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Blood, Vol. 96 No. 3 (August 1), 2000:
pp. 988-995
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Unit of Molecular Vascular Medicine, University of Leeds
School of Medicine, Leeds, UK; Department of Haematology, Imperial
College School of Medicine, Charing Cross Hospital Campus, London, UK;
and Department of Cell and Developmental Biology, University of
Pennsylvania School of Medicine, Philadelphia, PA.
Factor XIII on activation by thrombin cross-links fibrin. A common
polymorphism Val to Leu at position 34 in the FXIII A subunit is under
investigation as a risk determinant of thrombosis. Because Val34Leu is
close to the thrombin cleavage site, the hypothesis that it would alter
the function of FXIII was tested. Analysis of FXIII subunit proteolysis
by thrombin using sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and high-performance liquid chromatography showed that
FXIII 34Leu was cleaved by thrombin more rapidly and by lower doses
than 34Val. Mass spectrometry of isolated activation peptides confirmed
the predicted single methyl group difference and demonstrated that the
thrombin cleavage site is unaltered by Val34Leu. Kinetic analysis of
activation peptide release demonstrated that the catalytic
efficiency
(kcat/Km) of thrombin
was 0.5 for FXIII 34Leu and 0.2 (µmol/L)
On cleavage of fibrinopeptides A and B by thrombin,
fibrin spontaneously polymerizes into a network of multimeric strands, initially held together by noncovalent interaction. Blood coagulation factor XIII (FXIII) is activated by thrombin, and activated FXIII (FXIIIa) covalently cross-links the fibrin clot to increase resistance to chemical, mechanical, and proteolytic insults. FXIII is a tetrameric protransglutaminase consisting of 2 A subunits, which contain the
active site, and 2 B subunits, which serve a carrier function for the A
subunit in plasma.1,2 FXIII is also found in platelets as
an A-subunit dimer.2 Platelets do not release FXIII on
activation, but lysis of platelets entrapped in the blood clot may
increase local concentrations of FXIII.3
FXIIIa catalyzes the introduction of A common polymorphism with an allele frequency of around 25% has been
identified in the FXIII A subunit (Val34Leu), 3 amino acids from the
thrombin activation site.12 Recent studies have reported
that the prevalence of the Leu encoding allele is lower in patients
with myocardial infarction,13,14 deep vein
thrombosis,15,16 and cerebral infarction17 when
compared with matched control groups. These clinical studies suggest
that this polymorphism may be a risk determinant of thrombosis in both
the arterial and venous systems. Paradoxically, ex vivo and in vitro
studies have suggested that possession of the Leu allele leads to
increased cross-linking rates by FXIIIa.18-20 The
mechanisms by which this occurred remained unclear. In view of the
close proximity of the Val34Leu polymorphism to the activation site, we
tested the hypothesis that this polymorphism alters the activation rate
of FXIII by thrombin and affects fibrin structure.
Blood sampling and processing
Determination of the FXIII Val34Leu genotype
Purification of the FXIII Val34Leu variants Buffy coats from 34 outdated donations of human platelet-poor plasma were obtained from the regional blood transfusion center; genomic DNA was extracted and genotyped for the Val34Leu polymorphism. There were 19 homozygous FXIII 34Val samples, 1 homozygous 34Leu sample, and 14 heterozygous samples. FXIII 34Val was purified from a pool of 10 of the homozygous plasma donations (vol = 2.170 L) and homozygous FXIII 34Leu from a single plasma donation (vol = 0.205 L). In addition, FXIII was purified from the plasma of 40 outdated, mixed (unknown) genotype donations (vol = 11.895 L). Purification of FXIII was performed using a method adapted from previous publications.21,22 In brief, plasma was subjected to repeated precipitations with ammonium sulfate: 20% saturation at room temperature pH 7.0, 16% saturation at 4°C pH 5.4, 16% saturation at 4°C pH 7.0, and 36% saturation at 4°C pH 7.5. EDTA was added at 1 mmol/L to all the buffers used to prevent inopportune activation of FXIII. The final precipitate was resuspended in 1/1000 plasma volume of 0.05 mol/L Tris-HCl pH 7.5, 1 mmol/L EDTA, dialyzed against the same buffer and further purified by gel filtration on a Sepharose 6B column (2.6 × 40 cm), equilibrated and developed with 0.05 mol/L Tris-HCl pH 7.5, 1 mmol/L EDTA. Peak fractions containing both FXIII A and FXIII B subunit were pooled, concentrated on aquacide (Calbiochem Corp, La Jolla, CA) and extensively dialyzed against 0.05 mol/L Tris-HCl pH 7.5, 1 mmol/L EDTA. Purity and activity of the preparations were tested with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), in-house FXIII A- and B-subunit sandwich enzyme-linked immunosorbent assays,23 and 5-(biotinamido) pentylamine incorporation assay.23,24 Concentration of the preparations was measured by absorbance at 280 nm using an extinction coefficient of E1 mg/ml280 nm = 1.38.22Cross-linking assay for FXIII The FXIIIa-specific cross-linking activity was determined with a microtiter assay using fibrinogen and 5-(biotinamido) pentylamine as substrates, as previously described.23,24 The assay is based on the incorporation of 5-(biotinamido) pentylamine by FXIIIa into microtiter plates coated with fibrinogen, following activation with thrombin. The amount of cross-linked 5-(biotinamido) pentylamine is detected by measuring phosphatase activity after incubation with a streptavidin-alkaline phosphatase conjugate. FXIIIa activity was measured before and after preincubation with human -thrombin (Sigma
Chemical, St Louis, MO) in plasma samples from subjects homozygous for FXIII 34Val, FXIII 34Leu, heterozygous, and in pooled
normal plasma obtained from 47 healthy donors. Plasma samples (150 µL) were preincubated with 150 µL -thrombin at a final
concentration of 5 U/mL in microtiter plates, after which
FXIIIa activity was measured with the pentylamine-incorporation assay
in a separate plate. Samples were diluted 1/10 in Tromethamine-buffered
saline (TBS) (40 mmol/L Tris-HCl, 140 mmol/L NaCl, 0.02% ([w/v])
NaN3, pH 8.3) containing 0.3 mg/mL of the synthetic peptide
Gly-Pro-Arg-Pro-Ala-amide (Sigma) to prevent inopportune polymerization
of fibrin in the preincubation mixture, which would interfere with the
following subsampling procedure. After 30 minutes of preincubation,
aliquots of 20 µL were subsampled into the pentylamine-incorporation
plate containing 80 µL of the reaction mixture of 1.25 mmol/L
5-(biotinamido) pentylamine, 0.63 mmol/L dithiothreitol, and 0.12 mol/L
CaCl2 in TBS. The pentylamine-incorporation assay was
further performed as described.23
FXIII proteolysis studies using SDS-PAGE The SDS-PAGE procedure was performed using a Miniprotean 3 (Biorad, Hercules, CA) electrophoresis unit. Gels were cast at a polyacrylamide concentration of 8% (bis/acrylamide ratio of 1:37.5) in 1.5 mol/L Tris-HCl, pH 8.8, and run at 150 V for 80 minutes. Gels were stained with Coomassie blue (2.5 g/L in 45% methanol, 10% acetic acid) for 30 minutes at room temperature and destained for 1 to 2 hours in 45% methanol, 10% acetic acid with 3 changes of destaining solution. Dose response of FXIII proteolysis by thrombin was studied by incubating 4.0 µmol/L of purified FXIII variants with 0 to 10 U/mL human-thrombin (Sigma) in 0.05 mol/L Tris-HCl, 1 mmol/L EDTA, pH 7.5 for 1 hour at 37°C. The reactions were stopped
by the addition of equal (1:1) volume of reducing loading
buffer 100 mmol/L Tris-HCl, 0.1 mol/L DTT, 4% SDS, 0.2% bromophenol
blue, 20% glycerol, pH 6.8 with immediate boiling for 5 minutes, and
25 µL was loaded on the gel. The time course of FXIII proteolysis was
studied by incubating 4.0 µmol/L of purified FXIII variants with 0.4 U/mL human -thrombin at 37°C. The reaction was
stopped at 0 to 240 minutes as described for the dose-response
reactions, and 25 µL was loaded on the gel.
Reverse-phase high-performance liquid chromatography (HPLC) Reverse-phase HPLC was performed to analyze the FXIII activation peptide release using a 0.46 × 25 cm silica C18 (5 µm, 300 Å) column (Pepmap C18; Perseptive Biosystems Inc) on a Biocad Sprint automated chromatography system (also from Perseptive Biosystems, Framingham, MA), according to a method previously described by Janus and coworkers.25 The column was equilibrated with 1 column volume of 85% buffer A/15% buffer B. The sample was applied and eluted with a linear gradient from 85% buffer A/15% buffer B to 100% buffer B in 7 column volumes. The column was further washed with 2 column volumes of 100% buffer B followed by a linear gradient from 100% buffer B to 85% buffer A/15% buffer B in 1 column volume. Flow-rate was set at 1 mL/min throughout the experiment. Buffer A consisted of 10% acetonitrile/90% 0.083 mol/L sodium phosphate pH 3.1 and buffer B of 40% acetonitrile/60% 0.083 mol/L sodium phosphate pH 3.1. All reagents were HPLC grade and solutions were filtered through 0.22 µm to eliminate particulates. Elution of peptides was detected by absorbance at 205 nm.Kinetics of activation peptide release from FXIII Molar quantities of the activation peptides analyzed by reverse-phase HPLC were calculated from the chromatograms by integration of the respective peak areas. The area under the curve was converted into molar quantity by comparison to that of a calibration FXIII activation peptide loaded at known concentration. Purified FXIII was dialyzed against 9.47 mmol/L sodium phosphate, 137 mmol/L NaCl, 2.5 mmol/L KCl, 0.1% PEG, pH 7.4. Dose-response kinetics of FXIII activation peptide release by thrombin were performed by incubating 4.0 µmol/L of dialyzed FXIII with 0, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, and 10 U/mL (1 U/mL = 9.16 nmol/L) human-thrombin (Sigma) for 1 hour at 37°C. The reactions were stopped
by the addition of 1:10 (vol/vol) of 3 mol/L HClO4,
centrifuged for 10 minutes at maximum speed in an Eppendorf centrifuge
and 180 µL injected onto the C18 column. Time course of the
activation peptide release reaction was studied by incubation of 4.0, 2.0, and 1.0 µmol/L of FXIII with 0.5 U/mL human
-thrombin at 37°C. The reactions were stopped at 0, 2, 5, 10, 20, 30, 60, 120, 180, and 240 minutes by subsampling 10 volumes into 1 volume of 3 mol/L HClO4. The samples were centrifuged and
180 µL injected onto the C18 column. A similar method was used for
studying the time course of FXIII activation peptide release in the
presence of fibrin or fibrin and Gly-Pro-Arg-Pro-Ala-amide. In the
presence of fibrin alone, 2.7 µmol/L of FXIII was incubated with 3.1 µmol/L human fibrinogen (Sigma) and 0.2 U/mL human -thrombin. At
this concentration of fibrinogen a clot formed, which was defined in
size and did not interfere with the subsampling procedure. The reaction
was stopped after 0.25, 0.5, 1, 2, 3, 4, 8, and 16 minutes of
incubation and 270 µL injected onto the C18 column. In the presence
of fibrin and the antipolymerizing peptide Gly-Pro-Arg-Pro-Ala, 2.7 µmol/L of FXIII was incubated with 3.1 µmol/L human fibrinogen
(Sigma), 2 mg/mL Gly-Pro-Arg-Pro-Ala-amide (Sigma), and 0.5 U/mL human -thrombin. In these experiments, the
reaction was stopped at 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22 minutes and 270 µL injected.
Catalytic efficiency Catalytic efficiencies of the FXIII activation peptide release reaction by thrombin were calculated by fitting the data from the time-course and dose-response experiments analyzed with reverse-phase HPLC to the following equation25:
Mass spectrometry Fractions from reverse-phase HPLC containing the FXIII activation peptides were collected for each purified FXIII Val34Leu variant. Molecular weights were analyzed with a single quadrupole, bench top mass spectrometer (Platform II, Micromass, Cheshire, UK). Samples were infused into the ionization source at a flow rate of 4 mL/min using a Harvard syringe pump. The mass spectrometer was fitted with a standard electrospray ionization source. Positive electrospray ionization was affected with a probe tip voltage of 3.5 kV, and a counter electrode voltage of 0.5 kV. Nitrogen was used as both the nebulizing and the drying gas; typical flow rates were a nebulizing gas flow rate of 20 L/h and a drying gas flow rate of 200 L/h. The sampling cone voltage was set to 50 volts. Data were acquired over the m/z range 500 to 2500. Spectra were transposed onto a true molecular mass scale using Maximum Entropy techniques. An external calibration was applied on a separate introduction of horse heart myoglobin (molecular weight, 16 951.49 d).Analysis of rates of - and  -chain cross-link formation were studied by
incubating 6.1 µmol/L human fibrinogen (Sigma) with 0.1 µmol/L purified FXIII, 0.5 U/mL human thrombin
(Sigma) and 10 mmol/L calcium in 0.05 mol/L Tris-HCl, 0.15 mol/L NaCl, pH 7.5. The reaction was stopped by the addition
of equal (1:1) volume of reducing loading buffer with immediate boiling
for 5 minutes, and 25 µL was loaded on an 8% polyacrylamide
gel. SDS-PAGE was further performed as described for the
FXIII proteolysis studies.
Turbidity measurements at 350 nm Plasma samples from subjects homozygous for either FXIII 34Leu (n = 3) or FXIII 34Val (n = 3) were diluted 2/3 with 0.05 mol/L Tris-HCl, 0.15 mol/L NaCl, pH 7.5 and incubated with 1 U/mL human thrombin (Sigma) and 16 mmol/L calcium in a 0.5-mL cuvette. Immediately on addition of thrombin/calcium, absorbancy was read every 3 seconds at 350 nm for 5 minutes with a Perkin-Elmer Lambda 4B spectrophotometer. Parameters such as lag phase before start of fibrin polymerization, slope of the polymerization curve (A/min) and maximum absorbancy at full polymerization were recorded. Three replicate measurements were performed for each sample.Clot permeation measurements Plasma samples (100 µL) from subjects homozygous for either FXIII 34Leu (n = 3) or FXIII 34Val (n = 3) were incubated to allow clot formation with 1 U/mL human thrombin (Sigma) and 20 mmol/L calcium in open tubes for 2 hours at room temperature in a wet chamber. The tubes containing the clots were connected via plastic tubing to a reservoir containing 0.05 mol/L Tris-HCl, 0.15 mol/L NaCl, pH 7.5 with a pressure drop of 4 cm. After washing the clots, flow rates of buffer through the fibrin gels were measured by timing 6 drops for each tube and weighing each drop for exact volume. Four replicate clots of each sample were analyzed in this way. The permeation coefficient or Darcy constant, which represents the surface of the gel allowing flow through a network and thus provides information on the pore structure, was calculated using the following formula29:
 (10 2 poise) flowing through a
clot with length L (1.3 cm) and a cross-sectional area
A (0.049 cm2) in time t (seconds) under
pressure  P (dyne/cm2). The unit of the resulting
Darcy constant is cm2.
Scanning electron microscopy of fibrin clots After permeation experiments, the clots were fixed by permeating them with a 2% (v/v) glutaraldehyde solution overnight. Clots were recovered from the tubes and further processed by dehydration using a stepwise ethanol gradient, critical point drying, and sputter coating with gold-palladium as previously described.30 Plasma clots from 3 homozygous FXIII 34Leu subjects and 3 homozygous FXIII 34Val subjects were observed and photographed digitally in at least 6 different areas using a scanning electron microscope (XL 20, Philips Electron Optics, Eindhoven, The Netherlands).
Effect of preactivation on FXIIIa cross-linking activities Plasma samples from subjects homozygous for FXIII 34Leu showed greater activity in a pentylamine-incorporation assay, in which there is thrombin activation during the course of the assay, than plasma samples from subjects homozygous for FXIII 34Val or heterozygous subjects. Cross-linking rates in this assay were typically higher for FXIII 34Leu than for FXIII 34Val, with an intermediate response to heterozygous samples and pooled normal plasma (Figure 1A). After preincubation with 5 U/mL human-thrombin for 30 minutes at 37°C, however, this
difference disappeared, and cross-linking activities of plasma samples
from subjects homozygous for FXIII 34Leu or FXIII 34Val and
heterozygous subjects were similar (Figure 1B).
Increased proteolysis of FXIII 34Leu by thrombin Proteolysis of FXIII subunits by thrombin was studied using SDS-PAGE. Electrophoresis of FXIII on SDS-polyacrylamide gels under reducing conditions showed 2 bands for the A and B subunit migrating very closely together. Activation by thrombin produced a distinct band for the activated A subunit that migrated faster in the gel. We first examined activation of purified FXIII 34Leu and FXIII 34Val by increasing doses of human-thrombin. Figure 2 shows that FXIII 34Val (panel B)
proteolysis started with 0.3 to 0.4 U/mL of thrombin after
1 hour incubation at 37°C, whereas FXIII 34Leu (panel A)
proteolysis started with 0.1 to 0.2 U/mL of thrombin and
was complete with 1 U/mL. We next investigated FXIII
cleavage by adding 0.4 U/mL human -thrombin and
stopping the reaction after increasing times. Proteolysis of FXIII
34Val (panel D) started after 60 to 90 minutes, whereas FXIII 34Leu cleavage (panel C) by the same dose of thrombin started after 10 to 20 minutes and was complete after 120 minutes.
Analysis of FXIII activation peptides The preactivation and SDS-PAGE studies suggested that release of the activation peptide from FXIII 34Leu is more rapid than that from FXIII 34Val, but the incomplete resolution of the bands in SDS-PAGE did not allow quantification. Reverse-phase HPLC was used to quantify the amount of peptide released from each form of FXIII by thrombin. When activating purified FXIII 34Val with 10 U/mL human-thrombin for 1 hour at 37°C, one distinct peak for the
activation peptide was detected. Activating FXIII purified from a mixed
genotype, however, produced 2 peaks, and analysis of activated purified
FXIII 34Leu indicated that the 2nd peak represented the 34Leu
activation peptide (Figure 3). This
separation of the 2 species of activation peptides on the C18 column
was confirmed by mass spectrometry. HPLC fractions containing the respective peptide peaks were collected and analyzed for the molecular weight of their peptides. The first peak contained a peptide of molecular weight of 3951.5 d and the second peak a peptide of 3965.0 d.
These results confirmed that the first peak contains the FXIII 34Val
activation peptide and the 2nd peak the FXIII 34Leu activation peptide.
The difference in molecular weight between the peptides can be
accounted for by the expected single methyl group (14.0 d)
difference between the Leu and Val side chains. They also
demonstrated that the thrombin cleavage site between Arg37 and Gly38 is
unaltered by the Val34Leu polymorphism.
Kinetics of FXIII activation The separation of the FXIII 34Val from the FXIII 34Leu activation peptide on reverse-phase HPLC allowed us to carry out kinetic analysis of the activation reaction for each form of FXIII using purified FXIII from a mixed genotype. Purified FXIII was incubated at 4.0 µmol/L with increasing concentrations of human-thrombin. HPLC was performed on the incubation mixtures. Peak areas
of the FXIII 34Val and FXIII 34Leu activation peptides were calculated from the chromatograms and transformed in molar quantities. More FXIII
34Leu activation peptide was released than FXIII 34Val activation peptide after incubation with thrombin concentrations between 0.01 and
0.5 U/mL for 1 hour at 37°C (Figure
4A), confirming the findings from the gel
electrophoresis studies that FXIII 34Leu is activated by lower doses of
thrombin than FXIII 34Val. Time-course experiments were also performed.
Purified FXIII at 4.0, 2.0, and 1.0 µmol/L was activated with 0.5 U/mL human -thrombin at 37°C and the reaction was
stopped at increasing times. More FXIII 34Leu activation peptide was
released than FXIII 34Val activation peptide early in the reaction
(Figure 4B). This faster initial rate was observed regardless of the
relative concentrations of FXIII and thrombin; kinetic data are
summarized in Table 1.
Effect of fibrin on the activation peptide release reaction Fibrinogen was added at a concentration of 3.1 µmol/L to 2.7 µmol/L purified FXIII and 0.2 U/mL human-thrombin. The inset of Figure 5 shows a
typical chromatogram from these experiments, with defined peaks for
fibrinopeptide A, fibrinopeptide B, and FXIII 34Val and 34Leu
activation peptides. In the presence of fibrin(ogen), the release of
both FXIII activation peptides by thrombin was greatly accelerated.
FXIII 34Leu activation peptide release remained significantly faster
than that of FXIII 34Val. The FXIII 34Leu activation peptide was
released at a similar rate as fibrinopeptide A, followed by release of
the FXIII 34Val peptide and finally fibrinopeptide B (Figure 5).
Catalytic efficiency of FXIII activation Catalytic efficiencies of thrombin-dependent activation peptide release from the different polymorphic forms of FXIII were calculated in the absence of fibrin, in the presence of fibrin alone, and in the presence of both fibrin and Gly-Pro-Arg-Pro-amide. The 34Leu form showed twice the catalytic efficiency, regardless of the experimental conditions (Table 1). The presence of fibrin greatly enhanced FXIII activation peptide release by thrombin and this catalytic effect was reduced when polymerization of fibrin was inhibited with Gly-Pro-Arg-Pro-amide. The difference in catalytic efficiency between FXIII 34Val and FXIII 34Leu remained in the presence of polymerizing fibrin.Effect of FXIII 34Leu on - and -chains by the 2 forms of FXIII was studied using reducing SDS-PAGE. Cross-linking of both -chains and -chains appeared earlier when incubating a mixture of fibrinogen and thrombin with purified FXIII 34Leu than with
FXIII 34Val (Figure 6). The fibrinogen
preparation used for these experiments had some contamination with
fibronectin and FXIII. Due to traces of FXIII in the preparation, some
-chain dimers were already present without incubation with the
isolated FXIII variants. Nonetheless, more -chain dimers and
-chain polymers were formed by incubation with FXIII 34Leu than
FXIII 34Val after 5 minutes. After 20 minutes incubation FXIII 34Leu
produced more of high molecular weight -chain polymers that did not
enter the gel and remained in the loading well (Figure 6).
FXIII Val34Leu and fibrin structure Plasma samples from subjects homozygous for either form of FXIII Val34Leu were used to study effects of this polymorphism on fibrin structure. The lag phase in formation of turbidity at 350 nm was shorter for homozygous FXIII 34Leu (1.08 ± 0.11 minutes) than for homozygous FXIII 34Val samples (1.55 ± 0.15 minutes). The slope of the turbidity curves did not differ, but maximum absorbancy after 5 minutes was slightly lower for the FXIII 34Leu (1.83 ± 0.04) than FXIII 34Val (1.93 ± 0.03), indicating the presence of thinner fibrin fibers in the FXIII 34Leu samples. Consistent with these results, permeation of fibrin clotted for 2 hours was found to be slower in clots from FXIII 34Leu samples when compared with FXIII 34Val. The Darcy constant, which reflects the surface area of the clot available for flow, was 3.6 ± 0.5 × 109 cm2 for
FXIII 34Leu and 8.7 ± 4.4 × 109
cm2 for FXIII 34Val. These structural differences were
confirmed by scanning electron microscopy. Clots prepared from
plasma samples homozygous for FXIII 34Val (Figure
7A) showed a fibrin meshwork with thick
fibers and large pores, whereas clots prepared from plasma samples
homozygous for FXIII 34Leu showed a finer fibrin meshwork with thinner
fibers and reduced space between the fibrin strands (Figure 7B).
The FXIII gene is known to be highly polymorphic, with several common nucleotide sequence variations within the population, which encode amino acid substitutions. Although these amino acid sequence changes could alter the levels and activity of FXIII, to date there has been little formal examination of their effects. We report here the functional characterization of the Val34Leu polymorphism in the A subunit. This polymorphism is located adjacent to the thrombin cleavage activation site of FXIII. Presence of the 34Leu encoding allele has been associated with a protective effect against thrombotic disease in the arterial and venous systems.13-17
The authors wish to thank Dr S. MacLennan and Mr E. Lee from the Regional Blood Transfusion Centre of Yorkshire, Leeds, UK, for the provision of outdated transfusion plasma and buffy coat, and Dr A. E. Ashcroft for mass spectrometry. Mr I. Moss and Ms K. Standeven, Dr Y. Veklich, and Dr R. Marchi are gratefully acknowledged for their assistance with the Biocad Sprint, turbidity measurements, and permeation measurements, respectively. We also wish to thank Dr J. P. Collet for helpful discussion of the effects of FXIII Val34Leu on fibrin structure.
Submitted December 12, 1999; accepted May 15, 2000.
Supported by the British Heart Foundation (PG/98104), the Special Trustees of Charing Cross Hospital and the National Institutes of Health (HL30954).
Reprints: Robert A. S. Ariëns, Unit of Molecular Vascular Medicine, University of Leeds, G-Floor, Martin Wing, Leeds General Infirmary, Leeds LS1 3EX, UK; e-mail: r.a.s.ariens{at}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.
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  S. E. Iismaa, B. M. Mearns, L. Lorand, and R. M. Graham Transglutaminases and Disease: Lessons From Genetically Engineered Mouse Models and Inherited Disorders Physiol Rev, July 1, 2009; 89(3): 991 - 1023. [Abstract] [Full Text] [PDF]
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 D. M. O. Pruissen, A. J. C. Slooter, F. R. Rosendaal, Y. van der Graaf, and A. Algra Coagulation factor XIII gene variation, oral contraceptives, and risk of ischemic stroke Blood, February 1, 2008; 111(3): 1282 - 1286. [Abstract] [Full Text] [PDF]
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R. Ajjan, B. C. B. Lim, K. F. Standeven, R. Harrand, S. Dolling, F. Phoenix, R. Greaves, R. H. Abou-Saleh, S. Connell, D. A. M. Smith, et al. Common variation in the C-terminal region of the fibrinogen -chain: effects on fibrin structure, fibrinolysis and clot rigidity Blood, January 15, 2008; 111(2): 643 - 650. [Abstract] [Full Text] [PDF]
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K. F. Standeven, A. M. Carter, P. J. Grant, J. W. Weisel, I. Chernysh, L. Masova, S. T. Lord, and R. A. S. Ariens Functional analysis of fibrin {gamma}-chain cross-linking by activated factor XIII: determination of a cross-linking pattern that maximizes clot stiffness Blood, August 1, 2007; 110(3): 902 - 907. [Abstract] [Full Text] [PDF]
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A. Undas, K. E. Brummel-Ziedins, and K. G. Mann Antithrombotic properties of aspirin and resistance to aspirin: beyond strictly antiplatelet actions Blood, March 15, 2007; 109(6): 2285 - 2292. [Abstract] [Full Text] [PDF]
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C. Infante-Rivard and C. R. Weinberg Parent-of-Origin Transmission of Thrombophilic Alleles to Intrauterine Growth-Restricted Newborns and Transmission-Ratio Distortion in Unaffected Newborns Am. J. Epidemiol., November 1, 2005; 162(9): 891 - 897. [Abstract] [Full Text] [PDF]
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D. A. Lane, H. Philippou, and J. A. Huntington Directing thrombin Blood, October 15, 2005; 106(8): 2605 - 2612. [Abstract] [Full Text] [PDF]
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E. M. Scott, R. A.S. Ariens, and P. J. Grant Genetic and Environmental Determinants of Fibrin Structure and Function: Relevance to Clinical Disease Arterioscler. Thromb. Vasc. Biol., September 1, 2004; 24(9): 1558 - 1566. [Abstract] [Full Text] [PDF]
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E. J. Dunn, R. A. Ariens, M. de Lange, H. Snieder, J. H. Turney, T. D. Spector, and P. J. Grant Genetics of fibrin clot structure: a twin study Blood, March 1, 2004; 103(5): 1735 - 1740. [Abstract] [Full Text] [PDF]
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B. Voetsch and J. Loscalzo Genetic Determinants of Arterial Thrombosis Arterioscler. Thromb. Vasc. Biol., February 1, 2004; 24(2): 216 - 229. [Abstract] [Full Text]
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J.P. Collet, C. Nagaswami, D.H. Farrell, G. Montalescot, and J.W. Weisel Influence of {gamma}' Fibrinogen Splice Variant on Fibrin Physical Properties and Fibrinolysis Rate Arterioscler. Thromb. Vasc. Biol., February 1, 2004; 24(2): 382 - 386. [Abstract] [Full Text]
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 R Diz-Kucukkaya, V S Hancer, M Inanc, M Nalcaci, and Y Pekcelen Factor XIII Val34Leu polymorphism does not contribute to the prevention of thrombotic complications in patients with antiphospholipid syndrome Lupus, January 1, 2004; 13(1): 32 - 35. [Abstract] [PDF]
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A. V. Cooper, K. F. Standeven, and R. A. S. Ariens Fibrinogen gamma-chain splice variant {gamma}' alters fibrin formation and structure Blood, July 15, 2003; 102(2): 535 - 540. [Abstract] [Full Text] [PDF]
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A. P. Reiner, S. R. Heckbert, H. L. Vos, R. A. S. Ariens, R. N. Lemaitre, N. L. Smith, T. Lumley, T. D. Rea, L. A. Hindorff, G. D. Schellenbaum, et al. Genetic variants of coagulation factor XIII, postmenopausal estrogen therapy, and risk of nonfatal myocardial infarction Blood, July 1, 2003; 102(1): 25 - 30. [Abstract] [Full Text] [PDF]
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 K. F. Standeven, P. J. Grant, A. M. Carter, T. Scheiner, J. W. Weisel, and R. A.S. Ariens Functional Analysis of the Fibrinogen A{alpha} Thr312Ala Polymorphism: Effects on Fibrin Structure and Function Circulation, May 13, 2003; 107(18): 2326 - 2330. [Abstract] [Full Text] [PDF]
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A. Undas, W. J. Sydor, K. Brummel, J. Musial, K. G. Mann, and A. Szczeklik Aspirin Alters the Cardioprotective Effects of the Factor XIII Val34Leu Polymorphism Circulation, January 7, 2003; 107(1): 17 - 20. [Abstract] [Full Text] [PDF]
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V. Schroeder, H. P. Kohler, K. E. Brummel, and K. G. Mann Factor XIII activation by thrombin depends on FXIIIVal34Leu genotype Blood, January 1, 2003; 101(1): 371 - 371. [Full Text] [PDF]
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J. D. Mills, R. A.S. Ariens, M. W. Mansfield, and P. J. Grant Altered Fibrin Clot Structure in the Healthy Relatives of Patients With Premature Coronary Artery Disease Circulation, October 8, 2002; 106(15): 1938 - 1942. [Abstract] [Full Text] [PDF]
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R. A. S. Ariens, T.-S. Lai, J. W. Weisel, C. S. Greenberg, and P. J. Grant Role of factor XIII in fibrin clot formation and effects of genetic polymorphisms Blood, July 18, 2002; 100(3): 743 - 754. [Abstract] [Full Text] [PDF]
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N. Aleksic, C. Ahn, Y.-W. Wang, H. Juneja, A. R. Folsom, E. Boerwinkle, and K. K. Wu Factor XIIIA Val34Leu Polymorphism Does Not Predict Risk of Coronary Heart Disease: The Atherosclerosis Risk in Communities (ARIC) Study Arterioscler. Thromb. Vasc. Biol., February 1, 2002; 22(2): 348 - 352. [Abstract] [Full Text] [PDF]
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 K. F. Standeven, R. A.S. Ariens, P. Whitaker, A. E. Ashcroft, J. W. Weisel, and P. J. Grant The Effect of Dimethylbiguanide on Thrombin Activity, FXIII Activation, Fibrin Polymerization, and Fibrin Clot Formation Diabetes, January 1, 2002; 51(1): 189 - 197. [Abstract] [Full Text] [PDF]
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M. Weger, W. Renner, O. Stanger, O. Schmut, H. Deutschmann, T. C. Wascher, and A. Haas Role of Factor XIII Val34Leu Polymorphism in Retinal Artery Occlusion Stroke, December 1, 2001; 32(12): 2759 - 2761. [Abstract] [Full Text] [PDF]
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A. P. Reiner, S. M. Schwartz, M. B. Frank, W.T. Longstreth Jr, L. A. Hindorff, G. Teramura, F. R. Rosendaal, L. K. Gaur, B. M. Psaty, D. S. Siscovick, et al. Polymorphisms of Coagulation Factor XIII Subunit A and Risk of Nonfatal Hemorrhagic Stroke in Young White Women Editorial Comment Stroke, November 1, 2001; 32(11): 2580 - 2587. [Abstract] [Full Text] [PDF]
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P. E. Morange, M. Henry, D. Brunet, M.-F. Aillaud, I. Juhan-Vague, A. J. Catto, R. A. S. Ariens, and P. J. Grant Factor XIIIV34L is not an additional genetic risk factor for venous thrombosis in Factor V Leiden carriers Blood, March 15, 2001; 97(6): 1894 - 1896. [Full Text] [PDF]
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C. Sadasivan and V. C. Yee Interaction of the Factor XIII Activation Peptide with alpha -Thrombin. CRYSTAL STRUCTURE OF ITS ENZYME-SUBSTRATE ANALOG COMPLEX J. Biol. Chem., November 17, 2000; 275(47): 36942 - 36948. [Abstract] [Full Text] [PDF]
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| Copyright © 2000 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
-chains appeared earlier when fibrin was
incubated with FXIII 34Leu than with 34Val. Fully activated 34Leu and
34Val FXIII showed similar cross-linking activity. Analysis of fibrin
clots prepared using plasma from FXIII 34Leu subjects by turbidity and
permeability measurements showed reduced fiber mass/length ratio and
porosity compared to 34Val. The structural differences were confirmed
by electron microscopy. These results demonstrate that Val34Leu
accelerates activation of FXIII by thrombin and consequently affects
the structure of the cross-linked fibrin clot.
(Blood. 2000;96:988-995)
-lysine peptide
bonds between fibrin 
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