Anti-beta2-glycoprotein I (GPI) autoantibodies, annexin V binding and the anti-phospholipid syndrome

We examined the role of autoantibodies to beta2-GPI and prothrombin (PT) in the inhibition of annexin V binding to cardiolipin (CL) and the association with clinical manifestations of the anti-phospholipid syndrome (APS). Plasma samples from 59 patients with anti-phospholipid (aPL) antibodies were studied. Affinity purification of total IgG and IgG anti-ss2-GPI antibodies was performed using staphylococcal protein A and phospholipid liposomes. Annexin V binding to CL was significantly inhibited by 31/59 (53%) aPL+ plasma samples. There was a significant association between annexin V inhibition and elevated levels of IgG anti-cardiolipin (aCL) (r = -0.62; P < 0.001), IgG anti-ss2-GPI (r = -0.67; P < 0. 001) and a weaker association with lupus anti-coagulant (r = -0.27; P = 0.05). There was no association with other isotypes of aCL and anti-ss2-GPI or with anti-PT of any isotype. In patients with clinical manifestations of the APS there were higher levels of IgG aCL (median (range) Z score): 10.0 (0-17.6) versus 5.0 (0-16.1); P = 0.03), IgG anti-ss2-GPI (4.5 (0-11.3) versus 0.9 (0-9.7); P = 0.02) and greater inhibition of annexin V binding to CL (-3.4 (-11.4-0.6) versus -1.1 (-10.8-1.2); P = 0.22). Odds ratios for the laboratory assays and the presence of clinical manifestations of the APS varied between 0.38 and 4.16, with the highest values for IgG aCL (4.16), IgG anti-ss2-GPI (3.28) and annexin V inhibition (2.85). Additional experiments with affinity-purified IgG antibodies indicated that inhibition of annexin V binding was dependent upon the concentration of ss2-GPI and anti-ss2-GPI antibodies. These results indicate that inhibition of annexin V binding to procoagulant phospholipid surfaces is dependent upon anti-ss2-GPI antibodies and suggest a role for annexin V in the pathogenesis of the APS.     

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.     

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.     

Anti-beta 2-glycoprotein I and antiphosphatidylserine antibodies are predictors of arterial thrombosis in patients with antiphospholipid syndrome

The predictive value (PV) and association of 4 antiphospholipid antibodies with clinical manifestations of the antiphospholipid syndrome (APS) were evaluated in 90 patients with systemic lupus erythematosus (SLE) and 100 with APS. Patients with APS were classified into arterial thrombosis, venous thrombosis, and pregnancy morbidity subgroups. IgG, IgM, and IgA anticardiolipin (aCL), antiphosphatidylserine (aPS), anti-beta 2-glycoprotein I (anti-B2GPI), and antiprothrombin (aPT) antibodies were determined by enzyme-linked immunosorbent assay. Individually, anti-B2GPI and aPS antibodies had the strongest PV for APS (86.4%-94.1%; P < .001) in patients with SLE. The PV for APS reached 100% when 2 or more antibodies were present. Similarly, anti-B2GPI and aPS antibodies had a stronger PV and association for arterial thrombosis (87%-95%; P < .001) compared with venous thrombosis (80%-92%; P = .01). Weak PV and association with pregnancy morbidity were seen with all antibodies. These results suggest an important pathogenic role of anti-B2GPI antibodies in arterial thrombosis. In addition, anti-B2GPI and aPS antibodies seem to provide the best diagnostic value for the laboratory assessment of APS.     

Conformationally altered beta 2-glycoprotein I is the antigen for anti-cardiolipin autoantibodies

Anti-cardiolipin autoantibodies (aCL) induce thrombosis and recurrent fetal death. These antibodies require a 'cofactor', identified as beta 2-glycoprotein I (beta 2-GPI), to bind phospholipids. We show here that aCL can bind beta 2-GPI in the absence of phospholipid. Binding of aCL to beta 2-GPI is dependent upon the beta 2-GPI being immobilized on an appropriate surface including cardiolipin, irradiated polystyrene and nitrocellulose membrane. This effect cannot be explained by increased antigen density of beta 2-GPI immobilized on these surfaces. Rather, conformational changes that occur following the interaction of beta 2-GPI with phospholipid render this protein antigenic to aCL. Liquid-phase beta 2-GPI was not antigenic for aCL. Thus, aCL cannot bind circulating beta 2-GPI. These findings may explain why patients with aCL can remain healthy for many years but then undergo episodes of thrombosis or fetal loss without changes in their circulating aCL profile, as the triggering event for these pathologies can be predicted to be one that renders beta 2-GPI antigenic for aCL.     

Anti-beta(2)-glycoprotein I autoantibody expression as a potential biomarker for strokes in patients with anti-phospholipid syndrome

Anti-phospholipid syndrome (APS) is an autoimmune disease. Cerebral ischemia associated with APS occurs at a younger age than typical atherothrombotic cerebrovascular disease, is often recurrent, and is associated with high positive IgG anti-phospholipid (GPL) unit levels. This study sought to determine the frequency rates of anti-cardiolipin (aCL) dependent on the presence of beta(2)-GPI, anti-beta(2)-glycoprotein I (abeta(2)-GPI), and anti-phosphatidyl serine (aPS) IgG autoantibodies among stroke patients, and thus demonstrate the importance of testing for abeta(2)-GPI autoantibodies. For these study, stroke patients and control subjects recruited from Mosul, Erbil, and Dohuk provinces in Northeren Iraq between March 2004 and March 2005 were evaluated. All cases were under 50 years-of-age and had no recognizable risk factors. Using ELISA to evaluate the presence of IgG isotype of aCL, abeta(2)-GPI, and aPS autoantibodies in their blood, the results indicated that the frequency of abeta(2)-GPI was 14/50 (28%), aCL was 11/50 (22%), and aPS was 9/50 (18%) among stroke patients. In contrast, aCL was detected in 2/30 (6.7%) of control subjects; each of the other anti-phospholipid antibodies (APLA) was never observed. Of all the abeta(2)-GPI(+) cases, the incidence of stroke patients having the combined profile of abeta(2)-GPI + aCL was 11/14 (78.6%) and of abeta(2)-GPI + aPS was 9/14 (64.3%). Only 2/14 (14.3%) of these abeta(2)-GPI(+) patients also expressed aCL in the absence of aPS. The frequency of patients expressing all three markers was only 9/14 (64.3 %). In none of the APS/stroke patients were aCL or aPS expressed in the absence of the abeta(2)-GPI. Conversely, abeta(2)-GPI as a sole marker was seen in 3/14 (21.4%) of these patients (i.e., in absence of either other marker). It can be concluded from these studies that the among the three major forms of APLA examined, the presence of abeta(2)-GPI IgG autoantibodies appeared to correlate best with stroke in patients who were concurrently suffering APS.     

Anti-beta 2-glycoprotein I antibodies of IgM class are linked to thrombotic disorders in young women without autoimmune disease

Antiphospholipid autoantibodies particularly antibodies against beta2-glycoprotein I (anti-beta2GPI) are casually associated with thromboses in patients with autoimmune diseases. However, their exact prevalence and role in the pathogenesis of thromboses in the absence of autoimmune disease is still inconclusive. They might be particularly important when other risk factors of thrombosis are absent. We investigated antiphospholipid antibodies in 68 young women (aged <45yr at onset of the event, without autoimmune disease and with an otherwise low risk of thrombosis) in the stable period following myocardial infarction (MI), lacunar cerebral infarction (LACI) or deep vein thrombosis (DVT) and in 37 healthy age-matched controls. Patients had increased IgM anti-beta2GPI compared to controls (36.0, 11.5-49.5 vs. 17.50, 3.50-30.0 arbitrary units (AU), p<0.001), whereas no difference was obtained in other measured antibodies (anticardiolipin and antiphosphatidylserine (aPS) antibodies of IgG and IgM). IgM anti-beta2GPI positively correlated with some markers of increased coagulation potential and negatively with BMI (r=-039, p<0.005) and other parameters of the metabolic syndrome. In conclusion, we found that levels of IgM anti-beta2GPI are increased in young women suffering arterial or venous thromboses in the absence of other known autoimmune diseases and also in the absence of pronounced classical risk factors. We found that IgM anti-beta2GPI positively correlated with some markers of increased coagulation potential and negatively with parameters of the metabolic syndrome. Thus, it appears that elevated levels of IgM anti-beta2GPI are linked to thrombotic disorders in young women (without autoimmune disease) particularly when classical risk factors or the metabolic syndrome are absent.     

Oxidized low-density lipoprotein/beta2-glycoprotein I complexes and autoantibodies to oxLig-1/beta2-glycoprotein I in patients with systemic lupus erythematosus and antiphospholipid syndrome

Oxidized low-density lipoprotein (oxLDL) interacts with beta2-glycoprotein I (beta2-GPI) via oxLDL-derived specific ligands (oxLig-1) forming complexes. The prevalence and significance of oxLDL/alpha2-GPI complexes and antibodies to oxLig-1/alpha2-GPI were evaluated in patients with systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS). The oxLDL/beta2-GPI complex was 69% positive (above mean + 3 SD of control subjects) in 97 consecutive patients with SLE, 62% in 40 patients with SLE with secondary APS, and 60% in 50 control patients with SLE without APS. IgG anti-oxLig-1/beta2-GPI antibody was positive in 31 (32%) of 97 consecutive patients with SLE, in 26 (65%) of 40 patients with SLE with secondary APS, and in 6 (19%) of 32 control patients with SLE. Anti-oxLig-1/beta2-GPI antibodies were 93.7% specific with a positive predictive value of 90.0% for APS, better than anticardiolipin antibodies (80.0% specific, 71.4% predictive value). These results confirm that oxLDL/beta2-GPI complexes are common in SLE and suggest a possible immunogenic role in APS. In contrast, IgG anti-oxLig-1/beta2-GPI antibodies not only are associated with but also are clinically useful risk factors for APS.     

The involvement of beta2-glycoprotein I (beta2-GPI) in human and murine atherosclerosis.

Atherosclerosis is a multifactorial process, the hallmark of which is fat deposition in the vessel wall. Autoimmune factors have recently been shown to play an important role in the initiation and progression of atherosclerosis; candidate autoantigens are oxidized lipids and heat shock proteins. beta2-glycoprotein I (beta2-GPI) is a highly glycosylated plasma protein that serves as a major antigenic target for autoimmune type antiphospholipid antibodies. Its major relevant property is binding to negatively charged phospholipids/surfaces. In the set of studies presented in this paper, we provide evidence pointing towards beta2-GPI as an influential determinant in murine and human atherogenesis. Thus, immunization of transgenic atherosclerosis-prone mice (apolipoprotein E and low-density lipoprotein receptor knockouts) with human beta2-GPI results in a brisk and sustained respective response that extends to cross-react with the 'self' murine beta2-GPI. Atherosclerosis is accelerated in both strains concomitant with the infiltration of CD4 lymphocytes in the aortic sinus of the mice. When human plaques were studied, it was found that beta2-GPI resides in the subendothelial regions and co-localizes with CD4 lymphocytes. Thus, the immune response towards beta2-GPI may play an important role in atherogenesis, serving as a possible target for antigen specific therapies.     

Anticardiolipin and anti-beta-2-glycoprotein I antibodies

The anticardiolipin (aCL) antibody test was first established in 1983, using cardiolipin (negatively charged phospholipid) as an antigen in a solid-phase immunoassAy. It was first applied to the study of systemic lupus erythematosus patients, and was found associated with thromboses and recurrent pregnancy losses. The wide use of this test was determinant in the definition of the "aCL or antiphospholipid syndrome" (APS).Later, it was demonstrated that aCL antibodies do not recognize anionic phospholipids but are directed against plasma proteins bound to anionic phospholipids, mainly beta-2-glycoprotein I, which is now considered as the autoantigen in APS. Anti-beta-2-glycoprotein I (anti-beta2GPI) is not yet accepted as a serological criterion for APS, but most investigators would consider a patient with anti-beta2GPI antibodies and clinical features of APS to have the syndrome. aCL and anti-beta2GPI are a heterogeneous group of antibodies with different clinical significances and can be present in different autoimmune diseases as well as in infectious diseases.     

The effect of phospholipids on the formation of immune complexes between autoantibodies and beta2-glycoprotein I or prothrombin

In the last decennium, it became clear that antiphospholipid antibodies found in patients with antiphospholipid syndrome (APS) are in fact antibodies against lipid-bound plasma proteins. The most frequently occurring antigens are beta2-glycoprotein I and prothrombin, although several other lipid-bound plasma proteins have been reported as antigen for antiphospholipid antibodies. Both proteins bind to anionic phospholipids, mainly phosphatidylserine, which becomes exposed at the surface of activated platelets, apoptotic cells, or cell-derived microparticles. The binding of beta2-glycoprotein I and prothrombin to these cell surfaces or to artificial lipid vesicles with comparable amounts of anionic phospholipids is rather weak. Antiphospholipid antibodies from patients are predominantly of low affinity regarding their interaction with beta2-glycoprotein I or prothrombin in solution. In the presence of a suitable phospholipid surface, however, this interaction is strongly enhanced. There is now strong evidence that formation of bivalent, trimolecular immune complexes at the lipid membrane essentially contributes to the binding of these intrinsically low affinity patient antibodies. Depending on the affinity, the epitope specificity, and the polyclonality of a particular IgG preparation, multimeric structures of lipid-bound immune complexes may form a lattice with multiple interactions on the lipid (cell) surface. It is hypothesized that the functional activity, that is, the ability of antibodies to interfere with lipid-dependent reactions, not only depends on their affinity for the antigen, but also on their ability to form multiple interconnected bivalent trimolecular complexes at the lipid (or cell) surface. It is further proposed that the rate of desorption of immune complexes may present a better indicator for the functional properties of the antibodies than the amount of adsorbed immune complexes.     

Structure-function studies on beta 2-glycoprotein I

Human beta 2-glycoprotein I is a heavily glycosylated plasma protein which has been implicated in the binding of antiphospholipid antibodies to negatively charged phospholipids; a process considered as an important risk factor for the development of thrombosis. We have solved the crystal structure of beta 2-glycoprotein I. In this review we will discuss what the three-dimensional structure teaches us about the role of beta 2-glycoprotein I in the pathogenesis of the antiphospholipid syndrome.     

Avidity of anti-beta-2-glycoprotein I antibodies

The terms affinity and avidity are often used indiscriminately, despite clearly differing. Since affinity refers to monovalent binding of antibodies to a monovalent epitope, the majority of data on the binding of anti-beta2-glycoprotein I antibodies (anti-beta2-GPI) characterized their avidity rather than affinity. Anti-beta2-GPI were generally believed to be of low avidity, but heterogeneous avidity of patients' IgG anti-beta2-GPI has been demonstrated. High avidity anti-beta2-GPI monoclonals were reported to possess higher pathogenicity than low avidity anti-beta2-GPI. Polyclonal high avidity anti-beta2-GPI were found to be more common in patients with antiphospholipid syndrome (APS) and associated with thrombosis. Some conformational changes of beta2-GPI are required for the binding of polyclonal anti-beta2-GPI to the antigen: neither high density of the antigen nor high avidity of the anti-beta2-GPI alone is sufficient for the recognition. Avidity of anti-beta2-GPI should be considered in any attempt of inter-laboratory standardisation and/or evaluation of anti-beta2-GPI enzyme-linked immunosorbent assay (ELISA).     

Heat shock protein 60/65, beta 2-glycoprotein I and oxidized LDL as players in murine atherosclerosis

We have made consecutive studies to prove that autoimmune factors can influence the progression of atherosclerosis in inbred and transgenic mice. C57BL/6 as well as LDL-receptor deficient mice were immunized with heat shock protein 65. LDL-RD and apolipoprotein E knockout (apoE KO) mice were immunized with human B-glycoprotein I. ApoE KO mice were immunized with oxidized LDL. In all immunized mice, a sustained humoral response to the provided antigen was elicited evident by high titers of antibodies by ELISA. A primary cellular immune response was also shown by thymidine incorporation studies employing the antigens in vitro. Immunization with hsp-65 and with beta 2-GPI served to enhance the progression atherosclerosis and led to an increase in the infiltration of CD3 in the subendothelial regions of the early plaques. Transfer of hsp-65 and beta 2-GPI reactive lymphocytes to syngenic mice led to enhancement of fatty streak formation. However, immunization with homologous oxLDL in apoE KO mice led to attenuation of lesion progression concomitant with the production of anti-oxLDL antibodies. Thus, autoimmune factors appear to influence early artherosclerosis progression in mice. If proven in humans these antigen specific responses may be harnessed for selective immunomodulation of the atherosclerotic plaque.     

Anti-cardiolipin and anti-beta 2-glycoprotein I antibodies in celiac disease

Aims

To determine the frequency of anti-cardiolipin (aCL) and anti-β2-glycoprotein I antibodies (aβ2GPI) in celiac disease (CD) patients.

Patients and methods

Sixty-three untreated CD patients and 40 healthy blood donors (HBD) were studied. IgG, IgA and IgM aCL and aβ2GPI were detected by Elisa.

Results

The frequency of antiphospholipid antibodies (aPL) (aCL and/or aβ2GPI) was significantly higher in CD patients (12 out of 63) than in HBD (two out of 40) (19% vs 5%, P = 0.04). Six CD patients out of 63 (9.5%) and one HBD out of 40 (2.5%) had aCL. Ten CD patients (15.9%) and two HBD (5%) had aβ2GPI. Only aβ2GPI-IgA was significantly more frequent in CD patients than in HBD (14.3% vs 2.5%, P = 0.048). In CD patients, aβ2GPI-IgA (nine out of 63) was significantly more frequent (14.3%) than aβ2GPI-IgG (1.6%) and IgM (1.6%) (P = 0.008). In CD patients, the frequency of aCL-IgA and IgM was 6.3% (four out of 63) and aCL-IgG were not detected. Simultaneous presence of positive antibodies was found in four CD patients: one patient had four aPL, one had three aPL and two had two aPL. The four patients who had aCL-IgA had also aβ2GPI-IgA and three of them had a titer higher than 50 units. Among nine patients with aβ2GPI-IgA, four had a titer higher than 100 units. The highest titers were found in adults.

Conclusions

aPL and particularly aβ2GPI-IgA are frequent in CD. The significance of these antibodies has to be determined.     

Clinical significance of anticardiolipin and anti-beta2-glycoprotein I antibodies

BACKGROUND: Anticardiolipin (aCl) and anti-beta2-glycoprotein I (anti-beta2-gpI) antibodies are autoantibodies associated with the antiphospholipid syndrome (APS), which is characterized by both arterial and venous thrombosis and miscarriages. The scope of this study was to explore the clinical characteristics of patients with aCl and anti-beta2-gpI antibodies. METHODS: ACl were tested in 3,600 consecutive sera in our laboratory between January 1999 and June 2001. The clinical diagnosis and prevalence of thrombosis and pregnancy morbidity were retrospectively reviewed in aCl-positive patients. Furthermore, the frequency of anti-beta2-gpI antibodies, lupus anticoagulant (LA), prolonged activated partial thromboplastin time (aPTT), and thrombocytopenia were investigated in aCl-positive patients. RESULTS: 147 aCl-positive patients, 110 women and 37 men with a mean age of 41 years (range 7.8-82.5), were identified. 42 (28.6%) aCl-positive patients fulfilled the criteria for APS which was secondary to a connective tissue disorder in 8 patients. The frequency of anti-beta2-gpI antibodies and LA, prolonged aPTT, and thrombocytopenia in aCl-positive patients was 23.8, 27.2, 25.7 and 9.2%, respectively. The presence of both aCl and anti-beta2-gpI antibodies was strongly associated with clinical symptoms of APS (p = 0.007) compared to p = 0.008 for LA. CONCLUSION: Our data suggest that assessment of anti-beta2-gpI antibodies in addition to aCl is a valuable diagnostic tool in the workup of patients with APS.     

Anti-beta1-adrenergic receptor autoantibodies in patients with chronic Chagas heart disease

Chronic Chagas heart disease (cChHD), a chronic manifestation of the Trypanosoma cruzi infection, is characterized by high antibody levels against the C-terminal region of the ribosomal P proteins (i.e. peptide R13, EEEDDDMGFGLFD) which bears similarity with the second extracellular loop of beta1-adrenergic receptor (beta1-AR, peptide H26R HWWRAESDEARRCYNDPKCCDFVTNR). Because it has not been demonstrated clearly that IgGs from cChHD patients bind to native human beta1-AR, the aim of this study was to investigate further the physical interaction between cChHD IgGs and the human beta1-AR. Immunofluorescence assays demonstrated the binding of these antibodies to the receptor expressed on stably transfected cells, together with a beta1-AR agonist-like effect. In addition, immunoadsorption of the serum samples from cChHD patients with a commercially available matrix, containing peptides representing the first and the second extracellular loop of the beta1-AR, completely abolished reactivity against the H26R peptide and the physiological response to the receptor. The follow-up of this specificity after in vitro immunoadsorption procedures suggests that this treatment might be used to diminish significantly the serum levels of anti-beta1-AR antibodies in patients with Chagas heart disease.     

Antigenic structures recognized by anti-beta2-glycoprotein I auto-antibodies

Beta2-glycoprotein I (beta2-GPI) is a major antigen for anti-cardiolipin antibodies and their epitopes are cryptic. Conformation of each domain of beta2-GPI was optimized from its crystal structure by energy minimization and by molecular dynamics simulation. Three electrostatic interactions, i.e. D193-K246, D222-K317 and E228-K308, were observed between domains IV and V in the optimized structure that was constructed based on the consensus sequences obtained by the phage-displayed random peptide library. Antigenic structures determined by the epitope mapping mainly consisted of hydrophobic amino acids located on two discontinuous sequences in domain IV. These amino acid clusters, as an epitope, were covered by domain V and were of a hidden nature. A similar but incomplete counterpart to the epitopic clusters was found in domain I but was not in domains II or III. Binding of anti-beta2-GPI auto-antibodies to solid-phase beta2-GPI was significantly reduced either by L replacement for W235, a common amino acid component for the epitopes, or by V replacement for all of D193, D222 and E228. Structural analysis indicated a hypothesis that these electrostatic interactions between domains IV and V retained exposure to W235 and that epitope spreading occurred in the region surrounding W235. Thus, epitopic structures recognized by anti-beta2-GPI auto-antibodies are cryptic and inter-domain electrostatic interactions are involved in their in exposure.