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Blood, Vol. 93 No. 12 (June 15), 1999:
pp. 4242-4247
Familial Overexpression of  Antithrombin Caused by an Asn135Thr
Substitution
By
T.A. Bayston,
A. Tripodi,
P.M. Mannucci,
E. Thompson,
H. Ireland,
A.C. Fitches,
L. Hananeia,
R.J. Olds, and
D.A. Lane
From the Imperial College School of Medicine, London, UK; "A.
Bianchi Bonomi" Hemophilia and Thrombosis Center, IRCCS Maggiore
Hospital and University, Milan, Italy; and the Dunedin School of
Medicine, University of Otago, Dunedin, New Zealand.
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ABSTRACT |
We have investigated the basis of antithrombin deficiency in an
asymptomatic individual (and family) with borderline levels ( 70%
antigen and activity) of antithrombin. Direct sequencing of amplified
DNA showed a mutation in codon 135, AAC to ACC, predicting a
heterozygous Asn135Thr substitution. This substitution alters the
predicted consensus sequence for glycosylation, Asn-X-Ser, adjacent to
the heparin interaction site of antithrombin. The antithrombin isolated
from plasma of the proband by heparin-Sepharose chromatography
contained amounts of  antithrombin (the very high affinity fraction)
greatly increased (20% to 30% of total) above the trace levels
found in normals. Expression of the residue 135 variant in both a
cell-free system and COS-7 cells confirmed altered glycosylation
arising as a consequence of the mutation. Wild-type and variant protein
were translated and exported from COS-7 cells with apparently equal
efficiency, in contrast to the reduced level of variant observed in
plasma of the affected individual. This case represents a novel cause
of antithrombin deficiency, removal of glycosylation concensus
sequence, and highlights the potentially important role of antithrombin in regulating coagulation.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
INHERITED ANTITHROMBIN deficiency results
in a predisposition to venous thromboembolism. Appreciable progress has
been made in defining the molecular basis of deficiency, which mostly occurs in individuals heterozygous for point mutations in the antithrombin gene.1,2 As with many other deficiency states, antithrombin deficiency has been divided into two major types. Type I
deficiency is characterized by a reduction in the antigen level usually
to 50% of normal, a reduction paralleled by a decrease in
functional activity. Type II deficiency is characterized usually by
(near) normal levels of antithrombin antigen, but reduction, usually to
50%, in activity. Type II deficiency can be further subclassified and
a number of schemes have been used.3-7 That adopted in
recent reports of the antithrombin database of mutations applies a
classification involving the functional domains.2 Thus,
type II deficiency may be caused by mutations that alter the function
of the reactive site, the heparin binding site, or that have multiple
(or pleiotropic) effects on function. This latter group includes cases
that present with reduced levels of antithrombin antigen, between 50%
to 100%, with a disproportionate reduction in functional levels,
usually 50%.
In most cases of deficiency, simple assays of function and activity are
sufficient for diagnosis. Complication may arise when the levels of
either assay are borderline to the normal range. Often this can be
resolved by investigation of the kindred, by consideration of the
clinical circumstances, or by genetic studies; although the latter is
usually impractical unless the region of the antithrombin gene involved
can be targeted for simple polymerase chain reaction (PCR) analysis.
We sought to clarify the reason for borderline levels of antithrombin
in a panel of patients with phenotypic deficiency, which has formed the
material used for a systematic investigation of the genetic basis of
deficiency (the results of this work are summarized along with the vast
majority of known mutations in Lane et al2). Once the
genetic defects in most cases with clear cut phenotypic deficiency had
been determined, those cases with borderline phenotypic values were
then investigated. One of these cases, the subject of this report, was
found to have a mutation that alters the glycosylation consensus
sequence located adjacent to the proposed heparin binding site on
antithrombin. The consequence of this mutation was an alteration in the
proportion of antithrombin distributed into the two plasma subfractions
of antithrombin,  antithrombin (with high-affinity for heparin), and
antithrombin (with very high-affinity for heparin).
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MATERIALS AND METHODS |
Kindred.
The proband, an Italian who at the age of 25 years had not experienced
any thrombosis, was investigated at the request of her gynecologist.
She was taking the oral contraceptive pill. A clinical history and
plasma samples were also taken from her sister, mother, and father:
there was no indication of venous thrombosis in the family, but the
father had suffered a myocardial infarction at the age of 58 years (see
Table 1).
Functional and immunoassays.
Antithrombin activity was determined both as antifactor IIa and
antifactor Xa inhibitory activities in the presence of heparin and the
chromogenic substrate S-2238 or S-2765 (Chromogenix, Molndal, Sweden).
The immunologic determination of antithrombin levels was determined
with a commercial radial immunodiffusion assay (Behring, Marburg,
Germany). Interassay coefficient of variation for the functional and
immunoassays was less than 5%.
Preparation of antithrombin.
Plasma antithrombin was extracted using the dextran sulphate
precipitation method8 followed by heparin-Sepharose
chromatography on a Pharmacia FPLC system (Amersham Pharmacia Biotech,
St Albans, Herts, UK) in 0.05 mol/L Tris buffer pH 7.4. Antithrombin was either eluted with a single step with 0.4 mol/L to 3 mol/L NaCl or with a linear gradient from 0.4 to 3 mol/L NaCl,
collecting 10 mL volumes of eluant.
Isoelectric focussing.
Isoelectric focussing was performed in a 0.75-mm thin polyacrylamide
gel (8.5 × 7.5 cm) containing a final concentration of 0.7%
Pharmacia Ampholine pH 4.0 to 6.5. The gels after fixation were stained
with the Bio-Rad Silver Staining kit (Bio-Rad Laboratories, Herts, UK).
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and immunoblotting.
SDS-PAGE was performed using a BioRad MiniProtean II
system. Forty nanograms of total antithrombin and the
elution fractions were separated. The gels were electroblotted using
the Bio-Rad Mini Trans-Blot cell onto Hybond ECL Nitrocellulose
membrane (Amersham Pharmacia Biotech). Antithrombin was located on the
membrane using a peroxidase-conjugated rabbit antihuman antithrombin
antibody (Dako Ltd, High Wycombe, UK) followed by ECL autoradiography
(Amersham International).
Genetic analysis.
All six exons of the antithrombin gene were amplified by polymerase
chain reaction (PCR) and sequenced using an automated sequencer (ABI
PRISM/373 Stretch XL DNA Sequencer; Perkin Elmer Biosystems, Foster City, CA). Primers were as described using one
biotinylated primer for each pair and single strand sequencing on
magnetic beads.9
Mutagenesis of AT cDNA.
Wild-type AT cDNA was cloned into the vector pCR II (Invitrogen,
Adelaide, Australia). The construct contained the complete AT coding
sequence, but none of the 5' or 3' untranslated region. Two
variant cDNAs were generated by site-directed mutagenesis (ExSite,
Stratagene, Christchurch, New Zealand) using inverse PCR. Mutagenic
primers were Asn135Thr forward
5'-ACCAAATCCTCCAAGTTAGTATC-3' (nt 501-523, numbering of AT
cDNA with the A of the initiation Met being nt 1), Ser137Ala forward
5'-AACAAAGCCTCCAAGTTAGTATC-3' (nt 501-523), with a common
reverse orientation primer 5'-GGCTTTTCGATAGAGTCGGCAG-3' (nt
500-479). After ligation with T4 DNA ligase, the variant cDNA vector
constructs were transformed in Epicurian Coli XL1-Blue supercompetent cells (Stratagene). DNA from the resulting colonies was
sequenced to check that the mutation was present and no further changes
had been introduced.
Cell-free expression of AT constructs.
RNA from wild-type and mutant AT cDNA was generated using the SP6
promoter of the pCR II vector. Plasmid DNA was linearized with
Not1 (New England Biolab, Christchurch, New Zealand) and incubated at 37°C for 30 minutes in a 50-µL reaction containing 10 µL 5xSP6 buffer, 5 µL 0.1 mol/L dithiothreitol
(DTT), 1 µL RNAguard (Pharmacia, Auckland, New Zealand),
10 µL guanosine triphosphate (GTP) mix (10 mmol/L
adenosine triphosphate [ATP], cytidine triphosphate [CTP], uridine
triphosphate [UTP], and 0.5 mmol/L GTP), 4 µL linear DNA (0.5 µg/µL), 2.5 µL CAP (New England
Biolabs) and 1.5 µL SP6 polymerase (Pharmacia). After this, 5 µL of
10 mmol/L GTP was added with the reaction proceeding for a further 30 minutes. After phenol chloroform extraction, RNA was precipitated with
3 mol/L sodium acetate and 100% ethanol. Variant and wild-type
antithrombins were produced using nuclease-treated rabbit reticulocyte
lysate (Promega, Auckland, New Zealand) in a volume of 26 µL containing 17.5 µL lysate, 0.5 µL amino acids minus
methionine, 5 µL 35S-methionine (>1,400 Ci/mmol, ICN,
Auckland, New Zealand) and 3 µL RNA, in the presence
(1.8 µL) or absence of canine pancreatic microsomal membranes
(Promega, Dade Diagnostics, Aukland, New Zealand) for 1 hour at 30°C. Some products were incubated with endoglycosidase H
(New England Biolabs), 500 U at 37°C for 60 minutes, to remove any
Asn-linked carbohydrate. The products were electrophoresed in 10%
acrylamide-bis (37.5:1) protein gels for 1 hour at 100 V, followed by
fixation and autoradiography for 48 hours.
Mammalian cell expression of AT constructs.
A 1.4-kb EcoRI fragment containing the wild-type or mutant AT
cDNA was isolated from pCR II and cloned into the EcoRI site of
the mammalian expression vector pcDNA3 (Invitrogen). After transformation into DH5 competent cells, clones with the
correct insert orientation were selected by screening with restriction enzyme digests. COS 7 cells at approximately 50% confluence were transfected with 1 µg of the pcDNA3 constructs using Tfx-20 (Promega) as described by the manufacturer. A reporter gene vector,
pSEAP2-Control (Clontech, Sydney, Australia), was cotransfected with
the AT constructs to act as a control of transfection efficiency.
Assays for the reporter gene product, alkaline phospatase secreted into
culture supernatant, were performed using a chemiluminescent assay
(Great EscAPe, Clontech, San Diego, CA) according to the
manufacturer's instructions. Cells were then grown at 37°C in 10%
CO2/air in Dulbecco's modified Eagle's medium (DMEM,
Gibco BRL, Auckland, New Zealand) with 2 mmol/L glutamine
and 10% fetal bovine serum (FBS, Gibco BRL). Approximately 48 hours
after transfection, culture supernatant was discarded and the cells
were washed in DMEM lacking FBS and methionine. After resuspension of
the cell pellet in the same media, newly synthesized cellular proteins
were radiolabelled by the addition of 50 µCi
35S-methionine for 3 hours. Culture supernatants
were immunoprecipitated with rabbit polyclonal antisera raised against
human AT (Dako) using formalin fixed Staphylococcus aureus
(Gibco BRL). Electrophoresis was performed as described above,
except in 12.5% acrylamide-bis (37.5:1) gels.
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RESULTS |
The proband, her father, and sister (but not her mother) all have
antithrombin levels determined by two different functional assays and
antigen assay that are lower than that expected for normal individuals
(Table 1). This suggested an inherited
abnormality leading to reduced levels. Interestingly, while the
reduction in antigen level to almost 70% might have suggested the
presence of a genetic mutation resulting in a pleiotropic effect, the
functional activity was not reduced to 50%. The parallel reduction in
plasma antithrombin antigen and activity in the proband, father, and sister was consistent with either production of antithrombin from one
allele at a reduced rate or increased clearance of the protein, but no
impairment of reactive site function. Furthermore, no slow-moving peak
indicative of an antithrombin population with reduced heparin affinity
was observed on crossed immunoelectrophoresis in the presence of
heparin. These observations collectively suggest that this case is not
similar to those with pleiotropic effects.
To investigate further the phenotype, plasma from the proband was
applied to and eluted from heparin-Sepharose. Initially, elution was
performed with a step gradient to recover all antithrombin in plasma.
The antithrombin isolated from normal plasma migrated in SDS-PAGE and
immunoblotting as two bands, the major band of antithrombin and a
barely visible antithrombin band. In contrast, plasma of the
patient contained a much greater apparent antithrombin fraction
than normally seen, as well as antithrombin (see
Fig 1 "Total AT"). Elution with a
salt gradient was able to resolve the two bands in the proband plasma.
Under these conditions, the antithrombin from the normal plasma eluted
as expected between a NaCl concentration of 0.82 and 1.06 mol/L. The
apparent antithrombin band from the proband eluted later, between
1.06 and 1.51 mol/L NaCl (Fig 1).
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| Fig 1.
Elution of antithrombin from heparin-Sepharose.
Antithrombin from proband (A) and normal (B) plasmas was isolated
either by a single-step ("Total AT") or a gradient increase in
NaCl concentration. Fractions were collected and analyzed by
immunoblotting, as described in Materials and Methods. For fractions
collected by gradient elution, the molar concentration of NaCl required
for their elution is indicated.
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On isoelectric focussing and silver staining of extracted normal
antithrombin, at least nine components could be observed. The
antithrombin extracted from the proband showed multiple bands in the
same pH range, but with a different concentration distribution (results
not shown).
Genomic DNA was isolated from the proband, and the entire coding region
was sequenced. A single nucleotide abnormality was identified in the
codon for 135 Asn, AAC to ACC, which predicts an amino acid
substitution Asn135Thr (see Fig 2).
Inheritance of the mutation was demonstrated by PCR and sequencing in
the case of the sister and the father (a sample could not be obtained from the mother). This nucleotide substitution was not found in more
than 100 normal alleles, indicating it is not a common polymorphism.
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| Fig 2.
Selected nucleotide sequence of proband and normal
PCR-amplified DNA. All coding regions of the antithrombin gene were
amplified and sequenced with an automated sequencer (see Materials and
Methods). Only one region was found with a sequence difference between
normal and patient products, and this difference is illustrated along
with its predicted amino acid change.
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The predicted amino acid substitution affects the consensus Asn-linked
glycosylation sequence, Asn-X-Ser, beginning at position 135, and was
predicted to block glycosylation at this site. The variant protein
would be expected to have an almost identical molecular weight
(MW) and properties to antithrombin. This is entirely
compatible with the results presented in Fig 1. One property that could
not be explained is the reduced amount of variant in the plasma. Normal
plasma contains 50% antithrombin from each of two normal alleles.
In affected members of this family, the variant allele is assumed to
have resulted in a reduced amount of plasma antithrombin, 20%
rather than 50%, giving a total of 70% antithrombin (see Table 1).
This estimate was compatible with one obtained by determining the
relative peak areas on elution from heparin-Sepharose: the proportion
of antithrombin was then 20% to 30% of total antithrombin in
the proband and her sister. The effects on glycosylation of the
substitution and the reason for the reduced plasma concentration of
antithrombin in the affected members of the family was examined by
analysis of protein expressed in vitro.
Site-directed mutagenesis was used to generate variant AT cDNA encoding
the Asn135Thr amino acid substitution identified in the proband. In
addition, to check whether any effects of the substitution were related
to effects on glycosylation of antithrombin at Asn135 or were
residue-specific (that is, related to Asn135 rather than
glycosylation), an additional variant cDNA was created encoding the
substitution Ser137Ala. This substitution alters the Asn-linked
glycosylation consensus sequence of Asn-X-Ser and was predicted to
inhibit glycosylation at Asn135.
Using rabbit reticulocyte lysate as a cell-free expression system, RNA
derived from wild-type and the two variant AT cDNAs were translated to
protein with approximately equal efficiency (see
Fig 3A). For all three proteins, a product
of approximately 52 kD was observed, consistent with
translation of the full-length antithrombin including signal peptide.
In addition, all three translations produce a 47-kD form, which results
from internal initiation of translation of the AT cDNA from Met17
(A.C.F. and R.J.O., unpublished results). In the presence of pancreatic
microsomes, posttranslational processing of the wild-type Asn135Thr and
Ser137Ala antithrombins was observed as larger MW products. Incubation
of the translation products with endoglycosidase H left intact the 52-kD and 47-kD forms, but removed the larger MW bands, indicating the
latter were the result of Asn-linked glycosylation of the protein.
Careful inspection of the bands representing glycosylated protein
showed the presence of a high MW band in wild-type antithrombin, which
is missing for the variants (see Fig 3A, lanes 1 through 3). This is
more clearly demonstrated after trypsin treatment, lanes 4 through 6, which removes nonglycosylated protein that has not been translocated
into the microsomes. This shows that while the Asn135Thr and Ser137Ala
variants are translated and can be glycosylated, complete glycosylation
of the mutant proteins is prevented.
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| Fig 3.
Expression of wild-type, As135Thr, and Ser137Ala
antithrombins. (A) Antithrombin variants were expressed using rabbit
reticulocyte lysate and coupled to posttranslational modification by
the addition of microsomal membranes (+). Products above 52 kD in the
coupled reactions represent N-linked glycosylation products. The
uppermost of the doublet glycosylation isoform (*) produced from the
wild-type protein (Wt) is absent in two variants with substitutions
affecting the N-linked consensus glycosylation site at residue 135 (N135T and S137A, labelled 135 and 137, respectively); see lanes 1 through 3, but more clearly in lanes 4 through 6, where trypsin has
been added (+) after protein has been incubated with microsomes, and
protein not translocated into the protective environment of the
microsomes has been degraded. Lanes 7 through 9 are control incubations
of wild-type and two variant antithrombins with endoglycosidase H (+)
and indicate that this enzyme strips the carbohydrate side chains from
the high MW forms. The band labelled 47 kD results from internal
initiation of translation at Met17. (B) Antithrombin was expressed in
COS-7 cells. Supernatant of COS-7 cells transfected with antithrombin
constructs was immunoprecipitated using polyclonal antithrombin
antibody. Glycosylated antithrombin was found in the supernatants with
a size of approximately 60 kD. At least two glycosylation isoforms were
observed for wild-type (WT) antithrombin (just above and below the
60-kD arrow), but the larger form was not observed in the N135T or
S137A variants. Several nonspecific bands are apparent in each lane and
are also seen in supernatants from mock-transfected COS cells (not
shown).
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The supernatants of cultured COS-7 cells transfected with wild-type and
the two variant antithrombin constructs were compared to determine the
expression efficiency of the proteins. Protein products of
approximately 60 kD were immunoprecipitated from the culture
supernatants of all three transfectants using a polyclonal antibody to
antithrombin. Figure 3B shows that in addition to the 60-kD species,
there are several additional weak bands in each lane (a single band
above and a triplet below the 60-kD species). Additional experiments
(not shown) demonstrated that these bands were also present in
supernatants from mock-transfected COS cells, suggesting that they are
proteins, which are nonspecifically precipitated by the antibody. In
further control experiments, we have shown with expressed wild-type
antithrombin that the 60-kD form is glycosylated, as incubation of the
cells with tunicamycin, which inhibits glycosylation, results in an
antithrombin of about 50 kD. The glycosylated wild-type antithrombin
synthesized by the mammalian cells was present in the electrophoretic
separations of immunoprecipitated products as a doublet band, in
contrast to the Asn135Thr and Ser137Ala variant proteins, which were
each clearly expressed as a single band (Fig 3B). It is likely that the
lower band of the doublet observed for wild-type AT represents AT.
Inspection of the products suggested wild-type antithrombin and the two
variant proteins were secreted from cells with apparently similar
efficiency, and comparison of the reporter gene product assays from the
supernatants confirmed an equal efficiency of cell transfection by the constructs.
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DISCUSSION |
In this study, we have identified in an asymptomatic individual and her
family a mutation in the antithrombin gene in codon 135, AAC to ACC,
which results in an amino acid substitution, 135Asn to Thr. This alters
the carbohydrate consensus sequence beginning at Asn135 and thereby
prevents normal glycosylation at this site. Using two distinct in vitro
transfection and expression systems, we have shown that glycosylation
is specifically inhibited by this amino acid substitution. The
consequence of the mutation is an increased concentration of the
fraction of antithrombin in plasma that binds with very high-affinity
to heparin, antithrombin. Although the concentration of this
fraction is greatly increased, it does not approach 50% (the
concentration resulting from a single normal allele) and there is
therefore a moderate deficiency, 70%, of total antithrombin in the
plasma of the kindred.
In normal plasma, most (95%) antithrombin is glycosylated at Asn135
( antithrombin) and a minor fraction is unglycosylated ( antithrombin).10,11 It has been shown that this normal
variable glycosylation arises from the presence of Ser in the consensus Asn-X-Ser sequence at this site.12,13 Presence of the Ser
residue reduces the efficiency of core glycosylation at position 135. A
consequence of the lack of glycosylation in antithrombin is increased affinity of this minor fraction for heparin and related glycosaminoglycans, such as heparan sulphate. Heparin is known to bind
to antithrombin in a two-step reaction, initially with low-affinity,
but then with high-affinity.14 High-affinity binding is
believed to occur due to a conformational change involving also the
reactive site loop. Recent rapid-kinetic studies of and antithrombins have suggested that lower affinity binding of the former
to heparin is not due to interference of the initial (weak) binding
step by the oligosaccharide side chain at position 135, but to
decreased conformation flexibility resulting from incorporation of the
oligosaccharide side chain.15 This decreases the rate
constant for the conformational change in the second binding step and
increases that for its reversal.
Estimates of the concentration of antithrombin in normal plasma
vary between 5% to 10%, but there is no precise means of determining
its concentration and this might be an overestimate for human plasma.
Electrophoretic analysis is at best semiquantitative, and the low
relative concentration makes calculation of its recovery from
precipitation and chromatography procedures difficult. Our data suggest
that presence or absence of glycosylation at Asn135 will not
significantly affect the export of antithrombin from hepatocytes. The
finding that the concentration of the variant Asn135Thr in plasma is
less than that of normal antithrombin in the present family (compared
with 20% to 30% for the variant with 50% expressed by a normal
single allele), therefore suggests that antithrombin has a larger
distribution volume, or higher clearance rate from the vasculature, or
possibly it competes with antithrombin for heparan sulphate within
and outside of the circulation. This suggestion is compatible with
results of turnover studies of antithrombin fractions (differing in
heparin affinity) performed in the rabbit.16 Consequently,
the level of antithrombin in normal plasma may be determined by
both efficiency of glycosylation and by events that occur at the
vascular interface after export of the antithrombin into the circulation.
The physiological and pathological roles of antithrombin have yet
to be fully defined. Nevertheless, there is accummulating evidence that
heparan sulphate- antithrombin interaction could be important.
First, there is much evidence that the normal endothelium synthesizes
heparan sulphate, which is anticoagulantly active.17 This
heparan sulphate contains some of the residues identified as being
critical in the essential pentasaccharide of heparin, necessary for
high-affinity binding to antithrombin. Second, there is a rapid
reacting form of antithrombin, observable in a rat perfusion heart
model system, which interacts with factor Xa at a rate indicative of
heparin (-like) enhancement.18 Third, in models of vessel
injury, antithrombin associates more readily than antithrombin
with the vessel wall and preferentially inhibits thrombin coagulant
activity.19,20 From consideration of these experimental
studies, together with the kinetic data, it can be concluded
tentatively that the anticoagulant function of antithrombin at the
endothelial surface may be primarily determined by its antithrombin content.
The considerations above on the potential role of the heparin-
antithrombin interaction bear on the current case of antithrombin deficiency. Antithrombin is lowered in the affected family to 70% of
the normal level, inviting the suggestion that this could be associated
with an increased risk of venous thromboembolism. That no venous
thromboembolism has occurred in the family members studied (although we
note the myocardial infarction in the father), could be due to a lack
of concurrent or transient risk factors of sufficient strength.
However, it could also be due to the protective effect of the
relatively increased concentration of antithrombin. Judging from
the immunoblotting experiments illustrated herein, we speculate that
the large increase in concentration of this fraction in this kindred
may be sufficient to increase anticoagulant activity to compensate for
the reduction in total antithrombin and to provide protection against
venous thromboembolic events. Additional studies on the pathophysiology
of antithrombin will be required to substantiate or refute this speculation.
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FOOTNOTES |
Submitted July 30, 1998; accepted February 8, 1999.
Supported by the Special Trustees of Charing Cross Hospital, the
Charing Cross and Westminster Medical School Research Committee, by a
small grant from the British Society of Haemostasis and Thrombosis and
the Health Research Council of New Zealand.
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 D.A. Lane, PhD, Department of
Haematology, Imperial College School of Medicine, Charing Cross
Hospital Campus, Hammersmith, London W6 8RP, UK; e-mail:
d.lane{at}ic.ac.uk.
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ABSTRACT
INTRODUCTION