ARTHRITIS & RHEUMATISM
Vol. 39, No. 9, September 1996, pp 1444-1454
1444
REVIEW
IMMUNOLOGY OF THE ANTIPHOSPHOLIPID ANTIBODY SYNDROME
ROBERT A. S. ROUBEY
Her name was Magill
And she called herself Lil
But everyone knew her as Nancy
John Lennon and Paul McCartney
Like the femme fatale in The Beatles’ Rocky
Raccoon, antiphospholipid antibodies (aPL) and the
syndrome with which they are associated (thrombosis,
recurrent fetal deaths, thrombocytopenia) have at-
tracted considerable attention despite a serious nomen-
clature problem. Along with increasing experience in the
optimal management of patients with the aPL syndrome
and growing evidence that autoantibodies may play a
direct pathophysiologic role, research over the past
several years demonstrates that a large proportion of
“antiphospholipid” autoantibodies do not, in fact, rec-
ognize phospholipids. These new data indicate that the
antigenic targets of antibodies detected in conventional
anticardiolipin and lupus anticoagulant assays are
phospholipid-binding plasma proteins, most notably, &-
glycoprotein I (&GPI) and prothrombin or complexes
of these proteins with phospholipids. Further, autoanti-
bodies to other phospholipid-binding plasma proteins
and certain molecules expressed on vascular endothe-
lium may also be associated with the antiphospholipid
antibody syndrome (APS)although they are not detect-
able in standard aPL assays.
This review summarizes recent information on
the specificities of autoantibodies associated with A P S
and discusses the insights these data offer into the
potential role of autoantibodies in the syndrome’s
pathophysiology.
Supported by NIH grants AR-30701 and AR-01920. Dr.
Roubey is the recipient of a Clinical Investigator Award from NIAMS.
Robert A. S. Roubey, MD: University of North Carolina at
Chapel Hill.
Address reprint requests to Robert A. S . Roubey, MD,
Division of Rheumatology and Immunology, CB #7280, Room 3330
Thurston Building, University of North Carolina, Chapel Hill, NC
27599-7280.
Submitted for publication January 24, 1996; accepted in
revised form July 3, 1996.
0 1996, American College of Rheumatology
Antigenic specificities of aPL: historical perspective
Current concepts of aPL date to the early part of
this century and the development of nontreponemal
serologic tests for syphilis (STS). In the early 1940s,
Mary Pangborn identified the antigenic component of
the tissue extracts used in these tests as a novel anionic
phospholipid, which she named cardiolipin (1). As a
result of widespread population screening for syphilis, in
the 1950s it was observed that individuals with chroni-
cally false-positive STS results, when followed up for
many years, often developed systemic lupus erythema-
tosus (SLE) (2). False-positive STS results were also
noted to be present in early reports of SLE patients with
acquired inhibitors of in vitro phospholipid-dependent
coagulation tests, in particular, the activation of pro-
thrombin (3). Laurel1 and Nilsson investigated this asso-
ciation and suggested that the anticoagulant effect and
the positive reactions with phospholipid antigen were
ascribable to the same causal factor (4). Additional
support for the hypothesis that these acquired coagula-
tion inhibitors were directed against phospholipid came
from studies demonstrating that the anticoagulant effect
was enhanced when the phospholipid in the test system
was diluted (5).
In contrast, a number of key features of the
inhibitors, subsequently termed lupus anticoagulants (6),
were not compatible with phospholipid specificity and
suggested an important role of plasma proteins. First,
certain lupus anticoagulants were species specific, for
example, they prolonged coagulation reactions of human
plasma but not bovine plasma (7). Second, certain lupus
anticoagulants did not inhibit phospholipid-dependent
reactions other than the activation of prothrombin, such
as the activation of factor X (8,9). Third, a cofactor
phenomenon was observed in mixing studies of some
lupus anticoagulants. Mixing certain lupus anticoagulant-
positive plasmas with normal plasma not only failed to
correct the prolonged clotting time, but actually pro-
longed it still further. Loeliger concluded that the cofac-
tor that enhances lupus anticoagulant activity was pro-
IMMUNOLOGY OF A P S
Table 1. Antibodies detected in conventional antiphospholipid anti-
body assays’
Anticardiolipin ELISAs
Anti-&GPI
Anticardiolipin
Antibodies to other cardiolipin-binding serum proteins
(speculative)
Lupus anticoagulant assays
Antiprothrombin
Anti-&GPI
Anti-factor V
Anti-factor X
Antiphospholipid
* ELISAs = enzyme-linked immunosorbent assays; anti-&GPI
anti-P,-glycoprotein I.
thrombin itself (10). In another patient, however, Yin
and Gaston demonstrated that lupus anticoagulant ac-
tivity required the presence of a protein, which is found
in both normal and SLE patient plasma, that was distinct
from prothrombin (9).
Despite these observations, the conceptualization
of A P S by Hughes in 1983 (11) and the development of
the anticardiolipin enzyme-linked immunosorbent assay
(ELISA) (12) focused attention on the apparent phos-
pholipid specificity of the autoantibodies. It is difficult to
interpret much of the data indicating phospholipid spec-
ificity, however, because nearly all assays were per-
formed in the presence of serum or plasma. In instances
where purified antibodies or assay systems were em-
ployed, potential contamination with proteins now
known to be important (e.g., P,GPI) was not assessed. In
clinical practice, the observed heterogeneity of antibod-
ies detected in conventional anticardiolipin and lupus
anticoagulant assays is not adequately explained by
phospholipid specificity. Patients with syphilis have anti-
bodies that are detectable in syphilis tests and often in
the anticardiolipin assay; however, these antibodies do
not have lupus anticoagulant activity, and patients with
syphilis do not develop A P S (13). Patients with A P S
often have both anticardiolipin antibodies and lupus
anticoagulants; however, a significant proportion have
one reactivity but not the other (14). In some patients
with both anticardiolipin antibodies and the lupus anti-
coagulant, a single antibody population seems to have
both activities (15), while in other patients, these anti-
bodies are separable (16).
During the last 6 years, numerous studies have
elucidated the specificity of aPL for phospholipid-
binding plasma proteins, as summarized in Table 1.
These data provide a clear explanation for the features
of lupus anticoagulants cited above and for the patterns
1445
of overlapping reactivity observed in STS and anticar-
diolipin and lupus anticoagulant assays.
Antibodies detected in anticardiolipin assays
Anti-P,GPI. In 1990, three groups independently
reported that affinity-purified “anticardiolipin” antibod-
ies did not bind to cardiolipin in the absence of serum or
plasma (17-19). The critical plasma component was
found to be P,GPI. The amino acid sequence of P,GPI
was determined by Lozier et a1 (20) and its complemen-
= tary DNA was cloned (21,22). P,GPI is a 50-kd glyco-
protein that is present in normal plasma at a concentra-
tion of -200 pglml. Its physiologic function is not
known. Structurally, P,GPI is a member of the comple-
ment control protein family, with 5 of the consensus
repeats (“sushi” domains) that characterize this group of
molecules (23). A fraction of P,GPI circulates in asso-
ciation with lipoproteins and it has also been termed
apolipoprotein H (24). P,GPI binds to anionic phospho-
lipids (25), and lysine-rich segments in the fifth domain
have been implicated as a phospholipid-binding region
(26,27).
In vitro, P,GPI inhibits prothrombinase activity
(28-30), contact pathway activation (31), ADP-induced
platelet aggregation (32), and factor Xa generation by
platelets (33). Although these data suggest that P2GPI
functions as a natural anticoagulant, neither the het-
erozygous nor the homozygous deficiency of the protein
is clearly associated with an increased risk of thrombosis
(34). Recent data from our laboratory indicate that
P,GPI binding to membranes containing physiologic
concentrations of anionic phospholipids is relatively
weak; thus, normal plasma levels of P,GPI probably
have little effect on hemostatic reactions in vivo (35).
Most, if not all, “anticardiolipin” antibodies as-
sociated with A P S are directed against epitopes ex-
pressed on P,GPI, not on cardiolipin. Initial reports
differed as to whether “anticardiolipin” antibodies
bound to P,GPI in the absence of phospholipid
(17,18,36). Subsequently, however, binding of antibodies
to &GPI alone has been observed by most research
groups, including several that did not at first detect such
binding (26,37-41). The basis for the initial discrepan-
cies is methodologic. In ELISA, the detection of anti-
P,GPI autoantibodies is dependent upon the type of
microtiter plate used (37,38). Little or no binding occurs
if P,GPI is immobilized on untreated polystyrene plates,
whereas specific antibody binding to P2GPI is observed
if “high-binding” polystyrene plates are used. High-
binding plates are produced commercially by treating
1446
polystyrene plates with high levels of ?-irradiation,
which causes oxidation of the polystyrene surface and
significantly enhances the capacity of the plastic to bind
certain proteins (42). Autoantibodies to &GPI have also
been detected in an ELISA utilizing polyvinyl chloride
microtiter plates (39) and in immunoblotting assays (43).
At physiologic concentrations of &GPI, antibody
binding in the fluid phase is weak (38). Data from our
laboratory indicate that this pattern of anti-P,GPI auto-
antibody reactivity is due to low intrinsic affinity of the
autoantibodies (Kd -10-6-10-5M for fluid-phase bind-
ing) (38). Clustering, or a high density of immobilized
antigen, is required in order to allow bivalent or multi-
valent antibody binding and subsequent detection in
ELISA or immunoblotting. It has also been proposed
that anti-P,GPI antibodies may recognize conforma-
tional epitopes on &GPI that are induced when P2GPI
binds to an anionic surface such as cardiolipin vesicles,
cardiolipin-coated plates, or high-binding plates (37).
While it is likely that some autoantibodies recognize
conformational epitopes, exclusive specificity for such
epitopes would not account for fluid-phase binding at
high &GPI concentrations or the affinity purification of
these antibodies utilizing P,GPI-agarose beads (44).
To understand the detection of anti-&GPI anti-
bodies in conventional anticardiolipin ELISAs, it is
necessary to review certain technical aspects of these
assays. There are 2 sources of &GPI in anticardiolipin
ELISAs: bovine &GPI from the bovine serum that is
commonly used in the blocking bufferhample diluent,
and human &GPI from the test serum or plasma
sample. Bovine &GPI is the more abundant species in
most conventional anticardiolipin assays and accounts
for the vast majority of antibody binding. There is,
however, sufficient endogenous human P,GPI to support
antibody binding in the absence of exogenous bovine
&GPI in many instances (assuming a normal serum
concentration of P,GPI, a 1:lOO dilution contains -2
CLg/ml).
A number of recent studies have compared con-
ventional anticardiolipin assays with ELISAs utilizing
phospholipid-free human &GPI as the antigen (36,45-
47). Sera from patients with A P S generally show excel-
lent correlation between the 2 assays (36), although the
discordant behavior of certain sera indicates the exis-
tence of a number of antibody subsets. A small group of
sera from patients with A P S are reactive in anti-P,GPI
assays but not in standard anticardiolipin assays. Possi-
ble explanations are that these antibodies are directed
against epitopes on P2GPI that are not accessible when
&GPI is bound to cardiolipin, that P,GPI bound to
ROUBEY
cardiolipin is not clustered in such a way as to allow
bivalent attachment of the antibodies, or that the anti-
bodies are species specific, recognizing human, but not
bovine, P,GPI. Other sera are reactive in anticardiolipin
assays but not in anti-P,GPI assays. This pattern of
reactivity does not appear to be associated with APS.
These sera may contain authentic anticardiolipin anti-
bodies (P,GPI-independent anticardiolipin antibodies),
antibodies that are species specific for bovine P,GPI, or
antibodies that react only with the p,GPI-phospholipid
complex.
Anticardiolipin. In addition to antibodies to
&GPI, conventional anticardiolipin assays detect au-
thentic anticardiolipin antibodies, i.e., antibodies that
bind to cardiolipin in the absence of serum proteins.
Human &GPI inhibits the binding of these antibodies to
cardiolipin, presumably by competing for similar phos-
pholipid structures. This inhibitory effect is much less
pronounced with bovine P,GPI, which explains why
these antibodies are detectable in conventional anticar-
diolipin assays in the presence of high levels of bovine
p,GPI (22). Authentic anticardiolipin antibodies are
associated with syphilis and other infectious diseases
(30,36) and may occur in apparently healthy individuals.
They do not appear to be strongly associated with
clinical manifestations of APS. Some investigators have
hypothesized that anticardiolipin antibodies in patients
with A P S are not directed against epitopes on P2GPI,
but recognize conformational phospholipid epitopes in-
duced by the binding of &GPI to phospholipid (48).The
evidence of antibody binding to &GPI cited above does
not preclude the existence of a subset of antibodies
reactive with the phospholipid portion of the &GPI-
phospholipid complex.
Antibodies to other cardiolipin-binding proteins.
Potentially, anticardiolipin ELISAs could detect anti-
bodies to cardiolipin-binding proteins other than P,GPI
in bovine or human sera. For example, Kertesz et a1
recently demonstrated that complement factor H, which
has a high degree of homology with &GPI, binds to
cardiolipin-coated microtiter plates under conditions
used in most anticardiolipin ELISAs (27). The possibil-
ity that some patients might have autoantibodies to
factor H has not been investigated.
Antibodies detected in lupus anticoagulant assays
Lupus anticoagulants are defined as immuno-
globulins that prolong 1 or more phospholipid-
dependent coagulation tests in vitro. Specific lupus
anticoagulant assays and technical aspects of their per-
IMMUNOLOGY OF A P S
formance have been reviewed elsewhere (49). It should
be noted that the phospholipid dependence of lupus
anticoagulants is entirely compatible with the specificity
of these antibodies for phospholipid-binding proteins.
Antiprothrombin. The identification of pro-
thrombin as a lupus anticoagulant “cofactor” (lo), along
with early reports of hypoprothrombinemia in a subset
of patients with lupus anticoagulants, suggested that
prothrombin might be the antigenic target of lupus
anticoagulants. Bajaj et a1 initially demonstrated the
presence of high-affinity autoantibodies to prothrombin
in 2 patients with lupus anticoagulants and hypopro-
thrombinemia (50). Circulating complexes of antibodies
and prothrombin were subsequently identified in the
undiluted plasma of patients with lupus anticoagulants
and normal prothrombin levels (51,52). Fleck et a1
affinity-purified antiprothrombin antibodies from 3 pa-
tients and demonstrated that these autoantibodies had
lupus anticoagulant activity (52).
More recently, Bevers et al (53) and Galli et al
(29) studied plasma from 16 patients with both lupus
anticoagulants and anticardiolipin antibodies. In 11
plasma samples, lupus anticoagulant activity could not
be absorbed with cardiolipin-containing vesicles. In a
purified prothrombinase assay, these lupus anticoagu-
lants were shown to be specific for phospholipid-bound
human prothrombin. In the remaining 5 plasma samples,
the lupus anticoagulants also had anticardiolipin activity
and were identified as autoantibodies to &GPI (see
below). Permpikul et al purified IgG fractions from 10
patients with lupus anticoagulants and clearly demon-
strated that lupus anticoagulant activity was due to
antiprothrombin antibodies in at least 9 of the samples
(54). Recently, Arvieux et a1 demonstrated by ELISA
that 77 of 139 patients with lupus anticoagulants (55%)
had autoantibodies to prothrombin (55). Antiprothrom-
bin antibodies were present in the sera of -70% of
patients with lupus anticoagulants associated with SLE
or the primary APS, but were found in only 20% of
patients with lupus anticoagulants associated with infec-
tion or malignancy (55). A similar percentage of
prothrombin-dependent lupus anticoagulants (14 of 25
patients, 56%) was also reported by Galli et a1 (56).
Like antibodies to &GPI, antiprothrombin anti-
bodies associated with APS appear to be of relatively low
intrinsic affinity (55). A high percentage of antipro-
thrombin antibodies are species specific for the human
protein (53,55). Antiprothrombin antibodies are not
detected in conventional anticardiolipin ELISAs for a
number of reasons, including low prothrombin concen-
tration in patient and bovine serum and low calcium ion
1447
concentration (prothrombin binding to anionic phos-
pholipids is calcium dependent).
Anti-P,GPI. A subset of autoantibodies to &GPI
exhibit lupus anticoagulant activity (30,31,57), as do a
number of polyclonal and monoclonal anti-&GPI anti-
bodies raised in other species (31,58). Interestingly,
anti-P,GPI lupus anticoagulants act by enhancing the
relatively weak anticoagulant effect of &GPI itself
(30,31). A proposed mechanism of this enhancement is
discussed below. It is not known why some autoantibod-
ies to &GPI have lupus anticoagulant activity but others
do not. Interestingly, certain lupus anticoagulant assays
are more sensitive and others are less sensitive to
prolongation by autoantibodies with particular antigenic
specificities. The kaolin clotting time is more sensitive
than the dilute Russell viper venom time to prothrombin-
dependent lupus anticoagulants, whereas anti-&GPI
lupus anticoagulants prolong the dRVVT to a greater
extent than the KCT (56).
Antibodies to other coagulation factors. Anec-
dotally, a lupus anticoagulant specific for factor X has
been identified in a patient with anticardiolipin antibod-
ies and a hemorrhagic diathesis (Dr. D. Triplett: per-
sonal communication). Two patients with thrombosis,
lupus anticoagulants, and acquired factor V inhibitors
have been described (59,60).
Antiphospholipid antibodies. The issue of
whether some lupus anticoagulants are true antiphos-
pholipid antibodies (binding directly to phospholipids in
the absence of plasma proteins) is controversial. Data in
support of such a specificity are limited (61,62). It is
difficult to interpret previous data indicating that lupus
anticoagulants are specific for hexagonal-phase phos-
pholipid (63) due to the presence of serum or plasma in
the assay systems.
Antibodies not detected in s t a n d a d aPL assays (Table 2)
Antibodies to components of the protein C path-
way. There has been considerable interest in the possi-
bility that APS-associated autoantibodies may inhibit the
protein C pathway, a clinically important physiologic
anticoagulant mechanism (64) (see Figure 1). Until
recently, however, relatively little attention has been
paid to the possibility that antibodies might be directed
against constituents of this pathway. Autoantibodies to
thrombomodulin (65), phospholipid-bound protein C,
and phospholipid-bound protein S (66) have been
reported.
1448
Table 2. Autoantibodies possibly associated with the antiphospho-
lipid antibody syndrome but not detected in conventional antiphos-
pholipid antibody assays
~~
Antibodies to constituents of the protein C pathway
Anti-protein C
Anti-protein S
Antithrombomodulin
Antivascular heparan sulfate proteoglycan/antiheparin
Anti-annexin V
Anti-CD36
Anti-high and/or anti-low molecular weight kininogens
Antiphospholipase A, (speculative)
Antivascular heparan sulfate proteoglycan (anti-
vHSPG)/antiheparin. Heparan sulfate proteoglycan is
expressed on vascular endothelium and plays an impor-
tant role in vascular structure and function. Vascular
HSPG is required for the activation and optimal antico-
agulant activity of antithrombin 111. Autoantibodies to
both the heparan sulfate moiety and the core protein of
vHSPG have been detected in certain lupus sera by Fillit
and coworkers (67,68). Antiheparin antibodies have also
been reported in association with A P S (69).
Anti-annexin V. Annexins are a family of calcium-
dependent, phospholipid-binding, intracellular proteins
thought to have an important function in membrane
processes such as exocytosis. Preliminary evidence sug-
gests that annexin V (placental anticoagulant protein-I),
Figure 1. The protein C pathway. Protein C (PC) is a vitamin
K-dependent plasma glycoprotein that circulates as a precursor to a
serine protease. Activation of protein C occurs when thrombin (IIa)
binds to thrombornodulin, a constitutively expressed protein on the
surface of vascular endothelial cells. On binding to thrombomodulin,
thrombin's procoagulant activities (e.g., cleavage of fibrinogen, activa-
tion of platelets) are inhibited, while its ability to activate protein C is
markedly enhanced. Activated protein C (APC) acts as an anticoagu-
lant by proteolytically inactivating factors Va and VIIIa, thereby
limiting the rate of thrombin generation. The most efficient inactiva-
tion of factors Va and VIIIa requires the cofactor activity of protein S
(PS), another vitamin K-dependent plasma glycoprotein, and factor
V. Protein S circulates in plasma both as a free protein and in a
bimolecular complex with the complement regulatory protein C4b-
binding protein (not shown). Only free protein S has cofactor activity
for activated protein C .
ROUBEY
the most abundant member of this family, may be
involved in A P S (70). Generally considered an intracell-
ular protein, annexin V has also been detected in
extracellular sites, such as blood and amniotic fluid. It is
not known whether extracellular annexin V is secreted
by cells or results from cell injury, or if it plays a role in
hemostasis (70). Autoantibodies to annexin V have been
observed by some groups of investigators (71) but not
others (72).
Antikininogens. Sugi and McIntyre reported that
high and/or low molecular weight kininogens are re-
quired for the binding of certain antibodies to phos-
phatidylethanolamine (73).
Anti-CD36. CD36, an 88-kd membrane glycopro-
tein expressed on platelets, endothelial cells, and a
number of other cell types, may be a target of some
antiplatelet antibodies in patients with A P S (74).
Antiphospholipase A2. Based on evidence that
immunoglobulin fractions from some A P S patients in-
hibit phospholipase A2 activity ( 7 9 , it has been hypoth-
esized that autoantibodies might be directed against a
phospholipase A,-phospholipid complex (76). There is
no direct evidence of such autoantibodies at present.
Immunopathogenesis of APS
Little is known about the immune response that
leads to the production of autoantibodies associated
with APS. Analyses of T cell subsets (77), HLA class I1
associations (78), and IgG subclass distribution (79) and
V gene analysis (80) suggest that T lymphocytes play an
important role in autoantibody production and disease
pathogenesis. It has recently been shown that T cells are
critical in the transfer of A P S by bone marrow trans-
plantation in an animal model (81).
Patients with APS often have autoantibodies to
more than 1 of the phospholipid-binding proteins dis-
cussed above (29,53,66). The fact that a number of these
antigens are physically associated in enzyme-cofactor-
substrate complexes assembled on phospholipid mem-
branes (e.g., protein C and protein S, Figure l), suggests
that such complexes could be in vivo immunogens that
are driving the autoimmune response in A P S . This is
analogous to the occurrence of linked sets of autoanti-
bodies in other autoimmune diseases such as SLE, in
which the antigens are colocalized in subcellular parti-
cles (e.g., the U1 snRNP) (82). The mechanisms by
which such complexes become immunogenic are not
known.
IMMUNOLOGY OF A P S
Table 3. Some potential effects of autoantibodies to phospholipid-
binding plasma proteins
1. Autoantibodies may directly inhibit antigen enzymatic or cofactor
function (neutralizing antibodies)
2. Autoantibodies may bind fluid-phase antigens and decrease
plasma antigen levels via the clearance of immune complexes
3. Autoantibodies and antigens may form immune complexes that
are deposited in vessel walls, leading to inflammation and
tissue injury
4. Autoantibodies may cause dysregulation of antigen-phospholipid
binding due to cross-linking of membrane-bound antigen
5. Autoantibodies may trigger cell-mediated events by cross-linking
of antigen bound to cell surfaces or cell surface receptors
Are autoantibodies associated with A P S pathologic?
The hypothesis underlying the majority of re-
search into the pathophysiology of APS is that autoanti-
bodies are not only markers of disease, but also directly
contribute to the development of thrombosis, fetal
death, and thrombocytopenia. General observations
which support this hypothesis are 1) many of the anti-
gens targeted by aPL are involved in thrombosis and
hemostasis; 2) the autoantibodies and antigens are ac-
cessible to one another in circulating plasma or on cell
surfaces exposed to circulating plasma (blood cells,
vascular endothelium, placental trophoblasts); and 3)
antibody levels correlate with clinical risk (83). Limited
direct data are provided by several animal models in
which the passive transfer of patients’ antibodies leads to
the development of clinical features that mimic APS
(84 -86).
The recent elucidation of the heterogeneous
specificities of aPL offers promise in addressing the issue
of pathogenesis, and in sorting out the wide variety of
mechanisms that have been proposed. If autoantibodies
play a role in pathogenesis, it is reasonable to assume
that different antibodies or sets of antibodies will display
distinct effects directly related to their antigenic speci-
ficities. Accordingly, particular autoantibodies or com-
binations of autoantibodies may explain the observed
clinical spectrum of APS, for example, venous thrombo-
sis versus arterial thrombosis.
Potential effects of autoantibodies to phospholipid-
binding plasma proteins
Before discussing specific pathophysiologic
mechanisms, it is useful to consider some of the possible
effects of autoantibodies to phospholipid-binding plasma
proteins in general (Table 3). A number of characteris-
tics of both the antibody (concentration, class/subclass,
valency, affinity, charge) and the antigen (concentration,
1449
size, valency, location, charge, chemical properties) will
play a role in determining which interactions occur in
vivo. Direct inhibition of antigen function by neutraliz-
ing antibodies and decreased antigen levels due to the
clearance of antigen-antibody immune complexes may
occur with high-affinity antibodies and are characteristic
of acquired factor inhibitors. Except for the small subset
of patients with lupus anticoagulants and hypoprothrom-
binemia, antibodies associated with A P S do not appear
to have such activity. The deposition of immune com-
plexes that leads to inflammation and tissue injury, for
example, in serum sickness and certain vasculitides, does
not appear to occur with acquired factor inhibitors or
with autoantibodies associated with APS.
The last 2 mechanisms listed in Table 3 involve
antibody binding to membrane-bound antigens and may
occur even if the intrinsic affinity of the antibodies is low.
Data indicating that autoantibodies to P,GPI are of low
affinity (38) and enhance the anticoagulant effect of
P,GPI (30,31) have led us to formulate the following
hypothesis: Antibody cross-linking of a membrane-bound
antigen decreases the rate at which the antigen dissociates
from the phospholipid membrane, thereby altering the
kinetics of phospholipid-dependent reactions in which the
antigen is involved. For example, it is thought that P,GPI
inhibits prothrombinase activity in vitro by binding to
anionic phospholipids, thereby decreasing the availabil-
ity of the phospholipid surface upon which the pro-
thrombinase complex may assemble (29-31). At physi-
ologic concentrations of P,GPI, this inhibitory activity is
weak. Anti-P,GPI antibodies could potentiate the inhib-
itory activity of P,GPI by cross-linking membrane-bound
P,GPI and markedly enhancing the avidity of the
P,GPI-phospholipid interaction. Similarly, low-affinity
antibodies to prothrombin could inhibit coagulation
reactions by enhancing the avidity of the prothrombin-
phospholipid interaction, thereby slowing the dissocia-
tion of the prothrombinase complex (prothrombin-
factor Xa-factor V-phospholipid) and the release of
thrombin from the membrane surface. It is also possible
that membrane binding of antibody-prothrombin com-
plexes could decrease the concentration of prothrombin
and/or phospholipid sites available for optimal assembly
of the prothrombinase complex.
It should be kept in mind that antibodies with
lupus anticoagulant activity in vitro may have procoagu-
lant effects in vivo. For example, P,GPI and antibodies
to P,GPI may inhibit phospholipid-dependent reactions
of the protein C pathway (Figure 1) in the same way in
which they inhibit the prothrombinase reaction. Finally,
antibodies to phospholipid-binding proteins may induce
1450 ROUBEY

Table 4. Proposed mechanisms of autoantibody-mediated thrombosis in the antiphospholipid antibody

syndrome*
~ ~ ~ ~

Selected Potentially associated

Mechanism references autoantibodies
Inhibition of anticoagulant reactions
Inhibition of the protein C pathway
Inhibition of protein C activation 98-100 Antithrombomodulin,
anti-protein C,
antithrombin
Inhibition of activated protein C 66,100-104 Anti-protein C, anti-
anticoagulant activity protein S,anti-factor
V, anti-&GPI
Inhibition of antithrombin 111 activity 69,105 Anti-vHSPG,
antiheparin, anti-
P,GPI
Inhibition of &GPI anticoagulant activity 33 Anti-p&PI

Inhibition of fibrinolysis
Increased PAI-1 106,107 Unknown
Inhibition of factor XII-dependent fibrinolysis 108,109 Anti-&GPI

Cell-mediated events
Enhanced endothelial cell procoagulant activity
Increased tissue factor expression 110,111 Unknown
Increased expression of adhesion molecules 44,89 Anti-&GPI
Dysregulation of eicosanoids
Inhibition of endothelial cell prostacyclin 75,99,112 Anti-phospholipase A,
production
Enhanced platelet thromboxane production 98, 113,114 Unknown
Enhanced PAF production 115 Unknown
Platelet activation/aggregation 96,116 An ti-p,GPI,
antithrombin
* Anti-&GPI = anti-p,-glycoprotein I; anti-vHSPG = antivascular heparan sulfate proteoglycan;
PAI-1 = plasminogen activator inhibitor 1; PAF = platelet-activating factor.

cell-mediated events by engaging antigens that are
bound to the cell surface.
Mechanisms of thrombosis
Nearly all studies of the mechanisms of thrombo-
sis in A P S were performed prior to the new understand-
ing of autoantibody specificities. Table 4 summarizes
these proposed mechanisms and lists particular autoanti-
bodies that might be involved. Among the many throm-
bogenic mechanisms implicated in APS, some of the
most consistent and reproducible data involve inhibition
of the protein C pathway (Figure 1). The clinical impor-
tance of the protein C system in normal hemostasis is
evidenced by the association of inherited abnormalities
of this pathway with thrombosis (87) and an increased
risk of fetal death (88). Autoantibodies to &GPI, throm-
bomodulin, protein C, and protein S have all been
implicated in inhibition of the pathway. Another prom-
ising area for further research is the autoantibody-
mediated enhancement of endothelial cell procoagulant
activity. Two recent studies demonstrate that autoanti-
body binding to the surface of endothelial cells is
dependent upon the presence of P,GPI. The nature of
the cell surface “receptor” for P,GPI is not known.
Antibody binding to &GPI on the endothelial cell
surface induced the expression of the adhesion mole-
cules E-selectin, vascular cell adhesion molecule-1, and
intercellular adhesion molecule-1 (44,89), and enhanced
monocyte adhesion to cultured endothelial cells (89).
Mechanisms of fetal loss
Fetal loss associated with A P S is thought to
represent a special case of thrombosis involving the
placenta. The immediate cause of fetal death related to
A P S is hypoxia due to insufficient uteroplacental blood
flow (90). Histologic studies of placentae reveal a vas-
culopathy of the maternal spiral arteries that leads to
placental infarction (91). Placental infarction may result
from decreased amounts of annexin V on the surface of
placental villi in women with A P S and recurrent fetal
loss (92). Autoantibodies reactive with trophoblast cells
have also been implicated (93). In a recently described
IMMUNOLOGY OF A P S
animal model of APS, gestational failure appeared to be
due to defective embryonic implantation (94). Such a
mechanism may be important in early fetal loss.
Mechanisms of thrombocytopenia
Thrombocytopenia associated with A P S is pre-
sumably due to antiplatelet autoantibodies. The reactiv-
ity of some “anticardiolipin” antibodies with activated
platelets requires the presence of P2GPI, suggesting that
these antibodies are directed against platelet-bound
P2GPI (95). Murine monoclonal antibodies to P2GPI
have also been shown to bind to platelets in the presence
of P2GPI (96). Shi et a1 reported that purified lupus
anticoagulants lacking “anticardiolipin” activity bind to
thrombin-activated, but not resting, platelets (95). Since
it is likely that such lupus anticoagulants are directed
against prothrombin (53), these antibodies may be bind-
ing to platelet-bound thrombin. As previously men-
tioned, CD36 may also be a target of antiplatelet anti-
bodies in APS (74).
Implications for clinical laboratory testing
The identification of P,GPI, prothrombin, and
other proteins as the targets of autoantibodies in A P S
may lead to improved clinical laboratory testing. Auto-
antibodies to &GPI are detectable in phospholipid-free
anti-P,GPI ELISAs (37,38,40,41,46) and in immuno-
blotting assays (43). An ELISA for antibodies to
prothrombin has recently been described (55). Immuno-
assays utilizing purified protein antigens have a number
of potential advantages over conventional aPL tests.
For example, a possible explanation for some of the
observed interlaboratory variation in anticardiolipin
assays (97) is that the quality and amount of P,GPI in
these test systems are not explicitly addressed. In addi-
tion to improved control of the relevant antigen, anti-
P,GPI assays may offer enhanced diagnostic specificity
because, unlike conventional anticardiolipin ELISAs,
they would not be expected to detect authentic (P,GPI-
independent) anticardiolipin antibodies, which do not
appear to be associated with A P S (47). Several studies
have demonstrated that positivity in anti-p,GPI assays is
more strongly associated with clinical manifestations of
APS than positivity in conventional anticardiolipin as-
says (43,45-47). Larger, prospective clinical studies in
which these new assays are performed in parallel with
conventional aPL tests are needed to determine whether
these new assays will, in reality, enhance our diagnostic
and prognostic capabilities. Such studies will also deter-
1451
mine whether the spectrum of autoantibodies will cor-
relate with the heterogeneity of the clinical manifesta-
tions of APS. At the present time, these assays should be
considered investigational.
Conclusion
Over the last several years, our understanding of
the antigenic specificities of aPL has been substantially
revised. Recent data indicate that many of the autoanti-
bodies associated with A P S are directed against a num-
ber of plasma proteins and proteins expressed on, or
bound to, the surface of vascular endothelial cells or
platelets. The involvement of these antigens in clinically
important hemostatic pathways may offer important
insights into the pathophysiology of APS. Studies of the
effects of specific autoantibodies on antigen function
should provide a sound basis for definitive investigations
of possible mechanisms of autoantibody-mediated throm-
bosis and fetal loss.
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