From www.bloodjournal.org by guest on June 9, 2016. For personal use only.

Review article

The significance of autoantibodies against 2-glycoprotein I

Philip G. de Groot1 and Rolf T. Urbanus1
1Department

of Clinical Chemistry and Haematology, University Medical Center, Utrecht, The Netherlands

The antiphospholipid syndrome (APS) is

defined by the persistent presence of
antiphospholipid antibodies in patients
with a history of thrombosis and/or pregnancy morbidity, including fetal loss. APS
is an autoimmune disease with a confusing name because the pathologic autoantibodies are shown to be directed against
the plasma protein 2-glycoprotein I and

not against phospholipids. In fact, autoantibodies that recognize phospholipids

themselves are not associated with thrombosis but with infectious diseases. One of
the intriguing questions is why autoantibodies against 2-glycoprotein I are so
commonly found in both patients and the
healthy. Several potential mechanisms
have been suggested to explain the in-

creased thrombotic risk in patients with

these autoantibodies. In this overview,
we will summarize our knowledge on the
etiology of the autoantibodies, and we
will discuss the evidence that identify
autoantibodies against 2-glycoprotein
I as the culprit of APS. (Blood. 2012;
120(2):266-274)

Introduction
The existence of autoantibodies against 2-glycoprotein I (2GPI)
was described for the first time in 1990, when 3 different groups
identified these antibodies as an important subpopulation of
autoantibodies in patients with the antiphospholipid syndrome
(APS).1-3 The plasma glycoprotein 2GPI consists of 326 amino
acids, arranged in 5 highly homologous complement control
protein domains, designated domain I to V from the N- to the
C-terminus. The first 4 domains consist of approximately 60 amino
acids each, whereas the fifth domain is larger because of a 6-residue
insertion and a 19-residue C-terminal extension that constitute a
phospholipid binding loop.4
APS is characterized by the presence of so-called antiphospholipid antibodies in plasma of patients experiencing thrombotic
complications or pregnancy morbidity.5 At the time of the identification of 2GPI as an antigen in the syndrome, 2 other subpopulations of autoantibodies had already been identified as serologic
markers: anticardiolipin antibodies, detected with an ELISA or a
radioimmunoassay, and lupus anticoagulant (LA), detected as a
prolongation of a phospholipid-dependent clotting test. The discovery that the anticardiolipin antibodies that are correlated with
thrombotic complications are in reality autoantibodies that recognize 2GPI, which has a high affinity for cardiolipin, has been an
important improvement in the development of diagnostics for the
syndrome. This finding enabled the discrimination between 2GPIdependent anticardiolipin antibodies that are correlated with thrombosis and fetal loss and 2GPI-independent anticardiolipin antibodies, whose presence seems to be innocent and that are often found
in connection with infectious diseases.6
Soon after this discovery, it was established that autoantibodies
against 2GPI can also have LA activity, highlighting the importance of antibodies directed against 2GPI for the syndrome.7,8
Although LA activity is caused by antiprothrombin antibodies in
some patients,9 in additional studies investigators have shown that
LA activity caused by anti-2GPI antibodies correlates much
stronger with a history of thrombotic complications.10,11 On the
basis of these data, the presence of anti-2GPI antibodies has been
Submitted March 13, 2012; accepted April 19, 2012. Prepublished online as
Blood First Edition paper, March 2, 2012; DOI 10.1182/blood-2012-03-378646.

266

added to the official classification criteria for APS as the third

serologic marker at a consensus meeting in Sydney in 2004.5
The classification criteria of APS are based on the combined
results of many patient studies in which correlations were established between the presence of antiphospholipid antibodies in
plasmas of patients and the occurrence of thromboembolic complications or pregnancy morbidity.5 Evidence for a causal connection
between the presence of antiphospholipid autoantibodies and the
observed clinical manifestations comes from experiments in animals in which the passive infusion of patient antibodies into
pregnant mice resulted in reabsorbance of the fetuses and an
increased thrombotic response after an experimental injury in
otherwise-healthy mice.12 Additional experiments have shown that
in particular autoantibodies against 2GPI are responsible for the
enhanced thrombotic response.13 These studies have convinced
most scientists that autoantibodies against 2GPI are the pathologic
subpopulation of the antiphospholipid antibodies. Here, we review
the evidence that anti-2GPI antibodies are responsible for both the
thrombotic and the pregnancy complications associated with APS,
and we discuss why these autoantibodies are so commonly detected
in both patients and the healthy.

The detection and specificity of

autoantibodies against 2GPI
The anticardiolipin antibody ELISA, the anti-2GPI antibody
ELISA, and the LA assays detect overlapping, but different,
populations of autoantibodies.14 The anticardiolipin antibody ELISA
detects both antibodies directed against cardiolipin itself and
antibodies against 2GPI, the anti-2GPI antibody ELISA measures only antibodies against 2GPI, whereas LA assays can detect
antibodies against both prothrombin and 2GPI, with the restriction
that only a subpopulation of autoantibodies directed to 2GPI is
able to induce a prolongation of a clotting assay. Because these
3 assays are used to measure different subpopulations of autoantibodies, they correlate differently with the clinical manifestations

2012 by The American Society of Hematology

BLOOD, 12 JULY 2012 VOLUME 120, NUMBER 2

From www.bloodjournal.org by guest on June 9, 2016. For personal use only.

BLOOD, 12 JULY 2012 VOLUME 120, NUMBER 2

that determine APS. Many researchers have shown that LA assays

are by far superior to the other 2 tests to detect the pathologic
subpopulation of antiphospholipid antibodies.15-17 The correlation
between thrombotic complications and anti-2GPI or anticardiolipin antibodies is much weaker, and there are indications that
patients with only a positive anti-2GPI antibody ELISA do not
have any clinical risk at all.17-19
This correlation is not easy to explain, considering the observation that passive transfer of anti-2GPI autoantibodies into mice
induces an increased thrombotic response.13 The most convincing
explanation for this discrepancy is that there are different subpopulations of anti-2GPI antibodies, each recognizing different epitopes
on 2GPI and differing greatly in their pathologic effects. Indeed, it
has been shown that antibodies that recognize domain I of 2GPI
correlate much stronger with a history of thrombosis and pregnancy morbidity than antibodies that recognize other domains of
2GPI.20-22 Antibodies against domain V of 2GPI, often observed
in leprosy, seem to be harmless.23 Autoantibodies against domain
IV-V of 2GPI can be detected in young children without any sign
of thrombotic events.24 Additional support for the importance of
antidomain I antibodies comes from studies in mice in which
injection of domain I attenuated the effects of antiphospholipid
antibodies in a mouse model of APS.25 Moreover, antidomain
I antibodies express LA activity.10 Apparently, antibodies that are
directed against this domain score positive in all 3 assays. It is
therefore not surprising that when all 3 assays for the detection of
antiphospholipid antibodies are positive, the risk of recurrence of
thrombotic complications is greatest.26 It should be noted that there
might be different epitopes present on domain I.27 It is unknown
whether autoantibodies specific for these individual epitopes
harbor the same risk.
An important reason for the discrepancy between experimental
data and epidemiologic studies is the poor standardization of the
assays that are currently used to detect anti-2GPI antibodies.
Several surveys have shown that only the very high-titer anti2GPI antibodies are reliably detected, which depends in part on
differences in protein preparations, surface immobilization, choice
of microtiter plate, and blocking reagent.28 In particular, the
presentation of 2GPI on the microtiter plate greatly influences the
results.29 A popular way to obtain purified 2GPI is by perchloric
acid precipitation, which might influence the integrity of the
molecule.30 However, the use of perchloric acidtreated 2GPI
resulted in steep calibration curves, indicating that the protein can
be used as antigen in anti-2GPI antibody ELISAs. It is important
to use hydrophilic ELISA plates because the binding of 2GPI to
these surfaces induces a conformational change that results in the
presentation of the cryptic epitope to which the pathogenic
antibodies are directed. The use of 2GPI from nonhuman sources
is strongly discouraged because antibodies against bovine proteins
are not uncommon in human blood.
The international classification criteria for APS include only the
presence of IgG and IgM anti-2GPI antibodies as part of the
serologic criteria.5 This decision was determined by the results of
epidemiologic and functional studies that were published at that
time. Recent publications have questioned this decision.31-35 Additional studies have now been published in which the authors
challenge the importance of IgM as a risk factor, and other studies
have shown that IgA is associated with venous thrombosis and
stroke. There are even studies in which investigators show that IgM
protects against some of the clinical manifestations such as lupus
nephritis.36 It is not easy to draw conclusions from all these
conflicting observations. Aside from the fact that the assays to

SIGNIFICANCE OF AUTOANTIBODIES AGAINST 2-GPI

267

detect these autoantibodies are poorly standardized, there may be a

difference in risk between patients with and without another
underlying immune disease.35 Moreover, there may be an ethnic
component to consider.37 Another factor that has to be taken into
account is the affinity of the autoantibodies. Different studies have
shown that particularly autoantibodies with greater affinity correlate with thrombosis.38,39 Some authors have suggested that the
presence of anti-2GPI antibodies should be measured in plasma
diluted in 500mM NaCl to avoid the interference of the clinically
irrelevant low-affinity antibodies.39,40 In summary, there is a
general consensus that IgG is the important autoantibody that
matters. In view of the conflicting publications, the importance of
measuring IgM and IgA is unclear. As long as there are no
conclusive studies, we suggest measuring them both but only
consider medium- and high-titer antibodies as relevant.
Given all these uncertainties, one should be careful to interpret
the results of an anti-2GPI antibody ELISA. A combination of a
positive anti-2GPI antibody ELISA and a positive LA will identify
patients at risk, whereas patients with a single positivity of
anti-2GPI antibodies should be considered at low risk for adverse
clinical events. We strongly discourage the diagnosis of a patient
only on the results of a single assay. The introduction of an ELISA
specific for domain I of 2GPI could increase the specificity of the
assay, probably at the expense of the sensitivity because we do not
know whether this assay will identify all pathogenic autoantibodies.

The etiology of autoantibodies against 2GPI

Infection-related antiphospholipid antibodies are thought to interact directly with cardiolipin, independent of the presence of 2GPI,
and they are considered not to be related to an increased risk of
thrombosis.36 An increasing number of publications also describe
autoantibodies against 2GPI in combination with infections.41-43
The eminence-based consensus is that these infection-related
antibodies are distinct from the autoantibodies identified in APS
patients,44 although there is not much scientific evidence that these
infection-related autoantibodies are indeed different from APSrelated autoantibodies. Infection-related anti-2GPI antibodies circulate for a short period of time. The increased risk of thrombosis
of patients with persistently present anti-2GPI antibodies can be
explained in part by the circulation time of the autoantibodies.
Gharavi et al have shown that immunization with peptides derived
from cytomegalovirus induced LA activity and resulted in thrombotic complications.41 Moreover, the presence of anti-2GPI antibodies has been reported in patients with cytomegalovirus infections who developed thrombosis.45
Many publications link the etiology of autoantibodies to a
molecular mimicry mechanism.46 Sequence similarities between
foreign and self-peptides are considered to be sufficient to activate
auto-reactive T and B cells with pathogen-derived peptides. Anti2GPI antibody formation is induced when mice are immunized
with viral peptides, suggesting sequence or conformation similarities between the bacterial and viral peptides and amino acid
sequences of 2GPI.47 However, mice and rabbits boosted with the
anionic phospholipids cardiolipin and phosphatidylserine or with
lipopolysaccharide also develop autoantibodies against 2GPI,
demonstrating that molecular mimicry cannot be the only explanation for the etiology of these autoantibodies.48,49 Considering the
ease with which these autoantibodies are formed after infections
with a large number of dissimilar microorganisms (Table 1),50,51

From www.bloodjournal.org by guest on June 9, 2016. For personal use only.

268

BLOOD, 12 JULY 2012 VOLUME 120, NUMBER 2

DE GROOT and URBANUS

Table 1. Infectious diseases associated with the presence of

autoantibodies against 2GPI
Viruses

Parvovirus B19
Cytomegalovirus
Human immunodeficiency virus
Varicella-zoster virus
Epstein-Barr virus
Hepatitis B/C
Adenovirus
Human T-lymphotropic virus type I

Bacteria

Streptococcus pyogenes
Staphylococcus aureus
Helicobacter pylori
Salmonella typhi
Mycobacterium leprae
Escherichia coli
Rickettsia typhi
Mycobacterium leprae
Mycobacterium tuberculosis
Coxiella burnetii
Chlamydophila psittaci

Mycoplasma

Mycoplasma pneumoniae

Parasites

Plasmodium falciparum
Borrelia burgdorferi
Leptospirosis
Leishmania

For additional information, see Sherer et al.47

there must be a more fundamental background for the frequent

presence of these autoantibodies in the circulation.
There is a growing body of evidence that antiphospholipid
antibodies, including anti-2GPI antibodies, belong to the natural
antibody repertoire.52-55 Natural antibodies are defined as antibodies that are present in the circulation before an antigen stimulus.
Natural antibodies have been described as poly-reactive, that is,
they can react with several unrelated antigens, including selfantigens. These natural antibodies seem to bind primarily to
pathogenic structures that are well conserved and present in many
species, such as clusters of charge, and they are usually of low
affinity. Antiphospholipid antibodies have similar characteristics.
As part of the innate immune system, the presence of natural
antibodies must have a fundamental benefit, although they may
cross-react with self-antigens.56 If anti-2GPI antibodies belong to
the natural antibody repertoire, this means that they are present for
a purpose and that they could play a role in the innate immune
response.54 Support for the identification of antiphospholipid
antibodies as natural antibodies comes from the observation that
healthy persons without APS can have memory B cells that
produce antiphospholipid antibodies.57 In several elegant studies,
Fleming et al have shown that natural anti-2GPI antibodies are
involved in complement-mediated mesenteric ischemia/reperfusioninduced injury.58,59 In addition, there are also indications that

natural antiphospholipid antibodies are involved in acute graft

rejection after renal transplantation.60 Natural antibodies are thought
to play a role in the clearance of apoptotic bodies and there is
convincing evidence that 2GPI and anti-2GPI antibodies are
essential for the clearance of these cell remnants.61,62 Recent
epidemiologic studies in a large cohort of patients with systemic
lupus erythematosus (SLE) have shown that the presence of
anti-2GPI IgM protects against lupus nephritis.35 Up to 5% of the
healthy population has antiphospholipid antibodies, and these
autoantibodies seem to be benign and of low affinity. All these
observations suggest that many healthy patients have low-titer and
low-affinity natural anti-2GPI antibodies in their circulation and
that the prevalence of these antibodies grows with age.63
The natural antibody repertoire may comprise 2 major subsets,
an overt antibody population and a cryptic or latent population.64
The unmasking of these cryptic natural antibodies in vitro has been
observed via the use of high-salt solution, low pH, or oxidative
agents. Indeed, patients who are negative for antiphospholipid
antibodies can become positive after oxidation-reduction reactions
and vice-versa.65 In addition, the plasma of healthy persons
becomes positive after it is heated to 56C.66 Apparently, small
changes or slight modulations within the antibody binding site can
change epitope recognition.
The cause of the transition of these natural antibodies against
2GPI from benign to pathogenic antibodies remains elusive.
Natural antibodies may, under certain circumstances, begin to
modulate to pathologic autoantibodies via an antigen-driven selection of certain populations of B cells. In some studies authors have
shown that infections contribute to the maturation of the immune
system from low-titer and low-affinity antibodies to full blown
autoantibodies.64 The further induction of autoantibodies against
2GPI may strengthen the efficacy of 2GPI as part of our defense
mechanism. This latter suggestion is supported by the observation
that the epitope recognized by the autoantibodies against 2GPI is
completely conserved in mammalian evolution.67 Loss of tolerance
can occur after sudden exposure to an antigen.
Interestingly, lymphocytes are not continuously exposed to
antigenic stimulation in healthy patients because the epitope within
2GPI that is recognized by the pathogenic subpopulations of
autoantibodies is cryptic. 2GPI is a protein that can adopt different
conformations (Figure 1).68,69 In the presence of anionic phospholipids, lipopolysaccharide, or hydrophilic surfaces, the circular
conformation of the protein unfolds, exposing antigenic determinants that are normally shielded from the circulation.70 One of
these cryptic antigenic determinants is the epitope for the
pathologic autoantibodies present in domain I of 2GPI.20-22
Infectious diseases can result in the binding of 2GPI to pathogens,
resulting in exposure of an epitope in domain I and stimulation of
T and B cells. This hypothesis is strengthened by the observation
that binding of 2GPI to protein H, a membrane protein of
Streptococcus pyogenes, results in the conformational change

Figure 1. 2-glycoprotein I changes conformation on

antibody binding. Scanning electron microscopy images showing plasma 2-GPI (A) and 2-GPI in complex
with antibodies against domain I of 2-GGI (B). Samples
were visualized using a Jeol JEM 1230 transmission
electron microscope operated at 60 kV accelerating
voltage and recorded with a Gatan Multiscan 791 CCD
camera using the software provided by the manufacturer
(magnification 200 000).

From www.bloodjournal.org by guest on June 9, 2016. For personal use only.

BLOOD, 12 JULY 2012 VOLUME 120, NUMBER 2

of 2GPI from the circular to the open conformation. Injection of

mice with protein H results in induction of autoantibodies against
murine 2GPI with LA activity.70 Further support comes from the
observation that injection with murine domain I induces autoantibodies against 2GPI in these mice, whereas injection with
domains II through V does not result in an immune response.71 All
these experiments show that exposure of mice to this cryptic
epitope within domain I of 2GPI results in an up-regulation of
autoantibody production.
There are 4 2GPI allelic variants known, of which the
Val247Leu variant has attracted most attention. A meta-analysis has
shown that patients with APS have a greater prevalence of the
Val/Val genotype and that the presence of this variant is correlated
with the presence of anti-2GPI antibodies.72 It has been suggested
that the tertiary structure of the Val247 variant of 2GPI differs
from that of the Leu247 variant. Model systems suggest a loss of
electrostatic interaction between Glu228 in domain IV and Lys308
in domain V in the Val247 variant, which may influence the
stability of the circular plasma conformation.73 An increased
tendency of 2GPI to open up and to expose the epitope for
anti-2GPI antibodies can explain the observed risk to develop
autoantibodies against 2GPI in Val/Val carriers.
Recently, evidence has been brought forward that a part of
2GPI circulates in a reduced form with free surface-exposed thiol
groups and that the percentage of reduced 2GPI is much greater in
patients with APS.74 Binding studies have shown that the avidity of
patient-derived autoantibodies is significantly greater for reduced
2GPI than for oxidized 2GPI.75 The authors hypothesize that
oxidative stress lowers the threshold for the development anti2GPI antibodies. Alternatively, oxidation of 2GPI might favor a
conformational change from the circular to the stretched conformation of 2GPI, resulting in exposure of cryptic epitopes and an
enhanced antigen driven B-cell maturation.

The pathophysiology of the autoantibodies

Thrombotic events only take place every now and then in patients
with antiphospholipid antibodies, despite the continuous presence
of these antibodies in the circulation. This finding suggests that
there are additional factors that determine whether thrombosis will
occur. The current consensus is that a second hit is necessary to
unveil the prothrombotic activity of antiphospholipid antibodies.
The exact nature of this priming event, a perturbation of the
endothelial lining of the vasculature, an increased inflammatory
state, or something completely different, is unclear. Experimental
data obtained in animal models of APS support the second hit
hypothesis. Passive infusion of human IgG that was purified from
patients with APS into mice only results in excessive thrombus
formation on induction of a mechanical or chemical vascular
injury.76,77 Likewise, rats only develop occlusive thrombi on
infusion with human antiphospholipid antibodies when primed
with endotoxin.78
Antiphospholipid antibody-associated thrombosis can occur
throughout the body in every type of vesselvein, artery, or
microcirculation, although deep-vein thrombosis and ischemic
stroke are most frequently observed.79 Thrombosis in APS has been
attributed to antibody-mediated interference with several aspects of
the coagulation system, such as the fibrinolytic pathway80 or the
protein C axis.81 These suggestions offer unlikely explanations for
the diffuse thrombotic diathesis observed in the APS because
plasminogen deficiency is not associated with a thrombotic ten-

SIGNIFICANCE OF AUTOANTIBODIES AGAINST 2-GPI

269

dency, and deficiencies of regulators of coagulation are generally

associated with vascular bedspecific thrombosis. However, we
cannot exclude the possibility that the presence of the autoantibodies disturbs different metabolic pathways, which may operate under
different conditions or in different patient groups. Nevertheless, a
much more likely target-candidate for antiphospholipid antibodies
would be a cellular component of the blood, including the
endothelial lining of the vasculature. Indeed, activation of platelets,
monocytes and endothelial cells by antiphospholipid antibodies in
general and anti-2GPI antibodies in particular, has been shown
extensively (Figure 2). The prominent read-out for cell activation
was the induction of the surface expression of procoagulant tissue
factor.82 An interesting option is that budding of tissue factor
containing microparticles from monocytes or endothelial cells
could explain a more generalized increase in a risk for thrombotic
complications.83
Transmission of activation signals across cell membranes
usually involves ligand-receptor interactions and it is therefore not
surprising that the identification of the receptor(s) that mediate the
prothrombotic effects of antiphospholipid antibodies is subject of
ongoing investigations. Several receptors have been reported thus
far to interact with the 2GPI-antibody complex, among others
TLR2,84 TLR4,85,86 TLR8,87 annexin A2,88 glycoprotein Ib,89,90
and the low-density lipoprotein (LDL)receptor family member
LDL receptorrelated protein (LRP)8, also known as ApoER2.91
Activation of cell lines or cells isolated from blood with antiphospholipid antibodies indicates involvement of each of these receptors in the induction of a prothrombotic cellular phenotype in a
2GPI-dependent manner. Studies with murine thrombosis models
confirm the roles of TLR4,92 annexin A2,93 or LRP8,76,77 as
inhibition or absence of individual receptors results in an ameliorated thrombotic phenotype on challenge with antiphospholipid
antibodies. Because none of these individual deficiencies reduces
the thrombotic phenotype to baseline, we cannot exclude a
cooperative effect between receptors in the pathophysiology of the
syndrome.
Few data are available on the mechanism behind the adverse
effects of antiphospholipid antibodies on pregnancy outcome.
Antiphospholipid antibodies are reported to interfere with placentation,94,95 which might lead to placental insufficiency. Increased
rates of placental thrombosis have been observed in women with
antiphospholipid antibodies,96,97 which might be explained by
either disruption of the annexin a5 shield that prevents the
interaction between the coagulation system and the phosphatidylserine exposing syncytiotrophoblast surface98 or by a disproportionate
inflammatory response that involves activation of the complement
system and formation of the anaphylatoxin C5a.99
Data that support a role for the complement system in the
pathophysiology of APS are more ambiguous. F(ab)2 fragments of
anti-2GPI monoclonal antibodies retain the same prothrombotic
potential as the full-length antibody in a hamster thrombosis
model.100 This renders involvement of the Fc-mediated classic
pathway of complement activation unlikely, although a contribution of this pathway to antiphospholipid antibody mediated effects
cannot be fully excluded. Murine models of fetal loss, however,
clearly indicate a role for the classic pathway in antiphospholipid
antibody mediated pathology.99 This discrepancy can be explained
to some extend by the high concentrations of total human IgG used
in these studies, although similar amounts of IgG obtained from
healthy controls had no effect on fetal loss. Moreover, complement
deposition on circulating immune complexes is a prominent feature
of SLE, a frequent comorbidity of APS. Further evidence for

From www.bloodjournal.org by guest on June 9, 2016. For personal use only.

270

DE GROOT and URBANUS

BLOOD, 12 JULY 2012 VOLUME 120, NUMBER 2

Figure 2. Sequence of events leading to cellular activation by 2GPI-antibody complexes. 2GPI is not recognized by pathologic anti-2GPI antibodies in the circulation.
When negatively charged, phospholipids become exposed, and 2GPI will bind to this surface and change conformation. This step exposes a cryptic epitope in domain I that is
recognized by the pathologic antibodies. The antibody fixes 2GPI in this conformation, and the antibody2GPI complex can subsequently interact with several surface
receptors, such as glycoprotein Ib (GPIb), LRP8, annexin A2, and several members of the TLR family (TLR2, -4, and -8). Because receptor and phospholipid binding are
mutually exclusive, the 2GPIantibody complex probably dissociates from the surface before it can interact with a surface receptor.

involvement of the complement system comes from studies that

show that deficiency of either C3 or C5 protects from both
antiphospholipid antibody-induced thrombosis101 and fetal loss.99,102
If there is a role for the complement system in APS, then the
sustained effects of F(ab)2 fragments of anti-2GPI antibodies in
combination with a role for C3 and C5 point in the direction of the
alternative pathway of complement activation. This notion is
strengthened by the protective effects of factor B deficiency on
fetal loss.99

residues are mutated. Moreover, the presence of antibodies that

interact with this region is strongly associated with thrombosis.10
Another putative antibody-binding site is located at the opposite
side of the domain, in the region surrounding Lys19. Mutation of
this residue has similar inhibitory effects on antibody binding
albeit less pronounced,103 but the relevance of antibodies that
recognize this region remains to be determined. Interestingly,
mutation of residues Asp8 and Asp9 in domain I increases antibody
binding in the fluid phase.22 In fact, injection of domain I mutated at
these residues neutralizes the prothrombotic effects of human
antiphospholipid antibodies in a murine thrombosis model more

Attenuation of the activity of the

autoantibodies
Currently prescribed thromboprophylaxis for patients with APS are
antiplatelet agents, heparins, vitamin K antagonists, or combinations thereof. All of these drugs share the major disadvantage of a
substantially increased risk of bleeding. This holds true for the use
of vitamin K antagonists, considering patients with antiphospholipid antibodies are often treated for a longer period and with a
target international normalized ratio of 3 or greater. It is therefore
not surprising that a large amount of effort is being put in the
development of better-tailored treatment strategies that do not have
the adverse side-effects associated with anticoagulants. Solving the
crystal structure of 2GPI has been the first step on the way to a deeper
understanding of the biochemical mechanisms behind the interaction
between 2GPI and antibodies, phospholipid surfaces, and receptors.
This has allowed the design of tools that specifically antagonize the
interaction between either 2GPI and antiphospholipid antibodies, or
receptors and 2GPI-antibody complexes.
The primary antigenic epitope of anti-2GPI antibodies resides
in domain I and spans residues Arg39 to Arg43 (Figure 3).22,103 The
identification is based on the strong attenuation of the binding of
patient-derived antibodies to immobilized 2GPI in which these

Figure 3. Residues in the crystal structure of 2-GPI that are involved in the
interaction with ligands. Domain I contains 2 primary antibody binding sites
(highlighted in yellow): the region around Lys19 and the region spanning Arg39 to
Arg43. Mutation of residues Asp8 and Asp9 (highlighted in red) enhances the binding
of antiphospholipid antibodies to domain I. Domain V contains the phospholipid
binding site, which overlaps with the site that interacts with LA1 of LRP8 (highlighted
in green).110

From www.bloodjournal.org by guest on June 9, 2016. For personal use only.

BLOOD, 12 JULY 2012 VOLUME 120, NUMBER 2

SIGNIFICANCE OF AUTOANTIBODIES AGAINST 2-GPI

effectively than infusion of wild-type domain I,25 which indicates

its potential applicability as a therapeutic agent. Nevertheless, even
though attenuation of the prothrombotic effects of antiphospholipid
antibodies at the level of the interaction between antigen and
antibody seems to be an attractive idea, it is important to realize
that injection of domain I might not be without risk because it has
been shown to lead to further immunologic priming toward
2GPI.71
The phospholipid binding site is located within the fifth domain
of 2GPI. It has been mapped to the hydrophobic loop at the
C-terminus of the domain and includes a large positively charged
patch of Lysine residues. Mutations in this region,104,105 or cleavage
of this hydrophobic loop by proteases such as plasmin,106 greatly
reduce the capacity of 2GPI to interact with phospholipids.
Interestingly, the lysine-rich region in 2GPI is highly homologous
to a 20-amino acid peptide derived from an uncharacterized protein
of cytomegalovirus (TIFILFCCSKEKRKKKQAAT). This peptide,
referred to as TIFI, effectively competes with 2GPI-antibody
complexes for binding sites on cellular surfaces.107 Coinjection of
TIFI and antiphospholipid antibodies prevents excessive thrombus
formation on a mechanical vascular injury,108 and TIFI-treatment of
pregnant mice that were injected with human antiphospholipid
antibodies significantly reduces fetal resorption rates.107 It should
be noted, however, that immunization of mice with TIFI induces
the generation of prothrombotic antiphospholipid antibodies,41
which precludes its applicability as a therapeutic agent in the
syndrome.
2GPI also interacts with cellular receptors via its fifth domain,
but this interaction has only been studied in detail for LRP8.
Biochemical analysis has shown that domain V interacts with the
first LDL type A (LA) module of LRP8.109 Solution nuclear
magnetic resonance spectroscopy has identified the residues in both
LRP8 and domain V of 2GPI that are perturbed when both
proteins interact.110 Interestingly, the epitope in domain V that
interacts with LA1 of LRP8 involves several positively charged
residues, including the positively charged patch of lysine residues
that are essential for phospholipid binding. In fact, receptor binding
and interaction with phospholipids are mutually exclusive. This has
led to the development of an artificial dimer of LA1 of LRP8,
which effectively inhibits the binding of 2GPI-antibody complexes to phospholipid surfaces.111 The efficacy of soluble LA1 of
LRP8 as an inhibitor of the prothrombotic effects of antiphospholipid antibodies has been shown extensively both in vitro77,90,112 and
in vivo.76 However, LDL-receptor family members are ubiquitous
and are involved in several important processes, including protein
clearance, lipoprotein metabolism and neuronal plasticity. Treatment
with isolated LA modules might interfere with any of these processes,
which makes unwanted off-target side-effects more than likely.
One of the most promising candidate drugs for treatment of APS
is hydroxychloroquine, an antimalarial agent already used extensively for the treatment of SLE. Hydroxychloroquine effectively
blocks the interaction between 2GPI and phospholipids113 and
prevents the binding of antibody-2GPI complexes to cells.114
Moreover, treatment with hydroxychloroquine ameliorates the

271

prothrombotic effects of antiphospholipid antibodies in a murine

thrombosis model,115 although these effects can also be mediated
by the antiplatelet properties of the drug.116 Clinical trials should
provide us with information on the efficacy of the drug as an
antithrombotic agent in APS.
Because APS is an antibody-mediated disorder, antiB-cell
therapy might hold promise for the future. Data on the efficacy of
antiB-cell agents such as rituximab, however, are mostly limited
to case reports. Further studies are needed before any conclusions
can be drawn.

Concluding remarks
There is an increasing body of evidence that the presence of
anti-2GPI antibodies has a physiologic relevance and that they
play different roles in innate immunity. These probably advantageous autoantibodies deteriorate into pathologic risk factors when
their residence time in the circulation becomes indefinite and their
avidity increases. The triggers that cause the transition from
low-affinity temporary antibodies to greater-affinity persistent
antibodies are unknown. Significant progress has been made since
1990, the year that the autoantibodies against 2GPI were first
described, in our understanding of how the presence of these
autoantibodies can cause the clinical manifestations that characterize APS. Experiments in animals have revealed that several cell
types, such as endothelial cells, monocytes, and platelets, change
their phenotype toward a more prothrombotic and proinflammatory
state in the presence of these autoantibodies. Fundamental insight
at the molecular level on how 2GPI interacts with its autoantibodies, the consequences of these interactions for the structure of
2GPI, and the newly acquired properties of structurally altered
2GPI to interact with phospholipids and cellular receptors is
necessary to design new drugs for a better-tailored treatment of
patients with APS.

Acknowledgments
The authors gratefully acknowledge Dr Matthias Morgelin (Lund
University, Lund, Sweden) for his help with the acquisition of
electron micrographs.
R.T.U. is supported by a grant from the Dutch Heart Foundation
(grant 2010T068).

Authorship
Contribution: P.G.d.G. and R.T.U. contributed equally to writing
this review article.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Philip G. de Groot, University Medical Center
Utrecht, Department of Haematology (G03.550), Heidelberglaan
100, 3584 CX Utrecht, The Netherlands; e-mail: ph.g.degroot
@umcutrecht.nl.

References
1. McNeil HP, Simpson RJ, Chesterman CN, Krilis
SA. Anti-phospholipid antibodies are directed
against a complex antigen that includes a lipidbinding inhibitor of coagulation: beta 2-glycoprotein I
(apolipoprotein H). Proc Natl Acad Sci U S A. 1990;
87(11):4120-4124.
2. Galli M, Comfurius P, Maassen C, et al. Anticar-

diolipin antibodies (ACA) directed not to cardiolipin but to a plasma protein cofactor. Lancet.
1990;335(8705):1544-1547.
3. Matsuura E, Igarashi Y, Fujimoto M, Ichikawa K,
Koike T. Anticardiolipin cofactor(s) and differential
diagnosis of autoimmune disease. Lancet. 1990;
336(8708):177-178.

4. Bouma B, de Groot PG, van den Elsen JM,

et al. Adhesion mechanism of human beta(2)glycoprotein I to phospholipids based on its crystal structure. EMBO J. 1999;18(19):5166-5174.
5. Miyakis S, Lockshin MD, Atsumi T, et al. International consensus statement on an update of the
classification criteria for definite antiphospholipid

From www.bloodjournal.org by guest on June 9, 2016. For personal use only.

272

BLOOD, 12 JULY 2012 VOLUME 120, NUMBER 2

DE GROOT and URBANUS

syndrome (APS). J Thromb Haemost. 2006;4(2):

295-306.
6. Chamley LW, McKay EJ, Pattison NS. Cofactor
dependent and cofactor independent anticardiolipin antibodies. Thromb Res. 1991;61(3):291-299.
7. Oosting JD, Derksen RH, Entjes HT, Bouma BN,
de Groot PG. Lupus anticoagulant activity is
frequently dependent on the presence of beta
2-glycoprotein I. Thromb Haemost. 1992;67(5):
499-502.
8. Roubey RA, Pratt CW, Buyon JP, Winfield JB.
Lupus anticoagulant activity of autoimmune
antiphospholipid antibodies is dependent upon
beta 2-glycoprotein I. J Clin Invest. 1992;90(3):
1100-1104.
9. Bevers EM, Galli M, Barbui T, Comfurius P,
Zwaal RF. Lupus anticoagulant IgGs (LA) are not
directed to phospholipids only, but to a complex
of lipid-bound human prothrombin. Thromb Haemost. 1991;66(6):629-632.
10. de Laat B, Derksen RH, Urbanus RT, de Groot PG.
IgG antibodies that recognize epitope Gly40Arg43 in domain I of beta 2-glycoprotein I cause
LAC, and their presence correlates strongly with
thrombosis. Blood. 2005;105(4):1540-1545.
11. De Laat B, Derksen RH, Reber G, et al. An international multicentre-laboratory evaluation of a
new assay to detect specifically lupus anticoagulants dependent on the presence of anti-beta2glycoprotein autoantibodies. J Thromb Haemost.
2011;9(1):149-153.
12. Pierangeli SS, Chen PP, Raschi E, et al. Antiphospholipid antibodies and the antiphospholipid syndrome: pathogenic mechanisms. Semin
Thromb Hemost. 2008;34(3):236-250.
13. Arad A, Proulle V, Furie RA, Furie BC, Furie B.
Beta-glycoprotein-1 autoantibodies from patients
with antiphospholipid syndrome are sufficient to
potentiate arterial thrombus formation in a mouse
model. Blood. 2011;117(12):3453-3459.
14. McNeil HP, Chesterman CN, Krilis SA. Binding
specificity of lupus anticoagulants and anticardiolipin antibodies. Thromb Res. 1988;52(6):609619.
15. Galli M, Luciani D, Bertolini G, Barbui T. Lupus
anticoagulants are stronger risk factors for thrombosis than anticardiolipin antibodies in the antiphospholipid syndrome: a systematic review of
the literature. Blood. 2003;101(5):1827-1832.
16. Urbanus RT, Siegerink B, Roest M, Rosendaal
FR, de Groot PG, Algra A. Antiphospholipid antibodies and risk of myocardial infarction and ischaemic stroke in young women in the RATIO
study: a case-control study. Lancet Neurol. 2009;
8(11):998-1005.
17. Lockshin MD, Kim M, Laskin CA, et al. Lupus anticoagulant, but not anticardiolipin antibody, predicts adverse pregnancy outcome in patients with
antiphospholipid antibodies [prepublished online
ahead of print January 24, 2012]. Arthritis
Rheum. doi:10.1002/art.34402.

22. Ioannou Y, Pericleous C, Giles I, Latchman DS,

Isenberg DA, Rahman A. Binding of antiphospholipid antibodies to discontinuous epitopes on
domain I of human beta(2)-glycoprotein I: mutation studies including residues R39 to R43.
Arthritis Rheum. 2007;56(1):280-290.
23. Forastiero R, Martinuzzo M, de Larranaga G,
Vega-Ostertag M, Pierangeli S. Anti-beta2glycoprotein
I antibodies from leprosy patients do not show thrombogenic effects in an in vivo animal model. J Thromb
Haemost. 2011;9(4):859-861.
24. Andreoli L, Nalli C, Motta M, et al. Anti-betaglycoprotein I IgG antibodies from 1-year-old
healthy children born to mothers with systemic
autoimmune diseases preferentially target
domain 4/5: might it be the reason for their
innocent profile? Ann Rheum Dis. 2011;70(2):
380-383.
25. Ioannou Y, Romay-Penabad Z, Pericleous C,
et al. In vivo inhibition of antiphospholipid
antibody-induced pathogenicity utilizing the antigenic
target peptide domain I of beta2-glycoprotein I: proof
of concept. J Thromb Haemost. 2009;7(5):833-842.
26. Pengo V, Ruffatti A, Legnani C, et al. Incidence of
a first thromboembolic event in asymptomatic
carriers of high-risk antiphospholipid antibody
profile: a multicenter prospective study. Blood.
2011;118(17):4714-4718.
27. Dienava-Verdoold I, Boon-Spijker MG, de Groot PG,
et al. Patient-derived monoclonal antibodies
directed towards beta2 glycoprotein-1 display
lupus anticoagulant activity. J Thromb Haemost.
2011;9(4):738-747.
28. Urbanus RT, de Groot PG. Antiphospholipid antibodieswe are not quite there yet. Blood Rev.
2011;25(2):97-106.
29. Reber G, Schousboe I, Tincani A, et al. Interlaboratory variability of anti-beta2-glycoprotein I
measurement. A collaborative study in the frame
of the european forum on antiphospholipid antibodies standardization group. Thromb Haemost.
2002;88(1):66-73.
30. Brighton TA, Dai YP, Hogg PJ, Chesterman CN.
Microheterogeneity of beta-2 glycoprotein I: implications for binding to anionic phospholipids.
Biochem J. 1999;340(Pt 1):59-67.
31. Ruffatti A, Tonello M, Visentin MS, et al. Risk factors for pregnancy failure in patients with antiphospholipid syndrome treated with conventional
therapies: a multicentre, case-control study.
Rheumatology (Oxford). 2011;50(9):1684-1689.
32. Mehrani T, Petri M. Association of IgA anti-beta2
glycoprotein I with clinical and laboratory manifestations of systemic lupus erythematosus.
J Rheumatol. 2011;38(1):64-68.
33. Sweiss NJ, Bo R, Kapadia R, et al. IgA antibeta2-glycoprotein I autoantibodies are associated with an increased risk of thromboembolic
events in patients with systemic lupus erythematosus. PLoS One. 2010;5(8):e12280.

18. Brey RL, Abbott RD, Curb JD, et al. Beta(2)glycoprotein 1-dependent anticardiolipin antibodies and risk of ischemic stroke and myocardial
infarction: the Honolulu heart program. Stroke.
2001;32(8):1701-1706.

34. Samarkos M, Davies KA, Gordon C, Loizou S.

Clinical significance of IgA anticardiolipin and
anti-beta2-GP1 antibodies in patients with systemic lupus erythematosus and primary antiphospholipid syndrome. Clin Rheumatol. 2006;25(2):
199-204.

19. Palosuo T, Virtamo J, Haukka J, et al. High antibody levels to prothrombin imply a risk of deep
venous thrombosis and pulmonary embolism in
middle-aged mena nested case-control study.
Thromb Haemost. 1997;78(4):1178-1182.

35. Mehrani T, Petri M. IgM anti-beta2 glycoprotein

I is protective against lupus nephritis and renal
damage in systemic lupus erythematosus.
J Rheumatol. 2011;38(3):450-453.

20. Iverson GM, Victoria EJ, Marquis DM. Anti-beta2

glycoprotein I (beta2GPI) autoantibodies recognize an epitope on the first domain of beta2GPI.
Proc Natl Acad Sci U S A. 1998;95(26):1554215546.
21. de Laat B, Derksen RH, van Lummel M,
Pennings MT, de Groot PG. Pathogenic antibeta2-glycoprotein I antibodies recognize domain
I of beta2-glycoprotein I only after a conformational change. Blood. 2006;107(5):1916-1924.

36. Se`ne D, Piette JC, Cacoub P. Antiphospholipid

antibodies, antiphospholipid syndrome and infections. Autoimmun Rev. 2008;7(4):272-277.

european forum on antiphospholipid antibodies.

Lupus. 2011;20(11):1166-1171.
39. de Laat B, Derksen RH, de Groot PG. Highavidity anti-beta glycoprotein I antibodies highly
correlate with thrombosis in contrast to lowavidity anti-beta glycoprotein I antibodies.
J Thromb Haemost. 2006;4(7):1619-1621.
40. Kouts S, Wang MX, Adelstein S, Krilis SA. Immunization of a rabbit with beta 2-glycoprotein
I induces charge-dependent crossreactive antibodies that bind anionic phospholipids and
have similar reactivity as autoimmune antiphospholipid antibodies. J Immunol. 1995;155(2):
958-966.
41. Gharavi AE, Pierangeli SS, Espinola RG, Liu X,
Colden-Stanfield M, Harris EN. Antiphospholipid
antibodies induced in mice by immunization with
a cytomegalovirus-derived peptide cause thrombosis and activation of endothelial cells in vivo.
Arthritis Rheum. 2002;46(2):545-552.
42. Blank M, Krause I, Fridkin M, et al. Bacterial
induction of autoantibodies to beta2-glycoprotein-I
accounts for the infectious etiology of antiphospholipid syndrome. J Clin Invest. 2002;109(6):797-804.
43. Krause I, Blank M, Cervera R, et al. Crossreactive epitopes on beta2-glycoprotein-I and
Saccharomyces cerevisiae in patients with the
antiphospholipid syndrome. Ann N Y Acad Sci.
2007;1108:481-488.
44. Wilson WA, Gharavi AE, Koike T, et al. International consensus statement on preliminary classification criteria for definite antiphospholipid syndrome: report of an international workshop.
Arthritis Rheum. 1999;42(7):1309-1311.
45. Delbos V, Abgueguen P, Chennebault JM,
Fanello S, Pichard E. Acute cytomegalovirus
infection and venous thrombosis: role of
antiphospholipid antibodies. J Infect. 2007;54(1):
e47-e50.
46. Albert LJ, Inman RD. Molecular mimicry and
autoimmunity. N Engl J Med. 1999;341(27):20682074.
47. Sherer Y, Blank M, Shoenfeld Y. Antiphospholipid
syndrome (APS): where does it come from? Best
Pract Res Clin Rheumatol. 2007;21(6):10711078.
48. Gotoh M, Matsuda J. Induction of anticardiolipin
antibody and/or lupus anticoagulant in rabbits by
immunization with lipoteichoic acid, lipopolysaccharide and lipid A. Lupus. 1996;5(6):593-597.
49. Subang R, Levine JS, Janoff AS, et al.
Phospholipid-bound beta 2-glycoprotein I induces
the production of anti-phospholipid antibodies.
J Autoimmun. 2000;15(1):21-32.
50. Uthman IW, Gharavi AE. Viral infections and antiphospholipid antibodies. Semin Arthritis Rheum.
2002;31(4):256-263.
51. Blank M, Shoenfeld Y. Beta-2-glycoprotein-I,
infections, antiphospholipid syndrome and
therapeutic considerations. Clin Immunol. 2004;
112(2):190-199.
52. Merrill JT. Do antiphospholipid antibodies develop
for a purpose? Curr Rheumatol Rep. 2006;8(2):
109-113.
53. Jordo ED, Wermeling F, Chen Y, Karlsson MC.
Scavenger receptors as regulators of natural
antibody responses and B cell activation in autoimmunity. Mol Immunol. 2011;48(11):1307-1318.
54. von Landenberg P, Doring Y, Modrow S, Lackner
KJ. Are antiphospholipid antibodies an essential
requirement for an effective immune response to
infections? Ann N Y Acad Sci. 2007;1108:578583.

37. Biggioggero M, Meroni PL. The geoepidemiology

of the antiphospholipid antibody syndrome. Autoimmun Rev. 2010;9(5):A299-304.

55. Kra-Oz Z, Lorber M, Shoenfeld Y, Scharff Y.

Inhibitor(s) of natural anti-cardiolipin autoantibodies. Clin Exp Immunol. 1993;93(2):265268.

38. Cucnik S, Kveder T, Ulcova-Gallova Z, et al. The

avidity of anti-beta2-glycoprotein I antibodies in
patients with or without antiphospholipid syndrome: a collaborative study in the frame of the

56. Dighiero G, Rose NR. Critical self-epitopes are

key to the understanding of self-tolerance and
autoimmunity. Immunol Today. 1999;20(9):423428.

From www.bloodjournal.org by guest on June 9, 2016. For personal use only.

BLOOD, 12 JULY 2012 VOLUME 120, NUMBER 2

57. Lieby P, Soley A, Knapp AM, et al. Memory B

cells producing somatically mutated antiphospholipid antibodies are present in healthy individuals.
Blood. 2003;102(7):2459-2465.

SIGNIFICANCE OF AUTOANTIBODIES AGAINST 2-GPI

lial cells, and regulation of oxidative stressinduced cell injury. Blood. 2010;116(11):19611970.

58. Fleming SD, Egan RP, Chai C, et al. Antiphospholipid antibodies restore mesenteric
ischemia/reperfusion-induced injury in complement receptor 2/complement receptor 1-deficient
mice. J Immunol. 2004;173(11):7055-7061.

75. Ioannou Y, Zhang JY, Qi M, et al. Novel assays of

thrombogenic pathogenicity in the antiphospholipid syndrome based on the detection of molecular oxidative modification of the major autoantigen
beta2-glycoprotein I. Arthritis Rheum. 2011;63(9):
2774-2782.

59. Fleming SD, Pope MR, Hoffman SM, et al.

Domain V peptides inhibit beta2-glycoprotein
I-mediated mesenteric ischemia/reperfusioninduced tissue damage and inflammation. J Immunol. 2010;185(10):6168-6178.

76. Romay-Penabad Z, Aguilar-Valenzuela R,

Urbanus RT, et al. Apolipoprotein E receptor 2 is
involved in the thrombotic complications in a
murine model of the antiphospholipid syndrome.
Blood. 2011;117(4):1408-1414.

60. McIntyre JA, Wagenknecht DR. Antiphospholipid

antibodies and renal transplantation: a risk assessment. Lupus. 2003;12(7):555-559.

77. Ramesh S, Morrell CN, Tarango C, et al.

Antiphospholipid antibodies promote leukocyteendothelial cell adhesion and thrombosis in mice
by antagonizing eNOS via beta2GPI and
apoER2. J Clin Invest. 2011;121(1):120-131.

61. Balasubramanian K, Schroit AJ. Characterization

of phosphatidylserine-dependent beta2-glycoprotein
I macrophage interactions. Implications for apoptotic
cell clearance by phagocytes. J Biol Chem. 1998;
273(44):29272-29277.
62. Abdel-Monem H, Dasgupta SK, Le A, Prakasam
A, Thiagarajan P. Phagocytosis of platelet microvesicles and beta2- glycoprotein I. Thromb
Haemost. 2010;104(2):335-341.
63. Shi W, Krilis SA, Chong BH, Gordon S,
Chesterman CN. Prevalence of lupus anticoagulant and anticardiolipin antibodies in a healthy
population. Aust N Z J Med. 1990;20(3):231-236.
64. Cheng HM, Chamley L. Cryptic natural autoantibodies and co-potentiators. Autoimmun Rev.
2008;7(6):431-434.
65. McIntyre JA, Wagenknecht DR, Faulk WP.
Redox-reactive autoantibodies: detection and
physiological relevance. Autoimmun Rev. 2006;
5(1):76-83.
66. Hasselaar P, Triplett DA, LaRue A, et al. Heat
treatment of serum and plasma induces false
positive results in the antiphospholipid antibody
ELISA. J Rheumatol. 1990;17(2):186-191.
67. Agar C, de Groot PG, Marquart JA, Meijers JC.
Evolutionary conservation of the lipopolysaccharide binding site of beta-glycoprotein I. Thromb
Haemost. 2011;106(6):1069-1075.
68. Agar C, van Os GM, Morgelin M, et al. Beta2glycoprotein I can exist in 2 conformations: implications for our understanding of the antiphospholipid syndrome. Blood. 2010;116(8):1336-1343.
69. Kuwana M, Matsuura E, Kobayashi K, et al. Binding of beta 2-glycoprotein I to anionic phospholipids facilitates processing and presentation of a
cryptic epitope that activates pathogenic autoreactive T cells. Blood. 2005;105(4):1552-1557.
70. van Os GM, Meijers JC, Agar C, et al. Induction of
anti-beta2-glycoprotein I autoantibodies in mice
by protein H of streptococcus pyogenes.
J Thromb Haemost. 2011;9(12):2447-2456.
71. de Laat B, van Berkel M, Urbanus RT, et al.
Immune responses against domain I of beta(2)glycoprotein I are driven by conformational
changes: domain I of beta(2)-glycoprotein I harbors a cryptic immunogenic epitope. Arthritis
Rheum. 2011;63(12):3960-3968.
72. Chamorro AJ, Marcos M, Miron-Canelo JA,
Cervera R, Espinosa G. Val247Leu beta2glycoprotein-I allelic variant is associated with
antiphospholipid syndrome: systematic review
and meta-analysis [prepublished online ahead of
print January 8, 2012]. Autoimmun Rev. doi:
10.1016/j.autrev.2011.12.006.
73. Yasuda S, Atsumi T, Matsuura E, et al. Significance of valine/leucine247 polymorphism of
beta2-glycoprotein I in antiphospholipid syndrome: Increased reactivity of anti-beta2glycoprotein I autoantibodies to the valine247
beta2-glycoprotein I variant. Arthritis Rheum.
2005;52(1):212-218.
74. Ioannou Y, Zhang JY, Passam FH, et al. Naturally
occurring free thiols within beta 2-glycoprotein I in
vivo: nitrosylation, redox modification by endothe-

273

the apolipoprotein E receptor 2. J Biol Chem.

2003;278(36):33831-33838.
92. Pierangeli SS, Vega-Ostertag ME, Raschi E,
et al. Toll-like receptor and antiphospholipid mediated thrombosis: in vivo studies. Ann Rheum Dis.
2007;66(10):1327-1333.
93. Romay-Penabad Z, Montiel-Manzano MG,
Shilagard T, et al. Annexin A2 is involved in
antiphospholipid antibody-mediated pathogenic
effects in vitro and in vivo. Blood. 2009;114(14):
3074-3083.
94. Di Simone N, Meroni PL, de Papa N, et al.
Antiphospholipid antibodies affect trophoblast
gonadotropin secretion and invasiveness by
binding directly and through adhered beta2glycoprotein I. Arthritis Rheum. 2000;43(1):140150.

78. Fischetti F, Durigutto P, Pellis V, et al. Thrombus

formation induced by antibodies to beta2glycoprotein I is complement dependent and
requires a priming factor. Blood. 2005;106(7):
2340-2346.

95. Di Simone N, Raschi E, Testoni C, et al. Pathogenic role of anti-beta 2-glycoprotein I antibodies
in antiphospholipid associated fetal loss: characterisation of beta 2-glycoprotein I binding to trophoblast cells and functional effects of anti-beta
2-glycoprotein I antibodies in vitro. Ann Rheum
Dis. 2005;64(3):462-467.

79. Cervera R, Piette JC, Font J, et al. Antiphospholipid syndrome: clinical and immunologic manifestations and patterns of disease expression in a
cohort of 1,000 patients. Arthritis Rheum. 2002;
46(4):1019-1027.

96. Van Horn JT, Craven C, Ward K, Branch DW,

Silver RM. Histologic features of placentas and
abortion specimens from women with antiphospholipid and antiphospholipid-like syndromes.
Placenta. 2004;25(7):642-648.

80. Krone KA, Allen KL, McCrae KR. Impaired fibrinolysis in the antiphospholipid syndrome. Curr
Rheumatol Rep. 2010;12(1):53-57.

97. Out HJ, Kooijman CD, Bruinse HW, Derksen RH.

Histopathological findings in placentae from
patients with intra-uterine fetal death and antiphospholipid antibodies. Eur J Obstet Gynecol
Reprod Biol. 1991;41(3):179-186.

81. Urbanus RT, de Laat B. Antiphospholipid antibodies and the protein C pathway. Lupus. 2010;
19(4):394-399.
82. Boles J, Mackman N. Role of tissue factor in
thrombosis in antiphospholipid antibody syndrome. Lupus. 2010;19(4):370-378.
83. Ambrozic A, Bozic B, Kveder T, et al. Budding,
vesiculation and permeabilization of phospholipid
membranes-evidence for a feasible physiologic
role of beta2-glycoprotein I and pathogenic actions of anti-beta2-glycoprotein I antibodies.
Biochim Biophys Acta. 2005;1740(1):38-44.
84. Satta N, Dunoyer-Geindre S, Reber G, et al. The
role of TLR2 in the inflammatory activation of
mouse fibroblasts by human antiphospholipid antibodies. Blood. 2007;109(4):1507-1514.

98. Rand JH, Wu XX, Quinn AS, Taatjes DJ. The

annexin A5-mediated pathogenic mechanism in
the antiphospholipid syndrome: role in pregnancy
losses and thrombosis. Lupus. 2010;19(4):460469.
99. Girardi G, Berman J, Redecha P, et al. Complement C5a receptors and neutrophils mediate fetal
injury in the antiphospholipid syndrome. J Clin
Invest. 2003;112(11):1644-1654.
100. Jankowski M, Vreys I, Wittevrongel C, et al.
Thrombogenicity of beta 2-glycoprotein Idependent antiphospholipid antibodies in a photochemically induced thrombosis model in the
hamster. Blood. 2003;101(1):157-162.

85. Raschi E, Testoni C, Bosisio D, et al. Role of the

MyD88 transduction signaling pathway in endothelial activation by antiphospholipid antibodies.
Blood. 2003;101(9):3495-3500.

101. Pierangeli SS, Girardi G, Vega-Ostertag M, Liu X,

Espinola RG, Salmon J. Requirement of activation of complement C3 and C5 for antiphospholipid antibody-mediated thrombophilia. Arthritis
Rheum. 2005;52(7):2120-2124.

86. Allen KL, Fonseca FV, Betapudi V, Willard B,

Zhang J, McCrae KR. A novel pathway for human
endothelial cell activation by antiphospholipid/
anti-beta2 glycoprotein I antibodies. Blood. 2012;
119(3):884-893.

102. Holers VM, Girardi G, Mo L, et al. Complement

C3 activation is required for antiphospholipid
antibody-induced fetal loss. J Exp Med. 2002;
195(2):211-220.

87. Doring Y, Hurst J, Lorenz M, et al. Human antiphospholipid antibodies induce TNFalpha in
monocytes via toll-like receptor 8. Immunobiology. 2010;215(3):230-241.

103. Iverson GM, Reddel S, Victoria EJ, et al. Use of

single point mutations in domain I of beta 2glycoprotein I to determine fine antigenic specificity of antiphospholipid autoantibodies. J Immunol.
2002;169(12):7097-7103.

88. Ma K, Simantov R, Zhang JC, Silverstein R,

Hajjar KA, McCrae KR. High affinity binding of
beta 2-glycoprotein I to human endothelial cells is
mediated by annexin II. J Biol Chem. 2000;
275(20):15541-15548.

104. Hunt J, Krilis S. The fifth domain of beta 2glycoprotein I contains a phospholipid binding site
(Cys281-Cys288) and a region recognized by
anticardiolipin antibodies. J Immunol. 1994;
152(2):653-659.

89. Shi T, Giannakopoulos B, Yan X, et al. Antibeta2-glycoprotein I antibodies in complex with

beta2-glycoprotein I can activate platelets in a
dysregulated manner via glycoprotein ib-IX-V.
Arthritis Rheum. 2006;54(8):2558-2567.

105. Horbach DA, van Oort E, Tempelman MJ,

Derksen RH, de Groot PG. The prevalence of a
non-phospholipid-binding form of beta2glycoprotein I in human plasmaconsequences
for the development of anti-beta2-glycoprotein I
antibodies. Thromb Haemost. 1998;80(5):791797.

90. Pennings MT, Derksen RH, van Lummel M,

et al. Platelet adhesion to dimeric betaglycoprotein I under conditions of flow is mediated by at least two receptors: glycoprotein ibalpha and apolipoprotein E receptor 2. J Thromb
Haemost. 2007;5(2):369-377.
91. Lutters BC, Derksen RH, Tekelenburg WL,
Lenting PJ, Arnout J, de Groot PG. Dimers of
beta 2-glycoprotein I increase platelet deposition
to collagen via interaction with phospholipids and

106. Guerin J, Sheng Y, Reddel S, Iverson GM,

Chapman MG, Krilis SA. Heparin inhibits the
binding of beta 2-glycoprotein I to phospholipids
and promotes the plasmin-mediated inactivation
of this blood protein. Elucidation of the consequences of the two biological events in patients
with the anti-phospholipid syndrome. J Biol
Chem. 2002;277(4):2644-2649.

From www.bloodjournal.org by guest on June 9, 2016. For personal use only.

274

BLOOD, 12 JULY 2012 VOLUME 120, NUMBER 2

DE GROOT and URBANUS

107. de la Torre YM, Pregnolato F, DAmelio F, et al.

Anti-phospholipid induced murine fetal loss: novel
protective effect of a peptide targeting the beta2
glycoprotein I phospholipid-binding site. Implications for human fetal loss. J Autoimmun. 2012;38:
J209-J215.
108. Ostertag MV, Liu X, Henderson V, Pierangeli SS.
A peptide that mimics the vth region of beta2-glycoprotein I reverses antiphospholipidmediated thrombosis in mice. Lupus. 2006;15(6):
358-365.
109. van Lummel M, Pennings MT, Derksen RH,
et al. The binding site in {beta}2-glycoprotein
I for ApoER2 on platelets is located in domain
V. J Biol Chem. 2005;280(44):36729-36736.
110. Lee CJ, De Biasio A, Beglova N. Mode of interaction between beta2GPI and lipoprotein receptors

suggests mutually exclusive binding of beta2GPI

to the receptors and anionic phospholipids. Structure. 2010;18(3):366-376.
111. Kolyada A, Lee CJ, De Biasio A, Beglova N. A
novel dimeric inhibitor targeting beta2GPI in
Beta2GPI/antibody complexes implicated in
antiphospholipid syndrome. PLoS One. 2010;
5(12):e15345.
112. Urbanus RT, Pennings MT, Derksen RH,
de Groot PG. Platelet activation by dimeric beta2glycoprotein I requires signaling via both glycoprotein ibalpha and apolipoprotein E receptor 2.
J Thromb Haemost. 2008;6(8):1405-1412.
113. Rand JH, Wu XX, Quinn AS, Chen PP,
Hathcock JJ, Taatjes DJ. Hydroxychloroquine
directly reduces the binding of antiphospholipid

antibody-beta2-glycoprotein I complexes to phospholipid bilayers. Blood. 2008;112(5):1687-1695.

114. Rand JH, Wu XX, Quinn AS, et al. Hydroxychloroquine protects the annexin A5 anticoagulant
shield from disruption by antiphospholipid antibodies: evidence for a novel effect for an old antimalarial drug. Blood. 2010;115(11):2292-2299.
115. Edwards MH, Pierangeli S, Liu X, Barker JH,
Anderson G, Harris EN. Hydroxychloroquine reverses thrombogenic properties of antiphospholipid antibodies in mice. Circulation. 1997;96(12):
4380-4384.
116. Prowse C, Pepper D, Dawes J. Prevention of the
platelet alpha-granule release reaction by
membrane-active drugs. Thromb Res. 1982;
25(3):219-227.

From www.bloodjournal.org by guest on June 9, 2016. For personal use only.

2012 120: 266-274

doi:10.1182/blood-2012-03-378646 originally published
online May 2, 2012

The significance of autoantibodies against 2-glycoprotein I

Philip G. de Groot and Rolf T. Urbanus

Updated information and services can be found at:

http://www.bloodjournal.org/content/120/2/266.full.html
Articles on similar topics can be found in the following Blood collections
Review Articles (638 articles)
Thrombosis and Hemostasis (975 articles)
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml

Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society
of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.