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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 3 (February 1), 1999:
pp. 897-905
Genotype/Phenotype Correlations for Coagulation Factor XIII: Specific
Normal Polymorphisms Are Associated With High or Low Factor XIII
Specific Activity
By
Rashida Anwar,
Louise Gallivan,
Stuart D. Edmonds, and
Alexander
F. Markham
From the Molecular Medicine Unit, University of Leeds, St James's
University Hospital, Leeds, UK.
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ABSTRACT |
Factor XIII is a transglutaminase essential for normal hemostasis.
We have studied the plasma FXIII levels and FXIII activity in 71 individuals and found these to be normally distributed. FXIII specific
activity is also normally distributed. However, we show that FXIII
activity is not directly dependent on FXIII levels, and individuals
with low FXIII levels may have high FXIII activity and vice versa. We
have determined the FXIIIA genotype in these individuals to assess
whether the variation observed in FXIII specific activity is dependent
on specific polymorphisms in the FXIIIA gene. Our data show that the
Leu34 and Leu564 variants give rise to increased FXIII specific
activity, while the Phe204 variant results in lower FXIII specific
activity. We also report preliminary evidence that the Phe204
polymorphism may be associated with recurrent miscarriage. Overall, we
have identified 23 unique FXIIIA genotypes. Certain specific FXIIIA
genotypes consistently give rise to high, low, or median FXIII specific
activity levels, while others appear to have little or no consistent
influence on the FXIII phenotype. These genotype to phenotype
relationships are discussed in light of the growing interest in the
role of FXIII in clinical problems involving an increased thrombotic tendency.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
FXIII IS A TRANSGLUTAMINASE, which plays
an essential role cross-linking fibrin in the final stages of the blood
coagulation pathway. Low levels of FXIII have been reported in a number
of clinical conditions, including inflammatory bowel disease (both Crohn's disease and ulcerative colitis), as well as several types of
malignancy.1-3 Increased transglutaminase activity is found in the Alzheimer's disease brain.4 In addition, tissue
transglutaminase is now known to be the autoantigen of celiac
disease.5 Several studies have also indicated that FXIII is
important in stimulation of connective tissue cells and the processes
of inflammation and wound healing.6-9 Inherited homozygous
FXIII deficiency results in serious bleeding complications, inefficient
wound healing, and a high risk of miscarriage in deficient
females.10,11
The distribution of FXIII is now known to be almost
ubiquitous.12 Intracellular FXIII exists as a homodimer of
two A subunits, while circulating FXIII is an
A2B2 tetramer.13 The A subunit constitutes the catalytic moiety and the B subunit is thought to play a
role in stabilization of the A subunit. On activation by thrombin and
Ca2+, the A and B subunits dissociate. The A subunit is
then cleaved to produce the catalytically active form of the protein,
FXIIIA*.14 FXIIIA* catalyzes the Ca2+-dependent
formation of intermolecular  -( -glutamyl) lysine bonds between
fibrin molecules leading to stabilization of the clot structure.15 FXIIIA* can also cross-link fibronectin,
vitronectin, collagen, and lipoprotein(a) in the extracellular matrix.
Deficiency of both the FXIII A and B subunits has been described.
However, in the majority of cases, inherited autosomal recessive FXIII
deficiency is due to defects in the gene for the FXIIIA subunit.16-18 Hence, the FXIIIA subunit has been studied
much more extensively than the FXIIIB subunit. The FXIIIA gene is
now known to be highly polymorphic.16 The known, normal
FXIIIA gene polymorphisms are presented in Table 1.
A number of methods have previously been used to determine the activity
of FXIII in plasma. These are based on measurements of clot
stability,19 ammonia production,20 and
incorporation of labelled amines into either polymers of
casein21,22 or fibrin.23 We describe a
modification of the activity assay of Song et al,23 based
on measuring incorporation of biotinylcadaverine into fibrinogen to
determine the plasma FXIII activity. We also present an enzyme-linked immunosorbent assay (ELISA) for determination of FXIII levels. Both
assays are performed in 96-well microtiter plates for speed and for
ease of handling of large sample numbers.
In this report, we show that FXIII activity is not directly dependent
on FXIII levels and discuss the variation we have observed in FXIII
specific activity in 71 unrelated healthy individuals. We also present
the FXIIIA gene sequence variations we have found in 113 individuals
(comprising 36 normal men, 42 normal women, and 35 women who have
suffered three or more recurrent miscarriages), who are all normal with
respect to FXIII, from the United Kingdom. This variation in genotype
is then compared with the variation in FXIII specific activity, in each
individual in each group, to assess the influence of specific amino
acid changes (inferred from the genotype data) and the combination of
FXIIIA polymorphisms on specific activity. We describe the first
examples of associations between normal FXIIIA variants and high or low
FXIII specific activity. We therefore provide the first evidence of a
genetic basis for the wide variation seen in normal plasma FXIII levels and activities. Women who are homozygously deficient in FXIII are known
to suffer spontaneous abortion.11 We now present data, which suggests an association between one specific FXIIIA normal genetic variant and risk of recurrent miscarriage in women who are
otherwise normal with respect to plasma FXIII.
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MATERIALS AND METHODS |
Materials.
All chemicals were purchased from Sigma (Poole, UK) unless otherwise
stated. Sheep anti-human FXIII was obtained from The Binding Site
(Birmingham, UK) and rabbit anti-human FXIII was purchased from
Calbiochem (La Jolla, CA). Biotinylcadaverine was obtained from Pierce
& Warriner (Chester, UK). Standard normal plasma (Behring Standard
Human Plasma) was obtained from Behring Diagnostics (Milton Keynes, UK).
Subjects.
Blood samples were analyzed from 113 consenting, healthy, unrelated
individuals who do not suffer from FXIII deficiency. The 113 individuals are subdivided as follows: 36 normal (N) men, 42 N women,
and 35 women who have suffered three or more recurrent miscarriages.
The mean age of these individuals was 30.3 years ± 5.5 years
(standard deviation), age range, 18 to 49 years. Ethical approval from
The Leeds (East) Medical Ethics Committee was obtained before
initiating this study.
Sample processing.
Blood was collected in citrated Monovette tubes (Sarstedt, UK). The
plasma was separated by centrifugation, aliquoted, and flash frozen in
liquid nitrogen for storage at  70°C. The buffy coat,
containing the peripheral blood mononuclear cells (PBMCs), was also
collected and stored at 70°C for isolation of genomic DNA.
FXIII ELISA.
FXIII levels were determined using an ELISA. Ninety-six-well
microtiter plates (Corning, High Wycombe, UK) were coated with 100 µL/well of sheep anti-human FXIII antisera (1:1,000 dilution of 10 mg/mL antisera, in 200 mmol/L citrate/phosphate buffer, pH 3.0) for 1 hour at 37°C. The plates were blocked for 1 hour at 37°C using
200 µL/well of blocking buffer (1% bovine serum albumin in 0.5 mol/L
NaCl, 20 mmol/L Tris.HCl, pH 7.5, containing 0.01% Tween-20 and 0.02%
azide). The plate was then washed twice with blocking buffer. For each
test plasma sample and the normal plasma standard, a range of dilutions
was performed in blocking buffer ensuring that the dilution range
covered both the maximum and the minimum possible response. A total of
100 µL of diluted plasma samples was then loaded into each well, in
triplicate, and the plate incubated for 2 hours at 37°C. The plate
was washed twice with blocking buffer, then twice with washing buffer
(0.5 mol/L NaCl, 20 mmol/L Tris.HCl, pH 7.5, containing 0.01% Tween-20 and 0.02% azide) before applying 100 µL of rabbit anti-human FXIII antisera (1:1,000 dilution in blocking buffer) to each well. After a
further incubation for 1 hour at 37°C, the plate was rinsed once
with blocking buffer followed by two rinses with washing buffer. A
total of 100 µL of anti-rabbit IgG conjugated to alkaline phosphatase
(1:20,000 dilution in blocking buffer) was added to each well and the
plate incubated for 1 hour at 37°C. The plate was then washed twice
with washing buffer.
To each well, 100 µL of alkaline phosphatase substrate, p-nitrophenol
phosphate (pNPP; 1 mg/mL in 1 mol/L diethanolamine, pH 9.8, containing
0.5 mmol/L MgCl2), was added and the color development
performed at 37°C. The reaction was stopped by the addition of 100 µL of 4 mol/L NaOH. The absorbance was then read at 405 nm.
Calculation of FXIII levels.
The relative amount of FXIII in each plasma sample was determined using
a relative quantitation method comparing 50% maximum binding
(IC50) using dilutional analysis for each plasma sample. In
this method, it is important that the maximum and minimum responses are
achieved. The absorbances at which these two responses occur are then
designated 100% and 0% response, respectively, and the absorbances
observed at all other dilutions of plasma are calculated as a
percentage of the maximum response. Figure 1A shows a graph of
percentage response versus dilution of plasma. A sigmoidal fit is
applied and the dilution at which the IC50 is achieved is
then calculated. The relative FXIII level in each test plasma sample is
determined by the ratio of the IC50 of the test plasma to
the IC50 of standard normal plasma.
FXIII activity assay.
FXIII activity was determined using a modification of the fibrinogen
and biotinylcadaverine assay described previously.23 Ninety-six-well microtiter plates were coated with fibrinogen (100 µL/well of a 40-mg/mL solution in TBS; 40 mmol/L Tris.HCl, pH 8.3, 140 mmol/L NaCl, 0.02% azide) for 40 minutes at 25°C. The plate
was then blocked with 0.5% nonfat dried milk in TBS (NFDM). After 20 minutes at 37°C, the plate was washed with TBS and the following
reaction components were added; 50 µL of TBS, 10 µL of 0.5 mmol/L
dithiothreitol (DTT), 10 µL of 40 mmol/L
biotinylcadaverine, 10 µL of a 1:10 dilution of test plasma, 10 µL
of 1 mol/L CaCl2, and finally 10 µL of thrombin solution
(200 U/mL in TBS). The reaction was performed at room temperature and
stopped at various time points by the addition of 200 µL of 200 mmol/L EDTA. For t = 0, EDTA was added to the wells before the addition
of the reagents. The plate was then rinsed with TBS and 100 µL of
streptavidin-alkaline phosphatase (SAAP) conjugate (1:100 dilution of a
1 mg/mL solution in NFDM) was added to each well. After incubation for
1 hour at 37°C, the plate was washed twice with TBS containing
0.01% Triton X100, followed by two rinses with TBS. The color
development step was performed as described for the FXIII ELISA above.
For each plasma sample, the initial rate of generation of product is
determined (Fig 1B), and the activity is calculated as a percentage of
the rate observed with standard normal plasma.
Statistical analysis.
This was performed using SPSS (SPSS UK, United Kingdom) and Clump
statistics software (Dave Curtis; www.hgmp.mrc.ac.uk).
The analysis performed on the distribution of FXIII levels, activity, and specific activity was done using the Kolmogorov-Smirnov
"Goodness of Fit" Test which compares a given distribution with a
normal distribution.
Genotype analysis.
Genomic DNA was isolated from PBMCs using standard procedures. Exons 2, 5, 12, and 14 of the FXIIIA gene, carrying the known normal
polymorphisms, were amplified by polymerase chain reaction (PCR) as
described previously.16 PCR products from exons 2, 5, and
14 were denatured and then subjected to single-strand conformational polymorphism (SSCP) analysis in GeneGel Excel polyacrylamide gels (122 × 110 × 0.5 mm; Pharmacia Biotech, St Albans, UK) cooled continuously at 15°C, using the Pharmacia Biotech PhorGene
electrophoresis unit. After electrophoresis at 600 V for 90 minutes,
the gels were silver-stained using the Pharmacia Biotech silver
staining kit. PCR products showing mobility shifts in SSCP analysis
were sequenced as described previously.16 The sequence
variation at codon 564 (leucine or proline) was determined by
amplification refractory mutation system-PCR (ARMS-PCR)17
using the forward primer (dTTGCCTGTCATTATCTCTGG) with both a leucine
specific reverse primer (dCTTCTTGAAYTCTGCCTTGA), and proline specific
reverse primer (dCTTCTTGAAYTCTGCCTTGG) in separate PCRs. The sequence
variation at codon 567 was determined by EcoRI restriction analysis of
the exon 12 PCR product.
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RESULTS |
Factor XIII levels.
An ELISA assay has been developed to determine the levels of FXIII in
plasma. Figure 1A shows the dilution
profiles for plasma samples from three different individuals and the
method for determining the relative levels in each. It is clearly
possible to distinguish plasma samples containing different
concentrations of FXIII. The plasma FXIII levels were determined in 73 unrelated individuals. Levels were found to vary between 47.9% and
243.9% of that of the standard normal plasma with a mean at 105% ± 37.64% standard deviations (Fig 2A).
This variation was analyzed and found to be consistent with a normal
distribution (Fig 2A). The range of FXIII levels was compared between
the three groups studied and no differences were found. The range of
FXIII levels found is comparable to results we obtained previously from
34 normal, unrelated individuals using a rocket
immunoelectrophoresis assay (unpublished results, 1983).
Our results also compare favorably with other, smaller studies of
Murdock24 (mean, 95; range, 60 to 130, n = 24) and
Shainoff25 (mean, 112; range, 50 to 200, n = 12).
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| Fig 1.
(A) The percentage response at different dilutions,
assayed in triplicate, of three separate plasma samples. The FXIII
level in each of the plasma samples is determined by extrapolating the
inverse of the dilution at the IC50 and calculating the
FXIII level as a percentage of the IC50 observed with
standard normal plasma. (B) Initial rates of incorporation of
biotinylated cadaverine into fibrin in three separate plasma samples
showing the different rates detected for each of these samples. Each
sample was assayed in triplicate for every time point.
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| Fig 2.
FXIII activity, levels, and specific activity (SA) within
the normal study population. The Kolmogorov-Smirnov Goodness of Fit
Test was used to test the hypothesis that the values obtained for FXIII
levels, activity, and SA match a normal distribution. The value of the
Kolmogorov-Smirnov test is based on the largest absolute difference
between the observed and the theoretical cumulative distributions. The
two-tailed probability value was used to decide whether the null
hypothesis should be rejected. This is the probability of obtaining
results as extreme as the ones observed, in either direction, when the
null hypothesis is true. Because the two-tailed P values
obtained are much higher than .05, the null hypothesis is not rejected,
and hence the variation observed in FXIII levels, activity, and SA fits
a normal distribution. For (A) to (C) the normal distribution curve is
also plotted for comparison. (A) This histogram illustrates the
variation seen in plasma FXIII levels in 73 unrelated individuals
comprising 22 normal men, 37 normal women, and 14 RM women. (B) The
distribution of plasma FXIII activity in 72 unrelated individuals
comprising 23 normal men, 35 normal women, and 14 from RM group. (C)
FXIII SA was defined as FXIII activity per unit FXIII level, this being
1.0 (ie, 100% activity/100% level) for the standard normal plasma. SA
of plasma FXIII in 71 unrelated individuals (comprising 22 normal men,
35 normal women, and 14 RM women) mean age, 30.2 years, ± 5.5 standard deviation; age range, 18 to 48. (D) A normal probability plot
was used to determine whether FXIII SA is normally distributed. The
cumulative proportion for a single numeric variable is plotted against
the cumulative proportion expected if the sample were from a normal
distribution. Because the points all cluster around a straight line,
this suggests that FXIII SA has a normal distribution within the
population studied. SD, standard deviation; N, numbers studied; KS,
Kolmogorov-Smirnov Test; P, two-tailed probability value.
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FXIII activity.
The FXIII activity in plasma was determined by monitoring the rate of
incorporation of biotinylated cadaverine into fibrin. Figure 1B shows
that a difference in the rates of incorporation can be detected in
different plasma samples. When the plasma FXIII activity was
investigated in a population of 72 unrelated individuals, it was found
to range between 53.2% and 221.3% of the standard normal plasma value
(Fig 2B). The mean was calculated to be 105% ± 28.56% standard
deviations. This variation was also found to indicate a normal
distribution (Fig 2B). Again, no difference was found in the range of
FXIII activities between the three groups studied. The range that we
have found for FXIII activity is similar to the 47% to 250% range we
had previously determined when measuring the rate of incorporation of
dansylcadaverine into casein and comparing it with normal pooled plasma
(data not presented). Our data are also comparable to the results of
Wagner et al,26 who found the FXIII activity range to be
between 0.51 to 1.52 of the pooled plasma value (in U/mL for n = 64).
FXIII specific activity.
The FXIII specific activity was calculated for all the individuals
studied. Figure 2C shows the distribution of specific activity in 71 unrelated individuals and this is compared with a normal distribution.
There was no significant difference in the range of specific activities
found in the three groups studied. The overall FXIII specific activity
range was found to be between 0.50 and 2.13, with the value for
standard normal plasma being set at 1.0. The mean was 1.08 ± 0.40 standard deviations. Both the Kolmogorov-Smirnov Test (Fig 2A through
C) and the normal probability plot (Fig 2D) suggested that the FXIII
specific activities we have found in our study fit a normal distribution.
FXIIIA genotype.
Analysis of the FXIIIA gene exons 2, 5, 12, and 14 was performed in 113 individuals. The polymorphisms Arg77Gly, Arg78Lys, and Phe88Leu in exon
3 were not examined because these have been identified only at the
amino acid or cDNA level and have never been reported at the genomic
DNA sequence level (Table 1). We have
sequenced exon 3 of the FXIIIA gene in 25 individuals and have not
found any to carry the codon 77, 78, or 88 variations.
The frequency of each of the normal sequence variations is presented in
relation to the number of chromosomes studied
(Tables 2 and
3) and to numbers of homozygotes and
heterozygotes for each amino acid change
(Table 4). Although the frequency of Leu at
codon 34 is about 20% (Table 2), only one individual from 113 (0.88%)
was found to be homozygous for Leu34 (Table 4). Also, there were no
homozygotes found for Phe at codon 204 or Ile at codon 650 (Table 4).
The GAG sequence at codon 567 was only observed in Pro564 homozygotes
and Pro564/Leu564 heterozygotes, but not in Leu564 homozygotes. Nine
different haplotypes were identified from 88 individuals, representing
176 chromosomes, who were either homozygous for each polymorphism or
heterozygous at only one locus (Table 3). The
Val34-Tyr204-Pro564-Val650-Glu651 (11111) haplotype appears to be the
most common. Twenty-three unique genotypes were identified in the
population studied, suggesting a high degree of genetic variability
(Table 5). Again the genotype
Val34/Val34-Tyr204/Tyr204-Pro564/Pro564-Val650/Val650-Glu651/Glu651 (11111) is the most common followed closely by
Val34/Leu34-Tyr204/Tyr204-Pro564/Pro564-Val650/Val650-Glu651/Glu651 (31111) and
Val34/Val34-Tyr204/Tyr204-Pro564/Leu564-Val650/Val650-Glu651/Glu651 (11311).
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Table 2.
Frequency of the Normal FXIIIA Polymorphisms Found at
Codons 34, 204, 564, 567, 650, and 651 in 113 Individuals,
Representing 226 Chromosomes
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Table 3.
Haplotypes Identified From 88 Individuals Who Were
Either Homozygous for All of the FXIIIA Normal Polymorphisms or
Were Heterozygous for Only One Polymorphism
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Table 5.
The Frequency of the FXIIIA Genotypes Identified in
113 Individuals Analyzed and Correlation of Each Genotype With
FXIII SA Found for That Genotype
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The genotype frequencies for each of the polymorphisms were then
compared between the different groups studied (Tables 2 and 4). The
Phe204 polymorphism appears to be more common in the recurrent
miscarriage (RM) group (Tables 2 and 4). A significant difference for
Phe204 frequency was discovered when the RM data were compared with all
normals (78 N men and women; P = .017), as well as when the RM
data were compared with the N women group (P = .053). There
were no significant differences found for codons 34, 564, 650, and 651 between N men versus N women, N men versus all women (N women + RM
women), N women versus the RM group, and the RM group versus all
normals (N men + N women).
FXIIIA genotype/phenotype correlation.
This analysis also includes some individuals from families we have
studied previously,16 known to be heterozygous for FXIIIA deficiency alleles and these were therefore not presented as part of
the normal population study (Fig 2). The influence of each specific
FXIIIA polymorphism on FXIII specific activity was assessed (Table 4).
Alleles coding for Leu34, Tyr204, or Leu564 are associated with high
FXIII specific activity. The effects of the polymorphisms at codons 650 and 651 appear to be almost minimal to normal FXIII specific activity.
The FXIII specific activity found for each of the different genotypes
(representing codons 34, 204, 564, 650, and 651) is presented in Table
5 and Fig 3. It is clear that specific
genotypes can be associated with a tendency to give rise to FXIIIA
molecules with high, median, or low specific activities (Fig 3). Some
FXIIIA genotypes appear to have no influence on the resultant FXIII
specific activity, the same genotype resulting in variable FXIII
specific activity, indicating there may be other factors additional to
the genotype, which also affect this (Fig 3). The influence of Leu564
and Pro564 on FXIII specific activity is also clearly visible in Fig 3.
Leu564 is associated with a higher FXIII specific activity compared
with Pro564.
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| Fig 3.
The range of FXIII specific activity (SA) observed for
the different FXIIIA genotypes presented in Table 5. The genotype
codes, explained in Table 4 and in the legend to Table 5, are 5'
to 3' for codons 34-204-564-650-651. Individuals with FXIII SA of
2.43 and 2.58 (genotypes 11211 and 11311) are known to be heterozygous
for FXIIIA deficiency and were not included in the normal population
presented in Fig 2. Therefore, only one allele of genotype 11211 in
both of these cases is probably responsible for the FXIII SA
observed.
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DISCUSSION |
We have developed reliable assays for the determination of both the
activity and the levels of FXIII in plasma. These have enabled us to
study the variation in FXIII specific activity in a population of 71 unrelated individuals. There are no previous reports in the literature
on determination of the specific activity of FXIII in different
individuals. We now show that the specific activity in different
individuals does vary considerably, while displaying a normal
distribution for the population. Our data show that FXIII levels
are not directly indicative of FXIII activity. Hence, individuals may
have very high levels of the FXIII protein, but very low levels of
FXIII activity, and vice versa.
Our genotyping data confirm the high genetic variability of the FXIIIA
gene. The frequency of each of the amino acid polymorphisms at residues
34, 204, 564, 650, and 651 we have found in the normal UK population
(normal men and women) correlates with those found for other smaller
white populations studied (Finnish, n = 26; Russian, n = 21; and
German, n = 14).27 Genotype frequency analysis between the
three groups studied shows that the Phe204 residue is significantly
more common in the RM group compared with normal men and women. This is
also true when our RM data for Phe204 are compared with the study of
Suzuki et al,27 who report only one Phe204 allele in 128 normal alleles from the white population, giving P = .013.
It has been known for many years now that autosomal recessive FXIII
deficiency in women leads to spontaneous miscarriage unless the
patients receive plasma FXIII supplementation through FXIII concentrate
injections.10,11 It is also well established that only
0.5% to 2% of normal FXIII levels are sufficient to achieve normal
hemostasis.11 It is therefore very interesting to find the
Phe204 polymorphism to be associated with RM, particularly when this
variant gives rise to almost normal FXIII specific activity when
assaying for fibrin cross-linking. It is possible that FXIII may
cross-link substrates other than fibrin during its role in the
maintenance of pregnancy, and the Phe204 association with recurrent
miscarriage may correlate with FXIII structure and may indeed be
independent of its level and activity. In fact, three-dimensional analysis of the FXIIIA protein molecule28 in this region
using the RASMOL software (Roger Sayle;
www.umass.edu/microbio/rasmol/) shows that the polar side chain of Tyr
at position 204 is able to form a hydrogen bond with Arg333, while Phe,
with a nonpolar side chain, at position 204 is unable to form this
interaction. This has implications for the final FXIIIA structure
achieved by the two variants. In addition, from the structure, it
appears that the hydrogen bond between Tyr204 and Arg333 has to be
broken before the molecule can be activated. This may be the basis of the difference observed in FXIII specific activity between alleles with
Phe204 and Tyr204.
The comparative frequency of each polymorphism analyzed in the UK
population of normal men and women is Leu564 > Gln651 > Leu34 > Ile650 > Phe204. The presence of five amino acid polymorphisms in
apparently normal FXIIIA genes translates into 32 possible haplotypes.
Although we could identify nine unique FXIIIA haplotypes in the UK
population, we were unable to determine complete haplotypes for all of
the people studied because 25 individuals proved to be heterozygous at
more than one locus.
The genotype to phenotype correlations clearly indicate that Leu34,
Leu564, and Tyr204 are associated with high FXIII specific activity.
Activated FXIII (FXIII*) stabilizes the fibrin gel through two sets of
(-glutamyl) lysine covalent bonds: two in the -chains and
four in the  -chains, giving rise to -chain dimers and high molecular weight -chain polymers. The structure of the -polymer is further complicated by cross-linking to it of other plasma proteins
including 2-plasmin inhibitor and fibronectin. In vitro studies have
shown that the rate and extent of -polymer formation are critically
dependent on and significantly influenced by FXIII concentrations above
those found in normal plasma.29 This may be clinically
significant in the context of thrombus removal. Also, the local FXIII
concentration is increased at sites of injury due to platelet
aggregation and degranulation. In such regions in vivo, formation of
large fibrin polymers may significantly influence pathological
thrombotic and thromboembolic phenomena, particularly in individuals
with high plasma FXIII concentrations. This would be of particular
concern for individuals who carry the Leu34, Tyr204, Leu564 variants
either alone or particularly in combination on both alleles.
A very recent report has suggested that the Val34 FXIIIA haplotype may
be associated with myocardial infarction (MI).30 However,
no FXIIIA protein data were presented from the study groups. We
actually find the Leu34 variant to result in higher FXIII specific
activity and therefore would expect Leu34 to be associated with MI in
individuals who have high levels of the protein. Without FXIIIA protein
data from the study of Kohler et al,30 it is difficult to
understand the basis of the association they suggest. Clearly, further
work is required to resolve this question.
Apart from the actual cross-linking reaction, factors that influence
the activity of the FXIIIA enzyme in plasma include the efficiency of
enzyme activation and the specificity and efficiency of substrate
binding. The Leu34 polymorphism is three residues away from the
activation peptide cleavage site and may contribute toward more
efficient activation of FXIII. The Leu564 residue lies on the exposed
surface of the barrel 1 domain.28 This may play a role in
substrate binding. It is possible that these single amino acid changes
in the protein sequence lead to minor changes in protein structure,
which account for the variation observed in FXIII specific activity.
This may be especially true for FXIIIA molecules with Leu564 compared
with Pro564. Proline is an amino acid with properties very different
from leucine and can significantly affect the three-dimensional
structure of the protein.
Interestingly, the variable levels of other components of the
coagulation pathway have also been found to have a genetic basis. Examples include factor V and factor VII levels,31,32 high levels of both plasminogen-activator inhibitor-1, and plasma
fibrinogen, which are associated with an increased risk of
MI,33,34 and elevated plasma prothrombin levels, which are
associated with an increase in venous thrombosis (VT).35 It
is probable that the risk of cardiovascular problems, due to increased
thromboembolic activity, is much higher for individuals who carry
specific polymorphisms, which give rise to high activity proteins for
more than one component of the blood clotting pathway, the effects
probably being additive. It is therefore important that when assessing
genetic risk for thrombotic problems such as MI and VT, all of the
variable coagulation components are considered, including FXIII.
Although there is a clear effect of the FXIIIA genotype on FXIII
specific activity, there are probably other factors, such as the levels
of FXIIIB subunit in plasma, which may also influence the final levels
and activity of plasma FXIII. FXIII levels, activity, or specific
activity were not found to be dependent on age. Furthermore, there were
no differences observed in either plasma FXIII levels or its activity
(or in FXIIIA genotypes) between men and women. Plasma FXIII levels are
becoming accepted as an important marker of disease activity in various
inflammatory conditions including Crohn's disease, ulcerative colitis,
and bacterial infection, as well as in patients with benign and
malignant gynecological tumors.1-3 Also, transglutaminase
activity is increased in the Alzheimer's disease brain, and tissue
transglutaminase has been identified as the autoantigen of celiac
disease.4,5 As yet, there is no evidence whether FXIIIA may
also be involved in the covalent cross-linking of gliadin in celiac disease.
In conclusion, our findings strongly suggest that for accuracy of
diagnosis, the determination of both FXIIIA activity and levels is
important in clinical conditions for which FXIII may be used as a
diagnostic marker of disease activity. The assays we have described
provide a simple, but accurate and reproducible, means of determining
the plasma FXIII activity, FXIII level, and therefore the FXIII
specific activity, in large numbers of individuals. The studies we have
presented provide the first evidence of a genetic basis for the massive
variation that is observed in plasma FXIII levels and activity. They
also define specific genotypes, which are associated with FXIII
variability and these may serve as valuable tools for molecular
diagnostics in the growing number of clinical situations where FXIII is
recognized as an important marker. Our finding that the Phe204 FXIII
variant may be associated with RM in women who otherwise have normal
plasma FXIII highlights the importance of defining a structure/function
relationship for FXIII in its additional biological roles.
| |
NOTE ADDED IN PROOF |
Since the submission of this article, Kangsadalampai and Board
published a report on the factor XIIIA Val34Leu
polymorphism.36
| |
ACKNOWLEDGMENT |
We thank Dr A. Rutherford, Dr R. Oodit, and R. Newton from the Assisted
Conception Unit at the Leeds General Infirmary, UK, for providing blood
samples for the RM group. We are grateful to Prof S.E.V. Phillips and
Dr J. Jaeger for assistance with the structural implications of the
FXIII Phe204 variant. We also thank Dr K.J.A. Miloszewski for helpful discussions.
| |
FOOTNOTES |
Submitted April 28, 1998; accepted September 28, 1998.
Supported by the Northern and Yorkshire Regional Health Authority and
the West Riding Medical Research Trust.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Rashida Anwar, PhD, Molecular
Medicine Unit, University of Leeds, Clinical Sciences Building, St
James's University Hospital, Leeds LS9 7TF, UK; e-mail:
MSJRA{at}STJAMES.LEEDS.AC.UK.
| |
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Top
Abstract
Introduction