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Blood, Vol. 93 No. 3 (February 1), 1999:
pp. 897-905
By
From the Molecular Medicine Unit, University of Leeds, St James's
University Hospital, Leeds, UK.
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.
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 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.
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 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.
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.
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.
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).
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.
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.
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.
Since the submission of this article, Kangsadalampai and Board
published a report on the factor XIIIA Val34Leu
polymorphism.36
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.
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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92:2766, 1998 This article has been cited by other articles:
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Top
Abstract
Introduction
-(
-glutamyl) lysine bonds between
fibrin molecules leading to stabilization of the clot structure.
70°C. The buffy coat,
containing the peripheral blood mononuclear cells (PBMCs), was also
collected and stored at 
-chains, giving rise to 
association with factor V levels in plasma.
Thromb Haemost
75:45, 1996
