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Objective: Primary antiphospholipid syndrome (PAPS) is a rare autoimmune disease characterized by the presence of antiphospholipid antibodies and the occurrence of thrombotic events and pregnancy complications. Our study aimed to identify novel genetic susceptibility loci associated with PAPS., Methods: We performed a genome-wide association study comprising 5,485 individuals (482 affected individuals) of European ancestry. Significant and suggestive independent variants from a meta-analysis of approximately 7 million variants were evaluated for functional and biological process enrichment. The genetic risk variability for PAPS in different populations was also assessed. Hierarchical clustering, Mahalanobis distance, and Dirichlet Process Mixtures with uncertainty clustering methods were used to assess genetic similarities between PAPS and other immune-mediated diseases., Results: We revealed genetic associations with PAPS in a regulatory locus within the HLA class II region near HLA-DRA and in STAT1-STAT4 with a genome-wide level of significance; 34 additional suggestive genetic susceptibility loci for PAPS were also identified. The disease risk allele near HLA-DRA is associated with overexpression of HLA-DRB6, HLA-DRB9, HLA-DQA2, and HLA-DQB2 in immune cells, vascular tissue, and nervous tissue. This association is independent of the association between PAPS and HLA-DRB1*1302. Functional analyses highlighted immune-related pathways in PAPS-associated loci. The comparison with other immune-mediated diseases revealed a close genetic relatedness to neuromyelitis optica, systemic sclerosis, and Sjögren syndrome, suggesting co-localized causal variations close to STAT1-STAT4, TNPO3, and BLK., Conclusion: This study represents a comprehensive large-scale genetic analysis for PAPS and provides new insights into the genetic basis and pathophysiology of this rare disease., (© 2024 The Author(s). Arthritis & Rheumatology published by Wiley Periodicals LLC on behalf of American College of Rheumatology.)

The present review reports the history of our scientific collaboration with Professor Shoenfeld's group. The collaboration started at the end of the 80s and was mainly focused on studies on the pathogenetic mechanisms of the anti-phospholipid syndrome (APS). Following the initial collaborative studies on antibodies against endothelium in systemic autoimmune vasculitis, we were able to use a similar strategy in APS. This line of research has resulted in the characterization of beta 2 glycoprotein I (β2GPI)-dependent anti-phospholipid antibodies (aPL) as mechanisms capable of mediating an endothelial perturbation crucial for the pathogenesis of APS. Thanks to these studies, the collaboration has led to the characterization of the membrane receptors for β2GPI and the cellular signaling resulting from antibody binding. This mechanism has also been shown to mediate the aPL effect on other cell types involved in APS pathogenesis. Finally, the exchange of information made it possible to replicate and extend the setting of animal models of the syndrome, which proved to be valuable tools for understanding the pathogenesis of the syndrome. It has been a long story recently refueled by common studies on the similarity of pro-inflammatory and pro-coagulant endotheliopathy in APS and in COVID-19., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Pier Luigi Meroni reports financial support was provided by IRCCS Istituto Auxologico Italiano. Pier Luigi Meroni reports a relationship with IRCCS Istituto Auxologico Italiano that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Antibodies against phospholipid (aPL)-binding proteins, in particular, beta 2 glycoprotein I (β2GPI), are diagnostic/classification and pathogenic antibodies in antiphospholipid syndrome (APS). β2GPI-aPL recognize their target on endothelium and trigger a pro-thrombotic phenotype which is amplified by circulating monocytes, platelets and neutrophils. Complement activation is required as supported by the lack of aPL-mediated effects in animal models when the complement cascade is blocked. The final result is a localized clot. A strong generalized inflammatory response is associated with catastrophic APS, the clinical variant characterized by systemic thrombotic microangiopathy. A two-hit hypothesis was suggested to explain why persistent aPL are associated with acute events only when a second hit allows antibody/complement binding by modulating β2GPI tissue presentation. β2GPI/β2GPI-aPL are also responsible for obstetric APS, being the molecule physiologically present in placental/decidual tissues. Additional mechanisms mediated by aPL with different characteristics have been reported, but their diagnostic/prognostic value is still a matter of research., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Antiphospholipid syndrome (APS) is a systemic autoimmune disorder characterized by recurrent vascular thrombosis and miscarriages in the absence of known causes. Antibodies against phospholipid-binding proteins (aPL) are pathogenic players in both clotting and pregnancy APS manifestations. There is sound evidence that antibodies specific for beta2 glycoprotein I (β2GPI) trigger thrombotic and pregnancy complications by interacting with the molecule on the membranes of different cell types of the coagulation cascade, and in placenta tissues. In addition to the humoral response against β2GPI, both peripheral and tissue CD4 + β2GPI-specific T cells have been reported in primary APS as well as in systemic lupus erythematosus (SLE)-associated APS. While adaptive immunity plays a clear role in APS, it is still debated whether innate immunity is involved as well. Acute systemic inflammation does not seem to be present in the syndrome, however, there is sound evidence that complement activation is crucial in animal models and can be found also in patients. Furthermore, neutrophil extracellular traps (NETs) have been documented in arterial and venous thrombi with different etiology, including clots in APS models. Keeping in mind that β2GPI is a pleiotropic glycoprotein, acting as scavenger molecule for infectious agents and apoptotic/damaged body constituents and that self-molecules externalized through NETs formation may become immunogenic autoantigens, we demonstrated β2GPI on NETs, and its ability to stimulate CD4 + β2GPI-specific T cells. The aim of this review is to elucidate the role of β2GPI in the cross-talk between the innate and adaptive immunity in APS., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Grossi, Capitani, Benagiano, Baldari, Della Bella, Macor, Tedesco, Borghi, Maugeri, D’Elios and Meroni.)

Antiphospholipid antibodies (aPL) are mandatory for the diagnosis but are also a risk factor for the antiphospholipid syndrome (APS) clinical manifestations. Lupus anticoagulant (LA), anticardiolipin (aCL), and anti-beta2 glycoprotein I (β 2 GPI) assays are the formal laboratory classification/diagnostic criteria. Additional nonclassification assays have been suggested; among them, antiphosphatidylserine-prothrombin (aPS/PT) and antidomain 1 β 2 GPI antibodies are the most promising ones although not yet formally accepted. aPL represent the example of a laboratory test that moved from dichotomous to quantitative results consistent with the idea that reporting quantitative data offers more diagnostic/prognostic information for both vascular and obstetric manifestations. Although the general rule is that the higher the aPL titer, the higher the test likelihood ratio, there is growing evidence that this is not the case for persistent low titers and obstetric events. LA displays the highest diagnostic/prognostic power, although some isolated LAs are apparently not associated with APS manifestations. Moreover, isotype characterization is also critical since IgG aPL are more diagnostic/prognostic than IgA or IgM. aPL are directed against two main autoantigens: β 2 GPI and PT. However, anti-β 2 GPI antibodies are more associated with the APS clinical spectrum. In addition, there is evidence that anti-β 2 GPI domain 1 antibodies display a stronger diagnostic/prognostic value. This finding supports the view that antigen and even epitope characterization represents a further step for improving the assay value. The strategy to improve aPL laboratory characterization is a lesson that can be translated to other autoantibody assays in order to improve our diagnostic and prognostic power., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Meroni and Borghi.)

Background: Critically ill patients with coronavirus disease 2019 (COVID-19) have a profound hypercoagulable state and often develop coagulopathy which leads to organ failure and death. Because of a prolonged activated partial-thromboplastin time (aPTT), a relationship with anti-phospholipid antibodies (aPLs) has been proposed, but results are controversial. Functional assays for aPL (i.e., lupus anticoagulant) can be influenced by concomitant anticoagulation and/or high levels of C reactive protein. The presence of anti-cardiolipin (aCL), anti-beta2-glycoprotein I (anti-β 2 GPI), and anti-phosphatidylserine/prothrombin (aPS/PT) antibodies was not investigated systematically. Epitope specificity of anti-β 2 GPI antibodies was not reported., Objective: To evaluate the prevalence and the clinical association of aPL in a large cohort of COVID-19 patients, and to characterize the epitope specificity of anti-β 2 GPI antibodies., Methods: ELISA and chemiluminescence assays were used to test 122 sera of patients suffering from severe COVID-19. Of them, 16 displayed major thrombotic events., Results: Anti-β 2 GPI IgG/IgA/IgM was the most frequent in 15.6/6.6/9.0% of patients, while aCL IgG/IgM was detected in 5.7/6.6% by ELISA. Comparable values were found by chemiluminescence. aPS/PT IgG/IgM were detectable in 2.5 and 9.8% by ELISA. No association between thrombosis and aPL was found. Reactivity against domain 1 and 4-5 of β 2 GPI was limited to 3/58 (5.2%) tested sera for each domain and did not correlate with aCL/anti-β 2 GPI nor with thrombosis., Conclusions: aPL show a low prevalence in COVID-19 patients and are not associated with major thrombotic events. aPL in COVID-19 patients are mainly directed against β 2 GPI but display an epitope specificity different from antibodies in antiphospholipid syndrome., (Copyright © 2020 Borghi, Beltagy, Garrafa, Curreli, Cecchini, Bodio, Grossi, Blengino, Tincani, Franceschini, Andreoli, Lazzaroni, Piantoni, Masneri, Crisafulli, Brugnoni, Muiesan, Salvetti, Parati, Torresani, Mahler, Heilbron, Pregnolato, Pengo, Tedesco, Pozzi and Meroni.)

In the present study, we evaluated two novel technologies, the chemiluminescent immunoassay (CIA) QUANTA Flash on BIO-FLASH (Inova Diagnostics, San Diego, CA, USA) and the addressable laser bead immunoassay (ALBIA) on BioPlex™ 2200 (Bio-Rad, Hercules, CA, USA) for the detection of anti-cardiolipin IgG/IgM (aCL) and anti-β2-glycoprotein IgG/IgM (aβ2GPI) antibodies. The study was performed on 134 samples from consecutive patients (59 males and 75 females, mean age 54 ± 10 years) who consulted a rheumatologist because thrombosis and/or pregnancy complications were present or another immunological disease (Sjogren's syndrome, inflammatory arthritis). Fourteen patients of the total fulfilled 25the Sydney criteria for APS and for these patients previous results of aPLs were available. Sera were tested for aCL and aβ2GPI of IgG and IgM isotypes using CIA (BIO-FLASH) and ALBIA (BioPlex™ 2200). Overall agreement between CIA and ALBIA ranged from 88.1% (aCL IgG) to 97.8% (aβ2GPI IgG). Cohen's kappa coefficient ranged from 0.53 to 0.91, implying moderate to almost perfect agreement. Almost perfect agreement was found between BioPlex™ 2200 and BIO-FLASH aβ2GPI IgG and aCL IgM with Cohen's kappa of 0.91 and 0.88, respectively. On the other hand, moderate agreement was found between BioPlex™ 2200 and BIO-FLASH aCL IgG and β2GPI IgM assays with Cohen's kappa of 0.57 and 0.53, respectively. The two novel technologies look promising and comparable but further studies with larger cohorts are needed to contribute to the better understanding of the new aPLs antibodies assays performance.

Systemic lupus erythematosus is frequently associated with antiphospholipid syndrome. Patients with lupus-antiphospholipid syndrome are characterized by recurrent arterial/venous thrombosis, miscarriages, and persistent presence of autoantibodies against phospholipid-binding proteins, such as β2-Glycoprotein I. We investigated the cytokine production induced by β2-Glycoprotein I in activated T cells that infiltrate in vivo atherosclerotic lesions of lupus-antiphospholipid syndrome patients. We examined the helper function of β2-Glycoprotein I-specific T cells for tissue factor production, as well as their cytolytic potential and their helper function for antibody production. Lupus-antiphospholipid syndrome patients harbor in vivo activated CD4 + T cells that recognize β2-Glycoprotein I in atherosclerotic lesions. β2-Glycoprotein I induces T-cell proliferation and expression of both Interleukin-17/Interleukin-21 and Interferon-γ in plaque-derived T-cell clones. β2-Glycoprotein I-specific T cells display strong help for monocyte tissue factor production, and promote antibody production in autologous B cells. Moreover, plaque-derived β2-Glycoprotein I-specific CD4 + T lymphocytes express both perforin-mediated and Fas/FasLigand-mediated-cytotoxicity. Altogether, our results indicate that β2-Glycoprotein I is able to elicit a local Interleukin-17/Interleukin-21 and Interferon-γ inflammation in lupus-antiphospholipid syndrome patients that might lead, if unabated, to plaque instability and subsequent arterial thrombosis, suggesting that the T helper 17/T helper 1 pathway may represent a novel target for the prevention and treatment of the disease., (Copyright© 2019 Ferrata Storti Foundation.)

Antiphospholipid syndrome (APS) is a chronic and disabling condition characterized by recurrent thrombosis and miscarriages mediated by antibodies against phospholipid-binding proteins (aPL), such as beta 2 glycoprotein I (β 2 GPI). Complement is involved in APS animal models and complement deposits have been documented in placenta and thrombotic vessels despite normal serum levels. Analysis of circulating blood cells coated with C4d displays higher sensitivity than the conventional assays that measure soluble native complement components and their unstable activation products in systemic lupus erythematosus (SLE). As C4d-coated blood cell count has been reported to be more sensitive than serum levels of complement components and their activation products in systemic lupus erythematosus (SLE) patients, we decided to evaluate the percentage of C4d positive B lymphocytes (BC4d), erythrocytes (EC4d), and platelets (PC4d) in primary APS patients and asymptomatic aPL positive carriers as marker of complement activation in APS. We assessed by flow cytometry the percentages of BC4d, EC4d, and PC4d in primary APS (PAPS; n. 23), 8 asymptomatic aPL positive carriers, 11 APS-associated SLE (SAPS), 17 aPL positive SLE, 16 aPL negative SLE, 8 aPL negative patients with previous thrombosis, 11 immune thrombocytopenia (ITP) patients, and 26 healthy subjects. In addition, we used an in vitro model to evaluate the ability of a monoclonal anti-β 2 GPI antibody (MBB2) to bind to normal resting or activated platelets and fix complement. EC4d and PC4d percentages were significantly higher in PAPS and aPL carriers as well as aPL positive SLE and SAPS than in aPL negative controls. The highest values were found in PAPS and in SAPS. The EC4d and PC4d percentages were significantly correlated with serum C3/C4 and anti-β 2 GPI/anti-cardiolipin IgG. In vitro studies showed that MBB2 bound to activated platelets only and induced C4d deposition. The detection of the activation product C4d on circulating erythrocytes and platelets supports the role of complement activation in APS. Complement may represent a new therapeutic target for better treatment and prevention of disability of APS patients.

Clinical studies have reported different diagnostic/predictive values of antibodies to domain 1 or 4/5 of β 2 glycoproteinI in terms of risk of thrombosis and pregnancy complications in patients with antiphospholipid syndrome. To obtain direct evidence for the pathogenic role of anti-domain 1 or anti-domain 4/5 antibodies, we analyzed the in vivo pro-coagulant effect of two groups of 5 sera IgG each reacting selectively with domain 1 or domain 5 in lipopolysaccharide (LPS)-treated rats. Antibody-induced thrombus formation in mesenteric vessels was followed by intravital microscopy, and vascular deposition of β 2 glycoproteinI, human IgG and C3 was analyzed by immunofluorescence. Five serum IgG with undetectable anti-β 2 glycoproteinI antibodies served as controls. All the anti-domain 1-positive IgG exhibited potent pro-coagulant activity while the anti-domain 5-positive and the negative control IgG failed to promote blood clot and vessel occlusion. A stronger granular deposit of IgG/C3 was found on the mesenteric endothelium of rats treated with anti-domain 1 antibodies, as opposed to a mild linear IgG staining and absence of C3 observed in rats receiving anti-domain 5 antibodies. Purified anti-domain 5 IgG, unlike anti-domain 1 IgG, did not recognize cardiolipin-bound β 2 glycoproteinI while being able to interact with fluid-phase β 2 glycoproteinI. These findings may explain the failure of anti-domain 5 antibodies to exhibit a thrombogenic effect in vivo , and the interaction of these antibodies with circulating β 2 glycoproteinI suggests their potential competitive role with the pro-coagulant activity of anti-domain 1 antibodies. These data aim at better defining "really at risk" patients for more appropriate treatments to avoid recurrences and disability., (Copyright© 2019 Ferrata Storti Foundation.)

The relapse rate in antiphospholipid syndrome (APS) remains high, i.e. around 20%-21% at 5 years in thrombotic APS and 20-28% in obstetrical APS [2, 3]. Hydroxychloroquine (HCQ) appears as an additional therapy, as it possesses immunomodulatory and anti-thrombotic various effects [4-16]. Our group recently obtained the orphan designation of HCQ in antiphospholipid syndrome by the European Medicine Agency. Furthermore, the leaders of the project made the proposal of an international project, HIBISCUS, about the use of Hydroxychloroquine in secondary prevention of obstetrical and thrombotic events in primary APS. This study has been launched in several countries and at now, 53 centers from 16 countries participate to this international trial. This trial consists in two parts: a retrospective and a prospective study. The French part of the trial in thrombosis has been granted by the French Minister of Health in December 2015 (the academic trial independent of the pharmaceutical industry PHRC N PAPIRUS) and is coordinated by one of the members of the leading consortium of HIBISCUS., (Copyright © 2018. Published by Elsevier B.V.)

Recurrent thrombosis and miscarriages are the main clinical manifestations of antiphospholipid syndrome (APS). Although most patients display both clinical signs, some patients can have isolated vascular or obstetric variants. Emerging data raise the question of whether obstetric and vascular APS are the same or different diseases. An important difference between the two conditions is that a thrombophilic state is a common feature in vascular APS, whereas clot occlusions of the decidual spiral arteries are seldom observed in obstetric APS, and infarctions are found in only one-third of APS placentae. Conversely, inflammation, which is undetectable in vascular APS, is frequently observed in the placentae of patients with obstetric APS and has been documented in the placentae of pregnant mice with fetal loss mediated by antiphospholipid antibodies. Attempts to identify different antibodies or epitopes responsible for the two clinical manifestations of APS have so far been unsuccessful. Possible mechanisms exist that might explain the development of the two clinical presentations, including the tissue distribution and expression level of the main target antigen of antiphospholipid antibodies, β2 glycoprotein I (β2GPI). The identification of the factors that promote the onset of either obstetric or vascular APS has important diagnostic and therapeutic implications.

Antibodies against β2 glycoprotein I (anti-β2GPI) have been identified as the main pathogenic autoantibody subset in anti-phospholipid syndrome (APS); the most relevant epitope is a cryptic and conformation-dependent structure on β2GPI domain (D) 1. Anti-β2GPI domain profiling has been investigated in thrombotic APS, leading to the identification of antibodies targeting D1 as the main subpopulation. In contrast, scarce attention has been paid to obstetric APS, hence this study aimed at characterizing the domain reactivity with regards to pregnancy morbidity (PM). To this end, 135 women with persistently positive, medium/high titre anti-β2GPI IgG, without any associated systemic autoimmune diseases and at least one previous pregnancy were included: 27 asymptomatic carriers; 53 women with obstetric APS; 20 women with thrombotic APS; and 35 women with both thrombotic and obstetric complications. Anti-D1 and anti-D4/5 antibodies were tested using a chemiluminescent immunoassay and a research ELISA assay, respectively (QUANTA Flash ® β2GPI Domain 1 IgG and QUANTA Lite ® β2GPI D4/5 IgG, Inova Diagnostics). Positivity for anti-D1 antibodies, but not anti-D4/5 antibodies, was differently distributed across the 4 subgroups of patients (p < 0.0001) and significantly correlated with thrombosis (χ2 = 17.28, p < 0.0001) and PM (χ2 = 4.28, p = 0.039). Patients with triple positivity for anti-phospholipid antibodies displayed higher anti-D1 titres and lower anti-D4/5 titres compared to women with one or two positive tests (p < 0.0001 and p = 0.005, respectively). Reactivity against D1 was identified as a predictor for PM (OR 2.4, 95% confidence interval [CI] 1.2-5.0, p = 0.017); in particular, anti-D1 antibodies were predictive of late PM, conveying an odds ratio of 7.3 (95% CI 2.1-25.5, p = 0.022). Positivity for anti-D1 antibodies was not associated with early pregnancy loss. Anti-D4/5 antibodies were not associated with clinical APS manifestations. As a whole, our data suggest that anti-D1 antibodies are significantly associated not only with thrombosis, but also with late PM, while positive anti-D4/5 antibodies are not predictive of thrombosis or PM., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Domain I (DI) of beta-2-glycoprotein I (β2GPI) contains the immunodominant epitope for pathogenic antiphospholipid antibodies (aPL). DI is exposed in the linear form of the molecule but not in the circular form that comprises 90% of serum β2GPI. The majority of circulating β2GPI is biochemically reduced with two free thiols in Domain V. However, increased levels of oxidised β2GPI are found in patients with antiphospholipid syndrome (APS). It is not known whether oxidation of β2GPI favours the linear form of the molecule and thus promotes development of anti-DI antibodies. We investigated whether the proportion of oxidised β2GPI associates with the presence of anti-DI in APS patients. Serum samples from 44 APS patients were screened for IgG, IgM and IgA anti-DI, anti-β2GPI, anti-cardiolipin (anti-CL) and biochemically reduced β2GPI. A negative correlation was found between the proportion of β2GPI in the biochemically reduced form and IgG anti-DI levels (r = -0.54, p = 0.0002), but not with IgM or IgA anti-DI. Moreover, the proportion of β2GPI in the reduced form was lower in IgG anti-DI positive than anti-DI negative APS patients (p = 0.02). The relative amount of reduced β2GPI was no different between patients who were positive or negative for IgG, IgM and IgA anti-β2GPI or anti-CL. This study demonstrates that oxidised β2GPI lacking free cysteine-thiol groups most closely associates with IgG anti-DI positivity compared to IgG anti-CL and anti-β2GPI. Future studies are required to ascertain the directionality of this association to define causation.

Antiphospholipid syndrome (APS) is characterized by recurrent arterial/venous thrombosis and miscarriages in the persistent presence of autoantibodies against phospholipid-binding proteins (aPLs), such as β2 glycoprotein I (β2GPI). In addition to the aPL thrombophilic effect, arterial thrombosis was related to accelerated atherosclerosis in animal models; however, contrasting findings were reported in primary APS patients with regard to the increased number of plaques or abnormal arterial wall thickness. We investigated the cytokine production induced by β2GPI in activated T cells that infiltrate in vivo atherosclerotic lesions of primary APS patients with atherothrombosis. We also examined the helper function of β2GPI-specific T cells for monocyte matrix metalloproteinase-9 and tissue factor production, as well as their cytolytic potential and their helper function for Ab production. APS patients with atherothrombosis harbor in vivo-activated CD4 + T cells that recognize β2GPI in atherothrombotic lesions. β2GPI induces T cell proliferation and IFN-γ expression in plaque-derived T cell clones. β2GPI-specific T cells display helper function for monocyte matrix metalloproteinase-9 and tissue factor production and promote Ig production in autologous B cells. Moreover, plaque-derived β2GPI-specific CD4 + T lymphocytes express perforin-mediated and Fas/Fas ligand-mediated cytotoxicity. β2GPI, and especially the DI domain, drive a local Th1 inflammatory response, with subsequent plaque instability that eventually favors atherothrombosis. This finding may explain the association between aPLs and arterial thrombosis, despite the lack of evidence of surrogate markers for atherosclerosis in primary APS., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Background: Antiphospholipid antibodies (aPL) can be detected in asymptomatic carriers and infectious patients. The aim was to investigate whether a novel line immunoassay (LIA) differentiates between antiphospholipid syndrome (APS) and asymptomatic aPL+ carriers or patients with infectious diseases (infectious diseases controls (IDC))., Methods: Sixty-one patients with APS (56 primary, 22/56 with obstetric events only, and 5 secondary), 146 controls including 24 aPL+ asymptomatic carriers and 73 IDC were tested on a novel hydrophobic solid phase coated with cardiolipin (CL), phosphatic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, beta2-glycoprotein I (β2GPI), prothrombin, and annexin V. Samples were also tested by anti-CL and anti-β2GPI ELISAs and for lupus anticoagulant activity. Human monoclonal antibodies (humoAbs) against human β2GPI or PL alone were tested on the same LIA substrates in the absence or presence of human serum, purified human β2GPI or after CL-micelle absorption., Results: Comparison of LIA with the aPL-classification assays revealed good agreement for IgG/IgM aß2GPI and aCL. Anti-CL and anti-ß2GPI IgG/IgM reactivity assessed by LIA was significantly higher in patients with APS versus healthy controls and IDCs, as detected by ELISA. IgG binding to CL and ß2GPI in the LIA was significantly lower in aPL+ carriers and Venereal Disease Research Laboratory test (VDRL) + samples than in patients with APS. HumoAb against domain 1 recognized β2GPI bound to the LIA-matrix and in anionic phospholipid (PL) complexes. Absorption with CL micelles abolished the reactivity of a PL-specific humoAb but did not affect the binding of anti-β2GPI humoAbs., Conclusions: The LIA and ELISA have good agreement in detecting aPL in APS, but the LIA differentiates patients with APS from infectious patients and asymptomatic carriers, likely through the exposure of domain 1.

Purpose of Review: Many advancements in our understanding of the pathogenic mechanisms of the antiphospholipid syndrome (APS) have been accomplished over the recent months. Such progresses are paralleled by the development of innovative pharmacological tools that could provide novel therapeutic windows in APS management. The most recent and innovative findings about the biologic effects of antiphospholipid antibodies (aPLs) and the treatment APS will be hereby critically appraised., Recent Findings: Antibodies against the domain I of β2 glycoprotein I (β2GPI) are increasingly recognized as the main pathogenic subset; pioneer therapeutic options exploiting the pathogenicity of anti-domain I antibodies have been developed. AnnexinA2 and toll-like receptor (TLR)4 have been identified as the main receptors for β2GPI/anti-β2GPI antibodies on target cells; additional co-receptors might include TLR1, TLR2 and TLR6. Upon binding, aPLs engage intracellular mediators as nuclear factor kappa B and mammalian target of rapamycin, which provide potential therapeutic targets. Current innovative treatment options include novel oral anticoagulants and the complement inhibitor eculizumab. The addition to standard treatment of pleiotropic agents such as hydroxychloroquine, statins and vitamin D could allow better disease control., Summary: The lively and intense research in the APS field opens new frontiers in aPL pathogenic mechanisms, as well as diagnosis and treatment of the syndrome., Video Abstract: http://links.lww.com/COR/A26.

Objective: It has been suggested that only antibodies against domain 1 (D1) of β2 -glycoprotein I (β2 GPI) are pathogenic and diagnostic. The role of antibodies against other β2 GPI domains is still debated. This study was undertaken to evaluate the clinical relevance of domain specificity profiling of anti-β2 GPI IgG antibodies in antiphospholipid syndrome (APS) patients and in control groups of patients with systemic autoimmune rheumatic diseases and in asymptomatic antiphospholipid antibody (aPL) carriers., Methods: We evaluated 159 subjects with persistently positive, medium or high-titer anti-β2 GPI IgG, including 56 patients with thrombotic (obstetric or nonobstetric) primary APS, 31 women with obstetric primary APS, 42 aPL-positive patients with systemic autoimmune rheumatic diseases, and 30 asymptomatic aPL carriers. One hundred healthy donors were included. Anti-β2 GPI D1 and D4/5 IgG were tested on research enzyme-linked immunosorbent assays containing recombinant β2 GPI domains., Results: As compared to other groups, aPL carriers displayed higher frequency/titer of anti-D4/5 IgG. Unlike anti-D4/5, anti-D1 IgG antibodies were more frequent and at higher titer in triple than in single or double aPL-positive subjects. An anti-D1 to anti-D4/5 ratio of ≥1.5 was predictive of systemic autoimmunity (odds ratio 3.25 [95% confidence interval 1.45-7.49], P = 0.005). Neither anti-D1 nor anti-D4/5 antibodies were associated with APS clinical criteria., Conclusion: Anti-D1 IgG is the preferential specificity not only in vascular and obstetric primary APS, but also in patients with systemic autoimmune rheumatic disease with no clinical features of APS. Conversely, aPL carriers do not have a polarized profile toward D1. Combined testing for anti-β2 GPI IgG with different domain specificity allows a more accurate aPL profiling, with polarization toward anti-D1 IgG as a possible fingerprint of systemic autoimmunity., (© 2015, American College of Rheumatology.)

The international consensus for the classification of antiphospholipid syndrome (APS) requires clinical and laboratory criteria to be considered at an equal level for diagnosing APS. Thus, detection of antiphospholipid antibodies (aPL) being a hallmark of APS has been the object of intensive investigation over the past 40 years. However, appropriate detection of aPL still remains a laboratory challenge due to their heterogeneity comprising autoantibodies reactive to different phospholipid-binding plasma proteins, such as beta-2 glycoprotein I (β2GPI) and prothrombin. The relevance of aPL interacting with phospholipids other than cardiolipin (CL, diphosphatidylglycerol), such as phosphatidylserine (PS), remains elusive with regard to the diagnosis of APS. Recently, the concept of aPL profiling has been introduced to assess the risk of thrombotic complications in patients with APS. New assay techniques, apart from enzyme-linked immunosorbent assays (ELISAs) recommended by the international consensus for the classification of APS, have been proposed for multiplexing of aPL testing. Line immunoassays (LIAs) employing a novel hydrophobic solid phase for the simultaneous detection of different aPL seem to be an intriguing alternative. We evaluated a novel multiplex LIA employing a hydrophobic membrane coated with different phospholipid (PL)-binding proteins or PLs. The performance characteristics of this new multiplexing assay technique demonstrated its usefulness for aPL profiling., (© The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.)

A single-chain fragment variable (scFv) recognizing β2-glycoprotein 1 (β2GPI) from humans and other species was isolated from a human phage display library and engineered to contain an IgG1 hinge-CH2-CH3 domain. The scFv-Fc directed against β2GPI domain I-induced thrombosis and fetal loss, thus mimicking the effect of antibodies from patients with antiphospholipid syndrome (APS). Complement is involved in the biological effect of anti-β2GPI scFv-Fc, as demonstrated by its ability to promote in vitro and in vivo complement deposition and the failure to induce vascular thrombosis in C6-deficient rats and fetal loss in C5-depleted mice. A critical role for complement was also supported by the inability of the CH2-deleted scFv-Fc to cause vessel occlusion and pregnancy failure. This antibody prevented the pathological effects of anti-β2GPI antibodies from APS patients and displaced β2GPI-bound patient antibodies. The CH2-deleted antibody represents an innovative approach potentially useful to treat APS patients refractory to standard therapy., (© 2014 by The American Society of Hematology.)

Among the diagnostic assays for anti-phospholipid syndrome (APS), lupus anticoagulant (LA) is the strongest predictor of thrombosis; however, it presents several limitations as interference with anticoagulant therapy and poor inter-laboratory agreement. Two-thirds of LA activity is apparently due to antibodies against prothrombin (PT), usually detectable by ELISA. Binding of PT to phosphatidylserine (PS) has been shown to enhance solid-phase anti-PT assay sensitivity. To determine the prevalence of antibodies against PS/PT (aPS/PT) in APS, we tested the semiquantitative QUANTA Lite(®) aPS/PT ELISA in a cohort of 80 APS patients. The prevalence of aPS/PT was 81.3%, rising to 87.6% when considering LA-positive subjects only. We observed a strong correlation between aPS/PT and LA (p = 0.006). To note, APS patients with thrombotic manifestations displayed significantly higher IgG aPS/PT titers compared to 20 aPL asymptomatic carriers (p = 0.012). To rule out a possible cross-reactivity of anti-β2 glycoprotein I antibodies (aβ2GPI) with PS/PT complex, we tested two monoclonal aβ2GPI antibodies and an affinity-purified (AP) polyclonal aβ2GPI IgG obtained from the serum of a patient reacting against both β2GPI and PS/PT. The two monoclonal antibodies did not show any reactivity against PS/PT complex, similarly the AP IgGs did not react toward PS/PT antigen while preserved their aβ2GPI activity. Our findings suggest that aPS/PT are a definite antibody population in APS. Moreover, the good correlation between aPS/PT ELISA and LA may support its use as a surrogate test for LA, particularly useful to overcome the technical limitations of the functional assay.

Beta2 glycoprotein I (β2GPI)-dependent antiphospholipid antibodies (aPLs) are the main pathogenic autoantibody population and at the same time the laboratory diagnostic tool for the antiphospholipid syndrome (APS). These antibodies are responsible for both the vascular and the obstetric manifestations of the syndrome but the pathogenic mechanisms behind these manifestations are not the same. For example, thrombotic events do not appear to play a major role in APS miscarriages and a direct reactivity of β2GPI-dependent aPLs on decidual and trophoblast cells was reported. A local expression of β2GPI on these tissues was reported both in physiological conditions and in APS women, thus explaining the local tropism of the autoantibodies. The two hit hypothesis was suggested to explain why the vascular manifestations of APS may occur only occasionally in spite of the persistent presence of aPLs. This is not apparently the case for the obstetric variant of the syndrome, making the difference even more striking. A different pathogenesis may also provide the rationale for the well-known fact that the vascular and the obstetric manifestations may occur independently although in a minority of cases.

Antiphospholipid antibodies (aPL) are both diagnostic markers for, and pathogenic drivers of, antiphospholipid syndrome (APS). Although the presence of aPL is a necessary pre-condition, APS-associated clotting is seemingly triggered by an additional 'second hit', frequently related to innate inflammatory immune responses. β(2) glycoprotein I (β(2)GPI)-dependent aPL, the most important subset of these antibodies, mediate several--not necessarily alternative--thrombogenic mechanisms, mainly on the basis of their reactivity with β(2)GPI expressed on the membrane of cells that participate in the coagulation cascade. Recurrent pregnancy complications associated with aPL cannot be explained solely by thrombosis, and alternative pathogenic mechanisms have been reported. Although one in vivo model of fetal loss suggests a mechanism of aPL-mediated acute placental inflammation, other models and the histopathological examination of APS placentae do not support a widespread inflammatory signature. β(2)GPI-dependent aPL are thought to recognize their antigen on placental tissues, inhibit the growth and differentiation of trophoblasts, and eventually cause defective placentation. Why antibodies with similar antigen specificity produce different clinical manifestations is not clear. Characterization of the molecular basis of the pathogenic mechanisms involved, including the putative second hits and the role of complement activation, might offer an answer to this question.

Objective: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) plays a role in blastocyst implantation and is down-regulated in preeclampsia and in hypertensive pregnancy disorders associated with defective extravillous trophoblast invasion. Defective placentation and severe preeclampsia are also features of the antiphospholipid syndrome (APS). The purpose of this study was to investigate whether abnormal HB-EGF expression plays a pathogenic role in antiphospholipid antibody (aPL)-mediated defective placentation., Methods: HB-EGF expression in placental tissue was evaluated by Western blotting and messenger RNA analysis in normal and APS placentae. Polyclonal IgG fractions or monoclonal beta(2)-glycoprotein I-dependent aPL and their respective controls were investigated for the following 4 features: their binding to human trophoblast monolayers, as determined by cell enzyme-linked immunosorbent assay (ELISA); their effect on HB-EGF expression by Western blotting in trophoblast cell extracts as well as by ELISA as a protein secreted in the culture supernatants; their inhibitory effect on in vitro trophoblast invasiveness, as evaluated by Matrigel assay; and their inhibitory effect on matrix metalloproteinase (MMP) levels, as measured by gelatin zymography. Experiments were also performed in the presence of serial concentrations of heparin or recombinant HB-EGF., Results: Placental APS tissue displayed reduced expression of HB-EGF. Polyclonal and monoclonal aPL bound to trophoblast monolayers and significantly reduced the in vitro synthesis and secretion of HB-EGF. Heparin inhibited aPL binding and restored HB-EGF expression in a dose-dependent manner. Addition of recombinant HB-EGF reduced the in vitro aPL-induced inhibition of Matrigel invasiveness as well as MMP-2 levels., Conclusion: These preliminary findings suggest that the reduction of aPL-mediated HB-EGF represents an additional mechanism that is responsible for the defective placentation associated with APS and that heparin protects from aPL-induced damage by inhibiting antibody binding.

Background: There is strong evidence that antiphospholipid antibodies (aPL) perturb endothelium both in vitro and in experimental animal models. by inducing a vasculopathy and an endothelial pro-inflammatory/coagulant phenotype. However, few contrasting studies raised the issue about the possibility to detect a comparable endothelial perturbation in anti-phospholipid syndrome (APS) patients. The aim of this observational case-control study was to evaluate several parameters of endothelial perturbation in patients with APS and without any other atherosclerosis risk factor., Patients and Methods: We investigated plasma levels of soluble adhesion molecules (s-ICAM-1, s-VCAM-1, s-E-selectin), soluble thrombomodulin (sTM), von Willebrand factor (vWF) and tissue plasminogen activator (t-PA) by solid-phase assays in 40 selected APS patients and 40 age- and sex-matched healthy subjects. In addition, we evaluated circulating endothelial cells by flow cytometry and brachial artery flow-mediated vasodilation. Patients and controls were free of conditions known to affect both the biological and the functional endothelial parameters., Results: Plasma levels of sTM, s-E-selectin and s-VCAM-1 did not differ from controls, while a significant increase in s-ICAM-1 (P = 0.029), t-PA (P = 0.003) and vWF titres (P = 0.002) was found. Circulating mature endothelial cells were also significantly higher in patients than in controls (P = 0.05) and decreased during both vitamin K antagonists (P = 0.001) and antiplatelet (P = 0.032) treatments. Mean brachial artery flow-mediated vasodilation responses were significantly impaired compared to healthy subjects (P = 0.0001)., Conclusions: As a whole these findings indicate that APS patients display an endothelial perturbation in the absence of other detectable traditional risk factors for atherosclerosis., (Copyright 2009 Elsevier Ltd. All rights reserved.)

The research projects of the European Forum on Antiphospholipid Antibodies are representative of how dynamic is this area of investigation. The present review is focused on the most recent projects of the Forum on the aetiopathogenic aspects of the antiphospholipid syndrome (APS). Studies on the genetic background of the APS are ongoing in order to better define the proximity between APS and full-blown systemic lupus erythematosus. However, the analysis of the polymorphisms of genes coding for inflammatory mediators may offer new information on the role of inflammatory processes in triggering thrombotic events as well as the whole susceptibility for developing the vascular manifestations. A systematic and wide detection of serological markers of infectious processes will give new insight on the role of infectious agents in favouring autoimmunity in APS. Owing to the well-known role of vitamin D(3) defect in autoimmune disease, the detection of vitamin plasma levels in APS patients will offer the rationale for a possible therapeutic supplementation. Additional projects are aimed to better characterize the diagnostic/prognostic value of antiphospholipid antibodies (aPL) by defining their epitope specificity and binding avidity. Pregnancy complications represent the obstetric side of APS. Research projects are focussed on the role of complement activation in placenta damage and on the potential ability of aPL to affect the fertility. Finally, a study has been planned in order to draw definitive conclusions on the associations between aPL and atherosclerosis.

Objective: Primary antiphospholipid syndrome (APS) is formally classified by the presence of antiphospholipid antibodies, recurrent thrombosis, and/or pregnancy morbidity in the absence of any underlying full-blown systemic autoimmune disease. However, systemic manifestations in patients with primary APS have been recently reported, as has the presence of serologic markers in common with systemic lupus erythematosus (SLE). In spite of similarities between the 2 diseases, only a minority of cases of primary APS evolve into full-blown SLE, even after a long followup period. The aim of this study was to investigate whether the analysis of SLE susceptibility genes may provide at least a partial explanation for such a discrepancy., Methods: One hundred thirty-three patients with primary APS classified according to the Sydney criteria and 468 healthy control subjects from the same geographic area were recruited. We genotyped 3 single-nucleotide polymorphisms (SNPs) in IRF5 (rs2004640, rs2070197, and rs10954213), 4 SNPs in STAT4 (rs1467199, rs3821236, rs3024866, and rs7574865), 2 SNPs in BANK1 (rs10516487 and rs3733197), and 1 SNP in BLK (rs2736340)., Results: STAT4 and BLK displayed a strong genetic association with primary APS (for rs7574865, odds ratio [OR] 2.19, P=5.17x10(-7); for rs2736340, OR 2.06, P=1.78x10(-6)), while a weak association with IRF5 and no association with BANK1 were observed., Conclusion: The presence of a strong genetic association with only a few SLE susceptibility genes and the absence of a more complex gene association may contribute to the lack of cases of full-blown SLE developing in patients with primary APS, in spite of the clinical and serologic similarities between SLE and primary APS.

Objective: Inflammation might represent a second hit for anti-phospholipid antibody (aPL)-mediated thrombosis. Inflammatory responses have been linked to gene polymorphisms of several cytokines and Toll Like Receptors (TLRs). We examined IL1 beta, TNFalpha, TGFbeta, IL6, IFN gamma, IL10, tlr4 gene polymorphisms in a family with several members positive for IgG anti-beta2 glycoprotein I (beta 2GPI) antibodies but with recurrent thrombosis in one member only., Methods: Lupus anticoagulant, anti-cardiolipin, anti-beta 2GPI IgG/IgM antibodies, IL1beta, TNFalpha, TGF beta1, IL6, IL10, IFN gamma, tlr4 gene polymorphisms (by allele-specific polymerase chain reaction) in addition to standard thrombophilic risk factors and cytokine serum levels (IL-1 beta, TNFalpha, IL-10) were evaluated., Results: Recurrent thrombotic events was reported only in the proband, but not in three healthy siblings persistently positive for IgG anti-beta2GPI antibodies, respectively. The wild type tlr4 gene and cytokine polymorphisms associated with a high pro-inflammatory response (IL-1 beta promoter-511C/T; TNFalpha G/A; TGFbeta+10T/C, +25C/G; IL-6 -174C/G) were found only in the proband. Serum cytokine levels were normal., Conclusion: This case report confirms that protective tlr4 gene polymorphisms are more frequent in asymptomatic aPL carriers. In line with the role of inflammatory mediators as second hits for aPL-associated thrombosis, the polymorphisms of cytokines linked to higher inflammatory response were found in the proband only.

The anti-phospholipid syndrome (APS) is a systemic autoimmune disease characterized by an adaptive immune response against self phospholipid (PL)--binding proteins. Although APS is considered as an autoantibody-mediated disease, there is now evidence that anti-phospholipid antibodies (aPL) are necessary but not sufficient to trigger some of the clinical manifestations of the syndrome. For example, mediators of the innate immunity are recognized to be additional second hits able to induce the thrombotic events in the presence of aPL. Finally, environmental agents - in particular infectious ones - were reported to act as triggers for the production of autoantibodies cross-reacting with PL-binding proteins as well as inflammatory stimuli that potentiate the aPL thrombogenic effect. Altogether, these findings suggest a role for the innate immunity in APS pathogenesis. Toll-like receptors (TLR) are receptors that induce prompt inflammatory responses and mediate functional activation in immune effector cells. There is evidence that aPL, and in particular anti-beta(2) glycoprotein I (beta(2)GPI) antibodies, may activate endothelial cells and monocytes through TLR-4-dependent signalling. Whether or not TLR may behave as surface receptors for beta(2)GPI is still matter of research. Drugs or molecules able to interfere with TLR involvement may represent new therapeutic approaches for APS.

Anti-phospholipid antibodies (aPL) are risk factor for recurrent pregnancy loss and obstetrical complications. The mechanisms of aPL-mediated pregnancy failure are still a matter of research. Although aPL are associated with thrombosis, thrombotic events cannot explain all the miscarriages. There is evidence for a direct in vitro aPL effect on the trophoblast as shown by their binding; reduction of proliferation, human chorionic gonadotrophin release, in vitro invasiveness, adhesion molecule expression; and increased apoptosis. Such a direct reactivity is supported by the expression of beta2 glycoprotein (beta 2GP) I on trophoblast cell membranes. aPL/anti-beta 2GPI antibodies also bind to human decidual/endometrial cells in vitro and induce a pro-inflammatory phenotype. APL-mediated inflammatory processes at the placental level are apparently responsible for fetal loss at least in animal models. Both complement activation and pro-inflammatory cytokine/chemokine secretion have been shown to play a role. More recently, complement-induced tissue factor expression on infiltrating neutrophils was described as an additional pathogenic mechanisms mediated by aPL. As a whole, these findings do suggest that aPL may induce a defective placentation by acting at different levels without involving necessarily thrombotic events.

Antiphospholipid antibodies (Abs) are associated with thrombosis and are a risk factor for recurrent pregnancy loss and obstetric complications in patients with the antiphospholipid syndrome. It is generally accepted that the major autoantigen for aPL Abs is beta (2) glycoprotein I, which mediates the binding of aPL Abs to target cells (i.e., endothelial cells, monocytes, platelets, trophoblasts, etc.) leading to thrombosis and fetal loss. This article addresses molecular events triggered by aPL Abs on endothelial cells, platelets, and monocytes and complement activation, as well as a review of the current knowledge with regard to the putative receptor(s) recognized by aPL Abs on target cells as well as novel mechanisms that involve fibrinolytic processes. A section is devoted to the description of thrombotic and inflammatory processes that lead to obstetric complications mediated by aPL Abs. Based on experimental evidence using in vitro and in vivo models, new targeted therapies for treatment and/or prevention of thrombosis and pregnancy loss in antiphospholipid syndrome are proposed.

Objective: A study was undertaken to investigate the in vivo pathogenic role of Toll-like receptor 4 (TLR-4) in the antiphospholipid syndrome (APS) by studying the thrombogenic antiphospholipid (aPL) activity in lipopolysaccharide (LPS) non-responsive (LPS-/-) mice and the association between tlr4 gene polymorphisms and APS in patients., Methods: IgGs from two patients with APS, one with aPL negative systemic lupus erythematosus (SLE) and one with normal human serum (NHS), were evaluated for thrombosis, tissue factor (TF) activity and endothelial cell activation in LPS-/- mice displaying a tlr4 spontaneous mutation vs LPS responsive (LPS+/+) mice. Human tlr4 Asp299Gly and Thr399Ile polymorphisms were evaluated by allele-specific PCR in 110 patients with APS with arterial/venous thrombosis and in 220 controls of the same ethnic origin., Results: IgG-APS produced significantly larger thrombi and more leucocytes (WBC) adhering to endothelial cells in the cremaster muscle microcirculation of LPS+/+ mice than IgG-NHS or aPL negative SLE-IgG. These effects were abrogated after absorption of the anti-beta(2)glycoprotein I activity by an affinity column. The two IgG-APS induced significantly smaller thrombi and fewer WBC adhering to endothelial cells in LPS-/- mice than in LPS+/+ mice. IgG-APS induced higher TF activity in carotid artery homogenates of LPS+/+ mice than in LPS-/- mice. The prevalence of Asp299Gly and Thr399Ile tlr4 polymorphisms was significantly lower than in controls., Conclusions: These findings in LPS-/- mice and the reduction in the "protective" polymorphism in patients with APS with thrombosis suggest that TLR-4 is involved in the interaction of aPL with endothelial cells in vivo.

Antiphospholipid syndrome (APS) is characterized by the presence of recurrent venous/ arterial thrombosis and fetal losses associated with a family of auto-antibodies directed against phospholipid (PL)-binding proteins. Among them, beta2 glycoprotein I (beta2GPI) is the most important. As a plasma cationic protein, beta2GPI binds to anionic PLs involved in several fluid-phase coagulation steps, and more importantly, it can be expressed on the surface of different cell types. Anti-beta2GPI antibodies recognize the molecule expressed on endothelial cells, platelets, monocytes, and trophoblast cells. Once bound, the antibodies trigger in vitro cell signaling that modulates biological responses potentially responsible for pathogenic mechanisms. Experimental animal models have supported the in vivo pathogenic role of anti-beta2GPI antibodies in both thrombosis and fetal loss models.

Antiphospholipid antibodies are mainly directed against beta 2 glycoprotein I (beta2GPI), a plasma phospholipid-binding protein expressed on endothelial cells of different anatomical localizations. Anti-beta2GPI antibodies recognize the molecule on endothelial monolayers in vitro, and, once bound, might activate the cells both in vitro and in vivo experimental models inducing a proinflammatory and a procoagulant phenotype. Cell activation is associated with nuclear factor-kappaB (NF-kappaB) translocation and with a signaling cascade comparable to that triggered by the toll-like receptors (TLRs)-4. The cell membrane receptor(s) for beta2GPI adhesion is still under investigation. It has been suggested that beta2GPI might adhere through electrostatic interaction between its cationic phospholipid binding site and anionic structures on the cell membrane; however, binding to annexin II-the endothelial cell receptor for tissue plasminogen activator-plays also a role. Because annexin II does not display any transmembrane protein, it has been suggested that it requires a yet unknown "adaptor" protein to signal the cells. Because of the molecular mimicry between beta2GPI and viral/bacterial structures-the natural ligands for TLRs-antibodies might cross-link the molecule associated to annexin II and TLR-4 eventually triggering the signaling.

Anti-phospholipid syndrome is an autoimmune systemic disease characterized by the persistent presence of anti-phospholipid antibodies and by the occurrence of thrombosis, fetal loss and thrombocytopenia. Anti-phospholipid antibodies are widely accepted as pathogenic antibodies mainly directed against the phospholipid-binding protein beta 2 glycoprotein I. Beta 2 glycoprotein I can be expressed on the endothelial cell membranes of different anatomical localizations and recognized by the autoantibodies. The antibody binding might induce an endothelial activation both in vitro and in vivo experimental models, that was suggested to represent one of the pathogenic mechanisms leading to the prothrombotic state of the syndrome. Beta 2 glycoprotein I endothelial adhesion was found to take place through the interaction of the cationic phospholipid binding site of the molecule with anionic endothelial structures and through annexin II, the endothelial cell receptor for tissue plasminogen activator. Anti-beta 2 glycoprotein I antibodies can directly activate the cells via NF-kB translocation and the signaling cascade triggered by toll like receptors. It has been suggested that beta 2 glycoprotein I might be associated with toll like receptors because of its molecular mimicry with bacterial structures, the natural ligands of toll like receptors. The binding of the antibodies is thought to cross-link beta 2 glycoprotein I and the toll like receptors, eventually switching their signaling pathway.

Beta2-glycoprotein I (beta2GPI) is a cofactor for anti-phospholipid (aPL) binding to cardiolipin (CL)-coated plates. Beta2GPI is also able to bind to endothelial cell (EC) membranes as supported by in-vivo as well as by in-vitro studies. The PL-binding site in the fifth domain of the molecule is involved in the adhesion to endothelium. Actually, specific mutations in this molecular portion abolish endothelium binding and a synthetic peptide spanning the sequence Glu274-Cys288 of the CL-binding site displays comparable adhesion to EC monolayers. Heparan sulphate appears to be one of the anionic EC membrane structures with which cationic beta2GPI interacts, as supported by studies with heparitinase-treated EC. Beta2GPI binding to EC might be related to its activity as endothelial growth factor or as a lipid-carrying glycoprotein. Adhesion of beta2GPI to endothelial membranes offers suitable epitopes for circulating aPL that, once bound, can induce cell activation

Objective: To investigate the role of antibodies reacting with beta 2 glycoprotein I (beta 2GPI) in the antiendothelial cell binding activity present in sera from patients with the anti-phospholipid syndrome., Methods: Sera positive for anti-phospholipid, anti-endothelial and anti-beta 2 GPI antibodies were studied for their binding activity on endothelial monolayers cultured in the presence or absence of media containing bovine serum as a source of beta 2GPI. Anti-endothelial activity was also evaluated on endothelial cells cultured without serum and supplemented with exogenous human purified beta 2GPI. Affinity purified anti-beta 2 GPI antibodies were investigated under the same experimental conditions. Finally, the effect of the incubation of these affinity purified fractions on the expression of adhesion molecules (ELAM-1) was studied., Results: The reactivity of the sera decreased on endothelial cells incubated in serum-free medium, while endothelial cell binding was restored in a dose dependent manner after the addition of exogenous purified human beta 2 GPI. Affinity purified anti-beta 2 GPI antibodies obtained from the same sera retained their endothelial cell binding and were able to activate endothelial cells by inducing the ex novo surface expression of adhesion molecules (ELAM-1)., Conclusions: These findings indicate that the close association between anti-endothelial and anti-phospholipid antibodies is sustained by antibodies which recognize beta 2 GPI adhering to the endothelial cells, and can promote their activation.

Antiphospholipid syndrome (APS) is a systemic autoimmune disease characterized by thrombotic events and/or pregnancy morbidity in the persistent presence of antiphospholipid antibodies (aPL). aPL targeting β2 glycoprotein I (anti-β2GPI Abs) provide the main pathogenic autoantibody subset. In monocytes, platelets and endothelial cells (ECs), the interaction of circulating aPL with membrane-bound β2GPI results in cell activation, and EC perturbation provides an important player in clotting. Several receptors have been suggested to mediate β2GPI/EC binding. AnnexinA2 provides a high-affinity binding site for β2GPI but, since it does not span the cell membrane, an adaptor protein is required to trigger signal. Consistent evidence supports the role of Toll-like receptor (TLR) 4 as co-receptor for β2GPI on ECs. β2GPI was recently reported to behave as lipopolysaccharide (LPS) scavenger. In monocytes, TLR4 activation was shown to be apparent, due to LPS/β2GPI complexes. Conversely, our findings in ECs demonstrate that β2GPI interacts directly with TLR4, and that such interaction may contribute to β2GPI-dependent aPL-mediated EC activation. LPS enhanced anti-β2GPI Ab binding to EC only at cell-activating concentrations, able to up-regulate TLR4. This in vitro model may explain why LPS behaves as a second hit increasing the expression of β2GPI in vascular tissues and triggering aPL-mediated thrombosis in vivo. [ABSTRACT FROM PUBLISHER]