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INTRODUCTION |
2-Glycoprotein I
(
2GPI),1 also known as
apolipoprotein H, is a major protein constituent of plasma where its
concentration approaches 0.2 mg/ml (1). Whereas the physiologic
function of
2GPI has not been deduced,
2GPI interacts
specifically with lipoprotein(a) (2) and the endothelial cell protein
annexin II (3). At least in vitro,
2GPI acts as an
inhibitor of the intrinsic blood coagulation pathway (4), ADP-mediated
platelet aggregation, and the prothrombinase activity of activated
platelets (5).
2GPI binds to negatively charged cell surfaces such
as those on activated platelets, probably by binding to negatively charged molecules such as heparan sulfate cell surface proteoglycans and anionic phospholipids. It is this latter property that has been
proposed to be most clinically relevant. Interest in
2GPI increased
dramatically shortly after it was discovered that this plasma
protein is the most common antigen in patients with the "anti-phospholipid syndrome" (APS). (6). The term
"anti-phospholipid" is a misnomer because most of the antibodies
generated in this human autoimmune disorder are not directed against
phospholipids as first thought but rather against the
2GPI component
of the macromolecular complex. The presence of anti-
2GPI antibodies in these patients correlates well with thrombosis, miscarriages, and
thrombocytopenia (6). Finally,
2GPI has been implicated in apoptosis
(7).
2GPI is a single chain, 50-kDa protein consisting of 326 amino
acids. It contains large numbers of Pro and Cys residues, and it is
heavily glycosylated.
2GPI is a member of the complement control,
short consensus repeat superfamily of proteins. The first four
homologous repeat regions consist of ~60 amino acids with 4 conserved
Cys residues that form 2 disulfide bonds in each domain. The fifth
domain in
2GPI differs in that it contains 80 amino acids and 3 disulfide bonds. The amino acid sequences of mouse and human
2GPI
are 76% identical, and the
2GPI transcript in both species is
~1.2 kb in size. The nucleotide sequence of the entire mouse
2GPI
gene has been deduced (8). It is ~18 kb in size and consists of eight exons.
By using a homologous recombination approach, we now describe the
generation and initial characterization of transgenic mice unable to
express
2GPI.
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EXPERIMENTAL PROCEDURES |
Generation of
2GPI Null Mice--
A mouse
2GPI genomic
clone spanning ~18 kb that was previously isolated and sequenced from
a 129/Sv genomic P1 library (Genome Systems, Inc., St. Louis, MO) (8)
was used to disrupt the
2GPI gene. The targeting vector contained
PGK-Neo as the positive selection marker and herpes simplex virus
thymidine kinase gene (HSV-tk) as the negative selection marker. The 5'
portion of the construct contained a 3.5-kb
HindIII-AccI fragment, with exon 1 and part of
exon 2. The 3' portion of the construct contained a 3.5-kb XhoI-XhoI fragment, with exon 4. The HSVtk,
PGK-Neo, and vector backbone were from the PGKNeo cassette (9). Thirty
micrograms of the targeting vector was linearized with NotI,
electroporated into 2.5 × 107 embryonic stem
(ES) cells (129/Sv). Clones were selected with G418 and ganciclovir
according to the method described previously for other genes (10).
Single clones were selected after 10 days. Isolated DNA was digested
with XbaI and separated by routine gel electrophoresis, and
the resulting DNA blots were analyzed with a 32P-labeled
mouse
2GPI probe (0.4 kb), located within intron 4 just outside the
targeting vector (named probe A).
An ES cell clone that had undergone homologous recombination was
injected into C57BL/6 mouse blastocysts (10), and the resulting chimeric males were bred with C57BL/6 females. Germ line transmission of the disrupted allele was determined by the presence of agouti mice
in the offspring. Mice were genotyped by isolating genomic DNA from
tail biopsies and analyzed by Southern blotting using probe A.
For RNA blot analysis, total RNA was prepared from mouse livers by
using Trizol reagent (Life Technologies, Inc.). Ten micrograms of
denatured total RNA was separated by formaldehyde-agarose gel electrophoresis, transferred to a positively charged nylon membrane (HybondTM-N+, Amersham Pharmacia Biotech), and cross-linked to the
membrane by ultraviolet light. The membrane was hybridized with a
0.5-kb mouse
2GPI cDNA probe corresponding to exons 1-5.
SDS-PAGE/Immunoblot Analysis--
Plasma from wild type (+/+),
heterozygote (+/
), and homozygote (
/
) mice were separated on 12%
SDS-polyacrylamide gels and transferred onto nitrocellulose membranes
(Amersham Pharmacia Biotech). Each membrane was incubated in
Tris-buffered saline (TBS), 0.1% Tween 20, 10% skim milk to minimize
nonspecific binding. The membrane was then incubated with a 1/1000
dilution of affinity-purified rabbit polyclonal anti-
2GPI antibody
in 1% bovine serum albumin/TBS. Bound protein was detected by
ECL-enhanced chemiluminescence (Amersham Pharmacia Biotech), using a
1/1000 dilution of a goat anti-rabbit IgG horseradish peroxidase
conjugate (Sigma) in 1% bovine serum albumin/TBS. Normal rabbit IgG at
the same concentration as the rabbit anti-
2GPI antibody was used as
a negative control. SDS-PAGE/immunoblot analysis was performed on five
separate occasions using plasma from 10 mice (5 female and 5 male) from
each group aged from 4 to 8 weeks old. Quantitation of
2GPI protein
in +/+ and +/
mice was also assayed by serial dilution of pooled
plasma from each group of mice by Western blot analysis using rabbit
anti-
2GPI antibody. Purified mouse
2GPI at 5, 10, and 20 ng per
lane was used as a standard.
Histological Analysis--
Histological examination was
performed on 5 male and female homozygous mutant animals aged 4-9
months, and a total of 5 normal wild type and heterozygous littermates.
Tissues were fixed in 10% neutral buffered formalin, processed for
paraffin embedding according to standard methods, and sections were
stained with hematoxylin and eosin.
Platelet Counts--
Blood was recovered by cardiac puncture
into EDTA-coated Capiject tubes (Terumo) from female virgin or day-18
pregnant
2GPI +/
and
2GPI
/
mice at 12-16 weeks of age
after anesthesia with avertin (1 mg/ml tribromoethyl alcohol in
tertiary amyl alcohol (Sigma) diluted to 2.5% v/v in saline; 15 µl/g
body weight injected intraperitoneally). The concentration of platelets
in blood was determined using a Technicon H2 automated hematology
analyzer (Bayer Diagnostics, Tarrytown, NY).
Breeding Experiments--
Mice were housed in groups of 3-5 per
cage, kept in a pathogen-free environment, and raised under standard
conditions, 23 ± 1 °C, 12-h light/12-h dark cycles with free
access to food (normal chow diet) and water ad libitum. The
mice were hybrids between the C57BL/6 and 129/Sv strains. All
procedures were conducted with the approval of the University of New
South Wales Animal Care and Ethics Committees.
Adult females (10-12 weeks,
2GPI +/+,
2GPI +/
or
2GPI
/
) were housed 2:1 with adult stud males (
2GPI +/+ or
2GPI
/
, as specified in the text) and allowed to mate naturally. Females were separated from males and housed in groups of 2-3 on the day at
which a copulation plug was evident, nominated day 1 of pregnancy. Pregnant mice were sacrificed on day 18 of pregnancy for determination of numbers of implantation sites and platelet counts or allowed to
proceed to term for determination of litter size, individual pup
weight, and survival to weaning.
Coagulation Assays--
All coagulation tests were performed on
an Automated Coagulation Machine (ACL-3000 Plus, Coagulation System
Instrumentation Laboratory, Milano, Italy).
Mouse blood samples from 15 homozygous mutant mice and equal numbers of
age- and sex-matched wild type and heterozygous controls were collected
in 0.11 M sodium citrate (9:1, v/v) via direct cardiac
puncture as described previously. Plasma was centrifuged at 3,000 × g for 20 min, collected, filtered through a 0.22-µm filter to remove platelet fragments, and stored at
70 °C until analysis.
The following coagulation tests was performed with each genotype
plasma: dilute kaolin clotting time (dKCT); dilute Russell Viper venom
time (dRVVT); activated partial thromboplastin time (aPTT); and a
protein C pathway screening test.
The dKCT was performed according to Exner et al. (11). The
dRVVT was performed according to Thiagarajan et al. (12)
using the LA screen and LA confirm reagents from Gradipore (Gradipore, North Ryde, Australia). The aPTT was performed according to the manufacturers' instructions using cephalin to activate the intrinsic pathway of coagulation (Sigma). A protein C pathway screening test
(GradiThrom PCP) using a dRVVT methodology and purified protein C
activator from Agkistrodon contortrix (Gradipore) was
performed according to the manufacturers' instructions.
Assay of Coagulation Factor Activities--
The activity of the
coagulation factors II, V, and VII-XII was determined as a clotting
time after mixing the murine plasma with human plasma, deficient in the
specific factor, and the addition of appropriate activator (13). A
standard curve was constructed by log-log plot of the clotting time
(either aPTT or PT) of various dilutions of pooled
2GPI+/+ plasma
(assumed to represent 100% activity). The specific factor activity of
2GPI +/
and
2GPI
/
plasma was derived as the mean activity
of at least two dilutions extrapolated from the standard curve. All
pro-coagulant activities were expressed as a percentage of the
pro-coagulant activity in a pooled plasma of adult wild type mice.
In Vitro Thrombin Generation Assay Using a Chromogenic
Substrate--
A chromogenic assay was used to determine the rate of
thrombin generation over time. The plasma used in this assay was
defibrinated as follows. Plasma was spun at 3,000 × g
for 20 min and filtered through 0.22-µm filter. Aliquots were
collected in 1.5-ml Eppendorf tubes and placed into a shaking water
bath at 53 °C for 20 min and then centrifuged at 10,000 × g for 10 min. The supernatant was collected and stored at
70 °C for use. All reagents in the thrombin generation assay were
diluted in 0.9% NaCl. A mixture of 25 µl of diluted (1:9)
thromboplastin (Sigma), 25 µl of 0.9% NaCl, and 50 µl of 1:1
dilution of defibrinated plasma from the three groups of mice were
added to wells of a microtiter plate and pre-warmed to 37 °C for 10 min. Then 50 µl of 1 mM spectrozyme, a chromogenic
substrate for thrombin (American Diagnostica), and 50 µl of 30 mM calcium chloride were added sequentially. Background thrombin generation was determined in the absence of thromboplastin. The plates were read immediately and every 30 s thereafter at 405 nm at room temperature in an automated enzyme-linked immunosorbent assay plate reader (Molecular Devices Spectro Max 250 with Softmax Pro
1.2 software) until thrombin generation had reached a plateau, usually
after 20 min. Plotting thrombin generation over time yielded a
sigmoidal curve. Alteration in the rate of thrombin generation by
heterozygous or homozygous mice was examined in duplicate wells, and
the result was expressed as a percentage of the wild type optical
density at the time that the wild type curve reached half-maximal OD.
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RESULTS |
Generation of
2GPI-Null Mice--
An 18-kb
2GPI genomic
clone obtained from a mouse 129/Sv library was used to construct the
targeting vector (Fig. 1A) for homologous recombination. A
2GPI-null allele was produced in the
targeting vector by replacing a 4.7-kb portion of the gene containing
part of exon 2 and the entire exon 3 with a neomycin resistance
cassette. Two correctly targeted ES clones were identified among 700 G418/ganciclovir-resistant clones. Correct integration of the targeting
construct on the opposite side was confirmed by DNA blot analysis using
a probe adjacent to the targeting construct (data not shown). Analysis
of the blot with the neomycin resistance gene revealed no additional
integration event in the two clones (data not shown).

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Fig. 1.
A, 2GPI gene and targeting construct.
Line A represents the map of the endogenous murine 2GPI
gene and its flanking sequence. The open boxes denote exon
sequence, and the line represents flanking and intron
sequence. Restriction endonuclease sites used for cloning and screening
are shown. The position of the probe A (closed box) used to
screen for homologous integrants is indicated. Line B
represents the vector used to target the 2GPI locus. The solid
blocks represent portions of the 2GPI genomic locus used as
recombination arms; arrows represent the direction of
transcription of the neomycin resistance and thymidine kinase gene
(HSV-tk, herpes simplex virus thymidine kinase gene;
NEO, neomycin resistance gene). Line C denotes
the predicted organization of the locus after homologous recombination.
B, Southern blot to determine genotypes of mice. Tail tip
genomic DNA was digested with XbaI and hybridized with the
probe shown in A. DNA size is indicated in kilobases. Wild
type, +/+; heterozygous, +/ ; and homozygous-deficient, /
mice.
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One of the successfully targeted ES clones heterozygous for disruption
of the
2GPI locus was injected into blastocysts derived from C57BL/6
females to generate chimeric mice. Chimeric mice were bred with each
other, and subsequent mice of the three expected genotypes
2GPI +/+,
2GPI +/
, and
2GPI
/
were obtained by interbreeding of the
heterozygous offspring. Southern blot analysis of DNA obtained from the
tails of these animals was used to determine their genotype (Fig.
1B). Restriction enzyme digestion of the wild type
2GPI
mouse locus with XbaI generates a 10.5-kb fragment for the
129/Sv strain or a 2.8-kb fragment for the C57BL/6 strain, whereas the
correctly targeted locus generates a 5.7-kb fragment (Fig.
1B).
Analysis of the
2GPI Mutation--
Northern blot analysis was
performed to confirm the loss of
2GPI gene expression in the
surviving
2GPI
/
mice. As shown in Fig.
2A, no transcript was observed
in RNA samples isolated from liver tissue of
2GPI
/
mice when
hybridized with a highly specific radiolabeled cDNA probe encoding
exons 1-5 (Fig. 2A). In contrast, the expected 1.2-kb
full-length transcript was present in the liver of
2GPI +/+ and +/
mice when analyzed in parallel. A 
actin probe was hybridized to
the same blot after removal of the
2GPI probe to assess the amount
of RNA loaded in each lane (lower panel in Fig.
2A).

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Fig. 2.
A, Northern blot. Northern blotting
analysis of total RNA (10 µg/lane) isolated from the liver from
2GPI +/+, +/ , and / littermates. The same blot was probed with
a  actin probe demonstrating equal amount of RNA applied.
B, Western blot for 2GPI. Western blotting analysis of
control mouse 2GPI: wild type, +/+; heterozygous, +/ ; and
homozygous-deficient, / mice. Plasma from the three genotype mice
were run on a 12% SDS-polyacrylamide gel, blotted, and probed with a
rabbit anti- 2GPI antibody. C, quantitation of 2GPI in
+/+ and +/ mice by Western blot analysis using rabbit anti- 2GPI
antibody. Purified murine 2GPI was used as a standard. Lane
1, 5 ng of 2GPI; lane 2, 10 ng of 2GPI;
lane 3, 20 ng of 2GPI; lane 4, 2 µl of
plasma from +/+ at 1:4 dilution; lane 5, 2 µl of plasma
from +/+ at 1:8 dilution; lane 6, 2 µl of plasma from +/+
at 1:16 dilution; lane 7, 2 µl of plasma from +/ at 1:4
dilution; lane 8, 2 µl of plasma from +/ at 1:8
dilution; lane 9, 2 µl of plasma from +/ at 1:16.
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Based on the structure of the gene targeting construct, we expected
that mice homozygous for the mutation would be unable to produce
functional
2GPI protein. Plasma from 6-week-old mice was tested by
Western blot using a rabbit polyclonal anti-
2GPI antibody. The
antibody reacted with the expected 50-kDa protein band present in wild
type and
2GPI +/
mice (Fig. 2B). However, no such
immunoreactive band was detected in the plasma from
2GPI
/
mice.
Furthermore, analysis of serial dilutions of plasma from +/+ and +/
mice (Fig. 2C) revealed that the heterozygotes contained
approximately half the concentration of
2GPI in their plasma
relative to wild type mice. Thus, an abnormality in
2GPI expression
was even seen in +/
mice.
Phenotypic Characterization and Reproductive Performance of
2GPI-Null Mice--
Six pairs of male and female
2GPI +/
mice
were caged separately and allowed to breed naturally for periods
ranging from 4 to 8 months. All females carried at least four viable
pregnancies during this period, with a mean ± S.D. of 1.2 ± 0.3 litters of month. The mean ± S.D. litter size was 9.2 ± 2.2 pups (total of 39 litters), of which 97% (347/357) were viable at
24 h of age. Genotypes of the progeny were determined at 3 weeks
of age. Of the 336 successfully genotyped offspring, 121 were wild
type, 185 were heterozygous, and only 30 were homozygous for the
disrupted allele, which is a statistically significant
(p < 0.005) deviation from the expected 1:2:1 ratio.
Gross histologic examination of heart, lung, thymus, spleen, lymph
nodes, liver, gallbladder, kidneys, urinary bladder, reproductive
tract, stomach, small intestine, cecum, colon, pancreas, brain, eyes,
and skeletal muscle did not reveal any pathological changes
associated with the homozygous mutation.
The effect of homozygous mutation on reproductive performance was
investigated in further experiments. Initially,
2GPI
/
and
2GPI +/+ male mice were mated with C57BL/6 female mice. Each of six
males from each genotype mated successfully with females and sired
pregnancies. Female
2GPI
/
and
2GPI +/+ mice were then mated
naturally with adult stud males of the same genotype, and pregnancies
were allowed to proceed to term. Neither the interval between placing
with males and discovery of a vaginal plug nor the proportion of
plugged females delivering live pups was affected by the
2GPI
genotype. The duration of pregnancy, the number of pups born, and their
viability and weight at 24 h and at weaning were comparable in
2GPI
/
and
2GPI +/+ pregnancies (Table I), indicating a normal reproductive
potential for
2GPI
/
mice.
To determine the effect of
2GPI deficiency on platelet counts in
pregnancy, blood was recovered by cardiac puncture from adult virgin
2GPI
/
and
2GPI +/+ mice on day 18 of pregnancy and from
2GPI
/
and
2GPI +/+ female mice mated naturally with adult
stud males of the same
2GPI status. Whereas pregnancy was associated
with an increase in mean blood platelet count of 35%, there was no
significant effect of
2GPI deficiency in either the virgin or
pregnant state (Table II). Furthermore,
there was no effect of
2GPI deficiency on the number of implantation
sites at day 18 (mean ± S.D. = 9.3 ± 2.9 in
2GPI +/+
mice and 8.2 ± 2.8 in
2GPI
/
mice, n = 6 per group) or on the proportion of resorption sites (5/56 in
2GPI
+/+ mice and 1/49 in
2GPI
/
mice, n = 6 per
group).
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Table II
The effect of genetic deficiency in 2GPI on platelet numbers in
blood of virgin and day 18 pregnant mice
Data are mean + S.D. × 10 3 platelets/µl. The number
of mice per group are given in parentheses. 2GP1 +/+ or +/ females
were mated with 2GP1 +/+ males, and 2GP1 / females were mated
with 2GP1 / males.
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Characterization of the Coagulation Profiles of
2GPI +/+,
2GPI +/
, and
2GPI
/
Mice--
The role of
2GPI in
coagulation profiles was examined using a number of hematologic
parameters. Analysis of pooled plasma from
2GPI+/+,
2GPI+/
, and
2GPI
/
mice (5 mice in each group; the experiment has been
repeated on three occasions) revealed no significant differences in
dKCT, dRVVT, aPTT nor protein C pathways among the three groups of
animals (Table III).
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Table III
Coagulation profile of plasma from 2GPI +/+, +/ , and / mice
Data represents mean ± S.D. of three separate experiments using
pooled plasma samples from five mice of each genotype expressed as the
clot time in seconds.
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The activity of coagulation factors II, V, and VII-XII was assessed in
the three mouse genotypes. Specific factor assays in normal and
defibrinated plasma revealed similar levels for the factors assayed in
all three genotypes (data not shown).
In the in vitro chromogenic assay of thrombin generation,
the pooled plasma samples (Fig.
3A) or the individual plasma
samples (Fig. 3B) from
2GPI
/
mice had significantly
less thrombin generation compared with that obtained from
2GPI +/+
or
2GPI +/
mice. Fig. 3A demonstrates that the average
time point required to reach half-maximal optical density
(OD405 nm = 0.55) was 1050 s for
2GPI +/+ mice and
2100 s for the
2GPI +/
mice. In contrast, plasma from
2GPI
/
mice did not reach the half-maximal optical density even after
3000 s. Similar results were obtained when plasma from two further
groups of 15 mice were analyzed in the thrombin generation assay.
Mixing equal parts of
2GPI +/+ and
2GPI
/
mouse plasma
produced a thrombin generation curve similar to that of the
heterozygote plasma (data not shown).

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Fig. 3.
In vitro thrombin generation.
A, in vitro thrombin generation of pooled plasma
from 2GPI +/+, +/ , and / (n = 5) littermates
detected with a chromogenic substrate over 50 min (for details see
"Experimental Procedures"). B, in vitro
thrombin generation of individual plasma from 2GPI +/+, +/ , and
/ mice. Plotted values represent the mean ± S.D. of
determinations in 10 individual animals measured at 1000 s.
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The mean value (OD405 nm) of thrombin generation of 10 individual plasma samples from each population of animals (Fig. 3B) was measured at 1000 s. The mean value for
2GPI
/
was 0.175, which represents 69% less than the result for
2GPI
+/+ mice and 40% less than that for
2GPI +/
mice. The difference
in thrombin generation between
2GPI +/+ and
2GPI
/
mice was
highly significant by one-way analysis of variance (p = 0.0051).
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DISCUSSION |
We generated
2GPI null mice to determine the function of this
plasma protein in hemostasis and reproduction. Evidence in support of
the view that we targeted the appropriate gene was obtained by DNA,
RNA, and SDS-PAGE/immunoblot analysis. The obtained data confirm that
there is only one
2GPI gene in the mouse and that this gene was
inactivated in the ES cell lines and the subsequent mutant mice.
2GPI-null mice were born at significantly lower Mendelian ratios
with only 8.9% of the offspring of heterozygous crosses being
homozygous (
/
) for the disrupted allele. This finding suggested
that
2GPI might play an important role in embryonic development or
implantation; therefore, further experiments were undertaken to
investigate reproductive function in those
2GPI-null mice that
proceeded to develop on a C57BL/6 background. Reproductive outcomes
were indistinguishable from control mice in both males and females
carrying the
2GPI null mutation, in terms of the proportion of
animals that bred successfully and the number of viable pups born at
term and surviving to weaning. Furthermore, there was no evidence of
altered blood platelet counts in either virgin or pregnant mice.
Together, these data show that
2GPI is not essential for normal
reproductive function. However, the data from heterozygote pregnancies
indicate that
2GPI deficiency might pose a selective disadvantage to
survival of a conceptus gestating in a
2GPI-replete maternal
environment. Since litter sizes were comparable in heterozygote and
wild type pregnancies, any loss of
2GPI
/
embryos might occur
early in pregnancy at, or prior to, the time of implantation. However,
because ganciclovir can induce nonspecific point mutations in genes, we
cannot rule out the possibility that the initial fetal viability
problem was caused by ganciclovir-induced alteration of another gene.
In addition if a more severe fetal viability phenotype than the initial
2-GPI null mice was obtained after backcrossing with either the
129/Sv or BALB/c mouse strains, this would indicate an important role for
2-GPI in fetal development.
One of the most striking observations of the
2GPI
/
mice is that
they have a significantly diminished rate of thrombin generation
compared with
2GPI +/+ and
2GPI +/
mice. However, no
significant differences in clotting time were observed in plasma from
these three genotypes when measured by dRVVT, dKCT, aPTT, and protein C
pathway assays. Our data demonstrate that the reduction or absence of
2GPI diminishes thrombin generation in a dose-dependent manner. A similar prolongation of thrombin generation was observed following the addition of anti-
2GPI antibodies to normal human plasma, regardless of whether the antibody was of mouse monoclonal or
APS patient origin (14).
The conventional coagulation assays used in this paper (aPTT, dRVVT,
dKCT) measure the time to generate a thrombin-dependent clot. This usually takes less than 1 min in plasma using the above tests. However, the time to form a clot is a poor indicator of thrombin
generation because it occurs before peak thrombin production (15). The
use of a colorimetric thrombin substrate in defibrinated plasma gives
more reliable information about thrombin generation over time. This may
be of importance clinically as APS patients have evidence of a
continuously elevated level of thrombin (16) and an ongoing tendency to thrombosis.
Schousboe (4) demonstrated that
2GPI inhibits the contact activation
of the intrinsic blood coagulation pathway due to its ability to
interact with negatively charged surfaces, which in turn are necessary
for the activation of factor XII. On the other hand, Mori and
co-workers (17) showed that
2GPI can inhibit the anticoagulant
activity of activated protein C. Thus, currently it is not clear
whether
2GPI in vivo has anticoagulant or procoagulant properties. It has recently been demonstrated the
2GPI-dependent activation of human umbilical vein
endothelial cells by IgG autoantibodies from patients with APS, as
measured by increased expression of adhesion molecules (18). Thus, it
is possible that autoantibodies directed against
2GPI induce
endothelial cell activation, which in the presence of some other insult
may trigger a thrombotic event (18).
2GPI has also been shown to
bind preferentially oxidized low density lipoproteins, thereby
providing a link between anti-
2GPI antibodies and atherogenesis
(19).
The physiological and clinical significance of the in vitro
inhibition of thrombin generation is unclear at this time. The generation of thrombin is important for both thrombus formation and for
the initiation of the protein C anticoagulation pathway. It has been
reported that
2GPI deficiency in humans is not common in
patients with thrombosis (20) and does not result in a
significant perturbation of lipoprotein metabolism (21). However,
population studies of the effect of
2GPI deficiency have not been performed.
In summary, analysis of our
2GPI-null mice reveal a possible role of
this plasma protein in early embryonic fetal development in some mouse
strains. However, any function of
2GPI is likely to be limited to
providing a selective advantage at implantation since normal
reproductive function was observed in crosses between null mutant male
and female mice. Because mutating the
2GPI gene results in
significantly less thrombin generation in vitro,
2GPI may
have a prothrombotic role in vivo. However, since this
decrease in thrombin generation has only been demonstrated in
vitro, it still remains to be determined if this also holds
in vivo. The
2GPI-null mice generated in this study will
provide a valuable in vivo model system for exploring the
role of
2GPI in disease pathogenesis.