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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 12 (June 15), 1999:
pp. 4248-4255
Some Anticardiolipin Antibodies Recognize a Combination of
Phospholipids With Thrombin-Modified Antithrombin, Complement
C4b-Binding Protein, and Lipopolysaccharide Binding Protein
By
Josiane Arvieux,
Gilles Pernod,
Véronique Regnault,
Luc Darnige, and
Jérôme Garin
From the ETS Grenoble, Grenoble, France; Laboratoires
d'Hématologie, CHU Grenoble, Grenoble, France and Faculté
de Médecine Nancy, Nancy, France; the Département de
Biologie Clinique, CH Compiègne, Compiègne, France; and
CEA-Grenoble, Laboratoire de Chimie des Protéines, Grenoble,
France.
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ABSTRACT |
The standard enzyme-linked immunosorbent assay (ELISA) for
anticardiolipin antibodies (ACA) detects a heterogenous group of antibodies against cardiolipin on its own,
 2-glycoprotein I (2GPI), and,
potentially, other phospholipid-binding plasma proteins from bovine or
human origin. In an attempt to identify new proteic targets of ACA, we
selected 6 patients who possessed cofactor-dependent ACA but no
antibody to human or bovine 2GPI detectable in the 2GPI-ELISA. Three of these samples proved to recognize
2GPI in combination with cardiolipin, but not
2GPI directly immobilized on  -irradiated polystyrene
or agarose beads. In the other cases, the component required for ACA
binding was purified from adult bovine serum or plasma by means of
ammonium sulfate precipitation and chromatography on Phenyl-Sepharose,
diethyl aminoethyl (DEAE)-cellulose, heparin-Ultrogel, and
Sephacryl S-300 columns. Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) analysis coupled to N-terminal amino acid
microsequencing identified the cofactors of patients no. 4, 5, and 6 ACA as lipopolysaccharide binding protein (LBP), complement C4b-binding
protein (C4BP), and the thrombin-antithrombin (AT) complex,
respectively. Adsorption of each of these cofactor preparations with
cardiolipin liposomes led to suppression of ACA reactivity, concomitant
with the loss of bands from SDS gels corresponding to sequenced
material. Bacterial lipopolysaccharide (which forms high-affinity
complexes with LBP) specifically neutralized the cofactor activity of
the LBP preparation in a concentration-dependent manner. Bovine serum
and plasma, as well as the C4BP preparation, optimally supported the
binding of a rabbit anti-C4BP antiserum to immobilized cardiolipin. The binding of a rabbit anti-AT antiserum to solid-phase cardiolipin was
sustained by the thrombin-AT preparation and bovine serum, but neither
by bovine plasma nor by native AT, thus reproducing the behavior of
patient no. 6 ACA. Taking advantage of the restricted recognition by
the latter ACA of a cofactor from bovine origin appearing upon
clotting, we studied the generation of such activity in human plasma
supplemented with bovine AT or bovine prothrombin before clotting. In
these conditions, patient no. 6 antibody binding to cardiolipin
required the addition of bovine AT, whereas addition of bovine
prothrombin alone was ineffective. We therefore concluded that those
ACA targeted bovine AT once it has been modified/cleaved by thrombin.
These findings underline the wide heterogeneity of ACA and the links
that may exist between various coagulation pathways, inflammation and
the complement system.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
ANTIPHOSPHOLIPID antibodies (aPL) consist
of a heterogenous group of autoantibodies that have different
specificities for phospholipids and phospholipid-binding plasma
proteins, especially 2-glycoprotein I
(2GPI), and prothrombin for those antibodies detectable
in standard anticardiolipin (ACA) and lupus anticoagulant (LA)
assays.1 So as to improve the reliability of ACA detection by enzyme-linked immunosorbent assay (ELISA) using solid-phase cardiolipin, the addition of 10% animal (usually bovine) serum or
plasma in the blocking buffer/patient sample diluent has long been
recommended. More recently, the serum component required was found to
be 2GPI,2,3 and its addition was reported to either enhance ACA binding (samples from autoimmune patients) or
depress it through a competition mechanism (cases of
infection-associated true ACA). The identification of the antigenic
target of most ACA associated with the antiphospholipid syndrome (APS)
also led to the development of a more straightforward and specific
assay for anti-2GPI antibodies that uses purified
2GPI in the absence of phospholipids, the
2GPI-ELISA.4
In addition to detecting antibodies to 2GPI and to
cardiolipin proper, the standard ACA assay could potentially detect
antibodies to other bovine or human serum proteins that bind to
cardiolipin-coated wells independently of calcium.1,5 For
further insight into the spectrum of ACA specificities, 6 patients were
selected whose antibodies showed an absolute serum requirement in the
ACA assay but were not detectable in the 2GPI-ELISA
using the human and bovine proteins. Three of these antibodies proved
to recognize the 2GPI-cardiolipin complex, involving
human (patient no. 1) or bovine 2GPI (patients no. 2 and
3). In the 3 other cases (patients no. 4, 5, and 6), a series of
experiments was conducted to characterize the component responsible for
ACA cofactor activity leading to the identification of
lipopolysaccharide binding protein (LBP), complement C4b-binding
protein (C4BP), and thrombin-modified antithrombin (AT), respectively.
These results demonstrate for the first time that a variety of
phospholipid binding proteins in addition to 2GPI are
involved in epitope formation of some ACA. Also, the finding of new ACA
cofactors offers a broader view of the so-called aPL, establishing
links between various coagulation pathways, inflammation, and the
complement system.
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MATERIALS AND METHODS |
Patients.
Six female patients were selected on the basis of the presence of
serum-dependent ACA in the absence of antibodies to human or bovine
2GPI detectable in the 2GPI-ELISA.
Pertinent clinical and laboratory findings of these patients are
summarized in Table 1. None fulfilled the
classification criteria for definite antiphospholipid syndrome, either
primary or secondary to systemic lupus erythematosus. Indeed, patient
no. 1 (88 years of age) presented other well-established risk factors
for ischemic stroke, including hypertension and prosthetic heart valve,
in addition to the presence of ACA. Also, the two pregnancy losses (1 fetal loss and 1 neonatal death) in patient no. 3 (now 59 years of age)
were not consecutive (2 uncomplicated pregnancies in between) and
occurred long before the search of aPL.
ELISAs for ACA and anti-2GPI antibodies.
The standard ELISA for ACA was performed using cardiolipin- or methanol
(sample blank)-coated PolySorp plates (Nunc, Roskilde, Denmark) and
10% adult bovine serum (ABS) or adult bovine plasma (ABP) in
phosphate-buffered saline (PBS) for saturation and patient serum
dilution (1:100), essentially as described.4 The cut-off points for positivity were set at 15 GPL and 15 MPL using eight standards (from the Antiphospholipid Standardization Laboratory, University of Louisville, Louisville, KY) and after testing 100 blood
donors as controls (96th percentiles).
To investigate the role of cofactors in ACA binding and monitor their
purification, the standard assay was modified by replacing bovine serum
or plasma by 0.6% gelatin (from cold water fish skin; Sigma, St Louis,
MO) as blocking agent and sample diluent. The patients'
antibody binding step was performed with PBS/gelatin alone or
containing one of the following: bovine or human 2GPI (10 µg/mL) purified from plasma,4 crude ABS or ABP (10%,
unless otherwise specified), and column fractions being analyzed for ACA cofactor activity. The lysine residues in 2GPI were
modified by carbamylation, resulting in complete loss of binding to
anionic phospholipids.6
The ELISAs for anti-2GPI antibodies were performed using
purified human and bovine 2GPI, -irradiated
polystyrene plates (Maxisorp; Nunc), and PBS/0.1% Tween buffer, as
previously described.4
Purification of the ACA cofactors.
The ACA cofactors were purified from ABP and ABS by ammonium sulfate
precipitation (25% saturation), followed by hydrophobic chromatography
of the supernatant on a Phenyl-Sepharose column equilibrated in PBS
25% saturated with ammonium sulfate, pH 7.2. The column was eluted by
a linear gradient of decreasing ammonium sulfate concentration and
simultaneously increasing ethylene glycol concentration (final
concentrations, 0% and 60%, respectively). Fractions containing ACA
cofactor activity were pooled, dialysed against 50 mmol/L Tris-HCl, pH
8, and then applied to a diethyl aminoethyl
(DEAE)-cellulose column run in this buffer. Adsorbed material was eluted by a linear gradient from 0 to 0.3 mol/L NaCl in 50 mmol/L Tris-HCl, pH 8. Again, fractions were pooled according to ACA
cofactor activity. The individual pools were adjusted to the desired
ionic strength (0.125 mol/L NaCl) with distilled water or NaCl and
applied to a heparin-Ultrogel column (IBF, Villeneuve-la-Garenne, France) washed in PBS, and bound cofactors were recovered
with a linear gradient from 0.125 to 0.5 mol/L NaCl. After
concentration on a PM-10 membrane, the material was further separated
by chromatography over Sephacryl S-300 equilibrated with PBS, and the
fractions were pooled as described before.
Protein concentrations were determined by using the BCA (Pierce,
Rockford, IL) reagents and BSA as standard.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
and amino acid sequencing.
SDS-PAGE was performed in 7.5% homogenous resolving gels and 3%
stacking gels. Samples were boiled for 2 minutes in 2% (wt/vol) SDS
containing 0.1 mol/L Tris/HCl, pH 6.8, 20% (vol/vol) glycerol, 0.2%
(wt/vol) bromophenol blue. Reduction of samples was performed in the
presence of 1% (wt/vol) dithiothreitol. Gels were stained with
Coomassie blue R250.
The N-terminal amino acid sequence analysis of proteins separated by
SDS-PAGE was performed on a Procise 492A sequencer (Applied Biosystems,
Foster City, CA) according to the manufacturer's recommendations.
Inhibition experiments.
Cardiolipin liposomes were prepared as a stock 2.5 mg/mL suspension by
vortexing dried cardiolipin in PBS. Varying amounts of liposomes were
incubated with crude ABS or ABP (1:5 dilution in PBS) or with the
purified cofactors (5 to 50 µg/mL) for 30 minutes at 20°C. After
centrifugation at 20,000g for 20 minutes, the supernatants were
assayed for residual cofactor activity in the modified ACA ELISA.
Fluid-phase lipopolysaccharide (LPS), obtained from Escherichia
coli K12 D31m4 (gift of S. Chesne, INSERM U238, Grenoble, France),
was studied as an inhibitor of ACA binding by premixing serial
dilutions with crude ABS or ABP or the cofactor under study (5 to 50 µg/mL) for 10 minutes before adding this mixture together with
patient serum to cardiolipin-coated, gelatin-blocked wells.
Other methods.
IgG depletion of human serum and citrated plasma pools from healthy
blood donors was performed by passage through a protein G-Sepharose
(Pharmacia, Uppsala, Sweden) column.
The ability of crude ABS, ABP, or the appropriate purified cofactors to
support the binding to cardiolipin of polyclonal rabbit antisera raised
against human C4BP (gift of M.B. Villiers, INSERM U238, Grenoble,
France) and human AT (Diagnostica Stago, Asnières, France) but
cross-reacting with their bovine counterparts was studied by
substituting each antibody for patient serum in the modified ACA ELISA.
Bound antibodies were detected by the use of peroxidase-conjugated goat
antirabbit Ig (Bio-Rad, Hercules, CA).
The dilute Russell viper venom time (dRVVT) of human citrated plasma
spiked with bovine AT (Diagnostica Stago) and/or bovine prothrombin
(Kordia, Leiden, The Netherlands) was performed using Diagnostica Stago
reagents for Stypven-cephalin clotting time.
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RESULTS |
Binding of patient antibodies in the modified ACA assay.
Six patients who possessed serum- and/or plasma-dependent ACA but no
antibody to human or bovine 2GPI directly immobilized on
-irradiated plates (2GPI-ELISA) were selected for
further study. Binding was little changed by replacing cardiolipin with phosphatidylserine or phosphatidylglycerol, whereas substitution by the
neutral phospholipid, phosphatidylethanolamine, suppressed antibody
binding from all six patient sera. In the modified ACA ELISA using
gelatin buffer alone (Fig 1), no antibody
binding was observed when serum from patients no. 2 through 6 was
diluted 1:100. A higher serum dilution (1:600 or greater) was required for patient no. 1 so as to avoid the influence of the endogenous cofactor. Optimal ACA binding was ensured in this assay by adding 10 µg/mL purified 2GPI of human (patient no. 1) or bovine
(patients no. 2 and 3) origin, and a concentration-dependent effect was observed at lower 2GPI concentrations. Increasing
fluid-phase concentrations of 2GPI (human or bovine, as
appropriate) up to 1.5 mg/mL had no inhibitory effect on patients no.
1, 2, and 3 antibody binding. If the lysine residues in
2GPI were first modified by carbamylation to suppress
its phospholipid-binding capability, ACA binding was abolished (not
shown). Furthermore, the antibodies from these 3 patients were not
retained at all on affinity columns using 2GPI (human or
bovine, as appropriate) covalently linked to agarose beads, contrasting
with the usual behavior of anti-2GPI antibodies.7 As a whole, these results indicate that
solid-phase presentation of 2GPI on anionic
phospholipids, but neither on -irradiated polystyrene and agarose
beads nor in the fluid-phase, is required to induce epitope formation
for ACA from patients no. 1, 2, and 3.
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| Fig 1.
IgG binding in the modified ACA ELISA of the patient
sera, diluted 1:600 (no. 1) or 1:100 (others). The antibody binding
step was performed in the presence of gelatin buffer ( ), 10 µg/mL
purified 2GPI of
human ( )
or bovine ( ) origin, or 10% bovine plasma ( ) or serum
( ). The means ± SD of specific absorbance values
from three independent experiments are shown.
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In the 3 other cases, a cofactor distinct from 2GPI was
provided by ABS only (patient no. 6) or both ABS and ABP (patients no.
4 and 5; Fig 1). Antibodies from patient no. 4 were not species specific, because ABS or ABP could be replaced by IgG-free 10% human
serum or plasma. By contrast, antibody recognition was restricted to
bovine cofactors for patients no. 5 and 6. Figure 2 shows the absorbance in the
modified ACA ELISA as a function of ABP or ABS concentrations. The
requirement for relatively high (~5%) ABP or ABS concentrations to
sustain half-maximal binding of ACA from patient no. 4, in comparison
with the two others, may indicate less abundant cofactor protein
concentration. It also explains why the contribution to ACA binding
made by endogenous cofactor was negligible at a 1:100 dilution of
patient serum. Finally, the interaction between involved cofactors and
cardiolipin appears to be independent of calcium ions, because the
addition of EDTA or CaCl2 (5 mmol/L each) to the assay
buffers did not change the results.
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| Fig 2.
Bovine plasma or serum requirement for IgG ACA binding.
Sera from patients no. 4, 5, and 6 and a negative control (dashed
lines) were diluted 1:100 in PBS/gelatin containing different
concentrations of bovine plasma ( ) or serum ( ), and ACA
reactivity was measured. Results are from a representative
experiment.
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Purification and identification of ACA cofactors of patients no. 4, 5, and 6.
These cofactors were purified from ABS for patient no. 6 and from ABP
for the 2 other patients using ammonium sulfate precipitation followed
by four successive chromatographic steps. Both cofactors no. 4 and 5 were recovered in the eluant of the Phenyl-Sepharose column around 40%
ethylene glycol, whereas no. 6 cofactor was detected in the
fall-through fraction. The cofactors were totally retained on the next
two columns (DEAE-cellulose and heparin-Ultrogel) and were eluted at
different salt concentrations, eg, from the latter column 0.25, 0.3, and 0.45 mol/L NaCl for cofactors no. 4, 5, and 6, respectively. The
concentrated heparin column eluents were applied to a Sephacryl S-300
gel-filtration column and the resultant fractions were submitted to
electrophoretic separation on SDS-polyacrylamide gels
(Fig 3A) and pooled according to cofactor activity (Fig 3B). The S-300 column yielded a coincident protein (A280) and activity peak that eluted at the estimated
molecular weight (Mr) of 61 kD and close to the
void volume for cofactor preparations no. 4 and 5, respectively. The
latter high-Mr material (>200 kD on SDS-PAGE
under nonreducing conditions) showed an approximate 80-kD band upon
reduction. Seventeen N-terminal amino acids were identified from this
80-kD band and a sequence alignment search showed 100% correspondence
with the N-terminus of the  chain of bovine C4BP
(Table 2). N-terminal microsequencing of
the no. 4 cofactor preparation was determined on the 61-kD band from a nonreduced SDS gel and yielded two sequences. One of them closely matched the N-terminus of human LBP8 (bovine LBP was absent from current data base releases) with 8 identities among the 9 positions identified, whereas the second sequence, deduced, could not
be assigned (Table 2). The S-300 elution profile displayed by no. 6 cofactor preparation was at first puzzling, in that cofactor activity
emerged shortly after the void volume and then trailed to culminate
around 60 kD. Furthermore, individual fractions spanning this broad
activity peak exhibited essentially the same pattern on SDS gels. This
pattern comprised, in addition to faint bands in the region of 60 to 71 kD, three prominent bands at 86, 54, and 36 kD under nonreducing
conditions that migrated slightly faster after reduction at 80, 50 (closely spaced doublet), and 33 kD, respectively (Fig 3). It was
established by N-terminal sequencing, successfully performed on the
three dominant bands (reduced), that bovine AT and thrombin B chain
comigrated in the higher 80-kD band, whereas free AT and thrombin B
chain accounted for the 50- and 33-kD bands, respectively (Table 2).
These findings have to be taken in context with the known mechanism of
inhibition of serine proteinases (such as thrombin) by AT. This
involves two steps, an initial weak association in a reversible
Michaelis-type complex followed by the formation of a highly stable
bond between the reactive center of AT and the active site of thrombin
located on the B chain.9,10 It has also been demonstrated
that the final products of thrombin inhibition during blood clotting
are ternary complexes together with vitronectin, an adhesion
glycoprotein of 65 to 75 kD in human plasma.11,12 In this
respect, the faint bands observed around 60 to 71 kD on nonreduced and
reduced SDS gels may testify to the presence of vitronectin in high
Mr thrombin-AT complexes from the S-300 column, the
dissociation of which occurred in the presence of SDS.
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| Fig 3.
SDS-PAGE analysis (A) of purified ACA cofactors of
patients no. 4, 5, and 6 recovered after Sephacryl S-300 chromatography
(B). The three effluents from this column were monitored for protein
content and cofactor activity as described in Materials and Methods.
Fractions were pooled as indicated by the horizontal bars. The elution
profile of plasma proteins is depicted at top of (B). Electrophoresis
was performed in 7.5% nonreducing and reducing polyacrylamide gels and
then stained with Coomassie blue. Arrows indicate molecular weight
markers.
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Target recognition of ACA of patients no. 4, 5, and 6.
Further evidence for the involvement of those identified cofactors in
patient antibody binding to cardiolipin was obtained by inhibition
experiments and by using polyclonal antibodies (when available) against
the above-noted cofactor proteins as probes in the modified ACA ELISA.
It appears from Fig 4A that absorption of
purified cofactors of patients no. 4, 5, and 6 with cardiolipin liposomes led to suppression of ACA reactivity. Concomitantly, the loss
of bands from SDS gels corresponding to sequenced material was noticed.
Because LPS forms high-affinity complexes with LBP in acute-phase serum
and in solution when purified LBP and LPS are mixed,13
purified LPS was preincubated with cofactor preparations to exclude the
possibility that the unidentified contaminant of LBP was actually
responsible for cofactor activity of patient no. 4 ACA. LPS
specifically neutralized the cofactor activity of the LBP preparation
with dose-response effect (Fig 4B). For both experimental designs
(using cardiolipin liposomes and LPS as inhibitors), similar results
were obtained when using ABS or ABP as a source of cofactors (not
shown). Patient no. 3 antibodies, specific for bovine
2GPI complexed with cardiolipin, served as control in
these experiments. The purified no. 5 cofactor (bovine C4BP), ABS, and
ABP supported equally well the binding of a rabbit anti-C4BP antiserum
to immobilized cardiolipin (not shown). The behavior of a rabbit
anti-AT antiserum resembled that shown in Fig 1 for patient no. 6 ACA,
as judged by the absorbances (mean ± SD) in the modified ACA ELISA
with this reagent together with buffer (0.138 ± 0.034), ABP (0.161 ± 0.067), ABS (1.603 ± 0.102), no. 6 cofactor preparation (1.854 ± 0.176), and commercially prepared bovine AT (0.149 ± 0.076).
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| Fig 4.
Inhibition of patient antibody binding to immobilized
cardiolipin by preincubation of the corresponding purified cofactor (10 µg/mL) with increasing concentrations of cardiolipin containing
liposomes (A) or fluid-phase LPS (B). Results are from a representative
experiment. Patients no. 3 (), 4 ( ), 5 (), and 6 ().
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We also investigated which component of the thrombin-AT complex was
targeted by antibodies of patient no. 6. To this end, we took advantage
of the restricted recognition by those ACA of a cofactor from bovine
origin that is generated upon clotting. A coagulation assay (dRVVT) of
human plasma spiked with physiological concentrations of bovine AT
and/or bovine prothrombin was thus performed, and the resultant serum
was assayed for cofactor activity. In these conditions, patient no. 6 ACA bound only when supplementing human plasma with bovine AT or bovine
AT plus bovine prothrombin, but not bovine prothrombin alone (mean
absorbance after addition of the following: AT, 0.820; AT plus
prothrombin, 1.358; prothrombin, 0.104; none, 0.113). We concluded that
ACA from this patient recognized bovine AT, but only when it had been
modified by thrombin.
Finally, the purified ACA cofactors were used as coating antigens for
the development of direct ELISAs, ie, in the absence of cardiolipin
(Table 3). Whatever the combination of
plate and assay buffer chosen, the serum from patients no. 4 and 5 did
not react with their respective cofactor in such an ELISA system. To
the contrary, specific binding was observed between patient no. 6 ACA
and the corresponding purified preparation and was optimal when using
irradiated polystyrene plates and Tween-containing buffer as the
blocking agent and sample diluent. The commercial preparation of bovine
AT did not serve as an antigen for patient no. 6 antibodies, unless it
was preincubated with purified thrombin before coating (not shown).
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Table 3.
Reactivity of Sera From Patients No. 4, 5, and 6 Towards
Their Respective Purified Cofactors, Directly Immobilized on ELISA
Plates Under Various Experimental Conditions
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DISCUSSION |
Since the widespread recognition of the APS, aPL continue to be
scrutinized, with their very definition and significance being questioned. An important step has been the recognition of
2GPI as the antigenic target of most ACA associated with
a thrombotic diathesis.2,3 In the present study, we have
focused on a small series of patients with diverse clinical conditions
whose antibodies exhibited a rare pattern, characterized by an absolute plasma and/or serum requirement in the standard ACA assay but no
reactivity to human or bovine 2GPI directly immobilized
on -irradiated plates. Despite such negative results in the
2GPI-ELISA, 3 of 6 patient antibodies actually targeted
2GPI but only when presented on a cardiolipin-coated
surface. Most anti-2GPI antibodies are able to react
with 2GPI when the protein is immobilized on certain
surfaces represented by anionic phospholipids, -irradiated polystyrene, or even agarose beads.1,5,7 Two hypotheses have been proposed to account for those reactivities: (1) antibody recognition of neo-epitopes exposed when 2GPI interacts
with a suitable surface; and (2) alternatively, low intrinsic affinity of the antibodies thus requiring high density or clustering of 2GPI to allow their bivalent attachment.1
The latter possibility is strongly supported by the recent
demonstration, using various experimental approaches,14-16
that more 2GPI binds to phospholipid surfaces in the
presence of bivalent (but not monovalent) anti-2GPI antibodies, the affinity of which increases upon dimerization of
2GPI.17 Another reason why patients no. 1, 2, and 3 antibody binding requires both components of the
2GPI-cardiolipin complex may stem from the report by
Hörkkö et al18 that the neo-epitopes for some
aPL are covalent adducts formed between breakdown products of oxidized
phospholipid and associated proteins such as 2GPI. However, the hypothesis that our patient sera would be directed at such
oxidation-dependent structures appears unlikely, because it is
difficult to reconcile with the restricted species specificity of their
antibodies (Fig 1). It is likely that there is much more heterogeneity
among anti-2GPI antibodies, in particular with respect
to species and epitope specificities, than previously thought.7 An observation similar to ours has been made in
the case of most antiphosphatidylethanolamine antibodies shown to react
with high or low molecular weight kininogens or kininogen-binding proteins (eg, factor XI or prekallikrein) provided in combination with
this neutral phospholipid.19 The investigators conclude that binding of kininogens to phosphatidylethanolamine induces novel
antigenic epitopes.
By means of purification procedures, monitored by cofactor activity
measurement in a modified ACA ELISA and coupled to N-terminal amino
acid sequencing, it was possible to identify three hitherto unrecognized proteic targets of ACA, namely LBP, C4BP, and
thrombin-modified AT. The three of them behaved as heparin-binding
proteins in the purification process, in keeping with their known
affinity for various lipids or membranes. It should be emphasized that
the detection of cofactor activity depends on two successive or
concurrent phenomena, a protein-cardiolipin interaction and antibody
recognition of the available epitopes. A way to ascertain that putative
ACA cofactors do bind to cardiolipin under the very conditions of the
ACA assay (ie, from serum or plasma) is to use polyclonal antisera
raised against the proteins being studied as probes because of the
number and diversity of epitopes recognized by those highly specific
reagents. In this respect, the thrombin-AT preparation (patient no. 6 ACA cofactor) and bovine serum, but neither bovine plasma nor native
AT, supported the binding of a rabbit anti-AT antiserum to immobilized
cardiolipin, with such a reactivity pattern being identical to the one
of patient no. 6 ACA. This finding, together with evidence for the role
of bovine AT in mixing experiments with human plasma before clotting,
led us to conclude that those patient ACA do indeed target bovine AT
once it has been modified/cleaved by thrombin.
AT is a single-chain glycoprotein of 58 kD that belongs to the serpin
(serine proteinase inhibitors) superfamily and inhibits most
proteinases of the coagulation cascade (including thrombin) by forming
denaturant-stable, equimolar complexes with the enzymes.9 The massive change in AT conformation that occurs upon complex formation with thrombin would offer the opportunity to acquire cardiolipin-binding properties and/or generate neoepitopes for ACA. It
also probably accounts for the greatly reduced heparin affinity
compared with native AT, as judged by the salt concentrations for
elution from matrix-linked heparin: 1 mol/L NaCl for free AT and 0.15 mol/L NaCl for the thrombin-AT complex, with the value increasing to
0.45 mol/L NaCl upon ternary complex formation with vitronectin.11,20 Interestingly, patient no. 6 ACA cofactor activity eluted from the heparin-Ultrogel column around the latter ionic strength, which, together with the S-300 column activity profile,
may indicate the presence of such ternary complexes in the final
preparation and their participation in cofactor activity. It is also
conceivable that interactions to the heparin affinity column and/or to
solid-phase cardiolipin were actually mediated by the thrombin moiety
of the thrombin-AT complex, because thrombin contains two positive
poles to the east and northwest of the active site referred to as the
anion-binding exosite and the heparin-binding site,
respectively.21
C4BP, formerly called proline-rich protein,22 functions as
an inhibitor of the classical pathway of the complement cascade and
also regulates the protein C anticoagulant pathway through protein S
binding.23 The major form of C4BP is composed of seven identical  chains (~70 kD in human and 80 kD in the mouse) and one
chain (45 kD) linked by disulphide bridges.23 The
latter chain, being highly glycosylated, has been noticed to stain
poorly with Coomassie blue,23 probably explaining the
absence of visible band in the 45-kD region when patient no. 5 ACA
cofactor preparation was run on reduced SDS gels. Because circulating
C4BP is known to be involved in calcium-dependent and high-affinity
interactions with protein S (84 kD) and serum amyloid P component (5 noncovalently bound 25-kD subunits),24 their copurification
in the C4BP preparation would have been expected. However, this did not
prove to be the case possibly for the following reasons: (1) the weaker
affinity for the bovine C4BP-protein S interaction than for the human
complex,25 (2) the capability of heparin to disrupt the
C4BP-serum amyloid P component interaction,24 and, most
importantly, (3) the absence of calcium during the purification
process. The presence of C4BP in triglyceride-rich lipoproteins (ie,
chylomicrons and VLDL)26 is consistent with the property
(shared with 2GPI) of binding to
Intralipid,22 a lecithin-stabilized artificial triglyceride emulsion that behaves metabolically in a very similar way to
chylomicrons. Both and chains of C4BP have been shown to
contain several structural elements, referred to as short consensus or
complement repeats (SCR), which are also found in approximately half of
all complement proteins, including factor H, as well as in
2GPI.23 It is interesting to note the recent
finding by Kertesz et al27 that complement factor H, which
has a high degree of homology with 2GPI, binds to
cardiolipin-coated plates under conditions used in the standard ACA
assay. In addition, a high frequency of antibodies to several human
complement regulatory proteins, including factor H and C4BP, has been
reported in antiphospholipid syndrome patients using direct
ELISAs.28 The possibility that these antibodies actually
behave as cofactor-dependent ACA has not been investigated.
Finally, the antibodies of the last patient (no. 4) were shown to
recognize LBP in combination with cardiolipin, and their binding was
competed by fluid-phase purified LPS (or endotoxin, a glycolipid
present in the outer membrane of all gram-negative bacteria) (Fig. 4B).
LBP is a trace plasma glycoprotein of 60 kD that binds with high
affinity to the lipid A moiety of rough and smooth forms of
LPS13 and plays an important intermediary role in
host-endotoxin interaction. LBP functions as an opsonin by bridging
LPS-coated particles to the CD14 receptor of macrophages, thereby
promoting tumor necrosis factor- (TNF-) secretion.29 The interaction between LBP and a number of negatively charged polymeric substances, including DNA, RNA, heparin, and phospholipids, has been reported to be poor to nonobservable, stressing the high degree of specificity for the lipid A portion of LPS.13
More recently, a novel function for LBP and soluble CD14 has been
defined in transport of mammalian phospholipids both with purified
recombinant proteins and in whole plasma.30 Of note, a
valine residue at position 7 of the mature protein was identified in
bovine LBP (versus alanine in human LBP; Table 2) as well as in a
structurally related LPS binding protein found in granulocytes, human
bactericidal/permeability-increasing protein.8
The exact causes of the development of antibodies against these newly
identified ACA cofactors are unclear. However, both C4BP and LBP are
known to behave as acute-phase reactants, the levels of which increase
during various diseases, and an inflammation process might be involved
in the generation of these antibodies. In addition, the quantitation of
thrombin-AT complexes or modified AT in plasma is now available for the
diagnosis of preclinical states of coagulation activation, and high
values have been found in such conditions, eg, venous thromboembolism
or inherited thrombophilia.31 The present study has not
addressed two important issues: (1) the proportion and predictive value
in various clinical settings of ACA corresponding to antibody binding
to epitopes derived from the above-mentioned proteins, and (2) the
putative biological effects of such antibodies on the functions of
targeted proteins, as exemplified by antibodies to prothrombin and to
inhibitors of coagulation, protein C, protein S, and annexin
V.32-34 The isolated presence of ACA specificities of the
kind described here appears quite uncommon, being detected through the
systematic screening in the ACA- and 2GPI-ELISAs of
thousands of consecutive serum samples sent to our laboratory for aPL
testing. Obviously, patients could have more than one type of ACA, in
which case it may prove difficult to determine the relative
contribution made to binding in the standard ACA assay by the different
antibody subsets for the following reasons: (1) the possible role of
the endogenous cofactors from patient sera, (2) a competition
phenomenon between various phospholipid-binding proteins for access to
solid-phase cardiolipin, and, finally, (3) the influence of specific
antibodies on the equilibrium between bound and fluid-phase cofactors.
Although the involvement of these new ACA cofactors is for the moment
limited to a few patient samples, the reported experimental approach is increasing our insight into the numerous ACA specificities. The real
significance of those ACA is still elusive and clinical complications, such as thrombosis, seem uncommon, at least in our small series of patients.
| |
FOOTNOTES |
Submitted July 21, 1998; accepted February 8, 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 Josiane Arvieux, MD,
Laboratory of Immunology, Brest University Medical School Hospital, BP
824, F 29609 Brest Cedex, France.
| |
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