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(Circulation. 1995;91:284-290.)
© 1995 American Heart Association, Inc.

Articles

Association of Fibrinolytic Parameters With Early Atherosclerosis

The ARIC Study

V. Salomaa, MD; V. Stinson JD; J.D. Kark, MD; A.R. Folsom, MD; C.E. Davis, PhD; K.K. Wu, MD

From the Department of Epidemiology and Health Promotion (V. Salomaa), National Public Health Institute, Helsinki, Finland; Division of Hematology (V. Stinson, K.K.W.), University of Texas (Houston); Hadassah School of Public Health (J.D.K.), Hebrew University, Jerusalem, Israel; Division of Epidemiology (A.R.F.), School of Public Health, University of Minnesota (Minneapolis); and Collaborative Studies Coordinating Center (C.E.D.), Department of Biostatistics, University of North Carolina at Chapel Hill.

Correspondence to Kenneth K. Wu, MD, Professor and Director, Division of Hematology, University of Texas Medical School at Houston, 6431 Fannin, MSB 5.016, Houston, TX 77030.

	Abstract

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Background Thrombosis, provoked by a rupture of an atherosclerotic plaque, plays a crucial role in precipitating a coronary heart disease event. Its role at the early stage of atherosclerosis has, however, been unclear, but it has been hypothesized that thrombosis or defective fibrinolysis contributes to the progression of atherosclerotic lesions.

Methods and Results We studied the association of plasminogen activator inhibitor antigen (PAI-1), tissue-type plasminogen activator antigen (TPA), and D-dimer with early atherosclerosis in a cross-sectional case-control study involving 457 pairs chosen from the biracial cohort of the Atherosclerosis Risk in Communities (ARIC) Study. As examined by B-mode ultrasound, patients (cases) had intima-media thickness of carotid arteries above the 90th percentile and control subjects had thickness below the 75th percentile of the ARIC cohort. Persons with a history of heart disease, stroke, or claudication were excluded from the case-control selection. PAI-1, TPA, and D-dimer were higher in patients than in control subjects (P.001, Wilcoxon signed rank statistic). In conditional logistic regression analyses, the odds ratios of carotid atherosclerosis were, for PAI-1, for example, 1.22, 1.54, and 1.60 in the second, third, and fourth quartiles compared with the first quartile (P<.0001, test of linear trend, adjusting for age, systolic blood pressure, total cholesterol, acetylsalicylic acid use, and time of blood draw). Corresponding tests for D-dimer and TPA also showed an increasing trend (P<.0001).

Conclusions The findings support the hypothesis that thrombosis and fibrinolysis play a role at the early stage of the atherosclerotic process.

Key Words: thrombosis • blood cells • coagulation • atherosclerosis

	Introduction

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It is now widely accepted that thrombosis, provoked by a rupture of an atherosclerotic plaque, plays a key role in triggering a coronary heart disease (CHD) event.^{1 2} For years, it has been hypothesized that the initiation, or at least the progression, of the atherosclerotic plaque is brought about by thrombosis or defective fibrinolysis.^{3 4 5} The evidence has, however, been scarce, and this hypothesis has been overshadowed by the large body of literature on lipids and lipoproteins.

Recent clinical evidence suggests that low fibrinolytic activity is a determinant of major CHD events in young and middle-aged men.⁶ In a more specific study, high plasminogen activator inhibitor activity and low plasminogen activator capacity were risk factors for reinfarction in young, male survivors of myocardial infarction.^{7 8} Another study, also carried out in persons with severe CHD, found that tissue plasminogen activator antigen, but not plasminogen activator inhibitor activity, was predictive of total mortality during the follow-up of 7 years.⁹ This evidence is prospective and important, but it is so far based only on restricted population segments and limited laboratory techniques. Furthermore, some other studies have been less convincing.^{10 11} Thus, there is an obvious need for more versatile information on the role of coagulation and fibrinolysis in the atherosclerotic process.

The present study was undertaken to examine the association of plasminogen activator inhibitor antigen (PAI-1), tissue-type plasminogen activator antigen (TPA), and a fibrin breakdown product, D-dimer, with early atherosclerosis in male and female participants of the Atherosclerosis Risk in Communities (ARIC) Study.¹²

	Methods

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This was a cross-sectional case-control study. Patients with atherosclerosis and control subjects who were free of atherosclerosis were selected from the biracial cohort of the ARIC Study. ARIC is a prospective, multicenter study on the etiology and natural history of atherosclerosis. Its design and sampling strategies have been published previously.¹² In brief, a probability sample of residents aged 45 to 64 years was drawn from four communities in the United States: Forsyth County, NC; Jackson, Miss; the northwest suburbs of Minneapolis, Minn; and Washington County, Md. The participation rate was 46% in Jackson, where exclusively blacks were sampled, and 65% to 66% in the other three counties.

Patients and control subjects were selected mainly on the basis of the ultrasound investigation of carotid and popliteal arteries. The far wall (intima plus media) thickness was measured from three segments of both carotid arteries and one popliteal artery using a high-resolution B-mode ultrasound imaging method. The technique and quality control procedures of the ultrasound investigation have been documented.^{13 14} Patients had intima-media thickness measurements of the far wall of carotid arteries exceeding the 90th percentile of the respective carotid artery segment of the ARIC cohort. In practice, this meant that they had at least two measurements of carotid artery far wall thickness >2.5 mm, or bilateral thickening corresponding to a maximum intima-media thickness of at least 1.7 mm in the internal carotid, and/or at least 1.8 mm in the carotid bifurcation, and/or at least 1.6 mm in the common carotid arteries. Control subjects had no evidence of atherosclerotic thickening, ie, a maximum far and near wall thickness below a value approximating the 75th percentile of intima-media thickness on all carotid artery segments visualized and the popliteal artery.

After selection of candidate patients and control subjects, individuals with self-reported manifestations of cardiovascular diseases, such as a history of angina on effort, physician-diagnosed heart attack, transient ischemic attack or stroke, or intermittent claudication, were excluded. Patients were pair-matched to control subjects within strata defined by study center, race, sex, and 10-year age group.

Laboratory Methods
The blood collection and processing methods of the ARIC Hemostasis Study have been documented in detail.¹⁵ In brief, the blood was drawn in the morning after the participants fasted overnight from an antecubital vein with the participant sitting. The success of venipuncture was monitored by recording the filling time of the first tube. Specimens were collected into vacuum tubes containing various types of anticoagulants according to an organizational plan previously described.¹⁶ For TPA, PAI-1, and D-dimer measurements, blood was collected in tubes containing 3.8% sodium citrate, mixed, and centrifuged at 3000g for 10 minutes. Plasma was collected and placed into aliquots. The samples were shipped on dry ice to the central laboratory and stored at -80°C until assay.

The hemostasis laboratory personnel were blinded with regard to the case-control status of the samples. D-dimer and TPA were measured by sandwich EIA described in detail in the ARIC Manual of Procedures.¹⁷ The assays were performed using kits obtained from American Bioproducts Co, Diagnostica Stago. In brief, a 96-well plate was precoated with monoclonal antibody directed against D-dimer or TPA. Standard materials or plasma samples were added, and another monoclonal antibody conjugated to peroxidase was added. Peroxidase activity was measured spectrophotometrically by using ABTS (2,2'-azino-di[3-ethyl-benzthiazoline sulfonate]) as the substrate for the enzyme. In each assay, a calibration curve was established and the concentration of an unknown sample was determined by relating the optic density of the unknown sample to the calibration curve. PAI-1 was also measured by sandwich EIA using an assay kit obtained from American Diagnostica. The detailed PAI-1 procedure is also described in the ARIC Manual of Procedures.¹⁷ For each assay, a lyophilized pooled plasma was included as quality control material.¹⁷

A special study was carried out to determine the variabilities of PAI-1, TPA, and D-dimer due to methods, including blood collection, processing, sample shipping, storage, and laboratory assay procedures, and due to within-person biological variation.¹⁸ Blood samples were collected and processed from 16 men and 23 women aged 45 to 64 years three times at 1- to 2-week intervals according to the ARIC protocol. The results demonstrated that the proportion of variance attributable to the method was small for each of these parameters (0.03 for PAI-1, 0.12 for TPA, and 0.09 for D-dimer). Also, the within-person variabilities were reasonably small (0.25 for PAI-1, 0.07 for TPA, and 0.18 for D-dimer), and, correspondingly, the reliability coefficients (proportion of variance attributable to between-person variability) were high (0.72 for PAI-1, 0.81 for TPA, and 0.73 for D-dimer).

Total cholesterol and triglyceride levels were measured by enzymatic methods,¹⁹ and high-density lipoprotein (HDL) cholesterol was measured after dextran-magnesium precipitation.²⁰ Body mass index (BMI, kg/m²) was computed from height and weight measured with the participant in light clothing and without shoes. Sitting blood pressure was measured three times from the right arm of seated participant after a rest period of 5 minutes. A random zero sphygmomanometer was used; the mean of the last two measurements was used in the analyses. Cigarette smoking and medication use were assessed by a questionnaire. Current smoking was used in the analyses.

Statistical Analysis
The total number of case-control pairs identified from the cohort was 492. Four pairs were excluded from data analyses because one member of the pair was using oral anticoagulants. Another four pairs with venipuncture problems and/or long filling time of tube 1 in one member were also excluded. In 27 pairs for TPA and D-dimer and 29 pairs for PAI-1, the sample volume was insufficient or the sample was thawed. One person with a D-dimer value of 20 778 ng/mL was, together with his pair, excluded as an extreme outlier. An additional 6 pairs had missing information for at least one of the covariates and were therefore excluded from the odds ratio calculations.

The distributions of PAI-1 antigen, TPA, and D-dimer were considerably skewed and Wilcoxon signed rank statistics were used for comparing their mean values between patients and control subjects. Differences in the mean values and proportions of conventional risk factors were tested using paired t tests or McNemar's tests. Because of matching, these data should be interpreted as adjusted for the matching factors (10-year age group, race, sex, study center, and 6-month examination period). Spearman rank order correlation coefficients were used to examine the associations of PAI-1 antigen, TPA antigen, and D-dimer with established CHD risk factors and other relevant factors. These coefficients should be interpreted with caution, since patients and control subjects were pooled for this analysis. The results were not substantially different, however, when examined separately for patients and control subjects. Conditional logistic regression models based on matched pairs were used for calculating univariate and multivariate odds ratios of being a case. The main focus was in models adjusting for age, systolic blood pressure (SBP), total cholesterol, acetylsalicylic acid use, and time of day when the blood was drawn. Further adjustments were made for smoking, BMI, triglyceride, and HDL cholesterol, but these variables share a common biological pathway with factors of hemostasis and fibrinolysis and cannot be considered as confounders. The odds ratios were analyzed by race and, in white participants, also by sex. Because the differences between black and white participants as well as between men and women were nonsignificant, the results are pooled in most of the tables. Statistical computations were performed using STATISTICAL ANALYSIS SYSTEM (SAS).²¹

	Results

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The number of case-control pairs available for final analyses was 455 for PAI-1 and 457 for TPA and D-dimer, the majority being white men. The distribution of participants by race and sex is given in Table 1. Despite matching, patients were 1.2 years older than control subjects (Table 2) and had significantly higher levels of established cardiovascular risk factors. Time of day when the blood was drawn was the same in patients as in control subjects (mean±SD, 9.7±3.0 versus 9.6±3.0 hours, P=.80). The average fasting time was 13.0±2.7 hours in patients and 13.3±2.1 hours in control subjects. Although this difference of 0.3 hours (18 minutes) was statistically significant (P=.04), it is biologically trivial. The filling time of tube 1 was similar in patients and in control subjects (21.8±4.3 versus 21.4±4.1 seconds, P=.17). Among patients, 132 persons (28.7%) were using acetylsalicylic acid, 17 (3.7%) were using oral antidiabetic medications, and 13 (2.8%) were receiving insulin injections. Among control subjects, the corresponding numbers were 136 (28.7%), 7 (1.5%), and 2 (0.4%).

View this table: [in this window] [in a new window]   Table 1. Number of Case-Control Pairs by Race, Sex, and Fibrinolysis Parameter

View this table: [in this window] [in a new window]   Table 2. Characteristics of Study Participants by Case-Control Status

PAI-1 and TPA were strongly correlated with most of the conventional cardiovascular risk factors and with each other (Table 3), whereas D-dimer was correlated only with age and SBP and weakly with smoking (cigarette years), total cholesterol, and TPA. PAI-1 was also negatively associated with the time of day when the blood was drawn, and TPA was positively associated with filling time of tube 1.

View this table: [in this window] [in a new window]   Table 3. Spearman Rank Order Correlation Coefficients of Plasminogen Activator Inhibitor Antigen, Tissue-Type Plasminogen Activator Antigen, and D-Dimer With Established Risk Factors, Filling Time of Tube 1, and Time of Blood Drawing

In univariate analyses, patients had significantly higher PAI-1, TPA, and D-dimer values than control subjects (Table 4), and the differences tended to become more pronounced at the upper ends of the distributions.

View this table: [in this window] [in a new window]   Table 4. Mean Values and Distributions of Plasminogen Activator Inhibitor Antigen, Tissue-Type Plasminogen Activator Antigen, and D-dimer by Case-Control Status

In conditional logistic regression analyses comparing the race- and sex-specific uppermost quartile of the fibrinolysis parameter with the lower three quartiles, univariate models showed significant increases in the estimated risk of carotid atherosclerosis: 49% in PAI-1, 79% in TPA, and 60% in D-dimer (Table 5). This association originated exclusively from the white examinees; no clear increase was observed in blacks. After adjustment for age, SBP, total cholesterol, acetylsalicylic acid use, and time of day, the carotid atherosclerosis odds ratio remained higher by approximately one third but was no longer statistically significant. Further adjustment for BMI and current smoking removed the excess risk when the total sample was considered, although in white women the uppermost quartile of PAI-1 and TPA remained associated with an increase in the risk of atherosclerosis of 74% and 86%, respectively.

View this table: [in this window] [in a new window]   Table 5. Univariate and Multivariate Odds Ratios of Carotid Atherosclerosis Comparing Race- and Sex-Specific Uppermost Quartile of D-Dimer, Tissue-Type Plasminogen Activator Antigen, and Plasminogen Activator Inhibitor Antigen With the Lower Three Quartiles

Dose-response relations between the concentrations of the fibrinolysis parameters and the risk of carotid atherosclerosis were investigated by computing the odds ratios for each quartile of the fibrinolysis parameter using the lowest quartile as the reference category. For PAI-1 and D-dimer, there was a clearly linear increase of odds ratios (Figure), with 1.60-fold and 1.53-fold increases in risk in the uppermost quartiles of PAI-1 and D-dimer, respectively. The association between TPA and atherosclerosis was somewhat more irregular with the highest risk in the second quartile (1.85-fold), but a statistically significant (P<.0001) linear trend was present.

View larger version (28K): [in this window] [in a new window]   Figure 1. Bar graphs of odds ratios of carotid atherosclerosis by quartiles of plasminogen activator inhibitor antigen (PAI-1), tissue-type plasminogen activator antigen (TPA), and D-dimer. Test of linear trend across the quartiles (adjusting for age, systolic blood pressure, total cholesterol, acetylsalicylic acid use, and time of day when the blood was drawn): P<.0001 for all three parameters.

	Discussion

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Originally, von Rokitansky in 1852³ and later Duguid in 1946⁴ proposed that the intimal thickening in initial atherosclerosis resulted from mural thrombosis and fibrotic organization. Some later studies have supported these hypotheses.^{5 22} The examination of the early stages of atherosclerosis in vivo has, however, become possible in larger scale only recently due to the development of modern, noninvasive ultrasound technology.²³

The ultrasound scanning¹³ and reading¹⁴ methods of the ARIC Study have shown a high degree of repeatability and reliability. It has also been reported that patients with atherosclerosis, chosen on the basis of intima-media thickness measurements, have higher levels of established cardiovascular risk factors²⁴ and fibrinogen²⁵ than the control subjects. Thus, we can be confident that the selection process used in this study has produced a set of patients with atherosclerosis and of control subjects without early changes of atherosclerosis.

Our findings are consistent with the results of the Northwick Park Heart Study⁶ as well as those of Hamsten and coworkers^{7 8} and further suggest that defective fibrinolysis may play a role in the early progression of atherosclerotic lesions, in addition to the clinical CHD events. A limitation of the present study was that we have investigated PAI-1 antigen instead of activity, but it has been reported that results from these two assays are usually highly correlated.^{26 27}

Also, TPA antigen was positively associated with carotid atherosclerosis, which may seem surprising at first glance. There are, however, data to show that in several clinical conditions with decreased fibrinolytic activity, TPA antigen levels are increased due to circulating inactive TPA-inhibitor complexes.^{9 28} Another possible explanation is that increased TPA antigen may be a marker of endothelial injury. This alternative was suggested by a case-control study of Ridker and coworkers.²⁹ They showed that TPA antigen was associated with the risk of future myocardial infarction in US male physicians. Multivariate adjustment for other risk factors abolished the significance of the association, which was interpreted to suggest that the increased TPA concentration is a consequence of preclinical atherosclerosis. More recently, the same group demonstrated that the TPA antigen concentration predicted the risk of stroke and in this case the prediction was independent of other risk factors.³⁰ The investigators concluded that the activation of the endogenous fibrinolytic system occurs many years in advance of arterial vascular occlusion. Our findings are in good agreement with that conclusion.

Relatively little data exist on D-dimer. It is a breakdown product formed when plasmin acts on cross-linked fibrin.³¹ Therefore, it can be considered as a marker of fibrin production and plasmin activity. Relatively small, hospital-based studies have demonstrated that D-dimer levels are higher in patients with acute myocardial infarction, unstable angina, or acute stroke than in healthy control subjects.^{32 33 34} Among patients with claudication, D-dimer was associated with coronary events and deterioration in peripheral arterial disease during the follow-up of 1 year.³⁵ The present study extends these findings to persons with early, asymptomatic atherosclerosis. Higher concentrations of D-dimer in patients than in control subjects suggest a higher turnover of fibrin in these individuals. It fits well with the concept of a hypercoagulable state that has been hypothesized to precede clinical CHD events.^{31 36 37}

The cross-sectional nature of the present study does not permit causal inferences. However, we speculate that differences in the levels of PAI-1, TPA, and D-dimer may be causally related to atherogenesis, since the sensitive ultrasound technique enabled us to investigate asymptomatic individuals who have not changed their lifestyle as a result of manifestations of existing atherosclerotic disease. Furthermore, the risk of carotid atherosclerosis is gradually increased in higher levels of PAI-1 and D-dimer and, somewhat more irregularly, in higher TPA levels. This suggests a dose-related response and supports a putative causal relation. Increased PAI-1 and TPA levels may reflect excessive endothelial cell stimulation by thrombin and also endothelial damage during atherogenesis. We cannot exclude the possibility that they may represent a compensatory production as a consequence of rapid fibrin turnover.

Other possible mechanistic explanations, such as lipoprotein(a) [Lp(a)] and use of medications, also need to be taken into account when considering causality. A previous report of the ARIC Study has shown higher Lp(a) concentration in patients than in control subjects.³⁸ Although the role of Lp(a) as a cardiovascular risk factor is disputable at the moment,^{39 40} there is evidence that it can act as a competitive inhibitor of plasminogen activation by TPA.⁴¹ Nevertheless, our data on D-dimer suggest that the breakdown of cross-linked fibrin is enhanced in patients compared with control subjects, but it is possible that the TPA and PAI-1 antigen concentrations are affected by complex interactions of Lp(a) with the fibrinolytic system. Also, the use of insulin and oral antidiabetic medications was somewhat more prevalent among patients than control subjects. They may influence the concentrations of fibrinolytic variables measured, although their effects are insufficiently known at the moment. The difference in the prevalence was, however, quite small to explain the systematic shift of whole distributions to the right in patients compared with control subjects.

Our findings were consistent in white men and women, whereas in black participants no increase in risk was seen with increasing concentrations of PAI-1, TPA, and D-dimer. It is conceivable that the fibrinolytic mechanism in black individuals may be different from that in whites for genetic reasons. It has been shown that the Lp(a) concentration is higher in blacks than in whites,^{38 42} which may antagonize the effects of plasminogen in blacks. On the other hand, the 95% confidence intervals of the odds ratios of black and white participants were overlapping to a considerable degree and the number of black pairs was small. Therefore, the possibility of a chance phenomenon in blacks is substantial, and we can conclude only that PAI-1, TPA, and D-dimer are associated with carotid atherosclerosis in white individuals.

PAI-1, TPA, and, to a lesser extent, D-dimer were correlated with conventional cardiovascular risk factors, and, as expected, adjustment for a large variety of risk factors attenuated the odds ratios almost to unity. It is probable that the increased tendency to thrombosis and hypofibrinolysis mediates the effects of smoking, obesity, dyslipidemia, and diabetes in the development of atherosclerosis. Accordingly, preventive measures directed to lipid lowering, avoidance of obesity, and smoking cessation may reduce CHD risk, in part by reducing the risk of thrombosis and improving fibrinolysis.

In conclusion, our findings support the hypothesis that thrombosis and fibrinolysis have a role at the early stage of the atherosclerosis development. Further research on hemostatic factors could improve our knowledge of the pathogenesis of atherosclerosis and its complications. It may also help us understand better the effects of different preventive measures and find new ways to prevent atherosclerotic diseases.

	Acknowledgments

 
This work was supported by contracts N01-55015, N01-HC-55016, N01-HC-55018, N01-HC-55019, N01-HC-55021, and N01-HC-55022 from the National Heart, Lung, and Blood Institute, National Institutes of Health. The four field centers, Coordinating Center, Ultrasound Reading Center, Central Hemostasis and Lipid Laboratories of the ARIC Study, and their institutions, principal investigators and coinvestigators, and staff who contributed to this report are as follows:

Field Centers

The Johns Hopkins University, Baltimore, Md: Moyses Szklo, MD, DrPH; Carol Christman; Sonny Harrell; Joel Hill; Joan Nelling.

University of North Carolina at Chapel Hill: Gerardo Heiss, MD, PhD; Carol Summers; Catherine Burke; Deanna Horwitz; Carmen Woody.

University of Minnesota (Minneapolis): Aaron Folsom, MD; Virginia Wyum; Margaret Skelton; Shriley Van Pilsum; Karen Birkholz.

University of Mississippi (Jackson): Richard Hutchinson, MD; Cora L. Walls; Dorothy P. Washington; Mattye L. Watson; Nancy G. Wilson.

Central Laboratories

Hemostasis-The University of Texas Medical School (Houston): Kenneth K. Wu, MD; Valarie Stinson; Pam Pfile; Hoang Pham; Beverly Curbello.

Lipid-Methodist Hospital, Houston, Tex: Wolfgang Patsch, MD; Maria L. Mecci; Val Creswell; Julita Samora; Wanda Wright.

Ultrasound Reading Center

Bowman-Gray School of Medicine, Winston-Salem, NC: Ralph Barnes, PhD; Regina de Lacy; Delilah Cook; Carolyn Bell; Teresa Crotts; Suzanne Pillsbury.

Coordinating Center

University of North Carolina at Chapel Hill: Lloyd E. Chambless, PhD; Duanping Liao, MD; Ding-Yi Zhao; Doris L. Jones; Sharon Kerick; Mark Park; Debbie Rubin-Williams.

National Heart, Lung, and Blood Institute

Project Office: Richey Sharrett, MD, PhD.

Received April 18, 1994; accepted August 19, 1994.

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