T Cell Proliferative Responses and IgG Antibodies to β2GPI in Patients with Diabetes and Atherosclerosis.
Monjezi Mohammad R,Fouladseresht Hamed,Farjadian Shirin,Gharesi-Fard Behrouz,Khosropanah Shahdad,Doroudchi Mehrnoosh
Endocrine, metabolic & immune disorders drug targets
BACKGROUND:Diabetes increases the risk of myocardial infarction (MI) by 2 to 3 folds. Tlymphocytes play a role in atherosclerosis, which is the main pathology behind MI. Cellular immune responses to beta-2 glycoprotein I (β2GPI) are shown in carotid atherosclerosis. OBJECTIVE:To investigate the self-reactive, β2GPI-specific T-lymphocytes in patients with and without diabetes and atherosclerosis. METHODS:Collectively, 164 subjects with and without diabetes that underwent coronary angiography were divided into four groups based on their diabetes status and coronary stenosis. Group I=Diabetic with ≥50% stenosis: A+D+ (n=66); Group II=Non-diabetic with ≥50% stenosis, A+D- (n=39); Group III=Diabetic with <50% stenosis: A-D+ (n=28); and Group IV=Non-diabetic with <50% stenosis: AD- (n=31). All groups were evaluated for anti-β2GPI IgG antibody by ELISA method. Then, PBMCs were isolated from 18 subjects and were stimulated with β2GPI-derived peptides to assess their proliferation in accordance with their HLA-DRB1 alleles. RESULTS:Mean β2GPI IgG levels were higher in groups with ≥50% stenosis (A+) compared to those with <50% stenosis (A-), (P=0.02). The co-presence of diabetes in A+ individuals increased mean β2GPI-specific IgG. Auto-reactive β2GPI-specific T cells were detected in the repertoire of T-lymphocytes in all groups. β2GPI-peptides showed promiscuous restriction by various HLADRB1. CONCLUSION:β2GPI is the target of cellular and humoral immune responses in patients with atherosclerosis. Since the T cell responses but not antibodies were detectable in A-D+ and A-D- groups, it is reasonable to assume that cellular responses preceded the humoral responses. Post-translation modifications of β2GPI under oxidative and glycemic stresses may have increased the IgG levels in patients with diabetes. Finally, identification of antigens that trigger immuno-pathogenesis in atherosclerosis and diabetes may help the development of immunomodulation methods to prevent or treat these debilitating diseases.
Beta-2-Glycoprotein-I Deficiency Could Precipitate an Antiphospholipid Syndrome-like Prothrombotic Situation in Patients With Coronavirus Disease 2019.
Serrano Manuel,Espinosa Gerard,Lalueza Antonio,Bravo-Gallego Luz Yadira,Diaz-Simón Raquel,Garcinuño Sara,Gil-Etayo Javier,Moises Jorge,Naranjo Laura,Prieto-González Sergio,Ruiz-Ortiz Estibaliz,Sánchez Beatriz,Moreno-Castaño Ana Belen,Díaz-Pedroche Carmen,Viñas-Gomis Odette,Cervera Ricard,Serrano Antonio,
ACR open rheumatology
OBJECTIVE:Patients with coronavirus disease 2019 (COVID-19) present coagulation abnormalities and thromboembolic events that resemble antiphospholipid syndrome (APS). This work has aimed to study the prevalence of APS-related antigens, antibodies, and immune complexes in patients with COVID-19 and their association with clinical events. METHODS:A prospective study was conducted on 474 adults with severe acute respiratory syndrome coronavirus 2 infection hospitalized in two Spanish university hospitals. Patients were evaluated for classic and extra-criteria antiphospholipid antibodies (aPLs), immunoglobulin G (IgG)/immunoglobulin M (IgM) anticardiolipin, IgG/IgM/immunoglobulin A (IgA) anti-β2-glicoprotein-I (aβ2GPI), IgG/IgM antiphosphatidylserine/prothrombin (aPS/PT), the immune complex of IgA aβ2GPI (IgA-aβ2GPI), bounded to β2-glicoprotein-1 (β2GPI) and β2GPI levels soon after COVID-19 diagnosis and were followed-up until medical discharge or death. RESULTS:Prevalence of aPLs in patients with COVID-19 was as follows: classic aPLs, 5.8%; aPS/PT, 4.6%; IgA-aβ2GPI, 15%; and any aPL, 21%. When patients were compared with individuals of a control group of a similar age, the only significant difference found was the higher prevalence of IgA-aβ2GPI (odds ratio: 2.31; 95% confidence interval: 1.16-4.09). No significant differences were observed in survival, thrombosis, or ventilatory failure in aPL-positive versus aPL-negative patients. β2GPI median levels were much lower in patients with COVID-19 (15.9 mg/l) than in blood donors (168.8 mg/l; P < 0.001). Only 3.5% of patients with COVID-19 had normal levels of β2GPI (>85 mg/l). Low levels of β2GPI were significantly associated with ventilatory failure (P = 0.026). CONCLUSION:β2GPI levels were much lower in patients with COVID-19 than in healthy people. Low β2GPI-levels were associated with ventilatory failure. No differences were observed in the COVID-19 evolution between aPL-positive and aPL-negative patients. Functional β2GPI deficiency could trigger a clinical process similar to that seen in APS but in the absence of aPLs.
Specific domain V reduction of beta-2-glycoprotein I induces protein flexibility and alters pathogenic antibody binding.
Buchholz Ina,McDonnell Thomas,Nestler Peter,Tharad Sudarat,Kulke Martin,Radziszewska Anna,Ripoll Vera M,Schmidt Frank,Hammer Elke,Toca-Herrera Jose L,Rahman Anisur,Delcea Mihaela
Beta-2-glycoprotein I (β2GPI) is a blood protein and the major antigen in the autoimmune disorder antiphospholipid syndrome (APS). β2GPI exists mainly in closed or open conformations and comprises of 11 disulfides distributed across five domains. The terminal Cys288/Cys326 disulfide bond at domain V has been associated with different cysteine redox states. The role of this disulfide bond in conformational dynamics of this protein has not been investigated so far. Here, we report on the enzymatic driven reduction by thioredoxin-1 (recycled by Tris(2-carboxyethyl)phosphine; TCEP) of β2GPI. Specific reduction was demonstrated by Western blot and mass spectrometry analyses confirming majority targeting to the fifth domain of β2GPI. Atomic force microscopy images suggested that reduced β2GPI shows a slightly higher proportion of open conformation and is more flexible compared to the untreated protein as confirmed by modelling studies. We have determined a strong increase in the binding of pathogenic APS autoantibodies to reduced β2GPI as demonstrated by ELISA. Our study is relevant for understanding the effect of β2GPI reduction on the protein structure and its implications for antibody binding in APS patients.
Cardiolipin interacts with beta-2-glycoprotein I and forms an open conformation-Mechanisms analyzed using hydrogen/deuterium exchange.
Tang Kuo-Tung,Wu Ting-Yuan,Chen Hsin-Hua,Lin Chi-Chien,Hsu Yuan-Hao Howard
Protein science : a publication of the Protein Society
Beta-2-glycoprotein I (β GPI) is the major antigen for the antiphospholipid antibodies in the antiphospholipid syndrome. The exposed epitope in domain I of β GPI can be recognized by the anti-β GPI antibody. Here, we prepared the anionic di-oleoyl-phosphatidylserine (DOPS) and cardiolipin (CL) liposomes to interact with the β GPI. The conformational changes of β GPI upon binding with the liposomes were analyzed using hydrogen/deuterium exchange mass spectrometry. The exchange level of sequences 21-27 significantly increased after β GPI had interacted with DOPS. This change indicated a reduced interaction between domain I and domain V, inferring to a protrusion of the sequences 21-27 from the ring conformation. After β GPI had interacted with CL for 30 min, the exchange levels in 4 of the 5 domains increased significantly. The deuteration levels of sequences 1-20, 21-27, 196-205, 273-279 and 297-306 increased, suggesting that these regions had become more exposed, and the domain I was no longer in contact with domain V. The increasing deuteration levels in sequences 70-86, 153-162, 191-198, 196-205 and 273-279 indicated β GPI undergoing conformational changes to expose these inner regions, suggesting a structural transition. Overall, DOPS and CL induced minor conformational changes of β GPI at sequences 21-27 and forms an intermediate conformation after 10 min of interaction. After a complete protein-lipid interaction, high negatively charged CL membrane induced a major conformation transition of β GPI.
The fifth domain of beta 2-glycoprotein I contains a phospholipid binding site (Cys281-Cys288) and a region recognized by anticardiolipin antibodies.
Hunt J,Krilis S
Journal of immunology (Baltimore, Md. : 1950)
We have identified a phospholipid binding site in the fifth domain of beta 2-glycoprotein I (beta 2-GPI). Using synthetic peptides spanning the fifth domain of beta 2-GPI, we have shown that the presence of the sequence Glu274-Cys288 caused a decrease in the binding of purified anticardiolipin (aCL) antibodies in a modified cardiolipin (CL)-ELISA by inhibiting the binding of beta 2-GPI to CL. This peptide bound to and could be eluted from a CL affinity column in a manner similar to native beta 2-GPI. Peptides corresponding to other regions of the fifth domain had no inhibitory effect. The inhibitory activity was restricted to the sequence Cys281-Lys-Asn-Lys-Glu-Lys-Lys-Cys288. Peptides in which the two flanking cysteine residues were deleted or substituted with serine residues possessed no inhibitory activity, indicating that the conformation of this highly positively charged sequence may be critical for phospholipid binding. aCL antibodies purified from patients with autoimmune disease were shown to bind directly to wells coated with native beta 2-GPI but not to wells coated with a preparation of beta 2-GPI cleaved between Lys317 and Thr318. The integrity of this sequence is therefore critical for these antibodies to recognize beta 2-GPI, and the putative epitope for aCL antibodies is most likely to be in this region.
Amino acid sequence and location of the disulfide bonds in bovine beta 2 glycoprotein I: the presence of five Sushi domains.
Kato H,Enjyoji K
beta 2 glycoprotein I is a plasma protein with the ability to bind with various kinds of negatively charged substances. The complete amino acid sequence and the location of all the disulfide bonds of bovine beta 2 glycoprotein I were determined. Bovine beta 2 glycoprotein I consists of 326 amino acid residues with five asparagine-linked carbohydrate chains. Homology with the human protein was calculated to be 83%. Eleven disulfide bonds in bovine beta 2 glycoprotein I constitute four characteristic domains, Sushi domains, and one modified form of a Sushi domain.
Anti-β₂-glycoprotein I IgG antibodies from 1-year-old healthy children born to mothers with systemic autoimmune diseases preferentially target domain 4/5: might it be the reason for their 'innocent' profile?
Andreoli Laura,Nalli Cecilia,Motta Mario,Norman Gary L,Shums Zakera,Encabo Susan,Binder Walter L,Nuzzo Monica,Frassi Micol,Lojacono Andrea,Avcin Tadej,Meroni Pier-Luigi,Tincani Angela
Annals of the rheumatic diseases
BACKGROUND:Anti-β₂-glycoprotein-I (anti-β₂GPI) were demonstrated to be pathogenic in the antiphospholipid syndrome (APS). However, they can be detected in patients with no features of APS, especially those affected by systemic autoimmune diseases (SAD), and so in healthy children. It has been suggested that anti-β₂GPI against domain 1 (D1) associate with thrombosis, while those recognising domain 4/5 (D4/5) are present in non-thrombotic conditions. OBJECTIVE:To evaluate the fine specificity of anti-β₂GPI in adults and infants. METHODS:Three groups were examined-group A: 57 1-year-old healthy children born to mothers with SAD; group B: 33 children with atopic dermatitis; group C: 64 patients with APS. SUBJECTS:were selected based on positive anti-β₂GPI IgG results. Serum samples were tested for anti-β₂GPI IgG D1 and D4/5 using research ELISAs containing recombinant β₂GPI domain antigens. RESULTS:Children (A and B) displayed preferential IgG reactivity for D4/5, whereas patients with APS were mainly positive for D1. No thrombotic events were recorded in groups A and B. CONCLUSIONS:The specificity for D4/5 suggests that anti-β₂GPI IgG production in children born to mothers with SAD is a process neither linked to systemic autoimmunity nor related to the maternal autoantibody status. This unusual fine specificity might, at least partially, account for the 'innocent' profile of such antibodies.
Resistance to annexin A5 anticoagulant activity in women with histories for obstetric antiphospholipid syndrome.
Hunt Beverley J,Wu Xiao-Xuan,de Laat Bas,Arslan Alan A,Stuart-Smith Sara,Rand Jacob H
American journal of obstetrics and gynecology
OBJECTIVE:The objective of the study was to investigate whether resistance to annexin A5 anticoagulant activity (AnxA5) occurs in women with histories for obstetric complications of antiphospholipid syndrome (Obs-APS) and whether this correlates with antibody recognition of domain 1 of β2-glycoprotein. STUDY DESIGN:One hundred thirty-six women with antiphospholipid antibodies, including 70 with histories for Obs-APS and 30 controls, were investigated. RESULTS:Women with Obs-APS showed resistance to AnxA5 activity (median, 216%; range, 130-282% vs controls; median, 247%; range, 217-283%; P < .0001) and elevated levels of anti-domain I immunoglobulin (Ig) G (optical density: median, 0.056; range, 0.021-0.489 vs median, 0.042; range, 0.020-0.323; P = .002). Those in the lowest tertile of AnxA5 anticoagulant ratios had an odds ratio for Obs-APS of 58.0 (95% confidence interval, 3.3-1021.5). There was an inverse correlation between levels of annexin A5 anticoagulant activity and anti-domain I IgG. CONCLUSION:Resistance to AnxA5 anticoagulant activity is associated with antibody recognition of domain I of β2-glycoprotein I and identifies a subset of women with histories for Obs-APS.
Autoantibodies to domain 1 of beta 2 glycoprotein 1: a promising candidate biomarker for risk management in antiphospholipid syndrome.
Mahler Michael,Norman Gary L,Meroni Pier Luigi,Khamashta Munther
Antiphospholipid syndrome (APS) is an autoimmune disorder characterized by frequent clotting in arteries and veins and/or miscarriages. Autoantibodies to phospholipids and to beta 2 glycoprotein 1 (β(2)GP1) play an important role in the pathogenesis of APS. Antibodies to the domain 1 of β(2)GP1 (β(2)GP1-D1) have been suggested as a risk marker for thrombosis and to a lesser extent for pregnancy complications in patients suffering from APS. Despite significant interest in anti-β(2)GP1-D1 antibodies and a considerable research history, the number of studies is still limited and acceptance of the clinical significance of this biomarker is still evolving. The present review summarizes the current knowledge of anti-β(2)GP1-D1 antibodies and provides insights on recent discoveries. Moreover, we present a suggested guideline for future studies to better understand and verify the clinical utility of anti-β(2)GP1-D1 antibodies.
Use of single point mutations in domain I of beta 2-glycoprotein I to determine fine antigenic specificity of antiphospholipid autoantibodies.
Iverson G Michael,Reddel Stephen,Victoria Edward J,Cockerill Keith A,Wang Ying-Xia,Marti-Renom Marc A,Sali Andrej,Marquis David M,Krilis Steven A,Linnik Matthew D
Journal of immunology (Baltimore, Md. : 1950)
Autoantibodies against beta(2)-glycoprotein I (beta(2)GPI) appear to be a critical feature of the antiphospholipid syndrome (APS). As determined using domain deletion mutants, human autoantibodies bind to the first of five domains present in beta(2)GPI. In this study the fine detail of the domain I epitope has been examined using 10 selected mutants of whole beta(2)GPI containing single point mutations in the first domain. The binding to beta(2)GPI was significantly affected by a number of single point mutations in domain I, particularly by mutations in the region of aa 40-43. Molecular modeling predicted these mutations to affect the surface shape and electrostatic charge of a facet of domain I. Mutation K19E also had an effect, albeit one less severe and involving fewer patients. Similar results were obtained in two different laboratories using affinity-purified anti-beta(2)GPI in a competitive inhibition ELISA and with whole serum in a direct binding ELISA. This study confirms that anti-beta(2)GPI autoantibodies bind to domain I, and that the charged surface patch defined by residues 40-43 contributes to a dominant target epitope.
Clinical value of anti-domain I-β2Glycoprotein 1 antibodies in antiphospholipid antibody carriers. A single centre, prospective observational follow-up study.
Tonello M,Mattia E,Del Ross T,Favaro M,Calligaro A,Hoxha A,Bison E,Pengo V,Ruffatti A
Clinica chimica acta; international journal of clinical chemistry
BACKGROUND:There seems to be a clear correlation between antibodies against domain I (anti-DI) of β2Glycoprotein I and severe clinical profiles in antiphospholipid syndrome (APS) patients. We investigated the clinical significance of anti-DI antibodies in a cohort of aPL carriers. METHODS:One hundred and five carriers persistently positive for IgG anti-β2Glycoprotein 1 antibodies (a-β2GPI) and/or IgG anticardiolipin (aCL) and/or lupus anticoagulants (LAC) were tested for the presence of anti-DI antibodies using the QUANTA Flash® Beta2GPI-Domain I chemiluminescence immunoassay. RESULTS:Anti-DI antibodies were detected in 44 aPL carriers (41.9%) and they were significantly associated to triple aPL positivity (LAC plus IgG a-β2GPI plus IgG aCL antibodies). Isolated LAC and a-β2GPI antibodies were significantly associated to anti-DI negative aPL carriers. During a 82.2 month mean follow-up, ten aPL carriers (9.5%) developed a first thrombotic event so becoming APS patients. Anti-DI antibodies, triple aPL positivity, thromboembolic risk factors and autoimmune disorders significantly prevailed in carriers becoming APS. Logistic regression analysis showed that anti-DI positivity was an independent risk factor for thrombosis. CONCLUSIONS:Anti-DI antibody positivity can be considered a new risk factor predictive of the first thrombotic event in aPL carriers, instead, negative anti-DI may be useful to identify low-risk aPL carriers.
New insight into antiphospholipid syndrome: antibodies to β2glycoprotein I-domain 5 fail to induce thrombi in rats.
Durigutto Paolo,Grossi Claudia,Borghi Maria Orietta,Macor Paolo,Pregnolato Francesca,Raschi Elena,Myers Michael P,de Groot Philip G,Meroni Pier Luigi,Tedesco Francesco
Clinical studies have reported different diagnostic/predictive values of antibodies to domain 1 or 4/5 of βglycoproteinI 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 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 βglycoproteinI, human IgG and C3 was analyzed by immunofluorescence. Five serum IgG with undetectable anti-βglycoproteinI 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 βglycoproteinI while being able to interact with fluid-phase βglycoproteinI. These findings may explain the failure of anti-domain 5 antibodies to exhibit a thrombogenic effect , and the interaction of these antibodies with circulating βglycoproteinI 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.
IgG antibodies that recognize epitope Gly40-Arg43 in domain I of beta 2-glycoprotein I cause LAC, and their presence correlates strongly with thrombosis.
de Laat Bas,Derksen Ronald H W M,Urbanus Rolf T,de Groot Philip G
Anti-beta(2)-glycoprotein I antibodies are known to have a heterogeneous reactivity against beta(2)-glycoprotein I. We performed this study to characterize the epitope on beta(2)-glycoprotein I to which pathologic anti-beta(2)-glycoprotein I antibodies are directed. Plasma samples from 198 patients with various systemic autoimmune diseases were tested for the presence of lupus anticoagulant and anti-beta(2)-glycoprotein I immunoglobulin G (IgG) antibodies. The reactivity of the anti-beta(2)-glycoprotein I-positive samples was further tested by coating recombinant full-length beta(2)-glycoprotein I and 8 deletion mutants of beta(2)-glycoprotein I onto hydrophilic and hydrophobic enzyme-linked immunosorbent assay (ELISA) plates. Full-length beta(2)-glycoprotein I with point mutations in domain I at positions 8, 40, and 43 were used in inhibition experiments. Fifty-two patients with anti-beta(2)-glycoprotein I IgG antibodies could be divided into 2 patterns. Type A antibodies only recognize domain I when coated onto hydrophobic plates; they do not recognize domain I coated onto hydrophilic plates. Type B antibodies have heterogeneous reactivity for all domains. Type A antibodies recognize the epitope around amino acids Gly40-Arg43 and cause lupus anticoagulant activity. In contrast to type B antibodies, those of type A strongly correlated with thrombosis. In conclusion, antibodies directed at domain I (epitope comprising Gly40 and Arg43) have lupus anticoagulant activity and strongly associate with thrombosis.
Domain I of β2GPI is capable of blocking serum IgA antiphospholipid antibodies binding in vitro: an effect enhanced by PEGylation.
Albay A,Artim-Esen B,Pericleous C,Wincup C,Giles I,Rahman A,McDonnell T
OBJECTIVES:This study aims to inhibit antiphospholipid syndrome (APS) serum derived IgA anti-beta-2-glycoprotein I (aβ2GPI) binding using Domain I (DI). METHODS:Serum from 13 APS patients was tested for IgA aβ2GPI and Anti-Domain I. Whole IgA was purified by peptide M affinity chromatography from positive serum samples. Serum was tested for IgA aβ2GPI binding in the presence and absence of either DI or of two biochemically modified variants containing either 20 kDa of poly(ethylene glycol) (PEG) or 40 kDa of PEG. RESULTS:Significant inhibition with DI was possible with average inhibition of 23% ( = 13). Further inhibitions using 20 kDa PEG-DI and 40 kDa PEG-DI variants showed significant inhibition ( = 0.0001) with both the 40 kDa PEG-DI and 20 kDa PEG-DI variants showing increased inhibition compared with DI alone ( = 0.0001 and = 0.001, = 10). CONCLUSIONS:Inhibition of IgA aβ2GPI by DI is possible and can be enhanced by biochemical modification in a subset of patients.
Anti-beta-2-glycoprotein I domain 1 identifies antiphospholipid antibodies-related injuries in patients with concomitant lupus nephritis.
Sciascia Savino,Radin Massimo,Cecchi Irene,Fenoglio Roberta,De Marchi Andrea,Besso Luca,Baldovino Simone,Rossi Daniela,Miraglia Paolo,Rubini Elena,Roccatello Dario
Journal of nephrology
BACKGROUND:In this study we aimed to evaluate the usefulness of domain profiling of Beta-2-glycoprotein I(β2GPI)-Domain-1 (D1) antibodies in relation to antiphospholipid antibodies (aPL)-related nephropathy (aPL-N) in patients with biopsy-proven lupus nephritis (LN). METHODS:Of 124 consecutive patients (96 women, mean age 45.5 ± 12.3 years, mean disease duration 14.7 ± 9.6 years) fulfilling the 1982 criteria for systemic lupus erythematosus (SLE), we identified 39 patients (mean age 39.84 ± 8.6 years, mean disease duration 11.3 ± 7.7 years) with the following characteristics: (a) biopsy-proven LN; (b) no previous diagnosis of antiphospholipid syndrome (APS) according to the current classification criteria. RESULTS:Patients with both LN and aPL-N had higher median aβ2GPI-D1 antibody titres (220.1 CU, 25-75th IQ 29.1-334.2) as compared those with LN alone (46.5 CU, 25-75th IQ 12.5-75.1) (p = 0.0087). Median aβ2GPI-D1 antibody titres were higher in patients with acute thrombotic microangiopathy (aTMA) (N = 7) (250.1 CU, 25-75th IQ 61.2-334.2) vs. with LN alone (46.5 CU, 25-75th IQ 12.5-75.1 CU) (p = 0.0009). Having a Global Antiphospholipid Syndrome Score > 10 confers an increased probability of having acute features of aTMA (OR 6.25, 95%CI 1.2-31.8). As compared to other aPL, aβ2GPI-D1 antibodies have the best diagnostic accuracy for aTMA as evaluated by performances in Area Under the Curves in a ROC analysis. CONCLUSIONS:aβ2GPI-D1 antibodies detection might provide a second-line assay to be performed in aβ2GPI positive patients with LN, allowing more accurate stratification of the renal vascular involvement risk, thus potentially leading to a more tailored management.
Binding of antiphospholipid antibodies to discontinuous epitopes on domain I of human beta(2)-glycoprotein I: mutation studies including residues R39 to R43.
Ioannou Yiannis,Pericleous Charis,Giles Ian,Latchman David S,Isenberg David A,Rahman Anisur
Arthritis and rheumatism
OBJECTIVE:Pathogenic antiphospholipid antibodies (aPL) bind the self antigen N-terminal domain (domain I) of beta(2)-glycoprotein I (beta(2)GPI), with residues G40-R43 being important. However, peptides homologous to other regions of domain I have also been shown to bind aPL. Furthermore, there are no published reports of the effects of altering R39, which has greater surface exposure than the G40-R43 residues. METHODS:We used a novel, efficient method of production and purification of human domain I by Escherichia coli to create multiple mutants of domain I. These domain I mutants were then screened for binding to a range of polyclonal IgG purified from patients with antiphospholipid syndrome, using both solid-phase and fluid-phase assays. RESULTS:E coli-expressed purified domain I selectively bound IgG derived from patients with antiphospholipid syndrome. In region R39-R43, the R39S mutation had the greatest effect in terms of reducing binding to a panel of aPL in the fluid phase (mean +/- SD inhibition 14 +/- 18.5% versus 44.1 +/- 31.7% for G40E and 62.9 +/- 25.7% for wild-type domain I). Conversely, altering both D8 and D9 to S8 and G9, respectively, had the effect of enhancing binding to aPL in the fluid phase. Adding the remainder of the domain I-II interlinker resulted in enhanced binding over wild-type in the solid phase but not the fluid phase. CONCLUSION:The binding of aPL to beta(2)GPI domain I is complex and likely to involve discontinuous epitopes that include R39 in addition to G40-R43, the domain I-II interlinker, and possibly D8 and D9. Domain I variants with enhanced binding to aPL compared with wild-type domain I may aid in the development of novel therapies.
[Fine specificity of anti-β2glycoprotein I antibodies in systemic autoimmune diseases is mostly directed against domain 1].
Nalli C,Andreoli L,Motta M,Norman G L,Shums Z,Binder W L,Nuzzo M,Frassi M,Lojacono A,Meini A,Medeghini V,Avcin T,Meroni P L,Tincani A
OBJECTIVE:Anti-β2 GPI are a formal laboratory criterion for the antiphospholipid syndrome (APS). They were demonstrated to be a risk factor for thrombosis and fetal losses but can also be detected in patients with systemic autoimmune disease (SAD), in healthy adults individuals and pre-school children. It has been suggested that different subpopulations of anti-β2GPI may carry different pathogenetic potential: autoantibodies against Domain1 seem to be associated with thrombosis; autoantibodies against Domain4/5 have been identified in patients with non-thrombotic conditions. METHODS:We studied 48 patients with SAD (32 systemic lupus erythematosus, 16 undifferentiated connettive tissue disease), 64 patients with APS, 57 one-year-old healthy children born to mother with SAD, 33 children with atopic dermatitis. All subjects were IgG anti-β2 GPI positive. The specificity of anti-β2 GPI was investigated using ELISA research products containing recombinant β2 GPI D1 and D4/5 antigens. Cut-off values are calculated as 95th percentile on 100 NHD. IgG anti-β2 GPI were tested at a validated home-made ELISA routinely performed in our laboratory. No thrombotic events were recordered in patients with SAD and in both groups of children. RESULTS:Patients with SAD and APS showed prevalent reactivity for D1 while children in both groups preferentially recognize D4/5. CONCLUSIONS:IgG anti-β2 GPI against D1 seem to cluster in patients with systemic autoimmune conditions. Their pathogenic potential in determine APS manifestations may be mitigated by adequate prophylaxis.
Anti-beta2 glycoprotein I (beta2GPI) autoantibodies recognize an epitope on the first domain of beta2GPI.
Iverson G M,Victoria E J,Marquis D M
Proceedings of the National Academy of Sciences of the United States of America
Anticardiolipin (aCL) autoantibodies are associated with thrombosis, recurrent fetal loss, and thrombocytopenia. Only aCL found in autoimmune disease require the participation of the phospholipid binding plasma protein beta2 glycoprotein I (beta2GPI) for antibody binding and now are called anti-beta2GPI. The antigenic specificity of aCL affinity purified from 11 patients with high titers was evaluated in an effort to better understand the pathophysiology associated with aCL. Seven different recombinant domain-deleted mutants of human beta2GPI, and full length human beta2GPI (wild-type), were used in competition assays to inhibit the autoantibodies from binding to immobilized wild-type beta2GPI. Only those domain-deleted mutants that contained domain 1 inhibited the binding to immobilized wild-type beta2GPI from all of the patients. The domain-deleted mutants that contained domain 1 inhibited all aCL in a similar but not identical pattern, suggesting that these aCL recognize a similar, but distinguishable, epitope(s) present on domain 1.
Beyond thrombosis: Anti-β2GPI domain 1 antibodies identify late pregnancy morbidity in anti-phospholipid syndrome.
Chighizola Cecilia Beatrice,Pregnolato Francesca,Andreoli Laura,Bodio Caterina,Cesana Laura,Comerio Chiara,Gerosa Maria,Grossi Claudia,Kumar Rajesh,Lazzaroni Maria Grazia,Mahler Michael,Mattia Elena,Nalli Cecilia,Norman Gary L,Raimondo Maria Gabriella,Ruffatti Amelia,Tonello Marta,Trespidi Laura,Tincani Angela,Borghi Maria Orietta,Meroni Pier Luigi
Journal of autoimmunity
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.
Evaluating the conformation of recombinant domain I of β(2)-glycoprotein I and its interaction with human monoclonal antibodies.
Pericleous Charis,Miles Jennifer,Esposito Diego,Garza-Garcia Acely,Driscoll Paul C,Lambrianides Anastasia,Latchman David,Isenberg David,Rahman Anisur,Ioannou Yiannis,Giles Ian
Pathogenic antiphospholipid antibodies (aPL) cause the antiphospholipid syndrome (APS) by interacting with domain I (DI) of beta-2-glycoprotein I (β(2)GPI). The aPL/β(2)GPI complex then exerts pathogenic effects on target cells. We previously described periplasmic bacterial expression of native and mutated variants of DI, and reported the presence of immunodominant epitopes at positions 8-9 (D8/D9) and position 39 (R39). Mutations at these positions strongly influenced the ability of recombinant DI to bind patient-derived IgG aPL and to inhibit pathogenic effects of these aPL in a mouse model of APS. We now describe an improved cytoplasmic bacterial expression system allowing higher yield of DI. We demonstrate that the nuclear magnetic resonance (NMR) spectra of a (15)N,(13)C-isotope-labelled sample of the recombinant DI protein exhibit properties consistent with the structure of DI in crystal structure of intact β(2)GPI. Mutations at D8/D9 and R39 had limited impact on the NMR spectrum of DI indicating maintenance of the overall fold of the DI domain. We investigated interactions between five variants of DI and ten monoclonal human IgG antibodies, all derived from the IgG aPL antibody IS4 by sequence manipulation and in vitro expression. Arginine residues at positions 100 and 100g in IS4V(H) CDR3 play a particularly important role in binding to DI, but this is unlikely to be due to electrostatic interactions with negatively charged amino acids on DI. Both the strength of binding to DI and the ability to discriminate different DI variants varies between the different IgG antibodies tested. There was no simple relationship between these binding properties and antibody pathogenicity.
Domain I: the hidden face of antiphospholipid syndrome.
Pericleous C,Rahman A
IgG antiphospholipid antibodies (aPL) against β2-glycoprotein I (β2GPI) can target any of its five domains; however, aPL against the N-terminal domain (anti-DI, aDI) are considered the most clinically relevant in the antiphospholipid syndrome (APS). Circulating levels of aDI are elevated in patients with APS compared with disease and healthy controls, and crucially aDI are prothrombotic in in vivo and in vitro models. In addition, human recombinant DI has been shown to abrogate aPL-induced thrombosis in vivo. Therefore, although the potential of utilizing DI for management of APS is not yet fully defined, there is promise that DI could prove valuable both as a diagnostic and therapeutic tool.
Antibodies to Domain I of β(2)Glycoprotein I are in close relation to patients risk categories in Antiphospholipid Syndrome (APS).
Banzato A,Pozzi N,Frasson R,De Filippis V,Ruffatti A,Bison E,Padayattil S J,Denas G,Pengo V
INTRODUCTION:Antiphospholipid Syndrome (APS) is characterized by the presence of circulating antiphospholipid antibodies in patients with thrombosis or pregnancy morbidity. Antibodies involved in these disorders are mainly those directed against β(2)-Glycoprotein I (β(2)GPI) with the major epitope apparently located on discontinuous antigen with several parts of Domain I (DmI) involved. The relation between anti-DmI antibodies and patients' risk categories is unknown. MATERIALS AND METHODS:The synthetic full-length and correctly-folded DmI (1-64) to set up a competitive inhibition enzyme-linked immunoadsorbent assays (ELISA) was used. Plasma of 22 patients with APS and triple positivity [Lupus Anticoagulant positive (LAC+), IgG anti-cardiolipin positive (aCL+), IgG anti-β(2)GPI positive (a β(2)GPI +)], 15 with double positivity (IgG aCL+, IgG aβ(2)GPI+), 9 with single positivity (IgG aβ(2)GPI+) and 20 controls were evaluated. RESULTS:Median of percentage inhibition was 25.5% [interquartile range (IQR)17.2-33.0] in triple positive patients. Significantly lower inhibition was observed in patients with double positivity, median inhibition 5.0% (IQR 0.0-27.0) and in patients with single positivity median inhibition was 2.0% (IQR 0.5-8.0) (p<0.0001). No inhibition was detected in control subjects or using β(2)GPI peptides (40-52 and 57-70), or when antithrombin, an insignificant control protein was used. CONCLUSIONS:High risk patients with APS and triple laboratory positivity as compared with double and single positivity patients have significantly higher titre of anti-DmI antibodies as evaluated by an inhibition test.
Immune responses against domain I of β(2)-glycoprotein I are driven by conformational changes: domain I of β(2)-glycoprotein I harbors a cryptic immunogenic epitope.
de Laat Bas,van Berkel Miranda,Urbanus Rolf T,Siregar Berdien,de Groot Philip G,Gebbink Martijn F,Maas Coen
Arthritis and rheumatism
OBJECTIVE:The presence of autoantibodies against a cryptic epitope in domain I of β(2)-glycoprotein I (β(2)GPI) is strongly associated with thrombotic events in patients with the antiphospholipid syndrome. We hypothesized that a conformational change could be a trigger for the formation of antibodies against domain I of β(2)GPI. Therefore, we investigated whether immune responses against β(2)GPI are related to its conformation. METHODS:Conformational changes in β(2)GPI were studied using various techniques, either upon binding to cardiolipin or after disruption of the internal disulfide bonds. The immunogenicity of β(2)GPI in different conformations as well as the individual domains of β(2)GPI were studied in vivo by monitoring the generation of antibodies after intravenous administration of β(2)GPI to mice. Furthermore, plasma samples from these mice were assessed for lupus anticoagulant activity and thrombin-antithrombin complex levels. RESULTS:We observed that the interaction of β(2)GPI with cardiolipin induced a conformational change in β(2)GPI: electron microscopy revealed that β(2)GPI assembled into polymeric meshworks. We next investigated the immunogenicity of both human and murine β(2)GPI in mice. Both human and murine β(2)GPI combined with cardiolipin and misfolded β(2)GPI triggered antibody formation against the native protein as well as against domain I of β(2)GPI, while native β(2)GPI was not immunogenic. In addition, we observed that anti-domain I antibodies developed in mice injected with domain I of β(2)GPI, and that antibodies did not develop in mice injected with domains II-V. The induced anti-domain I antibodies prolonged the dilute Russell's viper venom plasma clotting time. The plasma of mice with anti-domain I antibodies had increased levels of circulating thrombin-antithrombin complexes. CONCLUSION:The results of our studies indicate that the exposure of cryptic epitopes due to conformational changes in β(2)GPI can induce autoantibody formation.
IgA Antiphospholipid Antibodies and Anti-Domain 1 of Beta 2 Glycoprotein 1 Antibodies are Associated with Livedo Reticularis and Heart Valve Disease in Antiphospholipid Syndrome.
Cieśla Marek,Wypasek Ewa,Undas Anetta
Advances in clinical and experimental medicine : official organ Wroclaw Medical University
BACKGROUND:Antiphospholipid syndrome (APS) is an autoimmune disease associated with venous or arterial thrombosis and pregnancy loss, but also infrequently with non-criteria APS manifestations such as thrombocytopenia, livedo reticularis and heart valve disease. The occurrence of antiphospholipid antibodies is necessary to diagnose APS and includes the presence of lupus anticoagulant and anticardiolipin as well as anti-β2-glycoprotein I antibodies, both in IgM and/or IgG isotype. OBJECTIVES:The aim of this study was to evaluate the associations between antiphospholipid antibodies including IgA isotype and IgG anti-domain I of β2-glycoprotein I (β-2GPI-D1) and non-criteria-related manifestations of APS. MATERIAL AND METHODS:Thirty-three consecutive APS patients (26 women, 7 men, aged 44.1 ± 15 years), including 23 (69.7%) subjects with primary APS, were enrolled. Together with standard antiphospholipid antibodies, IgA anticardiolipin, IgA anti-β2-glycoprotein I and IgG anti-β-2GPI-D1 antibodies in serum samples were evaluated by chemiluminescence using the QUANTA Flash® System. RESULTS:Livedo reticularis (n = 8, 24.2%) was associated with increased levels of IgG anti-β-2GPI-D1 (p = 0.005), IgA anticardiolipin (p = 0.001) and IgA anti-β2-glycoprotein I (p = 0.002) antibodies. Heart valve disease (n = 9, 27.3%) was observed in patients with higher IgG anti-β-2GPI-D1 (p = 0.01). The associations of HVD with increased levels of IgA aCL and IgA anti-β-2GPI tended to be significant (p = 0.07). None of antiphospholipid antibodies showed association with thrombocytopenia (n = 6, 18.2%). CONCLUSIONS:Our study suggests that increased IgA antiphospholipid antibodies and IgG anti-β-2GPI-D1 antibodies may be involved in the development of livedo reticularis and heart valve disease in APS patients.
New tests to detect antiphospholipid antibodies: anti-domain I beta-2-glycoprotein-I antibodies.
Chighizola Cecilia Beatrice,Gerosa Maria,Meroni Pier Luigi
Current rheumatology reports
Beta-2 glycoprotein I (β2GPI) is the main antigenic target for antiphospholipid antibodies (aPL), the serological markers of antiphospholipid syndrome (APS). Domain I (DI) of β2GPI has lately been identified as the main epitope targeted by antibodies reacting against β2GPI. DI is a cryptic epitope, becoming available for autoantibody binding when β2GPI opens from a circular to a fish-hook configuration. Antibodies targeting β2GPI-DI are more frequently detected in patients with a full-blown syndrome than in asymptomatic aPL carriers or in patients with infectious diseases that have reactivity toward the whole molecule. Interestingly, anti-DI antibodies are strongly positively correlated with thrombotic and pregnancy manifestations, enabling identification of patients at higher risk of clinical events. However, available tests to detect anti-DI antibodies still lack standardization. Moreover, some APS patients develop antibodies reacting against β2GPI epitopes other than DI, suggesting that other anti-β2GPI antibody subsets may be clinically relevant. Available evidence on anti-DI antibodies in APS is herein critically reviewed.
Autoantibodies to domain 1 of beta 2 glycoprotein I determined using a novel chemiluminescence immunoassay demonstrate association with thrombosis in patients with antiphospholipid syndrome.
Mahler M,Albesa R,Zohoury N,Bertolaccini M L,Ateka-Barrutia O,Rodriguez-Garcia J L,Norman G L,Khamashta M
INTRODUCTION:Antibodies to the domain 1 of beta 2 glycoprotein I (β2GPI-D1) have been suggested as a risk marker for thrombosis in patients with the antiphospholipid syndrome (APS). This cross-sectional study aimed to analyze the clinical utility of a novel chemiluminescence assay for the detection of anti-β2GPI-D1 antibodies. PATIENTS AND METHODS:Sera collected from patients with primary or secondary APS (n = 106; 72 with and 34 without history of thrombosis) and controls (n = 272) were tested for anti-β2GPI-D1 IgG by chemiluminescence assay (QUANTA Flash) and by two anti-β2GPI IgG assays (QUANTA Lite and QUANTA Flash β2GPI IgG). RESULTS:Anti-β2GPI-D1 IgG titers were significantly higher in patients with thrombosis (P = 0.0032) than those without. At the cut-off of 20 units, which yielded a 99.5% specificity, 24 of 72 (34.9%) patients with thrombosis and four of 34 (11.8%) without thrombosis were anti-β2GPI-D1 IgG positive (odds ratio, OR = 4.0). By further optimizing the cut-off specifically for correlation with thrombosis, 20.8% of the patients with thrombosis and 2.9% of the patients without thrombosis were positive (OR = 8.7). The ORs were significantly lower for antibodies to the full-length β2GPI by either the chemiluminescence assay or ELISA. Using the anti-β2GPI chemiluminescence assay, the OR was 2.3 (recommended cut-off of 20 CU) or 4.1 (optimal cut-off 164.6 CU). Using the anti-β2GPI ELISA, the OR was 2.7 (recommended cut-off of 20 units) or 3.7 (optimal cut-off 7.6 units). CONCLUSION:These data indicate that anti-β2GPI-D1 IgG are present more frequently and in higher titers in APS patients with thrombotic complications than in those without.The novel β2GPI-D1 chemiluminescence assay appears to be superior to full-length β2GPI assays for the risk assessment of thrombotic events in APS patients.
Development of a high yield expression and purification system for Domain I of Beta-2-glycoprotein I for the treatment of APS.
McDonnell Thomas,Pericleous Charis,Laurine Emmanuelle,Tommasi Rita,Garza-Garcia Acely,Giles Ian,Ioannou Yiannis,Rahman Anisur
BACKGROUND:In this paper we describe a novel method to achieve high yield bacterial expression of a small protein domain with considerable therapeutic potential; Domain I of Beta-2-glycoprotein I (β2GPI). β2GPI is intrinsic to the pathological progression of the Antiphospholipid Syndrome (APS). Patients develop autoantibodies targeting an epitope located on the N-terminal Domain I of β2GPI rendering this domain of interest as a possible therapeutic. RESULTS:This new method of production of Domain I of β2GPI has increased the production yield by ~20 fold compared to previous methods in E.coli. This largely scalable, partially automated method produces 50-75 mg of pure, folded, active Domain I of β2GPI per litre of expression media. CONCLUSION:The application of this method may enable production of Domain I on sufficient scale to allow its use as a therapeutic.
The Significance of Antibodies against Domain I of Beta-2 Glycoprotein I in Antiphospholipid Syndrome.
Kelchtermans Hilde,Chayouâ Walid,Laat Bas de
Seminars in thrombosis and hemostasis
The antiphospholipid syndrome (APS) is characterized by vascular thrombosis and/or pregnancy morbidity with the persistent presence of antiphospholipid antibodies (aPLs). Progress is being made in understanding the pathogenesis of the syndrome, but difficulties persist in the identification of patients at risk for thrombosis and/or pregnancy morbidity. Beta-2 glycoprotein I (βGPI), a plasma protein consisting of five sushi domains, is thought to be the main antigenic target of aPLs. Antibodies recognizing domain I of βGPI are predominantly present in patients with an elevated risk of thrombosis, whereas antidomain IV/V antibodies are found in nonthrombotic autoimmune diseases. Indeed, domain I antibodies proved to be pathogenic in multiple studies. Retrospective studies have provided evidence for an added clinical value of antidomain I antibodies in the risk stratification of patients with APS. Still, wide ranges of odds ratio exist between studies, probably due to differences in the study and control population, and detection methods used. Despite the proven pathogenicity of antidomain I antibodies and their correlations with clinical manifestations of APS, heterogeneity of the current studies has prohibited their acceptance in the official diagnostic criteria. Well-designed large longitudinal prospective studies with available and new, preferentially functional, assays for the risk stratification of patients with APS are required.
PEGylated Domain I of Beta-2-Glycoprotein I Inhibits the Binding, Coagulopathic, and Thrombogenic Properties of IgG From Patients With the Antiphospholipid Syndrome.
McDonnell Thomas C R,Willis Rohan,Pericleous Charis,Ripoll Vera M,Giles Ian P,Isenberg David A,Brasier Allan R,Gonzalez Emilio B,Papalardo Elizabeth,Romay-Penabad Zurina,Jamaluddin Mohammad,Ioannou Yiannis,Rahman Anisur
Frontiers in immunology
APS is an autoimmune disease in which antiphospholipid antibodies (aPL) cause vascular thrombosis and pregnancy morbidity. In patients with APS, aPL exert pathogenic actions by binding serum beta-2-glycoprotein I (β2GPI) via its N-terminal domain I (DI). We previously showed that bacterially-expressed recombinant DI inhibits biological actions of IgG derived from serum of patients with APS (APS-IgG). DI is too small (7 kDa) to be a viable therapeutic agent. Addition of polyethylene glycol (PEGylation) to small molecules enhances the serum half-life, reduces proteolytic targeting and can decrease immunogenicity. It is a common method of tailoring pharmacokinetic parameters and has been used in the production of many therapies in the clinic. However, PEGylation of molecules may reduce their biological activity, and the size of the PEG group can alter the balance between activity and half-life extension. Here we achieve production of site-specific PEGylation of recombinant DI (PEG-DI) and describe the activities and of three variants with different size PEG groups. All variants were able to inhibit APS-IgG from: binding to whole β2GPI in ELISA, altering the clotting properties of human plasma and promoting thrombosis and tissue factor expression in mice. These findings provide an important step on the path to developing DI into a first-in-class therapeutic in APS.
Antiphospholipid Antibodies to Domain I of Beta-2-Glycoprotein I Show Different Subclass Predominance in Comparison to Antibodies to Whole Beta-2-glycoprotein I.
McDonnell Thomas,Artim-Esen Bahar,Wincup Chris,Ripoll Vera M,Isenberg David,Giles Ian P,Rahman Anisur,Pericleous Charis
Frontiers in immunology
Antiphospholipid antibodies (aPL), the serological hallmark of antiphospholipid syndrome (APS), are a heterogeneous group of autoantibodies raised against circulating blood proteins. Of these proteins, the phospholipid-binding b2-glycoprotein I (β2GPI) is considered to be the main autoantigen in APS. Indeed, IgG antibodies targeting b2GPI (ab2GPI) directly cause both thrombosis and pregnancy morbidity in several mouse models. While antibodies raised against all five domains of b2GPI have been reported, a subgroup of IgG ab2GPI raised against the first domain (DI) of b2GPI (aDI), strongly correlate with thrombotic APS, and drive thrombosis and pregnancy loss . Few studies have focused on determining the type of IgG subclass(es) for aPL. The subclass of an antibody is important as this dictates the potential activity of an antibody; for example, IgG1 and IgG3 can fix complement better and are able to cross the placenta compared to IgG2 and IgG4. It is unknown what subclass IgG aDI are, and whether they are the same as ab2GPI. To determine IgG subclass distribution for ab2GPI and aDI, we purified total IgG from the serum of 19 APS patients with known ab2GPI and aDI activity. Using subclass-specific conjugated antibodies, we modified our established in-house ab2GPI and aDI ELISAs to individually measure IgG1, IgG2, IgG3, and IgG4. We found that while IgG1, IgG2, and IgG3 ab2GPI levels were similar, a marked difference was seen in IgG subclass aDI levels. Specifically, significantly higher levels of IgG3 aDI were detected compared to IgG1, IgG2, or IgG4 ( < 0.05 for all comparisons). Correlation analysis of subclass-specific ab2GPI vs. aDI demonstrated that IgG3 showed the weakest correlation ( = 0.45, = 0.0023) compared to IgG1 ( = 0.61, = 0.0001) and IgG2 ( = 0.81, = 0.0001). Importantly, total subclass levels in IgG purified from APS and healthy serum ( = 10 HC = 12 APS) did not differ, suggesting that the increased IgG3 aDI signal seen in APS-derived IgG is antigen-specific. To conclude, our data suggests that aDI show a different IgG subclass distribution to ab2GPI. Our results highlight the importance of aDI testing for patient stratification and may point toward differential underlying aPL-driven pathogenic processes that may be subclass restricted.
A novel expression system of domain I of human beta2 glycoprotein I in Escherichia coli.
Ioannou Yiannis,Giles Ian,Lambrianides Anastasia,Richardson Chris,Pearl Laurence H,Latchman David S,Isenberg David A,Rahman Anisur
BACKGROUND:The antiphospholipid syndrome (APS), characterised by recurrent miscarriage and thrombosis, is a significant cause of morbidity and mortality. Domain I (DI) of human beta 2 glycoprotein I (beta2GPI) is thought to contain crucial antibody binding epitopes for antiphospholipid antibodies (aPL), which are critical to the pathogenesis of APS. Expressing this protein in bacteria could facilitate studies investigating how this molecule interacts with aPL. METHODS:Using a computer programme called Juniper, sequentially overlapping primers were designed to be used in a recursive polymerase chain reaction (PCR) to produce a synthetic DI gene. Specifically Juniper incorporates 'major' codons preferred by bacteria altering 41 codons out of 61. This was cloned into the expression plasmid pET(26b) and expressed in BL21(DE3) Escherichia coli (E. coli). By virtue of a pelB leader sequence, periplasmic localisation of DI aided disulphide bond formation and toxicity was addressed by tightly regulating expression through the high stringency T7lac promoter. RESULTS:Purified, soluble his-tagged DI in yields of 750 microg/L bacterial culture was obtained and confirmed on Western blot. Expression using the native human cDNA sequence of DI in the same construct under identical conditions yielded significantly less DI compared to the recombinant optimised sequence. This constitutes the first description of prokaryotic expression of soluble DI of beta2GPI. Binding to murine monoclonal antibodies that recognise conformationally restricted epitopes on the surface of DI and pathogenic human monoclonal IgG aPL was confirmed by direct and indirect immunoassay. Recombinant DI also bound a series of 21 polyclonal IgG samples derived from patients with APS. CONCLUSION:By producing a synthetic gene globally optimised for expression in E. coli, tightly regulating expression and utilising periplasmic product translocation, efficient, soluble E. coli expression of the eukaryotic protein DI of beta2GPI is possible. This novel platform of expression utilising pan-gene prokaryote codon optimisation for DI production will aid future antigenic studies. Furthermore if DI or peptide derivatives of DI are eventually used in the therapeutic setting either as toleragen or as a competitive inhibitor of pathogenic aPL, then an E. coli production system may aid cost-effective production.
Detection of anti-domain I antibodies by chemiluminescence enables the identification of high-risk antiphospholipid syndrome patients: A multicenter multiplatform study.
Yin Dongmei,Chayoua Walid,Kelchtermans Hilde,de Groot Philip G,Moore Gary W,Gris Jean-Christophe,Zuily Stéphane,Musial Jacek,de Laat Bas,Devreese Katrien M J
Journal of thrombosis and haemostasis : JTH
BACKGROUND:Classification of the antiphospholipid syndrome (APS) relies predominantly on detecting antiphospholipid antibodies (aPLs). Antibodies against a domain I (DI) epitope of anti-β2glycoprotein I (β2GPI) proved to be pathogenic, but are not included in the current classification criteria. OBJECTIVES:Investigate the clinical value of detecting anti-DI IgG in APS. PATIENTS/METHODS:From eight European centers 1005 patients were enrolled. Anti-cardiolipin (CL) and anti-β2GPI were detected by four commercially available solid phase assays; anti-DI IgG by the QUANTA Flash β2GPI domain I assay. RESULTS:Odds ratios (ORs) of anti-DI IgG for thrombosis and pregnancy morbidity proved to be higher than those of the conventional assays. Upon restriction to patients positive for anti-β2GPI IgG, anti-DI IgG positivity still resulted in significant ORs. When anti-DI IgG was added to the criteria aPLs or used as a substitute for anti-β2GPI IgG/anti-CL IgG, ORs for clinical symptoms hardly improved. Upon removing anti-DI positive patients, lupus anticoagulant remained significantly correlated with clinical complications. Anti-DI IgG are mainly present in high-risk triple positive patients, showing higher levels. Combined anti-DI and triple positivity confers a higher risk for clinical symptoms compared to only triple positivity. CONCLUSIONS:Detection of anti-DI IgG resulted in higher ORs for clinical manifestations than the current APS classification criteria. Regardless of the platform used to detect anti-β2GPI/anti-CL, addition of anti-DI IgG measured by QUANTA Flash did not improve the clinical associations, possibly due to reduced exposure of the pathogenic epitope of DI. Our results demonstrate that anti-DI IgG potentially helps in identifying high-risk patients.
Pharmacokinetics of LJP 993, a tetrameric conjugate of domain 1 of beta2-glycoprotein I for antiphospholipid syndrome.
Jia L,Gu Y,Zeng E,Linnik M D,Jones D S
beta2-glycoprotein I is the best-characterized antigenic target for antiphospholipid autoantibodies. We synthesized a tetrameric conjugate of the domain 1 of beta2-glycoprotein I (LJP 993) aimed at developing the conjugate as a Toleragen to suppress antiphospholipid syndrome. The present studies focused on determining the stability, tissue distribution, plasma concentration-time profile and excretion of the LJP 993 in mice. The stability of LJP 993 in mouse plasma was quantitatively evaluated using strong cation-exchange high performance liquid chromatography. ( 125)I-labeled LJP 993 was intravenously injected to mice, and levels of (125)I-labeled LJP 993 in plasma, tissues, urine and feces were determined at known intervals. Incubation of LJP 993 with mouse serum at 37 degrees C for 8 h resulted in a decrease by 34% of LJP 993 concentration. No degradation fragment was observed during the incubation. After a single intravenous administration of (125)I-LJP 993 (0.5 and 5 mg/kg) to mice, both C(max) and area-under-curve values increased in a dose-proportional manner, and blood radioactivity disappeared in a bi-exponential manner with the distribution half-lives equal to 1.7 min, and the elimination half-lives 188 and 281 min, respectively. The (125)I-LJP 993 was moderately distributed into organs and tissues with the exception that brain level of ( 125)I-LJP 993 was negligible. The major sites of (125)I-LJP 993 uptake were the kidney (at 30 min post dosing), and kidney, lung, liver, heart, spleen, skin, muscle and fat tissues (at 4 h post dosing). Cumulative urinary and fecal radioactivity for 0-48 h post dosing accounted for 44.7% and 4.2% of the administered dose, respectively, with the fast rate of urinal excretion occurring within the first 8 h. In summary, LJP 993 was fairly stable in mouse plasma. After administration to mice, (125)I-LJP 993 was taken up mainly by kidney and then distributed extensively to tissues except brain. Both C(max) and area-under-curve values increased in a dose-proportional manner. It was predominantly excreted in the urine with an elimination half-life longer than 3 h. Kidney is a major route to excrete the tetrameric conjugate.
Correlation between antiphospholipid antibodies that recognize domain I of beta2-glycoprotein I and a reduction in the anticoagulant activity of annexin A5.
de Laat Bas,Wu Xiao-Xuan,van Lummel Menno,Derksen Ronald H W M,de Groot Philip G,Rand Jacob H
The paradoxical correlation between thrombosis and the lupus anticoagulant (LAC) effect is an enigmatic feature of the antiphospholipid (aPL) syndrome. The Dutch authors previously reported that thrombosis-related anti-beta2-glycoprotein I (beta2GPI) antibodies recognize domain I and cause LAC. The American authors reported that aPLs disrupt an anticoagulant annexin A5 (AnxA5) crystal shield. We investigated whether antidomain I antibodies correlate with disruption of AnxA5-anticoagulant activity. We studied a well-characterized group of 33 patients including subgroups with beta2GPI-dependent LAC that recognize domain I (n=11), with beta2GPI-independent LAC (n=12), and lacking LAC (n=10). The effects on AnxA5-anticoagulant activity were determined with an AnxA5 resistance assay that measures coagulation times with and without AnxA5. Patients with beta2GPI-dependent LAC (group A, all with thrombosis) had significantly lower AnxA5-anticoagulant ratios than those with beta2GPI-independent LAC (group B, thrombosis n=4; 157.8% versus 235.6%, P<.001) and those without LAC (group C, thrombosis n=2; 157.8% versus 232.5%, P<.001). There was no difference in the ratios between groups B and C (P=.92). Plasmas with beta2GPI-dependent LAC that recognize domain I displayed significantly increased AnxA5 resistance, suggesting that specifically anti-beta2GPI antibodies compete with AnxA5 for anionic phospholipids. These results are consistent with a model in which aPL antibodies may promote thrombosis by interfering with the anticoagulant activity of AnxA5.
Elevated IgA antiphospholipid antibodies in healthy pregnant women in Sudan but not Sweden, without corresponding increase in IgA anti-β glycoprotein I domain 1 antibodies.
Elbagir S,Mohammed N A,Kaihola H,Svenungsson E,Gunnarsson I,Manivel V A,Pertsinidou E,Elagib E M,Nur M A M,Elussein E A,Elshafie A,Åkerud H,Rönnelid J
OBJECTIVE:The role of antiphospholipid antibodies (aPL) during apparently normal pregnancy is still unclear. IgA aPL are prevalent in populations of African origin. Our aim was to measure all isotypes of anticardiolipin (anti-CL) and anti-β glycoprotein I (anti-βGPI) in healthy pregnant and non-pregnant women of different ethnicities. METHODS:Healthy Sudanese pregnant women ( = 165; 53 sampled shortly after delivery), 96 age-matched Sudanese female controls and 42 healthy pregnant and 249 non-pregnant Swedish women were included. IgA/G/M anti-CL and anti-βGPI were tested at one time point only with two independent assays in Sudanese and serially in pregnant Swedes. IgA anti-βGPI domain 1 and as controls IgA/G/M rheumatoid factor (RF), IgG anti-cyclic citrullinated peptide 2 (anti-CCP2) and anti-thyroid peroxidase (anti-TPO) were investigated in Sudanese females. RESULTS:Pregnant Sudanese women had significantly higher median levels of IgA anti-CL, IgA anti-βGPI ( < 0.0001 for both antibodies using two assays) and IgM anti-βGPI (both assays; < 0.0001 and 0.008) compared with non-pregnant Sudanese. IgA anti-CL and anti-βGPI occurrence was increased among Sudanese pregnant women compared with national controls. No corresponding increase during pregnancy was found for IgA anti-βGPI domain 1 antibodies. Both IgG anti-CL and IgG control autoantibodies decreased during and directly after pregnancy among Sudanese. Serially followed Swedish women showed no changes in IgA aPL, whereas IgG/M anti-CL decreased. CONCLUSIONS:IgA aPL are increased in Sudanese but not in Swedish women, without corresponding increase in IgA domain 1. Whether due to ethnicity and/or environmental influences the occurrence of IgA aPL during Sudanese pregnancies, and its clinical significance, is yet to be determined.
Pathogenic anti-beta2-glycoprotein I antibodies recognize domain I of beta2-glycoprotein I only after a conformational change.
de Laat Bas,Derksen Ronald H W M,van Lummel Menno,Pennings Maarten T T,de Groot Philip G
Recently, we published the existence of 2 populations of anti-beta2-glycoprotein I (beta2-GPI) IgG antibodies. Type A antibodies recognize epitope G40-R43 in domain I of beta2-GPI and are strongly associated with thrombosis. Type B antibodies recognize other parts of beta2-GPI and are not associated with thrombosis. In this study we demonstrate that type A antibodies only recognize plasma-purified beta2-GPI when coated onto a negatively charged surface and not when coated onto a neutrally charged surface. The affinity of type B antibodies toward plasma-purified beta2-GPI was independent of the charge of the surface to which beta2-GPI was coated. Type A antibodies did not recognize plasma-purified beta2-GPI in solution, whereas they did recognize recombinant beta2-GPI both in solution and coated onto a neutrally charged plate. When the carbohydrate chains were removed from plasma-purified beta2-GPI, we found that type A antibodies did recognize the protein in solution. This supports the hypothesis that the difference in recognition of plasma-purified and recombinant beta2-GPI is caused by the difference in glycosylation and that epitope G40-R43 of plasma-purified beta2-GPI is covered by a carbohydrate chain. Type A anti-beta2-GPI antibodies can only recognize this epitope when this carbohydrate chain is displaced as a result of a conformational change. This finding has major implications both for the detection of pathogenic anti-beta2-GPI antibodies and the comprehension of the pathophysiology of the antiphospholipid syndrome.
Anti-domain 1 β2 glycoprotein antibodies increase expression of tissue factor on monocytes and activate NK Cells and CD8+ cells in vitro.
Manukyan Gayane,Martirosyan Anush,Slavik Ludek,Margaryan Sona,Ulehlova Jana,Mikulkova Zuzana,Hlusi Antonin,Papajik Tomas,Kriegova Eva
Auto- immunity highlights
BACKGROUND:β2-Glycoprotein I (β2GPI) represents the major antigenic target for antiphospholipid antibodies (aPL), with domain 1 (D1) being identified as a risk factor for thrombosis and pregnancy complications in APS. We aimed to analyse the ability of aPL, and particularly anti-D1 β2GPI, to stimulate prothrombotic and proinflammatory activity of immune cells in vitro. METHODS:Peripheral blood mononuclear cells (PBMCs) from 11 healthy individuals were incubated with: (1) "anti-D1(+)"-pooled plasma derived from patients suspected of having APS contained anticardiolipin antibodies (aCL), lupus anticoagulant (LA), anti-β2GPI and anti-D1 β2GPI; (2) "anti-D1(-)"-pooled plasma from patients suspected of having APS contained aCL, LA, anti-β2GPI, and negative for anti-D1 β2GPI; (3) "seronegative"-negative for aPL. RESULTS:The presence of anti-D1(+) and anti-D1(-) plasma resulted in increased HLA-DR and CD11b on monocytes. While only anti-D1(+) plasma markedly increased the percentage and median fluorescence intensity (MFI) of CD142 (tissue factor, TF) on monocytes in comparison with those cultured with anti-D1(-) and seronegative plasma. Anti-D1(+) plasma resulted in increased percentage and MFI of activation marker CD69 on NK and T cytotoxic cells. Expression of IgG receptor FcγRIII(CD16) on monocytes and NK cells was down-regulated by the anti-D1(+) plasma. CONCLUSIONS:Taking together, our study shows the ability of patient-derived aPL to induce immune cell activation and TF expression on monocytes. For the first time, we demonstrated the influence of anti-D1 β2GPI on the activation status of monocytes, NK and cytotoxic T cells. Our findings further support a crucial role of D1 epitope in the promotion of thrombosis and obstetrical complications in APS.
In vivo inhibition of antiphospholipid antibody-induced pathogenicity utilizing the antigenic target peptide domain I of beta2-glycoprotein I: proof of concept.
Ioannou Y,Romay-Penabad Z,Pericleous C,Giles I,Papalardo E,Vargas G,Shilagard T,Latchman D S,Isenberg D A,Rahman A,Pierangeli S
Journal of thrombosis and haemostasis : JTH
SUMMARY OBJECTIVES:In the antiphospholipid syndrome (APS), the immunodominant epitope for the majority of circulating pathogenic antiphospholipid antibodies (aPLs) is the N-terminal domain I (DI) of beta(2)-glycoprotein I. We have previously shown that recombinant DI inhibits the binding of aPLs in fluid phase to immobilized native antigen, and that this inhibition is greater with the DI(D8S/D9G) mutant and absent with the DI(R39S) mutant. Hence, we hypothesized that DI and DI(D8S/D9G) would inhibit aPL-induced pathogenicity in vivo. METHODS:C57BL/6 mice (n = 5, each group) were injected with purified IgG derived from APS patients (IgG-APS, 500 microg) or IgG from normal healthy serum (IgG-NHS) and either recombinant DI, DI(R39S), DI(D8S/D9G), or an irrelevant control peptide (at 10-40 microg). Outcome variables measured were femoral vein thrombus dynamics in treated and control groups following standardized vessel injury, expression of vascular cell adhesion molecule-1 (VCAM-1) on the aortic endothelial surface, and tissue factor (TF) activity in murine macrophages. RESULTS:IgG-APS significantly increased thrombus size as compared with IgG-NHS. The IgG-APS thrombus enhancement effect was abolished in mice pretreated with recombinant DI (P <or= 0.0001) and DI(D8S/D9G) (P <or= 0.0001), but not in those treated with DI(R39S) or control peptide. This inhibitory effect by DI was dose-dependent, and at lower doses DI(D8S/D9G) was a more potent inhibitor of thrombosis than wild-type DI (P <or= 0.01). DI also inhibited IgG-APS induction of VCAM-1 on the aortic endothelial surface and TF production by murine macrophages. CONCLUSION:Our findings in this proof-of-concept study support the development of recombinant DI or the novel variant DI(D8S/D9G) as a potential future therapeutic agent for APS.
The association between circulating antibodies against domain I of beta2-glycoprotein I and thrombosis: an international multicenter study.
de Laat B,Pengo V,Pabinger I,Musial J,Voskuyl A E,Bultink I E M,Ruffatti A,Rozman B,Kveder T,de Moerloose P,Boehlen F,Rand J,Ulcova-Gallova Z,Mertens K,de Groot P G
Journal of thrombosis and haemostasis : JTH
BACKGROUND:Diagnosis of the antiphospholipid syndrome (APS) is difficult as a result of limited specificity of existing assays for detecting clinically relevant antiphospholipid antibodies. Anti-beta2-glycoprotein I (beta 2GPI) antibodies play a central role in the disease process of APS. OBJECTIVES:We have investigated the relation between antiphospholipid antibodies with specificity for domain I of beta 2GPI and thrombosis/pregnancy morbidity in an international multicenter study. PATIENTS/METHODS:Four hundred and seventy-seven patients derived from nine different centres met the inclusion criterion of having anti-beta 2GPI antibodies in their plasma/serum. Clinical data and results of tests for lupus anticoagulant, anti-cardiolipin antibodies and anti-beta 2GPI antibodies were established at the different centres of inclusion. After being re-tested for the presence of IgG and/or IgM anti-beta 2GPI antibodies, the samples were tested for the presence of IgG-directed against domain I of beta 2GPI and results were correlated with the thrombotic and obstetric history. RESULTS:Re-testing for the presence of anti-beta 2GPI antibodies resulted in inclusion of 442/477 patients. IgG class anti-domain I antibodies were present in plasma of 243/442 patients (55%). 201/243 (83%) had a history of thrombosis. This resulted in an odds ratio of 3.5 (2.3-5.4, 95% confidence interval) for thrombosis. Anti-domain I IgG antibodies were also significantly correlated with obstetric complications [odds ratio: 2.4 (1.4-4.3, 95% confidence interval)]. CONCLUSION:In this multicenter study, the detection of IgG antibodies that are directed against domain I of beta 2GPI proved to be more strongly associated with thrombosis and obstetric complications than those detected using the standard anti-beta 2GPI antibody assay.
Domain I of beta2-glycoprotein I: its role as an epitope and the potential to be developed as a specific target for the treatment of the antiphospholipid syndrome.
Ioannou Y,Rahman A
Antiphospholipid syndrome (APS) represents one of the most common acquired causes of thrombophilia and recurrent miscarriages. The only treatment of proven benefit is anticoagulation, often required at high intensity and life-long duration. This therapy can be associated with side effects such as bleeding and is not always effective. Hence, there remains a need for safer, targeted and ideally more effective therapies. Antiphospholipid antibodies (aPL) are pathogenic and promote thrombosis. Independent groups, including our own, have show that the major epitopes that pathogenic aPL targets lie within domain I of the protein beta2-glycoprotein I (beta2GPI). This review focuses on the evidence presented thus far which characterizes the immunodominant epitopes within domain I, demonstrating that the epitope is a conformational one centred around residues R39-G43 and also involving other residues within domain I, such as residues D8 and D9. The hypothesis is proposed that a recombinant domain I molecule, and a recombinant mutant domain I with enhanced aPL binding properties, may be used as an inhibitor of aPL binding and thus inhibit aPL-induced pathogenicity. In vivo proof-of-concept studies within the murine femoral vein injury model are presented supporting this hypothesis, and the rationale as well as potential benefits and problems of employing such an approach to treat APS are discussed.
Variability in exposure of epitope G40-R43 of domain i in commercial anti-beta2-glycoprotein I IgG ELISAs.
Pelkmans Leonie,Kelchtermans Hilde,de Groot Philip G,Zuily Stephane,Regnault Veronique,Wahl Denis,Pengo Vittorio,de Laat Bas
BACKGROUND:A major problem for diagnosing the antiphospholipid syndrome (APS) is the high variability between commercial anti-β2glycoprotein I (β2GPI) assays. Predominantly antibodies reactive against cryptic epitope Glycine40-Arginine43 (G40-R43) in domain I are associated with an increased risk for thrombosis. Upon interaction with anionic surfaces β2GPI opens up, thereby exposing G40-R43. OBJECTIVES:To examine whether suboptimal exposure of epitope G40-R43 explains the variations in results observed between commercial assays. METHODS:Two patient-derived monoclonal antibodies were tested on neutral versus anionic plates. Antibody P1-117 reacts with G40-R43 in the open conformation while P2-6 recognizes β2GPI irrespective of its conformation. These antibodies were tested in commercial anti-β2GPI assays (A-E). RESULTS:In assay A, both antibodies showed equal reactivity towards β2GPI, indicating that all the β2GPI exposes G40-R43. In other assays P1-117 displayed lower reactivity than P2-6, demonstrating reduced G40-R43 availability. To exclude influences of other assay features, reactivity was re-examined on plates of assay A and B using the protocol/reagents from each assay. In all combinations, reactivity of both antibodies on a plate was comparable to results obtained with its own protocol/reagents, suggesting that the coating, rather than other assay components, accounts for the observed differences. In two patient cohorts we demonstrated that a number of domain I-reactive samples are missed in assays characterized by a decreased exposure of epitope G40-R43. CONCLUSIONS:Exposure of epitope G40-R43 on β2GPI is highly variable between commercial anti-β2GPI assays. As a consequence, patients can be falsely assigned negative in assays characterized by a reduced exposure of G40-R43.
Chemical synthesis and characterization of wild-type and biotinylated N-terminal domain 1-64 of beta2-glycoprotein I.
Pozzi Nicola,Banzato Alessandra,Bettin Samuele,Bison Elisa,Pengo Vittorio,De Filippis Vincenzo
Protein science : a publication of the Protein Society
The antiphospholipid syndrome (APS) is a severe autoimmune disease associated with recurrent thrombosis and fetal loss and characterized by the presence of circulating autoantibodies (aAbs) mainly recognizing the N-terminal domain (DmI) of beta2-glycoprotein I (beta2GpI). To possibly block anti-beta2GpI Abs activity, we synthesized the entire DmI comprising residues 1-64 of beta2GpI by chemical methods. Oxidative disulfide renaturation of DmI was achieved in the presence of reduced and oxidized glutathione. The folded DmI (N-DmI) was purified by RP-HPLC, and its chemical identity and correct disulfide pairing (Cys4-Cys47 and Cys32-Cys60) were established by enzymatic peptide mass fingerprint analysis. The results of the conformational characterization, conducted by far- and near-UV CD and fluorescence spectroscopy, provided strong evidence for the native-like structure of DmI, which is also quite resistant to both Gdn-HCl and thermal denaturation. However, the thermodynamic stability of N-DmI at 37 degrees C was remarkably low, in agreement with the unfolding energetics of small proteins. Of note, aAbs failed to bind to plates coated with N-DmI in direct binding experiments. From ELISA competition experiments with plate-immobilized beta2GpI, a mean IC(50) value of 8.8 microM could be estimated for N-DmI, similar to that of the full-length protein, IC(50)(beta2GpI) = 6.4 microM, whereas the cysteine-reduced and carboxamidomethylated DmI, RC-DmI, failed to bind to anti-beta2GpI Abs. The versatility of chemical synthesis was also exploited to produce an N-terminally biotin-(PEG)(2)-derivative of N-DmI (Biotin-N-DmI) to be possibly used as a new tool in APS diagnosis. Strikingly, Biotin-N-DmI loaded onto a streptavidin-coated plate selectively recognized aAbs from APS patients.
Autoantibodies directed against domain I of beta2-glycoprotein I.
de Laat Bas,de Groot Philip G
Current rheumatology reports
Patients diagnosed with the antiphospholipid syndrome typically suffer from vascular thrombosis, pregnancy morbidity, or a combination of the two. Due to the high prevalence of these clinical symptoms, the diagnosis of antiphospholipid syndrome is almost completely dependent on the detection of antiphospholipid antibodies in patient plasma. However, not every individual with antiphospholipid antibodies in his or her plasma suffers from thrombosis and/or pregnancy morbidity, which suggests the existence of different populations of antiphospholipid antibodies. Although many antigens have been identified in relation to the antiphospholipid syndrome, β2-glycoprotein I is regarded as clinically most significant. During the past decade, evidence has accumulated to suggest the presence of a dominant epitope on the first domain of β2-glycoprotein I. Several studies have detected a specific population of antibodies recognizing a cryptic epitope on domain I, at least comprising arginine 39 to arginine 43. In contrast to antibodies recognizing other domains of β2-glycoprotein I, anti-domain I antibodies are found to be highly associated with clinical symptoms. This review discusses several studies that have investigated a role for domain I within the antiphospholipid syndrome on a predominantly diagnostic level.
Antibodies against domain I of β2-glycoprotein I: the one and only?
Pelkmans L,de Laat B
The antiphospholipid syndrome (APS) is diagnosed by the occurrence of thrombosis and/or specific pregnancy morbidity. However, the diagnosis of APS is not easy and is hampered by several problems including high prevalence of clinical symptoms and high variability between different assays resulting in a high false-positive rate. Currently APS can be diagnosed for example by detecting anti-β2-glycoprotein I antibodies by ELISA. It has been reported that β2-glycoprotein I (β2GPI) changes its conformation from a native to an active form and thereby it opens up enabling antibodies to bind a specific epitope. We amongst others have shown that epitope glycine40-arginine43 of domain I of β2GPI is predominantly responsible for binding thrombosis related antibodies. Antibodies with affinity towards other epitopes have not been associated with thrombosis. Despite these results the question remains whether these domain I antibodies are the only antibodies of importance for the detection of APS.
Role of antiphospholipid score and anti-β2-glycoprotein I Domain I autoantibodies in the diagnosis of antiphospholipid syndrome.
Mondejar R,González-Rodríguez C,Toyos-Sáenz de Miera F J,Melguizo-Madrid E,Zohoury N,Mahler M,Romero Losquiño I,Fabiani F
Clinica chimica acta; international journal of clinical chemistry
BACKGROUND:Antiphospholipid syndrome (APS) is characterized by the presence circulating antiphospholipid (aPL) antibodies in patients with thrombosis or pregnancy morbidity. Recently it has been shown that multiple positive results define a higher risk of clinical manifestation in APS patients. However, utilizing combined results generates challenges for a physician. Therefore, the antiphospholipid score. (aPL-S), a new variable that encompasses all aPL assays, has been described. We analyze clinical performance of different aPL-Ss based on ELISA or chemiluminescent immunoassays (CIAs). METHODS:A total of 39 patients and 77 controls were included in this study. All patients were tested for lupus anticoagulant (LAC). In addition, IgM/IgG anticardiolipin (aCL) and anti-β2 glycoprotein 1 (aβ2GP1) autoantibodies were tested by ELISA and CIA. Anti-β2GP1 Domain 1 IgG (D1) autoantibodies were tested by CIA. Three aPL-Ss were calculated (ELISA, CIA and CIA with D1 instead of β2GP1 IgG) using the Otomo equation: aPL-S=5×exp([OR]-5)/4. RESULTS:IgG assays showed a good correlation while IgM assays showed moderate correlation. The relative risk of having clinical manifestation of APS was calculated for each aPL test. All three aPL-Ss were higher in individuals with thrombosis or pregnancy morbidity than in those without APS manifestations (p<0.001) and the prevalence of APS manifestations increased with increasing aPL-Ss. CONCLUSION:The CIAs are comparable with the ELISAs for the detection of aPL antibodies. aβ2GPI-D1 antibodies seem to represent a strong indicator for clinical manifestations of APS. Any of the aPL-Ss studied represents a useful quantitative index for APS diagnosis and could be helpful to physicians in managing APS.
Anti-Domain I β2-Glycoprotein I Antibodies and Activated Protein C Resistance Predict Thrombosis in Antiphospholipid Syndrome: TAC(I)T Study.
Zuily Stephane,de Laat Bas,Guillemin Francis,Kelchtermans Hilde,Magy-Bertrand Nadine,Desmurs-Clavel Hélène,Lambert Marc,Poindron Vincent,de Maistre Emmanuel,Dufrost Virginie,Risse Jessie,Shums Zakera,Norman Gary L,de Groot Philip G,Lacolley Patrick,Lecompte Thomas,Regnault Véronique,Wahl Denis
The journal of applied laboratory medicine
BACKGROUND:Antibodies binding to domain I of β2-glycoprotein I (aDI) and activated protein C (APC) resistance are associated with an increased risk of thrombosis in cross-sectional studies. The objective of this study was to assess their predictive value for future thromboembolic events in patients with antiphospholipid antibodies (aPL) or antiphospholipid syndrome. METHODS:This prospective multicenter cohort study included consecutive patients with aPL or systemic lupus erythematosus. We followed 137 patients (43.5 ± 15.4 year old; 107 women) for a mean duration of 43.1 ± 20.7 months. RESULTS:We detected aDI IgG antibodies by ELISA in 21 patients. An APC sensitivity ratio (APCsr) was determined using a thrombin generation-based test. The APCsr was higher in patients with anti-domain I antibodies demonstrating APC resistance (0.75 ± 0.13 vs 0.48 ± 0.20, P < 0.0001). In univariate analysis, the hazard ratio (HR) for thrombosis over time was higher in patients with aDI IgG (3.31 [95% CI, 1.15-9.52]; P = 0.03) and patients with higher APC resistance (APCsr >95th percentile; HR, 6.07 [95% CI, 1.69-21.87]; P = 0.006). A sensitivity analysis showed an increased risk of higher aDI IgG levels up to HR 5.61 (95% CI, 1.93-16.31; P = 0.01). In multivariate analysis, aDI IgG (HR, 3.90 [95% CI, 1.33-11.46]; P = 0.01) and APC resistance (HR, 4.98 [95% CI, 1.36-18.28]; P = 0.02) remained significant predictors of thrombosis over time. CONCLUSIONS:Our study shows that novel tests for antibodies recognizing domain I of β2-glycoprotein I and functional tests identifying APC resistance are significant predictors of thrombosis over time and may be useful for risk stratification.
Antiphospholipid syndrome: antibodies to Domain 1 of β2-glycoprotein 1 correctly classify patients at risk.
Pengo V,Ruffatti A,Tonello M,Cuffaro S,Banzato A,Bison E,Denas G,Padayattil Jose S
Journal of thrombosis and haemostasis : JTH
BACKGROUND:Determination of lupus anticoagulant (LA), anticardiolipin (aCL) and β2-Glycoprotein 1 (aβ2GP1) antibodies is mandatory to classify patients with antiphospholipid syndrome (APS) into risk categories. OBJECTIVES:To measure relevant antibodies, considered to be those of the IgG isotype directed towards β2GP1 and particularly those directed to Domain 1 (Dm1) of the molecule. PATIENTS/METHODS:In this cross-sectional study we measured IgG aβ2GP1-Dm1 by a chemiluminescent immunoassay in a group of individuals initially positive for IgG aβ2GP1 and classified as triple (LAC+, IgG aCL+, IgG aβ2GP1+, n = 32), double (LAC-, IgG aCL+, IgG aβ2GP1+, n = 23) or single positive (LA-, IgG aCL-, IgG aβ2GP1+, n = 10). RESULTS AND CONCLUSION:Geometric mean and standard deviation expressed as chemiluminescent units (CU) in triple, double and single positive groups were 273.0 ± 6.2, 18.2 ± 9.6 and 4.4 ± 2.2, respectively. The geometric mean obtained in 40 healthy subjects was 2.0 ± 2.0. Mean CU values were significantly different among groups and with respect to values found in 40 healthy subjects (P < 0.0001). Positive values of IgG aβ2GP1-Dm1 (above 14.2 CU) were found in 45 individuals while 20 individuals (20/65 = 30.8%) positive for IgG aβ2GP1 were negative for IgG aβ2GPI-Dm1. There was a significant association between positive IgG aβ2GP1-Dm1 and thromboembolic events (P = 0.001). Positive and negative values of IgG aβ2GP1-Dm1 were consistently confirmed after 12 weeks, with only three low positive values being negative after 12 weeks. In conclusion, IgG aβ2GP1-Dm1 seems a robust and reproducible test that in association with the classic tests may be useful in clinical practice in identifying individuals at high risk of developing thromboembolic events.
Clinical implications of the detection of antibodies directed against domain 1 of β2-glycoprotein 1 in thrombotic antiphospholipid syndrome.
Montalvão Silmara,Elídio Priscila Soares,da Silva Saraiva Sabrina,de Moraes Mazetto Bruna,Colella Marina Pereira,de Paula Erich Vinícius,Appenzeller Simone,Annichino-Bizzacchi Joyce,Orsi Fernanda Andrade
INTRODUCTION:Antibodies directed against domain 1 of β2 glycoprotein 1 (aβ2GP1-Dm1) have been involved in the immunopathogenesis of antiphospholipid syndrome (APS). However, the clinical relevance of aβ2GP1-Dm1 in thrombotic APS has not yet been fully explored. OBJECTIVES:To determine the frequency of aβ2GP1-Dm1 in a cohort of patients with thrombotic APS, and to evaluate whether testing for aβ2GP1-Dm1 could have a clinical impact upon the risk assessment of the disease. METHODS:Patients were tested for aβ2GP1-Dm1 antibodies by chemiluminescence (BioFlash/AcuStar®, ES). The presence of aβ2GP1-Dm1 was evaluated in different clinical presentations of the disease. RESULTS:Eight-four patients with a history of venous or arterial thrombosis were included. Forty-five (54%) patients had aβ2GP1 antibodies and 40% of them were positive for aβ2GP1-Dm1. Levels of aβ2GP1-Dm1 were higher in patients with systemic autoimmune disease (AUC=0.665; 95% CI=0.544-0.786; P=0.01), positive antinuclear antibody (AUC=0.654; 95% CI=0.535-0.772; P=0.01), triple antiphospholipid antibody (aPL) positivity (AUC=0.680; 95% CI=0.534-0.825; P=0.02) and positive lupus anticoagulant (AUC=0.639; 95% CI=0.502-0.776; P=0.07). In this cohort, aβ2GP1-Dm1 antibodies were not associated with the site of the first thrombosis (OR=0,62, 95% CI=0.20-1.94, P=0.42), thrombosis recurrence (OR=1.0, 95% CI=0.37-2.71, P=1.0) or pregnancy morbidity (OR=1.5, 95% CI=0.33-7.34, P=0.58). In multivariate analysis, positivity for aβ2GP1-Dm1 antibodies was associated with the diagnosis of systemic autoimmune disease (OR=4.01, 95% CI=1.14-14.2; P=0.03) and triple aPL positivity (OR=3.59, 95% CI=0.87-14.85; P=0.07). CONCLUSIONS:In the present cohort of thrombotic-APS patients, aβ2GP1-Dm1 antibodies were related to the diagnosis of systemic autoimmunity and complex serological profile of the disease, as triple aPL positivity and positive antinuclear antibody. Thus, our results suggest that testing for aβ2GP1-Dm1 antibodies may be useful for improving APS risk assessment.
Clinical characterization of antiphospholipid syndrome by detection of IgG antibodies against β2 -glycoprotein i domain 1 and domain 4/5: ratio of anti-domain 1 to anti-domain 4/5 as a useful new biomarker for antiphospholipid syndrome.
Andreoli Laura,Chighizola Cecilia B,Nalli Cecilia,Gerosa Maria,Borghi M Orietta,Pregnolato Francesca,Grossi Claudia,Zanola Alessandra,Allegri Flavio,Norman Gary L,Mahler Michael,Meroni Pier Luigi,Tincani Angela
Arthritis & rheumatology (Hoboken, N.J.)
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.
Prevalence and Thrombotic Risk Assessment of Anti-β2 Glycoprotein I Domain I Antibodies: A Systematic Review.
Radin Massimo,Cecchi Irene,Roccatello Dario,Meroni Pier Luigi,Sciascia Savino
Seminars in thrombosis and hemostasis
BACKGROUND: To date, the exact prevalence of anti-β glycoprotein I domain I (anti-βGPI-DI) antibodies in patients with antiphospholipid syndrome (APS) and their role when assessing thrombosis risk is uncertain. OBJECTIVES: To estimate the prevalence of anti-βGPI-DI in patients with APS and to determine whether anti-βGPI-DI-positive individuals are at greater risk of thrombosis, as compared with individuals without anti-βGPI-DI, by systematically reviewing the literature. METHODS: A detailed literature search was applied a priori to Ovid MEDLINE In-Process and Other Non-Indexed Citation 1986 to present and to abstracts from the European League Against Rheumatism (EULAR) and American College of Rheumatology (ACR)/Association for Rheumatology Health Professionals (ARHP ) Annual Meetings (2011-2015). RESULTS: A total of 11 studies, including 1,585 patients, were analyzed. Patients were distributed as follow: 1,218 patients APS (45.4% anti-βGPI-DI-positive; in more detail: 504 primary APS [55.4% anti-βGPI-DI-positive], 192 secondary APS [43.2% anti-βGPI-DI-positive], and 522 not specified), 318 with systemic lupus erythematosus (SLE; 26.7% anti-βGPI-DI-positive), 49 asymptomatic carriers of antiphospholipid antibodies (aPL) (30.6% anti-βGPI-DI-positive), and 1,859 healthy controls. When considering the five studies eligible for thrombotic risk assessment, four studies found a significant association of anti-βGPI-DI-positivity with thrombotic events, whereas one study found no predictive correlation with thrombosis (overall odds ratio [OR] for pooled data: 1.99; 95% confidence interval [CI]: 1.52-2.6; < 0.0001). CONCLUSION: We report an overall estimated median prevalence of anti-βGPI-DI antibodies of 44.3% in patients with APS and/or SLE and a significantly higher prevalence among patients with APS compared with SLE alone. Anti-βGPI-DI antibodies might represent a promising tool when assessing thrombotic risk in patients with APS.
The association between IgG and IgM antibodies against cardiolipin, β2-glycoprotein I and Domain I of β2-glycoprotein I with disease profile in patients with multiple sclerosis.
Filippidou Natalia,Krashias George,Pericleous Charis,Rahman Anisur,Ioannou Yiannis,Giles Ian,Demetriou Christiana,Anatolitou Afroditi,Christodoulou Christina,Pantzaris Marios,Lambrianides Anastasia
Antiphospholipid antibodies (aPL) occur in patients with multiple sclerosis (MS) with a number of studies reporting elevated levels; their exact prevalence and pathogenic role remain unclear. Epidemiological studies associate MS with an increased risk of deep venous thromboembolism and stroke; overlapping clinical features with APS. Antibodies against the first domain - Domain I (DI) - of β2glycoprotein I (β2GPI), show the most clinical significance and evidence for pathogenicity in the antiphospholipid syndrome (APS), but have not yet been investigated in MS. Serum from a well-defined cohort of 127 MS patients and 92 healthy controls were tested for IgM and IgG antibodies against cardiolipin (CL), β2GPI and DI. Higher frequency of IgM and IgG anti-CL were found in MS patients (18.1% and 21.3%), compared to controls (1.1% in both cases, p<0.0001). We report that anti-DI antibodies were associated with MS patients, with 6.3% and 7.1% positive for IgM and IgG, respectively, compared to controls, 1.1% (p<0.05). IgM anti-CL antibodies were elevated in secondary progressive MS and primary progressive MS compared to relapse-remitting MS, (p<0.005). This study enrolled the largest number of patients with definite MS for studying the association with aPL. Although we confirmed IgM and IgG anti-CL antibodies occur in patients with MS, this is the first study that identified anti-DI antibodies in MS patients. This new finding may prove valuable and future studies are required to evaluate its role as a potential risk factor of thromboembolic phenomena in MS.
Detection of IgG anti-Domain I beta2 Glycoprotein I antibodies by chemiluminescence immunoassay in primary antiphospholipid syndrome.
Meneghel Lauro,Ruffatti Amelia,Gavasso Sabrina,Tonello Marta,Mattia Elena,Spiezia Luca,Tormene Daniela,Hoxha Ariela,Fedrigo Marny,Simioni Paolo
Clinica chimica acta; international journal of clinical chemistry
BACKGROUND:IgG anti-Domain I (anti-DI) β2 Glycoprotein I (β2GPI) antibodies are associated to thrombotic risk in antiphospholipid syndrome (APS), but their detection is technically difficult. In this study, a chemiluminescent immunoassay (CLIA) was used to evaluate the clinical significance of IgG anti-DI in a large cohort of patients with primary APS (PAPS). METHODS:The study population included 88 patients with PAPS, 63 ELISA-negative subjects and 166 controls. IgG anti-DI, IgG anticardiolipin (aCL) and IgG anti-β2GPI antibodies were assayed using CLIA (HemosIL AcuStar®). RESULTS:The sensitivity and specificity of IgG anti-DI antibodies were comparable to those of IgG aCL and IgG anti-β2GPI antibodies. There was a significant agreement, association and titre correlation between IgG anti-DI and IgG aCL as well as IgG anti-β2GPI antibodies (p<0.001 for all). IgG anti-DI antibody showed lesser prevalence and mean titres in the pregnancy morbidity than in thrombotic and PAPS patients with both involvements (p<0.001). Regarding the conventional aPL antibody profiles, the triple positivity group had higher prevalence and mean titres than single and double positivity ones (p<0.001). CONCLUSIONS:This study provides further evidence that anti-DI antibodies can be considered a promising biomarker for risk assessment particularly in patients having vascular thrombosis and triple conventional aPL positivity.
Oxidation of β2-glycoprotein I associates with IgG antibodies to domain I in patients with antiphospholipid syndrome.
Raimondo Maria Gabriella,Pericleous Charis,Radziszewska Anna,Borghi Maria Orietta,Pierangeli Silvia,Meroni Pier Luigi,Giles Ian,Rahman Anisur,Ioannou Yiannis
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.
Clinical significance of anti-domain 1 β2-glycoprotein I antibodies in antiphospholipid syndrome.
Iwaniec Teresa,Kaczor Marcin P,Celińska-Löwenhoff Magdalena,Polański Stanisław,Musiał Jacek
BACKGROUND:Antiphospholipid syndrome (APS) is characterized by the presence of circulating antiphospholipid antibodies (aPL) in patients with thrombosis and/or pregnancy morbidity. In APS patients anti-domain 1 β2-glycoprotein I (anti-D1 β2GPI) IgG antibodies correlate strongly with thrombosis and to the lesser extent, with pregnancy complications. The aim of this study was to assess clinical utility of the anti-D1 β2GPI antibodies in the diagnosis and risk stratification of antiphospholipid syndrome. PATIENTS/METHODS:In this retrospective study 202 autoimmune patients were studied (primary APS - 58, secondary - 45 SLE - 99). Anticardiolipin (aCL) and anti-βGPI (aβGPI antibodies) (IgG and IgM class) together with anti-D1 IgG were tested with QUANTA Flash chemiluminescent immunoassay and lupus anticoagulant (LA) with coagulometric methods. RESULTS:The highest anti-D1 values were observed in triple positive patients as compared to patients with other antiphospholipid antibody profiles. A strong correlation was found between levels of anti-D1 IgG and a β2GPI IgG antibodies for all patients analyzed (Spearman's ρ=0.87; p<0.0001). Anti-D1 IgG antibodies increase specificity resulting from classic aPL positivity but at the expense of sensitivity. Anti-D1 test does not add accuracy in predicting APS thrombotic complications on the top of accuracy offered by classic aPL tests and their profiles. CONCLUSIONS:Anti-D1 IgG antibodies did not add diagnostic power to the standard laboratory aPL tests as assessed by this retrospective study. A true clinical significance of anti-D1 antibodies in thrombotic risk stratification of aPL positive patients will require a properly designed clinical prospective trials.
Tolerogenic dendritic cells specific for β2-glycoprotein-I Domain-I, attenuate experimental antiphospholipid syndrome.
Zandman-Goddard Gisele,Pierangeli Silvia S,Gertel Smadar,Blank Miri
Journal of autoimmunity
Tolerogenic dendritic cells (tDCs) have the potential to control the outcome of autoimmunity by modulating the immune response. The aim of this study was to uncover the tolerance efficacy attributed to beta-2-glycoprotein-I (β2GPI) tDCs or β2GPI domain-I (D-I) and domain-V (D-V)-tDCs in mice with antiphospholipid syndrome (APS). tDCs were pulsed with β2GPI or D-I or D-V derivatives. Our results revealed that β2GPI related tDCs phenotype includes CD80(high), CD86(high) CD40(high) MHC class II(high). The miRNA profiling encompass miRNA 23b(high), miRNA 142-3p(low) and miRNA 221(low). In addition the β2GPI related tDCs showed reduced secretion of IL-1β, IL-12 and IL-23. D-I tDCs treatment was more efficient than β2GPI tDCs in inducing of tolerance in APS mice, manifested by lowered titers of anti- β2GPI antibodies (Abs) and reduced percentage of fetal loss. Tolerance induction was accompanied by poor T cell response to β2GPI, high numbers of CD4 + CD25 + FOXP3 + T-regulatory cells (Treg), reduced levels of IFNγ, IL-17 and increased expression of IL-10 and TGFβ. Tolerance was successfully transferred by Treg cells from the tolerized mice to β2GPI immunized mice. We conclude that predominantly D-I-tDCs and β2GPI tDCs have the potential to attenuate experimental APS by induction of Treg cells, reduction of anti- β2GPI Abs titers and increased expression of anti-inflammatory cytokines. We suggest that β2-GPI-D-I-tDCs may offer a novel approach for developing therapy for APS patients.
Evaluation of the diagnostic potential of antibodies to beta2-glycoprotein 1 domain 1 in Chinese patients with antiphospholipid syndrome.
Zhang Shulan,Wu Ziyan,Chen Si,Li Jing,Wen Xiaoting,Li Liubing,Zhang Wen,Zhao Jiuliang,Zhang Fengchun,Li Yongzhe
In this study, we evaluated the clinical performance of anti-β2-glycoprotein 1 domain 1 antibodies (aβ2GP1-D1) in the diagnosis of antiphospholipid syndrome (APS). Sera from 229 subjects were tested, including 35 patients with primary APS, 51 patients with APS associated to other diseases, 30 patients with non-APS thrombosis, 32 patients with non-APS pregnancy-related morbidity, 42 patients with systemic lupus erythematosus, and 39 healthy controls (HC). Serum IgG aβ2GP1-D1, IgG/IgM anti-cardiolipin (aCL) and IgG/IgM aβ2GP1 were measured by a chemiluminescence assay. The levels of IgG aβ2GP1-D1 were significantly increased in patients with APS, compared with disease controls and HCs (p < 0.001). Significant correlation was identified between IgG aβ2GP1-D1 and IgG aβ2GP1 (p < 0.0001), indicating IgG aβ2GP1-D1 were the predominant domain-specific antibodies in IgG aβ2GP1 family. Importantly, aβ2GP1-D1, but not aβ2GP1 non-D1, was significantly correlated with thrombotic events. Interestingly, no significant correlation between IgG aβ2GP1-D1 and obstetric complications was observed. Additionally, significantly higher levels of IgG aβ2GP1-D1 were found in patients with triple aPL positivity, compared with patients with double and single aPL positivity. Our findings suggest a potential role of IgG aβ2GP1-D1 in identifying APS patients with high risk of thrombosis, shedding insight on the introduction of IgG aβ2GP1-D1 in China.
Measuring IgA Anti-β2-Glycoprotein I and IgG/IgA Anti-Domain I Antibodies Adds Value to Current Serological Assays for the Antiphospholipid Syndrome.
Pericleous Charis,Ferreira Isabel,Borghi Orietta,Pregnolato Francesca,McDonnell Thomas,Garza-Garcia Acely,Driscoll Paul,Pierangeli Silvia,Isenberg David,Ioannou Yiannis,Giles Ian,Meroni Pier Luigi,Rahman Anisur
INTRODUCTION:Currently available clinical assays to detect antiphospholipid antibodies (aPL) test for IgG and IgM antibodies to cardiolipin (aCL) and β2-glycoprotein I (aβ2GPI). It has been suggested that testing for IgA aPL and for antibodies to Domain I (DI), which carries the key antigenic epitopes of β2GPI, could add value to these current tests. We performed an observational, multicenter cohort study to evaluate the utility of IgG, IgM and IgA assays to each of CL, β2GPI and DI in APS. METHODS:Serum from 230 patients with APS (n = 111), SLE but not APS (n = 119), and 200 healthy controls were tested for IgG, IgM and IgA aCL, aβ2GPI and aDI activity. Patients with APS were further classified into thrombotic or obstetric APS. Logistic regression and receiver operator characteristic analyses were employed to compare results from the nine different assays. RESULTS:All assays displayed good specificity for APS; IgG aCL and IgG aβ2GPI assays however, had the highest sensitivity. Testing positive for IgA aβ2GPI resulted in a higher hazard ratio for APS compared to IgM aβ2GPI. Positive IgG, IgM or IgA aDI were all associated with APS, and in subjects positive for aCL and/or aβ2GPI, the presence of aDI raised the hazard ratio for APS by 3-5 fold. IgG aCL, aβ2GPI, aDI and IgA aDI were associated with thrombotic but not obstetric complications in patients with APS. CONCLUSION:Measuring IgG aDI and IgA aβ2GPI and aDI may be useful in the management of patients with APS, particularly thrombotic APS.
First-Line, Non-Criterial Antiphospholipid Antibody Testing for the Diagnosis of Antiphospholipid Syndrome in Clinical Practice: A Combination of Anti-β -Glycoprotein I Domain I and Anti-Phosphatidylserine/Prothrombin Complex Antibodies Tests.
Nakamura Hiroyuki,Oku Kenji,Amengual Olga,Ohmura Kazumasa,Fujieda Yuichiro,Kato Masaru,Bohgaki Toshiyuki,Yasuda Shinsuke,Atsumi Tatsuya
Arthritis care & research
OBJECTIVE:To assess the value of a combination of anti-β -glycoprotein I (anti-β GPI) domain I antibody and anti-phosphatidylserine/prothrombin complex (anti-PS/PT) antibody tests for the diagnosis of antiphospholipid syndrome (APS). METHODS:This cross-sectional study involved a cohort of the patients who visited our clinic from April 2005 to March 2013. Tests for anti-β GPI domain I antibodies, IgG anti-PS/PT antibodies, and IgM anti-PS/PT antibodies, together with tests for criteria-defined antiphospholipid antibodies (aPL), were performed in all patients. The total antiphospholipid score (aPL-S) was calculated for each patient according to titers of and positivity for aPL. RESULTS:The study enrolled 157 patients (51 patients with APS and 106 with non-APS autoimmune diseases). All 21 patients positive for both anti-β GPI domain I antibodies and IgG and/or IgM (IgG/IgM) anti-PS/PT antibodies had APS with a high total aPL-S (median 46, range 26-76), as did all of the 10 patients who were positive for anti-β GPI domain I antibodies but negative for IgG/IgM anti-PS/PT antibodies (median 22, range 4-39). Of the 14 patients who were positive for IgG/IgM anti-PS/PT antibodies but negative for anti-β GPI domain I antibodies, 11 (79%) had APS; these individuals also had high total aPL-S values (median 23, range 11-60). In contrast, only 9 of the 112 patients (8%) with none of these antibodies had APS. CONCLUSION:The combination of the IgG anti-β GPI domain I antibody and IgG/IgM anti-PS/PT antibody tests shows a high positive predictive value for the diagnosis of APS and a strong correlation with the aPL-S. This combination as the first-line test for aPL may contribute to the simple and definite identification of APS with a high risk of thrombosis in clinical practice.
The IgA Isotype of Anti-β2 Glycoprotein I Antibodies Recognizes Epitopes in Domains 3, 4, and 5 That Are Located in a Lateral Zone of the Molecule (L-Shaped).
Serrano Manuel,Martinez-Flores Jose Angel,Norman Gary L,Naranjo Laura,Morales Jose Maria,Serrano Antonio
Frontiers in immunology
Antiphospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy morbidity with presence of anti-phospholipid antibodies (aPL). The APS classification criteria only consider the aPL of IgG/IgM isotype, however testing of aPL of IgA isotype is recommended when APS is suspected and consensus aPL are negative. IgA anti-βeta-2 glycoprotein-I (B2GP1) has been clearly related with occurrence of thrombotic events. Antibodies anti-B2GP1 of IgG/M isotypes recognize an epitope in Domain 1 (R39-G43), the epitopes that recognize IgA anti-B2GP1 antibodies are not well-identified. To determine the zones of B2GP1 recognized by antibodies of IgA isotype from patients with APS symptomatology and positive for IgA anti-B2GP1. IgA antibodies to Domain-1(D1) and Domain-4/5(D4/5) of B2GP1 (ELISA) and epitope mapping on oligopeptide arrays of B2GP1 were evaluated in sera from a group of 93 patients with at least one thrombotic and with isolated positivity for IgA anti-B2GP1 antibodies (negative for other aPL). A total of 47 patients (50.5%) were positive for anti-D4/5 and 23(25%) were positive for anti-D1. When peptide arrays were analyzed, three zones of B2GP1 reactivity were identified for more than 50% of patients. The center of these zones corresponds to amino acids 140(D3), 204(D4), and 264(D5). The peptides recognized on D3 and D4 contain amino acid sequences sharing high homology with proteins of microorganism that were previously related with a possible APS infectious etiology. In the three-dimensional structure of B2GP1, the three peptides, as the R39-G43 epitope, are located on the right side of the molecule (L-shape). The left side (J-shape) does not bind the antibodies. Patients with thrombotic APS clinical-criteria, and isolated IgA anti-B2GP1 positivity appear to preferentially bind, not to the D1 or D4/5 domains of B2GP1, but rather to three sites in D3, D4, and D5. The sites on D3 and D4 were previously described as the target identified by human monoclonal antibodies derived from patients that were capable of inducing APS in animal models. The localization of these epitopes opens a new route to explore to increase understanding of the patholophysiology of the APS and to propose new alternatives and therapeutic targets.
Role of anti-domain 1-β2 glycoprotein I antibodies in the diagnosis and risk stratification of antiphospholipid syndrome.
De Craemer A-S,Musial J,Devreese K M J
Journal of thrombosis and haemostasis : JTH
UNLABELLED:Essentials Antibodies to domain 1 of β2 glycoprotein I (aD1) are a subset of antiphospholipid antibodies. We evaluated the added diagnostic value of an automated aD1 assay in antiphospholipid syndrome. AD1 IgG correctly classifies patients at risk for thrombosis. Agreement between aD1 and aβ2GPI IgG is high, limiting the added value of aD1 in our setting. Click to hear Professor de Groot's perspective on new mechanistic understanding in antiphospholipid syndrome SUMMARY:Background Laboratory diagnosis of antiphospholipid syndrome (APS) includes lupus anticoagulant (LAC), anticardiolipin (aCL) or anti-β2 glycoprotein I (aβ2 GPI) antibodies. Antibodies targeting domain 1 of β2 GPI (aD1) constitute a pathogenic subset of autoantibodies. Objectives In this cohort study, we determined the clinical performance characteristics, additional diagnostic value and the contribution to APS risk stratification of an automated aD1 assay. Patients/Methods LAC, aCL, aβ2 GPI and aD1 IgG were measured in 101 APS patients, 123 patients with autoimmune disorders, 82 diseased controls and 120 healthy controls. aD1 antibodies were detected by QUANTA Flash(®) Beta2GPI-Domain 1 chemiluminescence immunoassay. Results With a cut-off value of 20.0 CU, the aD1 IgG assay identifies APS patients in a clinically affected patient cohort with a sensitivity of 53.5% and specificity of 98.8%. It implied a high odds ratio (OR) for clinical events (OR, 17.0; 95% confidence interval [CI], 7.1-40.5). aD1 IgG did not add diagnostic value to the formal aPL panel because aβ2 GPI IgG was nearly as specific but more sensitive for APS (sensitivity 56.4%) with a higher OR for clinical events (36.2; 95% CI, 11.1-117.9). High aD1 titers identify triple-positive patients and patients with thrombosis in a β2 GPI-dependent LAC-positive population. Agreement between aD1 IgG and aβ2 GPI IgG was high (positive and negative agreement 91.7% and 98.4%, respectively). Conclusion Detection of aD1 IgG correctly classifies patients at risk of thrombosis. However, the contribution of aD1 IgG to APS diagnosis and risk stratification depends upon the solid phase assays used for aCL and aβ2 GPI detection.
Coexistence of anti-β2-glycoprotein I domain I and anti-phosphatidylserine/prothrombin antibodies suggests strong thrombotic risk.
Lee Jee-Soo,Gu JaYoon,Park Hee Sue,Yoo Hyun Ju,Kim Hyun Kyung
Clinical chemistry and laboratory medicine
BACKGROUND:Highly specific assays for measuring antiphospholipid antibodies (aPLs) are required for accurate assessment of thrombotic risk. aPLs against β2-glycoprotein I domain I (anti-β2GPIdI) and against prothrombin complexed with phosphatidylserine (anti-PS/PT) have been recently identified as being associated with a hypercoagulable state. This study evaluated the synergism between anti-β2GPIdI and anti-PS/PT for predicting thrombotic events. METHODS:A total of 180 patients with clinical suspicion of hypercoagulability were evaluated. The plasma levels of lupus anticoagulant (LA) and antibodies against anticardiolipin (anti-CL) (IgG and IgM), β2GPI (IgG and IgM), PS/PT (IgG and IgM), and β2GPI dI (IgG) were measured. RESULTS:IgG anti-β2GPIdI and LA were highly associated with thrombosis. Mean values and positivity rates of IgG anti-β2GPI dI and IgG anti-PS/PT were significantly higher in the triple-positive group (LA+, IgG anti-CL+, IgG anti-β2GPI+) than in the other groups. Interestingly, the thrombotic risk [odds ratio (OR) 24.400, 95% confidence interval (CI) 1.976-63.273, p<0.001] of the newly defined triple positive group (LA+, IgG anti-CL+, IgG anti-β2GPIdI+; OR 11.182, 95% CI 1.976-63.273, p=0.006) was more than twice that of the triple-positive group (LA+, IgG anti-CL+, IgG anti-β2GPI+). Double positivity for IgG anti-PS/PT and IgG anti-β2GPI also indicated significant thrombotic risk (OR 7.467, 95% CI 2.350-23.729, p=0.001). Furthermore, the thrombotic risk associated with double positivity for IgG anti-PS/PT and IgG anti-β2GPIdI was markedly elevated (OR 33.654, 95% CI 6.322-179.141, p<0.001). CONCLUSIONS:Our data suggest that simultaneous measurement of IgG anti-β2GPIdI and IgG anti-PS/PT may improve clinical decision-making for aPL-positive patients.
Anti-domain 1 of beta2-glycoprotein I aids risk stratification in lupus anticoagulant-positive patients.
Guo Han,Zhang Yuncong,Li Aiwei,Wang Chanjuan,Yang Shuo,Zhang Yinmei,Zhang Jie,Qiao Rui
Clinical and experimental medicine
Lupus anticoagulant (LA) is considered a risk factor for thromboembolism (TE) and adverse pregnancy outcomes (APOs). However, quite a few patients diagnosed with LA positivity do not suffer these adverse events. Further testing of anticardiolipin (aCL), anti-beta2-glycoprotein I (anti-β2GPI) or anti-domain 1 of β2GPI (anti-D1) may help to assess the occurrence risk of TE and APOs. Therefore, we aimed to study how to stratify LA-positive patients. In our study, 167 LA-positive patients were consecutively enrolled from January 2015 to December 2016. Serum aCL and anti-β2GPI (IgG, IgM and IgA) and anti-D1 IgG were simultaneously measured. Among these patients, 114 (68.3%) were followed for an average of 36.5 months for TE and APOs. The outcomes showed that 105 patients experienced TE and/or APOs, and 62 patients were LA carriers. Anti-D1 had good consistency with triple positivity (LA+, aCL+, anti-β2GPI+) (kappa = 0.742). Elevated anti-D1 was related to increased risks for TE [odds ratio (OR) 29.87, 95% confidence interval (CI) 8.05-110.74] and APOs (OR 8.73, 95% CI 3.41-22.31). Area under curve showed that the diagnostic power of anti-D1 for TE and APOs was 0.856 (95% CI 0.743-0.970) and 0.682 (95% CI 0.599-0.765), respectively. Survival analysis revealed that patients with high anti-D1 titres had a high cumulative incidence of APOs (hazard ratio 4.66, 95% CI 1.46-14.87). In conclusion, anti-D1, based on good consistency with triple positivity in LA-positive patients, has a stronger association with TE and APOs and, to some degree, could predict pregnancy outcomes. Therefore, anti-D1 may aid risk stratification in LA-positive patients.
Quantitation of Total and Free Thiol β-Glycoprotein I Levels for Diagnostic and Prognostic Purposes in the Antiphospholipid Syndrome.
Qi M,Weaver James C,Krilis S A,Giannakopoulos B
Methods in molecular biology (Clifton, N.J.)
β-Glycoprotein I is the major autoantigen in the antiphospholipid syndrome (APS), a prothrombotic disorder characterized by the occurrence of either venous or arterial thrombosis. In women it is also associated with an increased risk of obstetric complications such as recurrent miscarriages. We have identified that the plasma protein β-glycoprotein I in healthy individuals exists in an optimal ratio between two distinct forms, an oxidized and free thiol, reduced form. This ratio is disrupted in pathophysiological conditions associated with increased oxidative stress such as the APS, but also in the setting of age-related macular degeneration and gram-negative sepsis. We have developed assays that quantify plasma/serum levels of total and free thiol β-glycoprotein I which can potentially be used for risk stratification and prognostic purposes in the early stages of the aforementioned conditions.
Structural and functional characterization of β -glycoprotein I domain 1 in anti-melanoma cell migration.
Leu Shr-Jeng Jim,Lee Tzong-Yi,Cheng Shu-Wei,Tsai Meng-Ying,Lin Yu-Shan,Chiou Tzeon-Jye,Huang Kai-Yao,Chiang An-Na
We previously found that circulating β -glycoprotein I inhibits human endothelial cell migration, proliferation, and angiogenesis by diverse mechanisms. In the present study, we investigated the antitumor activities of β -glycoprotein I using structure-function analysis and mapped the critical region within the β -glycoprotein I peptide sequence that mediates anticancer effects. We constructed recombinant cDNA and purified different β -glycoprotein I polypeptide domains using a baculovirus expression system. We found that purified β -glycoprotein I, as well as recombinant β -glycoprotein I full-length (D12345), polypeptide domains I-IV (D1234), and polypeptide domain I (D1) significantly inhibited melanoma cell migration, proliferation and invasion. Western blot analyses were used to determine the dysregulated expression of proteins essential for intracellular signaling pathways in B16-F10 treated with β -glycoprotein I and variant recombinant polypeptides. Using a melanoma mouse model, we found that D1 polypeptide showed stronger potency in suppressing tumor growth. Structural analysis showed that fragments A and B within domain I would be the critical regions responsible for antitumor activity. Annexin A2 was identified as the counterpart molecule for β -glycoprotein I by immunofluorescence and coimmunoprecipitation assays. Interaction between specific amino acids of β -glycoprotein I D1 and annexin A2 was later evaluated by the molecular docking approach. Moreover, five amino acid residues were selected from fragments A and B for functional evaluation using site-directed mutagenesis, and P11A, M42A, and I55P mutations were shown to disrupt the anti-melanoma cell migration ability of β -glycoprotein I. This is the first study to show the therapeutic potential of β -glycoprotein I D1 in the treatment of melanoma progression.