Fibrinogen predicts ischaemic stroke and advanced atherosclerosis but not echolucent, rupture-prone carotid plaques

The Copenhagen City Heart Study

S.C. Kofoeda, H.H. Wittrupb, H. Sillesena and B.G. Nordestgaardb,c,*

a Department of Vascular Surgery, Gentofte University Hospital, Gentofte, Denmark
b Department of Clinical Biochemistry, Herlev University Hospital, Herlev, Denmark
c The Copenhagen City Heart Study, Bispebjerg University Hospital, University of Copenhagen, Copenhagen, Denmark

* Corresponding author. Tel.: +45-4488-3297; fax: +45-4488-3311
E-mail address: brno{at}herlevhosp.kbhamt.dk

revised 2 July 2002; accepted 3 July 2002


    Abstract
 Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Aims Whether the association between fibrinogen and cardiovascular events reflects an association with advanced atherosclerosis in general, or rupture-prone plaques in particular, is unclear. We examined whether fibrinogen predicts incidence of ischaemic stroke, advanced atherosclerosis (measured as carotid artery stenosis) and/or echolucent, rupture-prone plaques.

Methods and results Study 1—8755 Copenhagen City Heart Study stroke-free participants; we observed 235 ischaemic strokes during 6 years of follow-up. Study 2—318 carotid stenosis patients and 1584 age- and gender-matched controls. Study 3—159 patients with echolucent vs 159 patients with echo-rich carotid artery plaques. Fibrinogen above vs below the median value of 3gl–1predicted risk of ischaemic stroke (relative risk: 1.9; 95% CI: 1.4–2.5; 235 events). Significant risk was found in men (2.7; 1.7–4.2; 113 events) and with a similar trend in women (1.4; 0.9–2.0; 122 events), in young (5.2; 1.1–26; eight events) and middle aged (2.9; 1.6–5.4; 64 events) with a similar trend in the elderly (1.4; 1.0–2.0; 163 events). Fibrinogen levels in those with and without ischaemic stroke were 3.6 and 3.1gl–1(ANCOVA: ). Likewise, in those with and without carotid artery stenosis fibrinogen levels were 4.7 and 3.1gl–1; equivalent values for high-sensitive C-reactive protein were 3.6 and 1.4mgl–1. Finally, neither fibrinogen nor high-sensitive C-reactive protein levels differed between those with echolucent and echo-rich carotid artery plaques ( and ); the power to exclude a 15% increase in fibrinogen or a 50% increase in high-sensitive C-reactive protein was 98 and 54%, respectively.

Conclusions Elevated fibrinogen predicts future ischaemic strokes, particularly in men and in the young and middle aged. This is most likely a reflection of advanced atherosclerosis, rather than an association with rupture-prone plaques.

Key Words: Atherosclerosis • Carotid disease • Follow-up study • Stroke • Ultrasonics


    1. Introduction
 Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Prospective studies of healthy individuals have demonstrated that acute phase reactants like fibrinogen and high-sensitive C-reactive protein are independent markers for future cardiovascular events.1–8 The underlying disease behind most cardiovascular events is atherosclerosis, which is an inflammatory process.9 Inflammation leads to elevated levels of acute phase reactants. It is, however, unclear, whether the association between elevated acute phase reactants and cardiovascular events is a general reflection of advanced atherosclerosis, or more particularlyreflects the presence of rupture-prone plaques.

High-resolution ultrasound can easily diagnose moderate to severe carotid stenosis, a strong marker of generalised advanced atherosclerosis.10,11 Furthermore, plaque echogenicity assessedby ultrasound relates directly to histological findings.12,13 By using computerised ultrasound B-mode images carotid artery plaques can be divided into echolucent, lipid-rich, rupture-prone plaques and echo-rich, fibrotic, stable plaques: echolucent carotid artery plaques are the ones most prone to rupture, resulting in an increased risk of ischaemic stroke.14–16 Therefore, with the use of ultrasound, patients with carotid stenosis can be used to test whether elevated acute phase reactants aremarkers of advanced atherosclerosis, and/or echolucent, rupture-prone carotid plaques.

The purpose of this study was threefold. First, we wanted to investigate if a single, baseline fibrinogen measurement in a large cohort of healthy individuals representing the general population, the Copenhagen City Heart Study, could predict future ischaemic strokes. Second, we tested if fibrinogen or high-sensitive C-reactive protein are elevated in patients with advanced atherosclerosis (measured as carotid artery stenosis), compared with age- and gender-matched controls. Finally, we tested if fibrinogen and/or C-reactive protein levels could discriminate echolucent, rupture-prone carotid artery plaques from echo-rich, stable plaques.


    2. Methods
 Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
2.1. Overview
To test the three aforementioned hypotheses, three studies were designed (Fig. 1).



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Fig. 1 Study design. Study 1: Fibrinogen as a risk factor for ischaemic stroke was investigated prospectively in the Copenhagen City Heart Study cohort. Study 2: Fibrinogen and high-sensitive C-reactive protein levels in patients with carotid artery stenosis ≥50% were compared with age- and gender-matched controls from the Copenhagen City Heart Study. Study 3: Fibrinogen and high-sensitive C-reactive protein levels in patients with echolucent, rupture-prone plaques were compared with that in patients with echo-rich, stable plaques.

 
Study 1: To investigate if baseline fibrinogen levels during 6 years of follow-up could predict risk of ischaemic stroke, 8755 stroke-free participants in The Copenhagen City Heart Study cohort were studied. Stroke risk was calculated overall and stratified for levels of conventional cardiovascular risk factors.

Study 2: To investigate if baseline fibrinogen and/or high-sensitive C-reactive protein were elevated in patients with advanced atherosclerosis, 318 patients with carotid artery stenosis ≥50% were compared with 1584 (fibrinogen) or 318 (C-reactive protein) age- and gender-matched controls without ischaemic cerebrovascular and ischaemic heart disease from the Copenhagen City Heart Study.

Study 3: To investigate if baseline fibrinogen and/or high-sensitive C-reactive protein were particularly elevated in patients with echolucent, rupture-prone plaques, the 318 patients withcarotid artery stenosis ≥50% were divided into those with echolucent and echo-rich plaques.

2.2. Participants
2.2.1. Study 1
The Copenhagen City Heart Study (third examination, 1991–1994) is made up of 20- to 93-year-old women and men stratified into 10-year age groups and selected with the goal of obtaining a representative sample of the adult Danish general population.17,18 Each participant was randomly selected from the City of Copenhagen by using the Danish Central Population Registry. Of the 17 180 persons invited, 10 049 participated and 9259 gave blood for fibrinogen measurement. In the Copenhagen City Heart Study cohort, information on development of ischaemic cerebrovascular disease (WHO International Classification of Diseases, Eight Edition, codes 432–435) and ischaemic heart disease (codes 410–414) were collected and verified by experienced neurologists and cardiologists up until the end of 1997 by reviewing all hospital admissions and diagnoses entered in the Danish National Hospital Discharge Registry, all deaths entered in the Danish National Registry of Cause of Death, and medical records from hospitals and general practitioners.17,19 After exclusion of persons with cerebrovascular disease developed before entry into the study in 1991–1994 (or with missing fibrinogen value), 8755 individuals were available for follow-up (Fig. 1). The endpoint followed over 6years was ischaemic stroke, that is, sudden onset of focal neurological symptoms lasting ≥24h. Individuals with transient ischaemic attack (focal neurological symptoms lasting <24h), amaurosis fugax (transient monocular blindness) or haemorrhagic stroke (validated via CT-scan) were excluded as endpoints. We had 100% follow-up.

2.2.2. Study 2
From 1994 to 2000 we recruited 318 patients with carotid artery stenosis ≥50%, identified among approximately 1400 consecutive patients referred to outpatient ultrasonography. Ultrasound examinations of the internal carotid artery, including the bifurcation, were performed by two experienced ultrasonographers in the anterior and lateral plane, with subjects in the supine position. The degree of stenosis was separated into the following groups: 0–49 and 50–99% and total occlusion, determined by generally accepted Doppler criteria.20 The 318 patients had previously experienced an ischaemic stroke , transient ischaemic attack , amaurosis fugax or non-focal symptoms, such as vertigo , confirmed by experienced neurologists. At least one CT scan was performed to exclude cerebral haemorrhage or tumour. These 318 patients were each matched by age and gender with five or one Copenhagen City Heart Study controls free of ischaemic cerebrovascular andischaemic heart disease (Fig. 1); these controls were not ultrasound scanned. In six of the 318 patients only four appropriate controls wereidentified.

2.2.3. Study 3
The 318 patients with carotid artery stenosis ≥50% were divided into those with echolucent or echo-rich plaques (Fig. 1), using the carotid plaque grey-scale median value generated for each patient by computerised image analysis (Fig. 2). A single good B-mode image of the carotid artery plaque, and a corresponding colour Doppler image to define plaque borders, were stored for later analysis. Hereafter, the most stenotic plaque of the two sides was outlined using the computer mouse and the software program Image-Pro Plus (Media Cybernetics, version 1.2.01). In case of acoustic shadowing from the plaque, the outline did not include the shadow. The grey-scale value of each pixel in the outlined area was then used to generate a grey-scale median value of the plaque as an objective measure of plaque echogenicity (0–255; 0=black and 255=white). The median of the 318 grey-scale values was 51. Those patients with a grey-scale median value ≥51 were consideredecho-rich, while those with a value <51 echolucent (Fig. 2). Ultrasonographers were blinded to acute phase reactant measurements.



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Fig. 2 Ultrasound B-mode image of an echolucent, rupture-prone plaque (left panels) and an echo-rich, fibrotic plaque (right panels). Outlines show the plaque areas where grey-scale levels of all pixels are used to construct the histogram showing distribution of grey-scale level among pixels. Median value of grey-scale levels is used for further analysis.

 
2.3. Other covariates
Body mass index was calculated as weight divided by height2(kgm–2). Smokers were current smokers. Diabetes mellitus was taken as self-reported disease treated with oral hypoglycaemic agents, insulin or diet. Hypertension was current use of antihypertensive agents or blood pressure above 140/90mmHg.21 Pulse pressure was systolic minus diastolic blood pressure; participants on antihypertensive agents were excluded in this analysis. Cholesterol, high-density lipoprotein cholesterol and triglycerides (non-fasting) were measured by enzymatic methods (Boehringer Mannheim, Mannheim, Germany). Fibrinogen and high-sensitive C-reactive protein levels were measured using a colorimetric method (Boehringer Mannheim) and nephelometry (Dade Behring Diagnostica). Fibrinogen wasmeasured immediately after blood sampling, while high-sensitive C-reactive protein was measured after storage at –80°C.

In Study 1 all fibrinogen measurements were performed on the same autoanalyser immediately after blood was drawn. In Study 2, however,fibrinogen in cases was measured several years later on a different autoanalyser to the one used for controls from Study 1. C-reactive protein levels in cases as well as controls in Study 2 were all measured on a single autoanalyser after thawing of stored samples. In Study 3, both fibrinogen and C-reactive protein were each measured on a single autoanalyser.

2.4. Ethics
Study protocols were approved by the Medical Ethics Committees in Copenhagen County (KA 98175 g) and the City of Copenhagen and Frederiksberg (100 2039/91 and KF 01-062/94). Signed informed consent was obtained from all participants.

2.5. Statistics
Cumulative incidence of ischaemic stroke from 1991 until the end of 1997 was plotted using Kaplan–Meier curves for fibrinogen above and below the median value (Study 1). Differencesbetween curves were assessed by the log–rank test. Cox regression analysis adjusted for age was used to examine the role of fibrinogen (above and below the median, in tertiles and in quartiles) in predicting a first ischaemic stroke; results are given as relative risks and 95% confidence intervals (Study 1). Age-adjusted relative risk of ischaemic stroke for fibrinogen above vs below the median value was also calculated stratified for gender, age, body mass index, smoking, diabetes mellitus, ischaemic heart disease, hypertension, cholesterol, high-density lipoprotein cholesterol and triglycerides. Finally, interaction between fibrinogen and these variables adjusted for age on ischaemic stroke was examined using Cox regression. Comparison of fibrinogen and high-sensitive C-reactive protein levels between study groups were adjusted for potential confounders such as age, gender, body mass index and smoking habits by multifactorial analysis of covariance (ANCOVA) (Studies 1, 2 and 3). These four covariates explained 20% of the variance in fibrinogen levels in the Copenhagen City Heart Study, while the other covariates mentioned above only explained minor additional variance. Statistical significance was taken as a two-sided . Statistical analyses were performed with SPSS. Power calculation was performed using PASS 2000 (Kaysville, UT).


    3. Results
 Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Baseline characteristics of participants are shown in Table 1.


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Table 1 Baseline characteristics of participants

 
3.1. Study 1
During six years of follow-up, 235 individuals in the Copenhagen City Heart Study cohort developed an ischaemic stroke: 4% of individuals with fibrinogen level above 3gl–1developed ischaemic stroke compared with slightly more than 1% of those with lower fibrinogen levels (Fig. 3) (log–rank test: ). On Cox regression adjusted for age, this difference was equivalent to a relative risk of 1.9 (95% CI: 1.4–2.5) for fibrinogen above vs below the median (Table 2). Equivalent relative risks for upper vs lower tertile and upper vs lower quartile were 2.0 (1.4–2.6) and 2.1 (1.3–3.3). The relative risk of ischaemic stroke for fibrinogen above vs below the median, for upper vs lower tertile and for upper vs lower quartile after adjustment for age, sex, body mass index, smoking, diabetes mellitus, ischaemic heart disease, hypertension, cholesterol, HDL cholesterol and triglycerides were 1.8 (1.3–2.4), 1.9 (1.2–2.8) and 1.9 (1.2–3.0), respectively.



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Fig. 3 Kaplan–Meier survival curves showing incidence of ischaemic stroke during follow-up in the Copenhagen City Heart Study cohort (Study 1), for individuals with fibrinogen above and below the median value of 3.0gl–1. Numbers at risk at the beginning of each year is shown below the x-axis.

 

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Table 2 Risk of ischaemic stroke by fibrinogen levels according to Cox regression analysis adjusted for age

 
Fibrinogen above vs below the median predicted risk of ischaemic stroke in men (relative risk: 2.7; 95% CI: 1.7–4.2), with a similar trend in women (1.4; 0.9–2.0) (Table 3; interaction: ). Fibrinogen predicted risk in the young (20–51 years: 5.2; 1.1–26) and middle aged (52–65 years: 2.9; 1.6–5.4), with a similar trend in the elderly (>65 years: 1.4; 1.0–2.0) (interaction:). Risk factors like body mass index, smoking, pulse pressure, cholesterol. HDL cholesterol and triglycerides did not seem to influence the value of fibrinogen as a predictor of risk of ischaemic stroke. In those with diabetes mellitus or ischaemic heart disease, fibrinogen did not predict risk of ischaemic stroke; however, the number of individuals in the these groups were relatively low, the power to detect a significant risk was limited, and there was no evidence of significant interaction (Table 3; diabetes mellitus, interaction: , ischaemic heart disease, interaction: ). Finally, fibrinogen predicted risk of ischaemic stroke in those with hypertension (1.8; 1.3–2.4), with a similar trend in those without (1.8; 0.9–3.6); importantly, because of fewer number of strokes the statistical power was less in individuals without hypertension than in those with hypertension, and the interaction test was non-significant (Table 3; ).


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Table 3 Risk of ischaemic stroke for fibrinogen above (≥3gl–1) vs below (<3gl–1) the median value, stratified by baseline variables and adjusted for age

 
3.2. Studies 1, 2 and 3
Fibrinogen levels in individuals who later developed an ischaemic stroke in the Copenhagen City Heart Study was 3.6gl–1compared with 3.1gl–1in those who did not (, Table 4, Study 1). In patients with carotid artery stenosis ≥50% (and thus with advanced atherosclerosis) fibrinogenlevels were 4.7gl–1compared with 3.1gl–1in age and gender-matched controls (, Table 4, Study 2); the equivalent values for high-sensitive C-reactive protein were 3.6 and 1.4mgl–1.


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Table 4 Fibrinogen and high-sensitive C-reactive protein levels

 
In contrast, neither fibrinogen nor high-sensitive C-reactive protein levels differed between those patients with echolucent and echo-rich plaques( and , Table 4, Study 3). The power in Study 3 (at the level) to exclude a 15% increase in fibrinogen or a 50% increase in high-sensitive C-reactive protein from patients with echo-rich plaques to those with echolucent plaques was 98 and 54%, respectively; these increases are equivalent to a 1/3 of the increases seen in Study 2 between patients with advanced atherosclerosis vs controls.


    4. Discussion
 Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
The present study demonstrates that fibrinogen is a predictor of future ischaemic strokes in people from the general population, particularly in men and in the young and middle aged. Furthermore, fibrinogen and high-sensitive C-reactive protein are markers for advanced atherosclerosis, but not echolucent, rupture-prone plaques.

4.1. Fibrinogen and ischaemic stroke
Our data (based on 8755 men and women; 6years follow-up; 235 strokes), which suggests a doubling in risk of ischaemic stroke for high vs low fibrinogen, are in accordance with most previous prospective studies on fibrinogen and C-reactive protein. In the Physicians' Health Study (1086 men; 8years follow-up; 196 strokes) relative risk of an ischaemic stroke in the highest vs lowest quartile of C-reactive protein was 1.9 (95% CI: 1.1–3.3).8 Furthermore, in the Framingham Heart Study (1315 men and women; 12 years follow-up; 92 strokes), relative risk in the highest vs lowest tertile of fibrinogen was 1.9 for men and 2.0 for women, though neither was significant.3 In the Cardiovascular Health Study (5888 men and women; 5 years follow-up; 473 strokes/transient ischaemic attack) relative risk of stroke or transient ischaemic attack in the highest vs the lowest quintile of fibrinogen was 1.8 in men and 1.1 in women; fibrinogen was not significantly associated with events in women.22 In the Atherosclerosis Risk in Communities Study (15 792 men and women; 7 years follow-up; 191 strokes) relative risk of stroke was 1.2 (1.0–1.5) in men and 1.1 (0.9–1.3) in women.23 Finally, in the Edinburgh Artery Study (1592 men and women; 5 years follow-up; 45 strokes) the overall relative risk of stroke was 1.5 (1.2–2.0).24

Several arguments support our observation that a higher relative risk of ischaemic stroke in men than women (as a function of fibrinogen) is a real phenomenon. First, the relative risk was highly significant in men but non-significant in women despite more statistical power. Second, there was statistical evidence of interaction between fibrinogen and sex on risk of ischaemic stroke. Third, because men at the same age have more advanced atherosclerosis than women, mechanistically it is plausible that the association atherosclerosis–fibrinogen–stroke may be more pronounced in men than in women. Conversely, other arguments suggest that this gender difference may not exist: (1) If correction for multiple comparisons in interaction testing was performed, then gender and fibrinogen did not interact on stroke risk. (2) The 95% CI for women and men on stroke risk overlap.

The higher predictive value of fibrinogen on ischaemic stroke in the young and middle aged compared with the elderly was also somewhat surprising. The significant interaction between fibrinogen and age on ischaemic stroke is most likely explained by the lack of significant relative risk in the elderly. Due to the large overlap in 95% confidence intervals, relative risk in young and middle aged may not differ. A possible explanation for the lack of significant relative risk in the elderly, could be that in this age range atherosclerosis is not alone in determining who will develop an event as a number of other risk factors like diabetes mellitus, hypertension, etc. also play a significant role. Furthermore, among our group of elderly 61% were women, helping explain the lower relative risk in the elderly as women had lower risk than men.

4.2. Fibrinogen, C-reactive protein and advanced atherosclerosis
Elevated levels of fibrinogen and C-reactive protein in patients with carotid artery stenosis vs controls imply that these acute phase reactants are non-specific markers of advanced atherosclerosis. This is supported by the fact that carotid artery stenosis is a predictor of wide-spread atherosclerosis,10,11 and that atherosclerosis is an inflammatory process.9 In atherosclerosis macrophages produce cytokines such as interleukin-1 and 6 and tumour necrosis factor, all of which stimulate hepatocytes to produce fibrinogen and C-reactive protein.Furthermore, our findings are in accordance with those by Levenson et al., that fibrinogen was elevated in subjects with silent atherosclerosis, particularly in those with disease in several arterial beds.25 Finally, within the Copenhagen City Heart Study those with ischaemic heart disease and thus atherosclerosis had elevated fibrinogen.17

Alternatively, C-reactive protein could directly influence atherogenesis. Pasceri et al.26 have shown that C-reactive protein induces adhesion molecule expression in human endothelial cells and Zwaka et al.27 reported that C-reactive protein can mediate low density lipoprotein uptake by macrophages. Furthermore, Hashimoto et al.28 found that C-reactive protein is an independent predictor of the rate of increase of carotid atherosclerosis. Finally, fibrinogen may also be more than a marker, since it binds to platelets and contributes to platelet aggregation and fibrin formation.

4.3. Fibrinogen, C-reactive protein and echolucent, rupture-prone plaques
How can the lack of significant differences in fibrinogen and C-reactive protein between echolucent, rupture-prone plaques and echo-rich plaques be explained? Rupture-prone plaques are characterised by additional activation of macrophages at plaque shoulders,14 and consequently acute phase reactants could be additionally elevated in patients with echolucent plaques. However, activation of macrophages in a single plaque (like measured in the present study) may not accurately reflect the situation in the average plaque in the entire arterial system. In other words, acute phasereactants reflect the average status of the entire body and therefore may not be significantly modulated by changes at one arterial site. Alternatively, our results of no association of fibrinogen and C-reactive protein with echolucent vs echo-rich plaques could be a Type II error; however, we had 98 and 54% power to exclude a 15% and a 50% increase in fibrinogen and C-reactive protein with echolucent vs echo-rich plaques.

4.4. Limitations
A limitation of Study 2 could be that fibrinogen measurements were not performed simultaneously in Copenhagen City Heart Study controls and the 318 patients with carotid stenosis. We therefore additionally measured high-sensitive C-reactive protein in cases and controls, and confirmed the finding for fibrinogen. This excludes that the observed difference in fibrinogen should be measurement errors.

Studies 2 and 3 were cross-sectional studies and not prospective as Study 1. Therefore, fibrinogen and C-reactive protein in Studies 2 and 3 were only examined for marker status for atherosclerosis and echolucency, rather than for causal relationships.

Although a standard operation procedure was followed when calibrating the ultrasound units at the beginning of each scan, a limitation of Study 3 could be variance between the two ultrasonographers and between the two ultrasound units used. Nevertheless, we attempted to overcome this variance by using objective grey-scale median values generated by a computer, and not visual subjective classification.

In conclusion, elevated fibrinogen predicts future ischaemic strokes, particularly in men and in the young and middle aged, and is associated with advanced atherosclerosis rather than rupture-prone plaques. Thus, fibrinogen may contribute to better risk assessment in younger and middle-aged men, in combination with established methods.


    Acknowledgments
 
Supported by the Danish Heart Foundation and the Danish Medical Research Council. Hanne Damm is thanked for technical assistance.


    References
 Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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