Factor XIII Val 34 Leu
A Novel Association With Primary Intracerebral Hemorrhage
Andrew J Catto, BSc, MRCP; Hans P Kohler, MD; Sally Bannan, BSc; Max Stickland, AIBMS;
Angela Carter, BSc; Peter J Grant, MD, FRCP
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Background and Purpose—A common G-to-T point mutation (Val 34 Leu) in exon 2 of the a-subunit of the factor XIII
is strongly negatively associated with the development of myocardial infarction. This result suggests that factor XIII Val
34 Leu is interfering with the formation of cross-linked fibrin. The role of factor XIII Val 34 Leu in the pathogenesis
of cerebral infarction and primary intracerebral hemorrhage is unknown.
Methods—Six hundred twelve patients with acute stroke, defined by World Health Organization criteria and cranial CT,
and 436 age-matched control subjects free of cerebrovascular disease were genotyped for the factor XIII Val 34 Leu
mutation. Venous blood was drawn for the determination of hemostatic variables and lipids. Factor XIII genotype was
determined through a single-stranded conformational polymorphism technique and plasminogen activator inhibitor
(PAI)-1 4G/5G promoter genotype by allele-specific polymerase chain reaction.
Results—The mutation was more frequent in patients with primary intracerebral hemorrhage (n562) (54.8%; P5.05) than
in control subjects (41.7%) or in patients with cerebral infarction (n5529) (46.5%; P5.22). There was no relationship
between PAI-1 levels and the PAI-1 4G/5G genotype.
Conclusions—There was a slightly higher incidence of factor XIII Val 34 Leu in patients with PICH. This may be related
to impaired cross-linking of fibrin and/or coagulation proteins. (Stroke. 1998;29:813-816.)
Key Words: factor XIII n fibrin n intracerebral hemorrhage n mutation n risk factors
T
he importance of the hemostatic system in the pathogenesis of atherothrombotic disorders has been demonstrated in prospective studies. These have identified increased
concentrations of fibrinogen, factor VII, and suppressed
fibrinolysis resulting from elevated PAI-1 in relation to MI.1
Elevated levels of tissue plasminogen activator2 and fibrinogen3 are risk factors for CI. In MI, abnormal fibrin structures
have been reported,4 and factor XIII may have a role in this
process.
Factor XIII is activated by thrombin, cleaving the a-subunit between Arg37 and Gly38 releasing the N-terminal 37
residues (activation peptides). The a-subunit selectively
cross-links fibrin monomers and other substrates, including
a-2 antiplasmin.5 Cross-linked fibrin shows increased resistance to fibrinolysis and enhanced mechanical strength.6
We have studied a G-to-T point mutation in codon 34 of
exon 2 that codes for a valine-leucine (factor XIII Val 34 Leu)
change in the a-subunit, 3 amino acids from the thrombin
activation site,7 in 398 subjects examined for investigation of
coronary artery disease determined by angiography.8 The
prevalence of the mutation was lower in subjects with MI,
indicating that factor XIII Val 34 Leu is protective against
atherothrombotic disease. However, in subjects with factor
XIII Val 34 Leu and MI, higher PAI-1 levels and an increased
frequency of the PAI-1 promoter 4G/4G genotype suggest
that inhibition of fibrinolysis negates the protective effects of
factor XIII Val 34 Leu, which supports a functional role for
this mutation.
In view of the apparently protective role of factor XIII Val
34 Leu in the pathogenesis of MI, we hypothesized that
possesion of the mutation might either be protective against
CI or predispose to the development of PICH through the
formation of weaker fibrin structures. The aims of this study
were therefore to investigate (1) the association of the factor
XIII Val 34 Leu genotype and the phenotypes CI and PICH
and (2) the relationships between PAI-1 levels, the PAI-1
4G/5G genotype, and the factor XIII Val 34 Leu mutation in
the pathogenesis of these discrete phenotypes.
Subjects and Methods
Study Population
We recruited 612 consecutive inpatients with acute stroke defined by
World Health Organization Criteria9 (excluding subarachnoid hemorrhage) and 435 healthy control subjects closely matched for age,
sex, and domicile and free of clinically detectable cerebrovascular
disease, as described previously.10 All subjects gave informed consent according to a protocol approved by the Research Ethics
Committees.
Identification of Stroke Phenotype
To determine the stroke phenotype (CI or PICH), cranial CT was
performed within 10 days of stroke. Cases with CI were classified
Received November 18, 1997; final revision received January 12, 1998; accepted January 12, 1998.
From theUnit of Molecular Vascular Medicine, Research School of Medicine, University of Leeds, Leeds General Infirmary, Leeds, United Kingdom.
Correspondence to Professor Peter J Grant, Unit of Molecular Vascular Medicine, Leeds General Infirmary, Leeds LS1 3EX, UK.
E-mail [email protected]
© 1998 American Heart Association, Inc.
813
814
Factor XIII Gene Intracerebral Hemorrhage Risk Factors
CI
IC
MI
PAI
PCR
PICH
Selected Abbreviations and Acronyms
5 confidence interval
5 intracerebral hemorrhage
5 myocardial infarction
5 plasminogen activator inhibitor
5 polymerase chain reaction
5 primary intracerebral hemorrhage
TABLE 1. Comparison of Factor XIII Val 34 Leu Combined
Genotypes (Wild Type 1/1 vs. Mutants 1/2 vs. 2/2) in Cases by
Stroke Type (cerebral infarction n5529 and primary
intracerebral hemorrhages n562) With Controls (n5436)
Stroke Type
Factor XIII wild type
into subtypes according to the Oxfordshire Community Stroke
Project Classification.11
Analysis of Circulating PAI-1 Activity
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Nonfasting venous blood samples were taken from both cases
(within 10 days of stroke) and controls between 8 and 11 AM and
placed in 0.1 mol/L sodium citrate (9:1, vol/vol) in ice water for
assay of PAI-1 activity, then centrifuged at 2560g at 4°C for 30
minutes. Samples were stored at 240°C. PAI-1 activity was measured by a chromogenic assay (Spectrolyse, Biopool).12 The interand intra-assay coefficients of variation for the assay were 5.7% and
3.1%, respectively. Cholesterol and total triglyceride levels were
measured with the Hitachi 747 automated analyzer (Boehringer
Mannheim).
Extraction of DNA and Genotype Determinations
Genomic DNA was extracted from 10 mL venous blood and
anticoagulated with 1.6mg/mL EDTA, with use of a detergent/salt
exchange method described previously.13
Identification of the Factor XIII Val 34 Leu Genotype
A 183-bp fragment of exon 2/intron B of the factor XIII gene was
amplified by PCR, with 59-ACCCAGAGTGGTGGGGAAG as 59
primer and 59-GACCTTGTAAAGTCAAAAATGTC as the 39
primer. PCR reaction conditions were as described previously.8 To
detect the substitution of guanine to thymine (Val 34 Leu), we used
a single-stranded conformational polymorphism technique. Genotype was classified by two independent observers as wild type 1/1
(val/val), heterozygote 1/2 (val/leu), or homozygote mutant 2/2
(leu/leu). Confirmation of genotypes was carried out by sequencing
a random selection of samples using dye-labeled terminators (ABI
Prism™ Dye Terminator Cycle Sequencing Ready Reaction Kit,
Perkin-Elmer) on an ABI 373A sequencer. There were no discrepancies between genotypes determined by the two methods.
Identification of the PAI-1 4G/5G Promoter Genotype
The PAI-1 4G/5G promoter polymorphism was determined for each
subject by PCR amplification of genomic DNA with use of allelespecific primers and PCR conditions, as previously described.
Samples were classified into one of three possible genotypes
(4G/4G, 5G/5G, or heterozygous 4G/5G) and validated using known
genotypes verified by direct sequencing.
Statistical Methods
Body mass index, cholesterol, triglycerides and PAI-1 activity were
log transformed. PAI-1 levels in cases and controls were compared
using unpaired t tests. Levels were expressed as geometric means
and antilogged, with 95% CIs. x2 analysis was used to compare the
genotype distributions. The determinants of PICH were studied in a
logistic regression model, with the covariates age, sex, current
smoking history, past history of hypertension, diabetes mellitus,
ischemic heart disease, and factor XIII Val 34 Leu genotype.
Statistical analysis was performed with SPSS for Windows, version
6.1 (SPSS Inc).
Results
Characteristics of the Study Population
The median age of the case patients (n5612) and control
subjects (n5436) was 73.0 years. Conventional vascular risk
LACI
TACI
PACI/
POCI
99
69
115
4O (53.4%) O3
Factor XIII Val 34 Leu
77
51
118
4O (46.5%) O3
PICH
Control
28
254
(45.2%)
(58.3%)
34
182
(54.8%)
(41.7%)
Percentages refer separately to cerebral infarction, PICH, and controls.
Twenty-one cases of stroke were unclassified and thus not included in the
analysis. No differences were observed in genotype frequency between all
stroke cases (x2354.46; P5.22) or between cerebral infarcts and controls
(x2152.19; P5.14). There was an excess of the mutation in PICH cases
compared with controls (x2153.79; P5.05). LACI indicates lacunar infarction;
TACI, total anterior circulation infarction; PACI, partial anterior circulation
infarction; and POCI, posterior circulation infarction.
factors, namely, ischemic heart disease, MI, diabetes mellitus,
hypertension, and current smoking, were more common in
the case patients (P,.01). Cholesterol levels were lower in
cases than controls (5.1 mmol/L [95% CI, 3.4 to 7.3] and
5.8 mmol/L [CI, 5.6 to 5.9], respectively; P5.003).
Factor XIII Val 34 Leu genotypes were available in all
cases and controls; PAI-1 4G/5G genotypes were available in
558 cases and 172 controls. PAI-1 activity was measured in
the first 312 cases and in 213 controls. In cases, the median
PAI-1 level was 9.8 U/mL (95% CI, 8.8 to 10.9); in controls,
8.8 U/mL (CI, 7.8 to 10.1) (P,.0001).
Genotype Distribution in All Cases and Controls
The distribution of the factor XIII Val 34 Leu genotype in
cases was G/G, 326 (53.3%); G/T, 240 (39.2%); and T/T,46
(7.5%); in controls it was G/G, 254 (58.3%); G/T, 157
(36.0%); and T/T, 25 (5.7%). Distribution of the PAI-1
4G/5G genotypes in cases was 4/4, 148 (26.9%); 4/5, 270
(49.1%); and 5/5, 132 (24.0%); in controls it was 4/4, 60
(33.9%); 4/5, 84 (47.5%); and 5/5, 33 (18.6%). There was no
difference in the distribution of the factor XIII Val 34 Leu (or
PAI-1 4G/5G genotypes) between cases and controls (P5.21
and P5.13, respectively). There was no age relation with
genotype (case patients ,60 years (n5103) compared with
age-matched control subjects (n580); x250.16, P5.68), nor
was there any relation in controls when analyzed only by age
subdivided by decades (x2 [test for trend]50.19, P5.20).
Genotype Frequencies in PICH
Table 1 demonstrates an excess of the mutation in patients
with PICH (n562) compared with control subjects (P5.05).
In the logistic regression model, significant independent
variables for PICH were a past history of ischemic heart
disease (P5.006) and diabetes mellitus (P5.04) but not
factor XIII Val 34 Leu genotype (P5.22). In this model there
was no interaction between ischemic heart disease and factor
XIII Val 34 Leu genotype (P..05).
There was no difference in distribution of the PAI-1 4G/5G
genotype and factor XIII Val 34 Leu genotypes between
Catto et al
April 1998
815
TABLE 2. Selected Cardiovascular and Hemostatic Risk Factors in Patients With PICH (n572)
According to Factor XIII Val 34 Leu Genotype
Wild Type
(n528)
Mutant Val 34 Leu
(n534)
P
Body mass index, kg/m2
24.00 (22.23 to 24.03)
26.21 (26.19 to 24.03)
.06
Cholesterol level, mmol/L
4.76 (4.37 to 5.19)
5.27 (4.77 to 5.82)
.16
Triglyceride level, mmol/L
1.28 (1.02 to 1.60)
1.34 (1.05 to 1.72)
.76
262.4 (236.5 to 291.1)
236.7 (209.5 to 267.3)
.22
Acute
4.26 (3.71 to 4.90)
4.42 (3.92 to 4.97)
.71
Convalescent
3.35 (2.91 to 3.85)
3.81 (3.42 to 4.24)
.17
Acute
7.78 (5.21 to 11.62)
11.79 (7.51 to 18.52)
.20
Convalescent
8.62 (6.93 to 10.69)
10.87 (7.37 to 16.03)
.32
Platelet count
Fibrinogen, g/L
PAI-1 activity, U/mL
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History of hypertension, n (%)
9 (32.1%)
15 (44.1%)
.34
History of stroke, n (%)
3 (10.7%)
7 (20.6%)
.29
History of ischemic heart disease, n (%)
2 (7.1%)
5 (14.7%)
.35
Values shown are geometric mean (95% CI).
cases of both PICH and CI and no significant relationships
between factor XIII Val 34 Leu and other stroke risk factors.
Discussion
The results from this study indicate an association between
possession of a G-to-T point mutation coding for factor XIII
Val 34 Leu and PICH. We hypothesized that possession of
factor XIII Val 34 Leu mutation might favor the formation of
weaker fibrin structures, thereby protecting against CI or
predisposing to PICH. This study supports our previous
findings that factor XIII Val 34 Leu may play a significant
role in the pathogenesis of vascular disorders. The lack of
association of the mutation with ischemic stroke is interesting
when contrasted with MI. However, this may be accounted
for by the heterogeneous nature of ischemic stroke compared
with that of MI.
The precise role for fibrin in the pathogenesis of cerebrovascular disease has not been established. However, the
formation of cross-linked fibrin from fibrin monomer is
pivotal for the development of a stable thrombus, and
abnormalities of fibrin structure and architecture are associated with premature MI in men.4 This process involves the
action of thrombin on fibrinogen to produce soluble fibrin
and the activation of factor XIII, which cross-links fibrin,
thereby rendering it more resistant to fibrinolysis. Lysis of
fibrin is dependent on the binding of tissue plasminogen
activator and plasminogen to fibrin with local plasmin production in an environment protected from the circulating
PAI-1.
In our population, levels of PAI-1 were elevated in PICH
(median, 11.4 U/mL) as well as in CI (12.1 U/mL). Unlike in
MI, we did not demonstrate a relationship between the factor
XIII Val 34 Leu mutation and levels of PAI-1 or the 4G/5G
genotype, although there was a trend to higher levels of PAI-1
in subjects with PICH and the factor XIII Val 34 Leu
mutation than in those with PICH and the wild types (11.8
U/mL and 7.8 U/mL, respectively) (Table 2).
The results from this study indicate a slightly higher
incidence of the factor XIII Val 34 Leu mutation and risk of
PICH, although the present study does not provide direct
evidence that factor XIII Val 34 Leu results in direct
functional alteration in the protein. The findings of this study
have to be viewed in the light of the drawbacks associated
with a case-control study and the relatively small numbers of
PICH subjects available for study in such a cohort. However,
while this finding was not an independent relationship in this
population andthe findings could not be regarded as causal,
these observations are consistent with previous findings in
relation to MI, which indicates that this mutation justifies
further study. Attention should be directed to the heterogeneous nature of PICH, because the factor XIII Val 34 Leu
mutation may be more strongly related to the different
pathological variants of PICH, such as lipohyalinosis or
amyloid angiopathy. Furthermore, the influence of very early
mortality from PICH14 may be acting to dilute the strength of
the association, a possibility that should be considered in
future studies of the factor XIII Val 34 Leu mutation and
PICH. This might be addressed through use of archival DNA
methods in subjects who have died from PICH.
Acknowledgments
This study was supported by the Stroke Association. HPK is funded
by the Royal Society, London, United Kingdom.
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Factor XIII Val 34 Leu: A Novel Association With Primary Intracerebral Hemorrhage
Andrew J Catto, Hans P Kohler, Sally Bannan, Max Stickland, Angela Carter and Peter J Grant
Downloaded from http://stroke.ahajournals.org/ by guest on June 18, 2017
Stroke. 1998;29:813-816
doi: 10.1161/01.STR.29.4.813
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