© 1999 American Heart Association, Inc.
Original Contributions |
Polymorphisms of the Tissue Factor Pathway Inhibitor (TFPI) Gene in Patients With Acute Coronary Syndromes and in Healthy Subjects
Impact of the V264M Substitution on Plasma Levels of TFPI
From INSERM U479 and Service d'Hématologie et d'Immunologie (D.M., S.D., V.O., J.H., D.d.P.), Service de cardiologie A (P.S., M.C.A.), INSERM U409 (C.G., M.G., D.D.), Laboratoire de nutrition humaine, CHU Xavier Bichat (F.F.), and INSERM SC7 (O.P.), Paris, France; the MONICA Project, Bas-Rhin (D.A.), Lille (G.L.), and Haute-Garonne (J.-B.R.), France; and the MONICA Project, Belfast, Northern Ireland (A.E.).
Correspondence to D. de Prost, Hôpital Bichat, Service d'Hématologie et d'Immunologie Biologiques, 46, rue Henri Huchard, 75018 Paris, France. E-mail dominique.deprost{at}bch.ap-hop-paris.fr
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Abstract |
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Abstract—Mutations of the gene encoding tissue factor pathway inhibitor (TFPI), an inhibitor of TF-induced activation of the coagulation cascade, were screened for in 130 patients and 142 healthy controls to determine whether these variants contribute to acute coronary syndromes or modify plasma TFPI levels. The following 3 new polymorphisms were identified: 384T
C in exon IV, which does not
change the corresponding amino acid (tyrosine 57); -33CT in intron
7 (the T/T, C/T, and C/C genotypes were found in 
50%, 40%,
and 10% of subjects in both groups); and 874GA in exon IX
(GTGATG), which predicts a valine to methionine change (V264M) in
the carboxy-terminus tail of TFPI. The V264M polymorphism was found
in 9.2% of the cases and 4.9% of the controls; the associated odds
ratio (OR) for acute coronary syndromes was 2.0 (95%
confidence interval [CI], 0.7 to 5.1). The OR increased to 3.6 (95%
CI, 0.8 to 15.7) and 3.2 (95% CI, 0.9 to 11.8) in nonsmokers and
patients without other risk factors, respectively. The possible link
between the V264M polymorphism and coronary heart disease
was checked in a large case-control study of myocardial infarction
(Etude Cas-Témoins de l'Infarctus du Myocarde [the ECTIM
Study]). The results showed no link between the V264M polymorphism
and coronary syndromes. Interestingly, however, 5 patients
heterozygous for the V264M polymorphism had significantly lower
plasma TFPI levels than did 13 patients with the most common
genotype. Although our present results do not support an
association between TFPI polymorphisms and acute coronary
syndromes, the possibility that 1 of them, especially the exon IX
polymorphism, is associated with subtypes of myocardial infarction
or to evolutive particularities that were not assessed in this study,
cannot be excluded and is currently being evaluated.
Key Words: tissue factor pathway inhibitor • case-control studies • myocardial infarction • genetics
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Introduction |
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Thrombus formation on disrupted atherosclerotic lesions plays a fundamental role in the onset of acute coronary syndromes.1 Tissue factor (TF), an integral membrane glycoprotein, is the main initiator of the coagulation cascade.2 Recently, TF activity and antigen were detected in directional coronary atherectomy specimens.3 Moreover, circulating monocytes express TF in unstable angina.4 Finally, TF pathway inhibition prevents arterial thrombosis in several animal models, underlining the major role of TF in arterial thrombosis.5
Tissue factor pathway inhibitor (TFPI), a circulating, multidomain, Kunitz-type protease inhibitor, is thought to play a major role in the inhibition of TF–factor VIIa proteolytic activity in vivo. The TFPI molecule consists of a negatively charged NH2 terminus, 3 tandem Kunitz-type inhibitor domains, and a positively charged COOH-terminal tail.6 The DNA sequence, including the promoter region, has been elucidated7 8 9 ; the TFPI gene consists of 9 exons separated by 8 introns. Kunitz-type domains 1, 2, and 3 are encoded by exons IV, VI, and VIII, respectively. The NH2 terminus and COOH-terminal tail are encoded by exons III and IX, respectively.
Experimental data strongly suggest that TFPI plays an important role as a natural anticoagulant. Immunodepletion of TFPI lowers the threshold at which TF induces disseminated intravascular coagulation.10 Conversely, infusion of recombinant TFPI protects against thrombosis in numerous experimental models.5 Despite extensive screening of plasma from patients with arterial or venous thrombosis, no quantitative or qualitative hereditary TFPI abnormalities have so far been detected.
The aim of this study was to screen the TFPI gene for point sequence variations in selected subjects with a history of myocardial infarction (MI) or unstable angina in comparison with healthy subjects. Because the Kunitz-type domains and the carboxy-terminal tail of TFPI appear to have major functional importance, the exons coding for these domains were analyzed first. Plasma TFPI was assayed in a small subgroup of patients.
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Methods |
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Selection of Cases and Controls
The patients and controls gave their written, informed consent for the study, which was approved by the Pitié Salpétrière ethics committee. One hundred thirty unrelated patients were genotyped; all were under 65 years of age and had been admitted to Bichat Hospital coronary care unit with a diagnosis of MI or unstable angina. Acute MI was diagnosed on the basis of any 2 of the following criteria: typical chest pain lasting
30
minutes, unequivocal ECG changes, or an increase in at least 2 of the 3
serum cardiac enzymes to >2x the upper limit of normal, or an
increase in creatine kinase and MB isoenzyme. Unstable angina was
diagnosed on the basis of typical chest pain lasting >5 minutes (class
I to III of Braunwald's classification11 ) with or without
ST-T abnormalities and with no elevation of the cardiac enzymes above
the upper limit defined for MI. Among patients with unstable angina,
those with angiography-proven 50% diameter stenosis were
selected. Blood was taken for genotyping either at the time of the
acute coronary event (67% of patients) or 3 to 9 months later.
Similar genotyping analyses were performed on 142 unrelated
control subjects matched with the patients for age and sex and who had
no documented personal history of stable or unstable angina, MI,
peripheral arterial thrombosis, or stroke. The
control subjects had no family history of arterial
thrombosis in first-degree relatives before the age of 60 years. The
control subjects were recruited almost equally among hospital employees
and blood donors. Subjects (cases and controls) and their parents and
grandparents were all white; 30 cases (23%) and 13 controls (9%)
originated from North Africa (Maghreb).
All subjects completed a questionnaire including age; smoking habits;
and a history of hypertension,
hypercholesterolemia, or diabetes. Smokers were
defined as all subjects who reported current smoking or who had stopped
<5 years previously. Subjects were classified as diabetic,
hypertensive, or hypercholesterolemic if they reported
that they were currently taking prescribed drugs for these conditions
and as obese when their body mass index (BMI) was 27.3
kg/m2. These latter 4 variables were grouped
together as metabolic risk factors. Heredity was considered
positive when subjects had a family history of arterial
thrombosis.
The polymorphism identified in exon IX was also investigated in a large case-control study of MI, the ECTIM Study (Etude Cas-Témoins de l'Infarctus du Myocarde). The inclusion criteria for this study have been described in detail elsewhere.12 13 Men aged 25 to 64 years were recruited between 1988 and 1991 in regions covered by the WHO MONICA (World Health Organization MONItoring trends and determinants in CArdiovascular disease) registers in Northern Ireland and France.14 Cases were recruited to the study 3 to 9 months after the event, and they had to satisfy WHO criteria for definite acute MI (category I). Controls were randomly recruited from the same geographic areas, and age stratification was used to obtain an approximate match between the age distribution of the controls and cases. DNA extracted from 509 patients and 562 controls was screened for the polymorphism in exon IX. Coronary angiograms were available for 314 cases. The number of arteries with >50% stenosis was used to assess the degree of stenosis.
Genotyping Study
The screening strategy used a method comprising polymerase chain
reaction (PCR) amplification of exons IV, VI, VIII, and IX followed by
denaturing gradient gel electrophoresis (DGGE) of the amplified
fragments. Mutations were identified by direct sequencing of fragments
with abnormal electrophoretic behavior. The first 6 patients in whom we
detected a sequence variation in exon IX were screened for other
mutations in the remaining DNA coding sequences (exons III, V, and VII)
by means of direct sequencing; none were found. The cDNA sequence
reported in this article has been published by Wun et
al.15
PCR and DGGE
DNA was extracted from blood leukocytes by the method of Bell et
al.16 Enzymatic amplification of exons IV, VI, VIII, and
IX was performed with a set of oligonucleotide primers
from GIBCO Life Technologies designed for DGGE analysis
(Table 1
). Because it is composed
of 2 domains with different melting temperatures, exon VIII was studied
with 2 sets of primers. A standard PCR was performed in a 50-µL
reaction mixture containing 50 ng of genomic DNA, 0.4 pmol/µL of each
primer, 200 µmol/L of each deoxynucleotide
5'-triphosphate (dNTPs from GIBCO), and 0.25 U of Taq
polymerase (Super Taq from ATGC) in buffer containing 10 mmol/L
Tris-HCl, 5 mmol/L KCl, 1.5 mmol/L
MgCl2, and 0.01% gelatin, pH 9. The reaction
mixture was subjected to 35 cycles of PCR with denaturation at 94°C
for 20 seconds, annealing for 30 seconds at temperatures depending on
the primer set, and extension at 70°C for 45 seconds in a
Trio-Thermoblock thermal cycler (Biometra). The reaction mixture was
then held at 70°C for 7 minutes and 94°C for 5 minutes. The
annealing temperatures and the size of amplification products are
shown in Table 1.
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Computer analysis was performed with the MELT 87
(Bio-Rad) program.17 Because the sequence of interest must
be located within the first melting domain of the fragment, a psoralen
derivative (ChemiClamp) was attached to the 5' extremity of 1 of the
amplification primers18 (Table 1
).
Psoralen-modified oligonucleotides were purchased from
Eurogentec. Before DGGE, the PCR products were exposed to UV
radiation (365 nm) for 15 minutes. Amplification products were
subjected to electrophoresis at 160 V in 6.5% polyacrylamide
gel containing a gradient of denaturing agents. The 90% solution of
denaturing agent contained 6.3 mol/L urea and 36% formamide in 1x TAE
buffer (40 mmol/L Tris, 20 mmol/L acetic acid, and 1
mmol/L EDTA, pH 8.3). The gradients used for each DGGE run are shown in
Table 1. The bands corresponding to the normal and mutant
alleles were excised from the gel, reamplified, purified, and
automatically sequenced (Genome Express) by cycle fluorescent
sequencing.
Restriction Analysis of PCR-Amplified Products
Exon IX Polymorphism
The restriction sites of the sequence were not altered by the
G-to-A substitution. To confirm the exon IX variation, a modified
30-mer oligonucleotide with an I-to-G substitution was
synthesized (5'-TAA CAA AAA TTT CTT CAT ATG CTA TTG TCA-3';
the substituted base is underlined). This creates a restriction site
for the enzyme MaeIII (Boehringer Mannheim;
GTNAC/CANTG
). After amplification, the PCR products were
digested by the enzyme overnight at 55°C and checked on a 2.5%
agarose gel.
Intron 7 Polymorphism
Because the intron 7 polymorphism creates a restriction site
for the enzyme NdeI (CATATG/GTATAC), this enzyme was
used to confirm the intron 7 variation. The PCR products were
digested overnight at 37°C by NdeI (BioLabsinc) and
checked on a 2.5% agarose gel.
Allele-Specific Oligonucleotide Hybridization
ECTIM DNAs were genotyped by using allele-specific
oligonucleotides (ASOs), as described.19
The following ASOs were synthesized by Eurogentec: 5' TAT TTT CAC TCT
CTG CT 3' and 5' AGC AGA GAA TGA AAA TA 3', and were used to detect the
CTC (valine) and GAA (methionine) codons, respectively.
TFPI Assays
Blood was collected from 12-hour–fasted patients 3 to 9 months
after the acute coronary event. Blood was taken before and 90
minutes after a subcutaneous injection of 0.66 mg/kg body weight
enoxaparin.
TFPI Antigen Assay
Total and free TFPI contents were measured by using the
Asserachrom total and Asserachrom free TFPI ELISA kits kindly donated
by Diagnostica Stago. In both assays the capture antibody
is a monoclonal F(ab')2 fragment against the
second Kunitz domain of TFPI. The detecting monoclonal antibodies are
conjugated to peroxidase. The tag monoclonal antibody used for the free
TFPI assay is specific for the Kunitz 3 domain; the tag antibody used
for total TFPI assay is specific for total TFPI, as shown using
recombinant full-length and C-terminal–truncated forms. The
Asserachrom total TFPI assay measures free and bound, as well as intact
and truncated, forms of TFPI, whereas the Asserachrom free TFPI assay
measures only TFPI free of lipoprotein (personal communication from
J. Amiral, Serbio, Gennevilliers, France.) The within-run
coefficients of variation were 6.0% and 6.8% with the total ELISA and
free ELISA, respectively (n=10).
Chromogenic Assay of TFPI Activity
TFPI activity was measured in microplates with a 2-stage
amidolytic assay according to Sandset et al,20 with minor
modifications. TF (Neoplastine), bovine factor Xa, human factor X, and
the chromogenic substrate CBS 5244 were from
Diagnostica Stago; factor VIIa was ACSET kindly provided by
LFB Laboratories. Absorbance was read at 405 nm in a microplate reader
(Molecular Devices).
Other Assays
Total cholesterol and triglyceride
levels in plasma were measured by using commercial enzymatic kits (Kone
Instruments SA). Plasma anti–factor Xa activity was measured using the
Rotachrom heparin kit from Diagnostica Stago.
Statistical Analysis
Links between each polymorphism and MI or unstable angina
were examined by simple cross-tabulation and by calculating the odds
ratio (OR) as an approximation of the relative risk. The extent to
which the link between the TFPI V264M variation and disease onset was
modified by other characteristics was assessed by means of stratified
analyses. Confidence intervals (95% CIs) were calculated using
Woolf's method. An OR was considered statistically significant when
the lower limit of the 95% CI was >1.0. The
2 test (or Fisher's exact test, when sample
numbers were too small) was used to test for deviation of the
genotype distribution from Hardy-Weinberg equilibrium and to
identify significant differences in allele or genotype
frequencies between the cases and controls. Two-tailed probability
values of 0.05 or less were considered statistically significant. TFPI
levels in patients and controls were compared using the Mann-Whitney
test for unpaired data.
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Results |
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Characteristics of the Study Population
One hundred forty-two controls and 130 cases (15 with unstable angina and 115 with MI) were studied. Table 2
shows the characteristics of the
subjects and the prevalence of selected risk factors for
coronary heart disease among them. The 2 groups were matched
for age and sex, and there were, therefore, no significant differences
in these variables. All of the subjects were white, but 23% of the
cases were from North Africa, compared with only 9% of the controls.
As expected, risk factors for coronary heart disease (including
current smoking, hypercholesterolemia, diabetes
mellitus, hypertension, and obesity) were more frequent in the
cases.
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DGGE Migration and Restriction Patterns
No abnormal DGGE patterns were detected in exon VI, which encodes
the second Kunitz domain (data not shown). We found an abnormal
migration pattern for exon IV (Figure 1A)
in 4 patients and 2 controls. The 6 subjects were heterozygous for a
thermostabilizing variant, which was further characterized by
sequencing. The variation was located at position 384 (TATTAC) in
exon IV (384TC); it did not change the corresponding amino acid
(tyrosine 57) in the Kunitz 1 domain of TFPI.
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X174RF DNA/Hae III fragments 1353, 1078,
872, 603, 310, 281, 271, 234, 194, and 118 bp from top to bottom.
Exon VIII (coding for the third Kunitz domain) was studied by using 2
different DGGEs corresponding to the beginning and end of the exon. No
abnormality was detected in the end of exon VIII, but an abnormal
migration pattern was detected at a high frequency in amplified
fragments including the end of intron 7 and the beginning of exon VIII
(Figure 1B). Sequence analysis showed that the variation
was a C-to-T substitution at position -33 in intron 7 (intron 7
-33CT).7 This was confirmed by restriction site
analysis with NdeI (Figure 1D). The
restriction site for this enzyme was absent from the normal strand,
which remained undigested (198 bp); the homozygous mutated strand, in
contrast, was completely digested into 2 fragments of 167 and 31 bp; in
heterozygous subjects, both patterns, corresponding to the undigested
(198 bp) and digested (167 bp) fragments, were present.
The DGGE profiles obtained for exon IX are shown in Figure 1C. A
thermodestabilizing heterozygous variation was found in some patients
and controls. These subjects were shown to be heterozygous for a
missense mutation in nucleotide 1006 (GTGATG) in exon IX
(nucleotides numbered according to Wun et
al).15 This variation predicts a valine-to-methionine
change in the sequence (V264M), which is located in the
carboxy-terminus tail of the molecule. Restriction analysis
with MaeIII was used to confirm the sequence variation
(Figure 1E). When the normal strand was amplified and digested,
the fragment was split into 2 fragments of 100 and 31 bp (the latter
was not visible on the gel). The restriction site was absent from the
mutant fragment, which remained undigested (131 bp). Both patterns were
observed in heterozygotes. As shown in Figure 2, the predicted valine residue in
position 264 of the carboxy-terminal tail of human TFPI is highly
conserved relative to 3 other species.21 22 23
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Distribution of TFPI Genotypes in Patients With MI or
Unstable Angina and in Control Subjects
The distribution of TFPI genotypes for exon IV and
intron 7 among patients and controls is shown in Table 3. The neutral 384TC variant in exon
IV was found in 4 cases (3.1%) and 2 controls (1.4%). Five of these
subjects originated from North Africa and the other was French. No
statistical case-control difference in its frequency was observed. The
variant of intron 7 was frequent in both groups. The OR associated with
the T/T genotype was 1.2 (95% CI, 0.7 to 1.9) relative to the
C/T+C/C genotypes, a difference that was not statistically
significant. C/C homozygotes represented 10% of the
subjects in each group. The genotype distributions in cases and
controls conformed to Hardy-Weinberg equilibrium.
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In all of the subjects the TFPI V264M variation in exon IX was
identified by DGGE screening and confirmed by restriction
analysis. As shown in Table 4, the frequency of the
TFPI V264M variation was 9.2% (12/130) in the cases (of whom 11 had MI
and 1 had unstable angina) and 4.9% (7/142) of the controls. No
homozygotes were found. The OR for MI or unstable angina associated
with the TFPI V264M variation was 2.0 (95% CI, 0.7 to 5.1). The link
was unaffected when cases and controls originating from Maghreb were
excluded from the analysis (OR=2; 95% CI, 0.7 to 5.9;
P=0.18). In contrast, the risk increased to 2.6 (95% CI,
0.9 to 7.3, P=0.06) in patients over 45 years old. We then
explored whether the risk associated with the V264M variation was
different in subjects with other major risk factors or specific
combinations of risk factors. ORs were calculated for smokers and for
subjects with 1 or more metabolic risk factor. The
proportion of smokers was 79% among the patients and 38% among the
controls (Table 2). As shown in Table 5, current or recent smoking was
associated with an OR for acute coronary events of 6.8 in
comparison with nonsmokers with the V/V genotype (OR, 6.8; 95%
CI, 3.8 to 12.1; P<0.0001). One or more
metabolic risk factors (hypertension, diabetes mellitus,
hypercholesterolemia, or obesity) was
present in 59% of the patients and 30% of the controls, yielding
a 3.5-fold increase in the OR (OR, 3.5; 95% CI, 2.1 to 6.0;
P<0.0001) among patients with 1 or more of these risk
factors compared with patients with none. The V264M variation was
associated with an increase of 3.6 and 3.2 times the relative risk
among nonsmokers (95% CI, 0.8 to 15.7; P=0.09) and patients
without metabolic risk factors (95% CI, 0.9 to 11.8;
P=0.09), respectively. In smokers and subjects with risk
factors, the polymorphism increased the relative risk from 6.8 to
9.7 and from 3.5 to 4.2, respectively.
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Frequency of the V264M Genotype in the ECTIM Study
Population
Because our patient and control groups were relatively small, the
V264M polymorphism was screened for in 509 cases and 562 controls
from the ECTIM Study. This was done by using ASO hybridization. To
simplify the data presentation, the results from the 3
French centers were pooled after checking that the results were not
statistically different across the centers. Mean age was similar in the
cases and controls (54±8.2 and 53±8.5 years, respectively). As shown
in Table 6, the overall V264M
genotype and allele frequencies did not differ
significantly between the cases and controls. Two homozygous subjects
were identified in the control group and 1 in the patient group.
Genotype frequencies conformed to Hardy-Weinberg equilibrium in
all subgroups. Similar results were obtained in subjects under 45 years
of age. The genotype and allele frequencies did not differ
significantly between the Northern Irish and French subjects. In the
314 French cases for whom coronary angiographic data were
available, 10 were heterozygous and 1 was homozygous. No link was found
with the degree of stenosis (data not shown).
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TFPI Levels According to Genotype
To assess the effect of the V264M variation on plasma TFPI levels,
the latter were measured in 5 V/M and 13 V/V patients (Table 7). The 5 patients with the V264M
mutation were being treated with statins. Because the
cholesterol level is known to affect TFPI
levels,24 the 13 patients with the wild-type variant were
chosen for also being on statins. As shown in Table 7, the 2
groups of patients had similar ages and total cholesterol,
LDL cholesterol, triglyceride, and glycemia
levels. Total and free TFPI antigen levels were lower in the patients
heterozygous for the V264M mutation than in patients with the wild-type
variant (P<0.05). TFPI activity levels measured by a
chromogenic assay were also lower, but the difference did
not reach significance.
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To evaluate the impact of the V264M variation on
endothelial cell–bound TFPI, blood was drawn 90
minutes after a subcutaneous enoxaparin injection, because Bara et
al25 have shown that TFPI levels peak at this time. In the
2 groups of subjects, the mean (±SD) anti–factor Xa activity level at
90 minutes was 0.4 IU/mL. In both groups, total TFPI levels increased
1.5-fold while free TFPI increased 3-fold. The mean TFPI level in
the patients with the genetic variation remained 15% lower than in the
other patients, although the difference did not reach significance.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In patients with MI or unstable angina, TF appears to play a determining role in triggering thrombosis in contact with the atherosclerotic plaque.1 The search for genetic variations in TFPI thus appeared important. In this study, we identified 3 new polymorphisms in the TFPI gene. The polymorphism of exon IX, predicting a methionine instead of a valine in position 264 of the protein, emerged as the most interesting change for several reasons: (1) in the first group of subjects studied, its frequency was 4.9% in the controls and 9.2% in the cases; (2) the valine residue in position 264 of TFPI is highly conserved among species and is located within the heparin-binding site; and (3) it is this polymorphism that was associated with low plasma levels of TFPI.
The exon IV variation (384TC) identified herein did not modify the
protein sequence, because TAT and TAC both code for tyrosine; 5 of the
6 subjects carrying this polymorphism were from North Africa,
suggesting that the C allele frequency is higher in Maghreb (12%)
than in Europe (0.4%; P<0.0001). A common C/T
polymorphism in intron 7 was located 33 bp upstream from the
beginning of exon VIII. In the sequence described by van der Logt et
al,7 the base identified at this site is a C. Our
results clearly show, however, that the most frequent allele,
encountered in 70% of the patients and controls, is a T. No
significant difference in the frequencies of the 3 genotypes
was observed between the cases and controls.
In the first group of subjects studied, the frequency of the
polymorphism in exon IX (V264M) was higher in the cases (9.2%)
than in the controls (4.9%) (OR, 2; 95% CI, 0.7 to 5.1;
P=0.16). This trend toward an increased relative risk
associated with the mutation was stronger in patients who did not smoke
(OR, 3.6; 95% CI, 0.8 to 15.7; P=0.09) and in those without
other risk factors (OR, 3.2; 95% CI, 0.9 to 11.8; P=0.09).
Since these results showed a trend toward an increased OR and given the
relatively small size of the study group, we investigated the V264M
polymorphism in ECTIM, a large, multicenter, case-control study.
The frequency of heterozygotes (2.9% to 5.3%, according to the
geographic area) and the M264 allele frequency (0.017 to 0.028)
were similar in the cases and controls (Table 6); moreover, the
values were very similar to those found in the controls of our initial
study (V/M genotype frequency, 4.9%; M264 allele
frequency, 0.025). There was no difference in the distribution of
either genotype or allele frequencies in the high-risk
populations of Northern Ireland or France. Among the possible reasons
why the initial trend toward a link between the TFPI V264M
polymorphism and the occurrence of acute coronary events
was not confirmed in the ECTIM Study is the geographic origin of the
subjects. In ECTIM, it was limited to 3 French regions and North
Ireland, whereas our initial study included subjects originating from
all over Europe and North Africa. In addition, the initial study
included subjects of both sexes, whereas only men were included in
ECTIM. However, restricting the analysis to the male population
or to subjects originating from Europe (ie, excluding those from
Maghreb) and to the patients with MI (ie, excluding those with unstable
angina) did not change the results. Although our results for the ECTIM
Study population do not indicate a link between MI and the V264M
polymorphism, certain baseline characteristics (including the
severity of coronary disease) of our initial group of patients
may have differed from those of the ECTIM population. Our results for
the former subjects suggest that this polymorphism might increase
the severity of coronary thrombosis. In the small subgroup of
patients with unstable angina (n=15), only 1 patient was heterozygous
for the V264M polymorphism. However, by study design, patients with
unstable angina had a 50% stenosis. Therefore, our results
do not rule out the role of the polymorphism in patients with less
severe angiographic lesions who may be prone to plaque rupture as
well.26
The influence of the V264M genotype on TFPI levels was assessed
in a small subgroup of patients, all of whom were on statin therapy,
before and after an injection of low-molecular-weight heparin.
Interestingly, total and free TFPI antigen levels were significantly
lower before heparin in the heterozygous patients (Table 7).
TFPI activity and TFPI levels measured after heparin administration
were still lower in these patients, but the difference did not reach
significance. Val264 is located in the carboxy-terminus tail of TFPI,
within the positively charged basic sequence (KTKRKRKKQRVK) involving
residues 254 to 265. This sequence has been shown to mediate TFPI
binding to heparin.27 Because infusion of heparin
increases levels of circulating TFPI,28 it has been
postulated that TFPI, via its basic region, binds to
glycosaminoglycans on the
endothelial surface and that heparin, by competing for
this basic region, releases bound TFPI.28 Many other
functions of TFPI have been reported to be dependent on the presence of
the carboxy-terminus tail, including optimal inhibition of
activated factor X,27 binding to fibrin and
subsequent degradation by thrombin,29 and interaction with
hepatoma cells, which is followed by LDL-related protein–mediated
internalization and degradation of TFPI.30 A number of
these functions, including those involving TFPI clearance and
degradation, appear to depend on the interaction of the positively
charged residues of the carboxy terminus of TFPI with the negative
charges carried by glycosaminoglycans of the
vascular wall or by fibrin. Although the replacement of a valine by a
methionine is not expected to strongly modify the charge of the basic
sequence, it might increase the TFPI interaction with
glycosaminoglycans, thereby reducing circulating
free TFPI levels. The measurement of free TFPI antigen after heparin
injection is of particular interest in estimating TFPI bound to the
vessel wall, which is released by heparin.31 In the
patients carrying the V264M polymorphism, the decrease in total and
free TFPI levels observed before heparin injection was attenuated after
heparin, suggesting that the release process and the amount of
endothelial cell–bound TFPI were not strongly
affected. Free TFPI is thought to be the active part of TFPI in
inhibiting coagulation, and a fall in its concentration might thus
affect thrombotic potential. Moreover, evidence that the valine in
position 264 varies little across species (Figure 2) supports a
potential role in TFPI function.
In conclusion, we identified 3 new polymorphisms in the TFPI gene. We found no evidence supporting a link between TFPI polymorphisms and the risk of coronary heart disease, but further studies are underway to determine whether the exon IX polymorphism is associated with particular subtypes of MI or certain outcomes.
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Acknowledgments |
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This study was supported in part by Rhône-Poulenc Rorer Bellon Laboratories (to D.d.P.). and by a grant from Assistance Publique-Hôpitaux de Paris (CRC 95135) (to D.d.P., M.C.A., L.S., M.G., D.D.). We thank C. Lacombe and L. Sustendal for their excellent technical assistance; J. Amiral and M. Grosley (Serbio Laboratory, Gennevilliers, France) for providing the TFPI ELISA kits; and Drs A.M. Becker, S. Menasie, and R. Babou (E.T.S. de l'AP-HP, Site Transfusionnel, Hôpital Bichat-Claude Bernard) for collecting blood samples from the control subjects. We also thank Professor F. Cambien (INSERM SC7) for stimulating discussions and critical reading of the manuscript.
Received June 8, 1998; accepted August 6, 1998.
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