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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:529.)
© 2000 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Distinct Risk Profiles of Early and Advanced Atherosclerosis

Prospective Results From the Bruneck Study

Johann Willeit; Stefan Kiechl; Friedrich Oberhollenzer; Gregor Rungger; Georg Egger; Enzo Bonora; Manfred Mitterer; Michele Muggeo

From the Department of Neurology, Innsbruck University Clinic (J.W., S.K., G.R.), Austria; and the Department of Internal Medicine, Bruneck Hospital (F.O., G.E.), the Department of Endocrinology and Metabolism, University of Verona (E.B., M. Muggeo), and the Department of Hematology, Bolzano Hospital (M. Mitterer), Italy.

Correspondence to Dr Johann Willeit, Department of Neurology, Innsbruck University Clinic, Anichstraße 35, A-6020 Innsbruck, Austria. E-mail johann.willeit{at}uibk.ac.at

	Abstract

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Abstract—Most epidemiological surveys on risk factors of atherosclerosis were cross-sectional in design and did not consider the existence of pathologically distinct processes. The Bruneck Study is a prospective survey in the general community (age range, 40 to 79 years). The baseline examination and first reevaluation were performed in the summers of 1990 and 1995 (participation, 92%; follow-up, 96%). Carotid atherosclerosis was monitored with high-resolution duplex ultrasound. Early (incidence and/or extension of nonstenotic lesions) and advanced (incidence and/or progression of stenosis >40%) stages of atherogenesis were differentiated. The risk profile of early atherogenesis consists of traditional risk factors, such as hypertension, hyperlipidemia, and cigarette smoking (pack-years), supplemented by a variety of less well-established risk conditions, including high body iron stores, hypothyroidism, microalbuminuria, and high alcohol consumption. In contrast, the risk profile of advanced atherogenesis includes markers of enhanced prothrombotic capacity, attenuated fibrinolysis, and clinical conditions known to interfere with coagulation: high fibrinogen, low antithrombin, factor V Leiden mutation, lipoprotein(a) >0.32 g/L, high platelet count, cigarette smoking, and diabetes. Hyperlipidemia and hypertension were of only minor relevance. These findings, along with the epidemiological features of advanced atherogenesis and emergence of an elevated fibrin turnover, suggest atherothrombosis to be a key mechanism in the development of advanced stenotic atherosclerosis. Supplementary 6-category logistic regression models illustrate the changing association between major risk predictors and atherosclerosis of increasing severity and substantiate appropriateness of the 40% threshold applied for the definition of advanced stenotic atherosclerosis. Atherosclerosis is a heterogeneous process that subsumes etiologically and epidemiologically distinct disease entities. The multifactorial etiology of atherosclerosis, which goes far beyond the traditional risk factors, has not yet achieved adequate attention in clinical practice and disease prevention.

Key Words: atherogenesis • carotid arteries • risk factors • ultrasonics • epidemiology

	Introduction

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Preventive measures aimed at fighting cardiovascular disease are most effective when already introduced at the stage of asymptomatic atherosclerosis. Precise knowledge of the natural course and risk factors of atherosclerosis is mandatory for this purpose. A number of ultrasound-based population studies have attempted to clarify the etiology of atherosclerosis.^{1 2 3 4} However, the vast majority of previous surveys in this field were cross-sectional in design and thus did not consider the temporal sequence of risk exposure and disease occurrence. Furthermore, most risk analyses neglected the existence of pathologically distinct processes in human atherosclerosis.^{5 6 7 8} There is strong evidence of a dualism of conventional atherogenesis and atherothrombosis, with the latter process gaining increasing importance in advanced lesions.^{7 8} The present prospective study may well be the first to attempt to assess risk profiles for these different stages of carotid artery disease in the general community.

	Methods

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Study Subjects
The Bruneck Study is a prospective population survey aimed at investigating the epidemiology and etiology of carotid atherosclerosis.^{7 8 9 10 11 12 13 14 15} In 1990, the study population was recruited as a sex- and age-stratified random sample of all inhabitants of Bruneck 40 to 79 years old (125 women and 125 men in each of the fifth to eight decades of age; n=1000). A total of 93.6% participated, with the data assessment completed in 919 subjects. During the follow-up between the summers of 1990 and 1995, a subgroup of 62 individuals died (26 of myocardial infarction and stroke), and 1 moved away. In the remaining population, follow-up was 96.5% complete (n=826). All participants gave informed consent before entering the study.

Clinical History and Examination
The study protocol included a clinical examination with cardiological and neurological priority and standardized questionnaires on current and past exposure to candidate vascular risk factors.^{7 8 9} The average number of cigarettes smoked per day and the pack-years were noted for each smoker and ex-smoker. Alcohol consumption was quantified in grams per day and classified into 4 categories.¹¹ Systolic and diastolic blood pressures were means of 3 measurements, each of which was taken after 10 minutes of rest. Hypertension was defined as a blood pressure >160/95 mm Hg or the use of antihypertensive drugs.¹⁶ A standardized oral glucose tolerance test¹⁷ was performed in all subjects except those with well-established diabetes. Diabetes and impaired glucose tolerance (IGT) were diagnosed according to World Health Organization criteria.¹⁷

Laboratory Methods
Blood samples were taken from the antecubital vein after subjects had fasted and abstained from smoking for 12 hours.^{9 10} Total and HDL cholesterol (HDL-C) were determined enzymatically (CHOD-PAP method, Merck; coefficient of variation [CV] 2.2% to 2.4%), lipoprotein (Lp) (a) concentrations with an ELISA (Immuno; CV 3.5% to 6.3%), and apolipoproteins with a nephelometric fixed-time method (Behring; apolipoprotein AI, CV 5.7%; apolipoprotein B, CV 2.4%). LDL cholesterol (LDL-C) was calculated with the Friedewald formula.¹⁸ Fibrinogen was measured according to the method of Clauss,¹⁹ and serum ferritin and antithrombin with a fluorometric enzyme immunoassay (Diagnostic Products Corp; CV 5.0% to 5.9%) and chromogenic assay (CV 3.9% to 4.9%). Hypothyroidism was defined by a thyroid-stimulating hormone level exceeding the assay cutoff of 6.2 mIU/L (Diagnostic Products Corp; enzyme immunoassay, CV 3.9% to 13.8%) or prediagnosed disease status. Albumin was assessed in an overnight urine sample (Behring; nephelometry, CV 4.3%), and factor V Leiden mutation was detected by allele-specific polymerase chain reaction amplification.¹² Other parameters were measured as follows: C-reactive protein, immunonephelometry (Behring; CV 2.2% to 4.2%), ₁-antitrypsin, nephelometry (Behring; CV 3.0% to 3.2%), and D-dimer, enzyme immunoassay (Behring; CV 2.4% to 4.4%). Assessment of protein C and protein S has been detailed previously.¹²

Scanning Protocol and Definition of Ultrasound End Points
The ultrasound protocol involves the scanning of the internal (bulbous and distal segments) and common (proximal and distal segments) carotid arteries on either side with a 10-MHz imaging probe and 5-MHz Doppler.^{7 8 9} Atherosclerotic lesions were defined by 2 ultrasound criteria: (1) wall surface (protrusion into the lumen) and (2) wall texture (echogenicity). The maximum axial diameter of plaques was assessed in each of 16 vessel segments, and an atherosclerosis score was calculated by addition of all diameters.⁹ Scanning was performed in 1990 and repeated in 1995. During follow-up, information on the baseline evaluation was not available to the sonographer. Incident atherosclerosis was defined by the occurrence of new plaques in previously normal segments, and progression of nonstenotic lesions by a relative increase in the plaque diameter exceeding twice the measurement error of the method.^{7 8 11} In the present analysis, both processes were combined to a single outcome category called "early atherogenesis" for ease of presentation and the fact that these processes shared most of the risk factors described. "Advanced atherogenesis" was assumed whenever the progression criterion was met and a narrowing of the lumen >40% was achieved (1995). The cutoff of 40% was adopted from previous epidemiological analyses in this cohort.^{7 8}

Statistical Analysis
CVs describe the measurement error (e) of an assay as a percentage of the pooled mean (x) according to the formula CV=(e/x)x100%. The range of CVs presented is that for different standards, ie, different levels of the variable. Strength and type of association between baseline candidate risk attributes and various stages of atherogenesis were assessed by means of logistic regression analysis. Scale fitting as described elsewhere was performed to identify nonlinearity in the logit.^{10 20} A base model was adjusted for age and sex only (Table 1). Multivariate equations were fitted by a forward stepwise selection procedure (probability values for entry and removal, 0.05 and 0.10, respectively)²⁰ that allowed for all variables given in Table 1. Age and sex were additionally forced into these models to account for the age and sex structure of the population sample. Model 1 of the multivariate analysis (Tables 2 and 3) was adjusted for baseline atherosclerosis score, whereas model 2 did not consider this variable to avoid suppression effects on other risk attributes. Regression standardized risks of atherogenesis according to the number of risk predictors were calculated. The marginal method of the regression adjustment technique was used because it does not rely on the rare-disease assumption.²¹

View this table: [in this window] [in a new window]   Table 1. Means and Proportions of Candidate Vascular Risk Factors in Subjects With and Without Early/Advanced Atherogenesis

View this table: [in this window] [in a new window]   Table 2. Multivariate Logistic Regression Analysis of Early Atherogenesis on Age, Sex, and Candidate Vascular Risk Factors

View this table: [in this window] [in a new window]   Table 3. Multivariate Logistic Regression Analysis of Advanced Atherogenesis on Age, Sex, and Candidate Vascular Risk Factors

Changes in the association of selected (major) risk predictors and atherosclerosis of increasing severity were specified with 6-category logistic regression analyses. For this purpose, subjects were assigned to 1 of 6 categories (no change, 20%, 21% to 30%, 31% to 40%, 41% to 50%, and >50%) according to the degree of diameter stenosis of the severest incident/progressive plaque. The logistic regression models provide ORs for given variables in each of the outcome categories as a measure of the strength of association (reference group: no change 1990 to 1995) and thus permit individual thresholds in this association to be identified. Mathematical background and performance of the polychotomous logistic regression analysis were described in detail by Hosmer and Lemeshow.²⁰

	Results

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During the 5-year follow-up, 390 subjects out of 826 (47.2%) developed new atherosclerotic lesions or showed extension of nonstenotic lesions (early atherogenesis), and 92 of 326 individuals (28.2%) with preexisting plaques showed stenotic transformation (advanced atherogenesis). Table 1 depicts means or proportions of risk variables in the above outcome categories. Analyses of risk profiles of early atherogenesis were performed in the entire population sample, whereas those of advanced atherogenesis focused on subjects with preexisting atherosclerosis (Figure 1). Age- and sex-adjusted associations are given in Table 1. In the multivariate analysis, hypertension, pack-years (smoking), LDL-C and HDL-C, ferritin, Lp(a), alcohol consumption, hypothyroidism, microalbuminuria, and white blood cell count were risk predictors of early atherogenesis, whereas risk profiles for advanced stenotic atherosclerosis included diabetes, high fibrinogen, low antithrombin, high platelet count, smoking, alcohol consumption (low amounts protective), Lp(a) >0.32 g/L, and the factor V Leiden mutation (Tables 2 and 3). Risk profiles did not differ between sexes. Regression-standardized risks according to the number of major risk predictors are given in Figure 2. Subjects without any risk factors faced a low atherosclerosis risk, whereas those with a clustering of risk predictors almost obligatorily experienced early/advanced atherogenesis. Preexisting atherosclerosis predicted disease progression beyond the effect of the risk conditions identified in the present study (Figure 2A). In ex-smokers, the risk of early atherogenesis remained high up to 5 to 10 years after cessation (current smokers and ex-smokers: adjusted ORs, 1.80 each; P<0.05), whereas the risk of advanced atherogenesis normalized (OR, 3.5, P<0.001, and 1.3, P=0.5). Several attributes of hyperlipidemia predicted early atherogenesis almost equally well. In the multivariate analysis, LDL-C and HDL-C may be replaced by the total/HDL-C ratio (OR, 1.3 per 1-U change; P<0.001) without attenuating the overall fit of the model or composition of the risk profile.

View larger version (47K): [in this window] [in a new window]   Figure 1. Flow diagram characterizes the population samples used in distinct risk analysis.

View larger version (15K): [in this window] [in a new window]   Figure 2. Regression-adjusted risks of early (A) and advanced (B) atherogenesis according to the number of vascular risk attributes. The dotted lines in A symbolize subjects with (upper line) and without (lower line) baseline atherosclerosis. The risk variables considered were as follows: hypertension, hypothyroidism, heavy alcohol consumption (100 g/d), LDL-C (fifth quintile [Q5], >4.3 mmol/L), HDL-C (Q1, <1.19 mmol/L), microalbuminuria (Q5, >0.024 g/L), lipoprotein(a) (Q5, >0.27 g/L), and pack-years of smoking (Q5, >13) for early atherogenesis; and IGT/diabetes, lipoprotein(a) >0.32 g/L, smoking, factor V Leiden mutation, platelet count (Q5, >2.6x109), fibrinogen (Q5, >3.2 g/L), and antithrombin (Q1, <84%) for advanced atherogenesis.

As extensively discussed elsewhere,^{7 8} early and advanced atherogenesis emerge and progress independently of each other. Thus, there is no good reason to exclude subjects with early atherogenesis from analysis of incident/progressive stenotic lesions, and vice versa. Such a procedure would yield the very same results but entail the disadvantage of selection and limited representativity for the general community. Exclusion of subjects receiving aspirin (n=23 at the 1990 baseline) or antihypertensive (n=136), antidiabetic (n=19), or lipid-lowering (n=6) drugs did not essentially affect the results obtained.

Levels of various acute-phase reactants measured as part of the 1995 reevaluation of the Bruneck cohort were significantly increased in subjects with early atherogenesis (Table 1). Concentrations of D-dimer (1995), a marker of fibrin turnover, were higher in subjects with incident and/or progressive carotid stenosis than in those without (Table 1). Finally, in a small subsample of the Bruneck cohort (n=100), low protein S and protein C predicted an increased risk of advanced atherogenesis (ORs, 1.54 and 1.39 for a 10-U increase in protein C and S, respectively, P<0.05 each).

A total of 26 men and women died of myocardial infarction or stroke between 1990 and 1995. For most (n=21) of these subjects, interim ultrasound scans were available either at regular intervals or immediately before death. When this patient group was included in the analyses with the outcome category defined by changes in the vascular status between baseline and the last scanning, results were virtually unchanged.

Finally, we critically reevaluated the cutoff of 40% applied for the definition of advanced stenotic atherosclerosis. For that purpose, we assessed changes in the association between major vascular risk predictors and atherosclerosis of increasing severity (degree of diameter stenosis in 1995) by means of 6-category logistic regression analysis. Results for 8 selected variables are illustrated in Figure 3. Traditional risk factors showed a preferential association with less severe atherosclerosis (stenosis 40% for all variables except for LDL, stenosis 50; Figure 3), whereas markers of a procoagulant state gained predictive significance in severe atherosclerosis (stenosis >40 for all variables except for fibrinogen, stenosis >30%; Figure 3). Cigarette smoking and alcohol consumption were strong risk predictors of early and advanced atherogenesis (Tables 2 and 3) and, in analogy, showed consistently strong associations with each category of atherosclerosis severity (smoking, OR 2.0, 1.9, 2.5, 3.4, 2.5; P<0.05 each; light drinking, OR 0.6, 0.6, 0.6, 0.2, 0.5; P<0.05 each). In all, this approach confirmed the results of the original analysis (Tables 1 through 3) and substantiated the appropriateness of the 40% threshold applied for the definition of advanced stenotic atherosclerosis.

View larger version (21K): [in this window] [in a new window]   Figure 3. Changing association between major vascular risk predictors and atherosclerosis of increasing severity. ORs were derived from 6-category logistic regression analyses and represent estimates for the strength of association between variables of interest and each of the outcome categories. This analysis focused on the entire population.

	Discussion

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Histopathological studies suggest the existence of 2 distinct processes in atherogenesis^{5 6} : (1) In incipient and small to medium-sized lesions, slow and continuous plaque growth predominates, which was proposed to rely on a variety of complex biological step-by-step phenomena, such as lipid-induced atherogenesis or smooth muscle cell proliferation.²² (2) This type of plaque growth may occasionally be accelerated by plaque fissuring and formation and fibrous organization of mural thrombi.^{5 6 23} Atherothrombotic mechanisms gain increasing importance with advancing severity of atherosclerosis size and may even be the key event in the development of stenosis.⁶ The complex histopathological appearance of atherosclerosis stimulated the coining of several contradictory concepts about its etiology, most important of which are the response-to-injury hypothesis²² and the thrombosis or incrustation theory.^{24 25} Badimon and Fuster^{5 6} combined the "atherogenic" and "atherothrombotic" models into a single integrative hypothesis. The Bruneck Study provided the first in vivo evidence of such a dualism in human carotid atherosclerosis: Early nonstenotic atherosclerosis showed a slow and continuous type of plaque growth, diffuse spread of disease, and highly efficient vascular remodeling, which prevented significant lumen obstruction in most instances. Advanced stenotic atherosclerosis in turn was characterized by occasional marked increases in plaque size, no or inadequate vascular remodeling, and preferential manifestation at sites exposed to hemodynamic stress. Notably, some 95% of incident stenosis >40% exhibited this type of lesion extension (for details see References 7 and 8^{7 8} ). The present study assessed risk profiles of both processes.

Early Nonstenotic Atherosclerosis
Age and Sex
In analogy to previous reports, age turned out to be the strongest risk predictor of atherosclerosis. This may well be the expression of intrinsic effects of aging but more likely reflects the cumulative component in the exposure to various risk factors. The well-known sex difference in the incidence of atherosclerosis disappeared after adjustment for body iron stores.¹⁰ Our findings suggest iron overload to be a key mechanism in the sex-specific manifestation of atherosclerotic diseases but do not rule out the possibility of additional (hormonal) factors.²⁶

Baseline Atherosclerosis
Preexisting atherosclerosis predicted the risk of further disease progression independent of other risk factors (Figure 2A). These findings point to an autocatalytic component in human atherogenesis. Actually, previous investigations in this cohort yielded evidence of (auto)immune mechanisms and identified heat-shock protein 65 as a potential target antigen.¹³

Hypertension
The present study advocates a crucial role of hypertension in early atherogenesis and provides prospective confirmation for a variety of previous cross-sectional surveys.^{1 3 27}

Infectious Disease
The concept of an infectious risk factor in atherosclerosis is based primarily on seroepidemiological studies and the emergence of elevated acute-phase reactants in subjects with cardiovascular disease.^{28 29 30} In the present study, white blood cell count was a risk predictor of early atherogenesis, and levels of C-reactive protein, ₁-antitrypsin, and neutrophil counts all were significantly elevated in subjects with prevalent disease status. These findings add some indirect support to the infection hypothesis but by no means furnish proof of causality.

Cigarette Smoking
Most previous studies agree that smoking represents a main risk factor of atherosclerosis.^{3 31} In our survey, pack-years as a measure of cumulative exposure showed the strongest association with early carotid artery disease. Notably, the risk burden did not normalize within 5 to 10 years after cessation, which agrees with the findings of several prospective surveys^{3 31} and tempts us to speculate that not smoking itself but rather associated disorders, such as chronic infections, that are known to persist for years after quitting³² represent the true atherogenic culprit. Notably, in our study, increased risk of early atherogenesis was confined to smokers with high circulating bacterial endotoxin (unpublished data, 1998).

Hyperlipidemia and Iron Stores
Epidemiological research almost univocally suggests a crucial role of hyperlipidemia in early atherogenesis. In the present evaluation, LDL-C, total cholesterol, apolipoprotein B, the total/HDL-C ratio, and low HDL-C were all risk predictors of atherogenesis. In the multivariate analysis, models including LDL-C and HDL-C or the total/HDL-C ratio best fitted the data. Lp(a), another cholesterol-carrying lipoprotein, showed an independent dose-response relation with atherosclerosis risk as well.

There is substantial experimental evidence that native LDL experiences oxidative surface modifications before achieving full atherogenicity.³³ It is thus not surprising that high concentrations of tissue iron, a major prooxidant in vivo, represent an independent risk condition of early atherosclerosis and appear to augment the injurious capacity of LDL (effect modification¹⁰ ). The iron hypothesis is substantiated by a variety of experimental and epidemiological studies.^{34 35 36}

Alcohol Consumption
The association between regular alcohol intake and early atherogenesis was U-shaped, with light drinkers facing a lower risk than either heavy drinkers or abstainers (see Reference 11¹¹ ). Protection offered by alcohol consumption 50 g/d acts primarily through inhibition of the injurious action of high LDL cholesterol, which may be explained by an increased intake of antioxidant phenols contained in alcoholic beverages.³⁷

Hypothyroidism
Hypothyroidism was proposed to be a risk factor of atherosclerotic diseases on the basis of a variety of proatherogenic properties, including dyslipidemic effects and enhancement of plasma homocysteine.^{38 39} The present study provides the first epidemiological support for this concept. Subjects with hypothyroidism faced a 3-fold risk of early atherogenesis, which was independent of other risk factors.

Microalbuminuria
Microalbuminuria is a marker of systemic endothelial leakage for albumin and LDL.⁴⁰ So far, no consensus has been reached on whether increased endothelial permeability is a genetically determined risk burden or a consequence of acquired endothelial dysfunction. In our study, excess risk of early atherosclerosis in subjects with high albumin excretion could only in part be attributed to the adverse action of vascular risk attributes (residual relation, P<0.05), which replicates the findings of a recent survey.⁴¹

Advanced Stenotic Atherosclerosis
Fibrinogen
A variety of cross-sectional surveys revealed a strong relation between high fibrinogen levels and atherosclerosis.^{4 42} The question of causality, however, is a matter of ongoing dispute. Early atherosclerosis exhibits several pathological features consistent with chronic inflammation, and elevated fibrinogen was suggested to be an epiphenomenon of such a process.⁴³ The present study provided evidence in favor of a true association in that it documented (1) the temporal sequence of high baseline fibrinogen and subsequent disease progression, (2) a preferential relation with advanced atherogenesis (atherothrombosis) rather than with early (inflammatory) stages of disease, and (3) elevated fibrin turnover in patients with stenosis.

Antithrombin and Factor V Leiden Mutation
The antithrombin and protein C pathways are 2 main antithrombotic systems in human coagulation aimed at limiting excessive thrombin formation. It is thus not unexpected that low antithrombin and the factor V Leiden mutation, which causes activated protein C (APC) resistance,⁴⁴ were significant risk predictors of advanced atherogenesis in our survey. As detailed in a previous evaluation of the Bruneck cohort,¹² this finding extends to impaired action of APC not caused by the Leiden mutation. The condition recently called "acquired" or "factor V mutation–independent" APC resistance may derive from protein C deficiency, the presence of lupus anticoagulant, or prominent acute-phase reaction (for details see References 12 and 15^{12 15} ). Finally, there is preliminary evidence of an inverse association of protein C and S concentrations and risk of advanced atherogenesis in our population.

Smoking
Cigarette smoking initiates a variety of dose-dependent prothrombotic properties at both the platelet and coagulation levels,⁴⁵ and it emerged as a prominent risk factor of advanced stenotic atherogenesis. The risk burden of advanced atherogenesis normalized after smoking cessation, consistent with a reversible procoagulant state. This feature and the preferential association to the amount of current smoking rather than to cumulative measures contrasted with the conjecture in early atherogenesis.

Diabetes
The present study uncovered a particularly strong and selective association of diabetes and IGT with advanced atherosclerosis, thereby suggesting coagulation disorders in diabetic subjects to be a main atherogenic culprit. Actually, conditions of glucose intolerance are well established to cause a procoagulant state and to attenuate endogenous fibrinolysis.⁴⁶ Notably, in our study, injurious effects of diabetes on atherogenesis were independent of glucose control and medication and applied equally to newly diagnosed (n=19, OR 7.05; P=0.0007) and well-established (n=21, OR 4.28; P=0.012) cases.

Lipoprotein(a)
Lp(a) competes with plasminogen for binding to fibrinogen and fibrin and attenuates lysis of fibrin clots.⁴⁷ This effect may be confined to Lp(a) particles with low-molecular-weight apolipoprotein(a) phenotypes, which are those with high plasma concentrations. In our study, Lp(a) exceeding a predefined threshold of 0.32 g/L¹⁴ emerged as one of the strongest risk predictors of incident stenosis. This finding is substantiated by serial angiography evaluations of coronary arteries that identified elevated Lp(a) as one of the leading indicators of rapid disease progression.⁴⁸

Alcohol Consumption
Regular alcohol consumption interferes with coagulation and platelet aggregation in a complex way.⁴⁹ At least for light to moderate drinking, the net effect is antithrombotic. In our survey, alcohol consumption appeared to lower the risk of atherothrombosis at dosages 50 g/d.

Platelet Count
The crucial role of blood platelets in arterial thrombosis is beyond dispute. Platelets produce and secrete various mediators of coagulation, such as ADP, thromboxane A₂, and von Willebrand factor; expose the surface receptor GP IIb/IIIa when activated; and bind fibrinogen, von Willebrand factor, and fibronectin.⁵⁰ Thus, it is not unexpected that a high platelet count confers an increased risk of advanced atherogenesis.

Conclusions
The present study provides the first epidemiological evidence for the existence of etiologically distinct processes in human atherosclerosis. Early atherogenesis relies on classic vascular risk factors supplemented by several less well-established risk conditions, such as iron overload, hypothyroidism, and microalbuminuria (Table 2). With advancing severity of atherosclerosis, the composition of risk profiles undergoes substantial change. Most traditional risk variables lose their predictive significance when incident/progressive stenosis exceeds 40% lumen obstruction, whereas markers of enhanced thrombotic activity, attenuated fibrinolysis, and clinical conditions known to interfere with coagulation gain increasing importance (Figure 3, Table 3). These findings, along with the epidemiological features of advanced atherogenesis^{7 8} and emergence of elevated fibrin turnover, suggest that focal stenotic atherosclerosis originates primarily from atherothrombosis. Assessment of the various procoagulant risk predictors may assist in identifying subjects at high risk of incident or progressive stenosis (Figure 2B). This finding, which may be relevant to the management of asymptomatic carotid stenosis, awaits confirmation in further epidemiological surveys, especially in populations with a substantial number of high-grade stenoses.

Fighting the same standard risk factors in all patients, as is common clinical practice, ignores the complexity of atherogenesis and its multifactorial etiology. There is an urgent need to expand our knowledge of less well-established risk conditions, such as iron overload, and of the prevention of stenotic atherosclerosis, thereby focusing on antithrombotic and anticoagulant strategies.

Received March 8, 1999; accepted September 6, 1999.

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