(Circulation. 1999;99:2423-2426.)
© 1999 American Heart Association, Inc.
Clinical Investigation and Reports |
Association of the 
-Fibrinogen Thr312Ala Polymorphism With Poststroke Mortality in Subjects With Atrial Fibrillation
From the Unit of Molecular Vascular Medicine, Research School of Medicine, University of Leeds, Leeds General Infirmary, UK.
Correspondence to Peter J. Grant, Unit of Molecular Vascular Medicine, Research School of Medicine, G Floor, Martin Wing, Leeds General Infirmary, Leeds, LS1 3EX, UK. E-mail p.j.grant{at}leeds.ac.uk
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Abstract |
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 Top Abstract IntroductionMethodsResultsDiscussionReferences |
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Background—The
-fibrinogen
Thr312Ala polymorphism occurs in close proximity to several sites
important for factor XIIIa–dependent cross-linking, which raises the
possibility that it affects fibrin clot stability.
Methods and Results—We determined the association of this
polymorphism with ischemic stroke, stroke subtype, and
poststroke mortality. There was no significant difference in the
genotype distributions of patients with acute ischemic
stroke (n=519) and healthy control subjects (n=423), nor was there any
association of this polymorphism with stroke subtype. In a Cox
regression model, a significant interaction between Thr312Ala and
atrial fibrillation was identified in relation to poststroke mortality
(P=0.002). In subjects in sinus rhythm (n=418), there
was no difference according to genotype in the proportion of
subjects who survived (
60% in each group), whereas in subjects with
atrial fibrillation (n=101), there was decreased survival in those
possessing the A allele (TT=42.1%, TA=18%, AA=0%).
Conclusions—The Thr312Ala polymorphism may give rise to an increased susceptibility for embolization of intra-atrial clot, and these findings could have important implications for identifying subjects most at risk of developing thromboembolic complications.
Key Words: stroke • mortality • atrial fibrillation
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The conversion of fibrinogen to fibrin gives rise to the formation of fibrin protofibrils,1 and stabilization of the forming fibrin clot requires cross-linking of adjacent
- and
-chains by factor (F) XIIIa.2 The - crosslinks
contribute to 35% and - cross-links to 65% of clot
stability,2 highlighting the relative importance of
-fibrinogen cross-linking in determining overall clot stability.
A polymorphism of the -fibrinogen gene has been identified that
codes for a threonine-to-alanine amino acid substitution at position
312 (Thr312Ala).3 This polymorphism lies close to the
FXIIIa cross-linking site at position A 328.2 FXIIIa
also cross-links 2-antiplasmin to fibrin at
position A 303; this results in localization of
2-antiplasmin within the fibrin clot and helps
to protect it from cleavage by plasmin.4 In addition, the
region of fibrinogen encompassing -fibrinogen amino acid residues
242 to 424 enhances the activation of FXIII by reducing the
Ca2+ concentration required for the dissociation
of the FXIII a and b subunit dimers to
physiological levels.5 Thus, Thr312Ala
could influence clot strength and elasticity by interfering with these
FXIIIa-dependent cross-linking processes.
To investigate whether clinical studies supported these possibilities,
we determined the association of the Thr312Ala polymorphism with
ischemic stroke, stroke subtype, and poststroke mortality. In
addition, we identified possible interactions of this polymorphism
with classic risk factors and determined whether Thr312Ala influences
the incorporation of 2-antiplasmin into
fibrin.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Subjects
The recruitment and characteristics of patients and control subjects have been fully described.6 Briefly, 519 patients with ischemic stroke, confirmed by noncontrast cranial CT scan, were recruited. Patients were subclassified according to the Oxfordshire Community Stroke Project classification into those with probable small-vessel disease (lacunar infarction caused by microatheromatous occlusion of small perforating arteries) and those with probable large-vessel disease (total or partial anterior circulation infarction caused by either in situ thrombosis or cardiogenic embolism).7 The presence of atrial fibrillation was established on clinical examination at the time of the acute stroke and confirmed on a diagnostic 12-lead ECG. Age-matched healthy control subjects (n=423) residing in the same postal districts as the patients were recruited from local Family Health Services Authority general practice registers. All subjects gave informed consent according to a protocol approved by the United Leeds Teaching Hospitals NHS Trust.
DNA Analysis
The Thr312Ala genotype was determined by use of
the following oligonucleotide primers, which gave a
polymerase chain reaction (PCR) product of 190 bp: forward,
5'-CCTAGCAGTGCTGGAAGCTG-3' and reverse, 5'-GGCTCCCAGGGTTTTGGT-3'.
Fragments were amplified by standard PCR procedures using 25 pmol of
each primer, 2.0 mmol/L magnesium, and 64°C annealing
temperature. PCR products were digested overnight with 5 U
RsaI (New England Biolabs), and samples were separated by
2% agarose gel electrophoresis. Digestion with RsaI gave
constant bands of 25 and 48 bp; Ala312 was characterized by an
additional band of 117 bp, which was cleaved into bands of 78 and 39 bp
in the presence of Thr312, as shown in Figure 1
. Genotype was classified as TT
(Thr, Thr), TA (Thr, Ala), and AA (Ala, Ala).
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Mortality Data
All patients were registered with the Office of National
Statistics for notification of date of death. Mortality data available
up until the end of March 1998 are presented in this study.
2-Antiplasmin Incorporation
Citrated plasma (400 µL) was clotted with 40 µL of
thrombin/calcium mix (1:1, 200 U/mL thrombin,1 mol/L
CaCl2) and incubated at 37°C for 2 hours. The
clot was then removed by careful winding onto a wooden toothpick.
2-Antiplasmin in serum
(APs) and plasma (APp) was
then determined by chromogenic substrate assay (Coamatic
Plasmin Inhibitor, Chromogenix) using pooled normal plasma
as reference. The APs was adjusted for dilution,
and the proportion of 2-antiplasmin
incorporated into the fibrin clot was calculated as
(APp-APs)/APp.
Statistics
The 1-sample Kolmogorov-Smirnov goodness-of-fit test was used to
determine whether the distributions of continuous variables
deviated significantly from normal; nonnormally distributed
variables were log-transformed to allow analysis by
parametric tests. Differences in levels between 2 unrelated
groups were compared by unpaired Student's t tests and
between >2 groups by 1-way ANOVA with Scheffé post hoc
analysis. Results are expressed as mean or geometric mean and
95% CI. Differences in categorical variables between groups were
assessed by 
2 test. Genotype
differences in survival were assessed by the Kaplan-Meier log-rank
statistic. The association of genotype with survival after
acute stroke, taking into account covariates and including interaction
terms, was assessed by Cox regression analysis, and results
were presented as relative risk (RR) with 95% CIs. All
statistical analyses were performed with the SPSS statistics
package version 7.0 (SPSS Inc).
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Results |
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The genotype distributions of the patients with ischemic stroke and healthy control subjects did not differ significantly from Hardy-Weinberg equilibrium. There was no significant difference in the genotype distributions of patients (TT=58.0%, TA=35.8%, AA=6.2%) and control subjects (TT=60.6%, TA=32.8%, AA=6.6%), nor was there any association of Thr312Ala with stroke subtype (data not shown).
A total of 232 deaths in the patient group had been notified by March 31, 1998, representing a median follow-up of 2.8 (0.7 to 4.0) years. There was no significant difference in the genotype distributions of those who died (TT=55.6%, TA=37.9%, AA=6.5%) compared with those still alive (TT=59.8%, TA=34.3%, AA=5.9%). A Cox regression model using backward stepwise selection with the probability for removal from the model of P=0.1 was used to identify independent predictors of poststroke mortality, including Thr312Ala and factors associated with mortality in univariate analyses (age, sex, atrial fibrillation, stroke subtype, smoking history, previous stroke, and previous MI) as covariates, and interaction terms were created between Thr312Ala and other risk factors (introduced into the model in addition to the above-mentioned factors) to identify significant interactions. The only interaction term significantly associated with poststroke mortality was Thr312Alaxatrial fibrillation (P=0.002). Other factors independently associated with poststroke mortality in this model were age (RR for an increase of 10 years, 1.62 [1.42 to 1.85]; P<0.00001), stroke subtype (RR for large- compared with small-vessel disease, 2.37 [1.70 to 3.30]; P<0.0001), and previous stroke (RR for previous compared with first-ever stroke, 1.41 [1.08 to 1.85]; P=0.01).
The characteristics of patients in sinus rhythm (n=418) and those with
atrial fibrillation (n=101) are presented in the
Table. A greater proportion of
subjects with atrial fibrillation had died after the acute event, with
30-day and total mortality rates of 15.8% and 69.3%, respectively, in
those with atrial fibrillation compared with 5.0% and 38.8%,
respectively, in those in sinus rhythm (P<0.0001 for 30-day
and total mortality).
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On further investigation of the interaction of Thr312Ala with atrial
fibrillation, in subjects in sinus rhythm (n=418), there was no
significant difference in the genotype distributions of those
who subsequently died (n=162, TT=59.3%, TA=34.6%, AA=6.2%) compared
with those still alive (n=256, TT=57.6%, TA=35.7%, AA=6.7%). In
subjects with atrial fibrillation (n=101), however, there was a
significant difference in the genotype distributions of those
who had died (n=70, TT=47.1%, TA=45.7%, AA=7.5%) compared with those
still alive (n=31, TT=77.4%, TA=22.6%, AA=0%, P=0.01).
This association was confirmed by use of the Kaplan-Meier log-rank
statistic, as shown in Figure 2, which
indicated that in subjects with atrial fibrillation, possession of the
A allele was associated with poor outcome. The proportions of
subjects in sinus rhythm and with atrial fibrillation who were still
alive at the end of March 1998 classified by Thr312Ala genotype
are presented in Figure 3.
Approximately 60% of subjects in sinus rhythm survived in each
genotype group, whereas in those with atrial fibrillation, 24
of the 57 subjects with TT genotype (42.1%) survived, compared
with 7 of the 39 with TA genotype (18%) and none of the 5 with
AA genotype.
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There was no association of Thr312Ala genotype with the
proportion of 2-antiplasmin incorporated into
the fibrin clot: TT=39.0% (33.3% to 44.8%), TA=42.7% (37.8% to
47.7%), AA=42.0% (38.5% to 45.6%).
2-Antiplasmin incorporation was significantly
correlated with fibrinogen level (r=0.66,
P<0.0001); after adjustment for fibrinogen level, no
association of Thr312Ala with 2-antiplasmin
incorporation remained (data not shown).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The
-fibrinogen Thr312Ala polymorphism occurs in a region
of fibrinogen that is important for FXIIIa-dependent cross-linking,
suggesting that this polymorphism may influence clot
stability.2 4 5 In the present study, there was no
association of Thr312Ala with stroke or stroke subtype. However, a
significant interaction between Thr312Ala and atrial fibrillation was
identified in relation to poststroke mortality. Atrial fibrillation is the most common sustained cardiac arrhythmia, and the incidence of atrial fibrillation increases with advancing age, with a prevalence of 4% in subjects >60 years old and >10% in those >80 years old.8 Atrial fibrillation is associated with a 5-fold increased risk of developing stroke, probably as a result of embolization of left atrial thrombus.9 In the present study, 20% of subjects with ischemic stroke had atrial fibrillation, which is in keeping with the proportion of subjects with atrial fibrillation and stroke in the prospective Framingham study.10 In addition to an increased risk of stroke, subjects with atrial fibrillation have an increased mortality rate after stroke compared with those in sinus rhythm.10 11 12 In keeping with these findings, in the present study, a greater proportion of subjects with atrial fibrillation died after acute stroke compared with those in sinus rhythm. In the patients in sinus rhythm, there was no association of Thr312Ala genotype with survival. In the patients with atrial fibrillation, however, only 18% of subjects with TA genotype survived, and none of the subjects with AA genotype survived, compared with 42% of those with TT genotype.
Because no association was observed in those in sinus rhythm, these
findings suggest that Thr312Ala contributes to the pathogenesis of
thromboembolic complications associated with the presence of atrial
fibrillation. Whether this is related to an increased susceptibility to
form intra-atrial thrombus or an increased susceptibility for
embolization of intra-atrial clot is unclear from the present
study. However, we have also found a significant difference in the
Thr312Ala genotype distributions of 218 patients with venous
thromboembolism compared with 250 age- and sex-matched healthy control
subjects (Carter et al, unpublished observations, 1998). When patients
with venous thromboembolism were categorized into those with deep vein
thrombosis (DVT, n=120) and those with pulmonary embolism (PE,
n=98), only the genotype distribution of those with PE differed
significantly (P=0.02) from that of healthy control subjects
(PE: TT=0.49, TA=0.36, AA=0.15; DVT: TT=0.50, TA=0.42, AA=0.08;
controls: TT=0.60, TA=0.34, AA=0.06), related to an increased incidence
of the AA genotype in those with PE. These findings, therefore,
suggest that Ala312 may give rise to an increased susceptibility for
embolization of thrombus, possibly due to defective FXIII-dependent
cross-linking. We did not find any difference in the amount of
2-antiplasmin incorporated into fibrin clots
by Thr312Ala genotype; however, it is possible that this
polymorphism interferes either with the FXIII-dependent
-fibrin/-fibrin cross-linking or with the ability of
-fibrinogen residues 242 to 424 to enhance the dissociation of FXIII
a and b subunit dimers. In both of these situations, it might be
expected that this would result in a less tightly cross-linked fibrin
clot, which would reduce its mechanical strength and lead to an
increased tendency to embolization. A recent report by Curran et
al13 suggested that Thr312Ala is associated with
differences in in vitro fibrin gel structure parameters,
eg, gel porosity and density, lending further support to our findings.
Further studies are currently being undertaken to determine the effect
of this polymorphism on -fibrin cross-linking and FXIII a and b
subunit dimer dissociation.
Further prospective studies will be necessary to confirm these observations, in particular to determine whether possession of the A allele is related to an increased incidence of stroke in subjects with atrial fibrillation. The present study suggests that possession of Ala312 may prove to be an additional factor to be considered when stratifying risk of stroke in subjects with atrial fibrillation and may help to target subjects most at risk of thromboembolic complications in whom more aggressive therapy could be considered.
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Acknowledgments |
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This study was funded by the UK Stroke Association.
Received September 30, 1998; revision received January 29, 1999; accepted February 16, 1999.
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References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Ruggeri ZM. von Willebrand factor and fibrinogen. Curr Opin Cell Biol. 1993;5:898–906.[Medline] [Order article via Infotrieve]
2. Muszbek L, Adany R, Mikkola H. Novel aspects of blood coagulation factor XIII, I: structure, distribution, activation, and function. Crit Rev Clin Lab Sci. 1996;33:357–421.[Medline] [Order article via Infotrieve]
3.
Baumann RE, Henschen AH. Human fibrinogen
polymorphic site analysis by restriction endonuclease
digestion and allele-specific polymerase chain reaction
amplification: identification of polymorphisms at positions
A312 and Bß448. Blood. 1993;82:2117–2124.
4.
Kimura S, Aoki N. Cross-linking site in fibrinogen
for 2-plasmin inhibitor.
J Biol Chem. 1986;261:15591–15595.
5.
Credo RB, Curtis CG, Lorand L. -Chain domain
of fibrinogen controls generation of fibrinoligase (coagulation factor
XIIIa): calcium ion regulatory aspects. Biochemistry. 1981;20:3770–3778.[Medline]
[Order article via Infotrieve]
6.
Carter AM, Catto AJ, Bamford JM, Grant PJ.
Platelet GPIIIa PlA, and GPIb variable
number tandem repeat (VNTR) polymorphisms and markers of
platelet activation in acute stroke. Arterioscler Thromb Vasc
Biol. 1998;18:1124–1131.
7.
Dennis MS, Burn JPS, Sandercock PA, Bamford J, Wade
DT, Warlow CP. Long term survival after first-ever stroke: the
Oxfordshire Community Stroke Project. Stroke. 1993;24:796–800.
8. Singer DE. Anticoagulation to prevent stroke in atrial fibrillation and its implications for managed care. Am J Cardiol. 1998;81:35C–40C.[Medline] [Order article via Infotrieve]
9. The National Heart, Lung, and Blood Institute Working Group on Atrial Fibrillation. Atrial fibrillation: current understandings and research imperatives. J Am Coll Cardiol. 1993;22:1830–1834.[Abstract]
10.
Lin HJ, Wolf PA, Kelly-Hayes M, Beiser AS, Kase CS,
Benjamin EJ, D'Agostino RB. Stroke severity in atrial fibrillation:
the Framingham Study. Stroke. 1996;27:1760–1764.
11.
Jorgensen HS, Nakayama H, Reith J, Raaschou HO, Olsen
TS. Acute stroke with atrial fibrillation: the Copenhagen Stroke Study.
Stroke. 1996;27:1765–1769.
12.
Kaarisalo MM, Immonen-Raiha P, Marttila RJ, Salomaa V,
Kaarsalo E, Salmi K, Sarti C, Sivenius J, Torppa J, Tuomilehto J.
Atrial fibrillation and stroke: mortality and causes of death after the
first acute ischemic stroke. Stroke. 1997;28:311–315.
13.
Curran JM, Evans A, Arveiler D, Luc G, Ruidavets JB,
Humphries SE, Green FR. The fibrinogen T/A312 polymorphism
in the ECTIM study. Thromb Haemost. 1998;79:1057–1058.
Letter.[Medline]
[Order article via Infotrieve]
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