Blood, Vol. 94 No. 11 (December 1), 1999:
pp. 3814-3819
Procoagulant Effect of Anti-
2-Glycoprotein I Antibodies With Lupus
Anticoagulant Activity
By
V. Pengo,
T. Brocco,
A. Biasiolo,
P. Rampazzo,
P. Carraro, and
R. Zamarchi
From the Department of Clinical and Experimental
Medicine, Thrombosis Center, the Department of
Laboratory Medicine, and the Department of Oncology and
Surgical Sciences, University of Padova School of
Medicine, Padova, Italy.
 |
ABSTRACT |
Prothrombin time (PT) is routinely used to monitor oral
anticoagulant treatment in patients with the antiphospholipid antibody syndrome (APS). The fact that PT is a phospholipid (PL)-dependent coagulation test raises the possibility that lupus anticoagulant (LA)
might interfere with this test, thus complicating the control of
anticoagulant treatment. The effect of 6 affinity-purified preparations
of anti- (a)
2-glycoprotein I (GPI) antibodies with LA
activity on the PT was tested. Instead of prolonging PT as expected,
the a2-GPI antibodies reduced the PT of both normal plasma and
anticoagulated plasma by a mean of 2.4 seconds and 5.6 seconds,
respectively. This effect was also observed using other 5 commercially
available preparations of thromboplastin. The a2-GPI-mediated
reduction in PT was dose-dependent and was lost upon removal of
2-GPI. The failure of a2-GPI antibodies to express LA activity in
PT was found to depend on the fact that calcium ions were added
together with PL at the beginning of the assay. In fact, modification
of the standard diluted Russell viper venom time (dRVVT) test by adding
calcium ions together with PL resulted in a loss of a2-GPI
anticoagulant activity. The procoagulant effect was not as evident in
an assay that used stimulated monocytes as a source of thromboplastin.
These results show that a2-GPI antibodies exhibit an `in vitro'
procoagulant effect in PT and an anticoagulant effect in dRVVT only
when the interaction with their antigen and PL occurs in the absence of
calcium ions.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
THE TERM `lupus anticoagulant' (LA)
defines a group of immunoglobulins able to prolong the clotting time of
phospholipid (PL)-dependent coagulation tests.1 The
interest in detecting LA is related to the fact that its presence is
strongly associated with both arterial and venous
thromboembolism.2,3 Ten years after demonstration of the
antiphospholipid nature of these immunoglobulins in 1980,4
it was shown that autoimmune antiphospholipid (aPL) antibodies require
the plasma protein 2-glycoprotein I (2-GPI) to bind anionic
PL.5-7 It was subsequently recognized that 2-GPI is
essential for the expression of the LA activity of some aPL antibodies,8,9 and that its adsorption removes LA activity from most LA-positive plasma samples.10 Affinity-purified
a2-GPI antibodies from plasma of LA-positive patients behave as
classical LA, with their activity disappearing at increasing PL
concentrations.11 Moreover, some monoclonal antibodies to
2-GPI express LA activity,12,13 depending on the
specific domain against which they are directed.12 The
mechanism underlying LA activity of a2-GPI antibodies is related to
their ability to enhance binding of 2-GPI to PL, thus impeding
binding/activation of clotting factors.12
As suggested by the Scientific and Standardization Committee of the
International Society on Thrombosis and Haemostasis,14 the
laboratory diagnosis of LA requires the prolongation of at least one
PL-dependent coagulation test, and the demonstration that the inhibitor
is responsible for this effect (mixing test) by interfering with PL or
PL-bound proteins (confirmatory test). Numerous tests measuring
clotting parameters such as activated partial thromboplastin time,
dilute Russell viper venom time (dRVVT), kaolin clotting time, and
tissue thromboplastin inhibition (TTI) are currently used to detect LA.
As the use of a single screening test will fail to identify
approximately 30% to 40% of LA patients, a combination of two or more
tests in the initial evaluation step is recommended. Among PL-dependent
coagulation tests, prothrombin time (PT) is not included14
because it is rather insensitive15 and sometimes is
prolonged due to associated hypoprothrombinemia.16
On the other hand, PT is routinely used to monitor anticoagulation, the
mainstream treatment of patients with antiphospholipid antibody
syndrome (APS).17,18 As pointed out by Moll and
Ortel,19 this monitoring approach could be compromised by
the inhibitory effect of LA on PT. This possibility prompted us to test
the effect of affinity-purified human a2-GPI antibodies with LA
activity on PT.
| |
MATERIALS AND METHODS |
Blood sampling and screening.
Venous blood was collected in siliconized glass tubes containing
one-tenth volume of 3.8% trisodium citrate. Plasma was obtained by
centrifugation at 2,000g at room temperature. Anticardiolipin antibody enzyme-linked immunosorbent assay (ELISA), a2-GPI antibody ELISA, and dRVVT for the detection of LA activity were performed on
patient plasma as described elsewhere.20
Purification of IgG autoantibodies to 2-GPI.
Filtered plasma was loaded onto a prepacked column of Superose
(Pharmacia LKB Biotechnology, Uppsala, Sweden) that had been coupled
with purified human 2-GPI11 (5 mg of protein per
milliliter of Superose) according to the manufacturer's instructions.
After extensive washing with phosphate-buffered saline (PBS), a2-GPI antibodies were eluted from the column using 0.1 mol/L glycine 0.5 mol/L NaCl, pH 2.8,21 and immediately buffered with 1 mol/L Tris, pH 8.4. The purity of the resulting a2-GPI IgG preparations was checked by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE), and the protein concentration was measured
by optical density at 280 nm as previously described.22
Normal IgG from a healthy donor was purified from plasma by means of a
protein A-Sepharose column (Pharmacia LKB Biotechnology). Purified
a2-GPI antibodies and control IgG were tested after a 1:4 dilution
in a2-GPI IgG ELISA as previously described.22
Preparation of 2-GPI-depleted plasma.
Pooled normal plasma was passed through a CL-octyl Sepharose column as
previously reported.22 To estimate the quantity of residual
2-GPI in the flow through, 24-µL samples (diluted 1:2 in 20 mmol/L
Tris, 150 mmol/L NaCl, pH 7.4 [TBS]) were applied to 4.0-mm wells of
a 1% agarose gel containing 6% vol/vol polyclonal a2-GPI rabbit
antiserum, and subjected to single radial immunodiffusion. Purified
2-GPI was serially diluted with TBS, pH 7.4, and analyzed in
parallel to provide a calibration curve. Results of this analysis showed that 2-GPI was no longer detectable in the depleted plasma.
Coagulation studies.
Pooled plasma from 5 healthy subjects and pooled plasma from 5 patients
on stable oral anticoagulant treatment were used as coagulation
substrates. Clotting times were recorded using an automated fibrometer
(Mechrolab Clot-timer Model 202A; Heller Laboratories, Santa Rosa, CA).
LA activity of purified a2-GPI IgG preparations was evaluated by
dRVVT as previously described.22 Standard PT was assayed by
preincubating 100 µL of plasma at 37°C for 2 minutes, followed by
the addition of 200 µL of calcium thromboplastin and measurement of
clotting time. To introduce 100 µL of a2-GPI IgG into the test
without altering the proportions between reagents, tissue calcium
thromboplastin was reconstituted with one-half volume of the
appropriate diluting agent. Distilled water was used for the
thromboplastin preparations Innovin (Dade, Miami, FL), Thromborel S
(Istituto Behring, L'Aquila, Italy), Thromboplastin IS (Baxter
Diagnostic Division, Milan, Italy), and IL-HS (Instrumentation Laboratories, Milan, Italy). Recombiplastin (Ortho Clinical
Diagnostics, Milan, Italy) was reconstituted with 30 mmol/L
CaCl2. The final reaction mixture contained 100 µL of
pooled plasma, 100 µL of either TBS or normal pooled or a2GPI IgG
in Tris buffer, and 100 µL of concentrated calcium thromboplastin. In
experiments performed to test the importance of the sequence of reagent
addition, tissue thromboplastin was incubated with a2-GPI antibodies
in the presence or absence of calcium ions for 2 minutes before
addition of anticoagulated plasma. In some experiments, the
reconstituted Recombiplastin was further diluted with 30 mmol/L
CaCl2.
A modified dRVVT was performed by changing the sequence of added
reagents. In the dRVVT used to measure the LA activity of purified
antibodies, the addition sequence was (1) normal plasma; (2) TBS,
normal IgG, or affinity-purified a2-GPI IgG; (3) viper venom; (4)
PL, followed by incubation at 37°C for 30 seconds; and (5)
CaCl2, followed by measurement of clotting time. In a modified test the sequence was (1), (2), and (3), followed by the
addition of CaCl2 and PL and measurement of clotting time. A second modified dRVVT was performed by incubating (2), (3), (4), with
or without (5) (calcium ions) for 30 seconds followed by initiation of
the clotting reaction by adding (1) (normal pooled plasma). Some
experiments were performed by using a range of CaCl2 concentrations (3 mmol/L to 7 mmol/L).
Induction of tissue factor-like activity in monocytes.
Peripheral blood mononuclear cells (PBMCs) were obtained as described
elsewhere.23 Briefly, blood was collected in heparin from a
healthy staff volunteer. PBMCs were isolated by Ficoll-Hypaque gradient
centrifugation (Pharmacia LKB Biotechnology Inc, Piscataway, NJ) and
washed twice with RPMI. PBMCs were then resuspended in RPMI-15% fetal
calf serum (FCS) and incubated in a tissue-culture flask
(Falcon) for 1 hour at 37°C in a humidified, 5%
CO2 atmosphere. Nonadherent cells were then discarded, and
adherent cells were collected by scraping and incubated overnight in
RPMI-10% FCS. This method resulted in a 3-fold enrichment in
CD14+ mononuclear cells, as evaluated by cytofluorimetry
after staining with an anti-CD14 antibody. The cells were resuspended
at a concentration of 1 × 106 cells/mL, incubated for 6 hours with 10 µg/mL lipopolysaccharide (LPS) to stimulate monocytes,
and then centrifuged and resuspended in TBS containing 30 mmol/L
CaCl2. PT was measured by adding 50 µL of the stimulated
monocyte/CaCl2 preparation to 50 µL of normal pooled
plasma that had been preincubated with 50 µL of TBS or a2-GPI IgG
for 2 minutes at 37°C.
Patients.
Six patients (2 males and 4 females, 40 to 58 years of age) with APS
were studied; 4 patients had primary APS, and 2 patients had the
syndrome associated with systemic lupus erythematosus. All patients
tested positive for anticardiolipin IgG and LA activity. Two patients
also had low titers of anticardiolipin IgM. Qualifying thrombotic
events were venous thromboembolism in 3 patients and arterial
thromboembolism in the remaining 3 cases (1 had acute myocardial
infarction and 2 had acute arterial insufficiency in the lower limbs).
All patients tested positive for a2-GPI IgG.
| |
RESULTS |
Affinity-purified preparations of a2-GPI IgG antibodies were
obtained from plasma samples of 6 patients with APS. All 6 preparations showed LA activity in dRVVT, with prolongation of clotting time ranging
from 3.7 to 8.3 seconds over control IgG. All 6 preparations also
showed marked positivity in a2-GPI IgG ELISA, with the
OD405 ranging from 2.587 U for patient no. 5 to 3.492 U for
patient no. 1 (Table 1). These preparations
were then tested by PT (see Materials and Methods) with the
thromboplastin routinely used in our laboratory (Recombiplastin) and
pooled normal plasma as a substrate. Comparison of results obtained
with normal IgG showed that all of the a2-GPI antibody preparations
slightly shortened the PT, with the reduction ranging from 1.1 seconds
in patient no. 5 to 2.9 seconds in patient no. 2 (mean, 2.4 seconds)
(Fig 1). As shown in Fig
2, this procoagulant effect was also
observed in assays performed using pooled plasma from 5 patients on
stable long-term anticoagulant treatment (International Normalized
Ratio [INR] of pooled plasma was 2.28). Compared with normal IgG, the a2-GPI antibody preparations shortened the PT of the anticoagulated pool by a mean of 5.6 seconds (from a minimum of 4.5 seconds in patient
no. 5 to a maximum of 6.7 seconds in patient no. 6). Results were
comparable when using different pools of normal or anticoagulated plasma (data not shown).
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| Fig 2.
Effect of affinity-purified a2-GPI antibody
preparations on PT of plasma from patients on oral anticoagulant
treatment.
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We next performed additional PT assays to test the influence of the
tissue thromboplastin preparations and the sequence of reagent addition
on the observed reduction in clotting time. To limit experimental
variability, these assays were performed using a2-GPI preparations
(nos. 1A and 1B) obtained from one of the patients (no. 1).
Table 2 summarizes results of PT performed
using 4 additional preparations of tissue thromboplastin (ie, Innovin,
Thromborel S, Thromboplastin IS, and IL-HS) and either a2-GPI
preparation no. 1A (52 µg/mL) or normal IgG (100 µg/mL). The
a2-GPI IgG antibodies shortened the PT regardless of the
thromboplastin preparation used, yielding a mean reduction of 2.1 and
10.1 seconds using normal and anticoagulated plasma, respectively. The
reduction in PT was particularly evident in tests performed using
anticoagulated plasma and either Thromborel S or Thromboplastin IS (9.8 and 19.3 seconds, respectively).
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Table 2.
Effect of a2-GPI Antibodies From Patient
No. 1 on PT of Normal Plasma (NP) and Plasma From Patients on Oral
Anticoagulants (OA), Measured Using Different Thromboplastin
Preparations (INR of pooled anticoagulated plasma = 2.7)
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In the standard PT assay, plasma was preincubated at 37°C for 2 minutes before the addition of calcium-thromboplastin and measurement
of clotting time. Results of modified assays in which a2-GPI
preparation (no. 1B) and thromboplastin were incubated in the absence
(
Ca) or presence (+Ca) of calcium ions for 2 minutes before the
addition of anticoagulated plasma showed that the sequence of reagent
addition did not influence the procoagulating effects of the antibodies
with PT reduced by 9.2 seconds in standard assay, and by 7.6 and 10.1 seconds in the Ca and +Ca assays, respectively.
Results of PT assays performed using a pool of anticoagulated plasma
(INR = 3.0) and increasing amounts of a a2-GPI
preparation no. 1B showed that the reduction in PT was dose-dependent
(Fig 3). This IgG preparation shortened PT
by 3.2, 6.7, 7.5, 9 seconds when added at a concentration
of 5.8, 11.6, 23.2, and 46.5 µg/mL, respectively. The undiluted
preparation (93 µg/mL) shortened the PT by 9.2 seconds (ie, from 47.7 seconds using normal IgG to 38.5 seconds).
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| Fig 3.
Reduction in PT induced by concentrations of a2-GPI
antibody preparation no. 1B. A dose-response was obtained starting at
an IgG concentration of 5.8 µg/mL. The reduction in PT was
ascertained using pooled anticoagulated plasma (INR = 3.0).
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To ascertain the role of the target antigen, 2-GPI, in
a2-GPI-mediated PT shortening, PT assays were performed using
plasma that had been depleted of 2-GPI by affinity chromatography
(see Materials and Methods). Results showed that 2-GPI-depleted
plasma exhibited a substantial loss in the ability to shorten PT (ie, from a 9.2-second reduction to a 1.5-second reduction),
indicating that the procoagulant effect of a2-GPI antibodies is
dependent on the presence of their antigen. Moreover, in PT performed
by incubating pooled anticoagulated plasma with PL and
recombinant-tissue factor (r-TF) for 2 minutes before
adding calcium ions, a2-GPI IgG did not exhibit procoagulant effect
(ie, 45.8 seconds for a2-GPI preparation no. 1B v 47 seconds
for normal IgG). Therefore, procoagulant activity of a2-GPI IgG is
not detectable in PT when plasma is incubated with PL in the absence of
calcium ions.
Previous results had shown that the use of diluted tissue
thromboplastin in TTI yielded comparable PTs in the
presence or absence of a2-GPI IgG with LA activity.24
This prompted us to test whether diluting thromboplastin would affect
the procoagulant activity of a2-GPI IgG. As shown in Fig
4, dilution of Recombiplastin progressively
abolished the a2-GPI-mediated reduction in PT of both normal and
anticoagulated plasma. Therefore, the observed procoagulant effect also
depend on the PL/r-TF concentration.
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| Fig 4.
Effect of r-TF/PL (Recombiplastin) dilutions on reduction
of PT of normal or anticoagulated plasma measured using anti-2GPI
antibody preparation no. 1A.
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As both dRVVT and PT depend on PL, we reasoned that the distinct
abilities of these tests to disclose LA might be influenced by the
sequence in which the reagents are mixed. One major difference between
the PT and dRVVT protocols is that calcium ions are added together with
PL in the former, while they are added after PL in the latter. In fact,
modification of the dRVVT protocol by concomitant addition of calcium
ions and anionic PL (as in PT) resulted in normal clotting times (Fig
5). The same effect was observed when dRVVT
was performed by incubating all reagents for 30 seconds before adding
normal pooled plasma. In fact, while affinity-purified a2-GPI
antibody preparation no. 1B prolonged conventional dRVVT by 10.3 seconds, it showed no prolongation when normal pooled plasma was added
last. The same behavior, ie, no prolongation, was observed when
preparation no. 1B was used and calcium ions and normal pooled plasma
were added after 30 seconds of incubation of the other reagents.
Moreover, an increase in final concentration of calcium from 3 mmol/L
to 7 mmol/L completely abolished the LA activity of a2-GPI
antibodies (data not shown). These observations showed that detection
of a2-GPI LA in dRVVT assay requires the incubation of plasma with
PL in the absence of calcium ions.
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| Fig 5.
Effect of modification of the assay protocol on LA
activity expressed by affinity-purified a2-GPI antibody
preparations.
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Finally, we tested whether this procoagulant effect could be mediated
by monocytes, cells known to express tissue factor activity on their
surface when provided with specific stimuli. As shown in Table
3, compared with nonactivated monocytes,
activated monocytes shortened the clotting time of normal plasma from
220 to 42.6 seconds in the presence of Tris buffer and from 217 to 43.3 seconds in the presence of normal IgG; a2-GPI IgG preparations nos.
4 and 6 exhibited a modest procoagulant effect (ie, reduction in clotting time by 3 seconds and 2.4 seconds, respectively) in the presence of activated monocytes.
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Table 3.
PT of Normal Plasma in the Presence of
a2-GPI Antibody Preparations Measured Using Monocytes as a
Source of Tissue Factor
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DISCUSSION |
LA is a strong risk factor for thrombosis, and its association with
either arterial or venous thrombosis defines the APS
syndrome.25 Most patients with this condition are treated
with oral anticoagulants17,18,26 and undergo PT-INR
monitoring, the internationally accepted method used to assess oral
anticoagulant treatment. It has been reported that the prolongation of
PT in patients with LA reflects both the effect of warfarin and the
`in vitro' anticoagulant effect of their autoantibodies; therefore,
PT-INR values might not accurately reflect the true level of
anticoagulation.19,27 The present study was undertaken to
elucidate the effect of a2-GPI LA, a common LA in patients with APS
syndrome,11 on PT. Results showed that, instead of
prolonging PT, these LA accelerate coagulation of both normal plasma
and plasma from patients on oral anticoagulant treatment. This explains
the observation that (1) PT is seldom influenced in the presence of LA;
(2) PT with Simplastin is not affected by monoclonal antibodies against
2-GPI28; and (3) the TTI is not influenced by a2-GPI
LA.24
The mechanism by which a2-GPI antibodies shorten PT (with possible
implications between this phenomenon and the occurrence of
thromboembolic events) is difficult to explain. Our data show that
a2-GPI-mediated PT shortening exhibits the following
characteristics: (1) it is dose-dependent; (2) it requires the presence
of the plasma antigen, 2-GPI; (3) it is not dependent on the type of thromboplastin; (4) it disappears upon dilution of TF and the template
PL through which these antibodies recognize their
antigen11,24; and (5) it is abolished when calcium ions are
added last to initiate clotting. Therefore, a2-GPI, 2-GPI, and PL
in the presence of calcium ions accelerate thrombin formation when the
activator of coagulation is TF.
Some investigators suggest that a2-GPI antibodies determine a
PL-dependent reduction in the activity of tissue factor pathway inhibitor (TFPI).29 Given that extrinsic
factor Xa is more efficiently generated when 2-GPI,
TFPI, and anionic PL are all present,29 the accumulated
findings led to the hypothesis that anti2GPI-2GPI complexes
formed in the presence of anionic PL might inhibit TFPI by impeding the
building of the quaternary complex (TFPI/FXa/FVIIa/TF). Our data focus
attention on the tissue factor pathway of coagulation to explain the
paradox that `in vitro' anticoagulant antibodies have an `in vivo'
procoagulant effect.
The `in vitro' anticoagulant effect of a2-GPI antibodies is in
agreement with the observation that binding of a2-GPI/2-GPI complexes to PL membranes severely impairs the absorption or hinders the lateral mobility and activation of clotting
factors.12,30 It was recently shown that increasing ionic
strength and calcium ions markedly inhibit the binding of 2-GPI to
anionic PL in comparison with coagulation proteins30 and
that 2-GPI/a2GPI complex formation on the PL surface (plannar
membrane, PS/PC 20/80) is reduced in the presence of physiological
concentrations of calcium ions.31 In the same way, the
present study showed that the LA of a2-GPI IgG was no longer evident
when the dRVVT was modified by adding PL in the presence of calcium
ions or in any situation in which 2-GPI (ie, plasma), PL and
a2-GPI antibodies were allowed to interact in the presence of
calcium ions.
It was recently reported that aPL antibodies that induce APS are able
to stimulate mononuclear cells to produce a procoagulant activity (PCA)
that resembles tissue factor (TF).32 Interestingly, upregulation of the TF pathway with elevated circulating levels of TF
and TFPI was demonstrated in patients with APS, thus implicating the TF
pathway in the pathogenesis of aPL-related thrombosis.33 Our observation that TF is able to induce a procoagulant effect in
normal plasma in the presence of a2-GPI might be relevant in
relation of the increased circulating levels of TF shown in these
patients. On the contrary, TF produced by stimulated normal monocytes
did not produce a substantial procoagulant effect, with only 2 of 6 a2-GPI IgG preparations producing a mild reduction in PT in
comparison to normal IgG.
| |
FOOTNOTES |
Submitted January 28, 1999; accepted July 21, 1999.
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 V. Pengo, MD, Department of Clinical and
Experimental Medicine, Thrombosis Center, University of Padova School
of Medicine, `Ex Busonera' Hospital, via Gattamelata 64, I-35128
Padova, Italy; e-mail: pengo{at}ux1.unipd.it.
| |
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TOP
ABSTRACT
INTRODUCTION
