Human beta 2-glycoprotein I binds to endothelial cells through a cluster of lysine residues that are critical for anionic phospholipid binding and offers epitopes for anti-beta 2-glycoprotein I antibodies

Beta 2-Glycoprotein I (beta 2GPI) is a phospholipid-binding protein recognized by serum autoantibodies from the anti-phospholipid syndrome both in cardiolipin- and beta 2GPI-coated plates. We found that: 1) recombinant wild-type beta 2GPI bound to HUVEC and was recognized by both human monoclonal IgM and affinity-purified polyclonal IgG anti-beta 2GPI anti-phospholipid syndrome Abs; and 2) a single amino acid change from Lys286 to Glu significantly reduced endothelial adhesion. Double and triple mutants (from Lys284,287 to Glu284,287, from Lys286,287 to Glu286,287, and from Lys284,286,287 to Glu284,286,287) completely abolished endothelial binding. A synthetic peptide (P1) spanning the sequence Glu274-Cys288 of the beta 2GPI fifth domain still displayed endothelial adhesion. Another peptide (P8), identical with P1 except that Cys281 and Cys288 were substituted with serine residues, did not bind to HUVEC. Anti-beta 2GPI Abs, once bound to P1 adhered to HUVEC, induced E-selectin expression and up-regulated IL-6 secretion. Control experiments conducted with irrelevant Abs as well as with the P8 peptide did not show any endothelial Ab binding nor E-selectin and IL-6 modulation. Our results suggest that: 1) beta 2GPI binds to endothelial cells through its fifth domain; 2) the major phospholipid-binding site that mediates the binding to anionic phospholipids is also involved in endothelial binding; 3) HUVEC provide a suitable surface for beta 2GPI binding comparable to that displayed by anionic phospholipids dried on microtiter wells; and 4) the formation of the complex between beta 2GPI and the specific Abs leads to endothelial activation in vitr

Anti–Neutrophil Extracellular Trap Antibodies in Antiphospholipid Antibody–Positive Patients: Results From the Antiphospholipid Syndrome Alliance for Clinical Trials and InternatiOnal Networking Clinical Database and Repository

OBJECTIVE: This study aimed to elucidate the presence, antigen specificities, and potential clinical associations of anti–neutrophil extracellular trap (anti-NET) antibodies in a multinational cohort of antiphospholipid (aPL) antibody–positive patients who did not have lupus. METHODS: Anti-NET IgG/IgM levels were measured in serum samples from 389 aPL-positive patients; 308 patients met the classification criteria for antiphospholipid syndrome. Multivariate logistic regression with best variable model selection was used to determine clinical associations. For a subset of the patients (n = 214), we profiled autoantibodies using an autoantigen microarray platform. RESULTS: We found elevated levels of anti-NET IgG and/or IgM in 45% of the aPL-positive patients. High anti-NET antibody levels are associated with more circulating myeloperoxidase (MPO)–DNA complexes, which are a biomarker of NETs. When considering clinical manifestations, positive anti-NET IgG was associated with lesions affecting the white matter of the brain, even after adjusting for demographic variables and aPL profiles. Anti-NET IgM tracked with complement consumption after controlling for aPL profiles; furthermore, patient serum samples containing high levels of anti-NET IgM efficiently deposited complement C3d on NETs. As determined by autoantigen microarray, positive testing for anti-NET IgG was significantly associated with several autoantibodies, including those recognizing citrullinated histones, heparan sulfate proteoglycan, laminin, MPO–DNA complexes, and nucleosomes. Anti-NET IgM positivity was associated with autoantibodies targeting single-stranded DNA, double-stranded DNA, and proliferating cell nuclear antigen. CONCLUSION: These data reveal high levels of anti-NET antibodies in 45% of aPL-positive patients, where they potentially activate the complement cascade. While anti-NET IgM may especially recognize DNA in NETs, anti-NET IgG species appear to be more likely to target NET-associated protein antigens

Beta-2 glycoprotein1: function in health and disease

Beta-2 glycoprotein I (β2GPI) is the principal target of autoantibodies in the antiphospholipid syndrome (APS). It is abundant in human plasma and shares high homology between different mammalian species. Although the exact physiological function of β2GPI has not been fully elucidated, several interactions have been described with other proteins and with negatively charged surfaces, such as anionic phospholipids, dextran and heparin. β2GPI is involved in the coagulation pathway, exerting both procoagulant and anticoagulant activities. Plasma from β2GPI-deficient mice exhibits impaired thrombin generation in vitro. Recently, it has been demonstrated that β2GPI binds factor (F) XI in vitro at concentrations lower than those of the protein in human plasma, and this binding inhibits FXI activation to FXIa by thrombin and FXIIa. Proteolytic cleavage of the fifth domain of β2GPI abolishes its inhibition of FXI activation and results in reduced ability of the cleaved β2GPI to bind phospholipids. It retains its ability to bind FXI. In vivo activation of FXI by thrombin is thought to be an important mechanism by which coagulation is accelerated via components of the contact activation pathway. Thus β2GPI may attenuate the contact activation pathway by inhibiting activation of FXI by thrombin. Moreover, because β2GPI is the dominant autoantigen in patients with APS, dysregulation of this pathway by autoantibodies may be an important mechanism for thrombosis in patients with APS

Beta-2 glycoprotein I and its role in antiphospholipid syndrome: lessons from knockout mice

The antiphospholipid syndrome is characterized by the presence in serum of autoantibodies against h2GPI. Although the role of h2GPI in the pathogenesis of antiphospholipid antibody syndrome (APS) is well recognized, its exact physiological functions still remain undisclosed. Several interactions of h2GPI with components of the coagulation cascade have been proposed, resulting in both procoagulant and anticoagulant effects. Additionally, h2GPI has been implicated in the mechanism of recurrent fetal loss entailed in APS. Recently, using a homologous recombination approach, reproduction of mice homozygous for deletion of the b2GPI gene has been feasible. h2GPI knockout mice offer a valuable tool for revealing the physiological role of the protein. These mice show decreased in vitro ability for thrombin generation. Furthermore, although mice lacking h2GPI are fertile, the success of early pregnancy is moderately compromised and functional h2GPI is believed necessary for optimal implantation and placental morphogenesis

Antibodies to β2-glycoprotein-I: Urea resistance, binding specificity, and association with thrombosis

The aim of the present study was to evaluate the urea resistance and binding characteristics of anti-β2-glycoprotein I (antiβ2GPI) antibodies using standard anticardiolipin (aCL) and anti-β2GPI enzyme immunosorbent assays (ELISAs). Sera from patients with antiphospholipid syndrome (APS) (n = 22) and non-APS (n = 24), positive in a standard aCL ELISA, were tested in an anti-β2GPI ELISA performed in polystyrene-irradiated ELISA plates. Urea resistance aCL and anti-β2GPI ELISAs were performed by measuring the ability of antibodies to recognize antigen in the presence of 2 M urea. The serum dilution after urea treatment (D) expressed as a percentage of the serum dilution without urea treatment (D(o)) corresponding to the same optical density was defined as residual activity (RA = 100 D/D(o)). The higher the RA, the higher the resistance of the antibodies to urea. APS compared to non- APS sera had higher aCL binding (absorbance values ranging between 0.180 and 1.400; median, 0.717 vs 0.120-1.273; median, 0.250, respectively; P < 0.004). Six APS patients' sera had low aCL levels but they expressed RA ≥30%. Anti- β2GPI antibodies were detected in 15 of 22 APS vs 3 of 24 non-APS patients (P < 0.03); RA ≥30% was detected in 15 of 22 APS vs 1 of 23 non-APS patients (P < 0.004). Using a CL affinity column, antibodies were purified from three APS anti-β2GPI negative and three non-APS anti-β2GPI-positive patients and tested in a aCL ELISA, using highly purified bovine serum albumin (BSA) as a blocking agent (modified ELISA); reactivity was not detected in two APS and one non-APS sera. On the contrary, the reactivity of the purified antibodies was high when β2GPI was incubated with CL in the ELISA plates; thus some anti-β2GPI negative sera from APS patients recognized the CL/β2GPI complex, rather than CL or β2GPI alone. In conclusion, anti-β2GPI antibodies are common in the APS patients, but a number of such patients recognize the CL/β2GPI complex and not CL or β2GPI. Antibodies to either β2GPI or the CL/β2GPI complex derived from APS sera present a high resistance to urea. Anti-β2GPI antibodies of low urea resistance exist in a minority of non-APS patients with autoimmune disease