Steven A. Krilis, Department of Immunology, Allergy and Infectious Diseases, St. George Hospital, University of New South Wales, Sydney, Australia. Tel.: +612 91132955; fax: +612 91133981. E-mail: email@example.com
Summary. It has been well established that antiphospholipid antibodies and specifically those directed against beta 2 glycoprotein I (β2GPI) are pathogenic for the development of thrombosis in the antiphospholipid syndrome (APS). Several groups have shown that anti-β2GPI antibodies, in complex with β2GPI, elicit effects on blood cells and coagulation-fibrinolysis proteins, which prime the arterial and venous vasculature for the development of thrombosis. However, much less is known about the mechanism initiating the production of autoantibodies against β2GPI, a physiological abundant protein of blood. In the current review, novel findings are presented regarding the structure and oxidative post-translational modifications of β2GPI, which trigger the immune response. The majority of circulating β2GPI exists in a form containing unpaired cysteines (free thiols), which constitutes the reduced form of β2GPI. The free thiols exposed on β2GPI are involved in the interaction with platelets and endothelial cells. We propose that this abundant pool of free thiols may serve as an antioxidant reservoir protecting cells or critical molecules from oxidative stress. Oxidation of β2GPI confers an increase in its immunogenicity through a Th1 immunological mechanism. The clinical significance of these observations is that serum from patients with APS, assessed by a novel ELISA assay, have a significant increase in oxidised β2GPI. These findings hold promise, not only for the delineation of the role of β2GPI as an immunological target, but also for the development of improved diagnostic and prognostic assays for APS.
Antiphospholipid syndrome and antiphospholipid antibodies
The antiphospholipid syndrome (APS) is defined by clinico-laboratory criteria, which combine clinical features of pregnancy morbidity and venous or arterial thrombosis and the laboratory detection of circulating autoantibodies . Based on the capturing antigen used in ELISA diagnostic assays, the circulating antibodies necessary for the laboratory diagnosis of APS fall into two categories: anticardiolipin and anti-beta 2 glycoprotein I (anti-β2GPI) antibodies. Some of these antibodies interfere with the assembly of clotting factors in phospholipid-dependent clotting assays, a phenomenon termed lupus anticoagulant activity (LA). APS is the most common acquired thrombophilia with 24% of first episode idiopathic venous thromboembolism patients being detected with the syndrome in the RIETE study .
Although the presence of antiphospholipid antibodies is necessary to attribute a thrombotic event to the homonymous syndrome, how these antibodies actually lead to thrombosis is still an intense area of investigation. Autoantibodies in patients with the clinical features of the syndrome are generated also against other autoantigens. Furthermore, in systemic lupus erythematosus approximately 40% of antiphospholipid antibodies occur in conjunction with other autoantibodies . However, a seminal report in 1990 identified the major antigenic target of this heterogeneous group of antiphospholipid antibodies as the plasma protein β2GPI . The anti-β2GPI specificity of antiphospholipid antibodies is linked to the pathophysiology of APS as clinical and epidemiological studies have shown that the anti-β2GPI antibodies are a significant risk factor for thrombosis morbidity and mortality in young adults [5,6]. The clinical observations are also supported by in vivo thrombosis models in rodents where anti-β2GPI antibodies are thrombogenic [7,8]. These findings have spurred interest into understanding the function of β2GPI. Equally important is understanding why this physiological component of human blood becomes a target of pathogenic autoantibodies. Understanding the specific mechanism of anti-β2GPI production will also improve understanding of the pathophysiology of APS.
One of the antigens of pathogenic antiphospholipid antibodies is β2GPI, which is a protein with a single polypeptide chain with a molecular mass of approximately 50 kDa. Its plasma concentration is approximately 200 μg mL−1 and its major site of synthesis is the liver [9,10]. β2GPI consists of five domains (DI-V). The first four domains each contain four cysteines, with disulphide bridges joining the first to the third and the second to the fourth cysteine. The fifth domain has an extra 20 amino acid tail, with an unusual termination in a cysteine that forms a loop-back disulphide link with an extra cysteine found midway between the standard second and third cysteine positions . The V domain is critical for binding of β2GPI to anionic phospholipids .
A considerable amount of work has been done which supports a regulatory role for β2GPI in coagulation, fibrinolysis, angiogenesis and apoptosis (a recent review of the function of β2GPI can be found in ref. ). However, there are no overt phenotypic features in humans  or mice  deficient in β2GPI. Advances regarding the function of β2GPI are expected to evolve following recent discoveries of the effect of oxidative post-translational modification of β2GPI, which occurs under conditions of increased oxidative stress and is the subject of the current review. The contribution of oxidative stress to the development of APS has been studied in the context of lipid peroxidation and the formation of oxidised LDL/β2GPI complexes. These complexes are immunogenic and proatherogenic .
Post-translational modification of cysteine residues in proteins
One of the most functional groups in various proteins is the free sulfhydryl group (SH) contained in the amino acid cysteine. Sulfhydryl modifications can alter the function of proteins that contain cysteines within their catalytic centers or as part of the interface of protein-protein interaction. Post-translational modification of cysteines includes the addition of oxygen or nitrogen oxide (NO) or glutathione. These modifications can also alter the function of extracellular proteins (i.e. the S-nitrosylation of albumin) and are enhanced under conditions of oxidative or nitrosative stress, present in various diseases . Oxidative stress is characterised by the overproduction of reactive oxygen species (ROS), which readily react with cysteine residues, especially redox active cysteines, to form reversible or irreversible oxidised forms. NO bioactivity originating from NO synthases involves a number of reactive nitrogen species, including S-nitrosothiol (SNO). Elevated levels of nitrosothiols are characteristic of nitrosative stress. O2 can also react rapidly with NO to form peroxynitrite (ONOO-). S-nitrosylation, the covalent attachment of NO to cysteine sulphur that forms SNO, is a major mechanism through which NO modifies protein functions in many biological processes. On the other hand, S-nitrosoglutathione (GSNO), the main non-protein SNO in cells, can cause glutathionylation of a cysteine residue. A schematic overview of the oxidative, nitrosative and glutathionative modification of protein cysteines is presented in Fig. 1. The relative contribution of each modification for a given cysteine is unknown, however, S-nitrosylation is regarded as a rapid reversible process (generally resulting in inhibition of function) whereas S-glutathionylation may require the assistance of enzymes such as glutathione S-transferase and is more stable .
Post-translational modification of β2GPI includes the process of oxidation, nitrosylation of redox sensitive cysteine residues or proteolytic cleavage. In this review, two terms will be used: reduced β2GPI is used to define the form of β2GPI containing one or more unpaired cysteines whereas oxidised β2GPI as the form where all cysteines are linked in disulphide bonds. The existence of different post-translationally modified states leads to two consequences: first, the post-translational modification directly affects the function of β2GPI and secondly, the post-translational modification confers an increase in the immunogenicity of β2GPI.
New evidence regarding the structure of human β2GPI
Existence of circulating reduced form of human β2GPI
Recently there have been major findings that have changed the previously held conceptualisation of the structure and function of β2GPI and have implications for its role in APS. First, there has been the description of a reduced form of β2GPI. The original crystal structure of native human β2GPI, by two independent groups, described it as adopting a fishhook shape [19,20]. In the crystal structures no unpaired cysteines were identified. However, it should be noted that purification of β2GPI from plasma, in particular when using perchloric acid in the purification process, results in oxidation not only of cysteines but also of other amino acids such as methionines and lysines [S.A. Krilis, unpublished data]. A form of β2GPI with free thiols has been identified in the circulation of both human and murine blood . A novel highly specific and sensitive streptavidin capture ELISA was developed which detects the free thiol form of β2GPI in plasma (Fig. 2).
The reduced form of β2GPI constitutes approximately 80% of the total circulating β2GPI in normal plasma . Cysteines of the reduced form of β2GPI can be oxidised and nitrosylated [S. A. Krilis, unpublished data]. The reduced state of β2GPI may be maintained by the function of thiol oxidoreductases, which are secreted by platelets and endothelial cells. We have identified Cys288–Cys326 as the major disulphide bond reduced by oxidoreductases . Both the platelet and endothelial surface provide reducing activity to maintain β2GPI in the reduced form [21,23] (Fig. 3).
Existence of a circular form of human β2GPI
Using electron microscopy, β2GPI has been reported to exist in plasma in two different conformations, a circular and open form . In the circular form amino acids Lys19, Arg39 and Arg43 in domain I come to interface with Lys305 and Lys317 in domain V. It has previously been shown that Arg39 and Arg43 are the major epitopes and Lys19 a minor epitope in domain I for anti-β2GPI antibodies of APS patients . Therefore, when β2GPI circulates in the circular form, the major and minor domain I epitopes are cryptic to the immune system. However, when β2GPI is captured on anionic structures such as negatively charged phospholipids (i.e. on the surface of apoptotic cells) the open conformation is stabilised and the domain I epitope is exposed (Fig. 4). This finding also introduces the concept that the circulating form of β2GPI is immunologically inert whereas the immobilised form exposes the B cell epitopes for autoantibody binding.
New evidence regarding the consequences of post-translational modification of β2GPI
Effect of oxidation on function of β2GPI
The oxidative/reductive conversion of β2GPI may offer a means of regulation of its biological activity. An example of this is the dual effect of β2GPI on platelet adhesion to von Willebrand factor (VWF). The native purified form (oxidised) of β2GPI was shown by Hulstein and coworkers to inhibit platelet adhesion to immobilised VWF under flow conditions . We have observed that the reduced form of β2GPI binds to VWF and increases adhesion of platelets (via the glycoprotein Ibα complex) to VWF .
The reduced form of β2GPI also protects endothelial cells from oxidative stress induced by hydrogen peroxide . During the process of protection of endothelial cells by reduced β2GPI from oxidative stress, β2GPI was S-nitrosylated by the endothelial cells. β2GPI has been shown to protect macrophages and human coronary artery smooth cells against NO-induced apoptosis . The same group  has also reported that β2GPI may inhibit low density lipoprotein oxidation, raising the possibility that one general function of β2GPI may be to protect cells of the vasculature from free radical injury.
Effect of oxidation on immunogenicity of β2GPI
Increased affinity of anti-β2GPI antibodies to oxidised β2GPI Increased oxidative stress, present in a variety of pathological states (e.g. infection), can increase the amount of oxidised β2GPI in the circulation. The oxidation of β2GPI may increase the immunogenicity of the molecule. In systemic lupus erythematosus, autoantibodies have been described that recognise post-translationally oxidatively modified human serum albumin . When β2GPI is reduced by the oxidoreductase thioredoxin, rabbit polyclonal or murine monoclonal anti-β2GPI antibodies show decreased immunoreactivity to the reduced form compared to the oxidised form of β2GPI . However, the affinity of β2GPI to cardiolipin does not differ between the reduced or oxidised form of β2GPI [S.A. Krilis, unpublished data]. Mass spectrometry of β2GPI reduced by thioredoxin revealed that the solvent exposed disulphide bond Cys288–Cys326 was the predominantly reduced bond. However, patient antibodies are usually generated against domain I. The second most solvent accessible disulphide bond was the Cys32–Cys60 bond, which was also reduced by thioredoxin. This could explain the significant decrease in immunoreactivity of the anti-domain I monoclonal antibody towards reduced β2GPI compared to oxidised β2GPI . This is particularly relevant to patient anti-β2GPI antibodies, which may show increased affinity when the oxidised form of β2GPI is the predominant species in plasma. Patients with APS have increased amounts of circulating oxidised β2GPI (see below).
Th1 response to oxidised β2GPI Buttari et al. have shown that when β2GPI without free thiols is further oxidated (i.e. using non physiological oxidants and conditions) it is able to directly bind immature monocyte-derived dendritic cells and cause the cells to mature. β2GPI under these non-physiological in vitro conditions forms aggregates which have not been described in vivo. The mature cells secreted interleukin (IL)-12, IL-1, IL-6, IL-8, tumour necrosis factor-α and IL-10. Furthermore, the supra non-physiologically oxidised β2GPI-stimulated dendritic cells attained allostimulatory ability, and primed naive T lymphocytes, inducing T helper 1 polarisation. Interestingly, pre-treatment of β2GPI with the antioxidant α-tocopherol prevented dendritic cell maturation .
T Cell response to β2GPI bound to anionic phospholipids Generation of peroxynitrite by inflammatory cells induces post-translationally modified cysteines or tyrosines in domain V, which may be responsible for generation of autoreactive T cells. The T cells recognise nitrosylated or nitrated epitopes in the peptide encompassing 276–290 (Fig. 5) and induce domain I specific B cells to generate autoantibodies.
It has been noted that β2GPI-autoreactive CD4 T cells can be found in both the healthy population and in individuals with APS . In vitro, these HLA class II restricted T cells can be stimulated to multiply by exposure to native β2GPI bound to anionic phospholipids . Exposure to either constituent alone does not lead to their proliferation. Macrophages and dendritic cells partake an important role in antigen processing, presentation, and subsequent stimulation of the T cells . The relevant β2GPI epitope which the autoreactive T cells recognise lies between amino acid residues 276–290 within domain V . This is in the phospholipid binding region of β2GPI . This is in contrast to the B-cell epitope, which is contained within domain I [36,37]. Both domains I and V on the β2GPI molecule are, therefore, required to elicit the generation of a pathogenic autoantibody response.
Kuwana et al. have postulated that β2GPI bound to phospholipid may be shielded from degradation by proteases in the endocytic compartments of antigen presenting cells, which may also allow for efficient presentation of the intact domain V (i.e. the cryptic T cell epitope). Our studies strongly suggest that post-translationally modified β2GPI can break immune tolerance. One possible explanation could be that the modified β2GPI is not represented in the thymus and, therefore, reactive T cells escape tolerance and migrate into the periphery. A second mechanism in breakdown of self-tolerance to post-translational modified β2GPI is that the intracellular processing of the oxidised protein is different compared to the reduced form (Fig. 5).
Clinical studies supporting the role of oxidised β2GPI in APS The concept that post-translational oxidatively modified β2GPI may be the relevant autoantigen in APS is raised by novel clinical data. Our group has recently shown that the total amount of β2GPI and the relative amount of oxidised β2GPI is increased in APS patients with a thrombotic history compared to healthy volunteers, patients with a history of thrombosis without APS or patients with autoimmune disease but no thrombosis. This large, multicentre study of nearly 500 patients showed that post-translational modification of β2GPI via oxidation of cysteine residues is a highly specific phenomenon in the setting of APS thrombosis  (Fig. 6).
Clinical states associated with an increased oxidative load, such as pregnancy and infection, may lead to further increases in the levels of oxidised β2GPI in the plasma, potentially elevating the risk of thrombosis occurring in patients who are positive for anti-β2GPI antibodies. A recent study has shown that oxidative stress may drive β2GPI production in vivo through activator protein I and nuclear factor-kappa B mediated up-regulation of β2GPI gene promoter activity . Therefore, enhanced oxidative stress may increase antigenic load, potentially driving anti-β2GPI production in autoimmune prone subjects and lowering the threshold for a clinical event.
Activation of cells sustains the oxidative environment It has been demonstrated that the formation of the β2GPI/anti β2GPI complex leads to activation of cells in the vasculature, which makes them prone to thrombosis. This potentially may lead to the establishment of an amplification loop responsible for the generation of additional post-translational oxidised β2GPI autoantigen. Endothelial cells, platelets and monocytes produce NO and ROS. In the presence of anti-β2GPI and β2GPI, monocytes are activated to express tissue factor [39,40]. The receptors that have been implicated to be crosslinked to achieve this include annexin A2 and toll-like receptor 4 . Activated macrophages generate NO and superoxide radicals, which can further oxidise vulnerable targets such as β2GPI. Furthermore, endothelial cells and platelets secrete active thiol oxidoreductases, which may display a reducing or oxidising effect on extracellular proteins according to the redox potential. The co-existence of β2GPI and anti-β2GPI can also potentiate platelet activation . This effect may be caused by the crosslinking of the glycoprotein Ibα receptor  and the ApoER2′ receptor . Platelet-generated superoxide potentiates platelet recruitment.  Endothelial cells can be activated to express a procoagulant, pro-adhesive phenotype by anti-β2GPI in complex with β2GPI . In vivo models using mice suggest that priming of the endothelium, either with a photochemical injury , or by using lipopolysaccharide , is necessary for the antibodies to exert their prothrombotic influence. S-nitroso-albumin is an important in vivo reservoir in blood for endothelial NO from which low molecular weight S-nitroso-cysteine and S-nitroso-glutathione are derived. As reduced β2GPI is an electron reservoir, it is logical to assume that once the reducing ability of β2GPI is exceeded, the production of oxidised β2GPI increases which stimulates the production of autoantibodies in susceptible individuals. The generation of anti-β2GPI antibodies against oxidised β2GPI initiates a redox cycle that potentiates the production of oxidative and nitrosative species, which can further oxidise β2GPI. A hypothetical model is depicted in Fig. 7.
It has become evident that redox sensitive cysteines in β2GPI can be modified by disulphide bond linkage, oxidation or nitrosylation. The existence of a particular oxidative state of β2GPI depends upon the specific redox environment created by a multitude of coplayers (including reduced:oxidized glutathione ratio, oxidoreductases released by platelets and endothelial cells, ROS and NO). The study of the function of post-translationally modified forms of β2GPI is currently an exciting area of research. Insights obtained may potentially be applied for diagnostic and prognostic purposes in identifying subsets of APS who are at higher risk for thrombosis. The currently available laboratory methods are not adequate to provide prognostic information to clinicians treating patients with APS . The development of assays with better characteristics in terms of risk stratification will aid in the decision for treatment options offered to these thrombophilic patients. The modified ELISA for detection of reduced β2GPI presented in this review [21,22] may prove particularly helpful in defining the subset of antiphospholipid antibody positive patients who are at higher risk of developing a thrombotic event.
Disclosure of Conflict of Interest
Work described in this work was supported by NHMRC grants to SA Krilis.