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Antiphospholipid Antibodies Accelerate Plasma Coagulation by Inhibiting
Annexin-V Binding to Phospholipids: A ‘‘Lupus Procoagulant’’ Phenomenon
By Jacob H. Rand, Xiao-Xuan Wu, Harry A.M. Andree, J.B. Alexander Ross, Elena Rusinova,
Mayra G. Gascon-Lema, Cesare Calandri, and Peter C. Harpel
The antiphospholipid syndrome is a thrombophilic condition
marked by antibodies that recognize anionic phospholipidprotein cofactor complexes. We recently reported that exposure to IgG fractions from antiphospholipid patients reduces
the level of annexin-V, a phospholipid-binding anticoagulant
protein, on cultured trophoblasts and endothelial cells and
accelerates coagulation of plasma exposed to these cells.
Therefore, we asked whether antiphospholipid antibodies
might directly reduce annexin-V binding to noncellular phospholipid substrates. Using ellipsometry, we found that antiphospholipid IgGs reduce the quantity of annexin-V bound
to phospholipid bilayers; this reduction is dependent on the
presence of b2-glycoprotein I. Also, exposure to plasmas
containing antiphospholipid antibodies reduces annexin-V
binding to phosphatidyl serine-coated microtiter plates, frozen thawed washed platelets, activated partial thromboplastin time (aPTT) reagent and prothrombin time reagent and
reduces the anticoagulant effect of the protein. These studies show that antiphospholipid antibodies interfere with the
binding of annexin-V to anionic phospholipid and with its
anticoagulant activity. This acceleration of coagulation, due
to reduced binding of annexin V, stands in marked contrast
to the ‘‘lupus anticoagulant effect’’ previously described in
these patients. These results are the first direct demonstration of the displacement of annexin-V and the consequent
acceleration of coagulation on noncellular phospholipid surfaces by antiphospholipid antibodies.
r 1998 by The American Society of Hematology.
T
annexin-V binding to noncellular phospholipid-coated surfaces
and to phospholipid preparations used for conventional clinical
coagulation tests.
We therefore investigated the effects of aPL IgG preparations
and aPL plasmas on annexin-V adsorption to phospholipidcoated surfaces. We also studied the effects of aPL IgG fractions
and plasmas on the binding of annexin-V and coagulation on
frozen thawed washed platelets (as prepared for the activated
partial thromboplastin time [aPTT] test known as the ‘‘platelet
neutralization procedure’’) using methods similar to those we
had previously used with cultured trophoblasts and endothelial
cells.10 We then moved to studies using readily available
phospholipid reagents used for routine clinical coagulation
reactions.
HE PRESENCE of antibodies in blood, which recognize
anionic phospholipids or anionic phospholipid-protein
complexes, has been associated with a thrombophilic syndrome,
the antiphospholipid antibody (aPL) syndrome. This syndrome
is characterized by arterial and venous thrombosis or by
recurrent pregnancy loss, attributed to placental thrombosis.1-4
Paradoxically, these antibodies may manifest as ‘‘lupus anticoagulants’’ in vitro by inhibiting phospholipid-dependent blood
coagulation.3,5 Yet, in vivo, these ‘‘anticoagulants’’ are associated with thrombotic manifestations, and not with bleeding
disorders.
Annexin-V (placental anticoagulant protein-I, vascular anticoagulant-a) has potent anticoagulant properties in vitro that
are based on its high affinity for anionic phospholipids and its
capacity to displace coagulation factors from phospholipid
surfaces.6 We previously reported that annexin-V, which is
normally present on the apical surface of placental syncytiotrophoblasts,7 is markedly reduced on apical membranes of
placental villi from aPL patients.8,9 Also, IgG fractions from
aPL patients reduce the quantity of annexin-V on cultured
trophoblasts and endothelial cells and accelerate the coagulation
of plasma exposed to these cells.10 The latter findings raised the
possibility that cultured cells might not be necessary to observe
this effect and that aPL antibodies might similarly reduce
From the Department of Medicine, the Divisions of Hematology and
Thrombosis, and the Department of Biochemistry, Mount Sinai School
of Medicine, New York, NY.
Submitted November 20, 1997; accepted May 4, 1998.
Supported in part by Grants No. HL-29019 and AI-24671 from the
National Institutes of Health.
Address reprint requests to Jacob H. Rand, MD, Hematology
Division, Mount Sinai School of Medicine, Box 1079, 5 E 98th St, New
York, NY 10029; e-mail:j_rand @ smtplink.mssm.edu.
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.
r 1998 by The American Society of Hematology.
0006-4971/98/9205-0025$3.00/0
1652
MATERIALS AND METHODS
Sources of IgGs and plasmas. IgG antibodies were isolated from
the citrated plasma of three patients with severe antiphospholipid
syndrome (previously described in Rand et al10) and three normal
control subjects using protein G as described.11 For studies with
plasmas, citrated specimens were collected from 10 patients with aPL
syndrome and 10 non-aPL controls at Mount Sinai Medical Center. The
IgGs and plasmas were tested for the presence of antibodies against
b2-glycoprotein I (b2-GPI), prothrombin and annexin-V with standardized nitrocellulose (Schleicher & Schuell, Keene, NH) dot-blots containing varying quantities of the proteins, up to 1 µg. All three of the purified
aPL IgGs recognized b2-GPI directly, one of the three recognized
purified human prothrombin, and none recognized annexin-V directly.
Of the 10 aPL plasmas, nine contained immunoglobulin that recognized
b2-GPI, one recognized prothrombin alone, and none recognized
annexin-V directly.
To provide the same standard plasma for the second stage of
coagulation tests, plasmas from three normal blood bank donors were
pooled, aliquotted and stored at 240°C for all coagulation studies.
Annexin-V. Annexin-V was purified from human placentas as
described.12 The identity of the protein was confirmed by immunoblot
analysis using a previously characterized affinity-purified monospecific
polyclonal rabbit anti-annexin–V IgG.7
For studies of annexin-V binding to phosphatidyl serine-coated
microtiter plates (below), the protein was labeled with biotin as
previously described.13 Annexin-V was dialyzed at 4°C against buffer
Blood, Vol 92, No 5 (September 1), 1998: pp 1652-1660
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INHIBITION OF ANNEXIN-V BINDING BY aPL ANTIBODIES
containing 0.05 mol/L boric acid, 0.1 mol/L NaCl, pH 8.5. Biotin-NHS
(Calbiochem-Novabiochem Corp, La Jolla, CA) was then added to the
annexin-V at a 1:2 molar ratio of annexin-V to biotin-NHS. The reaction
was performed for 30 minutes at 4°C and quenched with 10 mmol/L
glycine. The biotinylated annexin-V was then dialyzed against Trisbuffered saline (TBS) buffer (0.05 mol/L Tris, 0.1 mol/L NaCl, pH 7.4).
The concentration of biotinylated annexin-V was determined by
absorbance at 280 nm and aliquots were stored at 270°C.
Ellipsometry studies. We studied the effects of aPL antibodies on
phospholipid-bound annexin-V with computer-assisted ellipsometry
using methods similar to those previously published,6 as follows: planar
phospholipid bilayers were applied to silicon slides as described.14,15 A
5-mmol/L vesicle mixture of 30% 1,2-dioleoyl-sn-glycero-3-phosphatidylserine and 70% 1,2-dioleoyl-sn-glycero-3-phosphocholine (PS/PC)
(Avanti Polar-Lipids, Inc, Alabaster, AL) was dried under nitrogen and
sonicated (Sonic Dismembrator model F60; Fisher Scientific, Pittsburgh, PA) in HEPES buffer (0.01 mol/L HEPES, 0.14 mol/L NaCl, pH
7.5) at 0°C until the suspension was completely clear. Silicon slides of 1
by 4 cm and 0.4 mm thickness were cut from silicon wafers (Wacker
Chemie, Munich, Germany, n-type, phosphor-doped). The slides were
thoroughly cleaned with detergent (Sparkleen; Fisher Scientific) and
water. They were then kept overnight in 30% chromic sulfuric acid,
flushed with water, and stored in 50% alcohol-water until use, when
they were rinsed with distilled water and then dipped into a container
containing stirring HEPES buffer composed of 0.01 mol/L HEPES, 0.14
mol/L NaCl, 0.1% bovine serum albumin (BSA), 5 mmol/L CaCl2, pH
7.5. The sonicated PS/PC vesicles (final concentration 50 µmol/L) were
then added and stirred for 10 minutes. Each slide was flushed with the
latter HEPES buffer and transferred to an ellipsometer cuvette containing the stirring buffer. Adsorption of annexin-V (2 µg/mL), (b2-GPI) (2
µg/mL, kindly provided by Dr K. McCrae, Temple University, Philadelphia, PA) and IgG preparations (100 µg/mL) to the phospholipid
bilayers on the silicon slide were observed after the serial addition of
each of the proteins. The mass of the PS/PC bilayer (<0.4 µg/cm2) was
measured and subtracted for these curves. After adsorption had reached
equilibrium, residual annexin-V was desorbed from the phospholipid
bilayers on the silicon slide by the addition of 0.5 mol/L EDTA (to a
final concentration of 6 mmol/L) and measured. Addition of EDTA had
no effect on the adsorptions of IgGs or b2-GPI, alone or in combination.
The completeness of desorption of annexin-V from the surface by
EDTA was further confirmed by subsequently solubilizing the phospholipid bilayers with 0.1% sodium dodecyl sulfate (SDS). The SDSsolubilized samples were checked for annexin-V by standard immunoblot with a monospecific polyclonal rabbit anti–annexin-V IgG following
0.1% SDS-12% polyacrylamide gel electrophoresis, as described.7
Annexin-V binding to phosphatidyl serine-coated microtiter plates.
Microtiter plates (Nunc-Immuno Plate, MaxiSorp Surface) (Fisher
Scientific) were coated with phosphatidyl serine (PS) (Avanti PolarLipids, Inc) as previously described.16,17 Citrated plasma samples (50
µL) were added to each well in duplicate and incubated for 30 minutes
at room temperature. The wells were emptied and washed four times
with phosphate-buffered saline (PBS) buffer, pH 7.4. 50 µL of biotinylated-annexin V (1 µg/mL in TBS buffer containing 0.1 % BSA and 5
mmol/L CaCl2, pH 7.4) was then added to each well and incubated for
30 minutes at room temperature. The wells were washed four times with
PBS buffer, followed by 50 µL of phosphatase-labeled streptavidin (0.5
µg/mL in TBS buffer containing 0.1 % BSA) (Kirkegaard & Perry
Laboratories, Gaithersburg, MD), which was incubated for 30 minutes
at room temperature. The wells were then emptied and washed four
times with PBS buffer, pH 7.4. A total of 50 µL of p-nitrophenyl
phosphate substrate (1 mg/mL in DEA buffer) (Sigma Chemical Co, St
Louis, MO) was added and incubated for approximately 30 minutes at
room temperature, after which the optical absorbance was read at 405
nm with a kinetic microplate reader (Molecular Devices, Menlo Park, CA).
1653
Platelets. Platelets were prepared for coagulation studies after the
method previously described for the platelet aPTT test (‘‘platelet
neutralization procedure’’).18 Pooled platelets from blood bank donors
were washed three times in TBS buffer (0.15 mol/L NaCl, 0.02 mol/L
Tris) containing 1 mg/mL glucose, pH 7.4, resuspended to a density of
2 3 105/µL in the buffer, aliquotted, and stored at 270°C for all studies.
Annexin-V assays and coagulation studies with washed platelets.
The effects of aPL IgGs on the quantity of platelet-associated annexin-V
were performed with modified methods similar to those described for
cultured trophoblasts and endothelial cells.10 aPL and control IgGs were
added to normal citrated plasma to a final concentration of 5 mg/mL.
Aliquots of frozen thawed washed platelets (1 3 108) were incubated
with 100 µL of the plasmas at 4°C for 2 hours, as previously described.10
The platelets were washed three times and resuspended in HEPES
buffer containing 5 mmol/L CaCl2, pH 7.4. Annexin-V was then added
to the platelets in the HEPES buffer containing 5 mmol/L CaCl2 to a
final concentration of 20 µg/mL (determined after pilot studies with
varying concentrations of annexin-V) and incubated at 4°C for an
additional 15 minutes. The platelets were then centrifuged and washed
three times in the same buffer. After the final centrifugation, to
dissociate annexin-V, the platelets were resuspended in the HEPES
buffer containing 1 mmol/L EGTA. The quantity of platelet-associated
annexin-V was determined by an enzyme-linked immunosorbent assay
(ELISA),10 as described. The results of the assays were expressed as ng
of annexin-V/106 platelets.
We investigated the effects of aPL IgG on coagulation using platelets
exposed to aPL or control IgG. For these experiments the IgGs were
added to pooled normal plasma to a concentration of 5 mg/mL. Thawed
washed platelets (120 µL), at a density of 2 3 105/µL in the TBS buffer
described above, were added to 120 µL of IgG-containing plasma and
preincubated for 10 minutes at 37°C. The platelets were centrifuged,
washed two times, and resuspended in 120 µL of the TBS buffer. A
50-µL volume of the platelet suspension was then added to 50 µL of
pooled normal plasma, incubated for 30 seconds at 37°C in a ST4
Coagulation Instrument (American Bioproducts Co, Parsipanny, NJ). A
total of 50 µL of Celite (5 g/L in TBS buffer consists of 0.05 mol/L Tris,
0.1 mol/L NaCl, pH 7.4) was added and the mixture was incubated for
60 seconds. A volume of 50 µL of 0.02 mol/L CaCl2 or 0.02 mol/L
CaCl2 containing annexin-V at 20 µg/mL was then added, the times
until clot formation were measured, and the mean times of duplicate
tests were reported. Coagulation times of specimens without and with
annexin-V were recorded, along with the net prolongations (ie, coagulation time in the presence of annexin-V minus coagulation time in the
absence of annexin-V), which reflected the anticoagulant activity of the
annexin-V.
Annexin-V assays and coagulation studies with aPTT reagent. We
have studied the effects of aPL IgGs on the quantity of annexin-V
associated with aPTT reagent-phospholipid (Actin FS) (Baxter Diagnostics Inc, Deerfield, IL). A total of 100 µL of the aPTT reagentphospholipid was incubated with 100 µL of aPL patient plasma or
control plasma for 15 minutes at 37°C. The mixture of aPTT reagentphospholipid and plasma was sedimented with a microcentrifuge
(Model 5451; Brinkmann Instruments, Inc, Westbury, NY) at 15,000g
for 20 minutes at room temperature. The plasma-treated–phospholipid
pellets were washed three times with HEPES buffer (0.01 mol/L
HEPES, 0.14 mol/L NaCl, 0.1% BSA, pH 7.4) and resuspended in
HEPES buffer containing 5 mmol/L CaCl2. Annexin-V was then added
into the phospholipid suspension to a final concentration of 2 µg/mL and
incubated at 37°C for 10 minutes. After centrifugation, the phospholipid
pellets were washed three times in HEPES buffer containing 5 mmol/L
CaCl2. The phospholipid-associated annexin-V was then dissociated
from the aPTT reagent-phospholipid with HEPES buffer containing 5
mmol/L EDTA and assayed by ELISA as described above. The results
of the assays were expressed as nanograms of annexin-V/50 µL aliquot
of aPTT reagent-phospholipid.
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1654
The effect of aPL IgG on coagulation with aPTT reagentphospholipid was determined using a two-stage assay, in which the first
stage exposed phospholipid to potential aPL antibodies in the test
plasma, and the second stage measured coagulation times with the
phospholipid using a pooled normal plasma. We designed this assay
using the pooled normal plasma for the second stage to deal with the
variable baseline coagulation times of individual patients. A total of 50
µL of the aPTT reagent-phospholipid was added to 50 µL of aPL patient
or control citrated plasma. The mixture was sedimented with the
microcentrifuge, as described above. The pellets were then washed once
in the TBS buffer and resuspended in 220 µL of this buffer. A total of 50
µL of this plasma treated-phospholipid suspension was added to 50 µL
pooled normal plasma and incubated for 120 seconds at 37°C in the ST4
Coagulation Instrument, after which 50 µL of 0.02 mol/L CaCl2 or 0.02
mol/L CaCl2 containing annexin-V (30 µg/mL) was added. The times
until clot formation of duplicate specimens were monitored and
reported, as described for the platelets above.
Annexin-V assays and coagulation studies with prothrombin time
reagent. The effect of aPL IgG on the quantity of annexin-V
associated with prothrombin time (PT) reagent (tissue factorphospholipid complex) was also studied. PT reagent (Thromboplastin-C Plus; Baxter Diagnostics Inc) was washed three times with TBS
buffer by sedimentaion and resuspension with the microcentrifuge for
20 minutes at 15,000g, as described for aPTT reagent-phospholipid. A
total of 100 µL of the washed phospholipid of PT reagent was incubated
with 100 µL of aPL or control IgG-containing plasma (at a concentration
of 5 mg/mL, prepared as for the platelets described above) for 15
minutes at 37°C. The mixture was then centrifuged and the pellets of
IgG-treated PT reagent-phospholipid were then treated in the same way
as described above for aPTT reagent-phospholipid. The phospholipidassociated annexin-V was dissociated from the PT reagent-phospholipid by the HEPES buffer containing 5 mmol/L EDTA and was assayed
by ELISA, as described above. The results were reported as described
for the aPTT reagent above.
To investigate the effects of aPL IgG on coagulation with PT reagent,
a two-stage assay, similar to the aPTT assay described above, was used.
The PT reagent (50 µL) was washed three times in TBS buffer to remove
calcium and was added to 50 µL of aPL patient or control plasma. The
mixtures were centrifuged as described above and the pellets were
washed once in the TBS buffer and resuspended in 220 µL of this buffer.
A total of 50 µL of the suspension was then added to 50 µL pooled
normal plasma and incubated for 120 seconds at 37°C in the ST4
Coagulation Instrument. A 50-µL volume of 0.02 mol/L CaCl2 or 0.02
mol/L CaCl2 containing annexin-V (30 µg/mL) was added. The times
until clot formation of duplicate specimens were monitored and
recorded as described above.
Fluorimetric determination of annexin-V binding to phospholipid.
The effect of aPL plasma on the binding of fluorescein isothiocyanate
(FITC)-conjugated annexin-V to phospholipid was also determined. A
total of 50 µL of aPTT reagent-phospholipid was added to 50 µL of aPL
or control plasma and sedimented in the microcentrifuge as described
above. The pellets of plasma-treated–phospholipid were washed once in
the TBS buffer, pH 7.4, and then resuspended in 55 µL of the above
buffer containing 5 mmol/L CaCl2 and 1 µg/mL of FITC-conjugated
annexin-V (Clontech Laboratories Inc, Palo Alto, CA). The mixture was
incubated for 5 minutes at room temperature and centrifuged as
described above, after which the supernatants were collected and the
pellets were resuspended in 55 µL of the TBS buffer containing 10
mmol/L EDTA. The levels of labeled annexin V in the supernatant and
on the aPTT reagent-phospholipid were measured in terms of relative
fluorescence intensity with an SLM/Amico SPF-500C spectrofluorimeter (Milton Co, Rochester, NY). The samples were contained in 50 µL
quartz microcuvettes, thermostated at 20°C. Excitation was at 490 nm
with a 4-nm band-pass and emission was detected at 523 nm with a
20-nm band-pass. The results were reported as relative fluorescence
RAND ET AL
units (RFU). A standard curve of fluorescence intensity versus FITCannexin-V concentration was linear between 0 and 2 µg/mL of protein.
Statistical analysis. Statistical analyses were performed using the
Student’s two-tailed t-test.
RESULTS
Ellipsometry studies. The quantity of annexin-V adsorbed
to PS/PC phospholipid bilayers on silicon slides was determined by ellipsometry, and the amount of annexin-V remaining
on the phospholipid bilayers after addition of IgG preparations
was determined by adding EDTA to desorb the residual
annexin-V. All of the annexin-V, which bound to the phospholipid surface, was subsequently desorbed by addition of the
EDTA (Fig 1A). Because the adsorptions of IgGs and b2-GPI
were not affected by EDTA, we were able to use EDTA-induced
desorption to measure the amount of annexin-V remaining on
the phospholipid surface after addition of aPL IgG antibodies.
Measurement of the binding of three pairs of aPL IgG from aPL
patients to the planar phospholipid bilayers of PS/PC-bound
annexin-V showed that aPL IgG binding to the phospholipid
bilayers, in the presence of b2-GPI cofactor, displaced 0.115 6
0.014 µg/cm2 (mean 6 standard error of mean [SEM]) annexin-V from the phospholipid surface (Figs 1C and 2). In
contrast, control IgG with the cofactor did not displace annexin-V (0.005 6 0.007 µg/cm2, P 5 .002) (Fig 1D and 2).
Also, aPL IgG without the cofactor did not reduce adsorbed
annexin-V (Fig 1B). Immunoblotting of SDS-solubilized slides
after the EDTA desorption showed no residual annexin-V (data
not shown).
Annexin-V binding to phosphatidyl serine-coated microtiter
plates. PS-coated microtiter plates treated with aPL plasmas
bound significantly less biotin-labeled annexin-V than PScoated microtiter plates, which had been treated with control
plasmas (Fig 3). The mean optical density (OD; 6SEM) of the
aPL plasma-treated wells was 0.085 6 0.003 and 0.123 6 0.005
for the wells teated with control plasmas (n 5 10 for each
group, P , .0001).
Platelets. Frozen thawed washed platelets, which had been
incubated with annexin-V after preincubation with aPL IgG
preparations from three different patients, had significantly less
annexin-V bound to their surfaces (mean 6 SEM, 0.89 6 0.12
ng/106 platelets) than platelets, which had been preexposed to
control IgGs (2.01 6 0.38 ng/106 platelets, P 5 .05; n 5 3) (Fig
4A). Although annexin-V increased the coagulation times with
both aPL and control IgG treatments, the protein had less of an
anticoagulant effect with aPL IgG-treated platelets, ie, there was
significantly less prolongation with thawed washed platelets,
which had been preincubated with aPL IgG (mean 6 SEM,
33.2 6 0.9 seconds longer than coagulation time in absence of
annexin-V) compared with control IgG (50.4 6 4.1 seconds
longer than coagulation time in absence of annexin-V, P 5 .01;
n 5 3) (Fig 4B).
aPTT reagent. We found that aPTT reagent-phospholipid
preexposed to aPL plasma bound significantly less annexin V
(mean 6 SEM, 318 6 28 ng/50 µL aliquot of reagent) than
controls (656 6 80 ng/50 µL aliquot of reagent, P 5 .01; n5 4)
(Fig 5A). We then designed a two-stage test, in which aPTT
reagent-phospholipid was first incubated with individual test
plasma, then washed and, for the second stage, exposed to a
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INHIBITION OF ANNEXIN-V BINDING BY aPL ANTIBODIES
1655
Fig 1. Ellipsometry studies of effects of aPL IgG and cofactor on displacement of annexin-V from PS/PC phospholipid bilayers. (A) Shows the
rapid adsorption of annexin-V to the PS/PC (30%/70%) phospholipid bilayer. Treatment with EDTA and measurement of the desorption of this
protein can be used to measure the amount of annexin-V on the phospholipid surface. As shown, this calcium-dependent binding protein is
completely desorbed from the phospholipid surface by addition of 6 mmol/L EDTA. (B) Shows that incubation of the annexin-V–coated
phospholipid bilayer with a polyclonal human aPL IgG in the absence of b2-GPI does not displace the annexin-V, ie, the quantity of annexin-V
desorbed after treatment with EDTA matches the quantity of annexin-V, which had originally adsorbed. (C) Incubation of the annexin-V–coated
phospholipid bilayer with b2-GPI followed by polyclonal aPL IgG results in a significant reduction of the quantity of annexin-V on the bilayer. This
is reflected by the marked reduction of the amount of annexin-V, which desorbs after treatment with EDTA. (D) In contrast, treatment of the
phospholipid bilayer with the b2-GPI cofactor followed by a control (non-aPL) IgG fraction does not change the quantity of annexin-V on the
phospholipid surface at all, ie, the quantity of annexin-V that is desorbed by treatment with EDTA is the same as the quantity that had been
adsorbed in the first place.
pooled normal plasma, which was then recalcified in the
presence and absence of added annexin-V. We found that
preexposure of aPTT reagent-phospholipid to plasmas from aPL
syndrome patients significantly accelerated the coagulation of
pooled normal plasma in the presence of annexin-V (mean 6
SEM, 89.2 6 2.2 seconds) as compared with control plasmas
(mean 6 SEM, 102.5 6 2.6 seconds; P 5 .001, n 5 10) (Fig
5B). Also, there was a commensurate decrease in the annexin-V–
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1656
Fig 2. Quantitative displacement of annexin-V from the PS/PC
phospholipid bilayers by aPL IgG preparations in the presence of
b2-GPI cofactor. The combination of aPL IgG with b2-GPI significantly
displace annexin-V from the bilayers as compared with control IgG
with b2-GPI. The mean (6SEM) quantity of annexin-V displaced by
three different aPL syndrome patients’ IgG fractions was 0.115 6
0.014 mg/cm2 as compared with no significant displacement by three
different control IgG preparations (0.005 6 0.007 mg/cm2, P 5 .002).
These data demonstrate the displacement of annexin-V from the
phospholipid bilayer surface by aPL IgG in the presence of b2-GPI.
induced anticoagulant effect, as assessed by prolongation of the
coagulation time, of the aPL treated reagent as compared with
the reagent, which had been preincubated with control plasma
(mean 6 SEM, 13.6 6 1.8 seconds for aPL patients and 23.1 6
0.8 seconds for controls, P 5 .0002) (Fig 5B).
Fig 3. Annexin-V binding to microtiter plates coated with phosphatidyl serine (PS). PS-coated microtiter plate wells treated with aPL
plasmas bound significantly less biotin-labeled annexin-V than PScoated microtiter plate wells, which had been treated with control
plasmas. Annexin-V was detected by addition of phosphataselabeled streptavidin followed by p-nitrophenyl phosphate substrate.
The mean OD (6SEM) of the aPL plasma-treated wells was 0.085 6
0.003 and 0.123 6 0.005 for the wells treated with control plasmas
(n 5 10 for each group, P F .0001).
RAND ET AL
Prothrombin time reagent (tissue factor-phospholipid).
Similar experiments with tissue factor-phospholipid suspensions (PT reagent) also showed significantly less binding of
annexin V to PT reagent-phospholipid, which had been preexposed to aPL IgG fractions (mean 6 SEM, 82 6 4 ng/50 µL
aliquot of reagent) than controls (110 6 1 ng/50 µL aliquot of
reagent, P 5 .02, n 5 3) (Fig 6A). In the presence of annexin-V,
PT reagent, which had been preexposed to aPL plasmas,
accelerated the subsequent coagulation of pooled normal plasma
(mean 6 SEM, 35.0 6 0.8 seconds) as compared with controls
(38.3 6 1.2 seconds, P 5 .03, n 5 10) (Fig 6B). There was a
corresponding decrease in the annexin-V–induced anticoagulant effect with PT reagent, which had been preincubated with
aPL plasma (mean 6 SEM, 10.3 6 0.8 seconds) as compared
with controls (15.2 6 1.2 seconds, P 5 .004). In contrast, in the
absence of annexin-V, aPL-treated prothrombin time reagent
caused a small but significant slowing of coagulation compared
with prothrombin time reagent, which had been preincubated
with control plasma (mean 6 SEM, 24.7 6 0.5 seconds for
aPL-treated reagent compared with 23.1 6 0.1 seconds for
controls, P 5 .003) (Fig 6B).
FITC-Annexin-V binding to phospholipid. We found that
preexposure of aPTT reagent-phospholipid to aPL plasma
significantly reduced the amount of FITC-conjugated annexin-V, which subsequently bound to the phospholipid (mean 6
SEM, 0.083 6 0.008 RFU) as compared with control plasmas
(0.131 6 0.015 RFU, P 5 .01, n 5 10) (Fig 7), and increased
the amount of labeled annexin-V in the supernatant (mean 6
SEM, 0.070 6 0.008 RFU for aPL exposed phospholipid and
0.046 6 0.005 RFU for control plasmas, P 5 .02) (Fig 7).
DISCUSSION
We recently described a new thrombogenic mechanism in the
aPL syndrome—that IgG fractions from aPL patients reduce the
quantity of annexin-V on cultured trophoblasts and endothelial
cells and accelerate the coagulation of plasma added to these
cells.10 In this report, we extend our previous observations with
cultured cells and demonstrate that cultured cells are not
necessary to observe this effect, which also occurs with
phospholipid-coated surfaces, frozen thawed washed platelets,
and ordinary phospholipid suspensions used for conventional
clinical coagulation tests, which were treated similarly to the
cells in our previous study.10 It should be noted that in the
present study, the experiments with platelets were not designed
to study unactivated and activated platelets, but rather platelet
phospholipid for coagulation testing, as previously described.18
Using ellipsometry, we provide direct evidence that aPL IgG
preparations, in the presence of b2-GPI, displace annexin-V
from PS/PC phospholipid bilayers. Interestingly, both the aPL
IgG and b2-GPI are necessary for the displacement to occur. In
accordance with this finding, incubating PS-coated microtiter
plate wells with aPL patient plasmas also results in the
significant reduction of the quantity of labeled annexin-V,
which binds to that surface.
Similarly, exposing different sources of phospholipids used
for coagulation reactions, such as washed frozen thawed platelet
suspensions, aPTT reagent, and prothrombin time reagent, to
aPL antibodies reduces the quantity of annexin-V, which binds
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INHIBITION OF ANNEXIN-V BINDING BY aPL ANTIBODIES
1657
Fig 4. The effects of aPL IgG on the quantity of platelet surface annexin-V and plasma coagulation. (A) Washed human platelets were exposed
to three different aPL and control IgG preparations in plasma, following which the platelets were incubated with annexin-V (20 mg/mL), in the
presence of calcium, as described in Materials and Methods. Surface annexin-V was then dissociated with EGTA and measured by ELISA.
Platelets preexposed to aPL IgG had significantly less annexin-V on their surfaces (mean 6 SEM, 0.89 6 0.12 ng/106 platelets) as compared with
controls (2.01 6 0.38 ng/106 platelets, P 5 .05). (B) Plasma coagulation times were determined using platelets, which had been preexposed to the
aPL and control IgGs in plasma. The platelets were added to pooled normal plasma, which was recalcified in the presence and absence of added
annexin-V (20 mg/mL). Annexin-V lengthened the coagulation times of pooled normal plasma with both control and aPL IgG-treated platelets.
The net prolongation, compared with the coagulation time without annexin-V, was significantly less with the aPL-treated platelets (mean
prolongation 6 SEM, 33.2 6 0.9 seconds) as compared with controls (50.4 6 4.1 seconds, n 5 3, P 5 .01).
to these phospholipid surfaces. As with the previously reported
cell culture experiments,10 the aPL antibodies also accelerate
coagulation in the presence of annexin-V, reducing the potent
anticoagulant activity of this phospholipid-binding protein.
These results, which demonstrate accelerated coagulation by
aPL antibodies when annexin-V is present in coagulation
reactions, stand in contrast to the ‘‘lupus anticoagulant’’ phenomenon, which was first described in 1952.19 To our knowledge,
our results are the first direct demonstration of the displacement
of annexin-V and the acceleration of coagulation on noncellular
phospholipid surfaces by antiphospholipid antibodies and provide a methodology, which may prove suitable for clinical
testing. Because annexin-V has the highest affinity for phospholipid among the various members of the annexin family,20 we
speculate that antiphospholipid antibodies may also have a
similar effect on displacing the lower affinity annexins, such as
annexin-II, which might also have antithrombotic properties,21
from phospholipids. Previously, using a different system that
investigated the inhibition of activated protein C by aPL
antibodies, Smirnov et al22 were able to show accelerated
clotting in some aPL plasmas. They found that clotting initiated
by factor X activating enzyme from Russell’s viper venom was
faster in some aPL patient plasma when high concentrations of
activated protein C and phosphatidyl ethanolamine were present.
Because aPL antibodies are capable of reducing the binding
of annexin-V to anionic phospholipids in vitro and in cell
culture, we propose that this effect may occur in the in vivo
situations and processes where anionic phospholipids are exposed to flowing blood. These include the surface of placental
villi,23 activated platelets,24 apoptotic cells and cellular particles,25 and disrupted erythrocyte membranes.26 In all of these
settings, the presence of aPL antibodies could inhibit the
binding of circulating annexin-V, which would in turn result in a
membrane surface, which is more thrombogenic.
In light of these data, how are we to understand the
prothrombotic effect of the aPL antibodies? Even if aPL
antibodies displace annexin-V from the phospholipid surface,
what difference ought it make whether the phospholipid is
bound by annexin-V or by aPL antibody-cofactor complexes, ie,
Fig 5. The effects of aPL plasmas on annexin-V bound to aPTT reagent-phospholipid and plasma coagulation with this reagent. (A) aPTT
reagent-phospholipid was exposed to four different aPL and control plasmas and then to annexin-V (2 mg/mL), after which surface annexin-V
was dissociated with EDTA and measured by ELISA. aPTT reagent-phospholipid, which had been preexposed to aPL-plasmas, had significantly
less annexin-V (mean 6 SEM, 318 6 28 ng/50 mL aliquot of reagent) as compared with controls (656 6 80 ng/50 mL aliquot of reagent, P 5 .01). (B)
Plasma coagulation times were determined using aPTT reagent-phospholipid exposed to the aPL and control plasmas (n 5 10 for each group) in
the first stage and then in the second stage, to pooled normal plasma in the presence and absence of added annexin-V (30 mg/mL). Annexin-V
delayed the coagulation times of aPTT reagent exposed to both types of plasmas. In the presence of annexin-V, the coagulation times of the aPTT
reagent, which had been preexposed to aPL-plasma, was significantly faster (mean 6 SEM, 89.2 6 2.2 seconds) than reagent exposed to the
control plasma (102.5 6 2.6 seconds, P 5 .001). Also, there was a significant decrease in the net prolongation of the coagulation times induced by
annexin-V (mean 6 SEM, 13.6 6 1.8 seconds for aPL plasmas versus 23.1 6 0.8 seconds for controls, P 5 .0002).
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1658
RAND ET AL
Fig 6. The effects of aPL plasmas on annexin-V bound to prothrombin time reagent (tissue factor-phospholipid complex) and on plasma
coagulation. (A) Prothrombin time reagent was preexposed to three different aPL and control IgG preparations in plasma and then to annexin-V
(2 mg/mL), after which surface annexin-V was dissociated with EDTA and measured by ELISA. Prothrombin time reagent preexposed to aPL
IgG-containing plasmas had significantly less annexin-V (mean 6 SEM, 82 6 4 ng/50 mL aliquot of reagent) compared with controls (110 6 1
ng/50 mL aliquot of reagent, P 5 .02). (B) Plasma coagulation times were determined using prothrombin time reagent, which was exposed to the
aPL and control plasmas (n 5 10 for each group), in the first stage and in the second stage to pooled normal plasma in the presence and absence
of annexin-V (30 mg/mL). There was a small but significant prolongation of coagulation time when the prothrombin time reagent was exposed to
the aPL plasmas in the absence of annexin-V (P 5 .003). Addition of annexin-V resulted in prolongation of coagulation times with both types of
reagent (ie, exposure to control and aPL plasmas). However, in contrast to the results without annexin-V, the coagulation times of the
prothrombin time reagent, which had been preexposed to aPL-plasma, were significantly shortened (mean 6 SEM, 35.0 6 0.8 seconds compared
with 38.3 6 1.2 seconds for control plasmas, P 5 .03). There was a concomitant significant decrease in the net prolongation of the coagulation
times using PT reagent, which had been pretreated with aPL plasma (10.3 6 0.8 seconds compared with 15.261.2 seconds for control plasmas,
P 5 .004).
shouldn’t the phospholipid still remain unavailable for coagulation reactions? The mechanism by which aPL antibodies
accelerate coagulation in the presence of annexin-V is probably
due to topographical differences between the binding of the two
ligands to phospholipid. Annexin-V binds in clusters on the
phospholipid surface,14 while aPL antibodies disrupt this carpet
of annexin-V, which shields the surface and allow enough room
on the phospholipid surface for coagulation factors such as
prothrombin to bind.27 Thus, the prothrombotic effect of the aPL
antibodies is due to their increasing the net quantity of available
anionic phospholipid for coagulation reactions by displacing
annexin-V from the surface. We propose that the paradoxical
Fig 7. The effects of aPL plasmas on the binding of FITC–
annexin-V to aPTT reagent-phospholipid. aPTT reagent was incubated with aPL and control plasmas (n 5 10 for each group), after
which 1 mg/mL of FITC–annexin-V was added. Annexin-V bound to
the aPTT reagent and in the fluid phase were quantified by spectrofluorimetry, as decribed in Materials and Methods. There was a significant decrease of the amount of annexin-V associated with the aPTT
reagent, which had been preincubated with aPL plasmas (mean 6
SEM, 0.083 6 0.008 RFU/50 mL aliquot of reagent) compared with
controls plasmas (0.131 6 0.015 RFU/50 mL aliquot of reagent, P 5
.01). In contrast, there was a significant increase in the amount of
labeled annexin-V remaining in the supernatant of aPTT reagent,
which had been preincubated with aPL plasmas (mean 6 SEM,
0.070 6 0.008 RFU/50 µL aliquot of reagent) as compared with control
plasmas (0.046 6 0.005 RFU/50 µL aliquot of reagent, P 5 .02).
‘‘lupus anticoagulant’’ phenomenon is a reflection of the effects
of the aPL antibodies in the presence of coagulation protein, but
in the absence of annexin-V; in this situation, the antibodies will
prolong coagulation by decreasing the net quantity of phospholipids available for coagulation reactions (because annexin-V is
not present). In reflecting the relative inhibitory effects of high
affinity antiphospholipid-protein complexes, in the absence of
annexin-V, to the binding of coagulation factors, we propose
that the ‘‘lupus anticoagulant’’ phenomenon may indeed serve
as a surrogate marker for antibodies whose actual pathophysiogic function lies in their capacity to displace annexin-V.
We propose the following model (Fig 8), which advances the
one we had previously described10: Annexin-V plays a physiologic role in inhibiting blood coagulation reactions on vascular
surfaces by shielding highly thrombogenic anionic phospholipids from coagulation enzyme complexes. We hypothesize that
aPL syndrome patients have antibodies with a sufficient affinity
for anionic phospholipids (and/or presumptive cofactors such as
b2-GPI) to disrupt or prevent the assembly of this annexin-V
protective shield. This results in a net increase of exposed
phospholipid and permits a more procoagulant topography to be
available for coagulation reactions. Thus, coagulation systems,
which include annexin-V, will demonstrate a relative acceleration of coagulation, a ‘‘lupus procoagulant effect’’, due to
displacement of annexin-V by aPL syndrome antibodies. In
contrast, in the absence of added annexin-V, only the relatively
inhibitory effects of the antibodies on phospholipid-dependent
coagulation reactions are observed (as compared with when
antibodies are absent), thereby resulting in the classical ‘‘lupus
anticoagulant’’ effect.
In conclusion, we have demonstrated that, in addition to their
effects on cultured trophoblasts and endothelial cells, IgGs and
plasmas from patients with the aPL syndrome also reduce
annexin-V on noncellular phospholipid-coated surfaces and
accelerate coagulation with phospholipids used for standard
clinical coagulation assays. Further studies of large numbers of
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
INHIBITION OF ANNEXIN-V BINDING BY aPL ANTIBODIES
1659
Fig 8. Model for mechanisms of the ‘‘lupus anticoagulant effect’’ and the inhibition of annexin-V and acceleration of coagulation by
antiphospholipid antibodies. (A) Anionic phospholipids (negative charges), when exposed on the apical surface of the cell membrane bilayer,
serve as potent cofactors for the assembly of three different coagulation complexes: the tissue factor (TF)-VIIa complex, the IXa-VIIIa complex and
the Xa-Va complex, and thereby accelerating blood coagulation. The TF complexes yield either factor IXa or factor Xa, the IXa complex yields factor
Xa, and the Xa formed from both of these reactions is the active enzyme in the prothrombinase complex, which yields factor IIa (thrombin), which
in turn cleaves fibrinogen to form fibrin. (B) Annexin-V, in the absence of aPL antibodies, forms clusters, which bind with high affinity to the
anionic phospholipid surface and shield the surface from the assembly of the phospholipid-dependent coagulation complexes, thereby inhibiting
coagulation reactions. (C) In the absence of annexin-V, aPL antibodies can prolong the coagulation times, compared with control antibodies, by
reducing the access of coagulation factors to anionic phospholipids. This may result in a ‘‘lupus anticoagulation effect.’’ (D) In the presence of
annexin-V, antiphospholipid antibodies, either directly or via interaction with protein-phospholipid cofactors, disrupt the ability of annexin-V to
cluster on the phospholipid surface, resulting in a net increase of the amount of anionic phospholipid available for promoting coagulation
reactions. This manifests in the net acceleration of coagulation in vitro and in thrombophilia in vivo.
well-characterized aPL and control patients will be necessary to
learn whether the methods developed in this study may be
suitable for clinical tests.
ACKNOWLEDGMENT
This article is dedicated to the memory of Dr Richard Gorlin, the
consummate physician, scientist, and teacher.
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1998 92: 1652-1660
Antiphospholipid Antibodies Accelerate Plasma Coagulation by Inhibiting
Annexin-V Binding to Phospholipids: A ''Lupus Procoagulant''
Phenomenon
Jacob H. Rand, Xiao-Xuan Wu, Harry A.M. Andree, J.B. Alexander Ross, Elena Rusinova, Mayra G.
Gascon-Lema, Cesare Calandri and Peter C. Harpel
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