Autoantibody explosion in antiphospholipid syndrome

Antiphospholipid syndrome (APS) is characterized by thrombosis and pregnancy loss in the presence of antiphospholipid antibodies (aPL), mainly anticardiolipin, anti-beta2-glycoprotein I and lupus anticoagulant. However, similar to systemic lupus erythematosus, APS is also characterized by multiple other autoantibodies including 'non-classical' aPL, as well as other antibodies. Herein we describe the autoantigen properties, prevalence and clinical importance of 30 different antibodies in APS. Among the other antibodies characterizing APS are autoantibodies directed to platelets, glycoproteins, various coagulation factors, lamins, mitochondrial antigens and cell surface markers. Few of these autoantibodies are correlated with the presence of other antibodies, and some may have an additive role in the pro-thrombotic tendency of the syndrome. This autoantibody explosion might be important in early identification of the syndrome and its manifestations.     

Clinical and epidemiological aspects in the antiphospholipid syndrome

The antiphospholipid syndrome (APS) is defined by the occurrence of venous and arterial thromboses, often multiple, and pregnancy morbidity (mainly, recurrent fetal losses and premature births), frequently accompanied by a moderate thrombocytopenia, in the presence of antiphospholipid antibodies (aPL), namely lupus anticoagulant (LA), anticardiolipin antibodies (aCL), or both. Other autoantibodies have also been detected in many patients with an APS, such as anti-beta2 glycoprotein I (GPI), antimitochondrial (M5 type), antiendothelial cell, antiplatelet, antierythrocyte, and antinuclear antibodies. The APS can be found in patients having neither clinical nor laboratory evidence of another definable condition (primary APS) or it may be associated with other diseases. Systemic lupus erythematosus (SLE) is the disorder in which an APS is most commonly associated. Less frequently, aPL and, rarely, an APS may also be encountered in other groups of patients (Table 1) (1).     

New autoantigens in the antiphospholipid syndrome

The antiphospholipid syndrome (APS) is an autoimmune disease characterized by arterial and venous thrombosis, recurrent miscarriages or fetal loss, and circulating antiphospholipid antibodies (aPL). Enzyme-linked immunosorbent assays for anticardiolipin and anti-β2-glycoprotein I antibodies and clotting assays for the lupus anticoagulant are the tests recommended for detecting aPL. However, the aPL are a heterogeneous group of antibodies directed against anionic phospholipids but also toward phospholipid-binding plasma proteins or phospholipid-protein complexes. β2-glycoprotein I (β2GPI) is the playmaker antigen of APS, however during apoptosis, lysophospholipids can become exposed on the cell surface, and mainly through their interaction with β2GPI, they can become targets of aPL. Some CL metabolites are likely to escape from the remodeling cycle. This would account for the progressive loss of mitochondrial CL during apoptosis, as well as for the presence of CL and lyso-CL at the cell surface, where they can interact with β2GPI and become targets of aPL. Other recognized targets of aPL are represented by phosphatidylserine, lyso(bis)phosphatidic acid, Phosphatidylethanolamine, vimentin, and annexin A5. These molecules may allow improving the knowledge on the pathogenesis, and the early identification of APS. Although several studies have shown the presence of antibodies directed against other antigens in APS, their clinical relevance is still a matter of debate, and it needs to be confirmed with experimental data and longitudinal studies.     

Which are the best biological markers of the antiphospholipid syndrome?

The diagnosis of antiphospholipid syndrome (APS) requires the presence of both clinical and biological features. Due to the heterogeneity of anti-phospholipid antibodies (aPL) the laboratory approach for their detection includes clotting-based tests for lupus anticoagulant (LA) as well as solid-phase assays for anticardiolipin antibodies (aCL). In addition, as it has been shown that autoimmune aPL recognize epitopes on phospholipid (PL)-binding plasma proteins, assays detecting antibodies to beta 2-glycoprotein I (beta 2-GPI) or prothrombin have been developed. The association between venous or arterial thrombosis and recurrent fetal loss with the presence of conventional aPL (LA and/or aCL) has been confirmed by many studies. The LA and IgG aCL at moderate/high titre seem to exhibit the strongest association with clinical manifestations of the APS. Several reports indicate that LA is less sensitive but more specific than aCL for the APS. Assays against PLs other than CL as well as the use of mixtures of PLs have been proposed to improve the detection of APS-related aPL. Concerning antibodies to PL-binding proteins (detected in the absence of PLs), there is evidence that anti-beta 2-GPI are closely associated with thrombosis and other clinical features of the APS. Moreover, these antibodies may be more specific in the recognition of the APS and in some cases may be present in the absence of aPL detected by standard tests. Many issues are still under debate and are discussed in this review, such as the problems of standardization of anti-beta 2-GPI assays, detection of the IgA isotype of aCL and anti-beta 2-GPI, the coagulation profiles of LA in the recognition of the thrombotic risk and the association of particular markers with subsets of patients with APS.     

Antiphospholipid syndrome

Abstract. The antiphospholipid syndrome (APS) was reported in the early 1980s as the association of thrombosis, recurrent pregnancy loss in the presence of anticardiolipin antibodies (aCL) and/or lupus anticoagulant (LA). Since then, many other clinical manifestations have been associated with aPL. Almost any organ and tissue may be involved in the disease, including the brain, the heart, the kidneys, the placenta and many more. aPL are a heterogeneous group of autoantibodies that are detected by immunoassays and functional coagulation tests. The antigenic targets are negatively charged phospholipids and serum phospholipid-binding proteins. Despite the strong association between aPL and thrombosis, the pathogenic role of aPL in the development of thrombosis has not been fully elucidated. Proposed mechanisms include antibody-mediated interference with coagulation homeostasis, activation of platelets and endothelial cells and a T-cell immune response to serum phospholipid-binding proteins. The mainstay of therapy is anticoagulation, whereas immunosuppression seems to be ineffective. Recommendations for the management of thrombosis in the antiphospholipid antibody syndrome have been based largely on retrospective case series. Several prospective clinical trials are currently underway and their results will probably lead to a more precise therapeutic approach of this problem.     

Antiphospholipid syndrome

The antiphospholipid syndrome (APS) was reported in the early 1980s as the association of thrombosis, recurrent pregnancy loss in the presence of anticardiolipin antibodies (aCL) and/or lupus anticoagulant (LA). Since then, many other clinical manifestations have been associated with aPL. Almost any organ and tissue may be involved in the disease, including the brain, the heart, the kidneys, the placenta and many more. aPL are a heterogeneous group of autoantibodies that are detected by immunoassays and functional coagulation tests. The antigenic targets are negatively charged phospholipids and serum phospholipid-binding proteins. Despite the strong association between aPL and thrombosis, the pathogenic role of aPL in the development of thrombosis has not been fully elucidated. Proposed mechanisms include antibody-mediated interference with coagulation homeostasis, activation of platelets and endothelial cells and a T-cell immune response to serum phospholipid-binding proteins. The mainstay of therapy is anticoagulation, whereas immunosuppression seems to be ineffective. Recommendations for the management of thrombosis in the antiphospholipid antibody syndrome have been based largely on retrospective case series. Several prospective clinical trials are currently underway and their results will probably lead to a more precise therapeutic approach of this problem.     

Antiphospholipid syndrome

Antiphospholipid syndrome (APS) is a disease characterized by venous and arterial thromboses or spontaneous abortions and the repeated detection of antiphospholipid antibodies (aPL). APS may be associated with another autoimmune disease (secondary APS), particularly systemic lupus erythematosus (SLE), or unrelated to an underlying disease (primary APS). APS affects almost all organs. In addition to the clinical criteria, lupus anticoagulant testing and immunological aPL determinations are required to establish the diagnosis of APS.     

HLA class II alleles and genetic predisposition to the antiphospholipid syndrome

The antiphospholipid syndrome (APS) is an autoimmune disease characterized by the presence of antiphospholipid antibodies (aPL). Its etiology is linked to genetic predisposition, which is accounted for, at least in part, by genes of major histocompatibility complex (HLA system). The association of APS with human leukocyte antigen (HLA) alleles is a consequence of the association of aPL with HLA alleles. Some HLA alleles carry the risk to produce aPL, and this is independent of the clinical context. In fact, we find the same associations between HLA and aPL in primary APS and in APS secondary to systemic lupus erythematosus (SLE). The association of HLA-DR4, -DR7, -DRw53 and -DQB1*0302 with aCL that has been demonstrated in primary APS can also be found in SLE, a disease with a completely different pattern of HLA allele association (DR2, DR3, DRw52). In addition, the various aPL (anticardiolipin antibodies, lupus anticoagulant, anti-beta2GPI antibodies, antiphosphatidylserine/prothrombin antibodies) show similar HLA association, again independent of the clinical context (primary APS or SLE), and across various ethnic groups.     

Autoantibody-mediated atherosclerosis

Beta2-glycoprotein I (beta2-GPI) is a major antigen for antiphospholipid antibodies (aPL) present in patients with antiphospholipid syndrome (APS). Oxidized low-density lipoprotein (oxLDL) is subsequently targeted by beta2-GPI and anti-beta2-GPI autoantibodies. Ligands specific for beta2-GPI derived from oxLDL have been characterized as oxidized forms of cholesteryl linoleate, such as 7-ketocholesterol-9-carboxynonanoate, i.e. 9-oxo-9-(7-ketocholest-5-en-3beta-yloxy) nonanoic acid, (namely oxLig-1). The in vitro phenomenon that it is significantly increased in binding of oxLig-1 containing liposomes to macrophages via an interaction with beta2-GPI and an anti-beta2-GPI autoantibody (via the Fcgamma receptor) may propose a novel mechanism on 'autoantibody-mediated atherosclerosis'. Furthermore, autoantibodies against a complex of beta2-GPI and oxLig-1 are detected in sera of APS patients and appearance of the antibodies is associated with episodes of thrombosis, especially, arterial thrombosis. Thus, autoimmune atherogenesis linked to beta2-GPI interaction with oxLDL and autoantibodies may be present in APS.     

The Pathogenesis of Neural Injury in Animal Models of the Antiphospholipid Syndrome

Circulating antiphospholipid antibodies (aPL) are associated with central nervous system dysfunction in antiphospholipid syndrome (APS) patients and in a mouse model of APS. We propose a logical pathway of how experimental APS (eAPS) causes brain dysfunction: binding of the antibodies to the brain endothelium evoking microthrombosis, endothelial dysfunction, and IgG leakage through the blood–brain barrier (BBB), then secondary inflammatory cell spread around blood vessels and production of cytokines by these inflammatory cells leading to further disruption of the BBB. The diffuse brain endothelial dysfunction would result in extravasation of serum proteins including APS IgG and activated thrombin, which may induce the behavioral changes observed in the APS mice. We have collected data from the mouse eAPS model which supports this hypothesis. Elucidating the mechanism of the pathogenicity of aPL in vitro and in vivo will serve as a much needed basis for developing new therapeutic modalities in this important disorder.     

Antiprothombin antibodies and the diagnosis of antiphospholipid syndrome

The preliminary classification criteria for definite antiphospholipid syndrome (APS) include the presence of anticardiolipin antibodies (aCL) and/or lupus anticoagulant (LA) as laboratory criteria. However, antiphospholipid antibodies (aPL) are a heterogeneous group of antibodies comprising also antibodies against phospholipid-binding proteins or their complexes with phospholipids. Prothrombin is one of the antigen recognized by aPL. In the last decade, there has been increasing interest in antibodies against prothrombin alone and those against phosphatidylserine-prothrombin complex. The latter, phosphatidylserine-dependent antiprothrombin antibodies (aPT), have been closely associated with APS and LA. In this paper, we review the properties of antiprothrombin antibodies.     

Antiphospholipid antibodies: effects on trophoblast and endothelial cells

PROBLEM: Antiphospholipid syndrome (APS) may affect placental functions through several possible mechanisms. Interaction of antiphospholipid antibodies (aPL) with cells involved in the coagulation cascade is thought to produce a procoagulant state. Thrombotic placental pathology is however not specific for the APS. METHOD OF STUDY: An analysis of published data. RESULTS: It is now generally accepted that the clinically relevant aPL bind to proteins with affinity for phospholipids (PL), such as beta2-glycoprotein I (beta2-GPI). Following the attachment of beta2-GPI to trophoblast anionic PL, both molecules undergo conformational changes resulting in the exposure of cryptic epitopes within the structure of beta2-GPI. This may allow the subsequent binding of antibodies hence affecting trophoblast functions directly. Moreover anti-beta2-GPI antibodies induce the activation of endothelial cells (ECs), resulting in a proinflammatory state which favours the prothrombotic diathesis of the syndrome. CONCLUSION: Numerous ameliorations in the APS knowledge have been introduced in the last few years. To have clarified the mechanism of antibody mediated damage on trophoblast and ECs represents an important step to explain the cellular events leading to pregnancy complications.     

beta2-glycoprotein I, the playmaker of the antiphospholipid syndrome

From its discovery in the early 60s till the beginning of the 90s, there was not much interest in plasma protein beta2-glycoprotein I (beta2-GPI). The finding that beta2-GPI acts as an essential cofactor for the detection of antiphospholipid antibodies (aPL) tremendously increased the interest in beta2-GPI [Lancet 335 (1990) 1544; Lancet 336 (1990) 177; Proc. Natl. Acad. Sci. U. S. A. 87 (1990) 4120]. It is now generally accepted that autoantibodies directed towards beta2-GPI are not only a serological marker but that they are involved in the pathology of the antiphospholipid syndrome (APS). In this review, we will first discuss the biochemistry of the protein beta2-GPI and the influence that the antibodies have on the function of beta2-GPI. Next, we will discuss the problems that are faced when assays to detect the presence of the autoantibodies are performed, emphasizing the urgent need for standardization of the anti-beta2-GPI-ELISA. Finally, we will discuss our latest insights into beta2-GPI and its role in the pathology of APS. Thereby, we will focus on the role of dimerized beta2-GPI on platelet and endothelial cell function.     

Viral origin of antiphospholipid antibodies: endothelial cell activation and thrombus enhancement by CMV peptide-induced APL antibodies

Our observations and those from others, give further support to our hypothesis that "autoimmune aPL" may be generated by immunization with products from bacteria or viruses after incidental exposure or infection. We also were able to generate APS-like syndrome in a strain of mice susceptible to autoimmunity, indicating that other factors such as genetics are likely to be involved in the development of APS. Furthermore, not all aPL antibodies generated by immunization with bacterial or viral products are pathogenic. Based on the clinical experience and on the numerous reports indicating presence of aPL in a large number of infectious diseases, it may be expected that not all aPL antibodies produced during infection will be pathogenic. We hypothesize that a limited number aPL antibodies induced by certain viral/bacterial products would be pathogenic in certain groups of predisposed individuals. Identification of these bacterial and/ or viral agents may help to find strategies for the prevention of production of aPL "pathogenic" antibodies. Alternatively, free peptides may be used to induce tolerance against aPL production.     

Evaluation of novel assays for the detection of autoantibodies in antiphospholipid syndrome

Patients with antiphospholipid syndrome (APS) present with clinical features of recurrent thrombosis and pregnancy morbidity and persistently test positive for the presence of antiphospholipid antibodies (aPL). At least one clinical (vascular thrombosis or pregnancy morbidity) and one lab-based (positive test result for lupus anticoagulant, anticardiolipin antibodies and/or anti-β2-glycoprotein 1 antibodies) criterion have to be met for a patient to be classified as having APS. Nevertheless, the clinical variety of APS encompasses additional signs and symptoms, potentially affecting any organ, that cannot be explained exclusively by a prothrombotic state. Those manifestations, also known as extra-criteria manifestations, include haematologic (thrombocytopenia and haemolytic anaemia), neurologic (chorea, myelitis and migraine) manifestations as well as the presence of livedo reticularis, nephropathy and valvular heart disease. The growing body of evidence describing the clinical aspect of the syndrome has been paralleled over the years by emerging research interest focusing on the development of novel biomarkers that might improve the diagnostic accuracy for APS when compared to the current aPL tests. This review will focus on the clinical utility of extra-criteria aPL specificities. Besides, the promising role of a new technology using particle based multi-analyte testing that supports aPL panel algorithm testing will be discussed. Diagnostic approaches to difficult cases, including real-world case studies investigating the diagnostic added value of extra criteria aPL, particularly anti-phosphatidylserine/prothrombin, will also be examined.     

The emerging role of multiple antiphospholipid antibodies positivity in patients with antiphospholipid syndrome

Antiphospholipid syndrome (APS) is an autoimmune disorder characterized by clinical symptoms of vascular thrombosis and/or pregnancy morbidity in the presence of autoimmune antiphospholipid antibodies (aPL). Current laboratory APS criteria include the presence of at least one of the three relevant aPL: lupus anticoagulant, anticardiolipin antibodies and anti-β₂ glycoprotein I antibodies. Therefore, patients could have a single aPL pattern or combinations of aPL. Evidence arising from clinical experience indicates that patients having the highest aPL titer and simultaneous aPL detected by different tests have a worse prognosis and a higher probability of recurrence of the APS clinical features. In recent years, an emerging role of multiple aPL positivity in the identification of high-risk patients with aPL/APS is evident. This paper will review the current knowledge on the clinical relevance of having single or multiple aPL positivity.     

Atherosclerosis and antiphospholipid syndrome

Atherosclerosis is an autoimmune/inflammatory disease associated with infectious, inflammatory, and autoimmune factors. Both humoral and cellular immune mechanisms have been proposed to participate in the onset and/or progression of atheromatous lesions. Heat-shock protein (hsp), oxidized low-density lipoprotein (LDL), and β2-GPI have been reported to elicit humoral and cellular immune response in both experimental animals and humans. These autoantigens are expressed within atherosclerotic lesions. Immunization with the given autoantigens elicits an immune response that influences lesion progression. Atherosclerosis susceptibility can be transferred by autoantigen-sensitized lymphocytes from immunized animals. Patients with systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS) have a high risk for atherosclerotic cardiovascular events. The traditional risk factors fail to fully account for accelerated atherosclerosis in SLE and APS. Immunological alterations, such as antibodies to oxidized LDL, antiphospholipid antibodies (aPL), antibodies to β-2 Glycoprotein (anti-β2-GPL), anti-prothrombin antibodies, may play a role in premature atherosclerosis in SLE and APS. Paraoxonase (PON1) is an enzyme with antioxidant activity attached to the circulating high-density lipoprotein (HDL) in plasma. Its function is to prevent oxidation of LDL, thereby accounting for the antioxidant properties and the atherosclerotic protective effects of HDL. The relationship between PON1 and aPL has been recently suggested. IgG anti-HDL and IgG anti-β2-GPI antibodies were associated with reduced PON1 activity in patients with SLE and primary APS. The determination of classic and new factors, together with specific autoantibody titers and the use of Doppler carotid ultrasound, are useful methods to detect early atherosclerosis in SLE and PAPS. Therapeutic strategies, including early control of disease and other risk factors, are essential to reduce morbidity and mortality.     

Antiphospholipid antibodies mediate autoimmunity against dying cells

The Antiphospholipid Syndrome (APS) is characterized by thrombosis and pregnancy loss, clinical events mediated by pathogenic anti-phospholipid autoantibodies (aPL). β2-glycoprotein I (β2GPI) is the major autoantigens recognized by aPL. β2GPI is a cationic protein that binds to negatively charged surfaces such as those of apoptotic cells. This feature may lead to two major events: i) immunization with β2GPI fosters the Fc-receptor-mediated uptake by antigen presenting cells of apoptotic material decorated with β2GPIand the activation ofβ2GPI-specific T cells which in turn provide help to β2GPI-specific B cells for the production of anti-β2GPI; ii) apoptotic bodies decorated with β2GPI can be opsonized by anti-β2GPI and shifted towards a pro-inflammatory clearance by macrophages; epitope spread can occur with the generation of autoimmunity against nuclear autoantigens. In the presence of a predisposing genetic background and of a particular cytokine environment (type I interferons), the sequential emergence of autoantibodies can evolve into overt clinical disease. The spectrum of clinical phenotypes of the patients can be modulated by several factors affecting the pathogenicity of anti-β2GPI (e.g. domain specificity). We conclude that dying cells may play a dual role in APS: (I) as immunogen for the induction of aPL (etiology) and (II) as targets of aPL for the chronification of inflammation and the development of autoimmune diseases (pathology).