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REVIEW ARTICLE
Autoantibodies to Phospholipid-Binding Plasma Proteins: A New View of
Lupus Anticoagulants and Other “Antiphospholipid” Autoantibodies
By Robert A.S. Roubey
L
UPUS ANTICOAGULANTS, and “antiphospholipid”
erythematosus, and are associated with thrombosis, not
autoantibodies in general, are of considerable clinical
bleeding. Recently this problem has been exacerbated beimportance because of their strong association with thrombocause changes in terminology have not kept pace with resis, recurrent fetal loss, and thrombocytopenia, ie, the “antisearch developments, eg, “antiphospholipid” autoantibodies
phospholipid” antibody syndrome.’ This review focuses on
that are not directed against phospholipids. For the purposes
recent evidence that “antiphospholipid” autoantibodies are
of this review, the following definitions will apply:
not directed against anionic phospholipids, as has previously
Lupus Anticoagulants
been thought, but are part of a larger group of autoantibodies
against certain phospholipid-binding plasma proteins. At
Antibodies that inhibit certain in vitro phospholipid-depresent, the most common and best characterized antigenic
pendent coagulation reactions, typically the conversion of
targets are &glycoprotein I (P2GPI)’-* and prothr~mbin.~”~ prothrombin to thrombin, are lupus anticoagulants. This inOther phospholipid-binding proteins, particularly protein C
hibitory activity is thought to be directly related to the antiand protein S,” may be important targets as well. Autoantigenic specificity of these antibodies, rather thanto some
bodies with these specificities differ from acquired coagulaother property. The anticoagulant activity of these antibodies
tion factor inhibitors in that they preferentially bind antigens
is accentuated by lowering the phospholipid concentration,
that are immobilized on anionic phospholipid membranes or
eg, in the dilute Russell viper venom time assay, and inhibcertain synthetic surfaces. In most instances, antibody bindited by raising the phospholipid concentration or by preincuing to the antigens in the fluid-phase is weak or nondetectbation with excess phospholipid, eg, the platelet neutralizaable. Thus, such autoantibodies usually do not decrease
tion procedure. Mixing patient plasma with normal plasma
plasma antigen levels. Although direct evidence for a pathodoes not correct the prolongation of the coagulation reaction
physiologic role of “antiphospholipid” autoantibodies is
caused by lupus anticoagulants, distinguishing themfrom
lacking, it is hypothesized that autoantibodies to phosphofactor deficiencies. The various coagulation assays used for
lipid-binding proteins contribute directly to a thrombotic dithe diagnosis of lupus anticoagulants have beenrecently
athesis by interfering with hemostatic reactions that occur
reviewed elsewhere.” Lupus anticoagulant assays have been
on anionic phospholipid membranes in vivo. Interestingly,
thought to detect antibodies against anionic phospholipids.
the effect of autoantibodies on target antigen function may
However, these tests use whole plasma and recent data from
vary. For example, certain antibodies to P2GPI enhance its
prothrombinase assays usingpurified components indicate
activity,””’ whereas antibodies to phospholipid-bound actithat certain lupus anticoagulants are specific for either phosvated protein C are inhibitory.” Taken together, these new
pholipid-bound prothrombin or /32GPI.10.15
Thus, lupus antiobservations explain much of the confusion regarding the
coagulants are heterogeneous, and include autoantibodies to
laboratory tests used to detect “antiphospholipid” antibodat least two phospholipid-bound plasma proteins.
ies and are providing key insights into the pathophysiology
Anticardiolipin Antibodies
of the “antiphospholipid” antibody syndrome.
TERMINOLOGY
The nomenclature of ‘ ‘antiphospholipid” antibodies has
always been confusing. For example, lupus anticoagulants
occur in many individuals who do not have systemic lupus
From the Division of Rheumatology and Immunology, Thurston
Arthritis Research Center, The University of North Carolinaat
Chapel Hill.
Submitted January 19, 1994; accepted June 22, 1994.
Supported by National institutes of Health Grants No. AR-30701
und AR-01920. R.A.S.R. is the recipient of a Clinical Investigator
Award from the National Institute of Arthritis and Musculoskeletal
and Skin Diseases.
Address reprint requests to Robert A S . Roubey, MD, Division
of Rheumatology and Immunology, CB# 7280, Room 932 Faculty
Laboratory Office Building, University of North Carolina, Chapel
Hill, NC 27599-7280.
0 1994 by The American Society of Hematology.
0006-4971/94/8409-0001$3.00/0
2854
Anticardiolipin antibodies are antibodies detected in solidphase immunoassays, typically enzyme-linked immunosorbent assays (ELISAs), in whichin the putative antigen is
cardiolipin dried onto a microtiter plate. However, in addition to detecting antibodies to cardiolipin this assay also
detects antibodies to serum or plasma proteins that bind to
the cardiolipin coated on the plate, in particular, antibodies
to P2GPI. These proteins maybe present in the serum or
plasma samples and/orin bovine serum, which is often a
component of the blocking buffer and sample diluent. Because of the conditions of the assay, the relevant proteins
are those that bind to anionic phospholipids independently
of calcium. Thus, like lupus anticoagulants, the antibodies
detectable in the anticardiolipin assay are heterogeneous,
and include both antibodies to cardiolipin and antibodies to
cardiolipin-bound proteins.
Antiphospholipid Antibodies
Antiphospholipid antibodies are antibodies detected in lupus anticoagulant assays, anticardiolipin assays, and certain
Blood, Vol 84, No 9 (November l ) , 1994: pp 2854-2867
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ANTIBODIES
TO PHOSPHOLIPID-BINDINGPROTEINS
2855
Table 1. Reactivity of Autoantibodies to Phospholipid-Binding
Plasma Proteins in ”Antiphospholipid Antibody Assays
Reactiviv in Standard ”Antiphospholipid”
Assays
Antibody
Anti-fi2GPI
Anti-prothrombin
Anti-factor X
Anti-protein C
Anti-protein S
“Anticardiolipin”
No
No
No
No
Lupus Anticoagulant
YesINo
Yes
Yes
No
No
serologic tests for syphilis. This term will be used for ease
of discussion, with the understanding that such antibodies
actually may be directed against several phospholipid-binding proteins. In most instances, quotations marks will be
used to highlight this discrepancy, ie, “antiphospholipid”
antibodies.
Antiphospholipid Antibody Syndrome
The association of moderate- to high-titer “antiphospholipid” autoantibodies with arterial thrombosis, venous
thrombosis, recurrent fetal loss, or thrombocytopenia is
termed antiphospholipid antibody syndrome. The syndrome
often occurs in patients with systemic lupus erythematosus
or related autoimmune diseases. When it occurs in the absence ofan associated autoimmune disease, it has been
termed the primary antiphospholipid antibody
Antiphospholipid antibodies in patients with syphilis, detectable in serologic tests for syphilis and anticardiolipin assays,
are not associated with the antiphospholipid syndrome.
Autoantibodies to Phospholipid-Binding Plasma Proteins
This is a broader term encompassing “antiphospholipid”
autoantibodies as well as antibodies to other phospholipidbinding proteins that are not detectable in lupus anticoagulant or anticardiolipin assays, eg, antibodies to protein C and
protein S. These latter antibodies appear to be associated
with “antiphospholipid” antibodies and may have pathologic significance. This phrase more accurately describes the
group of autoantibodies associated with the “antiphospholipid” antibody syndrome. Table l lists the phospholipidbinding proteins known to be antigenic targets and the reactivity of autoantibodies to these proteins in “anticardiolipin”
and lupus anticoagulant assays.
INADEQUACY OF THEPHOSPHOLIPIDMODEL
Until recently it was held that “antiphospholipid autoantibodies” were directed against anionic phospholipids and, by
virtue of this specificity, inhibited phospholipid-dependent
reactions. However, this model does not adequately explain
the subsets of “antiphospholipid” autoantibodies observed
in current laboratory testing or the clinical heterogeneity of
the antiphospholipid antibody syndrome. In the laboratory,
sera from patients with syphilis are often reactive inthe
anticardiolipin assay, but do not have lupus anticoagulant
activity.” In contrast, “anticardiolipin” antibodies and lupus
anticoagulants are closely associated in patients with autoimmune diseases, although some patients clearly have one specificity but not the other.’ In some patients, “anticardiolipin”
antibodies and lupus anticoagulants appear tobe a single
antibody p o p u l a t i ~ n ,whereas
~ ~ - ~ ~ in other patients the two are
distinct and ~eparable.’~-~~
Heterogeneity also exists among
lupus anticoagulants. For example, Brandt” tested the ability
of lupus anticoagulant IgG fractions to inhibit the phospholipid-dependent formation of factor Xa and thrombin, using
the same phospholipid reagent. Most lupus anticoagulants
inhibited thrombin generation to a significantly greater degree than Xa generation, whereas one exhibited significantly
more inhibitory activity in the Xa assay.
More importantly, the basis of the heterogeneity of the
clinical features of the antiphospholipid syndrome is not well
understood. If one hypothesizes that these antibodies are
pathogenic on the basis of their antigenic specificity, it is
not clear why certain patients have venousthrombosis versus
arterial thrombosis versus thrombocytopenia versus recurrent fetal loss, or some combination of these manifestations.
It is also not clear why only about 30% of individuals with
“antiphospholipid” antibodies will develop the clinical syndrome,’ although greater risk is associated with higher antibody titers.’*.*’
SPECIFICITIES OF AUTOANTIBODIESTO
PHOSPHOLIPID-BINDINGPLASMAPROTEINS
This section will review recent data regarding the antigenic specificities of “antiphospholipid” and related autoantibodies. It should be remembered that most of these studies
are limited to relatively small numbers of patients with moderate- to high-titer IgG autoantibodies. In retrospect, several
early reports clearly suggest a role for plasma proteins in
the activity of lupus anticoagulants. In 1959, Loelige8’ was
among the first to describe the so-called lupus anticoagulant
“cofactor” phenomenon, ie, the addition of normal plasma
to patient plasma increased the inhibition of coagulation. He
concluded that the cofactor wasmostlikelyprothrombin
itself. In 1965, inan elegant and often overlooked report,
Yin and Gaston” purified a lupus anticoagulant, showed it
to be a 7s immunoglobulin (IgG), and clearly demonstrated
that its anticoagulant activity required a protein cofactor.
The cofactor, although not fully characterized, was present
in both normal and patient plasma and was notprothrombin.
&-Glycoprotein I (PZGPI)
In 1990 two independent reports challenged the conventional wisdom that antiphospholipid autoantibodies were directed against anionic phospholipids.’.’’ Both groups of investigators prepared affinity-purified IgG “anticardiolipin”
antibodies from patients sera and showed that these antibodies didnotbindto
solid-phase cardiolipin or cardiolipin
liposomes in serum-free assay systems. Addition of normal
serum restored antibody binding. The serum component required for the binding of “anticardiolipin” antibodies to
solid-phase cardiolipin was found to be P2GP1, a phospholipid-binding plasma protein.
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2856
P2GPI is a 50-kD protein present at -200 pg/mL in normal plasma. Although its physiologic role is not known, in
vitro data suggestthat P2GPI mayplay a role in coagulation.
p2GPI binds to anionic
phospholipids” and inhibitsthe contact phase of intrinsic blood c ~ a g u l a t i o n , adenosine
~ ~ - ~ ~ diphosphate (ADP)-dependent platelet aggregation:” and the
platelet^.^' Recent preliminary
prothrombinaseactivityof
data suggest that P2GPI may play a regulatory role in the
protein C pathway by inhibiting the interaction between pro’
these data sugtein S and C4b-binding p r ~ t e i n . ~Although
gest an anticoagulant role for P2GP1, deficiency of this proteinisnota
clear risk factorfor thrombosis.Population
and family studies indicate that the plasma concentration of
P2GPI is controlled by codominant
recent
A
study of patients with familial thrombophilia indicated that
heterozygouspartial
P2GPI deficiency (plasmalevels of
-60 to 140 pg/mL) was not associated with thrombosis.“
Two brothers with homozygous P2GPIdeficiency were identified; one hadahistory
of recurrentvenous thrombosis
whereas the otherwas asymptomatic at age 35. Patients with
“antiphospholipid” antibodies have normal4’ or somewhat
elevated levels of ,82GPI.47
Structurally, P2GPI is a noncomplement member of the
complement control protein family?8 P2GPI has five of the
consensus repeats, or so-called “sushi domains,” characteristic of such proteins. The fifth domain may contain a phospholipid-binding region.””.” Theaminoacidsequence
of
P2GPI was originally determined by Lozier et al,” and severallaboratories havereported
thecompletecDNA
sequence.5’-s5P2GPI has also been termed apolipoprotein H,”
as approximately 40% is bound to lipoproteins, although it
bears no structural similarity to other apolipoproteins. Genetic polymorphisms of P2GPI have been observed based
on isoelectric focusing pattern^,'^^^^ but not yet defined on a
DNA basis. An unrelated polymorphism, avaline-leucine
amino acid exchange at position 247, has recently been reported.5y
The observation that P2GPI is required for the binding
of “anticardiolipin” autoantibodies to cardiolipin has been
confirmed by several laboratories.”.‘”“’ However, some investigators have reported that P2GPI enhances the binding
of theseantibodies tocardiolipin, butisnotan
absolute
requirement for such binding?’@ Interestingly, the requirement for P2GPI clearly distinguishes the “anticardiolipin”
antibodies that occur in the setting of autoimmune disease
and the antiphospholipid syndrome from those that occur in
the setting of syphilis and other infectious diseases (Fig I).
Syphilis-associated anticardiolipin antibodies bind to cardiolipin in the absence of P2GPI and this binding to cardiolipin
is inhibited by human P2GP1, presumably because the antibodies andP2GPI
bind to similarphospholipidstructures.”,” This differencein antigenic specificity may explain
why the autoimmune type of “anticardiolipin” antibody is
associated with the lupus anticoagulant and thrombosis, fetal
loss, etc, whereas anticardiolipin antibodies associated with
infection are not.’”‘‘
In view of these data,potential antigenic targetsof autoimmune “anticardiolipin” antibodies are (1) P2GP1, (2) a com-
ROBERT A.S. ROUBEY
plex comprised of both P2GPI and anionicphospholipid, (3)
neo- or cryptic phospholipid antigens expressed as a result
of the binding of P2GPIto phospholipid, or (4) neo- or
crypticP2GPIantigens.
Based on theirobservation that
anticardiolipinantibodies fromtwo patientswere not retained by P2GPI bound to a heparin-agarose column, McNeil
et al’ concluded that the antibodies recognized a
complex
antigen that includes both 02GPI and anionic phospholipid.
to
In contrast,Galli et a12 demonstratedantibodybinding
P2GPI in an ELISA performed in the absence of phospholipid. They concluded that “anticardiolipin” autoantibodies
recognize P2GPI when it is bound to cardiolipin or absorbed
on a microtiter plate, but not in the fluid phase. Subsequent
studies have differed as to whether these antibodies recognize P2GPI in the absence of phospholipid. Our laboratory
and several other groups previously found no reactivity to
P2GpI alone. 17.63-65.67 Conversely, Arvieux et al,” Viard et
al,’ and Keeling et ald reported antibody binding to P2GPI
in the absence of phospholipid in a total of 44 patients.
Recent data from our laboratory may explain this discrepancy.’ Binding of autoantibodies to pure P2GPI wasdetected
by ELISA only under certain experimental
conditions, the
type of microtiter plate being the critical factor (Fig 2). Antibodies did not bind to P2GPI coated on plain polystyrene
plates, but did bind when P2GPI was coated on “high-binding” polystyrene plates. These plates are produced commercially by y-irradiation (typically 2 3 X 10‘ rad). This treatment
partially
oxidizesthe
polystyrene
surface,
which
renderstheplate
moreanionic andsignificantly enhances
protein binding capacity.6KTwo possible explanations are
(1) that the antibodiesrequirearelatively
high density of
immobilized antigen which cannot be achieved on untreated
polystyrene, or (2) that the antibodies are specific for a conformational epitope of P2GPI formed when the proteinis
bound to an anionic surface, such as irradiated polystyreneor
cardiolipin. Our datasupport the first explanation, suggesting
that antiCP2GPI autoantibodies are of intrinsically low affinity and bind to P2GPI only when the density of immobilized
antigen is sufficient to allow bivalent attachment. The low
affinity of a single IgG antigen binding site for P2GPl does
not mean that these antibodies are trivial or irrelevant with
regard to pathophysiology. The marked enhancement of the
antibody-antigen interaction provided by antibody bivalency
means thatthesearehigh-avidityantibodieswhen
P2GPI
is immobilized at a sufficiently
high density. In vivo such
antibodies may bind to P2GPI clustered on an anionic phospholipid surface, eg, an activated platelet or endothelial cell.
but would not bind to P2GPI in the circulation. In this way
antibodies to p2GPI are analogous to another autoantibody
to a plasma protein, rheumatoid factor. Rheumatoid factors
have low affinity for monomeric IgG in solution, but bind
avidly to IgG that is aggregated or bound to particles, because of the enhancement provided by multivalency.”’ Others have observed antibody binding to P2GPI on irradiated
polystyrene and support the alternative explanation, that the
antibodies recognize conformational epitopes formed when
P2GPI binds to an anionic surface.x These two possibilities
are not mutually exclusive.
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2857
ANTIBODIES TO PHOSPHOLIPID-BINDINGPROTEINS
1.6
1.6
B
A
A
Fig 1. Binding of autoimmune "anticardiolipin" antibodies to cardiolipin requires the
presence
of
p2GPI. The serumfree anticardiolipin ELSAwas
performed in the absence (A) or
presence (B) of 25 pglmL human
p2GPI. Purified IgG fractions
from representative patients
with the antiphospholipid antibody syndrome (A)and syphilis
(01, and from a normal individual
(0)
were assayed. See text for
references.
l.2
1.2
0.8
c3
0 0.8
0.4
0.4
0.0
6.25 12.5
50
25
100 200
IgG concentration (pg/ml)
Some, butnot all, autoantibodies to P2GPI have lupus
anticoagulant a~tivity.""'.~' This is interesting in light of the
fact that P2GPI itself inhibits prothrombinase activity in
vit1-0.~'P2GPI-dependent lupus anticoagulants appear to enhance the anticoagulant effect of P2GPI. Several monoclonal
and polyclonal antibodies to P2GPI have similar lupus anticoagulant-like activity.'3,7',72Enhancement of P2GPI's anticoagulant effect could be caused by the steric effect of antibodies bound to P2GPI on a phospholipid surface. Another
1.6
0
1.2
0
0
L3
0 0.8
0.6
0
0.4
0.2
0.0
,Normal
aCL
I
, ,Normal
aCL
I 1
0.0
IgG concentration (pg/ml)
possibility is that these antibodies could cross-link surfacebound P2GP1,increasing the affinity of the P2GPI-phospholipid interaction. Indeed, "antiphospholipid" autoantibodies
enhance the binding of P2GPI to cardiolipin-coated microtiter plates.73As will be discussed below, the effect of autoantibodies to P2GPI on the interaction of P2GPI with phospholipid surfaces may contribute to the pathogenesis of the
antiphospholipid antibody syndrome. The reason why some
anti-P2GPI antibodies have lupus anticoagulant activity
whereas others do not is unclear, but maybe relatedto
avidity and/or epitope specificity.
Prothrombin
1.4
1 .o
/
,
1
Untreated
Plate
High-binding
Plate
Fig2.
Detection of autoantibodies to p2GPI in the absenceof
phospholipid is dependent on the typeof ELBA plate. Sera from four
patients with "anticardiolipin" autoantibodies ( 0 )and four normal
individuals (01, diluted 1:100, were tested in an anti-p2GPI EUSA
performed using either a standard, untreated polystyrene microtiter
plate or a high-binding, yirradiated polystyrene plate. See text for
references.
The occurrence of acquired hypoprothrombinemia in a
subset of patients with lupus anticoagulants has been recognized for some time.30"4-76As previously mentioned, Loeliger3' suggested that prothrombin was the lupus anticoagulant cofactor in one such case. Bajaj et a177were the first to
demonstrate the presence of autoantibodies to prothrombin
in two patients with the lupus anticoagulant-hypoprothrombinemia syndrome. These antibodies were of relatively high
affinity and itis likely that hypoprothrombinemia was caused
by the clearance of prothrombin antigen-antibody complexes
from the circulation. Subsequently, circulating prothrombinantibody complexes were also observed in 38 of 55 patients
with lupus anticoagulants and normal prothrombin level^.^.^'
Although antibodies to prothrombin were first thought to
be non-neutralizing and distinct from lupus anticoagulant^,^^
Fleck et a19 showed that three affinity-purified antiprothrombin autoantibodies had lupus anticoagulant activity. These
investigators concluded that, in some patients, the autoantibodies to prothrombin were of relatively low affinity. This
was based on the observation that, although these antibodies
bound to immobilized prothrombin, the antibodies and prothrombin were not bound to each other in patient plasma.
More recently, others have demonstrated that some lupus
anticoagulants are directed against phospholipid-bound prothrombin. Bevers et al'' studied 16 patients with bothanticardiolipin antibodies and lupus anticoagulants. Plasma samples
were incubated with cardiolipin-containing liposomes fol-
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2858
lowed by centrifugation. In 11 of 16 patients, the lupus anticoagulant activity remainedinthe
supernatant whilethe
anticardiolipin antibody activity pelleted with the liposomes.
In a purifiedprothrombinase assay, these non-phospholipidbinding lupus anticoagulants were specific for human prothrombin bound to phospholipid. In the remaining5 patients,
lupus anticoagulant activity co-sedimented with the anticardiolipin antibodies; these lupus anticoagulants were antibodies to P2GPI." Oosting et all' showed that 4 of 22 lupus
anticoagulants inhibited endothelial cell-mediated prothrombinase activity. This inhibitory activity could beadsorbed by
prothrombin-coated cardiolipin vesicles, but not by vesicles
alone, or vesicles coated with P2GP1, factor Va, or factor
Xa.
Taken together these data strongly suggest thatsome lupus
anticoagulants aredirected against immobilized or phospholipid-bound prothrombin. Analogous to thediscussion of antibodies to P2GP1, these antibodies may be directed against
cryptic or neo-antigens formed when prothrombin binds to
anionic phospholipid, and/or they may be low-affinity antibodies binding bivalently to immobilized prothrombin. The
observation that the antibodies could be adsorbed by prothrombin bound to agarose beads favors the latter explanation.' The occurrence of hypoprothrombinemia in asmall
subset of patients with the lupus anticoagulant suggests that
this group of patients has higher affinity antibodies, or antibodies that recognize nativeprothrombin. These autoantibodies bind to prothrombin in the circulation leading to the
formation and clearanceof prothrombin-containing immune
complexes. It is not known whether some antibodies to prothrombin also bind to thrombin.
Protein C and Protein S
Limited data suggest that autoantibodies may be directed
against components of the protein C pathway, ie, protein C
and protein S. Although such antibodies are not detectable
instandardanticardiolipin
or lupusanticoagulant assays,
they are included here part
as of a broader view
of "antiphospholipid" autoantibodies asantibodies to phospholipid-binding plasma proteins. Oosting et all' demonstrated inhibition
of the protein C-mediated inactivation of factor Va by IgG
fractions from 7 of 30 patients with antiphospholipid antibodies andor a historyof thrombosis. This inhibitory activity
could be adsorbed by protein C-coated cardiolipin vesicles
in the rein 3 patients, and by proteinS-coatedvesicles
maining 4 patients. No inhibition was observed by P2GPIcoated vesicles or by vesicles alone. These investigators concluded that antibodies were specific for phospholipid-bound
protein C or protein S. In another recent report antibodies
to protein C were detected in 12 of 108 SLE serum samples
by an ELISA using high-binding microtiter plates7' These
antibodies were not associated with decreasedprotein C levels or functionalprotein C deficiency. In contrast, antibodies
to protein C or protein S were not detected by immunoblotting in 11 patients with the antiphospholipid antibody syndrome, 7 of whom had low levels of free protein S.'" As is
the case with antibodies to 02GP1, antibodies to protein C
ROBERT A.S. ROUBEY
and protein S may be detectable under certain experimental
conditions but not others.
Other Potential Targets
Inhibition of endothelial cell prostacyclin production has
been cited as a potential mechanism by which antiphospholipid autoantibodies may predispose to thrombosis. In view
of recent data indicating that thismay be causedby inhibition
of phospholipase A? activity,x' Vermylen and Amout" suggested that these antibodies might be directed against a phospholipaseA,-phospholipid complex. Adifficultywiththis
hypothesis is that the fraction of phospholipase A2 thought
to mediate arachidonic acid release is intracellular, ie, in the
cytosol or associatedwith the inner leaflet of the plasma
membrane. However, nonpancreatic secretory phospholipase
A? has also been reported to contribute to the synthesis of
arachidonic acid metabolite^.^'^^^ At the present time there
is no direct evidence of autoantibodies to any type of phospholipase A2.
Annexins are afamily of calcium-dependent phospholipid-bindingproteins thoughtto play importantroles in
membrane processes such as exocytosis.88 The potential interaction of antiphospholipid autoantibodies with annexin V
(human placental anticoagulant protein I) has recently been
studied.x9 Primarily an intracellular protein, very low levels
of annexin V are detectablein human plasma, amniotic fluid,
and conditionedmedium
of' culturedendothelial
cells.""
V
There are no data suggestingthatextracellularannexin
plays a physiologic role in coagulation. Because of its high
affinity for anionic phospholipid surfaces," annexin V
can
block the binding of "antiphospholipid" antibodies to anionic phospholipids in vitro.x" "Antiphospholipid" antibodies do not recognize annexin V.'"
Anecdotally, an antibodyto factorX with lupus anticoagulant activity has been identified in a patient
with high titer
"anticardiolipin"antibodies
and ahemorrhagicdiathesis
(Dr D. Triplett, personal communication, January 1994).
Autoantibodies with apparent specificity for phosphatidylethanolamine have been reported in a few patient^.^'.'^ Sugi
et ai" showed that the binding of these antibodies to phosphatidylethanolamine in ELISArequiresa
140-kD serum
glycoprotein, the precise identity of which is not yet known.
Antibodies to Endothelial Cell Surface Proteins
Although
not
phospholipid-binding
plasma
proteins,
thrombomodulin and vascular heparan sulfate proteoglycan
are included as potential antigenic targets
because of their
respective roles in the control of thrombosis. Autoantibodies
to thrombomodulin have been identified in few patients.95,y6
However, in a larger study of antithrombomodulin antibodies, Gibson et aIy7observed no significant differences among
patients with lupus anticoagulants,patients referred for lupus
anticoagulant testing but found to be negative, and normal
individuals.
Fillit et a1 have demonstrated autoantibodies to both heparan sulfate" and the protein core of vascular heparan sulfate pr~teoglycan'~in the sera of certain patients with sys-
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ANTIBODIES TO PHOSPHOLIPID-BINDINGPROTEINS
temic lupus erythematosus (SLE). Given that sera were used
in these assays and that P2GPI binds to heparin, it is possible
that the apparent ’Pecificity Of autoantibodies for heparan
sulfate is, in fact, caused by antibodies to P2GPI.
2859
A New Model: Autoantibodies to Phospholipid-Binding
Plasma Proteins
Taken together, recent d a b strongly suggest that phospholipid-binding proteins andor protein-phospholipid com-
plexes are the physiologically relevant targets of “antiphospholipid” autoantibodies.notSome, but
all, of these
autoantibodies are detectable in the “antiphospholipid” antiAre some “antiphospholipid” autoantibodies specific for
body assays currently in clinical use (see Table 1). Autoantianionic phospholipids? Previous studies indicating such a
bodies to P2GPI are detected in the “anticardiolipin”
specificity are difficult to interpret because of the fact that
ELISA, p2GPI being present in the bovine sera used in
most experiments were performed in the presence of serum
the blocking buffer and in the patient’s serum sample. Also
or plasma. Therefore, the possible role of plasma proteins
detected in this assay are “true” anticardiolipin antibodies
and/or protein-phospholipid complexes may not have been
seen in association with syphilis and other infections. A
appreciated. Additionally, contamination of purified antibodlupus anticoagulant assay, such as the dilute Russell viper
ies and other reagents with plasma proteins now known to
venom time, detects certain antibodies to P2GPI,’3”5as well
be important, eg, P2GP1, was not directly evaluated. A look
as antibodies to prothrombin and factor X. Antiprothrombin
at the key report by Pengo et a1” highlights these difficulties.
and anti-factor X antibodies are probably not detectable in
These investigators studied IgG lupus anticoagulants from
“anticardiolipin” ELISAs because of several factors includfive patients with lupus or lupuslike disease and/or a history
ing the use of serum rather than plasma and the low concenof thrombosis. Although the IgG fractions and F(ab‘), fragtration of Ca’+ in the assay. Autoantibodies to protein C and
ments were highly purified, anticoagulant activity was asprotein S are not detected in either assay. Thus, the detection
sessed in a dilute Russell viper venom time assay using
of different antigenic specificities in the currently used “antinormal plasma, and the anticardiolipin ELISA wasperphospholipid” assays appears to explain much of the heteroformed with 10% fetal calf serum as the blocking agent and
geneity and inconsistent overlap of “anticardiolipin” antisample diluent, as is often the case for this immunoassay.’”
bodies and lupus anticoagulants. For example, it clarifies
In the absence of plasma or serum, these lupus anticoagulants
why, in some patients, “anticardiolipin” antibodies and luinhibited the binding of prothrombin and factor X to solidpus anticoagulants are the same antibodies, whereas in others
phase phospholipids. As discussed above, prothrombin, and
they are clearly distinct antibodies.
possibly factor X, are the targets of some “antiphosphoImmunologically, it is of interest that autoantibodies to
lipid” autoantibodies. Further, this experiment wasperdifferent phospholipid-binding proteins occur together in
formed in the presence of bovine serum albumin, some comvarious combinations. As recently demonstrated by Oosting
mercial preparations of which may contain significant
et a],” some patients may have autoantibodies to P2GP1,
amounts of bovine P2GPI (unpublished observation, Decemprothrombin, protein C, and protein S , whereas others may
ber 1992). More recent evidence that some lupus anticoaguhave antibodies against one, two, or three of these proteins
lants may be directed against anionic phospholipid alone
in different combinations. Speculatively, this is similar to
is limited.‘“ As discussed above, some investigators have
certain linked sets of antinuclear antibodies that occur in
suggested that 02GPI is not the target of “anticardiolipin”
patients with systemic lupus erythematosus, eg, antibodies
autoantibodies, but rather enhances antibody binding to
to constituents of small nuclear ribonucleoprotein particles.
cardiolipin by modifying the phospholipid conformation.”’
Antibodies may be directed against different protein compoHowever, at present there are no direct data to support this
nents of such particles, such as Sm and U1-RNP proteins,
hypothesis. Anionic phospholipids do appear to be the target
suggesting that the immune response is antigen-driven.”’ By
of anticardiolipin antibodies associated with syphilis and
analogy, the co-occurrence of antibodies to different phosother infectious diseases, as well as those detected in many
pholipid-binding proteins suggests that the relevant antigenic
normal sera after heat
particle in this instance may consist of proteins bound in
Lupus anticoagulants have been reported to recognize nonclose proximity to one another on an anionic phospholipid
bilayer,hexagonal (U) phasephospholipid ~ t r u c t u r e ~ ~ ~surface,
~ ” ~ ~ perhaps
;
a damaged vascular endothelial cell or actihowever, this specificity has not been shown in the absence
vated platelet.
of serum or plasma. Binding of P2GPI tohexagonal (11)
phase phospholipid may explain some of these data.’0g
PATHOGENETICMECHANISMS
Although not antigenic targets, anionic phospholipids may
play an important role in vivo in the binding of autoantibodIf “antiphospholipid” autoantibodies play a role in the
ies to phospholipid-bound plasma proteins. As discussed,
pathogenesis of the antiphospholipid syndrome, the spectrum
these antibodies may be specific for protein conformations
of antigenic specificities discussed may offer an explanation
induced when the proteins bind to phospholipids. Further,
for the confusing variety of proposed pathophysiologic
antibody binding may be depend on the phospholipid surface
mechanisms as well as the heterogeneity of clinical manifesto provide a high local concentration of antigen, thereby
tations of the syndrome. Although no association between a
promoting high-avidity bivalent binding of intrinsically lowparticular antigenic specificity, a particular mechanism of
affinity antibodies.
action, and a particular clinical manifestation of the antiphosPhosvholiuids
‘ .
From www.bloodjournal.org by guest on March 6, 2016. For personal use only.
2860
ROBERTA.S.ROUBEY
Table 2. Potential Mechanismsof
Autoantibody-MediatedThrombosis
~~
Potential Mechanisms
Antibody
Anti-PZGPI
Inhibition
protein
Cof
activation
Inhibition of protein C/protein S-mediated
inactivation of factors Va and Vllla
Inhibition of factor Xll/prekallikreinmediated fibrinolytic activity
Inhibition of heparan sulfate-mediated
activation of antithrombin 111
Enhanced platelet activation
Enhanced factor Xa generation by
platelets
Decreased free protein S levels via
inhibition of theinteraction of p2GPI
and C4b-binding protein
Anti-prothrombin/
Anti-thrombin
Binding tothrombin-activated platelets
Inhibition of thrombin-mediated
endothelial cell prostacyclin release
Inhibition of protein C activation
Anti-protein C
Inhibition of protein C activation
Inhibition of protein Uprotein S-mediated
inactivation of Va and Vllla
Anti-protein S
Inhibition of protein C/protein S-mediated
inactivation of Va and Vllla
Anti-phospholipase A2Inhibitionof
endothelial cell prostacyclin
production
Anti-thrombomodulin
Inhibition of protein C activation
Anti-vascular heparan
sulfate proteoglycan
Inhibition of heparan sulfate-mediated
activation of
antithrombin Ill
pholipid syndrome has yet been established, it is reasonable
to speculate that antibodies to different proteins would have
different effects and may be associated with different types
of events. For example, it has been suggested that antibodies
which alter the prostacyclidthomboxane balance maybe
associated with arterial thrombosis, whereas antibodies that
inhibit the protein C pathway may be associated with venous
thrombosis."' Some specificities may not be related to any
disease manifestation, thus explaining whymany patients
are asymptomatic. In this section, the numerous mechanisms
proposed to explain how "antiphospholipid" autoantibodies
predispose to thrombosis and thrombocytopenia will be reviewed, with speculation as to the particular antigenic specificity or specificities which may be involved. The mechanisms potentially associated with certain autoantibodies are
summarized in Table 2.
Endothelial Cell Interactions
Inhibition of the protein C pathway. "Antiphospholipid" antibodies may inhibit phospholipid-dependent reactions of the protein C pathway: (1) the thrombidthrombomodulin activation of protein C, andlor (2) the activated protein
Uprotein S degradation of factors Va and VIIIa. Comp et
all" reported that IgG from two of seven patients with lupus
anticoagulants inhibited the activation of protein C both in
solution andon endothelial cells. Similar data have been
reported by other^.^^.^^.' I' Some of these reports have suggested that the antibodies may be directed against thrombor n ~ d u l i n ~ or
~ . "protein
~
C.' l6 Given that P2GPI-dependent
lupus anticoagulants appear to enhance the binding of P2GPI
to anionic phospholipid in the prothrombinase reaction, it
is possible that such antibodies could similarly inhibit the
activation of protein C. Although P2GPI has been shown to
inhibit protein C activation by thrombomodulin incorporated
in cardiolipin ~esicles,"~
there was little orno effect on
the endothelial cell-mediated activation of protein C, in the
presence or absence of anti-P2GPI autoantibodies."' This
discrepancy maybe explained by the fact that protein C
activation, while enhanced by anionic phospholipids, can
occur on neutral phospholipid membranes. The kinetics of
protein C activation on cultured endothelial cells are comparable to those observed with neutral synthetic membranes."'
Theoretically, antibodies to thrombin might also inhibit protein C activation.
Several laboratories have observed inhibition of the anticoagulant activity of activated protein C by "antiphospholipid" antibodies.".'20"23Marciniak and Romondl" reported
a decreased rate of factor Va degradation in the plasma of
15 patients with lupus anticoagulants. Exogenous protein C
did not correct this deficiency, leading these investigators to
conclude that the antibodies inhibited the formation of the
anionic phospholipidprotein C/protein S complex. However,
Malia et all2' found that IgG fractions from certain patients
prevented Va degradation only in the presence of protein S,
whereas others inhibited equally well with and without protein S. As previously discussed, similar results were obtained
by Oosting et al," who further demonstrated that the antibodies responsible for inhibiting Va degradation were directed
against phospholipid-bound protein C or protein S. Interestingly, one patient in this study had a history of thrombosis
and autoantibodies to phospholipid-bound protein S, in the
absence of "anticardiolipin" antibodies or lupus anticoagulant. Decreased levels of protein S have been reported in a
small number of patients with the antiphospholipid antibody
~ y n d r o m e . ~ " , ' Although
~ ~ . ' ~ ' this could be caused by antibodies to protein S itself, autoantibodies to P2GPI might alter
the level of free protein S by interfering with the proposed
interaction of P2GPI with C4b-binding pr~tein.~'
Inhibition of prostacyclinproduction.
Although some
investigators have demonstrated inhibition of endothelial cell
prostacyclin production by plasma or purified IgG from patients with "antiphospholipid" antibodies,"'.'2h"'' others
have reported no effect, enhanced prostacyclin production,
or mixed results.'i4~'3z-i34
True differences among patients
and small numbers of patients in these studies may explain
some of these differences. Cameras et al, in a recent review
of this field,'3shave suggested several methodologic reasons
for this discrepancy including the heterogeneity of the vascular systems studied, eg, rat aortic rings,126~,'ZR
bovine aortic
endothelial ~ e l l s , ' ~or
' ~human
' ~ ~ umbilical vein endothelial
ce~~s,l14,13Z-134 different cell culture conditions, differences in
agonists, and different methods of antibody of serudplasma
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ANTIBODIESTOPHOSPHOLIPID-BINDING
PROTEINS
2861
kallikrein generation would lead to decreased cleavage of
preparation. Another important factor in these studies is the
prourokinase, thus impairing
relative state of activation of the endothelial cells.”’ ActivaEnhanced platelet-activatingfactor production. Silver
tion of endothelial cells by cytokines and inflammatory meet
reported that “anticardiolipin” autoantibodies endiators significantly alters the expression of cell surface
hance the endothelial cell production of platelet-activating
proteins. For example, “antiphospholipid” antibodies pofactor. However, platelet-activating factor, like prostacyclin,
tentiate tumor necrosis factor-induced endothelial cell procois released by phospholipase A2-mediated hydrolysis. Acagulant activity.’36Inhibition of endothelial cell prostacyclin
cordingly, these data conflict with those of Schorer et a1,”
production could be explained by inhibition of phospholipase
who observed decreased platelet-activating factor, in accorA2 activity,” and it has been suggested that autoantibodies
dance with their findings that some antiphospholipid antibodmay be directed against phospholipase A2or a phospholipase
ies inhibit phospholipase AZactivity.
A2-phospholipidcomplex.82 Antibodies to thrombin could
potentially interfere with thrombin-induced prostacyclin proPlatelet Interactions
duction. At the present time, thereare no data regarding other
antigens, either phospholipid-binding proteins or endothelial
Thrombocytopenia. Autoimmune thrombocytopenia is
cell-surface molecules, which might explain antibody-medipart of the antiphospholipid antibody syndrome and thought
ated alteration of arachidonic acid metabolism.
to be caused by the antiplatelet activity of a subset of these
Increased tissue factor expression. As mentioned above,
antibodies.’ “Antiphospholipid” antibodies have been
it has been reported that six patientsera containing “antiphosshown to bind to freeze-thawed platelets and activated platepholipid’’ antibodies acted synergistically with tumor necrosis lets; however, there is little evidence for binding to fresh,
factor to enhance endothelial cell procoagulant activity.L36
The
unstimulated platelet^."^"^^
likely mechanism for this activity is upregulation of tissue
Recently Shi et all5’ studied the platelet-binding activity
factor expression. In support of this hypothesis, sera or IgG
of “anticardiolipin” antibodies and lupus anticoagulants
from 14 of 16 lupus patients have been shown to induce tissue separated by anticardiolipin affinity chromatography and
factor production in cultured endothelial cells.’37Whether this
other physicochemical techniques from the plasma of two
effect is caused by cell injury or by the engagement of specific
patients with both activities. The lupus anticoagulant fracendothelial cell antigens is not known.
tions bound to thrombin-activated but not resting platelets.
Inhibition of antithrombin I l l activity. Autoantibodies to
Together with the observation that lupus anticoagulants lackvascular heparan sulfate pr~teoglycan~’,~~
could contribute
ing “anticardiolipin’ ’ activity are directed against phosphoto a thrombotic diathesis by blocking the heparan sulfatelipid-bound prothrombin,” it is intriguing to speculate that
mediated activation of antithrombin 111.Monoclonal antibodthe antiplatelet activity of these lupus anticoagulants is
ies to heparan sulfate have been shown to have such an
caused by antibodies binding to thrombin. “Anticardiolipin”
activity.13*Compatible with this mechanism, Cosgriff and
antibody fractions bound to activated platelets as well, but
MartinI3’ described a patient with the lupus anticoagulant
required the presence of P2GPI. This suggests that plateletand recurrent thrombosis who had high antigenic, but low
bound P2GPI may be a physiologic target of these autoantifunctional, levels of antithrombin 111. More recently, Chambodies.
ley et alla showed that IgM purified from the serum of a
Effects on platelet activation. A number of studies sugpatient with “anticardiolipin” autoantibodies inhibited the
gest that “antiphospholipid” autoantibodies may alter plateheparin-dependent activation of antithrombin 111. In view of
let activity themselves or in association with other agonists.
the fact that P2GPI binds to heparin, this effect might be
Antiphospholipid antibodies have been reported to increase
caused by anti-P2GPI autoantibodies.
thromboxane A2 p r o d u c t i ~ n . ~Measuring
~ ~ ~ ~ ~ ~urinary
- l ~ ~ meImpairedjbrinolysis. Studies of tissue plasminogen actabolites of prostacyclin and thromboxane A2, two groups
tivator levels, before and after venous occlusion, have generhave observed increased average thromboxane:prostacyclin
ally been i n c o n ~ l u s i v e . ~The
~’”~
most
~ consistent finding is
ratios in a total of 56 patients with the antiphospholipid
an elevated level of plasminogen activator inhibitor- 1 (PAIThis imbalance was primarily caused by in1) in patients with SLE.L46-’48
However, there is no strong
creased platelet thromboxane A2. Further, IgG F(ab’), fragcorrelation between increased PAI-1, the presence or level
ments from six patients with elevated ratios were shown to
of “antiphospholipid” autoantibodies, and a history of
enhance platelet generation of thromboxane in ~ i t r 0 . l ~The
’
thrombo~is.’~~.‘~~
mechanism by which this occurs and the relevant platelet
The contact system may play an important role in fibriantigen are not known. There are conflicting data regarding
no1ysis.149
Two groups have observed inhibition of the factor
the effect of “antiphospholipid” on platelet aggregation.
XII-dependent fibrinolytic pathway in a total of 14 of 22
Some investigators have reported that “antiphospholipid”
patients, and suggested that this is a mechanism of lupus
autoantibodies may induce platelet aggregation,’ss316216’
anticoagulant-associated t h r o m b o ~ i s . ’In
~ ~view
. ~ ~of~ the fact
whereas others have found that lupus anticoagulants block
that P2GPI inhibits factor XI1 and prekallikrein activation
platelet aggregation in response to some agonists butnot
on anionic phospholipid surface^,^^^^'.'^^ anti-02GPI autoanothers.’30,’64In the study by Shi et alls7discussed above,
tibodies may amplify this inhibitory activity by enhancing
neither lupus anticoagulant nor “anticardiolipin” antibody
the binding of P2GPI to the phospholipid surface, as prefractions affected platelet degranulation or aggregation. Muviously discussed. Decreased activation of factor XI1 and
rine monoclonal antibodies to P2GPI have been shown to
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ROBERT A.S. ROUBEY
2862
bind to platelets in the presence of P2GPI and lead to platelet
activation in the presence of subthreshold concentrations of
weak a g 0 n i ~ t s . Both
I ~ ~ Fab and Fc portions of the antibodies
were required for platelet activation, suggesting a role for
the platelet IgG Fc receptor, F,,RII.
It has been reported that p2GPI inhibits the factor Xa
generating activity of platelets and that “anticardiolipin”
autoantibodies block this inhibitory activity.“‘ This would
result in unopposed Xa generation, which may contribute to
a thrombotic tendency. However, these data are in contrast
to those observed in the prothrombinase assay in which
“anticardiolipin” antibodies enhance the inhibitory activity
of ~ ~ G P I . ~ ~ - ~ ~ . ~ ~
CONCLUSIONS AND FUTUREDIRECTIONS
Recent data support the paradigm that “antiphospholipid”
autoantibodies are part of a linked set of autoantibodies directed against various phospholipid-binding plasma proteins.
This group includes autoantibodies that are detected in
“anticardiolipin” and/or lupus anticoagulant assays, eg,
anti-02GPI and anti-prothrombin antibodies, as well as antibodies not detected by currently used clinical laboratory
tests, eg, anti-protein C and anti-protein S antibodies. Additional specificities may well be discovered as research in this
field progresses. In contrast, antibodies specific for anionic
phospholipids occur in association with syphilis and other
infectious diseases, and do not appear to be associated with
autoimmune diseases or the “antiphospholipid” antibody
syndrome. Although evidence that “antiphospholipid” autoantibodies are directed against a number of proteins involved
in the control of thrombosis and hemostasis should stimulate
further investigation regarding the potential pathologic role
of these antibodies, it should be keptinmindthat
direct
evidence for such a role is not yetavailable. These antibodies
may represent an epiphenomenon rather than a causative
factor in the development of the “antiphospholipid” antibody syndrome.
This new model explains muchof the heterogeneity of
“antiphospholipid” autoantibodies observed in laboratory
testing. More importantly, it offers great promise in explaining the clinical heterogeneity and pathophysiology of
the “antiphospholipid” antibody syndrome. Future studies
are needed to determine if different antigenic specificities,
alone or in combination, are associated with particular clinical manifestations, or carry greater or less risk for the development of the syndrome. Important areas for investigation
include (1) the effect of different antibodies on the physiologic functions of their target antigens; ( 2 ) the location, accessibility, and density of the antigens on phospholipid membranes in vivo; (3) the role of titer, avidity, and epitope
specificity of the antibodies; and (4) characterization of the
immune response that leads to the development of these
autoantibodies. The use of purified antibodies and assay systems utilizing purified plasma components, rather than serum
or plasma, will be of critical importance.
If autoantibodies to particular phospholipid-binding proteins are shown to be associated with different clinical presentations or to confer different risks, then clinical testing
will need to be revised. Although current “anticardiolipin”
ELISAs and lupus anticoagulant assays are clinically useful,
these tests do not clearly differentiate antibodies with different specificities nor do they detect potentially important antibodies, such as those directed against protein C and protein
S. For example, “anticardiolipin” assays detect both antibodies to P2GP1, associated with the “antiphospholipid”
antibody syndrome, and antibodies to cardiolipin, which are
not associated with this syndrome. Thus, an assay that detects only antibodies against P2GPI would be expected to
have greater specificity for the “antiphospholipid” antibody
syndrome. Studies are needed to determine if it is clinically
important to differentiate between lupus anticoagulants that
are antibodies to P2GPI and those that are antibodies to
prothrombin. Antigenic and functional assays for the detection of autoantibodies to protein C and protein S maybe
shown to have clinical utility.
Finally, with regard tonomenclature, recent data highlight
the inadequacy of the current classification of antiphosphqlipid antibodies as anticardiolipin antibodies and lupus antihave suggested that anticoagulants. Vermylen and Amout**
phospholipid
antibodies
be renamed antiphospholipidprotein antibodies. Because antibody binding to ,B2GP12,4-R
and prothrombin’,’’ has been observed in the absence of
phospholipid, the term autoantibodies to phospholipid-binding plasma proteins has been used in this review. If warranted by future studies, a classification based on antigenic
specificity may prove to beuseful. For example, designations
such as anti-P2GPI antibodies or anti-prothrombin antibodies may confer more relevant information than the term lupus
anticoagulant.
NOTEADDED
IN PROOF
The protein required for the binding of autoantibodies to phosphatidylethanolamine, initially thought to be a 140-kD serum glycoprotein, has recently been identified as low molecular-weight kininogen
and/or nonfragmented high molecular-weight kinin~gen.’~’
ACKNOWLEDGMENT
The author thanks Drs Harold R. Roberts, Gilbert C. White 11,
John B. Winfield, and Dougald M. Monroe 111for helpful discussions
and suggestions.
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1994 84: 2854-2867
Autoantibodies to phospholipid-binding plasma proteins: a new view
of lupus anticoagulants and other "antiphospholipid" autoantibodies
[see comments]
RA Roubey
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