anti-β2GPI Autoantibodies Research Articles

Anti-β2-glycoprotein I autoantibodies influence thrombin generation parameters via various mechanisms

IntroductionAntiphospholipid syndrome (APS) is a systemic autoimmune disease characterised by recurrent thrombotic events, pregnancy loss and thrombocytopenia and the presence of antiphospholipid antibodies (APL). The exact pathomechanism of APS is still unknown, thus we investigated the effect of anti-β2-glycoprotein I (anti-β2GPI) on thrombin generation in different plasma samples. MethodsFor the separation of anti-β2GPI IgG, overall 12 APS patients were selected. The criteria were the existence of lupus anticoagulant, and the presence of anti-CL and anti-β2GPI, the latter exceeding at least 25 times the upper reference limit. We purified anti-β2GPI IgG antibodies from APS patients by affinity chromatography and added the antibodies to normal pooled, and heterozygous forms of inherited thrombophilia plasma samples (prothrombin G20210A, factor V Leiden). To further specify the mechanism of the effect, we also used factor deficient plasmas in the thrombin generation assay. ResultsIn normal pooled plasma, the anti-β2GPI significantly prolonged Lag Time according to the lupus anticoagulant effect, in contrast, it also elevated Peak Thrombin significantly, which suggests a procoagulant effect. The antibody was also able to exert this multi-faceted effect both in FVLeiden heterozygous plasma and prothrombin G20210A heterozygous polymorphism, however, the prolonging effect was more remarkable in the latter. By using factor deficient plasmas, it was found that FVII is required for the prolongation, while intrinsic factors are needed for the elevation of the Peak Thrombin. ConclusionThe anti-β2GPI autoantibodies exert their effect in both normal and thrombophilic plasmas via various mechanisms.

Distinct and overlapping effects of β2-glycoprotein I conformational variants in ligand interactions and functional assays

One of the most abundant coagulation proteins is β2-glycoprotein I (β2GPI) that is present in humans at a concentration of around 200 mg/L. Its physiological role is only partially understood, but it adopts several different structural forms the majority of which are the open and closed forms. We isolated native (circular) β2GPI and converted it into an open conformation. The effectiveness of these procedures was assessed by Western blot and negative-staining electron microscopy. We found that in coagulation assays the open form of β2GPI had a significant prolonging effect on fibrin formation in a dilute prothrombin time test (p < 0.001). In the dilute activated partial thromboplastin time test, both conformations had a significant prolonging effect (p < 0.001) but the open conformation was more effective. In a fluorescent thrombin generation assay both conformations slightly delayed thrombin generation with no significant effect on the quantity of formed thrombin. By using surface plasmon resonance assays, the equilibrium dissociation constants of both the open and closed conformations of β2GPI showed a similar and strong affinity to isolated anti-β2GPI autoantibodies (Kd closed β2GPI = 5.17 × 10−8 M, Kd open β2GPI = 5.56 × 10−8 M) and the open form had one order of magnitude stronger affinity to heparin (Kd = 0.30 × 10−6 M) compared to the closed conformation (Kd = 3.50 × 10−6 M). The two different forms of β2GPI have distinct effects in functional tests and in ligand binding, which may considerably affect the intravascular events related to this abundant plasma protein in health and disease.

Necroptotic cell binding of β2 -glycoprotein I provides a potential autoantigenic stimulus in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is characterized by the development of autoantibodies against diverse self-antigens with damage to multiple organs. Immunization with the SLE autoantigen β2 -glycoprotein I (β2 GPI) and lipopolysaccharide (LPS), a known trigger of necroptosis, induces a murine model of SLE. We hypothesized that necroptotic cells, like apoptotic cells, provide a "scaffold" of cellular self-antigens, but, unlike apoptotic cells, necroptotic cells do so in a proinflammatory and immunogenic context. We demonstrate that β2 GPI indeed binds to necroptotic cells and serves as a target for anti-β2 GPI autoantibodies. We further demonstrate that necroptotic, but not apoptotic, cells promote antigenic presentation of β2 GPI to CD4 T cells by dendritic cells. Finally, we show that β2 GPI/LPS-immunized mice deficient in RIPK3 (receptor-interacting serine/threonine-protein kinase 3) or MLKL (mixed lineage kinase domain like), and consequently unable to undergo necroptosis, have reduced SLE autoantibody production and pathology. RIPK3-/- mice had low levels of SLE autoantibodies and no renal pathology, while MLKL-/- mice produced low levels of SLE autoantibodies initially, but later developed levels comparable with wild type (WT) mice and pathology intermediate to that of WT and RIPK3-/- mice. Serum cytokine levels induced by LPS tended to be lower in RIPK3-/- and MLKL-/- mice than in WT mice, suggesting that impaired proinflammatory cytokine production may impact the initiation of autoantibody production in both strains. Our data suggest that self-antigen (i.e. β2 GPI) presented in the context of necroptosis and proinflammatory signals may be sufficient to overcome immune tolerance and induce SLE.

Oral administration of Domain-I of beta-2glycoprotein-I induces immunological tolerance in experimental murine antiphospholipid syndrome

It is well established that the humoral immunity in antiphospholipid syndrome (APS) is presented by circulating pathogenic anti-β2GPI autoantibodies targeting mainly domain I of the β2GPI protein, playing a major role in the disease pathogenesis. Previously, we have demonstrated that treatment of experimental APS mice with tolerogenic dendritic cells loaded with domain-I was more efficient in tolerance induction than with the whole molecule or domain-V. In the current study we had orally administered a domain-I derivative of the β2GPI molecule, as a new therapeutic approach to induce oral tolerance in this mouse model of APS. BALB/c mice immunized with β2GPI, were fed with either domain-I, domain-V derivative or the complete β2GPI protein. β2GPI immunized mice developed experimental APS which were fed with domain-I significantly had decreased fetal loss (p < 0.004), a lower size of thrombi (p < 0.001) and lower circulating anti-β2GPI Abs in comparison to mice fed with domain-V or PBS (p < 0.002). Likewise, Domain-I fed mice had a lowered inflammatory response, exhibited by decreased expression of inflammatory cytokines (IFNγ, IL-6, IL-17) and elevated production of IL-10 anti-inflammatory cytokine by splenocytes. Moreover, the anti-inflammatory response in the domain-I fed APS mice was associated with increased circulating miRNA variations (155, 146, 182, 98) by RT-PCR, which are associated with immunomodulation of the immune network.We propose that oral tolerance with domain-I can be a novel therapy for patients with APS.

Antiphospholipid Antibodies in Women with Recurrent Urinary Tract Infection

Background: A variety of infections, including acute and recurrent urinary tract infections (UTIs), can trigger production of antiphospholipid antibodies (aPL). These antibodies in women can lead to recurrent pregnancy loss. The aim of this study was to evaluate the prevalence of aPL in recurrent UTI patients.Materials and Methods: A total 52 subjects who had positive urine culture and 50 healthy individuals as controls were evaluated for presence of lupus anticoagulant (LA), anticardiolipin, anti-β2 GPI(anti-𝛽2-glycoprotein I) autoantibodies IgM and IgG and Interleukin-8 levels. Determination of lupus anticoagulant was done by Activated Partial Thromboplastin Time (aPTT). Anticardiolipin and anti-β2 GPI autoantibodies were evaluated by ELISA method. Interleukin-8 values were also evaluated using ELISA method. Results: Escherichia coli (86.61%) and Proteus mirabilis (1.92%) had the highest and lowest frequency respectively. The prevalence of anti- β2 GPI IgG and IgM isotypes and anticardiolipin IgG and IgM isotypes or LA in UTI patients and healthy controls was 0.0%. There was significant association between neutrophil counts and IL-8 levels at the p < 0.01.Conclusion: Our results showed that in the UTI group and controls evaluated antiphospholipid antibodies were not present. The production of antiphospholipid antibodies is influenced by various genetic and environmental factors and chronic urinary tract infection alone is not the cause. This can affect the prevalence of antiphospholipid antibodies in various populations. However, other factors, such as the type of antiphospholipid antibody, sampling season and methodology can affect the results.

Laboratory methods to detect antiphospholipid antibodies.

This chapter reviews several important themes pertaining to the antiphospholipid syndrome (APS), including a description of the clinical features, a discussion of the main autoantigen, beta 2-glycoprotein I (β2GPI), and insights into the characteristics of the pathogenic anti-β2GPI autoantibodies. Evidence-based considerations for when to test for APS are explored, along with the clinical significance of patients testing positive on multiple APS assays, so-called triple positivity. A detailed review of recently published laboratory guidelines for the detection of lupus anticoagulant and the solid-phase anticardiolipin and anti-β2GPI ELISAs is undertaken. Finally, a brief review of nonclassification criteria laboratory assays with potential future diagnostic utility is presented.

Involvement of TLR4 in oxidized LDL/β2GPI/anti-β2GPI-induced transformation of macrophages to foam cells.

It has been reported that oxidized low-density lipoprotein (oxLDL) forms a stable and non-dissociable complex with β2-glycoprotein I (β2GPI) and that IgG anti-β2GPI autoantibodies are able to recognize this complex, thus facilitating macrophage-derived foam cell formation in patients with antiphospholipid syndrome (APS). However, the immunopathological mechanisms of oxLDL/β2GPI complexes in promoting foam cell formation are not fully understood. In this study, we examined the role of toll-like receptor 4 (TLR4) in the oxLDL/β2GPI/anti-β2GPI complex-induced transformation of mouse peritoneal macrophages to foam cells. Oil red O staining and optical density (OD) measurements of intracellular stained oil red O solution were used to monitor the transformation of peritoneal macrophages to foam cells in TLR4-competent C3H/HeN and TLR4-mutant C3H/HeJ mice. During foam cell formation induced by the oxLDL/β2GPI/anti-β2GPI complex, the expression of TLR4 and activation of nuclear factor kappa B (NF-κB) were confirmed by analyzing the protein and mRNA levels of these compounds. Furthermore, the related active molecule expression during foam cell formation induced by the oxLDL/β2GPI/anti-β2GPI complex was examined in the presence or absence of TLR4. The data showed that treatment with the oxLDL/β2GPI/anti-β2GPI complex markedly increased foam cell formation, the TLR4 expression, NF-κB activation, the tissue factor (TF) expression and tumor necrosis factor-α (TNF-α) and monocyte chemotactic protein-1 (MCP-1) secretion in the C3H/HeN mice. However, the transformation of macrophages to foam cells and the expression levels of phosphorylated NF-κB, TF, TNF-α and MCP-1 were significantly reduced in the C3H/HeJ mice treated with the oxLDL/β2GPI/anti-β2GPI complex. In addition, compared with that achieved by oxLDL alone, the oxLDL/β2GPI complex decreased foam cell formation and the related signaling molecule expression in the C3H/HeN mice. Our results indicate that TLR4 plays an important role in the process of oxLDL/β2GPI/anti-β2GPI complex-induced transformation of macrophages to foam cells, which may accelerate the development of atherosclerosis in the setting of APS. However, β2GPI alone functions as an antiatherogenic protein by preventing the foam cell formation induced by oxLDL.

Pathophysiology of β2-glycoprotein I in antiphospholipid syndrome

Since beta(2)-glycoprotein I (beta(2)GPI) was described as the major antigenic target for antiphospholipid antibodies, many studies have focused their attention to the physiological role of beta(2)GPI and anti-beta(2)GPI antibodies on autoimmune-mediated thrombosis. Studies reporting the physiological role of beta(2)GPI have been numerous, but the exact mechanism of action(s) has yet to be completely determined. beta(2)GPI's epitopes for anti-beta(2)GPI autoantibodies have been characterized, however, not all of the heterogeneous anti-beta(2)GPI antibodies are pathogenic. The pathophysiologic role of beta(2)GPI has been reported in the fields of coagulation, fibrinolysis, angiogenesis, and atherosclerosis. Our understanding of the impact of beta(2)GPI, its metabolites and autoantibodies to beta(2)GPI on these physiological functions may contribute to the development of better therapeutic strategies to treat and prevent autoimmune-mediated atherothrombotic vascular disease.

Diluted Russell viper-venom time improves identification of antiphospholipid syndrome in a lupus anticoagulant-positive patient population

The objective of this retrospective study was to evaluate the potential ability of diluted Russell viper-venom time (dRVVT) to identify antiphospholipid syndrome (APS) in a lupus anticoagulant (LA)-positive patient population, already selected by other LA clotting tests. Our cohort of positive LA patients was first identified in our outpatients population by the following sensitive LA-detecting tests: Rosner index, diluted prothrombin time (dPT) and Rosove index. Then the 227 consecutive LA-positive patients were tested for dRVVT with the same blood sample. Anticardiolipin (aCL) and anti-beta(2)-glycoprotein-I (beta(2)GPI) autoantibodies assays were also performed. APS using Sapporo clinical criteria revised at Sydney, was found in 116 of these 227 consecutive LA-positive patients. Results of the different tests were analysed statistically. Using univariate analysis, dRVVT, dPT, IgG aCL and IgG anti-beta(2)GPI autoantibodies were significantly associated with APS. The receiver operating-characteristics (ROC) curve defined the best cut-off value for dRVVT ratio at 1.61 with a good specificity (78%) and a lower sensitivity (53%). A multivariate analysis using a binary logistic procedure, retained the dRVVT ratio (> or = 1.61) and IgG anti-beta(2)GPI autoantibodies (> 15 USG) as being associated with APS (p = 0.018; odds ratio [OR] 2.39; 95% confidence interval [CI] 1.2-4.7, and p = 0.0001; OR 3.2; 95% CI 1.5-6.5, respectively). To conclude, these results agree with the need for LA criteria favouring specificity over sensitivity. The use of a threshold around 1.6 for dRVVT ratio should help discriminate APS from non-APS patients.

Distinguished effects of antiphospholipid antibodies and anti-oxidized LDL antibodies on oxidized LDL uptake by macrophages

Several interpretations have been made regarding the specificity of antiphospholipid antibodies and antibodies against oxidized low-density lipoprotein (oxLDL), but these are still controversial. In the present study, we delineated specificity of these two types of antibodies and analyzed their regulatory effect on oxLDL and/or beta( 2)-glycoprotein I (beta(2)GPI) binding to macrophages. Scavenger receptor-mediated binding of oxLDL (or its beta(2)GPI complexes) to macrophages was observed and the binding was partly prevented by beta( 2)GPI. The IgG monoclonal anti-beta(2)GPI antibody (WB-CAL-1), which was derived from NZW x BXSB F1 mouse (a model of antiphospholipid syndrome), significantly increased the oxLDL/beta(2)GPI binding to macrophages. In contrast, IgM anti-oxLDL natural antibody, EO6 (derived from apoe( -/-) mouse), prevented the binding. Different antigenic specificity of these antibodies to oxLDL and its beta(2)GPI complexes was also confirmed in TLC-ligand blot and ELISA. Thus, IgG anti-beta(2) GPI autoantibodies contribute to lipid metabolism (housekeeping of oxLDL by macrophages) whereas IgM natural anti-oxLDL antibodies may protect against atherogenesis. In addition, in vitro data suggest that relatively high dose of intravenous immunoglobulin preparations (mainly contain IgG anti-oxLDL antibodies) might also prevent atherogenesis by inhibiting the oxLDL binding to macrophages.

β2-Glycoprotein I inhibits von Willebrand factor–dependent platelet adhesion and aggregation

AbstractPatients with antiphospholipid syndrome are characterized by the association of thrombosis or pregnancy morbidity and the presence of antiphospholipid autoantibodies. Particularly, anti-β2-glycoprotein (β2 GPI) autoantibodies correlate with thrombosis, suggesting an antibody-induced gain of prothrombotic function and/or an antibody-induced loss of antithrombotic function of β2 GPI. In the search for potential antithrombotic properties of β2 GPI, we found that β2 GPI inhibits von Willebrand factor (VWF)–induced platelet aggregation. In addition, platelet adhesion to a VWF-coated surface was decreased by 50% in the presence of β2 GPI (P < .03). β2 GPI binds to the A1 domain of VWF but preferably when the A1 domain is in its active glycoprotein Ibα-binding conformation. Anti-β2 GPI antibodies isolated from a subset of antiphospholipid syndrome patients neutralized the β2 GPI-VWF interactions and thus the inhibitory activity of β2 GPI. In comparison to healthy individuals, the amounts of active VWF in circulation were increased 1.5-fold (P < .001) in patients positive for lupus anticoagulant (LAC) due to anti-β2 GPI antibodies. Thus, β2 GPI is a biologically relevant inhibitor of VWF function by interfering with VWF-dependent platelet adhesion. Anti-β2 GPI autoantibodies neutralize this inhibitory function and are associated with increased levels of active VWF. This mode of action could contribute to the thrombosis and consumptive thrombocytopenia observed in patients with anti-β2 GPI antibodies.

Intracellular trafficking of β 2-glycoprotein I complexes with lipid vesicles in macrophages: Implications on the development of antiphospholipid syndrome

β 2-Glycoprotein I (β 2GPI) is known as a major autoantigen for antiphospholipid antibodies. Our recent data show that binding of β 2GPI to oxidized low-density lipoprotein (oxLDL) or to liposomes containing anionic phospholipid(s) may facilitate the presentation of β 2GPI's epitope by macrophages/dendritic cells to autoreactive T cells. In the present study, we investigated intracellular trafficking of β 2GPI and its complexes with oxLDL or liposomes containing phosphatidylserine (PS-liposomes) in mouse macrophage-like J774 cells. A relatively small amount of non-complexed β 2GPI was taken up and stagnated in the late endosome after incubating for 16 h. In contrast, β 2GPI complexes with oxLDL or PS-liposomes were transported into the lysosome. In the presence of the IgG anti-β 2GPI autoantibody, WB-CAL-1, β 2GPI/oxLDL complexes were rapidly incorporated into intracellular space and were finally localized in the lysosome. Interestingly, in vitro pulses by β 2GPI/oxLDL complexes together with WB-CAL-1 led to the expression of membranous CD36 as well as Fcγ type I receptors (FcγRI). These observations suggest that IgG immune complexes of β 2GPI/oxLDL provide not only FcγRI- but also scavenger receptor-mediated uptake of β 2GPI/oxLDL complexes by macrophages. Thus, β 2GPI/oxLDL complexes as a major atherogenic autoantigen and IgG anti-β 2GPI autoantibodies may facilitate antigen presentation and foam cell formation in antiphospholipid syndrome.

Requirement for dendritic cells in the establishment of anti-phospholipid antibodies

Objective: The cross-presentation of cell-associated autoantigens contributes to systemic autoimmune diseases, including systemic lupus erythematosus (SLE). Little is known about the regulation of the immune response against soluble autoantigens targeted in these diseases.Methods: We immunized the offspring of New Zealand Black and New Zealand White mice (NZB × NZW F1) with syngeneic dendritic cells (DC) that had macropinocytosed β2-glycoprotein 1 (β2GPI) during propagation in normal mouse serum or that had phagocytosed apoptotic thymocytes with syngeneic (murine) or xenogeneic (bovine) β2GPI, which was associated to plasma membrane of the cells. Mice were in parallel immunized with apoptotic thymocytes that had associated the β2GPI to their membranes in the absence of DC. The development of anti-β2GPI antibodies and clinical features were monitored.Results. Apoptotic cells alone, opsonized with β2GPI, failed to induce anti-β2GPI autoantibodies or clinical disease. In contrast, autoimmunity developed in the presence of DC. Furthermore, the syngeneic β2GPI was a more effective antigen than the xenogeneic protein in re-boosted animals.Conclusions. DC effectively initiate in NZB × NZW F1 mice self-sustaining autoimmunity against the β2GPI, either associated to apoptotic cells or macropinocytosed from the serum.

Patients with atherosclerotic syndrome, negative in anti-cardiolipin assays, make IgA autoantibodies that preferentially target domain 4 of β 2-GPI

Autoantibodies targeting β 2-glycoprotein l (β 2-GPI), a component of the atherosclerotic plaque, are commonly found in patients with acute ischemic syndromes. Serum samples from APS (antiphospholipid syndrome) patients and from cardiovascular patients exhibiting acute atherosclerotic syndromes were analyzed for IgG and IgA antibodies in both anti-β 2-GPI and anticardiolipin (aCL) ELISA assays. All of the APS samples used here were positive in both assays. Serum samples from 382 atherosclerosis patients were also analyzed for IgG and IgA antibodies in the same assays. In sharp contrast to the APS samples, we found that only 1% of the samples from atherosclerosis patients were positive for IgA aCL, and 1.6% positive for IgG aCL, whereas 35.6% were positive for IgA anti-β 2-GPI and only 1.6% for IgG anti-β 2-GPI. The antigenic specificity of 29 serum samples from atherosclerosis patients was evaluated. Six different recombinant domain-deleted mutants (DM) of human β 2-GPI and full-length human β 2-GPI (wild-type) were used in competitive inhibition assays to inhibit the autoantibodies from binding in the anti-β 2-GPI ELISA assays. Domain-deleted mutants D—345 and D---45 inhibited the binding in the IgA anti-β 2-GPI assay, suggesting that these autoantibodies recognize domain 4 of the β 2-GPI molecule. These results clearly show that IgA anti-β 2-GPI autoantibodies from atherosclerotic patients are distinct from IgA autoantibodies found in APS samples.

Adalimumab clinical efficacy is associated with rheumatoid factor and anti-cyclic citrullinated peptide antibody titer reduction: a one-year prospective study

Studies on autoantibody production in patients treated with tumor necrosis factor-α (TNF-α) inhibitors reported contradictory results. We investigated in a prospective study the efficacy of a treatment with human monoclonal anti-TNF-α antibody (adalimumab) in patients with rheumatoid arthritis (RA) and we evaluated the relationship between treatment efficacy and the incidence and titers of disease-associated and non-organ-specific autoantibodies. Fifty-seven patients with RA not responsive to methotrexate and treated with adalimumab were enrolled. Antinuclear, anti-double-stranded(ds)DNA, anti-extractable nuclear antigens, anti-cardiolipin (aCL), anti-β2 glycoprotein I (anti-β2GPI) autoantibodies, rheumatoid factor (RF) and anti-cyclic citrullinated peptide (anti-CCP) autoantibodies were investigated at baseline and after 6 and 12 months of follow-up. Comparable parameters were evaluated in a further 55 patients treated with methotrexate only. Treatment with adalimumab induced a significant decrease in RF and anti-CCP serum levels, and the decrease in antibody titers correlated with the clinical response to the therapy. A significant induction of antinuclear autoantibodies (ANA) and IgG/IgM anti-dsDNA autoantibodies were also found in 28% and 14.6% patients, respectively, whereas aCL and anti-β2GPI autoantibodies were not detected in significant quantities. No association between ANA, anti-dsDNA, aCL and anti-β2GPI autoantibodies and clinical manifestations was found. Clinical efficacy of adalimumab is associated with the decrease in RF and anti-CCP serum levels that was detected after 24 weeks and remained stable until the 48th week of treatment. Antinuclear and anti-dsDNA autoantibodies, but not anti-phospholipid autoantibodies, can be induced by adalimumab but to a lower extent than in studies with other anti-TNF blocking agents.

The Orientation of β2GPI on the Plate is Important for the Binding of Anti-β2GPI Autoantibodies by ELISA

(β2-glycoprotein I (β2GPI) is a plasma protein that plays an important role in the antigenic specificity of antiphospholipid autoantibodies (aPL). These antibodies are associated with an increased risk for thrombosis and recurrent foetal loss in humans. Crystallographic analysis of β2GPI showed that its five complement control protein (CCP) or ‘sushi’ domains are arranged in an elongated, fish-hook shape; yet the domain-specific location of epitopes recognized by these autoantibodies has remained the subject of considerable controversy. Investigators have used different forms of recombinant β2GPI and different ELISA methods to obtain conflicting results. One group mapped autoimmune epitopes to domain I using deletion mutants of β2GPI in a competitive inhibition ELISA on NUNC Maxisorp™ microplates. Another group mapped epitopes to domain IV using β2GPI with mutations in domain IV in a direct binding ELISA on polyoxygenated microplates. In an effort to resolve these discrepancies, a collaboration between the groups compared wildtype β2GPI with domain IV mutants in both types of ELISA. Autoantibodies bound very poorly to domain IV mutants coated on polyoxygenated plates, yet they bound very well to the same mutants coated on NUNC Maxisorp™ plates. The amount of protein adsorbed on to both types of plates was similar. In the competitive inhibition ELISA, no difference could be detected between wildtype β2GPI and domain IV mutants. These results strongly suggest that the orientation of β2GPI on the microplate, and not necessarily the lateral density, plays the predominant role in the binding of autoantibodies.

Bacterial induction of autoantibodies to β2-glycoprotein-I accounts for the infectious etiology of antiphospholipid syndrome

The antiphospholipid syndrome (APS) is characterized by the presence of pathogenic autoantibodies against β2-glycoprotein-I (β2GPI). The factors causing production of anti-β2GPI remain unidentified, but an association with infectious agents has been reported. Recently, we identified a hexapeptide (TLRVYK) that is recognized specifically by a pathogenic anti-β2GPI mAb. In the present study we evaluated the APS-related pathogenic potential of microbial pathogens carrying sequences related to this hexapeptide. Mice immunized with a panel of microbial preparations were studied for the development of anti-β2GPI autoantibodies. IgG specific to the TLRVYK peptide were affinity purified from the immunized mice and passively infused intravenously into naive mice at day 0 of pregnancy. APS parameters were evaluated in the infused mice on day 15 of pregnancy. Following immunization, high titers of antipeptide [TLRVYK] anti-β2GPI Ab’s were observed in mice immunized with Haemophilus influenzae, Neisseria gonorrhoeae, or tetanus toxoid. The specificity of binding to the corresponding target molecules was confirmed by competition and immunoblot assays. Naive mice infused with the affinity-purified antipeptide Ab’s had significant thrombocytopenia, prolonged activated partial thromboplastin time and elevated percentage of fetal loss, similar to a control group of mice immunized with a pathogenic anti-β2GPI mAb. Our study establishes a mechanism of molecular mimicry in experimental APS, demonstrating that bacterial peptides homologous with β2GPI induce pathogenic anti-β2GPI Ab’s along with APS manifestations.

Characterization of binding interactions in solution between β-2-glycoprotein I and monoclonal antibodies

The circulating so-called antiphospholipid antibodies associated with autoimmune thrombophilia appear in most cases to be directed against phospholipid-binding proteins, chiefly apolipoprotein H (β-2-glycoprotein I, β2gpI) or prothrombin. Accordingly, the determination of antibodies reacting with β2gpI in solid-phase immunoassays is useful in the diagnostic workup of thrombophilic patients. These antibodies are characterized by significant binding only when β2gpI is immobilized on binding, chemically activated, or phospholipid-coated surfaces. It is not yet known, however, if the binding of antibodies is a result of a specific conformation that β2gpI attains when immobilized on a surface or simply a reflection of a sufficiently high epitope density. We are establishing solution binding assays based on capillary electrophoresis to measure antibody-β2gpI interactions without immobilizing any of the interacting molecules. Here we describe the optimization of analysis conditions in uncoated and coated capillaries for native and succinylated human β2gpI. Monoclonal anti-β2gpI and the humanized monoclonal antibodies proposed as standards for solid-phase assays were used as model ligand systems and we demonstrate the measurement and quantitation of binding. The influence of changes in buffer conditions such as ionic strength, pH, and of buffer additives including heparin - a known β2gpI ligand - is easily assessed by this approach and minute amounts of biological material are consumed (nanoliter injection volumes). The set-up is well suited for determining weak affinity interactions and will be helpful in providing answers to the questions about solution affinities of patient antibodies for native β2gpI and thus for understanding the molecular pathology behind the thrombophilic effect of anti-β2gpI autoantibodies.