Anti-beta(2)-glycoprotein I antibodies in children with atopic dermatitis

beta(2)-Glycoprotein I (beta(2)GPI) appears to be the major antigen for antiphospholipid antibodies (aPL) in patients with antiphospholipid syndrome (APS). In early infancy, virtually all children initiate transient immune response to non-pathogenic nutritional antigens, which fails to terminate in children with atopic diseases. To examine the possibility that a prolonged immune response to beta(2)GPI could also spread to the human protein, antibodies against human beta(2)GPI (anti-beta(2)GPI) were determined in 93 randomly selected children with different allergic diseases. A high frequency (42%) of IgG anti-beta(2)GPI was found in children with atopic dermatitis (AD), but not in those with other allergic diseases. Anti-beta(2)GPI in children with AD were exclusively of the IgG1 subclass and bound to bovine beta(2)GPI as well, but not to either beta(2)GPI combined with the phospholipid cardiolipin. The epitopes were identified in domain V of beta(2)GPI and the antibody binding was abolished upon the specific proteolytic cleavage of the phospholipid-binding C-terminal loop in domain V of beta(2)GPI. These results indicated that the epitopes for anti-beta(2)GPI in children with AD most likely resided in close vicinity of the phospholipid-binding site of beta(2)GPI. The epitopic difference from anti-beta(2)GPI in APS may explain presumed non-thrombogenicity of anti-beta(2)GPI in children with AD.     

Effect of Antiphospholipid Antibodies on β2Glycoprotein I-Phospholipid Interaction

PROBLEM: β₂ glycoprotein I (β₂GPI) physiologically binds to negatively charged phospholipids (PLs) and is a natural regulator of the coagulation cascade. Thrombotic clinical complications and recurrent fetal loss associated with autoimmune antiphospholipid (aPL) antibodies are thought to be related to their binding to β₂GPI-PL complex and interference with the physiological function of β₂GPI. METHOD OF STUDY: To investigate the effect of aPL on β₂GPI-PL interaction, we studied the binding of biotinylated β₂GPI to cardiolipin (CL) by enzyme-linked immunosorbent assay (ELISA) in the presence and absence of purified aPL immunoglobulin G (IgG) antibodies. RESULTS: Adding five different aPL IgG antibodies with different levels of aPL activity isolated from the sera of five patients with aPL-associated recurrent fetal death greatly increased the binding of biotinylated β₂GPI to CL-coated plates. The optical densities (ODs) were 0.635, 0.890, and 1.265 in the presence of three aPL IgG antibodies, compared to 0.425 in the absence of aPL IgG. In contrast, normal human IgG had no enhancing effect. The OD was 0.480 and 0.425, respectively. The enhancement of β₂GPI binding to CL by aPL IgG correlated with the titers of aPL antibodies. The use of phosphate-buffered saline with increasing salt concentrations as a washing buffer for the ELISA resulted in more stable binding of β₂GPI to PL in the presence of aPL IgG. CONCLUSIONS: These findings suggest that the binding of autoimmune aPL antibodies to β₂GPI-PL complex results in abnormally tighter interaction between β₂GPI and PLs, which may lead to physiological dysfunction of β₂GPI as a regulator of coagulation.     

Interaction of giant phospholipid vesicles containing cardiolipin and cholesterol with beta2-glycoprotein-I and anti-beta2-glycoprotein-I antibodies

Antiphospholipid syndrome is characterized with thrombotic events and/or pregnancy morbidity and antiphospholipid antibodies (aPL). The most common antigen for aPL is beta2-glycoprotein-I (beta(2)GPI), a plasma protein binding to negatively charged phospholipids. The influence of aPL on coagulation is not well understood. Giant phospholipid vesicles (GPVs) are a convenient in vitro system for studying interactions between phospholipid membranes and proteins resulting in the change of the vesicles' configuration. We aimed to set up an in vitro model and to study changes in the morphology of GPVs with high content of cardiolipin upon addition of beta(2)GPI and/or IgG fraction of a patient with antiphospholipid syndrome (APS). Addition of the IgG fraction of the APS patient caused lateral segregation of the membrane inclusions and adhesion of GPVs. Addition of beta(2)GPI caused adhesion of GPVs. Addition of both, the patient IgG fraction and beta(2)GPI caused adhesion of vesicles to the glass slides and to each other, formation of pores and burst of vesicles. Our results indicate that adhesion of the cardiolipin-containing vesicles does not seem specific for added proteins, rather, it indicates electrostatic and curvature-mediated interactions between the membrane constituents.     

Antibodies against beta2-glycoprotein I complexed with an oxidised lipoprotein relate to intima thickening of carotid arteries in primary antiphospholipid syndrome

To explore whether antibodies against beta2-glycoprotein I (beta2GPI) complexed to 7-ketocholesteryl-9-carboxynonanoate (oxLig-1) and to oxidised low-density lipoproteins (oxLDL) relate to paraoxonase activity (PONa) and/or intima media thickness (IMT) of carotid arteries in primary antiphospholipid syndrome (PAPS). As many as 29 thrombotic patients with PAPS, 10 subjects with idiopathic antiphospholipid antibodies (aPL) without thrombosis, 17 thrombotic patients with inherited thrombophilia and 23 healthy controls were investigated. The following were measured in all participants: beta2GPI-oxLDL complexes, IgG anti-beta2GPI-oxLig-1, IgG anti-beta2GPI-oxLDL antibodies (ELISA), PONa, (para-nitrophenol method), IMT of common carotid (CC) artery, carotid bifurcation (B), internal carotid (IC) by high resolution sonography. Beta2GPI-oxLDL complex was highest in the control group (p < 0.01), whereas, IgG anti-beta2GPI-oxLig1 and IgG anti-beta2GPI-oxLDL were highest in PAPS (p < 0.0001). In healthy controls, beta2GPI-oxLDL complexes positively correlated to IMT of the IC (p = 0.007) and negatively to PONa after correction for age (p < 0.03). PONa inversely correlated with age (p = 0.008). In PAPS, IgG anti-beta2GPI-oxLig-1 independently predicted PONa (p = 0.02) and IMT of B (p = 0.003), CC, (p = 0.03) and of IC (p = 0.04). In PAPS, PONa inversely correlated to the IMT of B, CC and IC (p = 0.01, 0.02 and 0.003, respectively). IgG anti-beta2GPI-oxLig-1 may be involved in PAPS related atherogenesis via decreased PON activity.     

Evaluating the conformation of recombinant domain I of β(2)-glycoprotein I and its interaction with human monoclonal antibodies

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.     

Anti-beta(2)-glycoprotein I antibodies and the antiphospholipid syndrome

The mechanism of antiphospholipid syndrome (APS) development is still not completely understood. Accumulating evidence indicates that beta(2)-glycoprotein I (beta(2)GPI), is the major target antigen for antiphospholipid (aPL) antibodies, which play a crucial role in the pathogenesis of this autoimmune condition. Knowledge about the molecular structure, biological characteristics and function of beta(2)GPI has been expanding in recent years. In this review, we have focused on some recent important findings on beta(2)GPI and anti-beta(2)GPI antibodies in patients and animal models with APS.     

A new player in the antiphospholipid syndrome: the beta 2 glycoprotein I cofactor.

The study of antiphospholipid (aPL) antibodies has been greatly developed in recent years and conclusive evidence now exists concerning the correlation between aPL and clinical signs such as thrombosis, thrombocytopenia, abortion, and fetal loss. Several hypotheses have been put forward concerning the pathogenic mechanism of aPL, but none has received final confirmation from experimental data. Many studies have been devoted to characterizing the antigens recognized by the different aPL autoantibodies and to a cofactor involved in the binding of autoantibodies and phospholipids; this cofactor has been identified as an apolipoprotein, the beta 2 glycoprotein I (beta 2GPI) or APO-H. Direct evidence now exists which suggests that both the beta 2GPI and the phospholipid comprise the epitope to which aPL are directed. On the other hand anti-beta 2GPI antibodies have been identified in sera of patients suffering from SLE and primary Antiphospholipid Syndrome. beta 2GPI is normally present in human plasma/serum and possesses numerous inhibitory functions in multiple coagulation pathways. The amino acid sequence of beta 2GPI has been identified and found to consist of five repeating units that belong to the complement control protein (CCP) superfamily. This development of knowledge related to aPL has followed three steps respectively: 1. the standardization of the techniques of detection: 2. identification of the clinical signs related to the autoantibodies: and finally 3. the discovery of a new player, the beta 2GPI cofactor.     

Autoreactive CD4(+) T cells to beta(2)-glycoprotein I in patients with antiphospholipid syndrome

Antiphospholipid syndrome (APS) is characterized by recurrent thrombosis and intrauterine fetal loss in association with antiphospholipid antibodies (aPL). We have recently identified autoreactive CD4(+) T cells to beta(2)-glycoprotein I (beta(2)GPI) that promote aPL production in APS patients. beta(2)GPI-specific CD4(+) T cells preferentially recognize the antigenic peptide containing the major phospholipid-binding site in the context of DRB4*0103 (DR53). T-cell receptor beta chains of beta(2)GPI-specific T cells are highly restricted and mainly utilize rearranged Vbeta7 or Vbeta8 gene segments. T-cell helper activity that stimulates B cells to produce anti-beta(2)GPI antibodies is mediated through IL-6 and CD40-CD40 ligand engagement. beta(2)GPI-specific T cells respond to reduced beta(2)GPI and recombinant beta(2)GPI fragments produced in bacteria, but not to native beta(2)GPI, indicating that the epitopes recognized by beta(2)GPI-specific T cells are apparently cryptic. Activation of beta(2)GPI-specific T cells resulting in production of pathogenic anti-beta(2)GPI antibodies can be induced by the exposure to cryptic peptides of beta(2)GPI. Finally, beta(2)GPI-specific T cell is a reasonable target of potential therapeutic strategies that selectively suppress pathogenic aPL production in APS patients.     

Pathogenic role of anti-beta2-glycoprotein I antibodies on human placenta: functional effects related to implantation and roles of heparin

Most of the clinical manifestations of the antiphospholipid syndrome (APS) can be related to thrombotic events; however, placental thrombosis cannot explain all of the pregnancy complications that occur in women with this syndrome. In this regard, it has been hypothesized that antiphospholipid (aPL) antibodies can directly attack trophoblasts, but it is still unclear what pathogenetic mechanisms play a role and which aPL antibodies subpopulations are involved. Although it has been assumed that aPL antibodies are directed against anionic phospholipids (PLs), current advances in the field suggest that antibodies to PL-binding plasma protein such as beta2-glycoprotein-I (beta2-GPI) are the clinically relevant aPL antibodies. It appears that following the attachment of beta2-GPI to PLs, both molecules undergo conformational changes that result in the exposure of cryptic epitopes within the structure of beta2-GPI allowing the subsequent binding of antibodies. aPL antibodies detected by anti-beta2-GPI assays are associated with fetal loss. However, there is still debate on how the antibodies might induce the obstetrical manifestations. The significantly improved outcome of pregnancies treated with heparin has stimulated interest in the drug's mechanisms of action. Several mechanisms could explain its beneficial effects, because in addition to a direct effect of heparin on the coagulation cascade, it might protect pregnancies by reducing the binding of aPL antibodies, reducing inflammation, facilitating implantation and/or inhibiting complement activation. Further investigations are needed to better understand how aPL antibodies induce obstetric complications and to better clarify the functional role of heparin in the human placenta leading to more successful therapeutic options.     

Relation of preexisting anti-beta2GPI antibodies to infarct size in a rat model.

BACKGROUND: Anti-beta2-glycoprotein I (beta2GPI) antibodies (a subpopulation of antiphospholipid (aPL) antibodies) are associated with a procoagulant state in humans and with enhanced atherosclerosis in experimental animal models. Moreover, the presence of high titers of aPL antibodies in relatively young patients is associated with higher incidence of subsequent myocardial infarction. Herein, we evaluated the role of preexisting high levels of aPL antibodies in determining the size of the infarct induced by permanent ligation of the left anterior descending artery (LAD) in a rat model. METHODS AND RESULTS: A total of 11 Wistar rats were immunized and boosted with 10 microg of the phospholipid binding protein -beta2GPI (a method commonly applied for induction of aPL antibodies). Rats in the control group (n=9) were immunized and boosted with a Freund's adjuvant. Upon development of high anti-beta2GPI antibodies levels, myocardial infarction was induced by ligation of the LAD coronary artery. Rats were sacrificed 7 days later, their lymph nodes were collected for evaluation of cellular immunity to beta2GPI and their hearts were removed for assessment of infarct size and for immunohistochemical stains for iNOS and TGF-beta. beta2GPI-immunized rats exhibited high levels of aPL antibodies (mean optical density of 1.3+/-0.3) as compared with the control group (mean optical density of 0.12+/-0.03; P<.0001). Cellular immunity to beta2GPI was also pronounced as evident by an increased thymidine uptake and by increased interferon gamma secretion by the lymph node cells from beta2GPI-immunized rats. Myocardial infarct size has shown a tendency to be increased in rats induced to develop anti-beta2GPI antibodies (mean size 23+/-9%) as compared with controls (17+/-12%; P<.23). iNOS positive cells in the infarct area of beta2GPI-immunized rats were significantly increased in comparison to the control group (P<.01). Similarly, TGF-beta cell expression was significantly increased in the infarct area of the immunized rats in comparison to the control group (22.6+/-5.1 and 7+/-2.1 per 100 mononuclear inflammatory cells, respectively; P=.01). CONCLUSION: The presence of high levels of aPL antibodies is associated with higher expression of iNOS and TGF-beta and may contribute to myocardial damage.     

A peptide that shares similarity with bacterial antigens reverses thrombogenic properties of antiphospholipid antibodies in vivo

OBJECTIVE: The factors causing production of antiphospholipid (aPL) antibodies remain unidentified. Recently, studies have shown that aPL and anti-beta2Glycoprotein I (anti-beta2GPI) antibodies with pathogenic properties can be generated with peptides from bacterial and viral origin, that mimic regions of beta2GPI. These data suggest a molecular mimicry between bacterial/viral antigens and self-proteins. In this study we examined the ability of a synthetic peptide (named peptide A, NTLKTPRVGGC) that shares similarity with common bacterial antigens, to reverse aPL-mediated thrombosis in mice in vivo. Peptide A is also found in region I/II of beta2GPI. A scrambled form of peptide A (named scA, GTKGCPNVRLT) was used as a control. METHODS AND RESULTS: Sera from 29 patients with APS bound to peptide A but not to peptide scA by ELISA in a dose-dependent fashion. Cardiolipin (CL) liposomes inhibited the binding of IgG-APS by ELISA to peptide A by 35% and to CL by 56%. The inhibition of binding to cardiolipin and to peptide A was enhanced by addition of beta2GPI to the liposomes. CL/peptide A liposomes but not peptide A alone inhibited the binding of IgG-APS to peptide A. beta2GPI alone did not inhibit binding of IgG-APS to peptide A, to beta2GPI or to CL. For the in vivo experiments, CD1 mice in groups of 20 were injected with affinity purified aPL antibodies or with control IgG-NHS twice intraperitoneally. Seventy hours after the first injection, and 30 min before the surgical procedure (induction of experimental thrombus) mice were infused i.v. in each group with either peptide A or with peptide scA. The femoral vein of the anesthetized mice were dissected to examine the dynamics of an induced thrombus in treated and control mice. The mean aCL titer of mice injected with aPL was 60 GPL units. Mice treated with aPL and infused with peptide scA produced significantly larger thrombi when compared to mice treated with IgG-NHS and peptide scA (2466+/-462 microm2 vs 772.5+/-626.4 microm2). Treatment with peptide A significantly decreased thrombus size in mice injected with aPL antibodies (1063+/-890 microm2 compared to 2466+/-462 microm2). CONCLUSION: The data indicates that a synthetic peptide that shares similarity with common bacterial antigens and with regions of beta2GPI is capable to inhibit thrombogenic properties of aPL in mice. This may have important implications in designing new modalities of prevention and/or treatment of thrombosis in APS.     

Intracellular trafficking of beta2-glycoprotein I complexes with lipid vesicles in macrophages: implications on the development of antiphospholipid syndrome

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

Anti-phospholipid antibodies following vaccination with recombinant hepatitis B vaccine

This study was undertaken to evaluate the possible role of hepatitis B recombinant vaccine inducing the synthesis of IgG and IgM anti-cardiolipin antibodies (aCL), antibodies against beta(2)GPI (anti-beta(2)GPI), lupus anti-coagulant (LA), anti-nuclear antibodies and antibodies against extractable nuclear antigens (anti-ENA). The study population consisted of 85 healthy students (63 female, 22 male; mean age 20.8 years), vaccinated with three doses of recombinant DNA hepatitis B vaccine. One month after vaccination with the first dose of hepatitis B vaccine a minority of vaccinated individuals showed changes in IgG or IgM aCL or anti-beta(2)GPI or LA activity (P < 0.001). Among subjects in whom changes of IgG anti-beta(2)GPI were observed, a significantly higher number of increased (8/85) than decreased (2/85) values were found (P < 0.01). Analyses of paired data showed that differences in aCL or anti-beta(2)GPI levels before vaccination or 1 month later did not reach statistical significance. In two people aCL transitorily reached medium positivity after the first dose of hepatitis B vaccine with a drop 5 months later. Similar evident anti-beta(2)GPI fluctuation was also observed in one person. Another participant was initially low positive for IgG anti-beta2GPI and the levels were increasing after vaccination. Two participants became positive for anti-nuclear antibodies during 6 months' follow-up. There were no sex-dependent differences in tested antibodies observed and no associations between levels of aPL and levels of anti-HBV antibodies. We conclude that HBV can induce aPL, although rarely. In genetically susceptible individuals or together with some other triggers such combination might confer the risk of developing a continuous autoimmune response in an individual.     

Patients with atherosclerotic syndrome, negative in anti-cardiolipin assays, make IgA autoantibodies that preferentially target domain 4 of beta2-GPI

Autoantibodies targeting beta2-glycoprotein l (beta2-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-beta2-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-beta2-GPI and only 1.6% for IgG anti-beta2-GPI. The antigenic specificity of 29 serum samples from atherosclerosis patients was evaluated. Six different recombinant domain-deleted mutants (DM) of human beta2-GPI and full-length human beta2-GPI (wild-type) were used in competitive inhibition assays to inhibit the autoantibodies from binding in the anti-beta2-GPI ELISA assays. Domain-deleted mutants D--345 and D--45 inhibited the binding in the IgA anti-beta2-GPI assay, suggesting that these autoantibodies recognize domain 4 of the beta2-GPI molecule. These results clearly show that IgA anti-beta2-GPI autoantibodies from atherosclerotic patients are distinct from IgA autoantibodies found in APS samples.     

Laboratory evaluation of the antiphospholipid syndrome

The antiphospholipid syndrome (APS) was first described in 1986. The original association of this hypercoagulable state with anticardiolipin antibodies (aCL) resulted from the synthesis of evidence stemming from laboratory findings in systemic lupus erythematosus (SLE), ie, the frequent occurrence of false-positive VDRL tests and the paradoxical observation of the so-called "lupus anticoagulant" (LA), an increase in phospholipid (PL)-dependent clotting times. By the early 1990s, it was clear that a co-factor was involved in the reaction of antibodies to PL (aPL) in SLE patients with secondary APS and that this was a hitherto-obscure protein, beta-2 glycoprotein I (beta2GPI). In the intervening years, it has been established that beta2GPI and other PL-binding proteins such as prothrombin (PT) are relevant antigens in APS and assays for these antigens have been developed, standardized, and applied to subjects with both primary and secondary APS. Measurement and confirmation of LA activity is based on a stepwise approach and should follow the recommendations of the International Society of Thrombosis and Haemostasis. Although antibodies to various PL-binding proteins have been suggested as diagnostic targets for APS, the current (2006) consensus guidelines recognize only LA, aCL, and anti-beta2GPI for the classification of APS.     

Antiphospholipid antibodies, antiphospholipid syndrome and infections

Since the association between antiphospholipid antibodies (aPL) and syphilis was first described, many other viral, bacterial and parasitic infections have been shown to induce antiphospholipid antibodies, notably anticardiolipin antibodies (aCL). A review of the literature shows that while aCL occur frequently in viral infections, particularly in HIV (49.75%), HBV (24%) and HCV (20%), it is very rarely associated with anti-beta2 glycoprotein I antibodies (anti-beta2GPI) and is not correlated with thrombosis risk or hematological manifestations of the antiphospholipid syndrome (APS). Concerning bacterial infections, aCL is often present in leprosy (42.7%), where it is frequently associated with the presence of anti-beta2GPI (44.8%), and in syphilis infections (8 to 67%), though without correlation with thrombotic events. Though few individual patients with unequivocal infection-induced aPL satisfy criteria for APS, the lack of statistical association with thrombotic events strongly argues against the identification of a true APS subset in this context. However, physicians should keep in mind the fact that an infection, generally bacterial, in patients with confirmed APS, may lead to catastrophic antiphospholipid syndrome with a possible fatal outcome.     

Antiphospholipid antibodies bind ATP: a putative mechanism for the pathogenesis of neuronal dysfunction

Antiphospholipid antibodies (aPL) generated in experimental animals cross-react with ATP. We therefore examined the possibility that aPL IgG from human subjects bind to ATP by affinity column and an enzyme linked immunosorbent assay (ELISA). Sera with high levels of aPL IgG were collected from 12 patients with the antiphospholipid syndrome (APS). IgG fractions from 10 of 12 APS patients contained aPL that could be affinity-bound to an ATP column and completely eluted with NaCl 0.5 M. A significant (> 50%) inhibition of aPL IgG binding by ATP 5 mM was found in the majority. Similar inhibition was obtained with ADP but not with AMP or cAMP. All the affinity purified anti-ATP antibodies also bound beta2-glycoprotein-I (beta2-GPI, also known as apolipoprotein H) suggesting that, similar to most pathogenic aPL, their binding depends on this serum cofactor. We further investigated this possibility and found that the binding of beta2-GPI to the ATP column was similar to that of aPL IgG in that most was reversed by NaCl 0.5 M. Furthermore, addition of beta2-GPI to aPL IgG significantly increased the amount of aPL binding to an ATP column. We conclude that aPL IgG bind ATP, probably through beta32-GPI. This binding could interfere with the normal extracellular function of ATP and similar neurotransmitters.     

Effect of anti-beta2glycoprotein I Lupus Anticoagulants on fibrin polymerization and fibrinolysis

Anti-beta2-Glycoprotein I (beta2GPI) autoantibodies are the prominent laboratory feature of Hughes syndrome. By prolonging some coagulation tests in the presence of exogenous phospholipids (PL), they behave as classical Lupus Anticoagulants (LA). We investigated the effect of 3 affinity-purified anti-beta2GPI IgG preparations from patients with Hughes syndrome on fibrin polymerization and fibrinolysis of normal plasma, measured by comparing the optical densities of assay mixtures in the presence of the autoantibodies or normal IgG. The presence of anti-beta2GPI IgG in diluted Russell Viper Venom Time (dRVVT) assays, carried out using a PL dilution of 1:8 or 1:64, resulted in a delay in the onset of polymerization by 30-40 and 60-70s, respectively. Fibrin polymerization was complete after 250s for both anti-beta2GPI IgG and normal IgG. The inhibitory effect of the anti-beta2GPI antibodies was not observed in the presence of excess PL, as expected for LA. Anti-beta2GPI IgG increased the plateau level of polymerization when dRVVT was performed in the presence of 1.5 nM recombinant tissue plasminogen activator, but did not impair the fibrinolytic process, which was almost complete after 250 min. The autoantibodies did not delay the onset of fibrin polymerization in tests carried out using recombinant tissue factor. On the contrary, the autoantibodies enhanced polymerization in prothrombin time assays, and accelerated it in tissue thromboplastin inhibition tests, with no effect on fibrinolysis. These data provide evidence that anti-beta2GPI LA may act as either anticoagulants or procoagulants in different in vitro coagulation tests.     

Antiphospholipid antibodies as a possible risk factor for atherosclerosis in patients with rheumatoid arthritis

Atherosclerosis shares many similarities with inflammatory and autoimmune diseases, among them rheumatoid arthritis (RA). Anticardiolipin antibodies (aCL) and antibodies against beta2-glycoprotein I (anti-beta2GPI) have been detected in sera of RA patients in several studies. We demonstrated aCL and anti-beta2GPI in a selected group of 70 patients with RA (premenopausal women, non-diabetic, non-hypertensive) and compared them with age- and sex-matched controls. There was a significant higher internal carotid artery intima-media thickness and number of plaques in RA patients compared to controls. aCL of IgG and IgM classes were present in 15.7% of RA patients as compared to 5% in the control group. Thirty percent of RA patients had anti-beta2GPI of IgG, IgM and IgA classes compared to 7.5% in controls. Major differences were seen in IgG and IgA classes. Our results support the idea that aCL and anti-beta2GPI represent an important risk factor for atherosclerosis in RA patients. Elevated levels of phosphatidylserine-dependent antiprothrombin antibodies did not contribute significantly to the general prevalence of antiphospholipid antibodies.