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Methods
Study Population
This study is part of a large, ongoing French prospective study aimed
at evaluating the risk of dementia attributable to vascular disorder.
Detailed methodology has been previously described.13 Briefly,
Received on: October 21, 2010; final version accepted on: March 16, 2011.
From Institut National de la Sante et de la Recherche Medicale (INSERM), Centre for Research in Epidemiology and Population Health, U1018,
Hormones and Cardiovascular Disease, Villejuif, France (L.C., P.-Y.S.); University Paris-Sud, UMRS 1018, Villejuif, France (L.C., P.D., P.-Y.S.);
INSERM Unit 897 (L.C., J.-F.D.) and University Hospital Center (J.-F.D.), Universite Victor Segalen Bordeaux II, Bordeaux, France; Assistance Publique
des Hopitaux de Paris, Laboratoire dhematologie, Hopital Europeen Georges Pompidou, Paris, France (M.A.-G.); INSERM Unit 765, Paris, France
(M.A.-G.); Dijon Stroke Registry, EA4184, University Hospital and Faculty of Medicine of Dijon, Dijon, France (Y.B.); INSERM Unit 708,
Neuroepidemiology, Paris, France (C.S.); Department of Neurology, Hopital Lariboisie`re, Assistance Publique des Hopitaux de Paris, Paris, France (C.S.);
INSERM Unit 888, Montpellier, France (K.R.); University of Montpellier I, Montpellier, France (K.R.); Faculty of Medicine, Imperial College London
(K.R.).
Correspondence to Laure Carcaillon, INSERM-CESP U1018 Eq08, 16 av PV Couturier, 94807 Villejuif Cedex, France. E-mail laure.carcaillon@inserm.fr
2011 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
DOI: 10.1161/ATVBAHA.111.223453
1446
June 2011
between 1999 and 2001, 9294 men and women were recruited in 3
French cities (Bordeaux, Dijon, and Montpellier). Subjects had to be
registered on electoral rolls, aged 65, and not institutionalized. The
protocol of the Three-City study was approved by the Consultative
Committee for Protection of Persons Participating in Biomedical
Research of the University Hospital of Kremlin-Bicetre (Paris), and
all subjects signed a legal consent form.
Since their recruitment, subjects have been followed every 2 years
for the detection of cardiovascular disease and dementia. For the
present analysis, we used data up to the second follow-up (approximately 4 years after inclusion).
Event Ascertainment
At baseline, history of vascular disease and cardiovascular risk
factors were assessed during the face-to-face interview. History of
MI, angina pectoris, and stroke were identified, and subjects who
reported a past history of stroke underwent further examination. For
the study of CHD, subjects with previous angina pectoris or MI were
excluded (n115). Similarly, for the study of incident AIS, subjects
with previous stroke were excluded (n51).
An independent committee of trained physicians reviewed all
possible cases of CHD. A CHD case was defined as a hospitalized
stable or unstable angina pectoris, a coronary dilatation or artery
bypass, a hospitalized MI, or a validated CHD death (coded I210 to
I219, I251 to I259, I461, and R960 according to the International
Classification of Diseases 10). After 4 years of follow-up, the total
number of new CHD cases was 216. The 4-year cumulative
incidence of CHD in the whole cohort was 0.79%.
Similarly, an independent panel of experts reviewed all possible
cases of stroke. The definition of stroke is the rapid onset of a
neurological deficit confirmed by a lesion compatible with an acute
stroke on tomography or magnetic resonance imaging of the brain.
AIS and hemorrhagic stroke were differentiated using brain imaging.
In this analysis, we focused on the study of AIS. After 4 years of
follow-up, the total number of new AIS cases was 94. The 4-year
cumulative incidence of AIS in the whole cohort was 0.31%. In
addition, subtypes of AIS were classified according to the Trial of
Org10172 in Acute Stroke Treatment classification.14 In this study,
AIS was categorized as cardioembolic, atherothrombotic, lacunar
(simple or multiple), iatrogenic, or undetermined. Ischemic fatal and
nonfatal strokes were included in our analyses.
Case-Cohort Design
Recently, a case-cohort study15 has been established within the
Three-City study to investigate the effect of biological markers on
the risks of cardiovascular disease or dementia. The methodology
of this design has been described in detail elsewhere.16 In brief, a
case-cohort design involves a randomly selected sample from the
initial cohort, as well as all incident cases of this cohort. In practice,
we randomly selected 1254 subjects from the initial cohort, stratified
by center, sex, and age and for whom blood sample had been
collected. For the study of cardiovascular events, we added all
incident cases of CHD and stroke occurring within the first 4 years
of follow-up who had not already been selected in the random
sample (n166 and n103, respectively). Similarly, all remaining
incident cases of dementia were added to the study sample (n218)
but are not included in our present analysis.
Statistical Analysis
Characteristics of subjects included in the random sample are given
as meanSD for continuous variables and as frequency and percentages for categorical variables. Mean (SD) of ETP and peak height are
given according to subject characteristics. Associations were tested
using univariate linear regression models, with thrombin generation
measures as the dependent variable. ETP and peak height generation
were log-transformed to approximate normal distribution. Regarding
cardiovascular risk factors at inclusion, tobacco use was studied in 2
categories (never smoker or ever smoker). Hypertension was defined
as a systolic blood pressure 160 mm Hg, a diastolic blood pressure
95 mm Hg, or the subject taking an antihypertensive drug. Hypercholesterolemia was considered present if the level of cholesterol
was 6.20 mmol/L. Diabetes included a diagnosis of diabetes,
fasting blood glucose of 7 mmol/L, or a treatment for diabetes. BMI
was studied as a continuous variable or dichotomized as BMI 27.5
kg/m2 or 27.5 kg/m2, which has been recommended as a cut-off for
preobesity/obesity by the World Health Organization.18 In addition,
Pearson correlations were measured between hemostatic parameters
and thrombin generation. Risks of CHD and AIS associated with
ETP and peak height was assessed using weighted Cox proportional
hazards regressions with modification of the standard errors based on
robust variance estimates to take into account case-cohort design.15
The risk of incident CHD was measured for all CHD cases, as well
as by disease severity (MI/angina pectoris, coronary dilatation, or
artery bypass). Age was used as the time scale in all analyses; thus,
the hazard ratio (HR) was always adjusted for age. The model used
also took into account late entry.19 To assess the linearity of the
relation between both thrombin generation parameters and the risk of
CHD and AIS, we used tests based on the difference in the log
likelihood between 2 models of prediction (one with 3 dummy
variables corresponding to the quartiles of the parameter distribution
and the other including the continuous variable). All tests were not
significant, allowing us to not reject the hypothesis of linearity. HR
are thus displayed for a 1 SD increase of ETP or peak height in crude
and adjusted models. Adjustments were made for cardiovascular risk
factors, including sex, tobacco use, diabetes, hypertension, hypercholesterolemia, and BMI. Finally, the differential effect of ETP and
peak height according to gender was tested using both multiplicative
and additive interaction scale. Regarding the assessment of additive
interactions, we measured the relative excess risk due to interaction.20,21 SAS statistical software version 9.1 was used for statistical
analyses (SAS Institute Inc., Cary, NC).
Carcaillon et al
1447
Results
Thrombin Generation and
Subjects Characteristics
Table 1 displays ETP and peak height values according to
subjects characteristics. Overall, the mean of ETP was 1785
nmol/L per minute (SD288), and the mean of peak height
was 337 nmol/L (SD50.3). We found positive and significant associations of BMI, hypercholesterolemia, and prothrombin G20210A mutation with ETP and peak height. The
correlations between ETP and fibrinogen and D-dimer were
r0.21 (P0.0001) and r0.17 (P0.015), respectively.
Similarly, correlations between peak height and fibrinogen
and von Willebrand factor were r0.19 (P0.0001) and
r0.11 (P0.001), respectively.
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June 2011
Table 1. ETP and Peak Height According to Baseline Characteristics of the Subjects, Random
Sample (n1177)
Age, years
75
75
BMI, kg/m2
27.5
27.5
Sex
Male
Female
Diabetes
No
Yes
Hypertension
No
Yes
Hypercholesterolemia
No
Yes
Ever smoker
No
Yes
FV Leiden mutation
No
Yes
FII G20210A mutation
No
Yes
Atrial fibrillation
No
Yes
Hormone use
Nonuser
Oral estrogen use
Transdermal estrogen use
Prevalent diseases
Dementia
Stroke
CHD
Hemostatic Variables
Fibrinogen, g/L
D-Dimers, ng/mL
von Willebrand factor, %
Peak, nmol/L
Random
Sample, n (%)
Mean (SD)
P Value*
Mean (SD)
P Value*
677 (57.5)
500 (42.5)
1808 (282)
1574 (294)
0.0001
340 (48.3)
333 (52.6)
0.019
843 (72.2)
325 (27.8)
1763 (285)
1842 (286)
0.0001
335 (50.9)
343 (48.5)
0.017
456 (38.7)
721 (61.3)
1773 (285)
1793 (290)
0.325
332 (48.6)
341 (51.0)
0.024
1067 (89.8)
108 (9.2)
1789 (288)
1747 (292)
0.182
337 (50.2)
335 (51.0)
0.792
468 (39.8)
709 (60,2)
1775 (287)
1792 (289)
0.313
336 (50.7)
338 (50.0)
0.360
764 (64.9)
413 (35.1)
1749 (279)
1852 (293)
0.0001
334 (50.0)
343 (50.2)
0.006
724 (62.4)
436 (37.6)
1791 (289)
1782 (288)
0.735
341 (51.6)
333 (47.6)
0.070
1098 (95.6)
51 (4.4)
1788 (291)
1744 (240)
0.425
337 (51.0)
335 (36.0)
0.921
1108 (96.6)
39 (3.4)
1772 (268)
2189 (500)
0.0001
336 (47.8)
375 (89.3)
0.035
855 (97.6)
21 (2.4)
1796 (274)
1763 (345)
0.351
341 (46.5)
334 (66.2)
0.207
503 (82.6)
27 (4.4)
79 (13.0)
1801 (283)
1866 (280)
1775 (286)
0.350
341 (49.3)
356 (57.1)
340 (44.0)
0.376
24 (2.0)
51 (4.4)
115 (9.8)
1614 (243)
1780 (340)
1768 (300)
0.093
0.970
0.703
318 (51.5)
339 (46.4)
333 (49.2)
0.346
0.516
0.973
m (SD)
P Value*
P Value*
3.3 (0.7)
747.0 (763.4)
136.5 (41.3)
0.21
0.07
0.003
0.0001
0.015
0.907
0.19
0.04
0.11
0.0001
0.204
0.001
*P for association between log of ETP and the determinant, linear regression model.
Heterozygous.
Atrial fibrillation has been defined at inclusion by electrocardiogram criteria and by self-report of this condition.
Among women aged 80 years, P value from ANOVA.
P adjusted for age.
respectively) but not in men. Probability values for interaction measured on a multiplicative scale were 0.04 and 0.08
for ETP and peak height, respectively. We also found
evidence of a departure from additivity for ETP and sex. The
relative excess risk due to interaction of both sex and ETP
Carcaillon et al
Table 2.
1449
All CHD
Control
CHD
HR (95% CI)
Myocardial infarction
HR (95% CI)
Other causes
HR (95% CI)
GM, IQR
GM, IQR
Crude
Adjusted*
GM, IQR
Crude
Adjusted*
GM, IQR
Crude
Adjusted*
Peak, nmol/L
1000
186
88
98
Discussion
In the present analyses, we evaluated the risk of arterial
disease associated with thrombin generation. We found that
Table 3. Hazard Ratios of AIS for 1 SD Increment in ETP and Peak Height, for the Total
Population and Stratified by Sex
Ischemic stroke
Control
Ischemic stroke
HR (95% CI)
Men
Control
Ischemic stroke
HR (95% CI)
Women
Control
Ischemic stroke
HR (95% CI)
GM, IQR
GM, IQR
Crude
Adjusted*
1074
87
GM, IQR
GM, IQR
Crude
Adjusted*
408
42
GM, IQR
GM, IQR
Crude
Adjusted*
666
45
P for multiplicative interaction between ETP/peak height and sex on the risk of AIS was 0.04/0.08. Relative risk due
to interaction of ETP/peak height and sex was 1.7/1.8, P0.09/0.33. Significant associations are given in
boldface. GM indicates geometric mean; IQR, interquartile range.
*Adjusted for classical cardiovascular risk factors: center, sex (when appropriate), BMI 27.5 kg/m2, hypertension,
hypercholesterolemia, diabetes, tobacco use (never or ever smoker).
1450
June 2011
Sources of Funding
The Three-City Study is conducted under a partnership agreement
between the Institut National de la Sante et de la Recherche Medicale
(INSERM), the Victor Segalen-Bordeaux II University, and SanofiAventis. The Fondation pour la Recherche Medicale funded preparation and initiation of the study. The Three-City Study is also
supported by the Caisse Nationale Maladie des Travailleurs Salaries,
Direction Generale de la Sante, Mutuelle Generale de lEducation
Nationale, Institut de la Longevite, Conseils Regionaux of Aquitaine
and Bourgogne, Fondation de France, and Ministry of ResearchINSERM Programme Cohortes et collections de donnees biologiques. Biological assays regarding hemostatic parameters was
supported by a grant from the Agence Nationale de la Recherche
(ANR 2007-LVIE-005-01).
Disclosures
None.
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1451
Increased Thrombin Generation Is Associated With Acute Ischemic Stroke but Not With
Coronary Heart Disease in the Elderly: The Three-City Cohort Study
Laure Carcaillon, Martine Alhenc-Gelas, Yannick Bejot, Christian Spaft, Pierre Ducimetire,
Karen Ritchie, Jean-Franois Dartigues and Pierre-Yves Scarabin
Arterioscler Thromb Vasc Biol. 2011;31:1445-1451; originally published online March 31,
2011;
doi: 10.1161/ATVBAHA.111.223453
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
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