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 Ducimetie`re,
Karen Ritchie, Jean-Francois Dartigues, Pierre-Yves Scarabin
ObjectiveA high thrombin generation level has been associated with the risk of venous thrombosis. Whether changes in
this biomarker are relevant to arterial disease remains unknown. We investigated the association of thrombin generation
with coronary heart disease (CHD) and acute ischemic stroke (AIS) in the elderly.
Methods and ResultsWe used data from the Three-City study, a prospective cohort including 9294 subjects aged 65
years. After 4 years of follow-up, a case-cohort study was established. Using the calibrated automated thrombography
method, endogenous thrombin potential and peak height were measured in plasma samples of all CHD and AIS cases
and a random sample of 1177 controls. We did not find any significant association between thrombin generation and
CHD. In multivariate analyses, high levels of endogenous thrombin potential and peak height were associated with an
increased risk of AIS (hazard ratio1.16 [95% CI, 0.90 to 1.50] and 1.31 [95% CI, 1.01 to 1.69] for a 1 SD increase,
respectively). Data also suggested that these associations might be more important in women (hazard ratio1.55 [95% CI,
1.05 to 2.33] and 1.71 [95% CI, 1.11 to 2.63], respectively) than in men (P for interaction0.04 and 0.08, respectively).
ConclusionThrombin generation emerges as an independent predictor of AIS, particularly in women. Hypercoagulability
may have an important role in the pathogenesis of AIS. (Arterioscler Thromb Vasc Biol. 2011;31:1445-1451.)
Key Words: aging coronary heart disease epidemiology stroke thrombin generation

hrombin is the key product of the coagulation system and

is produced following activation of factor X by tissue
factor/factor VIIa. This in turn activates platelets and other
coagulation factors, leading to clot formation.1 Recently, an
efficient method to measure thrombin generation in vitro has
been developed.2 This assay is an automated method (calibrated automated thrombography) in which tissue factor,
phospholipids, and CaCI2 are used to trigger the coagulation.
Using the calibrated automated thrombography method
may provide an efficient tool to detect hypercoagulatibility,
and several studies have reported a relationship between
thrombin generation and the incidence of venous thrombosis3 6 and recurrent venous thrombosis.79 There is also
increasing evidence of an association between venous thrombosis and subsequent atherothrombosis,10,11 with both conditions sharing risk factors. Moreover, factors associated with
thrombin generation have been associated with the risk of

myocardial infarction (MI) and stroke.12 However, to date no

study has investigated whether thrombin generation may also
be associated with the risk of coronary heart disease (CHD)
and stroke. Establishing a relationship between thrombin
generation and cardiovascular diseases would not only contribute to our current understanding of the physiopathological
processes leading to coronary thrombosis and acute ischemic
stroke (AIS) but also suggest a new approach to prevention.
Using data from a large prospective cohort study (the
Three-City study), we have investigated the relationship
between thrombin generation and incidence of CHD and AIS.

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

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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.

Blood Collection and Hemostatic

Factors Measurements
At baseline, blood samples were collected for 90% of the entire
cohort. Subjects included in the case-cohort design had all agreed to
venipuncture. Citrated plasma samples were obtained after 1 centrifugation (3000g at 4C) and immediately stored at 80C in 500-L
plastic tubes. Fibrinogen was measured using the kinetic method of
Clauss (Dade Behring, Paris, France), and von Willebrand factor and
fibrin D-dimer by ELISAs from Stago (Asserachrom von Willebrand
factor and D-Di, Asnieres sur Seine, France).
DNA was extracted centrally by the salt precipitation method, and
purified genomic DNA was stored at 20C. Factor V Leiden

mutation and G20210A prothrombin mutation (rs1799963) were

systematically genotyped for the overall cohort. Genotyping was
centrally performed by KBioscience (Hoddesdon Herts, United
Kingdom) using the KBioscience competitive Allele-Specific polymerase chain reaction single-nucleotide polymorphism genotyping
system with fluorescence reasonance energy transfer quencher cassette oligos (KASPar).17

Thrombin Generation Measurements

Thrombin generation was measured at baseline using the calibrated
automated thrombography method as described by Hemker et al2 at
final concentration of 5 pmol/L tissue factor (Innovin, Siemens) and
4 mol/L phospholipids (MP reagent, Thrombinoscope). Thrombin
calibrator and the fluorogenic substrate FluCa were from Thrombinoscope (Maastricht, the Netherlands). After exclusion of subjects
taking anticoagulant therapy (n48) and subjects for whom thrombin generation could not be assessed (n29), thrombin generation
parameters were successfully measured for 1177 subjects from the
random sample, 157 additional cases of CHD, and 76 additional AIS
cases (Figure). Excluded subjects did not differ from the retained
sample in term of age, sex, body mass index (BMI), or other
cardiovascular risk factors.
We studied 2 thrombogram parameters: peak height of thrombin
generation and endogenous thrombin potential (ETP), which corresponds to the area under the thrombogram curve.

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).

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Thrombin Generation and Ischemic Stroke

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Figure. Flow chart representing the constitution

of the case-cohort study for the investigation of
the risk of CHD and AIS in relation to thrombin
generation.

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.

Thrombin Generation and CHD

During the 4 years of follow-up, among subjects included in
our analysis, 186 subjects had a CHD event (15.7%). Of
these, MI was present in 88 subjects, and the others presented
angina pectoris, coronary dilatation, or artery bypass. Overall,
36 events were fatal. We did not find a significant association
between thrombin generation and the HR of total CHD, MI,
or other CHD (Table 2). After adjustment for traditional
cardiovascular risk factors, the HR of CHD was 1.09 (95%

CI, 0.92 to 1.28) for a 1 SD elevation in ETP and 1.02 (95%

CI, 0.89 to 1.17) for a 1 SD elevation in peak height.
A sensitivity analysis consisting of the exclusion of prevalent cases of stroke (n49) from our sample did not show
different results. The HR values of CHD associated with a 1
SD increase in ETP and peak were 1.11 (95% CI, 0.93 to
1.33) and 1.03 (95% CI, 0.90 to 1.18), respectively.

Thrombin Generation and AIS

Overall, in our sample, 87 subjects had an AIS during the
follow-up (7.5%) 3 of which were fatal. We found a borderline significant association of ETP and peak height with the
HR of AIS (HR1.16 [95% CI, 0.90 to 1.48] and HR1.27
[95% CI, 1.00 to 1.62], respectively) (Table 3). Further
excluding prevalent CHD cases (n121) from our sample did
not product different results. The HR values of AIS associated with a 1 SD increase in ETP and peak were 1.21 (95%
CI, 0.90 to 1.62) and 1.34 (95% CI, 1.02 to 1.76),
respectively.
In addition, we found some evidence of an interaction
between sex and thrombin generation on the risk of AIS. The
adjusted HR of AIS associated with both ETP and peak
height was significantly increased in women (HR1.55 [95%
CI, 1.03 to 2.33] and HR1.71 [95% CI, 1.11 to 2.63],

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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, %

ETP, nmol/L per Minute

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

was 1.7 (95% CI, 38.0 to 0.26, P0.09). Regarding peak

height, the relative excess risk due to interaction was 1.8
(95% CI, 20.5 to 5.56, P0.33). These results indicate, for
example, that because of the interaction between sex and
ETP, the HR for AIS was 1.7 lower than expected from the

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Table 2.

Thrombin Generation and Ischemic Stroke

1449

Hazard Ratios of CHD for 1 SD Increment in ETP and Peak Height

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*

ETP, nmol/L per Minute

Peak, nmol/L

1000
186

1765 (1620 to 1940)

1772 (1604 to 1939)
1.06 (0.92 to 1.23)
1.09 (0.92 to 1.28)
1772 (1588 to 1940)
1.05 (0.86 to 1.27)
1.04 (0.85 to 1.28)
1785 (1610 to 1947)
1.05 (0.84 to 1.29)
1.12 (0.89 to 1.41)

333.0 (308.0 to 365.0)

330.3 (301.9 to 357.8)
1.00 (0.89 to 1.13)
1.02 (0.89 to 1.17)
333.0 (301.9 to 357.8)
1.03 (0.88 to 1.21)
1.03 (0.87 to 1.22)
331.0 (311.1 to 351.8)
0.95 (0.81 to 1.11)
0.99 (0.83 to 1.17)

88

98

GM indicates geometric mean; IQR, interquartile range.

*Adjusted for cardiovascular risk factors: center, sex, BMI 27.5 kg/m2, hypertension, hypercholesterolemia,
diabetes, tobacco use (never or ever smoker).
Other causes include angina pectoris, coronary dilation, or artery bypass.

addition of the separate effects of sex and ETP. Being male

seemed to protect subjects with increased level of ETP from
the occurrence of AIS.
Stratified analyses showed that the HR of AIS was consistently associated with an increased thrombin generation in all
subgroups of women at higher or lower risk for cardiovascular disease (data not shown). Finally, when we studied the
subtype of ischemic stroke, the risks associated with ETP and
peak height were particularly elevated for cardioembolic
stroke (adjusted HR2.05 [95% CI, 0.87 to 4.80] and 2.86
[95% CI, 1.08 to 7.52], respectively), although they were not
significant for other subtypes.

Discussion
In the present analyses, we evaluated the risk of arterial
disease associated with thrombin generation. We found that

elevated thrombin generation was an independent risk factor

for AIS but not for CHD. This result appeared more relevant
among women, although the interaction was borderline
significant.
Several methods have been proposed to measure thrombin
generation, and differences in assays may at least partially
explain discrepancies in previous studies. We did, however,
observe a number of expected associations between thrombin
generation and biological variables. Indeed, we noted a strong
relationship between prothrombin G20210A mutation and
higher level of thrombin generation. This result is consistent
with other studies4,6,8,22 and has strong biological support. In
reality, prothrombin G20210A mutation is associated with
higher levels of plasma prothrombin, which is a direct
determinant of thrombin generation. We also found that
thrombin generation was positively correlated with fibrino-

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)

ETP, nmol/L per Minute

Peak, nmol/L per Minute

GM, IQR
GM, IQR
Crude
Adjusted*

1074
87

1755 (1604 to 1939)

1791 (1588 to 1998)
1.16 (0.90 to 1.48)
1.16 (0.90 to 1.50)

333.6 (308.0 to 365.0)

340.4 (320.5 to 365.0)
1.29 (1.00 to 1.67)
1.31 (1.01 to 1.69)

GM, IQR
GM, IQR
Crude
Adjusted*

408
42

1755 (1620 to 1940)

1720 (1572 to 1978)
0.92 (0.68 to 1.24)
0.88 (0.63 to 1.24)

327.0 (304.9 to 357.8)

330.2 (301.8 to 361.4)
1.11 (0.86 to 1.43)
1.04 (0.81 to 1.34)

GM, IQR
GM, IQR
Crude
Adjusted*

666
45

1755 (1604 to 1940)

1863 (1636 to 1998)
1.45 (1.00 to 2.08)
1.55 (1.03 to 2.33)

333.6 (311.0 to 372.4)

357.8 (320.5 to 391.5)
1.61 (1.08 to 2.40)
1.71 (1.11 to 2.63)

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).

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gen, which is a positive determinant of thrombin formation.23

Regarding D-dimer, it is interesting to note that the correlation
with ETP was slightly lower than between fibrinogen and
ETP. This result is in accordance with recent findings
suggesting that ETP and D-dimer may reflect different aspects
of the coagulation system.7,8 ETP represents the amount of
thrombin that could potentially be generated, whereas
D-dimer is a marker of ongoing coagulation in vivo.
Most importantly, we found a significant association between thrombin generation and AIS. This result has never
been reported before and suggests an important role of
hypercoagulability in the pathogenesis of AIS. This result is
in accordance with some previous studies, which showed a
positive association between hemostatic variables and the risk
of subsequent stroke.24,25 However, we failed to find a
significant association between thrombin generation and the
risk of CHD. First, we cannot exclude a lack of statistical
power to detect a true but small association between thrombin
generation and the risk of CHD. Second, our result is in
accordance with evidence of discrepancy in risk factors
between AIS and CHD. For example, oral use of hormone
therapy among postmenopausal women, strongly associated
with a rise in coagulation factors, results in an increased risk
of stroke26 28 whereas a protective effect of these treatments
has been suggested regarding the risk of CHD.29 Finally, a
recent study of arterial disease among women30 reported that
the effect of lupus anticoagulants, a type of antiphospholipid
antibodies known to induce a prothrombotic state, was more
pronounced on the risk of AIS than on the risk of MI.
Together with our data, these results are in favor of a stronger
implication of a hypercoagulable state on the risk of AIS than
on the risk of CHD.
We found a possible differential association of thrombin
generation with the risk of AIS according to sex. Many
studies have emphasized sex-specific risk profiles of stroke,
acute stroke presentation, and stroke etiology.31 For example,
hormone therapy could explain this difference. As we mentioned above, hormone therapy increases the risk of
stroke.26 28 Moreover, oral estrogen use in postmenopausal
women has been associated with higher risk of venous
thromboembolism through increased coagulation and inflammation.32 In our study, we observed an increased level of
thrombin generation in women using oral estrogen versus
nonusers. However, because of the small number of women
using oral estrogen, we were unable to reliably evaluate the
impact of estrogen use on the association between thrombin
generation and AIS. This issue remains to be addressed in
future studies. Another important determinant of the sexspecific risk of stroke is atrial fibrillation (AF). Although it is
more prevalent in men than women,33 growing evidence
supports the idea that AF is a greater risk factor for stroke in
women than men.34 36 Our data are consistent with this result,
as we found an elevated risk of AIS associated with AF, and
this risk was more pronounced in women than in men (data
not shown). Combined with AF, a high potential of thrombin
generation may increase the risk of AIS. In this manner,
thrombin generation would be a marker of thromboembolism
rather than atherothrombosis. We could not test this hypothesis because there were too many missing value for the status

of AF. However, the fact that we found a positive association

between thrombin generation and cardioembolic stroke but
not with other subtypes in women gives additional support to
this hypothesis. Of note, if thrombin generation is a marker of
thromboembolism rather than atherothrombosis, it could also
explain why we did not find any association between thrombin generation and CHD.
Our study presents some strengths and some limitations.
First, the prospective nature of the design and the high
participation rate during the 4 years of follow-up (92%) are 2
major strengths. In addition, incident events were carefully
validated by an independent committee of experts. Regarding
the methodology used to assess thrombin generation, we
chose a concentration of tissue factor that was not sensitive to
contact factor activation but relatively highly efficient to
detect hypercoaguability.37 40 The main limitation of our
study is the small number of incident cases, which may result
in a lack of statistical power. In our study, the low incidence
of events may have been due to the restriction of our study to
community-dwelling residents with better physical and cognitive abilities than the general elderly population.13 As a
consequence, we may have underestimated the association of
thrombin generation with the risk of CHD. Therefore, investigating the association of thrombin generation with the risk
of arterial disease is needed in other studies. Finally, given
our study sample, the association found between thrombin
generation and AIS pertains only to subjects older than 65.
As growing evidence supports an association between
elevated thrombin generation and risk of venous thrombosis,
our results regarding AIS, particularly in women, constitutes
a new and interesting finding requiring validation in other
populations. If our results are confirmed, prevention of AIS
should target the diminution of the thromboembolic risk
together with prevention of atherosclerosis.

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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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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