Tissue Plasminogen Activator, Fibrin D-Dimer, and Insulin Resistance in the Relatives of Patients With Premature Coronary Artery Disease
Elevated levels of tissue-type plasminogen activator antigen (tPA), fibrinogen, and fibrin D-dimer predict coronary artery disease (CAD) events and stroke. These factors, possibly in association with insulin resistance, may be important in families in which CAD has become clinically apparent at a premature age. From 125 patients with angiographically confirmed, premature CAD, 175 healthy male relatives (age ≤65 years) were identified. One hundred seventy-five healthy volunteers of similar age and without any family history of CAD were recruited. There were no differences between relatives and controls in terms of conventional CAD risk factors, cigarette smoking, alcohol consumption, or cardiorespiratory fitness. Estimated insulin resistance and plasminogen activator inhibitor 1 levels were not increased in relatives. Fibrin D-dimer, tPA, and fibrinogen levels were elevated in relatives compared with controls, 55 (52 to 58) ng/mL versus 49 (45 to 53) ng/mL, P<0.01, for D-dimer; 8.0 (7.5 to 8.6) ng/mL versus 5.6 (5.2 to 6.1) ng/mL, P<0.001, for tPA; and 3.0 (2.9 to 3.1) g/L versus 2.8 (2.7 to 2.9) g/L, P<0.05, for fibrinogen. These differences remained after adjustment for correlates, including fibrinogen, age for D-dimer, and features of the insulin resistance syndrome for tPA. tPA and D-dimer levels are elevated in the healthy, male, first-degree relatives of patients with premature CAD. This association is independent of potential confounding factors.
Elevated levels of tissue-type plasminogen activator (tPA) predict the occurrence of coronary artery disease (CAD) events and stroke in healthy subjects1–6⇓⇓⇓⇓⇓ as well as recurrent coronary events and cardiovascular death in patients with established CAD.7–9⇓⇓ In addition, increased fibrin D-dimer (a marker of fibrinolytic activity) is associated with myocardial infarction (MI) in healthy, middle-aged men2,10–12⇓⇓⇓ and stroke in men and women.5 Impaired fibrinolysis, as determined by increased levels of plasminogen activator inhibitor 1 (PAI-1), elevated tPA and insulin resistance are known to cluster with other cardiovascular risk factors;8,13,14⇓⇓ however, the status of the fibrinolytic system and insulin resistance has yet to be determined in subjects with a strong family history of premature CAD. Elevated fibrinogen concentrations have been reported in the healthy members of families in which coronary disease has occurred prematurely.15,16⇓ Given the association between fibrinogen and insulin resistance,17 increased levels of fibrinogen may act as a potential confounder in studies of the insulin resistance syndrome in such families.
First-degree relatives of patients with premature CAD are themselves at increased risk of premature coronary disease.18,19⇓ The principle aim of this study was to investigate tPA and PAI-1 antigen levels, fibrin D-dimer, fibrinogen, insulin resistance, and physical fitness (which is known to affect insulin resistance and fibrinolysis20,21⇓) in the healthy, male relatives of CAD patients.
One hundred twenty-five male patients (probands) ≤65 years old at the time of diagnostic coronary angiography and with confirmed 2- or 3-vessel CAD (World Health Organization criteria of ≥50% stenosis in a major epicardial vessel) were identified via the surgical revascularization waiting list at the Yorkshire Heart Center, Leeds. From the 125 male patients, 185 of their male, first-degree relatives ≤65 years old at the time of recruitment and free from a personal history of CAD were contacted and interviewed. An equal number of male, community control subjects ≤ 65 years old and without a personal or family history of CAD or diabetes mellitus were recruited via the Leeds Health Authority Family Health Service register. Subjects with a personal history of hypertension and/or taking cardiovascular medication (including aspirin and lipid-lowering drugs) were subsequently excluded from the study. All remaining subjects (175 in each group) were white, North European, and they gave informed consent according to a protocol approved by the United Leeds Teaching Hospitals (NHS) Trust Research Ethics Committee.
At the time of recruitment, all subjects had fasted for a minimum of 10 hours overnight. Venous blood, 50 mL, was taken from an antecubital vein with a 19-gauge needle without venous stasis with the subject in a supine position. Blood was collected in lithium heparin for lipid fraction analysis, a 10-mL tube containing 1 mL 0.9% citrate (pH 8.8) at 4°C for assay of insulin, PAI-1, tPA, and fibrin D-Dimer and a similar citrate tube at room temperature for assay of fibrinogen. The citrate samples were centrifuged at 2560g at 4°C or room temperature for 30 minutes, and 0.5-mL aliquots of plasma supernatant snap-frozen in liquid nitrogen for storage at −40°C until assay. All subjects underwent 75-g oral glucose tolerance testing with blood collected in lithium fluoride for fasting and 2-hour glucose estimation. DNA was extracted from blood collected in EDTA for subsequent genotyping at the PAI-1 4G/5G promoter polymorphism by using a previously described method.22
Systolic and diastolic blood pressure (BP) measurements were performed manually to the nearest 2 mm Hg with subjects supine, and the measurements were calculated as a mean value of 3 consecutive readings. Relatives and control subjects with currently treated hypertension were excluded from subsequent BP analysis. Standard 12-lead electrocardiographs were recorded for all subjects, along with anthropometric data: body mass index (BMI), calculated from weight in kilograms divided by the square of the height in meters, and waist-to-hip ratio (WHR). All subjects enrolled had given negative responses to the Rose angina questionnaire and provided details concerning medical history, drug history, family history, smoking, and alcohol consumption.
A subset of first-degree relatives (n=74) and control subjects (n=90), who expressed a willingness to take part and who, in common with the remainder of the study subjects, were free from significant lung disease or other exercise limiting conditions, underwent assessment of their cardiorespiratory fitness. Subjects performed a Bruce protocol cardiopulmonary exercise test using the Medgraphics Cardio2 System for analysis of respiratory gas concentrations and flow rates. Cardiorespiratory fitness was estimated from maximal oxygen uptake (VO2max) with anaerobic threshold as a secondary measure.
Insulin, PAI-1 antigen, tPA antigen, and fibrin D-dimer were measured with commercially available ELISA kits from BioSource (insulin) and Biopool International (PAI-1, tPA, and D-dimer). Fibrinogen was measured by using the Clauss method.23 Inter-assay and intra-assay coefficients of variation were 3.8% and 2.5% for insulin, 9.8% and 4.4% for PAI-1, 10.4% and 7.0% for tPA, 5.3% and 3.5% for D-dimer, and 3.5% and 2.0% for fibrinogen. A glucose oxidase method was used for measurement of plasma glucose and a Hitachi 747 autoanalyzer (Boehringer Mannheim) for estimation of triglyceride and total cholesterol. HDL cholesterol was measured by a Hitachi 717 autoanalyzer after removal of LDL, chylomicrons, and VLDL by precipitation with phosphotungstic acid and magnesium chloride. LDL cholesterol was calculated by the Friedewald equation.
Values for age for the 3 study groups did not conform to a normal distribution and are presented as medians with 25th and 75th percentiles. Differences in age between groups were assessed by the Kruskal-Wallis test. To achieve a normal distribution for insulin, PAI-1, tPA, D-dimer, plasma fasting glucose, and triglyceride, their values were log-transformed and subsequent data presented as geometric mean with antilog 95% confidence intervals. Values for insulin resistance were estimated by use of the homeostasis model assessment, whereby insulin resistance=fasting glucose (mmol/L)×fasting insulin (mU/L)/22.5, and which assumes that normal-weight, healthy subjects <35 years old have 100% β-cell function and an insulin resistance of unity.24 Spearman bivariate correlations were used to assess the relationship among PAI-1, tPA, and D-dimer, and features of the insulin resistance syndrome and other continuous variables. Independent Student’s t test was used to assess differences in continuous and parametric data and a χ2 test for differences in category frequencies. One-way ANOVA was used to identify any relationship between 4G/5G genotype and PAI-1 levels. Multiple linear regression analysis was performed for tPA and D-dimer, and the data presented represents the best-fit model as determined by the R2 value. Statistical significance was taken as P<0.05, and all analyses were performed with the Statistics Package for Social Scientists software for Windows version 9.0.
Clinical, biochemical, and hemostatic assay data are shown in Table 1. D-dimer, tPA, and fibrinogen levels were significantly higher in relatives compared with controls, 55 (52 to 58) ng/mL versus 49 (45 to 53) ng/mL, P<0.01, for D-dimer, 8.0 (7.5 to 8.6) ng/mL versus 5.6 (5.2 to 6.1) ng/mL, P<0.001, for tPA, and 3.0 (2.9 to 3.1) g/L versus 2.8 (2.7 to 2.9) g/L, P<0.05, for fibrinogen. There was no difference in PAI-1 levels between the two groups. Apart from HDL cholesterol, 1.4 (1.3 to 1.4) mmol/L in relatives compared with 1.3 (1.2 to 1.3) mmol/L in controls, P<0.05, the relatives and controls were similar in terms of clinical data (including alcohol consumption and cigarette smoking) and biochemical parameter. There was a trend toward increased insulin resistance with higher BMI and systolic BP in relatives compared with controls although not statistically significant. There was no difference in the cardiorespiratory fitness of relatives and control subjects as measured by VO2max, 34.7 (33.1 to 36.2) mL · min−1 · kg−1 versus 34.1 (32.2 to 36.0) mL · min−1 · kg−1 or anaerobic threshold, 20.8 (19.8 to 21.9) mL · min−1 · kg−1 versus 21.1 (20.0 to 22.2) mL · min−1 · kg−1.
Bivariate correlation analysis confirmed the association between PAI-1 and tPA and the insulin resistance syndrome. For tPA and age, estimated insulin resistance, BMI, WHR, systolic and diastolic BP, total cholesterol, triglyceride, HDL cholesterol, PAI-1, and fibrinogen, |r|≥0.20 in relatives and controls. For PAI-1 and the same group of correlates, |r|≥0.19 in relatives and controls. Correlation data for fibrin D-dimer is shown in Table 2. There was a strong correlation with age, r=0.32 and 0.40 (P<0.01), and fibrinogen, r=0.24 and 0.29 (P<0.01) in relatives and control subjects, respectively. The subgroup of relatives and controls who underwent the cardiopulmonary exercise test were similar in terms of age (median, 42 years) and all conventional CAD risk factors. Relatives had increased levels of tPA, D-dimer, and fibrinogen compared with controls. Significant correlation data for VO2max is shown in Table 3.
PAI-1 4G/5G genotype was determined for all relatives and control subjects. The 4G/4G genotype was associated with elevated levels of PAI-1 in control subjects, 15.9 (12.5 to 20.2) ng/mL for 4G/4G versus 8.9 (6.6 to 12.0) ng/mL for 5G/5G control subjects, P=0.01. In the relatives group, there was a similar trend, 14.0 (10.9 to 18.1) ng/mL for 4G/4G versus 9.3 (6.4 to 13.7) ng/mL for 5G/5G. Genotype frequencies did not differ between the relatives and control groups.
Multiple linear regression models were used to calculate adjusted tPA and D-dimer levels using the significant covariates identified from bivariate correlation analysis. Only those covariates which either independentlypredicted tPA or D-dimer levels or contributed >1% variance were included in the final models (Tables 4 and 5⇓). Exclusion of the remaining correlates did not affect the R2 values or the adjusted levels of tPA or D-dimer. The final models accounted for 53.7% and 21.6% of the variance in tPA and D-dimer levels, respectively. Relative/control status and PAI-1 levels were the most influential contributors to tPA variance, and relative/control status, age, and fibrinogen were the only significant factors in the D-dimer model. From the calculated effect size, relatives had a 1.4-fold increase in tPA and a 1.1-fold increase in D-dimer levels. An increase in age by 20 years was associated with a 1.3-fold rise in both tPA and D-dimer levels. Adjusted mean tPA levels were 8.1 (7.7 to 8.6) ng/mL for relatives versus 5.8 (5.5 to 6.1) ng/mL for controls, P<0.001, and adjusted D-dimer levels were 55 (52 to 59) ng/mL for relatives and 49 (46 to 52) ng/mL for controls, P=0.006.
Data concerning the probands (patients with premature, multi-vessel CAD) are included for completeness (Table 1). As expected, probands were older than the relatives and controls with higher BMI and WHR and elevated levels of tPA, PAI-1, D-dimer, fibrinogen, and estimated insulin resistance. BP and lipid profiles were affected by concurrent cardiovascular medication, which the majority of these patients were receiving at the time of recruitment.
In this study, we have found elevated levels of tPA antigen, fibrin D-dimer, and fibrinogen in the healthy, male, first-degree relatives of patients with severe, premature CAD. tPA antigen and D-dimer remained higher in relatives compared with controls after adjustment for all significant correlates, including fibrinogen. Confounding factors cannot be ignored, but the principle strength of this study lies in the quality of subject recruitment, thereby avoiding many of the confounders that undermine studies of similar design. We have successfully enrolled more than 90% of the suitable relatives identified from our CAD patient group and simultaneously recruited a well characterized and highly comparable group of community control subjects. The lack of any significant differences between the groups in terms of history of smoking, alcohol consumption, clinical and biochemical parameters, and physical fitness would all mitigate against any significant confounding factors.
tPA: CAD Risk Factor
In prospective studies involving healthy, middle-aged subjects, increased levels of tPA antigen independently predict the development of coronary1,6⇓ and cerebrovascular5 events. In some studies, however, the identified association between tPA levels and CAD has been weakened by adjustment for other confounding risk factors,2–4⇓⇓ principally, features of the insulin resistance syndrome (BMI, BP, and HDL cholesterol). These findings may simply reflect differences in age, sex, and geographical distribution of subjects in these studies or, given the close association between tPA and the insulin resistance syndrome,8,25⇓ may represent the effect of multivariate modeling if BMI, BP, and HDL cholesterol levels differ significantly between cases and controls.
There are several plausible mechanisms that may explain the observed relationship between tPA and atherothrombotic vascular disease. In the context of atherosclerotic CAD, increased circulating levels of tPA may reflect increased endothelial tPA content and expression26 and enhanced plasmin-mediated breakdown of the extracellular matrix, resulting in plaque instability.27 In addition, tPA levels may reflect the acute phase response given the association between CAD and markers of chronic infection or inflammation.8,28⇓ Alternatively, increased tPA antigen may represent increased tPA/PAI-1 complex (because the majority of tPA circulates in this inactive, bound form) and therefore a net reduction in fibrinolytic capacity.29 Previous studies have identified strong correlations of tPA/PAI-1 complexes with both tPA and PAI-1 antigens.9,25⇓ In the present study, PAI-1 antigen levels were not elevated in relatives whereas tPA antigen remained higher in relatives compared with controls after multivariate analysis that included PAI-1. Data from the Northwick Park Heart Study suggests that impaired fibrinolysis (as measured by dilute blood clot lysis time) predicts CAD events in healthy subjects;30 however, this finding may represent increased levels of PAI-1 in subjects older than those found in the present study. Therefore, elevated levels of tPA (which are independent of PAI-1) may reflect enhanced rather than impaired fibrinolysis in the healthy relatives of CAD patients.
Fibrin D-dimer is a product of the action of plasmin on cross-linked fibrin and therefore reflects fibrinolytic activity and fibrin turnover.31 Fibrin D-dimer levels are elevated in patients with established atherothrombotic vascular disease32,33⇓ and predict arterial thrombotic events in prospective studies involving healthy, middle-aged subjects.2,5,10–12⇓⇓⇓⇓ It is possible that D-dimer levels merely reflect the underlying fibrinogen concentration; however, in the Caerphilly Study, adjustment for other CAD risk factors including fibrinogen did not affect the independent relationship between elevated D-dimer levels and the relative risk of CAD events.2 In this study, there was a significant correlation between fibrinogen and D-dimer levels, but the increased levels of D-dimer observed in relatives compared with controls remained independent of adjustment for other covariates, including fibrinogen. Tobacco smoking and leisure time activity are known to affect D-dimer levels and are potential confounding factors;34 however, history of smoking and cardiorespiratory fitness did not differ between relatives and controls in the present study. Increased fibrin D-dimer, in conjunction with elevated levels of tPA, supports the hypothesis that fibrinolytic activity is increased in the healthy relatives of patients with premature CAD.
Insulin Resistance Syndrome
The insulin resistance syndrome, which includes elevated levels of tPA antigen and PAI-1, is associated with the development and progression of atherothrombotic vascular disease.8,13,14,25,35–37⇓⇓⇓⇓⇓⇓ The healthy, first-degree relatives of patients with type II diabetes mellitus exhibit many of the features of the insulin resistance syndrome,38 and in the present study there was a nonsignificant trend toward increased BMI, systolic BP, and estimated insulin resistance in relatives compared with controls. Adjustment for these factors in multivariate analyses did not affect the observed differences in tPA and D-dimer levels between relatives and control subjects. Increased PAI-1 activity has also been found in the relatives of diabetic probands;38 however, in agreement with other studies which have examined PAI-1 in the offspring of patients with premature MI,15,39⇓ we did not find any difference in PAI-1 levels between relatives and controls. One small study has reported increased PAI-1 activity but not tPA antigen in children with a family history of premature MI. However, this finding was based on univariate analysis alone and no correlation was found between PAI-1 and tPA.40 In the present study, we have identified the well reported relationship between the 4G allele of the PAI-1 gene 4G/5G polymorphism and increased PAI-1 levels but found no difference in genotype frequency between relatives and controls. This is in contrast to a study by Margaglione et al39 which found the 4G/4G genotype to be independently associated with family history of MI. It is possible that the relative contribution of environmental and genetic factors to the development of coronary disease is different between an Italian and a UK population, and this, coupled with the much larger number of subjects in the Italian study, would make the identification of a link between a polymorphism and disease (or family history of disease) more likely. However, there are numerous studies reporting both positive and negative associations between the PAI-1 4G/5G polymorphism and atherothrombotic disease,41 and given this lack of consistency, it seems unlikely that genotyping for this polymorphism will improve the risk stratification of relatives of affected patients.
Increased cardiorespiratory fitness (as estimated by VO2max during treadmill exercise testing) has been shown to correlate negatively with features of the insulin resistance syndrome, including insulin-mediated glucose uptake and tPA and PAI-1 levels.20,21,42⇓⇓ In the present study, we have also identified a significant association between insulin resistance, associated hemostatic factors, and VO2max, but we find no difference in VO2max or anaerobic threshold between relatives and controls. Therefore, given the similarity between the groups in terms of cardiorespiratory fitness and other lifestyle factors, it seems unlikely that our findings concerning estimated insulin resistance and tPA and PAI-1 levels were significantly influenced by such potential confounders.
We have found increased levels of tPA antigen and D-dimer in a group of healthy, male subjects who are at increased risk of developing clinically apparent, premature CAD by virtue of their deleterious family history. These findings concur with data from prospective studies which have identified tPA antigen and fibrin D-dimer as independent predictors of CAD events. The measurement of circulating levels of tPA and fibrin D-dimer may improve the risk stratification of individuals who appear to be at low risk in terms of routinely measured cardiovascular risk factors.
This study was funded via a British Heart Foundation Junior Research Fellowship, FS/98063. We would like to thank May Boothby for her technical assistance regarding all the assays performed.
Received October 2, 2001; revision accepted November 19, 2001.
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