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To examine the strength of the associations of fibrinogen with subclinical atherosclerosis in healthy persons.
A population-based, prospective, observational study of black and white men and women (Coronary Artery Risk Development in Young Adults [CARDIA]). Fibrinogen levels were measured at year 7 (ages 25–37, n = 2969), and again at year 20 (ages 38–50, n = 2832). Measures of subclinical atherosclerosis (coronary artery calcification [CAC] and carotid intimal-medial thickness [CIMT]) were recorded at year 20.
Over the 13-year study interval (1992–1993 to 2005–2006), fibrinogen rose from a mean of 3.32 to 4.05 g L−1. After adjusting for age, gender and race, fibrinogen was positively associated with greater incidence of CAC and increased CIMT cross-sectionally as well as after 13 years of follow-up (all P-trend < 0.001). After further adjustment for field center, BMI, smoking, education, systolic blood pressure, diabetes, antihypertensive medication use, total and HDL cholesterol, and CRP, significant positive relationships between fibrinogen and incidence of CAC remained for the total cohort longitudinally (P-trend = 0.037), but not cross-sectionally (P-trend = 0.147).
This 13-year study demonstrates that higher levels of fibrinogen during young adulthood are positively associated with incidence of CAC and increased CIMT in middle-age, but the strength of the association declines with increasing age.
Many studies have confirmed an association between elevated fibrinogen levels and cardiovascular disease [1–7]. Studies have also shown an association of fibrinogen with the quantity of coronary artery calcification, a measure of subclinical coronary disease [8,9]. But whether fibrinogen contributes to the development or progression of atherosclerosis is less clear . Many years ago, it was shown that as atheromatous plaques form they incorporate fibrinogen and fibrin . The fibrin provides a scaffold for smooth muscle cell migration and proliferation, and fibrin degradation products are mitogenic for macrophages. A fibrin matrix could also provide a site for calcium deposition, analogous to the role of collagen . Recently, we reported that elevated levels of fibrinogen in persons aged 25–37 were independently associated with an increased incidence of subclinical coronary and carotid disease after 13 years of follow-up (ages 38–50) . To further examine the association of fibrinogen with subclinical disease, we greatly enlarged our investigation. We included participants at additional field centers to more than double the population studied, performed a second measurement of fibrinogen at a later time point, and used nephelometry to assay fibrinogen at both time points. Our goal was to determine whether the association of fibrinogen with subclinical atherosclerosis becomes stronger or weaker as participants grow older.
Participants in this study were from the Coronary Artery Risk Development in Young Adults (CARDIA) study, a multi-center longitudinal study designed to investigate the evolution of CVD risk factors and sub-clinical atherosclerosis. Details of the study design have been published previously . Briefly, the initial cohort included 5115 black and white adults aged 18–30 years at baseline (1985–1986) recruited from four field centers (Birmingham, AL; Chicago, IL; Minneapolis, MN; and Oakland, CA). Age, race, gender and education were roughly balanced by the sample design. To date, six follow-up examinations have been completed at years 2, 5, 7, 10, 15 and 20. In 1992 and 1993, as part of the year 7 CARDIA examination (termed baseline or Y7 in this article), fibrinogen was measured along with other clinical, demographic and health variables. Fibrinogen was again measured during the year 20 CARDIA examination in 2005 and 2006 (termed follow-up or Y20 in this article). Longitudinal analyses used data from both the Y7 and the Y20 examinations, and cross-sectional analyses used fibrinogen and covariate data from the Y20 examination.
Figure 1 is a flow chart showing the initial number of participants, the number analyzed at Y7 and Y20, and the exclusions. Of the 4024 participants returning for the Y7 examination, 808 did not return at Y20 and 40 of those who did return did not have CAC or CIMT studies. Fibrinogen measurements were not obtained in 168, and 39 were excluded because of pregnancy, heart disease or missing data. The final sample for longitudinal analyses included 2969 participants (517 black men, 788 black women, 816 white men and 848 white women). Of the 3048 persons returning at Y20, 104 were pregnant, persons with heart disease (heart attack, angina, stroke and TIA), or had missing covariates. Forty did not have CAC and CIMT examinations, and fibrinogen was not assayed in 72, leaving 2832 for the cross-sectional analyses. According to Y7 data, persons who had missing data or who were lost to follow-up were more likely to be black, younger, less educated or smokers, and had higher fibrinogen levels than participants in the study sample.
In the CARDIA study, all data collection technicians were centrally trained and certified. The CARDIA Coordinating Center and the CARDIA Quality Control Committee monitored data collection throughout the study. Informed consent was obtained from each participant at each examination. Participants’ age, race, gender and cigarette use were assessed by questionnaire. Anthropometric variables included height and weight, from which body mass index (BMI) was derived. Height and weight were measured using a balance beam scale and a vertical ruler, respectively, with the participant wearing light clothing and no shoes. BMI was calculated as the weight (kg) divided by the height in meters squared (m2). Right-arm blood pressure was measured three times after the participants rested in a quiet room for 5 min. The average of the second and third readings was used for analyses. Blood pressure was measured using a Hawksley random-zero sphygmomanometer until the examination at Y20, when concerns about mercury contained in the apparatus required a switch to the OmROn HEM907XL sphygmomanometer (Omron Corporation, Kyoto, Japan). A calibration study was performed and calibrated values were used for the Y20 measurements to ensure comparability. Biochemical variables included total cholesterol, high density lipoprotein (HDL) and low density lipoprotein (LDL) cholesterol, and fasting glucose. C-reactive protein (CRP) was measured using the BNII Nephelometer from Dade Behring (Deerfield, IL, USA) with a particle-enhanced immunonepholometric assay . HDL levels were quantified after dextran sulfate-magnesium precipitation , and LDL cholesterol was estimated using the Friedewald equation . Diabetes was defined as fasting glucose ≥ 126 mg dL−1 or current use of diabetic medications.
CAC was measured by computerized tomography (CT) of the chest . Electron beam CT (two centers) and multidetector CT (two centers) scanners were used to obtain 40 contiguous 2.5–3 mm-thick transverse images from the root of the aorta to the apex of the heart in two sequential scans. Participants were scanned over a hydroxyl-apatite phantom to allow monitoring of image brightness and noise and adjust for scanner differences. Data from both scans were transmitted electronically to the CARDIA CT Reading Center. A calcium score in Agatston units was calculated for each calcified lesion and the scores were summed across all lesions within a given artery and across all arteries (left anterior descending, left main, circumflex, and right coronary) to obtain the total calcium score for the participant. The mean scores of the two scans were used in the analyses.
High-resolution B-mode ultrasonography was used to capture images of the bilateral common carotid (CC) and internal carotid (IC) arteries using a Logiq 700 ultrasound machine (General Electric Medical Systems, Chicago, IL, USA). One longitudinal image of the CC and three longitudinal images of the IC were acquired. Measurements of the maximal CIMT were made at a central reading center by readers blinded to all clinical information. The maximum IMT of the CC and the bulb/IC was defined as the mean of the IMT of the near and far wall of both the left and right sides. The number of measurements that were available for averaging ranged from 1 to 4 for the CC and 1 to 16 for the IC. 
Blood was drawn between 07.00 and 10.00 from participants who were asked to fast for at least 8 h. Five milliliters were placed into tubes containing EDTA, mixed by repeated inversion, and spun at 3000 × g for 20 min at 4 °C in a refrigerated centrifuge. Within 90 min the plasma was placed in a −70 °C freezer. During 2003, plasma fibrinogen was assayed in samples stored since Y7 (1992–1993) in all examined participants using automated nephelometry as we have previously described , and this method was also used to measure fibrinogen at Y20. We have shown previously that there is no evidence of sample degradation when samples are stored at −70 °C . The intra-assay and inter-assay coefficients of variation (CVs) were 2.7% and 2.6% at Y7 and 3.1% and 4.2% at Y20. In our previous study, fibrinogen was measured by a clotting assay and the CV was higher, at 5.6% .
To examine the associations of changes in fibrinogen level over 13 years with subclinical cardiovascular disease, participants were classified into four mutually exclusive fibrinogen groups based on their fibrinogen tertile levels (low, middle, high) at Y7 and Y20: Group 1, those who were in the lowest tertile at both Y7 and Y20 (low-low); Group 2, those who were in the lowest or middle tertile at Y7 and Y20 (low-middle, middle-low, middle-middle); Group 3, those who were in the highest tertile at Y7 and in the lowest or middle tertile at Y20 (high-low, high-middle), or were in the lowest or middle tertile at Y7 and in the highest tertile at Y20 (low-high, middle-high); and Group 4, those who were in the highest tertile at both Y7 and Y20 (high-high). The purpose of this classification was to determine whether participants in Groups 3 and 4 would have a higher incidence of CAC and increased CIMT at Y20 compared with participants in Group 1 (referent).
Due to potentially large differences in associations of CAC and CIMT with fibrinogen by gender and race, all analyses were performed separately stratified by sex-race groups. First, baseline (Y7) characteristics were computed for the participants and differences between black people and white people within gender were assessed by either t-tests for continuous variables or chi-square tests for dichotomous variables or Wilcoxon rank sum test for skewed variables. Next, a series of models was fit, first with presence of CAC (mean total Agatston score > 0 at Y20) as the outcome and then with CIMT (common and internal carotid IMT at Y20) as the outcome. Linear regression models were used to examine the cross-sectional association between incidence of Y20 CAC and CIMT and Y20 fibrinogen levels (low, middle, high) based on tertiles of fibrinogen at Y20 within the entire cohort after adjustment for Y20 age, sex, race (African-American or not), BMI, smoking (yes/no), education (in years), systolic blood pressure, diabetes (yes/no), antihypertensive medication use (yes/no), total and HDL cholesterol, and CRP (log-transformed value was used to minimize the skewness of the distribution).Linear regression models were also used to evaluate the longitudinal associations between Y7 fibrinogen levels (low, middle, high) based on tertiles of fibrinogen at Y7 within the entire cohort and fibrinogen groups (as defined above) with Y20 incidence of CAC and CIMT, adjusted for Y7 age, sex, race (African-American or not),BMI, smoking (yes/no), education (in years), systolic blood pressure, diabetes (yes/no), antihypertensive medication use (yes/no), total and HDL cholesterol, and log-transformed CRP. The final models for the change in fibrinogen tertile group analyses also included Y7 fibrinogen as a covariate. The adjusted incidence (percentage) rates of CAC > 0 across fibrinogen tertiles (or fibrinogen groups) were computed using general linear models with the binary variable of CAC > 0 as the outcome, in which the covariate-adjusted least square means of the binary outcome provided the percentages of CAC > 0. To test for linear trend, we included fibrinogen as a continuous variable in multivariable models using linear regression for continuous outcomes (CIMT) and logistic regression for binary outcomes (incidence of CAC). Multivariable adjusted logistic regression was also used to test differences between the fibrinogen reference group (low-low) and other fibrinogen groups (represented by the coefficient for the dummy variable of that group).
To maximize the amount of information in each analysis, we used all available data for each outcome (cross-sectional, n = 2650 for CAC, n = 2710 for common carotid IMT, and n = 2566 for internal carotid IMT; longitudinal, n = 2682 for CAC, n = 2753 for common carotid IMT, and n = 2596 for internal carotid IMT; fibrinogen groups, n = 2652 for CAC, n = 2723 for common carotid IMT, and n = 2570 for internal carotid IMT) as opposed to restricting all analyses to the subset of participants who had non-missing data for all outcomes. All analyses were conducted using SAS statistical software (Version 9.2; SAS Institute Inc., Cary, NC, USA).
Of the 3856 participants with fibrinogen data at the Y7 examination, 887 eligible participants (i.e. 284 black men, 166 white men, 255 black women and 182 white women) were excluded from the analyses (808 were lost to follow-up and 79 because of exclusion criteria or missing data) (Fig. 1). We adjusted for selection bias (i.e. missing data due to lack of information on some covariates and thus excluded from analyses, dropouts, or losses to follow-up) using multiple imputation of missing data. The implementation of multiple imputation was based on the procedure described by Raghunathan et al.  using IVEware software (Institute for Social Research, University of Michigan, Ann Arbor, MI, USA) . The following analysis was performed: first, the variable with the least missing data (variable 1) was imputed conditional on all variables with no missing data. The variable with the second least missing data was then imputed conditional on the variables with no missing values and variable 1, and this process was continued until all of the variables had been cycled through (one ‘iteration’), and there were no longer any missing values in the data. Using this data set, the procedure was repeated multiple times to create ‘complete’ data sets. The analysis was then run separately on each data set, and the results were combined across data sets by using the multiple imputation combining rules . The resulting estimates account for both within- and between-imputation uncertainty.
Table 1 displays the characteristics of study participants according to gender-race groups at Y7. Black people were younger, had a higher BMI, fewer years of education, and more black people than white people were current smokers. Black people had higher mean systolic BP and more black women used antihypertensive medication. HDL cholesterol was lower in white men, but CRP was higher in black people than in white people. Mean fibrinogen was 3.32 g L−1, and was higher in the older members of the cohort (3.29 at age 24–28 and 3.44 at age 37–41). Over the course of 13 years, mean fibrinogen in the total group rose to 4.05 g L−1 (3.98 at age 37–41 and 4.24 at age 50–54). While fibrinogen was significantly higher in black people than in white people (P < 0.001), the magnitude of the increase (22%) was similar in all race/gender groups. Increases were also noted in CRP (median CRP, 1.05–1.12 µg mL−1) and BMI (26.5–29.4 kg m−2).
Table 2A shows associations between Y7 fibrinogen tertiles and Y20 CAC. CAC was more prevalent among men than women and white than black men. After adjustment for age, gender and race, the association of elevated fibrinogen with greater incidence of CAC was highly significant for the total sample (P-trend < 0.001) and for all race/gender subgroups, and remained significant for the total group (P-trend = 0.047), although not for the subgroups, after further adjustment for field center, BMI, smoking, education, systolic blood pressure, diabetes, antihypertensive medication use, and total and HDL cholesterol levels. Table 2B shows that associations between Y20 fibrinogen and Y20 CAC were significant for the total sample after age, gender and race adjustment (P-trend < 0.001), but not after multivariable adjustment.
We also looked for associations between fibrinogen and intima-media thickness (IMT) of the common and internal carotid arteries. While Y7 fibrinogen in the total group was significantly associated with Y20 common and internal carotid IMT after age, gender and race adjustment (P < 0.001), significance was not retained after multivariable adjustment (P > 0.1). Similarly, Y20 fibrinogen in the total group was significantly associated with Y20 common and internal carotid IMT after age, gender and race adjustment (P < 0.001), but not after multivariable adjustment (P > 0.1). In a supplemental regression analysis that singly included potential covariates one at a time into the age-, gender- and race-adjusted model, BMI and smoking substantially reduced the fibrinogen coefficient, and the P-value for the fibrinogen coefficient was no longer statistically significant at the 0.05 level.
Next, we performed analyses to examine whether individuals with fibrinogen in the highest tertile at either Y7 or Y20 (Group 3), or both (Group 4) would have higher incidence of CAC compared with participants with fibrinogen levels in the lowest tertile at both Y7 and Y20 (Group 1, referent). In age, gender and race adjusted analyses, participants in Group 4 had the most CAC, followed by those in Group 3; those whose fibrinogen was in the lowest tertile at both time points (Group 1) had the lowest incidence of CAC (Fig. 2A). The relationships between fibrinogen groups and age-adjusted incidence of CAC > 0 were consistent for each of the four race-gender groups. After further adjustment for other Y7 coronary risk factors, associations were only significant for Group 3 of the total sample and not significant for any subgroup (Fig. 2B). A similar analysis examining carotid IMT showed that common and internal carotid IMT were greater in Group 4 than in Group 1 (P < 0.05) after adjustment for age, gender and race, but not after multivariable adjustment.
We employed a multiple imputation method to adjust for the potential bias caused by dropouts and missing data. The two sets of estimates (imputed and not-imputed) were consistent, with the direction and magnitude of the relationship between fibrinogen and incidence of CAC and CIMT persisting across most of the various sensitivity analyses (data not shown), thus providing assurance that our results were likely to be robust despite some loss of participants due to dropouts or missing data.
This study, encompassing almost 3000 participants and using a different assay method for fibrinogen (nephelometry rather than clotting), confirms one observation of our initial small study  that an elevated fibrinogen level in youth is independently associated with subclinical cardiovascular disease in middle-age. However, we now find that the associations of fibrinogen with subclinical disease are attenuated over time, suggesting that fibrinogen might be important when atherosclerosis is developing, but that its predictive role declines when the disease is established. By the fourth decade, factors known to decrease fibrinogen synthesis, such as age  and smoking cessation , could modify the association. Our data are consistent with a recent report noting that although fibrinogen is independently associated with mortality risk in elderly men with peripheral arterial disease, the inclusion of fibrinogen in a set of other risk factors does not improve individual-level risk prediction . This situation is not unique for fibrinogen; studies have shown that another hemostatic factor, factor V Leiden, is associated with stroke in young but not in older individuals [28–30].
We also observed differences in the strength of the associations with CAC and with CIMT. In general, fibrinogen was more strongly associated with CAC than with CIMT. Fibrinogen has an affinity for binding to hydrophobic, atheromatous lipid surfaces, particularly those rich in cholesteryl esters , and accumulates in plaques. Calcification of these plaques may account for the association of fibrinogen concentration with the quantity of CAC as measured by electron beam computed tomography . Fibrinogen in the plasma and in the plaques serves as a substrate for thrombin, resulting in intraluminal and intramural fibrin generation. Increases in fibrinogen, especially above physiologic concentrations, affect the balance between fibrinolysis and clotting in favor of thrombosis . A study of young post-myocardial infarction patients with elevated fibrinogen levels noted that ex vivo fibrin clots were stiffer and lysed at a slower rate than those of healthy persons . These observations are likely to account for the association of fibrinogen with acute and chronic coronary disease.
Many studies have examined the cross-sectional associations of hemostatic factors with cardiovascular disease, but there have been few longitudinal analyses. Taylor et al.  studied 180 middle-aged men with measurements of CAC at 4.2-year intervals. CAC progression occurred in 60%, and those with progression had higher triglycerides, LDL and total cholesterol, but not fibrinogen. On the other hand, the Atherosclerosis Risk in Communities study found that over a 6-year period, adverse changes in cardiovascular risk factors were accompanied by increases in plasma levels of fibrinogen . It may be difficult to discern a role for fibrinogen in plaque progression when the window of observation is relatively brief and there are many other contributing factors.
The main strengths of our study include the relatively large sample size moderately well-balanced by age, race, sex and education, and the standardized data collection and rigorous quality control of the CARDIA study. A limitation of this study is that subclinical measures of atherosclerosis were not performed at Y7, so it is possible that some participants may already have had disease at that time. However, CAC was measured in this cohort at Y15, and the incidence was half that at Y20 (9.4% vs. 18.3%). This suggests that it would be quite low at Y7, and is consistent with the value in the literature of < 0.6% in otherwise healthy persons at age 25–37, the age of our participants at Y7 . As with any observational study, residual confounding could have influenced our findings.
In summary, our research shows that fibrinogen measured at age 25–37 is more closely associated with atherosclerosis detected at age 38–50 than is fibrinogen measured at the latter age. In these older adults, other cardiovascular risk factors such as obesity and current smoking weaken the associations of fibrinogen with subclinical atherosclerosis.
This study was supported by grant HL-43758 and contracts NO1-HC-48049 andNO1-HC-95095 from the National Heart, Lung, and Blood Institute, and grant AG032136 from the National Institute on Aging, National Institutes of Health. The funders had no role in: the design and conduct of the study; collection, management, analysis and interpretation of the data; and preparation, review or approval of the manuscript, except as required of all studies supported by the NHLBI. The authors had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.
Disclosure of Conflict of Interests
The authors state that they have no conflict of interest.