Dimers of beta 2-Glycoprotein I Mimic the in Vitro Effects of beta 2-Glycoprotein I-Anti-beta 2-glycoprotein I Antibody Complexes*

Bianca C. H. Lutters**Dagger §, Joost C. M. Meijers||, Ronald H. W. M. Derksen§§, Jef ArnoutDagger Dagger , and Philip G. de Groot**Dagger

From the Departments of ** Haematology and §§ Rheumatology and Clinical Immunology, University Medical Center, 3508 GA Utrecht and Dagger  Institute of Biomembranes, Utrecht University, 3508 TB Utrecht, The Netherlands,  Department of Vascular Medicine, Academic Medical Center, 1100 DD Amsterdam, The Netherlands, and the Dagger Dagger  Center for Molecular and Vascular Biology, University of Leuven, B-3000 Leuven, Belgium

Received for publication, September 8, 2000, and in revised form, October 16, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Anti-beta 2-glycoprotein I antibodies are thought to cause lupus anticoagulant activity by forming bivalent complexes with beta 2-glycoprotein I (beta 2GPI). To test this hypothesis, chimeric fusion proteins were constructed of the dimerization domain (apple 4) of factor XI and beta 2GPI. Both a covalent (apple 4-beta 2GPI) and a noncovalent (apple 4-C321S-beta 2GPI) chimer were constructed. As controls, apple 2-beta 2GPI and apple 4-C321S-beta 2GPI-W316S, in which beta 2GPI-W316S is not able to bind to phospholipids, were made. In a phospholipid binding assay, apple 4-beta 2GPI and apple 4-C321S-beta 2GPI were able to bind to phospholipids with an affinity 35 times higher than that of plasma-derived beta 2GPI and apple 2-beta 2GPI. Apple 4-C321S-beta 2GPI-W316S did not bind at all. Only apple 4-beta 2GPI and apple 4-C321S-beta 2GPI were able to bind to adhered platelets as shown by immunofluorescence. Using the prothrombin time, which was the most responsive coagulation assay, the clotting time was approximately doubled when 200 µg/ml apple 4-beta 2GPI or apple 4-C321S-beta 2GPI was added. Addition of 200 µg/ml plasma-derived beta 2GPI, apple 2-beta 2GPI, or apple 4-C321S-beta 2GPI-W316S did not affect clotting time. Clotting time could be corrected with the addition of extra phospholipids, which is indicative for lupus anticoagulant activity. An additional increase in clotting times for apple 4-beta 2GPI or apple 4-C321S-beta 2GPI was achieved by the addition of monoclonal antibodies against beta 2GPI. In conclusion, dimerization of beta 2GPI explains the in vitro observed effects of beta 2GPI-anti-beta 2GPI antibody complexes.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

beta 2-Glycoprotein I (beta 2GPI),1 also known as apolipoprotein H, is a single chain protein present in plasma at a concentration of ~200 µg/ml. It consists of 326 amino acids and has a molecular mass of 42 kDa. beta 2GPI is a member of the short consensus repeat or complement control protein superfamily. The first four domains of beta 2GPI consist of ~60 amino acids and contain two conserved disulfide bonds. The fifth domain, comprising 84 amino acids, is different with an extra C-terminal tail, resulting in a C-terminal loop and an extra disulfide bond (1-5). This domain is important for phospholipid binding, as has been shown by several studies. Experiments, using mutated forms of beta 2GPI and peptides spanning the fifth domain of beta 2GPI, indicated that residues Cys281 to Cys288 are important for phospholipid binding (6-8). Furthermore, analysis of a naturally occurring polymorphism, in which Trp316 is mutated to a serine (9) showed that Trp316 is essential for phospholipid binding. Mehdi et al. (10) showed that a hydrophobic sequence at position 313-316 in the fifth domain is crucial for cardiolipin binding. The recent elucidation of the crystal structure of beta 2GPI (11) showed that a patch of 14 positively charged amino acids in the fifth domain is important for electrostatic interaction with negatively charged phospholipids. It was also shown that the amino acids at positions 311-317 form a membrane-insertion loop, which yields specificity for lipid bilayers. At present, the in vivo function of beta 2GPI is unknown. In vitro, it has been shown to influence coagulation (both procoagulant and anticoagulant) (12-19), platelet function (20-23), lipoprotein metabolism (24-27), and apoptosis (24).

The persistent presence of antiphospholipid antibodies in plasma is a risk factor for thrombosis, pregnancy loss, and thrombocytopenia. These clinical and serological observations together are termed the antiphospholipid syndrome (28-33). Initially it was thought that the antiphospholipid antibodies were directed against phospholipids directly, but now it is generally accepted that these antibodies are directed against phospholipid-bound proteins, such as beta 2GPI (34-36) and prothrombin (37). Antiphospholipid antibodies are a very heterogeneous group of antibodies, which can be subdivided into lupus anticoagulants (LAC) and anticardiolipin antibodies, of which the latter can bind to beta 2GPI immobilized on cardiolipin surfaces (38, 39). Antibodies with LAC activity are detected by their ability to prolong phospholipid-dependent coagulation assays (40, 41). The prolongation cannot be corrected by the addition of normal pool plasma, but can be corrected by the addition of extra phospholipids. Some of these lupus anticoagulants depend on prothrombin to prolong clotting time in vitro (42), but most of them depend on beta 2GPI (43, 44).

Two possible explanations for the mechanism of action of beta 2GPI-anti-beta 2GPI antibody complexes are suggested. First, beta 2GPI undergoes a conformational change when it interacts with anti-beta 2GPI antibodies (45-50). Second, beta 2GPI is able to form bivalent complexes when it interacts with an anti-beta 2GPI antibody (51-53). The final result is a strongly increased affinity of beta 2GPI for negatively charged phospholipids. Due to the increased affinity, beta 2GPI is then able to compete with clotting factors for the phospholipid surface, resulting in the in vitro prolongation of coagulation (54).

Here, a covalent and a noncovalent chimeric dimer of beta 2GPI were constructed to determine the role of bivalent beta 2GPI complexes without the intervention of antibodies. It is shown that chimeric dimers of the dimerization domain of coagulation factor XI (apple 4) (55, 56) and beta 2GPI were able to induce a strongly increased binding to phospholipids, resulting in a LAC activity.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Construction of Expression Vectors-- Human beta 2GPI cDNA, kindly provided by Dr. T. Kristensen (University of Aarhus, Aarhus, Denmark) was subcloned into the BamHI site of pUC18. The nonphospholipid binding form beta 2GPI-W316S (9) was constructed by site-directed mutagenesis (QuickChange site-directed mutagenesis kit; Stratagene, La Jolla, CA) of pUC18-beta 2GPI with the primers beta 2GPI-f (CA GTT CTC TGG CTT TTT GCA AAA CTG ATG CAT CCG ATG) and beta 2GPI-r (CAT CGG ATG CAT CAG TTT TCG AAA AAG CCA GAG AAC TG). The sequences in italic indicate the mutated codon. The sequence encoding the mature beta 2GPI protein was amplified with the primers beta 2GPI-XhoI (C CCT CGA GGA CGG ACC TGT CCC AAG CC) and beta 2GPI-XbaI (GC TCT AGA AAA CAA GTG TGA CAT TTT ATG TGG A) in a PCR reaction. To construct chimeric fusion proteins of the dimerization domain of factor XI (apple 4) and beta 2GPI, the PCR product was cloned with XhoI and XbaI (underlined in beta 2GPI-XhoI and beta 2GPI-XbaI, respectively) into the vectors apple 4-tissue-type plasminogen activator (tPA)-S478A and apple 4-C321S-tPA-S478A (56). In this way apple 4-beta 2GPI and apple 4-C321S-beta 2GPI were constructed. As controls, the chimers apple 2-beta 2GPI and apple 4-C321S-beta 2GPI-W316, in which beta 2GPI-W316S is not able to bind to phospholipids, were made. For apple 2-beta 2GPI, the PCR product was cloned into apple 2-tPA-S478A with XhoI and XbaI (56). For apple 4-C321S-beta 2GPI-W316S, the PCR product was cloned into apple 4-C321S-tPA-S478A (56). Sequence analysis was performed to confirm correct amplification of the beta 2GPI cDNA.

Cell Culture, Transfection, Expression, and Purification of Fusion Proteins-- Transfection of baby hamster kidney cells was performed as described previously (55). Expression of all fusion constructs was performed in conditioned serum-free medium (Dulbecco's modified Eagle's medium/F-12 medium supplemented with 1% UltroserG; Life Technologies, Inc., Paisley, United Kingdom). Protein expression was measured using a total beta 2GPI-ELISA as described previously (9). Apple-beta 2GPI fusion proteins were purified using a monoclonal antibody to beta 2GPI (21B2) bound to CNBr-activated Sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden) (9). Bound fusion proteins were eluted with 0.1 M glycine (pH 2.7). The purified proteins were immediately neutralized with 1 M Tris, pH 9. Apple 4-beta 2GPI, apple 4-C321S-beta 2GPI, and apple 4-C321S-beta 2GPI-W316S containing fractions were dialyzed against 50 mM Na2HPO4, pH 7.0, containing 50 mM NaCl and were subjected to further purification on a mono S column using FPLC (Amersham Pharmacia Biotech). Fusion proteins were eluted with a linear salt gradient from 50 mM NaCl to 1 M NaCl. All chimers were dialyzed against TBS (50 mM Tris, 150 mM NaCl, pH 7.4). Protein concentrations were determined using a bicinchoninic acid (BCA) protein assay (Pierce) according to the instructions of the manufacturer, and with bovine serum albumin (BSA) as a standard. The yield varied from 5 to 10 µg of protein/ml of culture medium. Purified constructs were analyzed by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

Purification of Proteins-- beta 2GPI was isolated from freshly frozen citrated human plasma as described previously (33). In short, dialyzed human plasma was applied to a DEAE-Sephadex A50 column, subsequently followed by protein G-Sepharose, S-Sepharose, and finally heparin-Sepharose chromatography (all Sepharoses were obtained from Amersham Pharmacia Biotech). Bound proteins were eluted with a linear salt gradient. Afterward, beta 2GPI was dialyzed against TBS. Purity of the protein was checked with SDS-PAGE analysis.

Fab fragments of the monoclonal anti-beta 2GPI-antibody 23H9, which is directed against domain 2, were generated using an ImmunoPure Fab kit according to the instructions of the manufacturer (Pierce). In short, IgG was incubated with papain-coated beads overnight at 37 °C, and Fc fragments and uncut IgG were removed on a protein-A Sepharose column. After SDS-PAGE, 23H9 Fab appeared as a single band with a molecular mass of 25 kDa under reducing conditions. The Fab fragments still recognize beta 2GPI. Protein concentration was determined using a BCA protein assay.

Gel Filtration Studies-- Apple-beta 2GPI fusion proteins and beta 2GPI were applied to Superdex 200 gel filtration column using FPLC equipment. The column was equilibrated with TBS, pH 7.4, at a flow rate of 0.5 ml/min. The absorbance of the eluent was monitored at 280 nm. Molecular masses were determined by comparison to a standard curve of chymotrypsinogen A (25 kDa), ovalbumin (43 kDa), BSA (67 kDa), aldolase (158 kDa), and thyroglobulin (669 kDa).

Preparation of Phospholipid Vesicles-- Phospholipid vesicles containing 20% phosphatidylserine (PS), 40% phosphatidylcholine (PC), and 40% phosphatidylethanolamine (PE) or 20% PS and 80% PC (Sigma) were prepared according to Brunner et al. (57) with some modifications as described by van Wijnen et al. (58). The phospholipid content of the fractions was determined by phosphate analysis (59).

Binding of Apple-beta 2GPI Fusion Proteins to Phospholipids-- To measure binding of apple-beta 2GPI chimers and beta 2GPI to phospholipids, a solid phase binding assay according to Horbach et al. (9) with some modifications was used. In short, 25 µM phospholipids (20% PS/80% PC and 20% PC/40% PC/40% PE vesicles) were coated in 96-well ELISA plates (Costar, Cambridge, MA) overnight at 4 °C. Wells were blocked with TBS plus 0.5% gelatin for 2 h at 37 °C, followed by successive incubations with apple-beta 2GPI chimers or beta 2GPI (50 µl/well) for 1.5 h at 37 °C, and monoclonal antibody 2B2 (3 µg/ml; 50 µl/well; 1.5 h at 37 °C). Afterward, wells were incubated with a rabbit-anti-mouse peroxidase-conjugated antibody (1:1000 diluted; 50 µl/well; Dako, Glostrup, Denmark) and developed using o-phenylenediamine. All samples were diluted in TBS and 0.5% gelatin and after each incubation step, wells were washed three times with TBS. Nonspecific binding was determined in wells where phospholipids were absent.

Spraying of Coverslips-- Glass coverslips (18 × 18 mm; Menzel Gläser, Braunschweig, Germany) were soaked overnight in chromosulfuric acid, rinsed thoroughly with deionized water, and air-dried. Human placenta collagen type III (Sigma) was solubilized in 50 mM acetic acid (1 mg/ml) and sprayed on glass coverslips at a final density of 30 µg/cm2 with a retouching air-brush (Badger model 100; Badger Brush Co., Franklin Park, IL).

Perfusions-- Perfusions were performed in a single-pass perfusion chamber. The perfusions were performed under nonpulsatile flow conditions using a parallel plate perfusion with a slit height of 0.1 mm and a slit width of 2 mm. Experiments were done with a flow rate of 58 µl/min (shear rate 300 s-1) (60) and a perfusion time of 3 min with collagen type III as a surface. Prewarmed (5 min at 37 °C) citrated blood (1/10 volume, 3.2% trisodium citrate) was drawn through the perfusion chamber by an infusion pump (pump 22, model 2400-004; Harvard, Natick, MA). Afterward, the coverslips were removed and rinsed with 10 mM HEPES buffer, containing 150 mM NaCl, followed by fixation with 3% paraformaldehyde, 0.005% glutardialdehyde in PBS, pH 7.4, for 30 min at room temperature. Fixed glass coverslips were stored until use in PBS, 1% paraformaldehyde, 0.002% glutardialdehyde, pH 7.4.

Immunofluorescence Studies-- Coverslips were washed three times with PBS and blocked for 10 min with PBS, 1% BSA, 0.1% glycine, pH 7.4. After this, the coverslips were incubated for 1 h at 37 °C with apple-beta 2GPI fusion proteins (10 µg/ml) diluted in PBS, 1% BSA, 0.1% glycine, pH 7.4, followed by three washes with PBS and 10 min of blocking with PBS, 1% BSA, 0.1% glycine, pH 7.4, at room temperature. The coverslips were then incubated for 45 min with the monoclonal anti-beta 2GPI antibody 2B2 diluted in PBS, 1% BSA, 0.1% glycine, pH 7.4 (10 µg/ml), at 37 °C, followed by three washes with PBS and 10 min of blocking with PBS, 1% BSA, 0.1% glycine, pH 7.4, at room temperature. Afterward, the coverslips were incubated with a fluorescein isothiocyanate-labeled goat-anti-mouse antibody (Becton Dickinson, San Jose, CA), diluted 1:20 in PBS, 1% BSA, 0.1% glycine, pH 7.4, for 45 min at 37 °C, followed by three washes with PBS. Finally, the coverslips were mounted in Mowiol 4-88 (Calbiochem, La Jolla, CA) and 0.1% para-phenylenediamine (Sigma).

beta 2GPI-deficient Plasma-- beta 2GPI-deficient plasma was made using a monoclonal antibody to beta 2GPI (21B2) coupled to CNBr-activated Sepharose (Amersham Pharmacia Biotech) (9). Pooled normal plasma (10 ml) was applied to the column, and the flow-through plasma was collected in fractions of 0.5 ml and directly frozen in liquid nitrogen and stored at -70 °C. Levels of beta 2GPI were determined using a total-beta 2GPI ELISA as described previously (9), and fractions containing less than 10% beta 2GPI were considered deficient.

Coagulation Assays-- All assays were performed in a KC-10 coagulometer (Amelung, Lemgo, Germany). For prothrombin time (PT), 25 µl of pooled normal plasma and 25 µl of apple-beta 2GPI fusion protein or plasma-derived beta 2GPI (final concentration, 0-200 µg/ml) were incubated for 30 min at 4 °C, followed by an incubation of 1.5 min at 37 °C. Clotting was initiated by the addition of 50 µl of Innovin (Dade Behring, Marburg, Germany).

Activated partial thromboplastin time (PTT) was measured with a test that has been sensitized to aid the detection of lupus anticoagulants, the PTT-LA (Diagnostica Stago, Asnieres-sur-Seine, France). 25 µl of pooled normal plasma and 25 µl of apple-beta 2GPI fusion protein or plasma-derived beta 2GPI (final concentration, 0-200 µg/ml) were incubated for 30 min at 4 °C. After addition of 50 µl of PTT-LA reagent, the samples were incubated for 3 min at 37 °C. Clotting was initiated by the addition of 50 µl of 25 mM CaCl2.

Dilute Russell's viper venom time (dRVVT) was measured with LAC screen (Gradipore Ltd, North Ryde, Australia). 25 µl of pooled normal plasma and 25 µl of apple-beta 2GPI fusion protein or plasma-derived beta 2GPI (final concentration, 0-200 µg/ml) were incubated for 30 min at 4 °C, followed by an incubation of 1.5 min at 37 °C. Clotting was initiated by addition of 50 µl of LAC screen.

Correction of clotting time was performed using PT and extra addition of cephalin. 25 µl of pooled normal plasma and 25 µl of a mixture of apple-beta 2GPI fusion protein (final concentration, 0-200 µg/ml) and cephalin (final concentration was 3.2 times the concentration used in a PTT according to the instructions of the manufacturer; PTT reagent, Roche Molecular Biochemicals, Mannheim, Germany) were incubated for 30 min at 4 °C, followed by an incubation of 1.5 min at 37 °C. Clotting was initiated by the addition of 50 µl of Innovin.

The effect of monoclonal antibodies against beta 2GPI on clotting time in the presence of plasma-derived beta 2GPI or apple-beta 2GPI dimeric chimers was determined using PT. Two monoclonal anti-beta 2GPI antibodies with LAC activity were used. One was directed against domain four (19H9), and one was directed against domain two (23H9) of beta 2GPI (52). 25 µl of beta 2GPI-deficient plasma and 25 µl of a mixture of plasma-derived beta 2GPI or apple-beta 2GPI fusion protein (final concentration, 0 or 200 µg/ml) and a monoclonal antibody against beta 2GPI (final concentration, 100 µg/ml) or the Fab fragment of the monoclonal antibody 23H9 (final concentration, 100 µg/ml) were incubated for 30 min at 4 °C, followed by an incubation of 1.5 min at 37 °C. Clotting was initiated by the addition of 50 µl of Innovin (Dade Behring).

Results were indicated in clotting time (seconds; mean ± S.D., n = 3). Clotting times >=  mean clotting time + 20% were considered to be positive. For the coagulation assays using monoclonal antibodies against beta 2GPI, only one representative experiment was shown.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression and Purification of Fusion Proteins-- To study the effect of dimeric beta 2GPI on phospholipid binding and clotting time, chimeric constructs of beta 2GPI fused to the C terminus of the dimerization domain, apple 4, of factor XI were made (Fig. 1A). Both a covalent (apple 4-beta 2GPI) and a noncovalent (apple 4-C321S-beta 2GPI) chimeric dimer were made. As controls, apple 2-beta 2GPI and apple 4-C321S-beta 2GPI-W316S, in which beta 2GPI-W316S is not able to bind to phospholipids, were constructed. Baby hamster kidney cells were transfected with expression vectors containing chimeric apple-beta 2GPI constructs. Protein expression was confirmed with a total beta 2GPI-ELISA. The proteins were affinity-purified with a monoclonal antibody against beta 2GPI. Apple 4-beta 2GPI, apple 4-C321S-beta 2GPI, and apple 4-C321S-beta 2GPI-W316S were further purified by FPLC with a mono S column. After purification, the fusion proteins were applied to 10% SDS-PAGE under reducing and nonreducing conditions and stained by Coomassie Brilliant Blue (Fig. 1, B and C). Under nonreducing conditions apple 4-C321S-beta 2GPI and apple 4-C321S-beta 2GPI-W316S migrated as monomers with an apparent molecular mass of 47 kDa. Apple 2-beta 2GPI migrated slightly slower than apple 4-C321S-beta 2GPI and apple 4-C321S-beta 2GPI-W316S at 49 kDa, which is probably caused by glycosylation of apple 2. Apple 4-beta 2GPI migrated as a dimer under nonreducing conditions with a molecular mass of ~110 kDa. Plasma-derived beta 2GPI migrated with a molecular mass of 42 kDa under nonreducing conditions. Upon reduction, all fusion proteins migrated as monomers with a molecular mass of 59 kDa for apple 2-beta 2GPI and 57 kDa for the other fusion proteins. Plasma-derived beta 2GPI migrated with a molecular mass of 49 kDa under reducing conditions. Western blot analysis with polyclonal anti-beta 2GPI and anti-factor XI antibodies showed reactivity with all apple-beta 2GPI fusion proteins (results not shown).



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Fig. 1.   Apple-beta 2GPI fusion proteins. A, schematic representation of the chimeric proteins. The monomeric proteins beta 2GPI and apple 2-beta 2GPI are able to bind weakly to phospholipids. The covalently associated dimer (indicated by -S-S-) apple 4-beta 2GPI and the noncovalently associated dimer (indicated by ···) apple 4-C321S-beta 2GPI are able to bind with strongly increased affinity to phospholipids. The noncovalently associated dimer apple 4-C321S-beta 2GPI-W316S is not able to bind to phospholipids. B and C, SDS-PAGE analysis of purified plasma-derived beta 2GPI (lanes 1), apple 2-beta 2GPI (lanes 2), apple 4-beta 2GPI (lanes 3), apple 4-C321S-beta 2GPI (lanes 4), and apple 4-C321S-beta 2GPI-W316S (lanes 5) were analyzed on 10% polyacrylamide gels under reducing (B) and nonreducing (C) conditions. The proteins were visualized by Coomassie staining. The molecular masses of prestained markers are indicated in kilodaltons.

Gel Filtration Studies-- To determine whether apple 4-C321S-beta 2GPI was dimeric in the native state, gel filtration studies were performed. Apple 4-beta 2GPI eluted with a molecular mass of 115 kDa, indicating that it was a dimer, as was also shown by SDS-PAGE analysis. Apple 4-C321S-beta 2GPI eluted with molecular mass of 115 kDa, demonstrating that apple 4-C321S-beta 2GPI was also a dimer under a nondenaturing condition. Apple 2-beta 2GPI eluted with a molecular mass of 61 kDa, indicating that it was indeed a monomer. These results were consistent with the findings of Meijers et al. (56) for chimeric apple-tPA constructs. Plasma-derived beta 2GPI eluted with a molecular mass of 49 kDa, which is consistent with its known molecular mass.

Binding of Apple-beta 2GPI Chimers to Immobilized Phospholipids-- The phospholipid binding features of apple-beta 2GPI fusion proteins were tested in a solid phase binding assay. Phospholipid vesicles (25 µM, 20% PS/80% PC or 20% PS/40% PC/40% PE) were immobilized on 96-well ELISA plates, and binding of plasma-derived beta 2GPI and apple-beta 2GPI chimers was measured. As shown in Fig. 2A, half-maximal binding of apple 4-beta 2GPI and apple 4-C321S-beta 2GPI to phospholipid vesicles (20% PS/80% PC) occurred at concentrations as low as 0.9 and 3.7 µg/ml, respectively. For all proteins binding of apple-beta 2GPI fusion proteins to 20% PS/40% PC/40% PE vesicles (Fig. 2B) was stronger than binding to 20% PS/80% PC vesicles (Fig. 2A). Half-maximal binding of apple 4-beta 2GPI and apple 4-C321S-beta 2GPI to 20% PS/40% PC/40% PE vesicles was observed at concentrations of 0.3 and 1.4 µg/ml, respectively. Binding of apple 4-C321S-beta 2GPI at concentrations of 0.5-16 µg/ml was lower than binding of apple 4-beta 2GPI at these concentrations for both 20% PS/80% PC and 20% PS/40% PC/40% PE vesicles. For both vesicle types, plasma-derived beta 2GPI and apple 2-beta 2GPI only showed little binding to phospholipid vesicles at a concentration of 32 µg/ml. Apple 4-C321S-beta 2GPI-W316S was not able to bind to phospholipid vesicles (both 20% PS/80% PC and 20% PS/40% PC/40% PE vesicles) at concentrations as high as 32 µg/ml, which was expected, since beta 2GPI-W316S is not able to bind to phospholipid vesicles (9). To determine whether plasma-derived beta 2GPI and apple 2-beta 2GPI were able to bind to phospholipids (20% PS/80% PC vesicles) at higher concentrations, concentrations to 100 µg/ml were used. As shown in Fig. 2C, plasma-derived beta 2GPI and apple 2-beta 2GPI were able to bind to phospholipids at a concentration of 100 µg/ml. Half-maximal binding of plasma-derived beta 2GPI occurred at 32 µg/ml (results not shown). Even at a concentration of 100 µg/ml, apple 4-C321S-beta 2GPI-W316S was unable to bind to immobilized phospholipids (Fig. 2C). These results demonstrated that apple 4-beta 2GPI and apple 4-C321S-beta 2GPI were able to bind to phospholipids with increased affinity compared with plasma-derived beta 2GPI and apple 2-beta 2GPI.



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Fig. 2.   Binding of apple-beta 2GPI chimers to immobilized phospholipids. 20% PS/80% PC vesicles (A and C) or 20% PS/40% PC/40% PE vesicles (B) were coated on 96-well ELISA plates. A and B, increasing concentrations (0-32 µg/ml) of purified plasma-derived beta 2GPI (black-square), apple 2-beta 2GPI (), apple 4-beta 2GPI (), apple 4-C321S-beta 2GPI (open circle ), and apple 4-C321S-beta 2GPI-W316S (×) were assayed for phospholipid binding. C, binding of 1 and 100 µg/ml of plasma-derived beta 2GPI (white bar), apple 2-beta 2GPI (lined bar), apple 4-beta 2GPI (gray bar), apple 4-C321S-beta 2GPI (black bar), and apple 4-C321S-beta 2GPI-W316S (shaded bar) to phospholipids.

Binding of Apple-beta 2GPI Fusion Proteins to Platelets-- Platelets expose phosphatidylserine immediately after adhesion to collagen (61). To test the binding of apple-beta 2GPI chimers to physiological phospholipid membranes, perfusion experiments were performed. Whole blood was perfused over collagen type III-coated glass coverslips at a shear rate of 300 s-1 to allow platelets to bind to collagen. After fixation of the adhered platelets, plasma-derived beta 2GPI or apple-beta 2GPI fusion proteins (10 µg/ml) were allowed to bind to the platelets. Binding of fusion proteins was determined by immunofluorescence. As shown in Fig. 3, apple 4-beta 2GPI and apple 4-C321S-beta 2GPI were able to bind to the platelets, while plasma-derived beta 2GPI, apple 2-beta 2GPI, and apple 4-C321S-beta 2GPI-W316S were unable to bind.



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Fig. 3.   Binding of apple-beta 2GPI chimers to platelets adhered to collagen type III. No protein (A), plasma-derived beta 2GPI (B), apple 2-beta 2GPI (C), apple 4-beta 2GPI (D), apple 4-C321S-beta 2GPI (E), or apple 4-C321S-beta 2GPI-W316S (F) at a concentration of 10 µg/ml was bound to adhered platelets. Binding was determined using immunofluorescence.

Effect of Apple-beta 2GPI Fusion Proteins on in Vitro Coagulation Tests-- To study the effect of apple-beta 2GPI chimers on coagulation, several coagulation assays were performed. Increasing concentrations of plasma-derived beta 2GPI and apple-beta 2GPI fusion proteins (50-200 µg/ml), diluted in TBS, were mixed 1:1 with pooled normal plasma and incubated for 30 min at 4 °C, which was followed by measurement of PT, PTT-LA, or dRVVT. The most pronounced effect was seen with PT (Table I). In this assay prolongation of clotting time was already seen for apple 4-beta 2GPI and apple 4-C321S-beta 2GPI at a concentration of 50 µg/ml. The observed effect was concentration-dependent as shown in Table I. At a concentration of 200 µg/ml, which is the plasma concentration, apple 4-beta 2GPI and apple 4-C321S-beta 2GPI showed clotting times of 31.0 ± 4.2 and 25.8 ± 3.9 s, respectively. This corresponds for apple 4-beta 2GPI to a prolongation of clotting time of 15.9 s and for apple 4-C321S-beta 2GPI of 10.7 s. Plasma-derived beta 2GPI, apple 2-beta 2GPI, and apple 4-C321S-beta 2GPI-W316S did not induce prolonged clotting times. With PTT-LA a concentration of 200 µg/ml apple 4-beta 2GPI was able to prolong the clotting time from 45.2 ± 1.6 s (mean clotting time of pooled normal plasma) to 66.1 ± 8.1 s (Table II). For apple 4-C321S-beta 2GPI also prolongation of clotting time was observed. At a concentration of 200 µg/ml the clotting time was 65.2 ± 13.4 s. As expected, no prolongation of clotting times was observed for plasma-derived beta 2GPI, apple 2-beta 2GPI, and apple 4-C321S-beta 2GPI-W316S. dRVVT showed prolonged a clotting time for apple 4-beta 2GPI and apple 4-C321S-beta 2GPI with 57.1 ± 5.9 and 56.2 ± 6.3 s, respectively, at a concentration of 200 µg/ml (Table II). The mean clotting time of pooled normal plasma using dRVVT was 44.5 ± 1.7 s. Plasma-derived beta 2GPI, apple 2-beta 2GPI, and apple 4-C321S-beta 2GPI were not able to induce prolongation of clotting times. Thus, these results suggest that dimeric apple-beta 2GPI chimers were able to induce LAC activity.


                              
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Table I
Effect of apple-beta 2GPI fusion proteins on clotting assays
Plasma-derived beta 2GPI and apple-beta 2GPI chimers were diluted 1:1 with pooled normal plasma (mean PT 15.1 ± 1.2 s, n = 20), with or without the addition of extra phospholipids, followed by the measurement of the PT. Results represent mean clotting time ± S.D. in seconds (n = 3). Samples with a clotting time >= 18.1 s (mean PT + 20%) were considered to be positive. ND, not done. *, considered to be positive.


                              
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Table II
Effect of apple-beta 2GPI fusion proteins on clotting assays
Plasma-derived beta 2GPI and apple-beta 2GPI chimers were diluted 1:1 with pooled normal plasma (mean dRVVT 44.5 ± 1.7 s and mean PTT-LA 45.2 ± 1.6 s, n = 15) followed by the measurement of dRVVT or PTT-LA. Data represent mean ± S.D. (n = 3). Samples with clotting times >= 53.4 s for dRVVT and >= 54.2 s for PTT-LA were considered to be positive (mean clotting time pooled normal plasma + 20%). *, considered to be positive.

To confirm that the increased clotting times were caused by the binding of dimeric apple-beta 2GPI chimers to the catalytic phospholipids, correction experiments were done. Here, extra phospholipids were added to increasing concentrations of apple 4-beta 2GPI or apple 4-C321S-beta 2GPI (50-200 µg/ml) diluted in TBS, which was mixed afterward 1:1 with pooled normal plasma and incubated for 30 min at 4 °C. After this incubation PT was measured. As shown in Table I, the prolonged clotting times disappeared by the addition of extra phospholipids. For both apple 4-beta 2GPI and apple 4-C321S-beta 2GPI, clotting time was reduced to normal values at protein concentrations of 50 and 100 µg/ml. At a concentration of 200 µg/ml, clotting times were reduced to 25.1 ± 6.4 s for apple 4-beta 2GPI and 17.8 ± 0.9 s for apple 4-C321S-beta 2GPI. This was a reduction of 60% and 72% for apple 4-beta 2GPI and apple 4-C321S-beta 2GPI, respectively. This strongly suggests that the prolonged clotting times were caused by the binding of dimeric apple-beta 2GPI fusion proteins to phospholipids.

To determine whether addition of LAC-inducing monoclonal antibodies against beta 2GPI were able to induce extra prolongation of clotting time in the presence of apple 4-beta 2GPI or apple 4-C321S-beta 2GPI, coagulation assays using PT were performed. Two different monoclonal anti-beta 2GPI antibodies were used: 19H9, which is directed against domain four; and 23H9, which is directed against domain two of beta 2GPI. First, the effect of the monoclonal anti-beta 2GPI antibodies, at a concentration of 100 µg/ml, on clotting time in beta 2GPI-deficient plasma was determined. Both antibodies did not prolong clotting time, as was expected (Fig. 4). Second, the effect of the monoclonal antibodies (concentration 100 µg/ml), in the presence of plasma-derived beta 2GPI or apple-beta 2GPI fusion proteins (concentration of 200 µg/ml), on clotting time in beta 2GPI-deficient plasma was determined. As shown in Fig. 4, both monoclonal anti-beta 2GPI antibodies were able to prolong clotting time in the presence of plasma-derived beta 2GPI or apple 2-beta 2GPI, indicating that both monoclonal antibodies indeed were able to induce LAC activity. In the presence of apple 4-beta 2GPI or apple 4-C321S-beta 2GPI, the monoclonal anti-beta 2GPI antibodies were able to induce an extra prolongation of clotting time. For apple 4-beta 2GPI clotting time was 48.7 s in the absence of a monoclonal antibody and 74.9 and 93.3 s in the presence of 19H9 and 23H9, respectively. Addition of apple 4-C321S-beta 2GPI showed a clotting time of 40.0 s, which was prolonged to 72.1 and 62.2 s when 19H9 and 23H9 were added, respectively. When the Fab fragment of monoclonal antibody 23H9 was added (concentration 100 µg/ml), no prolongation of clotting time was observed compared with the clotting time in the presence of 200 µg/ml plasma-derived beta 2GPI or apple-beta 2GPI fusion proteins (Fig. 4). Addition of the Fab fragment to an incubation with the original antibody inhibited the prolongation induced by the antibody (results not shown). These observations suggest that multimerization of apple-beta 2GPI chimeric dimers induces an extra prolongation of clotting time.



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Fig. 4.   Effect of addition of monoclonal anti-beta 2GPI antibodies on clotting time. No protein (white bar), plasma-derived beta 2GPI (lined bar), apple 2-beta 2GPI (gray bar), apple 4-beta 2GPI (black bar), apple 4-C321S-beta 2GPI (shaded bar), or apple 4-C321S-beta 2GPI (hatched bar) were added to beta 2GPI-deficient plasma in the absence (no MoAb) or in the presence (19H9 and 23H9) of anti-beta 2GPI monoclonal antibodies or the Fab fragment (Fab 23H9) of the monoclonal anti-beta 2GPI antibody 23H9. This was followed by measurement of PT.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the antiphospholipid syndrome, persistently elevated levels of antiphospholipid antibodies can be detected. These antibodies are in most cases directed against phospholipid-binding proteins such as beta 2GPI and prothrombin (34-37). It is known that, when beta 2GPI interacts with anti-beta 2GPI-antibodies, it acquires a much higher affinity for phospholipid membranes. One of the postulated mechanisms of action for anti-beta 2GPI-antibodies is that, when anti-beta 2GPI-antibodies interact with beta 2GPI, beta 2GPI is able to form bivalent complexes with these antibodies, which results in a strongly increased affinity for phospholipid membranes (51-53). Another explanation is that beta 2GPI undergoes a conformational change when it interacts with such antibodies, resulting also in a strongly increased affinity for phospholipid membranes (45-49). Here, the role of bivalent beta 2GPI complexes, without the intervention of antibodies, is determined by the construction of dimeric chimers of the dimerization domain (apple 4) of coagulation factor XI and beta 2GPI. Introduction of the apple 4 domain at the N terminus of beta 2GPI resulted in the formation of a covalent dimer of beta 2GPI. A noncovalent dimer was made by fusion of a mutated apple 4 domain, apple 4-C321S, to beta 2GPI. Gel filtration studies demonstrated that both recombinant chimers formed dimers, as had also been shown for tPA (56). As controls, apple 2-beta 2GPI, a monomer, and apple 4-C321S-beta 2GPI-W316S, in which beta 2GPI-W316S is not able to bind to phospholipids, were made. We showed that dimeric apple-beta 2GPI fusion proteins bind with a 35 times increased affinity to phospholipid vesicles (Fig. 2). Apple 4-beta 2GPI dimers bind also spontaneously to collagen type III adhered platelets and dimeric apple-beta 2GPI chimers were able to induce LAC activity when added to normal plasma. Thus, the in vitro observed effects of anti-beta 2GPI antibodies can be explained by dimerization of beta 2GPI.

For apple 4-beta 2GPI, half-maximal binding to 20% PS/80% PC vesicles was achieved at a 35-fold lower concentration than for plasma-derived beta 2GPI, which indicates a strong increase of affinity for phospholipids of the dimeric apple-beta 2GPI chimers. Half-maximal binding for the dimeric chimers apple 4-beta 2GPI and apple 4-C321S-beta 2GPI to 20% PS/80% PC vesicles was achieved at concentrations of 0.9 and 3.7 µg/ml, respectively (Fig. 2A). The difference in concentrations for half-maximal binding between these two chimeric proteins could be caused by the fact that apple 4-C321S-beta 2GPI is a noncovalently associated dimer, which dissociates more easily than the covalently associated dimer apple 4-beta 2GPI. No large differences between binding of plasma-derived beta 2GPI and apple-beta 2GPI fusion proteins to phospholipid vesicles with and without PE were observed, since half-maximal binding of apple 4-beta 2GPI was achieved at 0.9 and 0.3 µg/ml for 20% PS/80% PC and 20% PS/40% PC/40% PE vesicles, respectively. The binding of beta 2GPI to negatively charged phospholipids appears to be relatively independent of the presence of PE. This finding is in contrast with the observation that PE plays an important role in the inhibition of activated protein C activity by antiphospholipid antibodies (62). beta 2GPI does not discriminate between PE-containing and PE-free phospholipid surfaces and therefore inhibits pro- and anticoagulant reactions independent of the composition of the negatively charged phospholipid surfaces.

The dimeric chimers were able to induce LAC activity without the intervention of anti-beta 2GPI antibodies. Three different coagulation assays were used, an activate partial thromboplastin time-based assay, a prothrombin time-based assay and a dilute Russell's viper venom time. The most responsive coagulation assay was the prothrombin time, which showed a doubling of the clotting time. The difference in responsiveness of the different coagulation assays is the result of different sensitivities of the assays for our dimeric chimers, probably due to differences in phospholipid composition of the tests. The concentrations of plasma-derived beta 2GPI or the apple-beta 2GPI fusion proteins used in the plasma-based assays are much higher than the concentrations used for the phospholipid binding assay. In the plasma-based assay, the dimeric fusion proteins must compete with other plasma proteins, particularly the clotting factors, for the phospholipid surface, while in the in vitro binding assays no competing proteins are present. Due to the high affinity of, e.g., factor Va for phospholipids higher concentrations of the dimeric fusion proteins are necessary. When the results of the coagulation assays are compared with what is observed in patients, the results are quite impressive. A patient with a clotting time that is 20% prolonged is considered to be LAC-positive when no other abnormalities are observed (e.g. coagulation factor deficiencies). A patient with a clotting time that is doubled is considered to be strongly LAC-positive. Extra prolongation of clotting time was achieved by the addition of monoclonal anti-beta 2GPI antibodies, which are able to induce LAC activity by themselves, in the presence of apple-beta 2GPI chimeric dimers. When the Fab fragment of monoclonal anti-beta 2GPI-antibody 23H9 was added, no prolongation of clotting time was observed. To all probability, the effect of the antibodies is caused by multimerization of the dimeric apple-beta 2GPI fusion proteins. The difference between dimerization and multimerization of beta 2GPI may be an explanation for the observed differences in LAC activities found in patients. When weak LAC activity is observed, a patient may have monoclonal anti-beta 2GPI-antibodies that dimerize beta 2GPI. When strong LAC activity is observed, a patient may have polyclonal anti-beta 2GPI antibodies directed toward different epitopes, which makes multimerization of beta 2GPI possible.

With the recombinant dimeric beta 2GPI constructs, it will be possible to discriminate between cellular activation caused by the interaction of an antibody with a Fcgamma RII receptor and by dimerization of beta 2GPI caused by binding of the antibody to beta 2GPI. In this way, the dimeric apple-beta 2GPI fusion proteins will be very helpful to use in animal models to investigate whether dimerization of beta 2GPI explains also the in vivo observed complications.

In conclusion, we showed that dimerization itself is enough to mimic the effect of anti-beta 2GPI antibody-beta 2GPI complexes in vitro. This indicates that a conformational change of beta 2GPI is not needed to induce an increased affinity of beta 2GPI for phospholipids. We cannot exclude, however, that a conformational change of beta 2GPI can also induce an increased affinity of beta 2GPI for phospholipids.


    FOOTNOTES

* This work was supported in part by Netherlands Heart Foundation Grant 98.060.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.

§ To whom correspondence should be addressed: Thrombosis and Haemostasis Laboratory, Dept. of Haematology, G03.647, University Medical Center, P.O. Box 85.500, 3508 GA Utrecht, The Netherlands. Tel.: 31-30-2506510; Fax: 31-30-2511893; E-mail: b.c.h.lutters@lab. azu.nl.

|| Established investigator of the Netherlands Heart Foundation (Grant D96.021).

Published, JBC Papers in Press, October 25, 2000, DOI 10.1074/jbc.M008224200


    ABBREVIATIONS

The abbreviations used are: beta 2GPI, beta 2-glycoprotein I; LAC, lupus anticoagulants; tPA, tissue-type plasminogen activator; BCA, bicinchoninic acid; BSA, bovine serum albumin; PS, phosphatidylserine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PT, prothrombin time; PTT, activated partial thromboplastin time; dRVVT, dilute Russell's viper venom time; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay; TBS, Tris-buffered saline; FPLC, fast protein liquid chromatography.


    REFERENCES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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