From the Departments of ** Haematology and
§§ Rheumatology and Clinical Immunology, University Medical Center,
3508 GA Utrecht and Institute of Biomembranes, Utrecht
University, 3508 TB Utrecht, The Netherlands, ¶ Department of
Vascular Medicine, Academic Medical Center, 1100 DD Amsterdam, The
Netherlands, and the
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
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ABSTRACT |
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Anti- 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
Two possible explanations for the mechanism of action of
Here, a covalent and a noncovalent chimeric dimer of
Construction of Expression Vectors--
Human
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 Purification of Proteins--
Fab fragments of the monoclonal anti- Gel Filtration Studies--
Apple- 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- 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 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- 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-
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-
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-
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-
The effect of monoclonal antibodies against
Results were indicated in clotting time (seconds; mean ± S.D.,
n = 3). Clotting times Expression and Purification of Fusion Proteins--
To study the
effect of dimeric Gel Filtration Studies--
To determine whether apple
4-C321S- Binding of Apple- Binding of Apple- Effect of Apple-
To confirm that the increased clotting times were caused by the binding
of dimeric apple-
To determine whether addition of LAC-inducing monoclonal antibodies
against 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
For apple 4- The dimeric chimers were able to induce LAC activity without the
intervention of anti- With the recombinant dimeric In conclusion, we showed that dimerization itself is enough to mimic
the effect of anti-2-glycoprotein I
antibodies are thought to cause lupus anticoagulant activity by forming
bivalent complexes with
2-glycoprotein I
(
2GPI). To test this hypothesis, chimeric fusion
proteins were constructed of the dimerization domain (apple 4)
of factor XI and
2GPI. Both a covalent (apple
4-
2GPI) and a noncovalent (apple 4-C321S-
2GPI) chimer were constructed. As controls,
apple 2-
2GPI and apple
4-C321S-
2GPI-W316S, in which
2GPI-W316S
is not able to bind to phospholipids, were made. In a phospholipid
binding assay, apple 4-
2GPI and apple
4-C321S-
2GPI were able to bind to phospholipids with an
affinity 35 times higher than that of plasma-derived
2GPI and apple 2-
2GPI. Apple
4-C321S-
2GPI-W316S did not bind at all. Only apple
4-
2GPI and apple 4-C321S-
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-
2GPI or apple 4-C321S-
2GPI was added.
Addition of 200 µg/ml plasma-derived
2GPI, apple
2-
2GPI, or apple 4-C321S-
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-
2GPI or apple 4-C321S-
2GPI was
achieved by the addition of monoclonal antibodies against
2GPI. In conclusion, dimerization of
2GPI
explains the in vitro observed effects of
2GPI-anti-
2GPI antibody complexes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-Glycoprotein I
(
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.
2GPI is a member of
the short consensus repeat or complement control protein superfamily.
The first four domains of
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
2GPI and peptides spanning the fifth domain of
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
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
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).
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
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
2GPI (43, 44).
2GPI-anti-
2GPI antibody complexes are
suggested. First,
2GPI undergoes a conformational change
when it interacts with anti-
2GPI antibodies (45-50).
Second,
2GPI is able to form bivalent complexes when it
interacts with an anti-
2GPI antibody (51-53). The final
result is a strongly increased affinity of
2GPI for
negatively charged phospholipids. Due to the increased affinity,
2GPI is then able to compete with clotting factors for
the phospholipid surface, resulting in the in vitro
prolongation of coagulation (54).
2GPI were constructed to determine the role of bivalent
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
2GPI were
able to induce a strongly increased binding to phospholipids, resulting
in a LAC activity.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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
2GPI-W316S (9) was constructed by site-directed
mutagenesis (QuickChange site-directed mutagenesis kit; Stratagene, La
Jolla, CA) of pUC18-
2GPI with the primers
2GPI-f (CA GTT CTC TGG CTT TTT GCA AAA CTG
ATG CAT CCG ATG) and
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
2GPI
protein was amplified with the primers
2GPI-XhoI (C CCT CGA
GGA CGG ACC TGT CCC AAG CC) and
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
2GPI, the PCR product was cloned with
XhoI and XbaI (underlined in
2GPI-XhoI and
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-
2GPI and
apple 4-C321S-
2GPI were constructed. As controls, the
chimers apple 2-
2GPI and apple 4-C321S-
2GPI-W316, in which
2GPI-W316S is
not able to bind to phospholipids, were made. For apple
2-
2GPI, the PCR product was cloned into apple
2-tPA-S478A with XhoI and XbaI (56). For apple 4-C321S-
2GPI-W316S, the PCR product was cloned into
apple 4-C321S-tPA-S478A (56). Sequence analysis was performed to
confirm correct amplification of the
2GPI cDNA.
2GPI-ELISA as described
previously (9). Apple-
2GPI fusion proteins were purified
using a monoclonal antibody to
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-
2GPI, apple
4-C321S-
2GPI, and apple
4-C321S-
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).
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,
2GPI was dialyzed
against TBS. Purity of the protein was checked with SDS-PAGE analysis.
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
2GPI. Protein concentration was determined using a BCA protein assay.
2GPI fusion
proteins and
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).
2GPI Fusion Proteins to
Phospholipids--
To measure binding of apple-
2GPI
chimers and
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-
2GPI chimers or
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.
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.
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-
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).
2GPI-deficient
Plasma--
2GPI-deficient plasma was made using a
monoclonal antibody to
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
2GPI
were determined using a total-
2GPI ELISA as described
previously (9), and fractions containing less than 10%
2GPI were considered deficient.
2GPI
fusion protein or plasma-derived
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).
2GPI fusion
protein or plasma-derived
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.
2GPI fusion protein or
plasma-derived
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.
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.
2GPI on
clotting time in the presence of plasma-derived
2GPI or
apple-
2GPI dimeric chimers was determined using PT. Two
monoclonal anti-
2GPI antibodies with LAC activity were
used. One was directed against domain four (19H9), and one was directed
against domain two (23H9) of
2GPI (52). 25 µl of
2GPI-deficient plasma and 25 µl of a mixture of
plasma-derived
2GPI or apple-
2GPI fusion
protein (final concentration, 0 or 200 µg/ml) and a monoclonal
antibody against
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).
mean clotting time + 20% were considered to be positive. For the coagulation assays using
monoclonal antibodies against
2GPI, only one
representative experiment was shown.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2GPI on phospholipid binding and
clotting time, chimeric constructs of
2GPI fused to the C terminus of the dimerization domain, apple 4, of factor XI were made
(Fig. 1A). Both a covalent
(apple 4-
2GPI) and a noncovalent (apple
4-C321S-
2GPI) chimeric dimer were made. As controls,
apple 2-
2GPI and apple
4-C321S-
2GPI-W316S, in which
2GPI-W316S
is not able to bind to phospholipids, were constructed. Baby hamster kidney cells were transfected with expression vectors containing chimeric apple-
2GPI constructs. Protein expression was
confirmed with a total
2GPI-ELISA. The proteins were
affinity-purified with a monoclonal antibody against
2GPI. Apple 4-
2GPI, apple 4-C321S-
2GPI, and apple
4-C321S-
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-
2GPI and apple
4-C321S-
2GPI-W316S migrated as monomers with an apparent
molecular mass of 47 kDa. Apple 2-
2GPI migrated slightly
slower than apple 4-C321S-
2GPI and apple
4-C321S-
2GPI-W316S at 49 kDa, which is probably caused by glycosylation of apple 2. Apple 4-
2GPI migrated as a
dimer under nonreducing conditions with a molecular mass of ~110 kDa. Plasma-derived
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-
2GPI and 57 kDa for the other fusion proteins.
Plasma-derived
2GPI migrated with a molecular mass of 49 kDa under reducing conditions. Western blot analysis with polyclonal
anti-
2GPI and anti-factor XI antibodies showed
reactivity with all apple-
2GPI fusion proteins (results
not shown).
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Fig. 1.
Apple- 2GPI fusion
proteins. A, schematic representation of the chimeric
proteins. The monomeric proteins
2GPI and apple
2-
2GPI are able to bind weakly to phospholipids. The
covalently associated dimer (indicated by -S-S-) apple
4-
2GPI and the noncovalently associated dimer (indicated
by ···) apple 4-C321S-
2GPI are able to bind with
strongly increased affinity to phospholipids. The noncovalently
associated dimer apple 4-C321S-
2GPI-W316S is not able to
bind to phospholipids. B and C, SDS-PAGE analysis
of purified plasma-derived
2GPI (lanes
1), apple 2-
2GPI (lanes
2), apple 4-
2GPI (lanes
3), apple 4-C321S-
2GPI (lanes
4), and apple 4-C321S-
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.
2GPI was dimeric in the native state, gel
filtration studies were performed. Apple 4-
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-
2GPI eluted with molecular mass of 115 kDa, demonstrating that apple 4-C321S-
2GPI was also a dimer under a nondenaturing
condition. Apple 2-
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
2GPI
eluted with a molecular mass of 49 kDa, which is consistent with its
known molecular mass.
2GPI Chimers to Immobilized
Phospholipids--
The phospholipid binding features of
apple-
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
2GPI and apple-
2GPI chimers was measured. As shown in Fig.
2A, half-maximal binding of
apple 4-
2GPI and apple 4-C321S-
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-
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-
2GPI and apple 4-C321S-
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-
2GPI
at concentrations of 0.5-16 µg/ml was lower than binding of apple
4-
2GPI at these concentrations for both 20% PS/80% PC
and 20% PS/40% PC/40% PE vesicles. For both vesicle types,
plasma-derived
2GPI and apple 2-
2GPI only
showed little binding to phospholipid vesicles at a concentration of 32 µg/ml. Apple 4-C321S-
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
2GPI-W316S is not able to bind to
phospholipid vesicles (9). To determine whether plasma-derived
2GPI and apple 2-
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
2GPI and apple
2-
2GPI were able to bind to phospholipids at a
concentration of 100 µg/ml. Half-maximal binding of plasma-derived
2GPI occurred at 32 µg/ml (results not shown). Even at
a concentration of 100 µg/ml, apple 4-C321S-
2GPI-W316S
was unable to bind to immobilized phospholipids (Fig. 2C).
These results demonstrated that apple 4-
2GPI and apple
4-C321S-
2GPI were able to bind to phospholipids with
increased affinity compared with plasma-derived
2GPI and apple 2-
2GPI.
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Fig. 2.
Binding of
apple- 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
2GPI (
), apple 2-
2GPI (
), apple
4-
2GPI (
), apple 4-C321S-
2GPI (
),
and apple 4-C321S-
2GPI-W316S (×) were assayed for
phospholipid binding. C, binding of 1 and 100 µg/ml of
plasma-derived
2GPI (white bar), apple
2-
2GPI (lined bar), apple
4-
2GPI (gray bar), apple
4-C321S-
2GPI (black bar), and apple
4-C321S-
2GPI-W316S (shaded bar) to
phospholipids.
2GPI Fusion Proteins to
Platelets--
Platelets expose phosphatidylserine immediately after
adhesion to collagen (61). To test the binding of
apple-
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
2GPI or apple-
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-
2GPI and apple
4-C321S-
2GPI were able to bind to the platelets, while
plasma-derived
2GPI, apple 2-
2GPI, and
apple 4-C321S-
2GPI-W316S were unable to bind.
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Fig. 3.
Binding of
apple- 2GPI chimers to platelets
adhered to collagen type III. No protein (A),
plasma-derived
2GPI (B), apple
2-
2GPI (C), apple 4-
2GPI
(D), apple 4-C321S-
2GPI (E), or
apple 4-C321S-
2GPI-W316S (F) at a
concentration of 10 µg/ml was bound to adhered platelets. Binding was
determined using immunofluorescence.
2GPI Fusion Proteins on in Vitro
Coagulation Tests--
To study the effect of
apple-
2GPI chimers on coagulation, several coagulation
assays were performed. Increasing concentrations of plasma-derived
2GPI and apple-
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-
2GPI and apple 4-C321S-
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-
2GPI and apple 4-C321S-
2GPI showed clotting times of 31.0 ± 4.2 and 25.8 ± 3.9 s,
respectively. This corresponds for apple 4-
2GPI to a
prolongation of clotting time of 15.9 s and for apple
4-C321S-
2GPI of 10.7 s. Plasma-derived
2GPI, apple 2-
2GPI, and apple
4-C321S-
2GPI-W316S did not induce prolonged clotting
times. With PTT-LA a concentration of 200 µg/ml apple
4-
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-
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
2GPI, apple 2-
2GPI, and apple 4-C321S-
2GPI-W316S.
dRVVT showed prolonged a clotting time for apple 4-
2GPI
and apple 4-C321S-
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
2GPI, apple 2-
2GPI, and apple
4-C321S-
2GPI were not able to induce prolongation of
clotting times. Thus, these results suggest that dimeric
apple-
2GPI chimers were able to induce LAC activity.
Effect of apple-2GPI fusion proteins on clotting assays
2GPI and apple-
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.
Effect of apple-2GPI fusion proteins on clotting assays
2GPI and apple-
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.
2GPI chimers to the catalytic
phospholipids, correction experiments were done. Here, extra
phospholipids were added to increasing concentrations of apple
4-
2GPI or apple 4-C321S-
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-
2GPI and apple 4-C321S-
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-
2GPI
and 17.8 ± 0.9 s for apple 4-C321S-
2GPI. This
was a reduction of 60% and 72% for apple 4-
2GPI and
apple 4-C321S-
2GPI, respectively. This strongly suggests that the prolonged clotting times were caused by the binding of dimeric
apple-
2GPI fusion proteins to phospholipids.
2GPI were able to induce extra prolongation of clotting time in the presence of apple 4-
2GPI or apple
4-C321S-
2GPI, coagulation assays using PT were
performed. Two different monoclonal anti-
2GPI antibodies
were used: 19H9, which is directed against domain four; and 23H9, which
is directed against domain two of
2GPI. First, the
effect of the monoclonal anti-
2GPI antibodies, at a
concentration of 100 µg/ml, on clotting time in
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
2GPI or apple-
2GPI fusion
proteins (concentration of 200 µg/ml), on clotting time in
2GPI-deficient plasma was determined. As shown in Fig.
4, both monoclonal anti-
2GPI antibodies were able to
prolong clotting time in the presence of plasma-derived
2GPI or apple 2-
2GPI, indicating that
both monoclonal antibodies indeed were able to induce LAC activity. In
the presence of apple 4-
2GPI or apple
4-C321S-
2GPI, the monoclonal anti-
2GPI
antibodies were able to induce an extra prolongation of clotting time.
For apple 4-
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-
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
2GPI or apple-
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-
2GPI chimeric dimers induces an extra prolongation of clotting time.
View larger version (23K):
[in a new window]
Fig. 4.
Effect of addition of monoclonal
anti- 2GPI antibodies on clotting
time. No protein (white bar), plasma-derived
2GPI (lined bar), apple
2-
2GPI (gray bar), apple
4-
2GPI (black bar), apple
4-C321S-
2GPI (shaded bar), or
apple 4-C321S-
2GPI (hatched bar)
were added to
2GPI-deficient plasma in the absence
(no MoAb) or in the presence (19H9 and
23H9) of anti-
2GPI monoclonal antibodies or
the Fab fragment (Fab 23H9) of the monoclonal
anti-
2GPI antibody 23H9. This was followed by
measurement of PT.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2GPI and prothrombin (34-37). It is known that, when
2GPI interacts with anti-
2GPI-antibodies,
it acquires a much higher affinity for phospholipid membranes. One of
the postulated mechanisms of action for
anti-
2GPI-antibodies is that, when
anti-
2GPI-antibodies interact with
2GPI,
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
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
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
2GPI. Introduction of the apple 4 domain at the N
terminus of
2GPI resulted in the formation of a covalent
dimer of
2GPI. A noncovalent dimer was made by fusion of
a mutated apple 4 domain, apple 4-C321S, to
2GPI. Gel
filtration studies demonstrated that both recombinant chimers formed
dimers, as had also been shown for tPA (56). As controls, apple
2-
2GPI, a monomer, and apple
4-C321S-
2GPI-W316S, in which
2GPI-W316S
is not able to bind to phospholipids, were made. We showed that dimeric
apple-
2GPI fusion proteins bind with a 35 times
increased affinity to phospholipid vesicles (Fig. 2). Apple
4-
2GPI dimers bind also spontaneously to collagen type III adhered platelets and dimeric apple-
2GPI chimers
were able to induce LAC activity when added to normal plasma. Thus, the in vitro observed effects of anti-
2GPI
antibodies can be explained by dimerization of
2GPI.
2GPI, half-maximal binding to 20% PS/80%
PC vesicles was achieved at a 35-fold lower concentration than for
plasma-derived
2GPI, which indicates a strong increase
of affinity for phospholipids of the dimeric apple-
2GPI
chimers. Half-maximal binding for the dimeric chimers apple
4-
2GPI and apple 4-C321S-
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-
2GPI is a noncovalently associated dimer, which
dissociates more easily than the covalently associated dimer apple
4-
2GPI. No large differences between binding of
plasma-derived
2GPI and apple-
2GPI fusion
proteins to phospholipid vesicles with and without PE were observed,
since half-maximal binding of apple 4-
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
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).
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.
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
2GPI or the
apple-
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-
2GPI antibodies, which are able to
induce LAC activity by themselves, in the presence of
apple-
2GPI chimeric dimers. When the Fab fragment of
monoclonal anti-
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-
2GPI fusion proteins. The difference between
dimerization and multimerization of
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-
2GPI-antibodies that dimerize
2GPI. When strong LAC activity is observed, a patient
may have polyclonal anti-
2GPI antibodies directed toward
different epitopes, which makes multimerization of
2GPI possible.
2GPI constructs, it will be
possible to discriminate between cellular activation caused by the interaction of an antibody with a Fc
RII receptor and by dimerization of
2GPI caused by binding of the antibody to
2GPI. In this way, the dimeric apple-
2GPI
fusion proteins will be very helpful to use in animal models to
investigate whether dimerization of
2GPI explains also
the in vivo observed complications.
2GPI antibody-
2GPI
complexes in vitro. This indicates that a conformational
change of
2GPI is not needed to induce an increased
affinity of
2GPI for phospholipids. We cannot exclude,
however, that a conformational change of
2GPI can also
induce an increased affinity of
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:
2GPI,
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.
![]() |
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