Additionally, a very low-density lipoprotein responsive element was found to partly overlap the guanine tract, which may explain the modification effect by triglycerides (22). The elevated plasma PAI-1 level in 4G/4G homozygotes may result in an increased risk of coronary heart disease due to a diminished fibrinolytic capacity. However, studies investigating this hypothesis have produced conflicting results (18,19,23-26).

If a deleterious effect of the ACE D-allele and the PAI-1 4G-allele is present, carriers of both risk alleles may be especially susceptible to disease due to the link between the renin-angiotensin system and fibrinolytic function. Infusion of angiotensin II increases plasma PAI-1 activity in humans (27), and two recent studies showed that the ACE D/D genotype was associated with elevated PAI-1 levels in plasma (28,29). Hence, the ACE and PAI-1 polymorphisms may be involved in cardiovascular disease in part by the same etiologic pathway.

In a cross-sectional analysis, the ACE and PAI-1 genotype distributions were compared between elderly subjects aged 85 years or older and young control subjects. To avoid false associations with the ACE and PAI-1 polymorphisms due to differences in geographic origin rather than age, only those aged 85 years or older who were born in Leiden (n=356, 55%) were compared with a control population consisting of 250 (139 men and 111 women) blood donors aged 18 to 40 years old of Dutch descent with either two Leiden-born parents or one Leiden-born parent and the other born within a 12-km distance from Leiden. Information regarding the birthplace of their grandparents was obtained from a written questionnaire.

The aim of the study was to assess whether the ACE and PAI-1 polymorphisms, either separately or in combination, are associated with mortality in the general population. This was done using two designs within a population-based study among subjects aged 85 years or older in Leiden between 1887 and 1901. Therefore, an investigation was made to determine if the young control population was likely to represent the Leiden genotype distribution of two generations before. The frequencies of the ACE D/D genotype were 27.1%, 27.0%, 27.5%, and 22.4% among young subjects with one or more (n=203), two or more (n=178), three or more (n=120), and four (n=76) Leiden-born grandparents, respectively. ForMortality Risks According to Plasminogen Activator Inhibitor-1 Genotype in Subjects 85 Years Old

All-Cause Cardiovascular Ischemic Heart Disease PAI-1 Genotype n RR* 95% CI RR 95% CI RR 95% CI

5G/5G 126 1 1 1

4G/5G 315 0.9 (0.7-1.1) 0.9 (0.6-1.2) 0.8 (0.4-1.6)

4G/4G 169 0.9 (0.7-1.1) 0.9 (0.6-1.3) 1.8 (0.9-3.7)

*Relative risk (RR) indicates the mortality risk as estimated by a Cox proportional hazard model adjusted for age at baseline.

CI confidence interval; other abbreviations as in Table 1.

The results of our study do not support an association between the ACE D/D genotype and (cardiovascular) mortality in elderly subjects, nor do they support an interaction between the ACE I/D and PAI-1 4G/5G polymorphisms with respect to mortality risks in elderly subjects. However, our results suggest that the PAI-1 4G/4G genotype may be associated with an increased risk of fatal ischemic heart disease in men, assuming a recessive effect. This finding needs to be confirmed in other studies, preferably with a larger number of events and subjects, and with information on potential confounding factors such as smoking, blood pressure and plasma lipid levels.

In conclusion, our study suggests that the ACE D/D genotype is not associated with increased mortality risks in elderly subjects, nor is there an interaction between the ACE I/D and PAI-1 4G/5G polymorphisms in this respect. The PAI-1 4G/4G genotype may be associated with an increased risk of fatal ischemic heart disease in men, but this finding needs to be confirmed in other studies.Mortality Risks According to Plasminogen Activator Inhibitor-1 Genotype in Subjects 85 Years Old: Cardiovascular Disease, Ischemic Heart Disease, Cerebrovascular Disease, and All-Cause Disease.

PAI-1 Genotype n RR* 95% CI RR 95% CI RR 95% CI RR 95% CI 

All subjects 644 n 575 n 242 n 61 n 78 
5G/5G 124 1 1 1 1 
4G/5G 340 0.8 (0.7-1.0) 0.9 (0.6-1.2) 0.9 (0.5-2.0) 0.9 (0.5-1.6) 
4G/4G 180 0.9 (0.7-1.1) 0.9 (0.6-1.3) 1.6 (0.8-3.3) 0.7 (0.4-1.4) 

PAI-1 plasminogen activator inhibitor-1; CI confidence interval; RR relative risk.

Men only: 

PAI-1 Genotype n RR* 95% CI RR 95% CI RR 95% CI RR 95% CI 

181 n 159 n 61 n 19 n 19 
5G/5G 32 1 1 1 1 
4G/5G 95 1.9 (0.8-1.8) 1.3 (0.6-2.6) 3.3 (0.4-27) 2.5 (0.6-11) 
4G/4G 54 1.2 (0.8-2.0) 1.6 (0.7-3.4) 8.2 (1.0-64) 1.9 (0.4-10) 

Women only: 

PAI-1 Genotype n RR* 95% CI RR 95% CI RR 95% CI RR 95% CI 

463 n 416 n 181 n 42 n 59 
5G/5G 92 1 1 1 1 
4G/5G 245 0.7 (0.6-0.9) 0.8 (0.5-1.1) 0.7 (0.3-1.5) 0.7 (0.4-1.3) 
4G/4G 126 0.8 (0.6-1.0) 0.7 (0.5-1.1) 0.9 (0.4-2.2) 0.6 (0.3-1.2) 

*Relative risk (RR) indicates the mortality risk as estimated by a Cox proportional hazard model adjusted for age at baseline.

ACE and PAI-1 Gene Variants and Mortality.

Studies have reported conflicting results regarding the association of ACE and PAI-1 gene variants with myocardial infarction and ischemic heart disease.