Impaired Thrombin Generation in beta 2-Glycoprotein I Null Mice*

Yonghua ShengDagger , Stephen W. ReddelDagger , Herbert Herzog§, Ying Xia WangDagger , Tim Brighton, Malcolm P. France||, Sarah A. Robertson**, and Steven A. KrilisDagger DaggerDagger

From the Dagger  Department of Medicine and the Department of Immunology, Allergy, and Infectious Disease, and the  Department of Haematology, University of New South Wales, The St. George Hospital, Sydney, New South Wales 2217, § Garvan Institute of Medical Research, St. Vincent's Hospital, Darlinghurst, New South Wales 2010, the || Department of Veterinary Anatomy and Pathology, the University of Sydney, Sydney, New South Wales 2006, and the ** Department of Obstetrics and Gynaecology and Reproductive Medicine Unit, Adelaide University, Adelaide, South Australia 5005, Australia

Received for publication, December 6, 2000, and in revised form, December 27, 2000




    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Autoimmune antibodies to beta 2-glycoprotein I (beta 2GPI) have been proposed to be clinically relevant because of their strong association with thrombosis, miscarriage, and thrombocytopenia. By using a homologous recombination approach, beta 2GPI-null mice were generated to begin to understand the physiologic and pathologic role of this prominent plasma protein in mammals. When beta 2GPI heterozygotes on a 129/Sv/C57BL/6 mixed genetic background were intercrossed, only 8.9% of the resulting 336 offspring possessed both disrupted alleles. These data suggest that beta 2GPI plays a beneficial role in implantation and/or fetal development in at least some mouse strains. Although those beta 2GPI-null mice that were born appeared to be relatively normal anatomically and histologically, subsequent analysis revealed that they possessed an impaired in vitro ability to generate thrombin relative to wild type mice. Thus, beta 2GPI also appears to play an important role in thrombin-mediated coagulation.




    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

beta 2-Glycoprotein I (beta 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 beta 2GPI has not been deduced, beta 2GPI interacts specifically with lipoprotein(a) (2) and the endothelial cell protein annexin II (3). At least in vitro, beta 2GPI acts as an inhibitor of the intrinsic blood coagulation pathway (4), ADP-mediated platelet aggregation, and the prothrombinase activity of activated platelets (5). beta 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 beta 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 beta 2GPI component of the macromolecular complex. The presence of anti-beta 2GPI antibodies in these patients correlates well with thrombosis, miscarriages, and thrombocytopenia (6). Finally, beta 2GPI has been implicated in apoptosis (7).

beta 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. beta 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 beta 2GPI differs in that it contains 80 amino acids and 3 disulfide bonds. The amino acid sequences of mouse and human beta 2GPI are 76% identical, and the beta 2GPI transcript in both species is ~1.2 kb in size. The nucleotide sequence of the entire mouse beta 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 beta 2GPI.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Generation of beta 2GPI Null Mice-- A mouse beta 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 beta 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 beta 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 beta 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-beta 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-beta 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 beta 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-beta 2GPI antibody. Purified mouse beta 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 beta 2GPI +/- and beta 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, beta 2GPI +/+, beta 2GPI +/- or beta 2GPI -/-) were housed 2:1 with adult stud males (beta 2GPI +/+ or beta 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 beta 2GPI+/+ plasma (assumed to represent 100% activity). The specific factor activity of beta 2GPI +/- and beta 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Generation of beta 2GPI-Null Mice-- An 18-kb beta 2GPI genomic clone obtained from a mouse 129/Sv library was used to construct the targeting vector (Fig. 1A) for homologous recombination. A beta 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, beta 2GPI gene and targeting construct. Line A represents the map of the endogenous murine beta 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 beta 2GPI locus. The solid blocks represent portions of the beta 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.

One of the successfully targeted ES clones heterozygous for disruption of the beta 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 beta 2GPI +/+, beta 2GPI +/-, and beta 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 beta 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 beta 2GPI Mutation-- Northern blot analysis was performed to confirm the loss of beta 2GPI gene expression in the surviving beta 2GPI -/- mice. As shown in Fig. 2A, no transcript was observed in RNA samples isolated from liver tissue of beta 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 beta 2GPI +/+ and +/- mice when analyzed in parallel. A beta -actin probe was hybridized to the same blot after removal of the beta 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 beta 2GPI +/+, +/-, and -/- littermates. The same blot was probed with a beta -actin probe demonstrating equal amount of RNA applied. B, Western blot for beta 2GPI. Western blotting analysis of control mouse beta 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-beta 2GPI antibody. C, quantitation of beta 2GPI in +/+ and +/- mice by Western blot analysis using rabbit anti-beta 2GPI antibody. Purified murine beta 2GPI was used as a standard. Lane 1, 5 ng of beta 2GPI; lane 2, 10 ng of beta 2GPI; lane 3, 20 ng of beta 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.

Based on the structure of the gene targeting construct, we expected that mice homozygous for the mutation would be unable to produce functional beta 2GPI protein. Plasma from 6-week-old mice was tested by Western blot using a rabbit polyclonal anti-beta 2GPI antibody. The antibody reacted with the expected 50-kDa protein band present in wild type and beta 2GPI +/- mice (Fig. 2B). However, no such immunoreactive band was detected in the plasma from beta 2GPI -/- mice. Furthermore, analysis of serial dilutions of plasma from +/+ and +/- mice (Fig. 2C) revealed that the heterozygotes contained approximately half the concentration of beta 2GPI in their plasma relative to wild type mice. Thus, an abnormality in beta 2GPI expression was even seen in +/- mice.

Phenotypic Characterization and Reproductive Performance of beta 2GPI-Null Mice-- Six pairs of male and female beta 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, beta 2GPI -/- and beta 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 beta 2GPI -/- and beta 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 beta 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 beta 2GPI -/- and beta 2GPI +/+ pregnancies (Table I), indicating a normal reproductive potential for beta 2GPI -/- mice.


                              
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Table I
Effect of genetic deficiency in beta 2GP1 on fertility and fecundity in mice
Data are mean + S.D.

To determine the effect of beta 2GPI deficiency on platelet counts in pregnancy, blood was recovered by cardiac puncture from adult virgin beta 2GPI -/- and beta 2GPI +/+ mice on day 18 of pregnancy and from beta 2GPI -/- and beta 2GPI +/+ female mice mated naturally with adult stud males of the same beta 2GPI status. Whereas pregnancy was associated with an increase in mean blood platelet count of 35%, there was no significant effect of beta 2GPI deficiency in either the virgin or pregnant state (Table II). Furthermore, there was no effect of beta 2GPI deficiency on the number of implantation sites at day 18 (mean ± S.D. = 9.3 ± 2.9 in beta 2GPI +/+ mice and 8.2 ± 2.8 in beta 2GPI -/- mice, n = 6 per group) or on the proportion of resorption sites (5/56 in beta 2GPI +/+ mice and 1/49 in beta 2GPI -/- mice, n = 6 per group).


                              
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Table II
The effect of genetic deficiency in beta 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. beta 2GP1 +/+ or +/- females were mated with beta 2GP1 +/+ males, and beta 2GP1 -/- females were mated with beta 2GP1 -/- males.

Characterization of the Coagulation Profiles of beta 2GPI +/+, beta 2GPI +/-, and beta 2GPI -/- Mice-- The role of beta 2GPI in coagulation profiles was examined using a number of hematologic parameters. Analysis of pooled plasma from beta 2GPI+/+, beta 2GPI+/-, and beta 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 beta 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.

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 beta 2GPI -/- mice had significantly less thrombin generation compared with that obtained from beta 2GPI +/+ or beta 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 beta 2GPI +/+ mice and 2100 s for the beta 2GPI +/- mice. In contrast, plasma from beta 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 beta 2GPI +/+ and beta 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 beta 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 beta 2GPI +/+, +/-, and -/- mice. Plotted values represent the mean ± S.D. of determinations in 10 individual animals measured at 1000 s.

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 beta 2GPI -/- was 0.175, which represents 69% less than the result for beta 2GPI +/+ mice and 40% less than that for beta 2GPI +/- mice. The difference in thrombin generation between beta 2GPI +/+ and beta 2GPI -/- mice was highly significant by one-way analysis of variance (p = 0.0051).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We generated beta 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 beta 2GPI gene in the mouse and that this gene was inactivated in the ES cell lines and the subsequent mutant mice.

beta 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 beta 2GPI might play an important role in embryonic development or implantation; therefore, further experiments were undertaken to investigate reproductive function in those beta 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 beta 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 beta 2GPI is not essential for normal reproductive function. However, the data from heterozygote pregnancies indicate that beta 2GPI deficiency might pose a selective disadvantage to survival of a conceptus gestating in a beta 2GPI-replete maternal environment. Since litter sizes were comparable in heterozygote and wild type pregnancies, any loss of beta 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 beta 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 beta 2-GPI in fetal development.

One of the most striking observations of the beta 2GPI -/- mice is that they have a significantly diminished rate of thrombin generation compared with beta 2GPI +/+ and beta 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 beta 2GPI diminishes thrombin generation in a dose-dependent manner. A similar prolongation of thrombin generation was observed following the addition of anti-beta 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 beta 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 beta 2GPI can inhibit the anticoagulant activity of activated protein C. Thus, currently it is not clear whether beta 2GPI in vivo has anticoagulant or procoagulant properties. It has recently been demonstrated the beta 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 beta 2GPI induce endothelial cell activation, which in the presence of some other insult may trigger a thrombotic event (18). beta 2GPI has also been shown to bind preferentially oxidized low density lipoproteins, thereby providing a link between anti-beta 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 beta 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 beta 2GPI deficiency have not been performed.

In summary, analysis of our beta 2GPI-null mice reveal a possible role of this plasma protein in early embryonic fetal development in some mouse strains. However, any function of beta 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 beta 2GPI gene results in significantly less thrombin generation in vitro, beta 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 beta 2GPI-null mice generated in this study will provide a valuable in vivo model system for exploring the role of beta 2GPI in disease pathogenesis.


    ACKNOWLEDGEMENTS

We thank Rosalie Gemmell for assistance with coagulation assays, Dr. Frank Köntgen for assistance in deriving the beta 2GPI knockout mice, and Dr. Jan Guerin for a critical review of this manuscript.


    FOOTNOTES

* This work was supported by the National Health and Medical Research Council (Australia) and the Clive and Vera Ramaciotti Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Dagger To whom correspondence should be addressed: Dept. of Immunology, Allergy, and Infectious Disease, St. George Hospital, South St., Kogarah, 2217 New South Wales, Australia. Tel.: 61-2-93502955; Fax: 61-2-93503981; E-mail: s.krilis@unsw.edu.au.

Published, JBC Papers in Press, January 5, 2001, DOI 10.1074/jbc.M010990200


    ABBREVIATIONS

The abbreviations used are: beta 2GPI, beta 2-glycoprotein I; APS, antiphospholipid syndrome; ES, embryonic stem; HSV-tk, herpes simplex virus thymidine kinase gene; dKCT, dilute kaolin clotting time; dRVVT, dilute Russell Viper venom time; aPTT, activated partial thromboplastin time; kb, kilobase pair; TBS, Tris-buffered saline; PAGE, polyacrylamide gel electrophoresis.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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