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(Circulation. 1999;99:614-619.)
© 1999 American Heart Association, Inc.
Clinical Investigation and Reports |
Poor Response to Activated Protein C as a Prominent Risk Predictor of Advanced Atherosclerosis and Arterial Disease
From the Department of Neurology, Innsbruck University Clinic, Innsbruck, Austria (S.K., A. Muigg, W.P., J.W.); the Departments of Laboratory Medicine (P.S., A. Mair), Internal Medicine (G.E., M.O., F.O.), and Neurology (A.G.), Bruneck Hospital, Bruneck, Italy; and the Department of Hematology, Bozen Hospital, Bozen, Italy (M.M.).
Correspondence to Dr S. Kiechl, Department of Neurology, Innsbruck University Clinic, Anichstraße 35, A-6020 Innsbruck, Austria. E-mail stefan.kiechl{at}uibk.ac.at
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Abstract |
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Background—The potential role of activated protein C (APC) resistance in arterial thrombosis and disease is a matter of ongoing controversy.
Methods and Results—In the present population-based survey, a random sample of 826 men and women underwent high-resolution duplex ultrasound scanning of the carotid and femoral arteries. Response to APC was expressed in APC ratios. Subjects were tested for the factor V Leiden mutation. The risk of carotid stenosis increased gradually with decreasing response to APC (adjusted OR [95% CI] for a 1-U decrease of response to APC, 1.6 [1.2 to 2.2]), as did the risk of femoral artery stenosis (1.7 [1.3 to 2.3]) and prevalent cardiovascular disease (1.4 [1.1 to 2.0]). The association between low APC ratio and atherosclerotic vascular disease applied equally to subjects with the factor V Leiden mutation and those without. Our study identified various nongenetic determinants of poor response to APC in the general population, including behavioral, hormonal, and environmental factors.
Conclusions—The present study revealed an independent and gradual association between low response to APC and both advanced atherosclerosis (stenosis) and arterial disease. Resistance to APC due to factor V Leiden mutation was only one facet of this relationship.
Key Words: proteins • atherosclerosis • arteries • cardiovascular diseases • thrombosis • ultrasonics
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Introduction |
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During normal hemostasis, activated protein C (APC) counteracts thrombin amplification and clot propagation by an enzymatic degradation of factors Va and VIIIa.1 In 1993, Dahlbäck and coworkers2 were the first to describe resistance to APC in patients with venous thromboembolic events. This condition is frequently3 4 5 but not exclusively6 caused by a point mutation in the cleavage region of factor V. Meanwhile, a broad consensus has been reached on the prominent role of APC resistance in idiopathic and familial venous thrombosis.7 8
As a key outstanding question, the transferability of this finding to arterial thrombosis remains to be elucidated. Atheroma-associated thrombus formation and organization are widely accepted as one main pathomechanism in the development of vessel stenosis and occlusion.9 The present study was designed to analyze the strength and type of association between response to APC expressed in APC ratios and both manifest arterial disease and advanced (stenotic) atherosclerosis in the carotid, femoral, and lower limb arteries. It addresses the role of factor V mutation as part of this association and attempts to further identify the main determinants of poor APC response in the general healthy population.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Study Population
APC ratios were measured as part of the first follow-up examination of the Bruneck Study. Special features of the survey area and study protocol have been detailed previously.10 In brief, at the 1990 baseline, the study population was recruited as an age- and sex-stratified random sample of all inhabitants of Bruneck (Bolzano province, Italy) 40 to 79 years old (125 women and 125 men in their 5th to 8th decades). A total of 93.6% participated, with data assessment completed in 919 subjects. Between 1990 and 1995, a subgroup of 63 individuals died or 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 systolic blood pressure was taken in a sitting
position after at least 10 minutes of rest (mean of 3 independent
measurements).10 The ratio of ankle to brachial
systolic blood pressure was assessed separately in both lower
limbs.11 The average number of cigarettes smoked per day
was noted for each smoker and ex-smoker. Regular alcohol consumption
was quantified in terms of grams per day.
Major Clinical Events
In an effort to identify all cardiovascular
events that had occurred in the Bruneck Study population, we performed
an extensive screening using the following sources of information: Rose
questionnaire,12 detailed self-reported medical and
medication history, cardiological and neurological examination, resting
ECG, medical records provided by general practitioners,
and medical records from Bruneck Hospital. The situation in this
geographically remote mountainous region is unique in that the whole
area is served by Bruneck hospital only, and the only facilities for
exercise ECG and echocardiography and the only
ambulatory service for cardiology and neurology are
located at the hospital. All medical records were collected from
Bruneck Hospital databases and thoroughly reviewed for diseases of
interest. Myocardial infarction was deemed confirmed when World Health
Organization criteria for definite disease status13 were
met, including compatible symptoms and either elevated cardiac enzymes
or ECG changes. Coronary heart disease was ascertained by means
of standard diagnostic criteria, including typical resting
(Minnesota codes 1.1 to 1.2, 4.1 to 4.2, 5.1 to 5.2, or 7.1) or
exercise ECG and/or pathological coronary angiography and/or
echocardiography. Stroke and transient
ischemic attack were classified according to the criteria of
the National Survey of Stroke.14 The diagnosis of
peripheral artery disease required a positive response to
the Rose questionnaire, with the vascular nature of complaints
confirmed by standard diagnostic procedures and/or a
documented history of previous aortofemoral or femoropopliteal bypass
surgery (n=10).
Laboratory Parameters
Blood samples were collected after subjects had fasted and
abstained from smoking for at least 12 hours.10 In 5
subjects with recent stroke or myocardial infarction, samples were
drawn at an interval of 3 months. For the coagulation assay, citrated
plasma was obtained by centrifugation at
2500g for 10 minutes (15°C) immediately after sampling.
Special care was exercised to prevent contamination of platelets
from the platelet layer. The plasma was kept frozen at -70°C for
a period of <3 months, and the response of plasma activated
partial thromboplastin time (aPTT) to APC was expressed as the ratio of
clot time of APC/CaCl2 to clot time of
CaCl2 (Coatest APC resistance,
Chromogenix).4 15 In an effort to assess the
reproducibility of this method, we performed triple measurements in
subjects with APC ratios of 
2.0 and 3.5 (n=30 each). Interassay
coefficients of variation were low, 5.2% and 3.2%. Plasma from 1
patient on heparin therapy was pretreated with hepzyme, a
heparinase that neutralizes heparin effects on PTT.4 16
Commercial factor V–depleted serum (Boehringer-Mannheim) was
used for standardized dilution of plasma samples (1:5) from patients
taking warfarin (n=10) to ensure reliable APC ratio estimates in this
subgroup. Plasma from 50 apparently healthy subjects without a family
history of venous thrombosis, regular medication, or evidence of recent
infections was pooled to serve as a reference plasma (mean APC ratio,
3.16) for the calculation of normalized APC ratios: n-APC ratio=APC
ratio/APC ratio of pool plasma.3 4 Levels of
ß-thromboglobin were assessed in a random subsample of the Bruneck
Study population17 (n=80, plasma frozen at –70°C for
2.5 years) and in 80 fresh plasma samples collected in 1998
(Asserachrom, Boehringer-Mannheim). Apolipoproteins B and A-I
were measured by a nephelometric fixed-time method (coefficient of
variation, 5.7% and 2.4%). Proteins C and S were assessed with
commercial assays (Protein S Asserachrom, Boehringer-Mannheim;
normal range, 70% to 140%; protein C chromogenic assay,
70% to 130%). Diagnosis of lupus anticoagulant was achieved by
measurement of lupus anticoagulant–sensitive aPTT
(Baxter/Laevosan; cutoff, >1.3) and the diluted Russel viper venom
time test (American Diagnostic Inc; cutoff, >1.8). An
ELISA was used to measure anti-cardiolipin (Diamedix; cutoff for IgG,
>10 U/L and IgM, >7.5 U/L) and
anti–ß2-glycoprotein antibodies
(Inova Diagnostics Inc; cutoff, >10 U/L). Genomic DNA was
prepared by the DNA Zol method.18 Samples were
investigated with 2 polymerase chain reaction (PCR) amplifications, one
amplifying the wild-type and the other amplifying the factor V point
mutation. Each PCR mix included a primer pair that amplified a fragment
of the human growth hormone gene (internal positive amplification
control3 18 ).
Evaluation of Vascular Status
Sonographic assessment was performed with a duplex ultrasound
system equipped with a 10-MHz imaging probe and a 5-MHz Doppler.
The scanning protocol included imaging of the right and left common
(proximal and distal segments), internal (bulbous and distal segments),
and external carotid arteries10 and of the femoral
arteries 40 mm proximal and 10 mm distal to the bifurcation
into the superficial and deep branches. Atherosclerotic lesions were
defined as echogenic lesions encroaching into the lumen. Doppler
criteria or, when no hemodynamic disturbances
were detectable, a diameter reduction of >40% in the B-mode images
was applied to define stenosis. The cutoff of 40% appeared to
be a biological threshold: in brief, nonstenotic
atherosclerosis showed a slow, continuous, and diffuse
type of lesion extension compensated by vessel dilation. This process
emerged as a domain of traditional risk factors, such as
hyperlipidemia. In contrast, the development of
stenosis >40% was characterized by occasional prominent
plaque growth, long stable periods, focal manifestation, and a
procoagulant risk profile indicative of underlying plaque thrombosis.
Failure in vascular remodeling and marked increases in plaque size
acted synergistically in producing significant lumen compromise.
Actually, some 95% of stenosis >40% originated from this
type of atherogenesis.
Statistical Analysis
The assumption of normality for the APC ratio distribution,
implicit in basing analysis on parametric statistics,
was checked and confirmed by detrended normal plots and the Lilliefors
test (P>0.20).19 The relation between APC
ratios and other variables was assessed by linear regression
analysis. A multivariate regression model was
built with a forward stepwise selection procedure (probability values
for entry and removal of variables, 0.15 and
0.2020 21 ). To evaluate the association between response
to APC and stenotic atherosclerosis, logistic
regression models were fitted with the test procedure on the basis of
maximum likelihood estimators.21 In an attempt to obtain
the most suitable parametric scale in the logit, 5 equally
spaced categories of APC ratios were modeled with indicator
variables in separate analyses. Trends were estimated by
visual inspection of plots of the logit against the midpoints of APC
categories and by application of orthogonal polynomials.21
For ease of presentation, the multivariate
analyses for all outcome variables were adjusted for the
same set of covariates by forced entry of these variables, and ORs
derived from these models are referred to as adjusted ORs in the text.
The list of covariates includes age and sex, the main determinants of
APC ratio (Table 1
);
cardiovascular risk factors; and drug therapy (see
Table footnote). Analogous logistic regression models were built by use
of a forward stepwise selection procedure. The 2 approaches yielded
virtually identical results.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In our population, responses to APC expressed in APC ratios ranged from 1.4 to 5.1 (n-APC ratio, 0.4 to 1.9). Mean APC activity was significantly higher in men than in women (Figure
). The APC ratio decreased with
increasing age by 0.07 per decade (95% CI, 0.4 to 1.0) (n-APC, 0.02
[95% CI, 0.01 to 0.03]). Further significant associations were found
for acute-phase reactants (C-reactive protein,
1-antitrypsin, coeruloplasmin, fibrinogen),
alcohol consumption, PTT, antithrombin III, and cholesterol
levels. A multivariate prediction model of APC activity
is given in Table 1
. All independent determinants in concert
accounted for 25% of the variability of APC ratios
(R2=0.23). Levels of
ß-thromboglobin, a platelet activation
marker,17 showed a rather low variability in 2
population samples, each containing 80 subjects (frozen plasma samples:
mean [interquartile ratio], 5878 IU/mL [4450 to 7604 IU/mL]; fresh
plasma samples: 5948 IU/mL [4680 to 7480 IU/mL]), and did not
correlate with APC ratios (r=0.023 and 0.013,
respectively).
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The allele frequency of the factor V mutation in our population was 1.5%. Functional tests for response to APC are frequently used to screen for the mutation. At the recommended cutoff for our coagulation assay (<2.03 4 8 ), the sensitivity for detection of the Leiden mutation was 75% and the positive predictive value 42%. In >50% of subjects with an APC ratio<2.0 (prevalence, 5.4% [95% CI, 3.9% to 6.9%]), DNA analysis remained negative. In a minority of these individuals, APC resistance may be explained by protein C deficiency (n=1), presence of lupus anticoagulant (n=1), or infectious diseases (n=3). None had a protein S level <20%.4 Anti-cardiolipin or anti–ß2-glycoprotein antibodies were found in 5 subjects with factor V–independent APC resistance and 4 subjects with the Leiden mutation (coincidence, 20% each).
Men and women with nonstenotic carotid or femoral
atherosclerosis and those without atherosclerotic
lesions did not differ in their mean responses to APC (APC ratio, 3.20
each). For ease of presentation, these subjects were
grouped together and compared with patients with stenotic
atherosclerosis, which is the group with a putative
pathogenetic relevance of arterial (plaque)
thrombosis.9 When this was done, the analysis
revealed a strong and independent association between APC ratio and the
risk of stenosis >40% in carotid or femoral arteries,
regardless of whether APC ratio was modeled as a categorical or
continuous variable (Table 2). The
scale fitting procedure described in the Methods section documented an
excellent fit of a linear (dose-response) relation across the entire
range of APC ratios (P<0.0001 for the linear component of
orthogonal polynomials21 ). The association remained
independently significant after control for potential confounders and
aspirin medication (Table 2). Separate models that were
additionally adjusted for use of ß-blockers (n=50) and ACE
inhibitors (n=76) yielded virtually identical results
(adjusted ORs [95% CIs], 1.8 [1.3 to 2.5] and 1.7 [1.2 to 2.4]
for carotid and femoral artery stenosis).
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Temporary APC resistance may occur in the course of infectious diseases
and introduce a bias in the association between APC ratio and
arterial disease.1 22 Thus, we repeated the
analysis after excluding subjects with laboratory evidence of
ongoing acute-phase reaction (C-reactive protein >2.5 mg/L; n=18).
This procedure tended to strengthen the association rather than to
dilute it (Table 2).
The association between poor response to APC and advanced
atherosclerosis emerged as independent of the presence
of factor V mutation: in particular, among subjects with APC ratios
<2.0, excess risk of carotid stenosis applied equally to those
with the Leiden mutation (adjusted OR [95% CI], 3.6 [1.1 to 11.7];
reference group with APC ratio 
3.5) and those without (3.9 [1.4 to
10.9]). Likewise, individuals with the factor V mutation and with
factor V–independent APC resistance faced a similar risk of femoral
artery stenosis (2.9 [1.1 to 7.8] versus 3.4 [1.4 to
8.4]).
In subjects on aspirin therapy that was initiated for reasons other than known carotid vessel pathology (n=80), the predictive significance of low APC ratio for advanced atherosclerosis was clearly less pronounced than in untreated individuals (adjusted OR [95% CI], 0.9 [0.4 to 2.1] versus 2.5 [1.7 to 3.7]; P=0.033 for effect modification).
An ankle/brachial pressure index 
0.8 is another validated
indicator of hemodynamically relevant stenosis
of the femoral and lower limb arteries.11 When this
outcome variable was substituted for the ultrasound end points, the
adjusted OR (95% CI) for a 1-U decrease in the APC ratio was 1.8 (1.1
to 3.1).
Finally, response to APC emerged as a significant risk predictor of
prevalent nonfatal cardiovascular disease (CVD) (Table 2). Subanalyses fitted separately for the different
types of arterial disease yielded results as follows:
transient ischemic attack and stroke (n=21): age- and
sex-adjusted OR (95% CI), 1.8 (1.1 to 4.7); coronary artery
disease (n=77): 1.4 (1.0 to 1.9); and peripheral artery
disease (n=37): 1.7 (1.0 to 2.8). These results should be interpreted
in the light of the low number of events in each disease category. In
subjects who suffered fatal CVD between 1995 and 1998 (n=15), APC
ratios were significantly lower (mean, 2.67) than in those who died of
causes other than CVD (n=33) (mean, 3.15) or those still alive without
manifest vascular illness (mean, 3.16; P<0.05 each).
Likewise, factor V mutation was overrepresented (7% versus
0% and 2%).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Response to APC and Advanced Atherosclerosis
As anticipated, plaque thrombosis is a main pathomechanism in the development of vessel stenosis and occurrence of arterial disease, and the protein C anticoagulant system ranks among the most potent control mechanisms of excess thrombosis in vivo.1 To date, however, the concept that poor anticoagulant response to APC is involved in advanced stages of atherogenesis has received limited attention in epidemiological research. The present survey is the first to demonstrate a (linear) dose-response relation between decreasing APC ratio and increasing risk of advanced atherosclerosis (stenosis) in the carotid, femoral, and lower limb arteries. The predictive significance of poor response to APC did not extend to nonstenotic atherosclerosis, corresponding to the fact that this type of early vessel pathology actually does not rely on plaque thrombosis.9
Poor response to APC due to factor V mutation, although per se a strong
risk condition of advanced atherosclerosis, was only
one (rare) facet of the overall relation, which emphasizes the need to
characterize further genetic and environmental determinants of APC
response in the general population. To date, such data are rather
sparse.23 In our population sample, regular alcohol
consumption was associated with increased APC ratios. Premenopausal
women exhibited lower APC ratios than did women after menopause or men.
This finding, taken together with the decrease of APC ratios during
pregnancy and estrogen supplementation, points to effects of hormone
status and hormonal shifts in the protein C system.24
Given that some regulatory components of the protein C pathway
(1-antitrypsin, C4bBP) increase in response to
infectious stimuli as part of the acute-phase
reaction,1 22 emergence of an inadequate APC action
(procoagulant state) confined to the period of acute disease was not
unexpected. In accordance with recent population surveys, greater age
and increased LDL cholesterol levels emerged as further
empirical predictors of inefficient APC action.4 23
Finally, APC ratios did not correlate with ß-thromboglobin levels.
Thus, variable platelet contamination and/or activation appears
not to be a major source for the variability of APC ratios in our
population.
Notably, fewer than 50% of APC-resistant subjects, who are considered potential carriers of factor V mutation, actually had this genetic disorder. In the remaining group, APC resistance was unknown in origin except for a few cases with protein C deficiency, lupus anticoagulant, or prominent acute-phase reaction. Possible underlying defects include as yet undescribed mutations of factors V and VIII and dysfunctional protein S molecules.5 6 Low rates of factor V mutation among subjects with APC resistance in our population contrast with previous reports that assessed rates up to 85% to 90%.3 4 These surveys, however, did not focus on randomly selected population samples.
Response to APC and CVD
As expected, in view of the association with advanced
atherosclerosis, poor response to APC emerged as a
significant risk predictor of nonfatal CVD. Subjects with the Leiden
mutation were at a particularly increased risk of arterial
disease. Predictive significance was not confined to this genetic
disorder but rather extended to poor response to APC of distinct
origin. Preliminary data suggested analogous results for fatal CVD.
The number of previous reports advocating a risk factor status of an impaired protein C anticoagulant pathway for arterial disease6 25 26 27 28 is approximately in balance with the number of those that failed to observe any relation.29 30 31 Inconsistency in the results observed may be explained by the profound differences in the study design: (1) A majority of studies applying coagulation assays reported a significant relation, whereas results from DNA-based analyses were contradictory. Analyses focusing on the Leiden mutation may lack statistical power because subjects with poor response to APC due to other genetic and environmental factors who are at an equally high risk of atherosclerosis were included in the reference group. (2) Long-term aspirin treatment appeared to level the predictive significance of low APC ratios for advanced atherosclerosis. Most previous studies did not control for this potential source of bias. One was designed as an intervention trial testing the efficacy of aspirin in the primary prevention of CVD.29 (3) Negative studies are in part hospital-based.30 31
Methodological Considerations
So far, no consensus has been reached on a standard laboratory
method for measuring response to APC. The present study provides
evidence against the use of DNA-based tests and clotting assays
specific to factor V (Arg506-Gln) mutation, given
that the predictive significance of poor response to APC for
arterial disease was not restricted to the Leiden mutation.
Our coagulation assay was standardized in a multicenter
trial15 and is widely used in clinical
research.2 8 15 25 Although interlaboratory variability
was found to be low,15 some residual variation may arise
from differences in plasma preparation and laboratory
equipment.4 15 23 Normalization of APC ratios to a
reference plasma has been suggested to overcome this
problem.3 4
In conclusion, the present epidemiological survey revealed an independent and gradual association between poor response to APC and the risk of arterial disease and suggests promotion of advanced atherosclerosis as a main underlying pathomechanism. Biological plausibility, a dose-response type of relation, and the excellent consistency of results for various vascular territories, along with distinct ultrasonographic, pressure index, and clinical end points, all favor significance beyond a purely mathematical relation. The APC ratio was associated with a variety of genetic, environmental, hormonal, and behavioral factors, some of which are potentially modifiable. From a preventive perspective, the absence of a relation between response to APC and advanced atherosclerosis in subjects under long-term aspirin therapy seems interesting, although the reliability of this finding is clearly limited by the observational study design.
Received June 1, 1998; revision received September 25, 1998; accepted October 9, 1998.
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 E. Bonora, S. Kiechl, J. Willeit, F. Oberhollenzer, G. Egger, R. C. Bonadonna, and M. Muggeo Carotid Atherosclerosis and Coronary Heart Disease in the Metabolic Syndrome: Prospective data from the Bruneck Study Diabetes Care, April 1, 2003; 26(4): 1251 - 1257. [Abstract] [Full Text] [PDF]
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 G. Sandusky, D. T. Berg, M. A. Richardson, L. Myers, and B. W. Grinnell Modulation of Thrombomodulin-dependent Activation of Human Protein C through Differential Expression of Endothelial Smads J. Biol. Chem., December 13, 2002; 277(51): 49815 - 49819. [Abstract] [Full Text] [PDF]
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 F-J Munoz-Rodriguez, J-C Reverter, J Font, D Tassies, G Espinosa, R Cervera, F Carmona, J Balasch, M Ingelmo, and A Ordinas Clinical significance of acquired activated protein C resistance in patients with systemic lupus erythematosus Lupus, November 1, 2002; 11(11): 730 - 735. [Abstract] [PDF]
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 P. Lochow, A. Schwartzbard, J. Guest, C. Ripps, D. Matalon, R. Gambetta, P. A. Tunick, and S. Sedlis Elevated Prothrombin and Activated Protein C Resistance in Patients with Thoracic Aortic Atheroma Angiology, July 1, 2002; 53(4): 423 - 428. [Abstract] [PDF]
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K. Kario, T. Sakata, T. Miyata, S. Kiechl, and J. Willeit Is Acquired Activated Protein C Resistance a Cardiovascular Risk? • Response Circulation, March 28, 2000; 101 (12): e120 - e120. [Full Text] [PDF]
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 D. A. Lane and P. J. Grant Role of hemostatic gene polymorphisms in venous and arterial thrombotic disease Blood, March 1, 2000; 95(5): 1517 - 1532. [Full Text] [PDF]
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 J. Willeit, S. Kiechl, F. Oberhollenzer, G. Rungger, G. Egger, E. Bonora, M. Mitterer, and M. Muggeo Distinct Risk Profiles of Early and Advanced Atherosclerosis : Prospective Results From the Bruneck Study Arterioscler Thromb Vasc Biol, February 1, 2000; 20(2): 529 - 537. [Abstract] [Full Text] [PDF]
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E.M. Faioni, F. Franchi, P. Bucciarelli, M. Margaglione, V. De Stefano, G. Castaman, G. Finazzi, and P.M. Mannucci Coinheritance of the HR2 Haplotype in the Factor V Gene Confers an Increased Risk of Venous Thromboembolism to Carriers of Factor V R506Q (Factor V Leiden) Blood, November 1, 1999; 94(9): 3062 - 3066. [Abstract] [Full Text] [PDF]
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