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Blood, Vol. 94 No. 10 (November 15), 1999:
pp. 3421-3431
Lupus Anticoagulants Form Immune Complexes With Prothrombin and
Phospholipid That Can Augment Thrombin Production in Flow
By
Susan L. Field,
Philip J. Hogg,
Elise B. Daly,
Yan-Ping Dai,
Barbara Murray,
Daniel Owens, and
Colin N. Chesterman
From The Centre for Thrombosis and Vascular Research, School of
Pathology, University of New South Wales, and the Department of
Haematology, Prince of Wales Hospital, Sydney, Australia; and the
Division of Immunology and Cell Biology, John Curtin School of Medical
Research, Australian National University, Canberra, Australia.
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ABSTRACT |
Lupus anticoagulants (LA) are a family of autoantibodies that are
associated with in vitro anticoagulant activity but a strong predisposition to in vivo thrombosis. They are directed against plasma
phospholipid binding proteins, including prothrombin. We found that a
murine monoclonal antiprothrombin antibody and 7 of 7 LA IgGs tested
enhanced binding of prothrombin to 25:75 phosphatidyl serine:phosphatidyl choline vesicles in a concentration-dependent manner. We hypothesized that enhanced binding of prothrombin to phospholipid in the presence of LA IgG might result in increased thrombin production when reactions are performed in flow. Thrombin production by purified prothrombinase components was measured in a
phospholipid-coated flow reactor. The flow reactor was incubated with
prothrombin, calcium ions, and the IgGs and then perfused with
prothrombin, calcium ions, the IgGs, factor Va, and factor Xa. A murine
monoclonal antiprothrombin antibody and 4 of 6 LA IgGs from patients
with a history of thrombosis increased thrombin production up to 100%
over control in the first 15 minutes. In summary, LA IgGs concentrate
prothrombin on a phospholipid surface that can augment thrombin
production by prothrombinase in flow. These observations suggest that
LA might propagate coagulation in flowing blood by facilitating
prothrombin interaction with the damaged blood vessel wall.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE LUPUS ANTICOAGULANT (LA) phenomenon
remains one of the conundrums of clinical medicine: a phenomenon that
is due to a family of autoantibodies that are associated with in vitro anticoagulant activity on the one hand and a powerful predisposition to
in vivo thrombosis on the other. There has been debate as to whether
the prothrombotic association with LA and anticardiolipin antibodies
(ACA) is fortuitous, with the antibodies not necessarily being
responsible for thrombosis, but rather being markers for as yet
undefined antibody activities. However, the weight of evidence, both
clinical and experimental, favors a causative role. Almost every
conceivable mechanism has been invoked in the effort to explain the
thrombotic tendency in patients with LA and ACA. These include
pathological activation of platelets,1 disturbance of
endothelial cell function,2-4 interference with protein C and protein S,5,6 and interaction with heparan sulphate
proteoglycans with consequent reduction in antithrombin
activity.7,8 None of these explanations has been entirely
convincing, and in most cases there has been no satisfactory mechanism
defined experimentally.
The discovery that what were regarded as antiphospholipid antibodies
were in fact directed at the phospholipid binding proteins  2-glycoprotein 1 (2-GP1) in the case of
ACA9,10 and prothrombin and/or 2-GP1 in the
case of LA11-13 opened the way to more logical approaches
to determining prothrombotic mechanisms. The focus has been largely on
demonstrating antibody binding to these proteins, with and without
phospholipid, and to defining potential inhibitory activity as a result
of such binding.14-17
Little attention has been given to the obverse, namely that LA Ig and
ACA Ig enhance the binding of phospholipid binding proteins to
phospholipid surfaces. In the case of prothrombin such binding may
result in concentration of prothrombin at the site of vascular injury
or platelet activation. A recent report described increased binding of
prothrombin to phospholipid and umbilical vein endothelial cell
monolayers in the presence of LA IgG.18
It is clear that LA IgGs prolong phospholipid dependent clotting time
in most in vitro assays. A feature common to in vitro coagulation tests
is the static conditions under which they are performed. In contrast,
thrombus formation occurs in flowing blood. We reasoned that the
effects of LA IgGs on coagulation might be different when examined
under flow conditions, which is more representative of the in vivo situation.
We hypothesized that enhanced binding of prothrombin to phospholipid in
the presence of LA IgG would result in increased thrombin production by
prothrombinase when reactions are performed in flow. We now report that
a murine monoclonal antiprothrombin antibody and 7 of 7 LA IgGs tested
enhanced binding of prothrombin to phospholipid vesicles and that the
monoclonal antibody (MoAb) and 4 of 6 LA IgGs from patients with a
history of thrombosis augmented thrombin production by purified
prothrombinase components in flow. These observations suggest that LA
might propagate coagulation in flowing blood by facilitating
prothrombin interaction with the damaged blood vessel wall.
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MATERIALS AND METHODS |
Reagents and proteins.
Human prothrombin (II), factor Xa, and factor V were from Enzyme
Research (South Bend, IN). Human  -thrombin (IIa) was a gift from Dr
Paul Bock (Vanderbilt University Medical Center, Nashville, TN)
1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) and
1,2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS) were from Avanti
Polar Lipids (Alabaster, AL), and
N-((6-(biotinoyl)amino)hexanoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (biotin-DHPE) was from Molecular Probes (Eugene, OR).
n-octyl--D-glucopyranoside and D-Phe-Pro-Arg-CH2Cl were
from Sigma (St Louis, MO).
Patients and control subjects.
IgG fractions were prepared from 7 patients with LA diagnosed on the
basis of the accepted criteria of the ISTH Subcommittee on Lupus
Anticoagulants/Phospholipid-dependent Antibodies19 and in
all cases displaying prolongation of the Kaolin clotting time (KCT) and
the dilute Russell's viper venom test (dRVVT). Blood samples were
obtained after informed consent, in accordance with NH&MRC guidelines.
The criteria for selection were repeated, unequivocal LA positivity;
all LAs were present for 0.5 to 3.5 years; availability and willingness
to provide blood; and patients were unselected, apart from LA5, who was
chosen because of the high titer antiprothrombin antibody. LA5 was
grossly hypoprothrombinaemic (0.08 U/mL) and, in fact, presented with
severe menorrhagia. Five control subjects were laboratory staff with no
evident illness. ACA, both IgG and IgM, were assayed with a
commercially available enzyme-linked immunosorbent assay (ELISA;
Medical Innovations, Artarmon, New South Wales, Australia) using the
Harris Standards as described in Shi et al.14 Binding of
the LA antibodies to plastic-adsorbed DOPS in the presence of
prothrombin, to plastic-adsorbed prothrombin, or to plastic-adsorbed
2-GP1 was measured exactly as described by Galli et
al,13 including the use of Linbro high-activated PVC plates.
Preparation of antibodies.
The purified antihuman prothrombin murine MoAb (anti-II MoAb) was from
Enzyme Research. The antibody recognizes prothrombin fragment 2 (residues 156-271) in solid-phase ELISA. A purified isotype-matched
murine MoAb against bovine protein disulfide isomerase was used as a
control. Total IgG was prepared from control and patient sera using
protein G sepharose (Pharmacia, Uppsala, Sweden). After application of
serum, the protein G sepharose was washed with 20 mmol/L sodium
phosphate, pH 7, buffer containing 1 mol/L NaCl. After re-equilibration
of the column with 20 mmol/L sodium phosphate, pH 7, the IgG was eluted
with 0.1 mol/L glycine, pH 2.7, and the pH was neutralized immediately
with 1 mol/L Tris, pH 9, buffer. The IgGs were aliquoted and stored at
 20°C.
Preparation of phospholipid vesicles.
DOPC, DOPS, and biotin-DHPE were combined in the molar ratio 74:25:1,
and the solvent was evaporated under argon. Unilamellar vesicles with
an average diameter of approximately 200 nm were prepared according to
Mimms et al.20 Phospholipid concentration was determined by
measuring organic phosphate as described by Bartlett.21
Binding of prothrombin to phospholipid vesicles.
Binding of iodinated prothrombin to phospholipid vesicles was measured
by separating bound from free prothrombin in an air-fuge.22 Prothrombin was iodinated using immobilized iodogen according to the
manufacturer's instructions (Pierce, Rockford, IL) to a specific
activity of 40,000 counts per minute (CPM)/µg.
125I-prothrombin (100,000 CPM), prothrombin (0.1 to 10 µmol/L), 74:25:1 DOPC:DOPS:biotin-DHPE vesicles (15 µmol/L), and
control or anti-II MoAb or LA IgG (0 to 50 µmol/L) was added to
polyallomer centrifuge tubes containing 50 mmol/L HEPES, 125 mmol/L
NaCl, 10 mmol/L CaCl2, 5 mg/mL bovine serum albumin (BSA),
pH 7.4, buffer. The reactions were incubated for 20 minutes and the
phospholipid vesicles were sedimented in an air-fuge (Beckman, Palo
Alto, CA) for 20 minutes. Measurement of the phospholipid
content21 of the supernatant after centrifugation indicated
that the air-fuge sedimented  95% of the phospholipid vesicles. The
supernatant was aspirated and bound, and free
125I-prothrombin was determined using a Packard Crystal II
 counter (Packard Instrument Co, Meriden, CT).
Nonspecific binding of 125I-prothrombin to control tubes
not containing phospholipid vesicles represented  20% of the binding
to phospholipid vesicles in the presence of 10 µmol/L control IgG.
The moles of prothrombin bound to phospholipid vesicles was calculated
from the total prothrombin and the ratio of bound versus total
125I-prothrombin. Fab fragments were prepared from control
and LA IgG using a Fab Preparation Kit (Pierce) according to the
manufacturer's instructions.
Activation of factor V.
Factor V (0.74 µmol/L) was activated by -thrombin (0.01 µmol/L)
for 15 minutes at room temperature in 25 mmol/L Tris, 5 mmol/L CaCl2, 0.5 mol/L NaCl, 50% glycerol, pH 7.5, buffer. The
reaction was quenched by the addition of
D-Phe-Pro-Arg-CH2Cl to a final concentration of 1 µmol/L,
and the factor Va stored at 20°C until use.
Measurement of thrombin production in flow conditions.
Glass capillaries (0.65 [internal diameter] × 127 mm; Brand, Wertheim, Germany) were cleaned as described by Billy et
al.23 Capillaries were dried under a stream of
nitrogen; filled with 100 µg/mL Neutralite avidin (Molecular Probes)
in 15 mmol/L Na2CO3, 35 mmol/L
NaHCO3, 0.02% NaN3, pH 9.6, buffer; and
incubated overnight at 4°C in a humid chamber. The capillaries were
drained, filled with approximately 1.5 mmol/L 74:25:1
DOPC:DOPS:biotin-DHPE vesicles, and incubated at room temperature for 2 hours before connection to a gas-tight syringe (Hamilton, Reno, NV).
Flow was controlled by a model sp100i syringe pump (World Precision
Instruments, Sarasota, FL). Capillaries were washed with 50 mmol/L
HEPES, 125 mmol/L NaCl, 3 mmol/L CaCl2, 0.5 mg/mL BSA, pH
7.4, buffer (HEPES buffer) for 2 minutes at a flow rate of 1 mL/min to
remove nonbound phospholipid. Three types of experiment were performed.
Phospholipid-coated capillaries were filled with prothrombin (0.1 µmol/L), factor Va (0.1 nmol/L), and control or anti-II MoAb (0.4 µmol/L) or LA IgG (5 to 50 µmol/L) in HEPES buffer and incubated
for 1 hour at room temperature. The capillaries were then perfused with
20 pmol/L factor Xa without or with the anti-II MoAb (0.4 µmol/L) or
LA IgG (5 to 50 µmol/L) in HEPES buffer at a flow rate of 30 µL/min. Alternatively, phospholipid-coated capillaries were incubated
with prothrombin (0.1 µmol/L) and control or anti-II MoAb (0.4 µmol/L) or LA IgG (5 to 50 µmol/L) and then perfused with factor Xa
(20 pmol/L) and factor Va (0.1 nmol/L) without or with the anti-II MoAb
or LA IgG.
Phospholipid-coated capillaries were filled with prothrombin (0.1 µmol/L) and control or anti-II MoAb (0.4 µmol/L) or LA IgG (5 to 50 µmol/L) in HEPES buffer and incubated for 1 hour at room temperature
and then perfused with prothrombin (0.1 µmol/L), factor Xa (1 nmol/L), factor Va (2 nmol/L), and control or anti-II MoAb (0.4 µmol/L) or LA IgG (5 to 50 µmol/L) in HEPES buffer at a flow rate
of 30 µL/min.
Normal human plasma was collected in citrate, depleted of IgG using
protein G sepharose, and dialyzed against 50 mmol/L Tris-HCl, 0.1 mol/L
NaCl, 1 mmol/L EDTA, pH 7.4, buffer to remove citrate. D-Phe-Pro-Arg-CH2Cl (10 µmol/L), CaCl2 (10 mmol/L), and control or LA IgG (25 µmol/L) was added to the plasma,
which was incubated in phospholipid-coated capillaries for 1 hour at
room temperature. The plasma was diluted 1:2 with 50 mmol/L Tris-HCl,
0.1 mol/L NaCl, pH 7.4, buffer. Capillaries were perfused with 2 capillary volumes of HEPES buffer at a flow rate of 30 µL/min to wash
away plasma and D-Phe-Pro-Arg-CH2Cl, followed by factor Xa
(1 nmol/L) and factor Va (2 nmol/L) in the HEPES buffer at the same
flow rate.
The reaction products were collected into wells of 96-well plates
containing 50 mmol/L Tris, 175 mmol/L NaCl, 20 mmol/L EDTA, 0.5 mg/mL
BSA, pH 7.9, buffer. Thrombin concentration was determined by adding
N-p-Tosyl-Gly-Pro-Arg-p-nitroanilide (TGPRpNA; Sigma) to a final
concentration of 100 µmol/L and measuring the initial rate of
p-nitroaniline formation at 405 nm using a Thermomax Kinetic Microplate
Reader (Molecular Devices, Menlo Park, CA).22
The results of the flow experiments were very reliable and reproducible
on a given day. There was some quantitative variation from day to day;
however, the qualitative differences were very consistent. All of the
experiments were performed at least twice, and on most occasions 3 to 6 times. A phospholipid capillary was only used once, because it was
difficult to ensure that all reactants from a previous experiment were removed.
Measurement of thrombin production in static conditions.
Phospholipid vesicles (5 µmol/L), prothrombin (0.1 µmol/L), factor
Va (0.1 nmol/L), and control or LA IgG (5 to 50 µmol/L) in HEPES
buffer was incubated for 1 hour at room temperature. Factor Xa was
added to a final concentration of 20 pmol/L to initiate the reaction.
Aliquots of the reaction were removed at discrete time intervals and
diluted 40-fold into 50 mmol/L Tris-HCl, 175 mmol/L NaCl, 20 mmol/L
EDTA, 0.5 mg/mL BSA, pH 7.9, buffer to quench the reaction. Thrombin
concentration was calculated as described above.
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RESULTS |
Characteristics of the anti-II MoAb and LA IgGs.
The antihuman II murine MoAb recognizes prothrombin fragment 2 (residues 156-271) adsorbed to ELISA plate wells. The effect of the
anti-II MoAb on the KCT of normal plasma was determined. The anti-II
MoAb prolonged the clotting of normal plasma in a concentration-dependent manner (Fig 1A). A
control MoAb had no effect.
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| Fig 1.
Effect of anti-II MoAb and LA IgGs on the KCT of normal
plasma. (A) A control MoAb or anti-II MoAb was added to an equal volume
of normal plasma and the KCT was determined.14 The normal
plasma was depleted of IgG using protein G sepharose. The anti-II MoAb
prolonged the KCT of normal plasma in a concentration-dependent manner,
whereas the control MoAb had no effect. (B) To confirm the LA activity
of the IgG preparations from the 7 patient plasmas, they were added to
an equal volume of normal plasma and the KCT was
determined.14 The normal plasma was depleted of IgG using
protein G sepharose. Values for LA IgGs are expressed as a proportion
of the KCT for normal plasma, with values greater than 1.2 (indicated
by a horizontal line) being positive for LA. Control values were
combined and are expressed as the mean ± SE (n = 5). The IgG
prepared from control subjects' serum showed no LA activity at the
concentrations tested, whereas all of the patients' IgGs resulted in
prolongation of the KCT at final concentrations of 20 µmol/L and
greater.
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The median age for 7 patients with LA was 32 years, with a range of 14 to 62 years (Table 1). Five healthy
subjects had no history of thrombosis, whereas 6 of the 7 patients had
a history of arterial (acute myocardial infarction [AMI] and
cerebrovascular accident [CVA]) or venous thrombotic
events, including deep vein thrombosis (DVT) with or without pulmonary
embolus (PE). The other patient (LA5) had no thrombotic history but
presented with a severe bleeding diathesis associated with
hypoprothrombinaemia. The KCT of a 20% patient:80% normal plasma
mixture as a ratio with the KCT of a normal plasma for each patient
varied from 1.3 to greater than 2 (ratios >1.2 are positive for LA).
IgG ACA were present in the plasma of 4 of the 7 patients (LA2, LA3,
LA4, and LA5), and 4 had mild to moderate thrombocytopenia. Using the
assay methodology of Galli et al,13 3 patients had
antibodies that reacted with either plastic-adsorbed DOPS in the
presence of prothrombin or plastic-adsorbed prothrombin (LA4, LA5, and
LA7), and 2 had antibodies that reacted with plastic-adsorbed
2-GP1 (LA2 and LA4). Only 2 patients (LA1 and LA5) were
found to have diagnostic lupus serology with positive antinuclear
factor and antibodies for double-stranded DNA.
The total IgG from the plasmas of the 7 patients with LA were purified
on protein G sepharose. The LA IgGs were not contaminated by
coagulation factors, as determined by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and functional
assays (not shown). The purpose of this study was to examine the net
properties of the LA IgGs on the binding and conversion of prothrombin
on phospholipid vesicles in both static and flow conditions. No attempt
was made to affinity purify the antiprothrombin Igs, because these Igs would have not necessarily reflected the net properties of the total Ig
population on prothrombin binding and conversion. For instance, only 3 of the 7 LA IgGs reacted with plastic-adsorbed DOPS in the presence of
prothrombin (Table 1); therefore, only 3 of the 7 LA IgG
antiprothrombin antibodies would have been expected to bind to an
immobilized DOPS column in the presence of prothrombin. The specificity
of effects in the experiments described herein was conferred by using
purified coagulation factors rather than by attempting to purify Ig populations.
To confirm the LA activity of the LA IgGs, they were added to normal
plasma and the KCT was determined. The IgG prepared from control
subjects' serum showed no LA activity at the concentrations tested,
whereas all of the patients' IgGs resulted in prolongation of the KCT
at final concentrations of 20 µmol/L and greater (Fig 1B). The effect
of the LA IgGs on the KCT was probably a combination of antiprothrombin
antibodies and anti-2GP1 antibodies (Table 1).
Effect of an anti-II MoAb and LA IgGs on binding of prothrombin to
phospholipid vesicles.
In addition to recognizing phospholipid-bound prothrombin, LA IgGs have
been shown to enhance interaction of prothrombin with plastic-adsorbed
phospholipid and to cultured human umbilical vein endothelial
cells.18 We found that the anti-II MoAb and LA IgGs also
enhanced binding of prothrombin to phospholipid bilayers.
Prothrombin was incubated with unilamellar 200-nm diameter 74:25:1
DOPC:DOPS:biotin-DHPE phospholipid vesicles in the presence of control
or anti-II or LA IgG and bound prothrombin determined by sedimenting
the vesicles in an air-fuge. The effect of increasing control, anti-II
MoAb, or LA IgG concentration on binding of prothrombin to phospholipid
vesicles is shown in Fig 2. The anti-II
MoAb and all of the LA IgGs enhanced prothrombin binding to
phospholipid vesicles in a concentration-dependent manner. Neither the
control MoAb nor the control IgG had any significant effect on
prothrombin binding. These results are similar to those reported by Rao
et al,18 who found that all 4 IgG preparations tested
increased the affinity of binding of 125I-prothrombin to
immobilized phospholipid.
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| Fig 2.
Effect of anti-II MoAb and LA IgGs on binding of II to
phospholipid vesicles. Binding of prothrombin to 200 nm 74:25:1
DOPC:DOPS:biotin-DHPE unilamellar vesicles was measured by separating
bound from free prothrombin in an air-fuge.22 (A) Effect of
control MoAb or anti-II MoAb on binding of 0.1 µmol/L prothrombin to
phospholipid vesicles. The data are expressed as the fraction of total
prothrombin bound. Data points and error bars represent the mean and SE
of 3 experiments. (B) Effect of control or LA IgGs on binding of 0.1 µmol/L prothrombin to phospholipid vesicles. The data are expressed
as the fraction of total prothrombin bound.
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The bivalency of the LA IgG was required to enhance binding of
prothrombin to phospholipid, because Fab fragments of LA2, LA4, and LA5
IgGs did not promote prothrombin binding (Chesterman et al, submitted
for publication). The other LA Fab fragments were not
tested. This dependence on LA antibody bivalency for prothrombin
binding parallels the requirement for ACA bivalency for high-affinity
binding of 2-GP1 to lipid membranes.24-26
Effect of the anti-II MoAb and LA IgGs on thrombin production in a
tubular flow reactor coated with phospholipid vesicles.
To investigate the effects of the anti-II MoAb and LA IgGs on thrombin
production and conversion in flow, tubular flow reactors were used. The
reactors consisted of glass capillaries whose interior surfaces were
coated with unilamellar 74:25:1 DOPC:DOPS:biotin-DHPE phospholipid
vesicles by linking the biotin-labeled PE to adsorbed avidin. Initially, we tested whether the effects of the anti-II MoAb
and LA IgGs on prothrombin binding to soluble phospholipid vesicles
shown in Fig 2 were reflected in the thrombin production on the
interior surface of the phospholipid-coated capillaries.
Phospholipid-coated capillaries were incubated with prothrombin, factor
Va, and control or anti-II MoAb or LA IgG and were then perfused with
factor Xa. Alternatively, the capillaries were incubated with
prothrombin and control or anti-II MoAb or LA IgG and were then
perfused with factor Xa and factor Va. The thrombin content of the
eluate from the capillaries was measured from the initial rate of
hydrolysis of the tripeptidyl p-nitroanilide substrate, TGPRpNA.
Reversing the introduction of factor Va did not significantly influence
the results with the anti-II MoAb and all LA IgGs tested (not shown).
The anti-II MoAb markedly increased thrombin production in the
phospholipid-coated capillary. The pattern of thrombin production is
shown in Fig 3 for an MoAb concentration of
0.4 µmol/L. Under the flow conditions of the assay, there was very
little thrombin production in the presence of the control MoAb, which
probably reflected the relatively weak binding of prothrombin to
phospholipid vesicles (dissociation constant, ~1
µmol/L27) and the concentration of prothrombin used in
the assays (0.1 µmol/L).
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| Fig 3.
Effect of anti-II MoAb on thrombin production in a
tubular flow reactor coated with phospholipid vesicles. Glass
capillaries were coated with phospholipid vesicles and incubated with
0.1 µmol/L prothrombin, 0.1 nmol/L factor Va, and 0.4 µmol/L of
control or anti-II MoAb for 1 hour. The capillaries were perfused with
20 pmol/L factor Xa at a flow rate of 30 µL/min. Two-minute fractions
were collected and the thrombin concentration was determined by the
rate of hydrolysis of N-p-Tosyl-Gly-Pro-Arg-p-nitroanilide.
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The LA IgGs also resulted in a net increase in thrombin production,
with the exception of LA2. The patterns of thrombin production are
shown in Fig 4 for selected LA IgG
concentrations. There was a small amount of thrombin production in the
presence of control IgG, which increased with increasing control IgG
concentration. However, there was a marked difference between control
and LA IgG for 6 of the 7 LA IgGs.
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| Fig 4.
Effect of LA IgGs on thrombin production in the
phospholipid-coated flow reactor. Glass capillaries were coated with
phospholipid vesicles and incubated with 0.1 µmol/L prothrombin, 0.1 nmol/L factor Va, and control or LA IgG for 1 hour. The LA IgG and
corresponding control IgG concentrations were 5 µmol/L for LA4 and
LA7; 10 µmol/L for LA2, LA5, and LA6; 20 µmol/L for LA1; and 50 µmol/L for LA3. The capillaries were perfused with 20 pmol/L factor
Xa at a flow rate of 30 µL/min. Two-minute fractions were collected
and the thrombin concentration was determined by the rate of hydrolysis
of N-p-Tosyl-Gly-Pro-Arg-p-nitroanilide.
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Capillaries were incubated with prothrombin, factor Va, and LA IgG for
either 5 minutes or 1 hour before perfusion with factor Xa, with the
timing having little effect on the outcome (not shown). The results
shown in Figs 3 and 4 were the same relative to control whether factor
Xa and factor Va concentrations of either 20 pmol/L and 0.1 nmol/L or 1 nmol/L and 2 nmol/L, respectively, were used (not shown).
The dependence of thrombin production on LA IgG concentration is shown
in Fig 5. The LA IgG concentration that
gave maximum thrombin production varied for the different patient IgGs.
Thrombin production peaked at 5 to 10 µmol/L for some LA IgGs,
whereas others peaked at 20 to 50 µmol/L LA IgG. Enhancements in
thrombin production of 1.2- to 5.6-fold (median, 2.8-fold; n = 7) over control IgG were observed. These results probably reflected enhanced binding of prothrombin to the phospholipid-coated capillary similar to
that observed using phospholipid vesicles (Fig 2).
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| Fig 5.
Effect of varying LA IgG concentration on thrombin
production in the phospholipid-coated flow reactor. Glass capillaries
were coated with phospholipid vesicles and incubated with 0.1 µmol/L
prothrombin, 0.1 nmol/L factor Va, and 5 to 50 µmol/L control or LA
IgGs for 1 hour. The capillaries were perfused with 20 pmol/L factor Xa
at a flow rate of 30 µL/min. Total thrombin production was calculated
for each LA IgG concentration from the area under the curve of profiles
of the type shown in Fig 4. Thrombin production is expressed as a
percentage of the mean control value for each IgG concentration. Data
points and error bars represent the mean and SE of 5 experiments.
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Effect of continual LA IgGs on thrombin production in the
phospholipid-coated flow reactor.
To determine whether thrombin production was perturbed by the continual
presence of the LA IgGs, phospholipid-coated capillaries were incubated
with prothrombin, factor Va, and control or LA IgG and then perfused
with both factor Xa and LA IgGs (Fig 6). The thrombin content of the eluate from the capillaries was measured from the hydrolysis of TGPRpNA.
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| Fig 6.
Effect of continual LA IgGs on thrombin production in the
phospholipid-coated flow reactor. Glass capillaries were coated with
phospholipid vesicles and incubated with 0.1 µmol/L prothrombin, 0.1 nmol/L factor Va, and control or LA IgG for 1 hour. The concentration
of LA IgG used in the experiment was that which resulted in maximal
prothrombin binding (see Fig 5): 5 µmol/L for LA4 and LA7; 10 µmol/L for LA2, LA5, and LA6; 20 µmol/L for LA1; and 50 µmol/L
for LA3. The capillaries were perfused with either 20 pmol/L factor Xa
or 20 pmol/L factor Xa and LA IgG at a flow rate of 30 µL/min.
Two-minute fractions were collected and the thrombin concentration was
determined by the rate of hydrolysis of
N-p-Tosyl-Gly-Pro-Arg-p-nitroanilide. Total thrombin production was
calculated for each LA IgG and is expressed as a percentage of the mean
control value for each IgG concentration (see Fig 5). Data points and
error bars represent the mean and SE of 5 experiments. ( ) Results
from perfusion with 20 pmol/L Xa alone; ( ) results from perfusion
with both 20 pmol/L Xa and LA IgG.
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The presence of LA IgG in the perfusate reduced thrombin production by
approximately half for LA7, to control levels for LA5, and to less than
control for LA2. In contrast, thrombin production increased 2-fold in
the presence of solution phase LA4. The presence of LA1, LA3, or LA6 in
the perfusate only marginally enhanced or inhibited thrombin
production. Therefore, the presence of solution phase LA IgGs
significantly changed the nature of prothrombin conversion for 4 of the
7 LA IgGs.
Effect of LA IgGs on thrombin production in flow.
A growing thrombus is continually supplied with prothrombin from the
flowing blood. To determine whether the anti-II MoAb and LA IgGs could
enhance thrombin production in flow in the presence of a continuous
supply of prothrombin, phospholipid-coated capillaries were
preincubated with prothrombin and LA IgGs and then perfused with factor
Xa, factor Va, LA IgGs, and prothrombin. The concentrations of
prothrombin, calcium ions, factor Va, and factor Xa were 0.1 µmol/L,
3 mmol/L, 1 nmol/L, and 2 nmol/L, respectively. The wall shear rate was
18.5 s 1, which is similar to that of large
veins.28
The anti-II MoAb was tested at a concentration of 0.4 µmol/L, whereas
all LA IgGs were tested at the concentrations that produced maximum
effects in the experiments shown in Figs 4 and 5. The anti-II MoAb
enhanced thrombin production by 70% over control in the first 10 minutes (Fig 7). LA1, LA4, and LA7 IgG
enhanced thrombin production up to 100% over control in the first 5 to 10 minutes, whereas LA3 enhanced both initial thrombin production and
the final steady state production of thrombin by approximately 100%
over control (Fig 8). LA2 and LA6 IgG were
not different from control, and LA5 IgG prolonged the approach to
steady state thrombin production. This is in keeping with the
inhibitory effect of LA5 IgG shown in Fig 6 and this patient's
clinical bleeding rather than thrombosis. The steady state rates of
thrombin production were not significantly different from control for
all LA IgG, except for LA3, which was increased by approximately 100%.
In addition, Fab fragments of LA3 IgG did not enhance thrombin
production (Chesterman et al, submitted for publication),
which is consistent with the requirement for antibody bivalency to
enhance prothrombin interaction with phospholipid vesicles.
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| Fig 7.
Effect of an anti-II MoAb on thrombin production in flow.
A glass capillary was coated with phospholipid vesicles and incubated
with 0.1 µmol/L prothrombin and 0.4 µmol/L control or anti-II MoAb
and was then perfused with 0.1 µmol/L prothrombin, 0.4 µmol/L
control or anti-II MoAb, 2 nmol/L factor Va, and 1 nmol/L Xa at a flow
rate of 30 µL/min (collecting 15-second fractions every 30 seconds).
Thrombin concentration was determined by the rate of hydrolysis of
N-p-Tosyl-Gly-Pro-Arg-p-nitroanilide.
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| Fig 8.
Effect of LA IgGs on thrombin production in flow. Glass
capillaries were coated with phospholipid vesicles and incubated with
0.1 µmol/L prothrombin and control or LA IgG and were then perfused
with 0.1 µmol/L prothrombin, control, or LA IgG; 2 nmol/L factor Va;
and 1 nmol/L Xa at a flow rate of 30 µL/min (collecting 15-second
fractions every 30 or 60 seconds). Thrombin concentration was
determined by the rate of hydrolysis of
N-p-Tosyl-Gly-Pro-Arg-p-nitroanilide. The concentration of LA IgG used
in the experiment was that which resulted in maximal prothrombin
binding (see Fig 5): 5 µmol/L for LA4 and LA7; 10 µmol/L for LA2,
LA5, and LA6; 20 µmol/L for LA1; and 50 µmol/L for LA3.
|
|
Effect of LA IgGs on thrombin production in static conditions.
The results of the experiments described in Fig 8 indicate that
prothrombin is a competent substrate for prothrombinase in flow despite
the presence of LA IgGs. Considering the anticoagulant effect of LAs in
in vitro clotting tests, we compared the results in flow with the
effect of the LA IgGs on progress curves for thrombin formation by
prothrombinase in static conditions. The LA IgGs were tested at the
concentrations that produced maximum effects in the experiments shown
in Figs 4 and 5, which were the same concentrations used in the flow
experiments in Fig 8.
LA2 and LA4 did not significantly effect the rate of thrombin
formation, whereas LA1, LA3, LA5, LA6, and LA7 all significantly inhibited the rate of thrombin formation
(Fig 9). LA5 was the most inhibitory of the
LA IgGs in the static conditions, which is in keeping with its
inhibitory effect in flow and this patient's bleeding diathesis.
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| Fig 9.
Effect of LA IgGs on thrombin production in static
conditions. Phospholipid vesicles (5 µmol/L), prothrombin (0.1 µmol/L), factor Va (0.1 nmol/L), and control or LA IgG (5 to 50 µmol/L) in HEPES buffer was incubated for 1 hour at room temperature.
Factor Xa was added to a final concentration of 20 pmol/L to initiate
the reaction. Aliquots of the reaction were removed at discrete time
intervals and thrombin concentration was determined by the rate of
hydrolysis of N-p-Tosyl-Gly-Pro-Arg-p-nitroanilide. The concentration
of LA IgG used in the experiment was that which resulted in maximal
prothrombin binding (see Fig 5): 5 µmol/L for LA4 and LA7; 10 µmol/L for LA2, LA5, and LA6; 20 µmol/L for LA1; and 50 µmol/L
for LA3. Data points and error bars represent the mean and SE of
triplicate determinations.
|
|
LA2 had little effect on thrombin formation in either static or flow
conditions, whereas LA4 did not effect thrombin formation in static
conditions but enhanced thrombin formation in flow. In contrast, LA1,
LA3, LA6, and LA7 inhibited thrombin formation in the static
conditions, but enhanced (LA1, LA3, and LA7) or did not effect (LA6)
thrombin formation in flow.
Effect of LA IgGs in plasma on thrombin production in the
phospholipid-coated flow reactor.
Our hypothesis is that LA IgG might propagate coagulation in flowing
blood by facilitating prothrombin interaction with the damaged blood
vessel wall. It was important, therefore, to determine whether the LA
IgGs would also promote thrombin production with the
phospholipid-coated flow reactor in the complex plasma environment.
Phospholipid-coated capillaries were incubated with normal plasma
containing calcium ions and physiological concentrations of control or
LA IgGs and were then perfused with factor Xa and factor Va. The
effects of control and LA IgGs on thrombin production is shown in
Fig 10. Similar effects of the LA IgGs as
shown in Fig 4 on thrombin production were observed when plasma was
used as the prothrombin source. Interestingly, the relative effects of
the LA IgGs differed when purified prothrombin rather than plasma
prothrombin was used (compare Figs 5 and 10). This probably reflected
the different IgG to prothrombin ratios used in the 2 experiments.
However, plasma proteins such as 2-GP1 may have also
contributed to the differing effects.
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| Fig 10.
Effect of LA IgGs in plasma on thrombin production in
the phospholipid-coated flow reactor. (A) Phospholipid-coated
capillaries were incubated with 50% normal plasma containing 10 µmol/L D-Phe-Pro-Arg-CH2Cl, 10 mmol/L CaCl2,
and 25 µmol/L control or LA IgG for 1 hour. The capillaries were
washed with 2 capillary volumes of HEPES buffer and were then perfused
with 1 nmol/L factor Xa and 2 nmol/L factor Va at a flow rate of 30 µL/min. One-minute fractions were collected and the thrombin
concentration was determined by the rate of hydrolysis of
N-p-Tosyl-Gly-Pro-Arg-p-nitroanilide. Results for LA1, LA5, and LA7 IgG
are shown. (B) Total thrombin production was calculated for each LA IgG
from the area under the curve of profiles of the type shown in (A).
|
|
| |
DISCUSSION |
Rao et al18 described increased binding of prothrombin to
plastic-adsorbed phospholipid and umbilical vein endothelial cell monolayers in the presence of LA IgG. We have extended these
observations to unilamellar 200-nm diameter synthetic phospholipid
vesicles comprising 74% DOPC, 25% DOPS, and 1% biotin-DHPE. An
anti-II MoAb and 7 of 7 LA IgGs tested enhanced prothrombin binding to phospholipid vesicles in a concentration-dependent manner. The precise
mechanism of this effect is unknown, although antibody bivalency was
essential for those LA IgGs tested (Chesterman et al, submitted for
publication). It is noteworthy that only 3 of the 7 LA IgGs reacted
with plastic-adsorbed DOPS in the presence of prothrombin (Table 1).
This probably reflected differences in presentation of prothrombin
epitope(s) for LA IgG when prothrombin was bound to plastic-adsorbed
DOPS versus bound to fluid bilayer phospholipid vesicles. Based on
these observations, we hypothesized that concentration of prothrombin
on a damaged blood vessel wall by LA might augment thrombin production
in flowing blood that could explain the link between LA and thrombosis.
To investigate the effects of the LA IgGs on thrombin production in
flow, tubular flow reactors coated with phospholipid were used. These
reactors were of similar design to those described by Andree et
al29 and Billy et al,23 with the
exception of how the phospholipid was immobilized. The flow reactors
consisted of glass capillaries whose interior surface was coated with
unilamellar 74:25:1 DOPC:DOPS:biotin-DHPE phospholipid vesicles by
linking the biotin-labeled PE to adsorbed avidin. The phospholipid
coated in this way presented a mobile unilamellar bilayer to the
flowing phase.
The ability of the anti-II MoAb and LA IgGs to promote prothrombin
binding to soluble phospholipid vesicles corresponded to enhanced
thrombin production in the phospholipid-coated reactor. The enhanced
thrombin production in the flow reactor was probably a consequence of
enhanced prothrombin binding to the phospholipid surface in the
presence of the anti-II MoAb and LA IgGs. The effect of LA IgG on
thrombin production in the phospholipid-coated reactor was dependent on
the antibody concentration. Measurement of thrombin production as a
function of LA IgG concentration resulted in bell-shaped isotherms for
5 of the 7 LA IgGs tested. This prozone effect is not uncommon in
antibody studies and is probably a consequence of antibody
bivalency.30 Importantly, prothrombinase converted prothrombin to thrombin in the phospholipid-coated reactor in the
presence of LA IgG. This finding led us to investigate whether the LA
IgGs could augment thrombin production in flow conditions resembling
the in vivo situation.
Thrombus formation proceeds in an environment that is continually
supplied with prothrombin from the flowing blood. In addition, we
anticipate that exposed phospholipid would be bathed in prothrombin and
LA IgGs before formation of factor Va and factor Xa and assembly of
prothrombinase. With this in mind, we tested whether the anti-II MoAb
and LA IgGs could augment thrombin production when phospholipid-coated capillaries were preincubated with prothrombin and LA IgGs and then
perfused with factor Xa, factor Va, LA IgGs, and prothrombin. The wall
shear rate was 18.5 s1, which approximated that of
large veins.28 The anti-II MoAb enhanced thrombin
production by 70% over control in the initial 10 minutes. Four of the
6 LA IgGs from patients with a history of thrombosis enhanced thrombin
production up to 100% over control in the initial 5 to 10 minutes, and
1 of these increased the final steady state production of thrombin by
approximately 100% over control. Therefore, the majority of the LA
IgGs tested could augment thrombin production in flow. The augmentation
in thrombin production was probably a consequence of enhanced
prothrombin binding to the phospholipid surface in the presence of the
anti-II MoAb and LA IgGs. Billy et al23
demonstrated that the rate of thrombin production in flow
is controlled by the rate of transfer of prothrombin to the
phospholipid surface when prothrombinase densities exceed 1 fmol/cm2. Preincubation of prothrombin with the LA IgGs
concentrated prothrombin on the phospholipid surface, thus
circumventing the limiting transfer of prothrombin in the initial phase
of the flow reaction. In effect, there was more prothrombin bound to
the phospholipid surface in the initial phase, which resulted in more
thrombin production.
Our results imply that prothrombin was a catalytically competent
substrate for prothrombinase in flow despite the presence of LA IgGs.
Four of the 7 LA IgGs inhibited thrombin formation in static conditions
but enhanced or did not effect thrombin formation in flow. Also, all of
the LA IgGs prolonged the KCT of normal plasma. The reason why
prothrombin was a competent substrate for prothrombinase in flow but
not in static conditions in the presence of LA IgG may relate to the
Km of prothrombin for prothrombinase. A striking feature of
prothrombinase assembly on phospholipid vesicles in flow is the low
Km for prothrombin (3 nmol/L), which is approximately
60-fold lower than the Km for prothrombin binding to
prothrombinase on small unilamellar phospholipid vesicles in a static
system (170 nmol/L).23 The concentration of prothrombin used in the studies herein was 0.1 µmol/L. Therefore, prothrombinase activity was anticipated to be zero order with respect to the prothrombin concentration in the flow system, ie,
prothrombin was saturating. This was in contrast to the static system
in which prothrombin concentration was anticipated to be less than the Km. This means that thrombin production in the static
system would have been sensitive to effects of LA IgG on Km
for prothrombin, whereas in flow an increase in Km by say
even 10-fold would have had negligible effects on thrombin production.
The concept that LA IgG can augment thrombin production in flow through
enhancement of prothrombin binding is worthy of investigation. An
attractive feature of this model is that it may be generally applicable
to LA antibodies, because the net effect of all 7 LA IgGs tested was to
promote prothrombin binding to phospholipid. The challenge now is to
determine whether this principle operates in flowing blood exposed to a
procoagulant phospholipid surface. The observation that LA IgGs
promoted thrombin production in the phospholipid-coated reactor when
incubated with plasma is one line of support for this proposal. Another
line of support is that thrombin production is increased in patients
with LA.31 In particular, Musial et al32 showed
that thrombin generation was enhanced in blood flowing ex vivo during
the 3 minutes after a skin bleeding time incision with antiphospholipid
antibody positive patients.
| |
ACKNOWLEDGMENT |
The authors thank Dr Kerry Taylor (Mater Hospital Brisbane, Queensland,
Australia) for supplying a patient blood sample and clinical details
and Margaret Aboud (Royal North Shore Hospital, New South Wales,
Australia) for supplying clinical details.
| |
FOOTNOTES |
Submitted January 12, 1999; accepted July 16, 1999.
Supported by grants from the National Health and Medical Research
Council of Australia, the National Heart Foundation of Australia, and
an Infrastructure Grant from the NSW Health Department.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Philip J. Hogg, PhD, Centre
for Thrombosis and Vascular Research, School of Pathology, University
of New South Wales, Sydney, NSW, 2052 Australia; e-mail:
p.hogg{at}unsw.edu.au.
| |
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TOP
ABSTRACT
INTRODUCTION