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Journal of Thrombosis and Haemostasis, 2: 445451

ORIGINAL ARTICLE

Homocysteine and markers of coagulation and endothelial cell


activation
V. E. A. GERDES,* H. A. KREMER HOVINGA, H. TEN CATE,* M. R. MACGILLAVRY,* A. LEIJTE,
P . H . R E I T S M A , D . P . M . B R A N D J E S * and H . R . B U L L E R O N B E H A L F O F T H E A M S T E R D A M V A S C U L A R
MEDICINE GROUP
*Departments of Internal Medicine and Clinical Chemistry, Slotervaart Hospital, Department of Vascular Medicine and Laboratory for
Experimental Internal Medicine, Academic Medical Center, Amsterdam, the Netherlands

To cite this article: Gerdes VEA, Kremer Hovinga HA, Ten Cate H, MacGillavry MR, Leijte A, Reitsma PH, Brandjes DPM, Buller HR on behalf of the
Amsterdam Vascular Medicine Group. Homocysteine and markers of coagulation and endothelial cell activation. J Thromb Haemost 2004; 2: 44551.

Introduction
Summary. Objective: In vitro studies suggest an inuence of
hyperhomocysteinemia on the coagulation system, but the
inuence of mild hyperhomocysteinemia in vivo is unclear.
Methods and results: We studied the relation between homocysteine and markers of coagulation activation and endothelial
cell activation in 279 patients with established atherosclerotic
disease. In addition, we performed an investigator-blinded
placebo-controlled cross-over study to investigate the inuence
of acute hyperhomocysteinemia by oral methionine load on
these markers in 20 healthy volunteers. In the atherosclerotic
patients prothrombin fragment F1+2 and soluble thrombomodulin (sTM) were associated with homocysteine in univariate analyses (P 0.003 and P 0.001, respectively), but not in
multivariate analyses. Age, creatinine and MTHFR C677T
polymorphism were major determinants of homocysteine
concentration. MTHFR C677T polymorphism status was not
associated with F1+2 and sTM. Median homocysteine
concentrations increased in the healthy volunteers after methione load. However, after methionine load or after placebo,
we did not observe different plasma concentrations of
F1+2 (0.9 nmol L)1 vs. 0.9 nmol L)1, P 0.39), d-dimer
(153 lg L)1 vs. 151 lg L)1, P 0.63) and von Willebrand
factor (103% vs. 107%, P 1.00). Conclusion: These results
provide evidence against a major effect of mild hyperhomocysteinemia on activation of the coagulation system and endothelial cell activation in vivo.
Keywords: atherosclerosis, coagulation, endothelium, homocysteine, methionine load.

Correspondence: V. E. A. Gerdes, Department of Vascular Medicine,


Academic Medical Center, Meibergdreef 9, Postbus 22660, 1100 DD
Amsterdam, the Netherlands.
Tel.: +31 20 566 9111; fax: +31 20 566 9158; e-mail: v.e.gerdes@amc.
uva.nl
Received 22 October 2003, accepted 5 November 2003
 2004 International Society on Thrombosis and Haemostasis

Mild hyperhomocysteinemia has been associated with arterial


and venous vascular disease [1]. Patients with a myocardial
infarction, ischemic stroke or peripheral arterial disease have
higher homocysteine levels, fasting or after a methionine load,
compared with healthy controls [2]. Higher homocysteine
concentrations have been associated with a greater risk of
future ischemic stroke and myocardial infarction, in asymptomatic individuals as well as patients with clinically manifest
atherosclerosis [36].
The pathophysiological mechanism that explains the association between homocysteine and cardiovascular disease is
unclear [1,5]. Since the coagulation system is an important
factor in both arterial and venous vascular disease, the
inuence of homocysteine on the coagulation system may be
one of the possible mechanisms. In vitro studies have shown
different procoagulant effects of homocysteine on vascular
endothelial cells, including activation of factor (F)V, inhibition
of thrombomodulin-dependent protein C activation, impairment of von Willebrand factor (VWF) secretion and induction
of tissue factor [710]. Tissue factor may be induced by
homocysteine on monocytes as well [11].
There are only sparse data about the relationship between
homocysteine and markers of coagulation in vivo. Acute
hyperhomocysteinemia after oral methionine load induced
increased concentrations of markers of coagulation and
endothelial cell activation [12]. An association between markers
of coagulation activation has been described in patients with
premature arterial disease, in patients with venous thrombosis
and in patients with acute coronary syndromes [1315]. A weak
association between hemostatic factors and homocysteine was
described in individuals free of arterial disease [16].
The methylenetetrahydrofolate reductase (MTHFR) C677T
polymorphism has a clear inuence on homocysteine concentrations. Homozygotes have approximately 25% higher homocysteine levels and the inuence of folate deciency on
homocysteine levels is increased in these persons [1719].
However, the role of this polymorphism in atherosclerosis is

446 V. E. A. Gerdes et al

controversial. The rst reports suggested a higher prevalence


of homozygotes among patients with a myocardial infarction
[2022]. This could not be conrmed by subsequent studies and
a meta-analysis and is in contrast with the clear association
between hyperhomocysteinemia and coronary artery disease
[17,2325]. A similar divergence in associations between
homocysteine and arterial wall thickness, and between
MTHFR and arterial wall thickness was reported [26,27]. This
possible discrepancy between homocysteine and the MTHFR
polymorphism, with a clear inuence on homocysteine concentrations, is intriguing. It can be interpreted as an argument
against a causal relationship between mildly elevated homocysteine and vascular disease.
We investigated the relation between homocysteine and
markers of coagulation and endothelial cell activation on the
one hand, and between homocysteine and several parameters
of atherosclerotic disease on the other hand in a cohort of
patients with established atherosclerotic disease. In addition,
we performed a placebo-controlled, investigator-blinded, randomized crossover trial in healthy individuals to test the
inuence of acute hyperhomocysteinemia after oral methionine
load on markers of coagulation activation and endothelial cell
activation.
Methods
Subjects

Atherosclerotic disease Consecutive patients with clinical


manifestations of atherosclerosis were recruited in two
teaching hospitals and classied according to the previous
event: myocardial infarction, ischemic stroke or peripheral
arterial disease. Only patients with a recent onset of myocardial
infarction ( 1 month) or ischemic stroke ( 6 months) were
included. Myocardial infarction was dened as typical chest
pain for more than 20 min in combination with laboratory or
ECG ndings consistent with myocardial infarction. Ischemic
stroke was dened as acute neurological decit persisting at
least 1 week. Intracerebral hemorrhage was ruled out by an
early computed tomography scan. Peripheral arterial disease
was dened as typical leg pain on walking and an ankle/arm
blood pressure ratio lower than 0.85 in either leg at rest or a
history of surgery for intermittent claudication. Smoking
status, hypertension dened as a diastolic blood pressure of
95 mmHg or current treatment, and hypercholesterolemia
dened as a total plasma cholesterol level of 7 mmol L)1 or
current treatment were registered. Patients receiving
anticoagulant treatment (coumarin or heparin) were not
included.
Healthy individuals (methionine load) Healthy volunteers
were seen on two visits with an interval of 1 week. They were
randomly assigned to oral methionine load (l-methionine
100 mg kg)1 in fruit juice) at the rst or second visit. Blood
samples were drawn after a 12-h overnight fast and 4 h after
methionine load or placebo (fruit juice only). Participants were

allowed to drink only water during the experiment.


Administration of methionine and placebo was done in a
separate room. The research nurses who took the venous blood
samples, laboratory personel and investigators were blinded
during the experiment and the code was broken after
laboratory investigations were completed.
Both studies were approved by the Ethical Review Boards of
the participating study centers and all patients gave their
written informed consent to participate.
Laboratory procedures

Homocysteine and coagulation markers were assessed in blood


samples drawn 3 months after the acute event. Blood samples
were collected after an overnight fast (> 10 h) by venepuncture from the antecubital vein and collected in tubes containing
EDTA for homocysteine and citrate for coagulation assays.
Homocysteine concentration was determined by high-performance liquid chromatography [28]. Commercial ELISAs for
soluble thrombomodulin (sTM) and prothrombin fragment
F1+2 (F1+2) were used (Diaclone, Besancon, France and
Dade Behring, Marburg, Germany, respectively). Lipids and
creatinine were assessed by routine laboratory methods.
Extraction of genomic DNA from peripheral leukocytes in
citrated blood was performed using a QIAamp blood kit
(Qiagen, Hilden, Germany). The primers and polymerase chain
reaction (PCR) conditions used to determine MTHFR genotype were described previously [29].
In the healthy individuals homocysteine and prothrombin
fragment F1+2 were measured with the same methods as in
the patients with atherosclerotic disease. Commercial ELISAs
for D-dimer (Bio Merieux Vidas, Marcy LEtoile, France),
VWF and thrombinantithrombin complexes (Dade Behring)
were used.
B-mode ultrasound

The B-mode ultrasound intima-media thickness (IMT) measurement procedures have been described elsewhere [30].
B-mode ultrasound scans were performed by one sonographer.
An ATL Ultramark IV (Advanced Technology Laboratories,
Bothell, WA, USA) with a High Resolution Linear Array
7.5-MHz transducer was used. Subjects were scanned in the
reclined position. Three right carotid, three left carotid, two
right femoral and two left femoral artery wall segments were
scanned. Images of each arterial wall segment were stored on
S-VHS video tape. IMT of the posterior wall segments was
measured off-line [31]. The average of the 10 segments was used
for analysis. The sonographer and image analyst were blinded
to the clinical status of the subjects.
Statistical analysis

All analyses were performed with the Statistical Package for


Social Science, version 10.0 (SPSS, Chicago, IL, USA). To
explore the distribution of risk factors and laboratory variables
 2004 International Society on Thrombosis and Haemostasis

Homocysteine and markers of coagulation and endothelial cell activation 447

the patients with atherosclerotic disease were divided into four


subgroups dened by quartiles of homocysteine concentration.
Frequencies were compared with v2 tests or Fishers exact tests
when applicable. For comparison of variables with a normal
distribution, Students t-test and analysis of variance tests were
used, whereas the MannWhitney U-test and KruskalWallis
test were used for variables with a skewed distribution. To test
for trend in the quartiles, tests for linearity were performed. We
subsequently performed linear regression analyses with F1+2
and sTM as dependent variables. Variables with a skewed
distribution were log-transformed and all variables with a
P-value < 0.10 were selected for the multivariate model. A
P-value of 0.05 was considered to be statistically signicant. In
the healthy volunteers study comparison of paired laboratory
variables was performed using the non-parametric Wilcoxon
signed ranks test.
Results
Homocysteine in patients with atherosclerotic disease

A total of 307 consecutive patients with manifest atherosclerotic disease were included. Homocysteine levels were determined in 279 patients from whom fasting plasma samples were
available. Of these 279 patients, 95 had a recent ischemic
stroke, 86 had a recent myocardial infarction and 98 had
peripheral arterial disease. The mean age was 62.9 years.
Median homocysteine concentration was 15.1 lmol L)1. We
divided the group in quartiles according to homocysteine

concentration. Patient characteristics of the whole study group


and the quartiles according to homocysteine concentration are
shown in Table 1. Age, creatinine, F1+2, and sTM values
were associated with homocysteine concentrations. Other
patient characteristics were not different between the quartiles.
The homocysteine concentrations were similar in patients with
different types of arterial disease: myocardial infarction,
ischemic stroke and peripheral arterial disease (medians
14.3 lmol L)1, 15.4 lmol L)1 and 15.2 lmol L)1, respectively; P 0.25, KruskalWallis test).
B-mode ultrasound IMT measurement was performed in
226 of the 279 patients, of whom homocysteine concentrations
were determined. A slight increase of the median IMT values in
the higher quartiles of homocysteine concentration was
observed. However, this was not statistically signicant, not
even in the trend analysis (P for trend 0.14). This slight
increase is probably attributable to difference in age among the
quartiles.
Homocysteine, F1 + 2 and sTM in patients with
atherosclerotic disease

To test whether the relationship between homocysteine and


F1+2 and sTM was independent, multivariate linear regression analyses with F1+2 and sTM as dependent variables were
performed. In the analysis for F1+2 the univariate model
selected homocysteine (B 0.18, SE 0.06, P 0.003), age, sTM,
gender, IMT and type of arterial disease as explanatory
variables of F1+2. In the multivariate model the association

Table 1 Characteristics: all patients and in quartiles according to homocysteine concentration

n
Age (years)
Male (%)
Diabetes (%)
Smoking (%)
Hypertension (%)
Creatinine (lmol L)1)
Total cholesterol (mmol L)1)
LDL (mmol L)1)
HDL (mmol L)1)
Triglycerides (mmol L)1)
F1+2 (nmol L)1)
sTM (ng mL)1)
IMT (mm)

All patients

II

III

IV

279
63
63
11
85
41
101.7
88.4113.2
6.1
5.46.9
4.0
3.54.7
1.11
0.961.35
1.7
1.22.3
1.33
1.041.73
8.9
7.011.5
0.94
0.771.11

68
58
54
15
78
38
96.0
85.9106.1
6.0
5.46.8
3.9
3.44.7
1.11
0.951.32
1.7
1.22.3
1.09
0.941.41
7.8
6.510.2
0.89
0.751.08

71
62
61
4
87
39
98.1
87.5112.3
6.3
5.77.0
4.0
3.34.6
1.12
0.961.43
1.8
1.22.4
1.34
1.101.67
8.5
6.611.5
0.92
0.721.09

69
64
77
19
86
46
106.0
92.8121.6
6.1
5.46.9
4.1
3.55.0
1.10
0.941.29
1.7
1.42.4
1.25
1.011.88
9.3
7.712.3
1.01
0.781.18

71
68
62
6
87
46
106.1
89.3125.5
6.2
5.46.8
4.1
3.54.7
1.16
0.981.32
1.6
1.32.3
1.47*
1.281.89
9.6*
7.212.8
0.97
0.811.14

sTM, Soluble thrombomodulin; IMT, intima-media thickness. For laboratory values and IMT medians and interquartile ranges are depicted. I:
Homocysteine < 11.8 lmol L)1; II: 11.815.1 lmol L)1; III: 15.118.8 lmol L)1; IV: 18.8 lmol L)1. IMT measurement was performed in 226
patients. P < 0.001; *P < 0.01 (KruskalWallis test).
 2004 International Society on Thrombosis and Haemostasis

448 V. E. A. Gerdes et al

between homocysteine and F1+2 was not statistically signicant any more, whereas age and type of arterial disease were the
main determinants of F1+2 in these patients. In the linear
regression analysis with sTM as dependent variable, univariate
analysis selected age, diabetes, homocysteine (B 4.26, 95%
condence interval 1.77, 6.76, P 0.001), lipoprotein(a),
F1+2, creatinine, IMT and type of arterial disease as variables
associated with sTM. In the multivariate model, homocysteine
was excluded and age, F1+2, lipoprotein(a) and peripheral
arterial disease had an independent relationship with sTM.
MTHFR polymorphism in patients with atherosclerotic disease

In 276 of the 279 patients MTHFR genotype could be


determined in available DNA samples. We observed an allele
frequency of 28%. The prevalence of homozygotes and
heterozygotes of the MTHFR polymorphism was 9% and
39%, respectively. The allele frequency was similar in patients
with different types of arterial disease. Median homocysteine
levels were 14.3 lmol L)1, 16.1 lmol L)1 and 19.8 lmol L)1
for the CC, CT and TT genotype, respectively. This difference
in homocysteine levels was signicant for homozygotes compared with patients with the wild type (P 0.007, Mann
Whitney U-test), as well when we compared the three groups in
Table 2 Homocysteine, markers of coagulation and endothelial cell
activation and intima-media thickness according to MTHFR
polymorphism status
MTHFR
)1

Hcy (lmol L )
F1+2 (nmol L)1)
sTM (ng mL)1)
IMT (mm)

CC (n 145)

TC (n 107)

TT (n 24)

14.3
11.717.5
1.33
1.011.71
9.4
7.112.2
0.96
0.741.14

16.1
11.719.1
1.32
1.061.69
8.2
6.911.1
0.91
0.791.09

19.8*
14.627.9
1.53
1.031.89
9.6
6.413.6
0.97
0.861.13

IMT, Intima media thickness; Hcy, homocysteine; sTM, soluble


thrombomodulin. The medians and interquartile ranges are depicted.
*P < 0.01 (KruskalWallis test).

a KruskalWallis test (P 0.008). In contrast, there was no


association between MTHFR status and F1+2, sTM or IMT
(Table 2). In none of the comparisons, either when we
compared these variables between three groups, or when we
compared these between homozygotes and carriers of the
wild type, were statistically signicant differences observed (all
P-values > 0.18).
Acute hyperhomocysteinemia in healthy volunteers

The group of healthy volunteers consisted of 10 male and 10


female individuals. The mean age was 33 (range 2451)
years. Median fasting homocysteine concentrations were
9.1 lmol L)1. After methionine load and placebo median
homocysteine concentrations were 30.1 and 9.2 lmol L)1,
respectively. Fasting homocysteine concentrations at the second visit were not inuenced by oral methionine load at the
rst visit. There was no effect of acute hyperhomocysteinemia
by the methionine load on VWF, F1+2, thrombinantithrombin complexes or D-dimer concentrations (Table 3).
Discussion
Acute hyperhomocysteinemia by methionine load did not
increase coagulation activation, or VWF concentration in the
healthy volunteers. In our patients with established atherosclerotic disease we did not observe associations between
homocysteine and markers of coagulation activation and
endothelial cell activation in the multivariate analysis. These
results provide evidence against a major direct effect of mildly
elevated homocysteine on activation of the coagulation system
and endothelial cell activation.
In vitro studies suggested a procoagulant effect of homocysteine on endothelial cells [710]. However, most effects on
endothelial cells were observed at higher homocysteine concentrations than those measured in patients with mild hyperhomocysteinemia. The in vitro studies may yield possible
pathophysiological mechanisms for patients with severe hyperhomocysteinemia, but the relevance for patients with mild
hyperhomocysteinemia is doubtful. A clear example are the

Table 3 Homocysteine and markers of coagulation in healthy volunteers, before and after methionine load or placebo
Placebo

Hcy (lmol L)1)


F1+2 (nmol L)1)
TAT (ng mL)1)
D-dimer

VWF
(%)

(lg L)1)

Methionine

t0

t4

t0

t4

8.9
8.210.3
1.00
0.701.16
1.88
1.443.26
146
140259
103
80122

9.2
8.211.0
0.86
0.621.11
1.59
1.252.86
153
123254
103
77121

9.3
7.910.7
1.00
0.691.55
2.36
1.173.07
158
102237
101
75122

30.1
26.737.8
0.86
0.681.27
1.85
0.953.23
151
107235
107
74124

0.000
0.39
0.91
0.63
1.00

Hcy, Homocysteine; TAT, thrombinantithrombin complexes; VWF, von Willebrand factor. Medians and interquartile ranges are depicted.
P-value for comparison of difference (value at t 4 ) value at t 0) after methionine and placebo.
 2004 International Society on Thrombosis and Haemostasis

Homocysteine and markers of coagulation and endothelial cell activation 449

studies on homocysteine and the protein C pathway. In vitro


studies indicated that activation of protein C was suggested to
be impaired in hyperhomocysteinemia [8,32]. Decreased
thrombomodulin activity, which is essential for protein C
activation, was observed in the aorta of hyperhomocysteinemic
monkeys and mice [33,34]. However, human in vivo studies
indicate that activation of protein C by thrombin and
inactivation of plasma FVa by activated protein C are not
impaired during moderate hyperhomocysteinemia [35,36].
Nappo and colleagues described increases of F1+2,
D-dimer, soluble intracellular adhesion molecule-1 and soluble
vascular cell adhesion molecule-1 after oral methionine load in
healthy individuals [12], according to a protocol that was
essentially identical to ours, except for an additional study arm
with antioxidant vitamins in their investigation. We have no
clear explanation for the discrepancy between their and our
results, although the use of parametric testing by Nappa et al.
on variables with a skewed distribution may have inuenced
the interpretation. Since others did not observe any inuence of
oral methionine loading on activated protein C resistance
[35,36], and we could not conrm the results of Nappo et al,
there is still no convincing evidence for a procoagulant effect of
mild hyperhomocysteinemia by an oral methionine load in
healthy individuals.
In our patients with established atherosclerotic disease we
observed, as shown previously, that homocysteine concentration was clearly associated with age, creatinine and MTHFR
polymorphism. IMT values were slightly higher in patients
with higher homocysteine concentrations in our cohort of
patients with atherosclerotic disease. Since age was a major
determinant of IMT in our study, the differences in age in the
different quartiles of homocysteine concentration explain the
observed differences in IMT. This is in agreement with a large
population-based study in which an observed association
between homocysteine and IMT disappeared after correction
for other risk factors [37]. In a few other ultrasound studies an
independent association between homocysteine and carotid
artery changes was found, but these studies focused on different
measures of atherosclerotic disease such as carotid plaque or
stenosis, not IMT [27,38,39].
Although MTHFR homozygosity was associated with higher
homocysteine concentrations in our patients, we did not detect
any inuence of MTHFR genotype on F1+2, sTM, or on IMT.
These ndings are compatible with other studies. McQuillan
and Spence reported no inuence of MTHFR on the carotid
artery in their ultrasound studies [26,27]. In a number of studies
and a meta-analysis a lack of association between MTHFR and
cardiovascular disease was observed [17,2325].
The independent associations of homocysteine and atherosclerotic disease found in cross-sectional and prospective trials
do not prove a causal relationship. Homocysteine concentration is inuenced by many factors. Mild hyperhomocysteinemia could be an epiphenomenon of atherosclerosis and
subclinical atherosclerotic disease may explain the reported
associations. Atherosclerosis is an inammatory disease [40].
We suggest that cytokines, the mediators of inammatory
 2004 International Society on Thrombosis and Haemostasis

disease, may contribute to the reported association between


homocysteine and atherosclerotic disease. When injected in
healthy volunteers, lipopolysaccharide causes an increase in
homocysteine levels [41], and homocysteine concentrations
have been associated particularly with interleukin (IL)-6 and
interleukin-8 concentrations in asymptomatic persons and
patients with premature atherosclerosis [42,43]. IL-6 induces
tissue factor, the main initiator of coagulation, in monocytes
and endothelial cells, and induces tissue factor-dependent
coagulation after endotoxin challenge in humans and primates
[44,45]. Such a link between inammation and coagulation
may also explain the observed relation between homocysteine
and F1+2 in the univariate analysis.
Another possibility is the presence of yet undened protective factors which counteract the direct inuence of homocysteine on endothelial cells. These factors may be sufcient to
regulate the effect of mild hyperhomocysteinemia as caused by
the MTHFR polymorphism, and yield an alternative explanation for the lack of association of MTHFR with atherosclerotic disease. Further studies need to elucidate if mild
hyperhomocysteinemia is an epiphenomenon or not.
Acknowledgements
We thank E. W. M. Vogels, Laboratory of Experimental
Internal Medicine, who did the MTHFR PCR assay, and
W. Mairuhu, who determined the homocysteine concentrations. H.t.C. is a Clinical Established Investigator from the
Netherlands Heart Foundation.
The Amsterdam Vascular Medicine Group

Academic Medical Center: R. J. G. Peters, Department of


Cardiology, J. Stam, Department of Neurology, H. R. Buller,
E. de Groot, Department of Vascular Medicine. Slotervaartziekenhuis: R. H. Bakker, Department of Cardiology, J. J. van
der Sande, V. I. H. Kwa, Department of Neurology, D. P. M.
Brandjes, H. ten Cate, V. E. A. Gerdes, Department of Internal
Medicine. Amstelveen Hospital: J. A. Lawson, Department of
Surgery. Apeldoorn Center Hospital: J. G. Kromhout,
Department of Surgery.
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