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Impaired Thrombin Generation in β2-Glycoprotein I Null Mice

2001; Elsevier BV; Volume: 276; Issue: 17 Linguagem: Inglês

10.1074/jbc.m010990200

1083-351X

Yonghua Sheng, Stephen Reddel, Herbert Herzog, Ying Xia Wang, Tim Brighton, Malcolm P. France, Sarah A. Robertson, Steven A. Krilis,

Cell Adhesion Molecules Research

Autoimmune antibodies to β2-glycoprotein I (β2GPI) have been proposed to be clinically relevant because of their strong association with thrombosis, miscarriage, and thrombocytopenia. By using a homologous recombination approach, β2GPI-null mice were generated to begin to understand the physiologic and pathologic role of this prominent plasma protein in mammals. When β2GPI 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 β2GPI plays a beneficial role in implantation and/or fetal development in at least some mouse strains. Although those β2GPI-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, β2GPI also appears to play an important role in thrombin-mediated coagulation. Autoimmune antibodies to β2-glycoprotein I (β2GPI) have been proposed to be clinically relevant because of their strong association with thrombosis, miscarriage, and thrombocytopenia. By using a homologous recombination approach, β2GPI-null mice were generated to begin to understand the physiologic and pathologic role of this prominent plasma protein in mammals. When β2GPI 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 β2GPI plays a beneficial role in implantation and/or fetal development in at least some mouse strains. Although those β2GPI-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, β2GPI also appears to play an important role in thrombin-mediated coagulation. β2-Glycoprotein I (β2GPI), 1The abbreviations used are: β2GPIβ2-glycoprotein IAPSantiphospholipid syndromeESembryonic stemHSV-tkherpes simplex virus thymidine kinase genedKCTdilute kaolin clotting timedRVVTdilute Russell Viper venom timeaPTTactivated partial thromboplastin timekbkilobase pairTBSTris-buffered salinePAGEpolyacrylamide gel electrophoresis also known as apolipoprotein H, is a major protein constituent of plasma where its concentration approaches 0.2 mg/ml (1Polz E. Kostner G.M. FEBS Lett. 1979; 102: 183-186Crossref PubMed Scopus (191) Google Scholar). Whereas the physiologic function of β2GPI has not been deduced, β2GPI interacts specifically with lipoprotein(a) (2Köchl S. Fresser F. Lobentanz E. Baier G. Utermann G. Blood. 1997; 90: 1482-1489Crossref PubMed Google Scholar) and the endothelial cell protein annexin II (3Ma K. Simantov R. Zhang J.-C. Silverstein R. Hajjar A. McRae K.R. J. Biol. Chem. 2000; 275: 15541-15548Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). At least in vitro, β2GPI acts as an inhibitor of the intrinsic blood coagulation pathway (4Schousboe I. Blood. 1985; 66: 1086-1091Crossref PubMed Google Scholar), ADP-mediated platelet aggregation, and the prothrombinase activity of activated platelets (5Nimpf J. Wurm H. Kostner G.M. Atherosclerosis. 1987; 63: 109-114Abstract Full Text PDF PubMed Scopus (193) Google Scholar). β2GPI 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 β2GPI increased dramatically shortly after it was discovered that this plasma protein is the most common antigen in patients with the "anti-phospholipid syndrome" (APS). (6Kandiah D.A. Sali A. Sheng Y. Victoria E.J. Marquis D.M. Coutts S.M. Krilis S.A. Adv. Immunol. 1998; 70: 507-563Crossref PubMed Google Scholar). 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 β2GPI component of the macromolecular complex. The presence of anti-β2GPI antibodies in these patients correlates well with thrombosis, miscarriages, and thrombocytopenia (6Kandiah D.A. Sali A. Sheng Y. Victoria E.J. Marquis D.M. Coutts S.M. Krilis S.A. Adv. Immunol. 1998; 70: 507-563Crossref PubMed Google Scholar). Finally, β2GPI has been implicated in apoptosis (7Balasubramanian K. Schroit A.J. J. Biol. Chem. 1998; 273: 29272-29277Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). β2-glycoprotein I antiphospholipid syndrome embryonic stem herpes simplex virus thymidine kinase gene dilute kaolin clotting time dilute Russell Viper venom time activated partial thromboplastin time kilobase pair Tris-buffered saline polyacrylamide gel electrophoresis β2GPI 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. β2GPI 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 β2GPI differs in that it contains 80 amino acids and 3 disulfide bonds. The amino acid sequences of mouse and human β2GPI are 76% identical, and the β2GPI transcript in both species is ∼1.2 kb in size. The nucleotide sequence of the entire mouse β2GPI gene has been deduced (8Sheng Y. Herzog H. Krilis S.A. Genomics. 1997; 41: 128-130Crossref PubMed Scopus (8) Google Scholar). 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 β2GPI. A mouse β2GPI genomic clone spanning ∼18 kb that was previously isolated and sequenced from a 129/Sv genomic P1 library (Genome Systems, Inc., St. Louis, MO) (8Sheng Y. Herzog H. Krilis S.A. Genomics. 1997; 41: 128-130Crossref PubMed Scopus (8) Google Scholar) was used to disrupt the β2GPI 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-kbHindIII-AccI fragment, with exon 1 and part of exon 2. The 3′ portion of the construct contained a 3.5-kbXhoI-XhoI fragment, with exon 4. The HSVtk, PGK-Neo, and vector backbone were from the PGKNeo cassette (9Tybulewicz V.L.J. Crawford C.E. Jackson P.K. Bronson R.T. Mulligan R.C. Cell. 1991; 65: 1153-1163Abstract Full Text PDF PubMed Scopus (1156) Google Scholar). 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 (10Köntgen F. Stewart C.L. Methods Enzymol. 1993; 225: 878-890Crossref PubMed Scopus (48) Google Scholar). 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 β2GPI 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 (10Köntgen F. Stewart C.L. Methods Enzymol. 1993; 225: 878-890Crossref PubMed Scopus (48) Google Scholar), 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 β2GPI cDNA probe corresponding to exons 1–5. 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-β2GPI 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-β2GPI 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 β2GPI 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-β2GPI antibody. Purified mouse β2GPI at 5, 10, and 20 ng per lane was used as a standard. 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. Blood was recovered by cardiac puncture into EDTA-coated Capiject tubes (Terumo) from female virgin or day-18 pregnant β2GPI +/− and β2GPI −/− 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). 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, β2GPI +/+, β2GPI +/− or β2GPI −/−) were housed 2:1 with adult stud males (β2GPI +/+ or β2GPI −/−, 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. 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. (11Exner T. Rickard K.A. Kronenberg H. Br. J. Haematol. 1978; 40: 143-151Crossref PubMed Scopus (350) Google Scholar). The dRVVT was performed according to Thiagarajan et al. (12Thiagarajan P. Shapiro S.S. De Marco L. J. Clin. Invest. 1980; 66: 397-405Crossref PubMed Scopus (319) Google Scholar) 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. 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 (13Rosen E. Chan J.Y. Idusogie E. Clotman F. Vlasuk G. Luther T. Jalbert L.R. Albrecht S. Zhong L. Lissens A. Schoonjans L. Moons L. Collen D. Castellino F.J. Carmeliet P. Nature. 1997; 390: 290-294Crossref PubMed Scopus (183) Google Scholar). A standard curve was constructed by log-log plot of the clotting time (either aPTT or PT) of various dilutions of pooled β2GPI+/+ plasma (assumed to represent 100% activity). The specific factor activity of β2GPI +/− and β2GPI −/− 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. 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 × gfor 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. An 18-kb β2GPI genomic clone obtained from a mouse 129/Sv library was used to construct the targeting vector (Fig. 1 A) for homologous recombination. A β2GPI-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). One of the successfully targeted ES clones heterozygous for disruption of the β2GPI 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 β2GPI +/+, β2GPI +/−, and β2GPI −/− 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.1 B). Restriction enzyme digestion of the wild type β2GPI 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.1 B). Northern blot analysis was performed to confirm the loss of β2GPI gene expression in the surviving β2GPI −/− mice. As shown in Fig.2 A, no transcript was observed in RNA samples isolated from liver tissue of β2GPI −/− mice when hybridized with a highly specific radiolabeled cDNA probe encoding exons 1–5 (Fig. 2 A). In contrast, the expected 1.2-kb full-length transcript was present in the liver of β2GPI +/+ and +/− mice when analyzed in parallel. A β−actin probe was hybridized to the same blot after removal of the β2GPI probe to assess the amount of RNA loaded in each lane (lower panel in Fig.2 A). Based on the structure of the gene targeting construct, we expected that mice homozygous for the mutation would be unable to produce functional β2GPI protein. Plasma from 6-week-old mice was tested by Western blot using a rabbit polyclonal anti-β2GPI antibody. The antibody reacted with the expected 50-kDa protein band present in wild type and β2GPI +/− mice (Fig. 2 B). However, no such immunoreactive band was detected in the plasma from β2GPI −/− mice. Furthermore, analysis of serial dilutions of plasma from +/+ and +/− mice (Fig. 2 C) revealed that the heterozygotes contained approximately half the concentration of β2GPI in their plasma relative to wild type mice. Thus, an abnormality in β2GPI expression was even seen in +/− mice. Six pairs of male and female β2GPI +/− 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, β2GPI −/− and β2GPI +/+ male mice were mated with C57BL/6 female mice. Each of six males from each genotype mated successfully with females and sired pregnancies. Female β2GPI −/− and β2GPI +/+ 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 β2GPI genotype. The duration of pregnancy, the number of pups born, and their viability and weight at 24 h and at weaning were comparable in β2GPI −/− and β2GPI +/+ pregnancies (TableI), indicating a normal reproductive potential for β2GPI −/− mice.Table IEffect of genetic deficiency in β2GP1 on fertility and fecundity in miceMale β2GP1 +/+Male β2GP1 −/−Female β2GP1 +/+ or +/−Female β2GP1 −/−No. of plugged mice pregnant at term (%)15/17 (88%)16/20 (80%)Litter size at birth8.2 + 1.17.9 + 1.6Weight of pups at birth (g)1.51 + 0.131.53 + 0.12Viability at 24 h (%)122/123 (99%)112/116 (97%)Viability at 3 weeks (%)120/123 (98%)112/116 (97%)Data are mean + S.D. Open table in a new tab Data are mean + S.D. To determine the effect of β2GPI deficiency on platelet counts in pregnancy, blood was recovered by cardiac puncture from adult virgin β2GPI −/− and β2GPI +/+ mice on day 18 of pregnancy and from β2GPI −/− and β2GPI +/+ female mice mated naturally with adult stud males of the same β2GPI status. Whereas pregnancy was associated with an increase in mean blood platelet count of 35%, there was no significant effect of β2GPI deficiency in either the virgin or pregnant state (Table II). Furthermore, there was no effect of β2GPI deficiency on the number of implantation sites at day 18 (mean ± S.D. = 9.3 ± 2.9 in β2GPI +/+ mice and 8.2 ± 2.8 in β2GPI −/− mice, n = 6 per group) or on the proportion of resorption sites (5/56 in β2GPI +/+ mice and 1/49 in β2GPI −/− mice, n = 6 per group).Table IIThe effect of genetic deficiency in β2GPI on platelet numbers in blood of virgin and day 18 pregnant miceβ2GP1 +/+ or +/−β2GP1 −/−Virgin697 + 109 (10)664 + 185 (7)Day 18 pregnant 2-ap = 0.001versus virgin, data compared by Student's unpairedt test.924 + 178 (6)928 + 226 (6)Data are mean + S.D. × 10−3 platelets/μl. The number of mice per group are given in parentheses. β2GP1 +/+ or +/− females were mated with β2GP1 +/+ males, and β2GP1 −/− females were mated with β2GP1 −/− males.2-a p = 0.001versus virgin, data compared by Student's unpairedt test. Open table in a new tab Data are mean + S.D. × 10−3 platelets/μl. The number of mice per group are given in parentheses. β2GP1 +/+ or +/− females were mated with β2GP1 +/+ males, and β2GP1 −/− females were mated with β2GP1 −/− males. The role of β2GPI in coagulation profiles was examined using a number of hematologic parameters. Analysis of pooled plasma from β2GPI+/+, β2GPI+/−, and β2GPI−/− 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).Table IIICoagulation profile of plasma from β2GPI +/+, +/−, and −/− mice+/++/−−/−dRVVT(s)17.8 ± 218.5 ± 1.619.3 ± 1.4dKCT(s)53.7 ± 6.259.5 ± 8.956.5 ± 2.5APTT(s)24 ± 0.523.1 ± 1.823.4 ± 2.8Protein C pathway(s)22.4 ± 1.423.7 ± 0.823.6 ± 0.2Data 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. Open table in a new tab 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.3 A) or the individual plasma samples (Fig. 3 B) from β2GPI −/− mice had significantly less thrombin generation compared with that obtained from β2GPI +/+ or β2GPI +/− mice. Fig. 3 A demonstrates that the average time point required to reach half-maximal optical density (OD405 nm = 0.55) was 1050 s for β2GPI +/+ mice and 2100 s for the β2GPI +/− mice. In contrast, plasma from β2GPI −/− 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 β2GPI +/+ and β2GPI −/− mouse plasma produced a thrombin generation curve similar to that of the heterozygote plasma (data not shown). The mean value (OD405 nm) of thrombin generation of 10 individual plasma samples from each population of animals (Fig.3 B) was measured at 1000 s. The mean value for β2GPI −/− was 0.175, which represents 69% less than the result for β2GPI +/+ mice and 40% less than that for β2GPI +/− mice. The difference in thrombin generation between β2GPI +/+ and β2GPI −/− mice was highly significant by one-way analysis of variance (p = 0.0051). We generated β2GPI 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 β2GPI gene in the mouse and that this gene was inactivated in the ES cell lines and the subsequent mutant mice. β2GPI-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 β2GPI might play an important role in embryonic development or implantation; therefore, further experiments were undertaken to investigate reproductive function in those β2GPI-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 β2GPI 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 β2GPI is not essential for normal reproductive function. However, the data from heterozygote pregnancies indicate that β2GPI deficiency might pose a selective disadvantage to survival of a conceptus gestating in a β2GPI-replete maternal environment. Since litter sizes were comparable in heterozygote and wild type pregnancies, any loss of β2GPI −/− 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 β2-GPI null mice was obtained after backcrossing with either the 129/Sv or BALB/c mouse strains, this would indicate an important role for β2-GPI in fetal development. One of the most striking observations of the β2GPI −/− mice is that they have a significantly diminished rate of thrombin generation compared with β2GPI +/+ and β2GPI +/− 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 β2GPI diminishes thrombin generation in a dose-dependent manner. A similar prolongation of thrombin generation was observed following the addition of anti-β2GPI antibodies to normal human plasma, regardless of whether the antibody was of mouse monoclonal or APS patient origin (14Hanly, J. G., Kandiah, D., Kouts, S., Sheng, Y., Koike, T., Ichikawa, K., Monestier, M., Krilis, S. A., Lupus, 7, 1998, 219, (Abstr. B066).Google Scholar). 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 (15Rand M.D. Lock J.B. van't Veer C. Gaffney D.P. Mann K.G. Blood. 1996; 88: 3432-3445Crossref PubMed Google Scholar). 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 (16Ginsberg J.S. Demers C. Brill-Edwards P. Johnston M. Bona R. Burrows R.F. Weitz J. Denburg J.A. Blood. 1993; 81: 2958-2963Crossref PubMed Google Scholar) and an ongoing tendency to thrombosis. Schousboe (4Schousboe I. Blood. 1985; 66: 1086-1091Crossref PubMed Google Scholar) demonstrated that β2GPI 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 (17Mori T. Takeya H. Nishioka J. Gabazza E.C. Suzuki K. Thromb. Haemostasis. 1996; 75: 49-55Crossref PubMed Scopus (89) Google Scholar) showed that β2GPI can inhibit the anticoagulant activity of activated protein C. Thus, currently it is not clear whether β2GPI in vivo has anticoagulant or procoagulant properties. It has recently been demonstrated the β2GPI-dependent activation of human umbilical vein endothelial cells by IgG autoantibodies from patients with APS, as measured by increased expression of adhesion molecules (18Del Papa N. Sheng Y. Raschi E. Kandiah D.A. Tincani A. Kamashta M.A. Hughes G.R.V. Koike T. Balestrieri G. Krilis S.A. Meroni P.L. J. Immunol. 1998; 160: 5572-5578PubMed Google Scholar). Thus, it is possible that autoantibodies directed against β2GPI induce endothelial cell activation, which in the presence of some other insult may trigger a thrombotic event (18Del Papa N. Sheng Y. Raschi E. Kandiah D.A. Tincani A. Kamashta M.A. Hughes G.R.V. Koike T. Balestrieri G. Krilis S.A. Meroni P.L. J. Immunol. 1998; 160: 5572-5578PubMed Google Scholar). β2GPI has also been shown to bind preferentially oxidized low density lipoproteins, thereby providing a link between anti-β2GPI antibodies and atherogenesis (19Matsuura E. Kobayashi K. Yasuda T. Koike T. Lupus. 1998; 7: 135-139Crossref PubMed Scopus (33) Google Scholar). The physiological and clinical significance of the in vitroinhibition 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 β2GPI deficiency in humans is not common in patients with thrombosis (20Bancsi L.F.J.M.M. van der Linden I.K. Bertina R.M. Thromb. Haemostasis. 1992; 67: 649-653Crossref PubMed Scopus (110) Google Scholar) and does not result in a significant perturbation of lipoprotein metabolism (21Hoeg J.M. Segal P. Gregg R.E. Chang Y.S. Lindgren F.T. Adamson G.L. Frank M. Brickman C. Brewer Jr., H.B. Atherosclerosis. 1985; 55: 25-34Abstract Full Text PDF PubMed Scopus (25) Google Scholar). However, population studies of the effect of β2GPI deficiency have not been performed. In summary, analysis of our β2GPI-null mice reveal a possible role of this plasma protein in early embryonic fetal development in some mouse strains. However, any function of β2GPI 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 β2GPI gene results in significantly less thrombin generation in vitro, β2GPI 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 holdsin vivo. The β2GPI-null mice generated in this study will provide a valuable in vivo model system for exploring the role of β2GPI in disease pathogenesis. We thank Rosalie Gemmell for assistance with coagulation assays, Dr. Frank Köntgen for assistance in deriving the β2GPI knockout mice, and Dr. Jan Guerin for a critical review of this manuscript.