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Blood, Vol. 96 No. 3 (August 1), 2000:
pp. 1177-1179
BRIEF REPORT
 -Fibrinogen Thr312Ala polymorphism and venous
thromboembolism
Angela M. Carter,
Andrew J. Catto,
Hans P. Kohler,
Robert A. S. Ariëns,
Max H. Stickland, and
Peter J. Grant
From the Academic Unit of Molecular Vascular Medicine, Research
School of Medicine, University of Leeds, Leeds General Infirmary,
Leeds, England.
 |
Abstract |
The A -fibrinogen Thr312Ala polymorphism, which occurs in a
region involved in factor XIII (FXIII)-dependent cross-linking processes, is associated with poststroke mortality in subjects with
atrial fibrillation, suggesting an influence either on intraatrial clot
formation or embolization. We have determined the association of
Thr312Ala with deep vein thrombosis (DVT) and pulmonary
embolism (PE) and have assessed the interaction of Thr312Ala
with the FXIII Val34Leu polymorphism in 122 patients with DVT, 99 patients with PE, and 254 healthy control subjects.
The genotype distribution of patients with PE
(TT = 49%, TA = 36%, AA = 15%), but not DVT (TT = 50%, TA = 42%, AA = 8%), differed significantly
from healthy control subjects (TT = 60%, TA = 34%,
AA = 6%, P = .02). A significant interaction of
Thr312Ala and Val34Leu was also identified (P = .01),
indicating an inverse association between Leu34 and Ala312. These
results support the hypothesis that Thr312Ala alters FXIII-dependent cross-linking, making formed fibrin clot more susceptible to embolization.
(Blood. 2000;96:1177-1179)
© 2000 by The American Society of Hematology.
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Introduction |
The hemostatic system maintains a delicate balance
between coagulation/anticoagulation, platelet activation/inhibition,
and fibrinolysis to maintain vascular patency. Abnormalities in
hemostatic factors may either lead to bleeding or to excessive fibrin
production and thrombosis. The FV Leiden and prothrombin G20210A
polymorphisms have been related to venous thromboembolism
(VTE),1,2 and we have shown that the Leu allele of the
factor XIII (FXIII) Val34Leu polymorphism is inversely associated with
risk of VTE3 and also of myocardial
infarction.4
The  -fibrinogen Thr312Ala5 polymorphism occurs in a
region important for FXIII-dependent cross-linking
processes6 and may, therefore, influence clot structure or
rigidity. We have found a significant association of Thr312Ala with
poststroke mortality in subjects with atrial fibrillation, with
increased mortality in subjects possessing Ala312 compared with those
homozygous for Thr312.7 These results indicate that
Thr312Ala may influence either intraatrial clot formation or embolization.
To further clarify the association of Thr312Ala with either thrombus
formation or embolization, the aim of the present study was to
determine the association of Thr312Ala with deep vein thrombosis (DVT)
and pulmonary embolism (PE) and additionally to identify any
significant interaction with FXIIIVal34Leu.
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Study design |
Subjects
In the patient group,3 122 (55%) subjects had sustained
a DVT, confirmed on doppler ultrasound venogram examination of calf,
popliteal, femoral, and iliac veins. Ninety-nine (45%) subjects had a
clinical diagnosis of PE, of which 17 had both DVT and PE (classified
as PE for analysis); the remaining 82 subjects with PE had no clinical
evidence of DVT. PE was confirmed by the presence of two or more areas
of ventilation-perfusion mismatch on a Technicium-99 lung scan or of
thrombus in the pulmonary vasculature, confirmed by contrast-enhanced
high resolution spiral computed tomography (reserved for subjects with
intermediate probability of PE on lung scanning [n = 15]).
Healthy control subjects (n = 254) were from the same geographical
area as patients and were free of a personal and family history of
VTE.3 The study was approved by the United Leeds Teaching
Hospitals Trust Research Ethics Committee.
DNA and biochemical analyses
Thr312Ala and FXIII Va134Leu genotypes were determined as previously
described.3,7 DNA from 3 patients and 4 control subjects
did not amplify for Thr312Ala. FXIII activity was determined as
previously described.8
Statistics
Genotype distributions were compared by  2 test.
Logistic regression models (backward stepwise selection) were used to
identify factors significantly associated with VTE (results presented
as odds ratios [OR] and 95% confidence interval [CI]). Interaction terms were created in these models to identify significant interactions of Thr312Ala with other risk factors. Statistical analyses were performed, using the SPSS statistics package V7.0 (SPSS Inc).
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Results and discussion |
As previously reported,3 FV Leiden was significantly
associated with VTE, and the FXIII Leu34 allele was inversely
associated with VTE (data not shown). There was a significant
difference in the Thr312Ala genotype distributions of patients
(TT = 49%, TA = 39%, AA = 12%) and healthy
control subjects (TT = 60%, TA = 34%, TT = 6%,
P = .04). However, in a logistic regression model including
age, sex, malignancy, FV Leiden, FXIII Val34Leu, and Thr312Ala, Thr312Ala was not independently associated with VTE. ORs for
factors independently associated with VTE were as follows: 1.17 (95%
CI = 1.04-1.32) for a 10-year increase in age; 2.93 (95% CI = 1.31-6.59) for those with versus without malignancy; 2.32 (95% CI = 1.12-4.79) for carriers of FV Leiden versus wild type; 0.37 (95% CI = 0.23-0.61) for subjects possessing FXIII Leu34 versus those homozygous
for Va134.
Subclassification of patients into those with DVT or PE
revealed a significant difference in Thr312Ala genotype distributions of patients with PE versus healthy control subjects, but no difference of patients with DVT versus control subjects, or patients with DVT
versus PE (see Table 1). In a logistic
regression model comparing patients with PE to healthy control
subjects, Thr312Ala was independently associated with PE (OR = 2.71 [95% CI = 1.23-6.01]) for subjects homozygous for Ala312 versus
those homozygous for Thr312. ORs for other factors significantly
associated with PE were as follows: 1.26 (95% CI = 1.08-1.49) for a
10-year increase in age; 0.58 (95% CI = 0.36-0.95) for subjects
possessing FXIII Leu34 versus those homozygous for Val34. We previously
reported no significant association of Val34Leu with PE in univariate
analyses;3 however, in this logistic regression model in
which the Val allele was considered to be recessive in relation to PE
(as previously shown for VTE), Leu34 was inversely associated with PE.
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Table 1.
Thr312Ala genotype distributions of patients with VTE
classified as those with DVT or PE compared with healthy control
subjects
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FXIII-dependent cross-linking processes are essential for
stabilizing fibrin and for forming a fibrin clot more resistant to
fibrinolysis. Therefore, it is not unreasonable to expect that any
factor interfering with these processes may adversely influence the
mechanical integrity or plasmin susceptibility of a fibrin clot.
Because Thr312Ala was related to PE but not to DVT (compared with
control subjects) in this study, it is plausible that Thr312Ala increases the susceptibility of fibrin clot to embolize, although further work on fibrin structure is required to test such a hypothesis.
To investigate the possible role of Thr312Ala in
FXIII-dependent cross-linking processes, we introduced the interaction
term Thr312Ala*Val34Leu (plus each individual term) into the PE versus control subject logistic regression model. A significant interaction between Thr312Ala and FXIII Val34Leu was indicated by this model (P = .01). Other factors significantly associated with PE
were age (OR for a 10-year increase = 1.28 [95% CI = 1.09-1.51]) and FXIII Val34Leu (OR for those possessing Leu34 versus those homozygous for Val34 = 0.33 (95% CI = 0.17-0.64]), but not cigarette smoking. To
investigate this interaction, the association of Val34Leu with PE was
determined separately in those homozygous for Thr312 and in those
possessing Ala312. These analyses indicated that, in subjects
homozygous for Thr312, possession of Leu34 is inversely associated with PE (2, P = .002), whereas, in
those possessing the Ala312 allele, the association between FXIII Leu34
and PE is lost (2, P = .95), as shown in
Figure 1. This finding supports the
possibility that Thr312Ala influences FXIII-dependent processes and
also points to a likely difference in the influence of each of these
polymorphisms on the stabilization of a fibrin clot.
View larger version (26K):
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| Fig 1.
Interaction of the fibrinogen Thr312Ala and FXIII
Val34Leu polymorphisms in relation to PE.
In subjects homozygous for Thr312, possession of Leu34 was associated
with a relative protection from PE compared with those homozygous for
Val34 (2, P = .002). However, in those
possessing Ala312, the protective effect of Leu34 is lost
(2, P = .95).
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FXIII Val34Leu occurs close to the thrombin cleavage site of
FXIII and may influence thrombin activation of FXIII. In 113 of the
healthy control subjects, FXIII cross-linking activity was available
and significantly associated with Val34Leu (Val/Val = 81.4%
[77.1%-86.0%]; Val/Leu = 124.4% [113.6%-136.2%];
Leu/Leu = 175.7% [152.2%-203.0%], P < .0001), as we
have previously demonstrated.8 This finding
indicates that Val34Leu influences FXIII activity or
activation rates, although it remains unclear at present how increased
activity in those individuals possessing Leu34 is related to protection from thrombosis.
Thr312Ala occurs close to the -fibrin/-fibrin
cross-linking site (A328) as well as to the
2-antiplasmin/-fibrin cross-linking site (A303)
and occurs within the region of -fibrinogen (A242-424) that has
been suggested to be involved in FXIII activation.6 Thus,
Thr312Ala could potentially interfere with any one of these processes.
We have previously shown no association of Thr312Ala with
2-antiplasmin incorporation.7 Similarly, we
found no association of Thr312Ala with FXIII activity (TT = 105.9%
[97.6%-114.8%]; TA = 105.0% [91.33%-120.8%]; AA = 92.2%
[70.5%-120.7%], P = ns), even when patients were
characterized by Val34Leu genotype (data not shown). Thus, it would
appear that Thr312Ala is most likely to influence -fibrin/-fibrin
cross-linking. Because -fibrin cross-linking contributes to
approximately 65% of overall clot stability,6 it would be
expected that any factor specifically influencing this cross-linking
would lead to a reduction in the overall clot strength and potentially
an increased likelihood of embolization. Further in vitro studies are
currently under way to determine the influence of Thr312Ala on
-fibrin/-fibrin cross-linking.
This study does have some limitations. In subjects classified
with DVT alone, it is possible that a small proportion of subjects (particularly those with more proximal venous thrombosis) would be
likely to embolize asymptomatically, and, therefore, the incidence of
PE might be underrepresented in this group. In addition, because of the
relatively small sample size, a larger study would be required to
confirm these findings.
| |
Footnotes |
Submitted October 19, 1999; accepted March 20, 2000.
Reprints: Angela M. Carter, Academic Unit of Molecular Vascular
Medicine, Research School of Medicine, G Floor, Martin Wing, Leeds
General Infirmary, Leeds LS1 3EX, UK; e-mail:
medamc{at}medphysics.leeds.ac.uk.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
References |
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Kohler HP, Stickland MH, Ossei-Gerning N, Carter AM, Mikkola H, Grant PJ.
Association of a common polymorphism in the factor XIII gene with myocardial infarction.
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The association of the -fibrinogen Thr312Ala polymorphism with post-stroke mortality in subjects with atrial fibrillation.
Circulation.
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Top
Abstract
Introduction