Anti-Beta 2-glycoprotein I antibody isotype and IgG subclass in antiphospholipid syndrome patients

Beta 2-glycoprotein I (beta2-GPI) is an antigenic target recognised by antiphospholipid antibodies found in association with the antiphospholipid syndrome (APS). In this study, the prevalence of Immunoglobulin M (IgM) and IgA anti-beta2-GPI antibodies was examined in APS patients and compared with IgG antibodies. In addition the value of measuring antibody isotypes and IgG subclass was investigated in the laboratory diagnosis of APS. A solid phase enzyme linked immunosorbent assay was established to measure IgG, IgM and IgA and IgG subclass antibodies to beta2-GPI in patients with APS and a variety of other thrombotic and non-thrombotic disorders. Raised levels of IgM anti-beta2-GPI antibodies were observed in 65% of patients with APS, 21% with systemic lupus erythematosus (SLE), 23% with rheumatoid factor, 4% with stroke, 5% carotid artery stenosis (CAS), 17% with a biological false positive serology for syphilis, 43% with infectious mononucleosis (IM) and 27% with human immunodeficiency virus (HIV). The median value for IgM antibodies to beta2-GPI for all these groups ranged from 2 to 7 arbitrary units (AU). Elevated levels of IgA antibodies to beta2-GPI were found in patients with APS (47%), SLE (13%), rheumatoid factor (26%), CAS (48%), stroke (25%), VDRL false positive serology for syphilis (33%), IM (47%) and HIV (7%). The median value of IgA antibodies to beta2-GPI in all of these groups ranged from 2 to 4 AU. Conversely the median value for IgG anti-beta2-GPI in APS patients was 112 AU compared to 1-4 AU in the other conditions examined. The presence of IgM and IgA antibodies to beta2-GPI was much less specific and sensitive for APS than IgG, with raised levels of these isotypes seen in a variety of thrombotic and non-thrombotic disorders. Elevated levels of IgG1, IgG2, IgG3 and IgG4 antibodies to beta2-GPI were detected in APS patients. While all four IgG anti-beta2-GPI antibody subclasses were represented in APS patients there appeared to be a significant overall skewing towards to the IgG2 subclass.     

Phospholipid specificity and requirement of beta 2-glycoprotein-I for reactivity of antibodies from patients with primary antiphospholipid syndrome.

Some disease manifestations are associated with serum antiphospholipid antibodies (aPL) in patients with systemic lupus erythematosus (SLE) in what has been termed antiphospholipid syndrome (aPLS). There are patients with aPLS who do not have SLE or any other illness who have been grouped under the term primary antiphospholipid syndrome (PAPS). However, patients with diverse infections, notably syphilis, may have aPL but do not develop the associated clinical manifestations. This has been attributed, at least in part, to the immunochemical features of their aPL, including the requirement for beta 2-glycoprotein-I (beta 2GP-I) for binding of aPL to phospholipids, but these have not been studied in sera from patients with PAPS. By ELISA we studied 95 sera from 17 patients with PAPS and 100 sera from clinically normal individuals for IgG and IgM antibodies to the main anionic and zwitterionic phospholipids and their related compounds, phosphatidic acid (PA) and synthetic phosphorylcholine (PRC). beta 2GP-I was present, either in newborn calf serum (NBCS) or purified, to block wells and to dilute samples, or was substituted by 0.3% gelatin. Inhibition studies with phospholipid micelles were used to confirm reactivities with the corresponding phospholipids. All 17 patients had IgG and 11 had IgM antibodies to cardiolipin. Antibodies to anionic phospholipids were primarily IgG whereas those to zwitterionic phospholipids were mainly, and often exclusively, IgM. We found a statistically significant difference in the mean levels of antibodies to all anionic phospholipids except aPTS, and to the haptene PA (P < 0.001) between patients and controls. The difference between levels of IgM antibodies to zwitterionic phospholipids was statistically significant with sphingomyelin (P < 0.001) and the haptene (P < 0.001). Levels of most IgG and most IgM aPL correlated significantly among them. The pattern and titers of reactivity are variable between patients, but stable within each patient. Requirement of beta 2GP-I for this reactivity was not an all-or-nothing phenomenon in individual sera. In general, as in lupus sera, antibodies to anionic phospholipids require that this cofactor be present coating the ELISA plates, whereas those to zwitterionic phospholipids do not. It would appear that patients with PAPS have polyclonal mixtures of antibodies that react with various phospholipids and have different requirements for beta 2GP-I for such reactivity.     

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.     

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.     

Immunologic characterization and functional properties of murine antibodies raised against deleted mutants of human beta 2-glycoprotein I

beta 2-Glycoprotein I (beta 2GPI) is a 50 kDa molecule proposed as a principal target of 'autoimmune' antiphospholipid antibodies (aPL). We have used deleted mutants (DM) representing different domains of beta 2GPI (I-IV, IV-V and V) for immunization of naive mice and studied the characteristics of the respective murine IgG preparations in comparison with affinity-purified IgG from two patients with primary antiphospholipid syndrome. Immunization with beta 2GPI and with the DM produced anti-beta 2GPI antibodies, part of which reacted with negatively charged phospholipids (PL), whereas reactivity with cardiolipin was evident only in the IgG from mice immunized with beta 2GPI. These results are consistent with the presumption that aPL are induced following the in vivo association of beta 2GPI (used for immunization) with resident negatively charged PL. Accordingly, DM which either lack the PL binding site or aPL attachment locus did not elicit, upon immunization, antibodies reactive with PL. Further, murine anti-beta 2GPI IgG and human 'autoimmune' aPL were similar, albeit not identical, in terms of DM requirement for PL binding and charge dependency. Murine antibodies and human aPL, regardless of their binding characteristics, were found to bind significantly to platelets upon their activation with thrombin and to promote platelet activation. The results of the current study emphasize the dissimilarities between human 'autoimmune' aPL and murine anti-beta 2GPI. Thus, anti-beta 2GPI antibodies to different DM as well as human aPL are capable of binding and activating human platelets provided beta 2GPI is present.      

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.     

Atherogenic autoantigen: oxidized LDL complexes with beta2-glycoprotein I

Beta2-Glycoprotein I (beta2-GPI) is a major antigen for antiphospholipid antibodies present in patients with antiphospholipid syndrome (APS). In 1997, we demonstrated that beta2-GPI specifically binds to Cu2+-oxidized low-density lipoprotein (oxLDL) and that the beta2-GPI-oxLDL complex is subsequently targeted by anti-beta2-GPI antibodies in vitro. Then ligands for beta2-GPI were purified from oxLDL and characterized as omega-carboxylated 7-ketocholesteryl esters, such as 7-ketocholesteryl-9-carboxynonanoate (oxLig-1) and 7-ketocholesteryl-12-carboxy (keto) dodecanoate (oxLig-2). These ligands mediate to form oxLDL-beta2-GPI complexes, and the complexes are taken up avidly by macrophages via anti-beta2-GPI autoantibody-mediated phagocytosis. We recently demonstrated that appearance of autoantibodies against a complex of beta2-GPI and oxLig-1 are highly associated with a history of arterial thrombosis. Serum oxLDL-beta2-GPI complex and their IgG immune complexes are also risk factors arterial thrombosis in APS patients. There is increasing circumstantial evidence of autoimmune mechanism involving beta2-GPI and oxLDL in the atherogenesis in APS.     

Antiphosphoplipid antibodies and retinal occlusive vasculopathy

PURPOSE: to determine the prevalence of antiphospholipid antibodies in patients with occlusive retinal vascular events, exempt from conventional risk factors of retinal thrombosis. METHODS: eleven patients with retinal vascular occlusion, free of main accepted risk factors for retinal thrombosis, were retrospectively screened for antiphospholipid antibodies (anticardiolipin and anti-beta2 glycoprotein 1 antibodies) by an Elisa method. Prevalence of antiphospholipid antibodies were compared with those in a homogenous control group of 100 patients. RESULTS: the prevalence of antiphospholipid antibodies in the study group was 27% (three of 11). Comparison with control group prevalence (3%) showed a statistically significant difference (p < 0,001). One patient in the study disclosed positivity for IgG anticardiolipine antibodies, one for IgM anticardiolipine antibodies and one for anti-beta2 glycoprotein 1 antibodies. CONCLUSION: our results lead us to recommend a systematic search for specific antiphospholipid antibodies in such young patients which could have an importance for the diagnosis of primary antiphospholipid syndrome.     

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.     

Antibodies to prothrombin, factor V, and beta2-glycoprotein I and vascular access thrombosis

We studied 88 hemodialysis patients for the presence of antibodies to human factor II (hFII), bovine factor V (bFV), and human beta2-glycoprotein 1 (beta2GPI). Forty-one patients had elevated anti-hFII antibodies, 17 had elevated anti-bFV antibodies, and 9 had elevated anti-beta2GPI antibodies. Fifty-two patients had elevated antibodies to one or more protein. Patients with PTFE grafts had elevated antibodies most frequently (21 [75%] vs. 20 fistulas [45%; p = 0.016 compared with PTFE] and 11 tunneled catheters [68.8%]). Twelve of 13 patients (92.3%) with PTFE grafts and thrombosis had elevated antibody levels, compared with 9 of 15 without thrombosis (60%; p = 0.049). The number of thromboses and mean thrombosis rates were significantly higher in PTFE patients with antibodies (1.24 vs. 0.14 thromboses, p < 0.01; 42.67 vs. 6.44 thromboses/100 patient years, p < 0.05). When analyzed individually, thrombotic complications occurred more frequently in patients with PTFE grafts and elevated anti-bFV antibodies (p = 0.016), but did not correlate with anti-hFII or anti-beta2GPI antibodies. Thrombotic complications did not correlate with elevated antibody levels in patients with AV fistulas or cuffed catheters. In conclusion, hemodialysis patients with PTFE grafts frequently have elevated antibodies to FII, FV, and beta2GPI, and the presence of elevated antibody levels to one or more of these proteins is associated with an increased thrombotic risk. Further studies are necessary to determine whether limiting exposure to bovine thrombin preparations will decrease the incidence of these antibodies and PTFE graft thrombosis.     

Binding of complement component C1q to anti-β2 glycoprotein I antibodies from patients with antiphospholipid syndrome

OBJECTIVE: To determine if anti-beta2 GPI reactive with surface-bound beta2 GPI can bind C1q, i. e. to determine whether surface-bound beta2 GPI-anti-beta2 GPI immune complexes can initiate the classical pathway of complement activation. METHODS: Beta2 GPI was bound to chemically-activated microtiter plates which had previously been shown to promote anti-beta2 GPI reactivity with bound beta2 GPI. Wells with surface-bound beta2 GPI (capped with bovine serum albumin) were then reacted with complement-inactivated sera from antiphospholipid syndrome patients (APS) or with control sera. Following removal of unbound serum components, the wells were incubated with biotinylated C1q and probed with peroxidase-conjugated avidin D. Bound C1q was detected at 450 nm using tetramethyl benzidine/peroxidase as a substrate system and expressed as absorbance units (Abs). RESULTS: The identified 20 APS with elevated anti-beta2 GPI: 4 with IgG only, 4 with IgM only, 1 with IgA only, 1 with IgG and IgA, 6 with IgG and IgM and 4 with IgG, IgA and IgM. C1q binding from 20 healthy controls was 0.039 +/- 0.029 (SD). Of the APS, 17/20 (85%) had Abs >5 SD above controls. The 3 APS with C1q Abs within normal limits had, respectively, IgM only (1), IgA only (1), and both IgG and IgM (1). Statistical analyses (Kruskal-Wallis followed by Dunn's post test) suggest differences in IgG and IgG + IgM groups compared to con (Kruskal-Wallis: p = 0.0002; Dunn's: con vs. IgG, p < 0.05; con vs. IgG + IgM, p < 0.01). CONCLUSIONS: Anti-beta2 GPI from APS appear to have a variable degree of C1q affinity. Those patients with strong C1q binding responses are likely to have an inflammatory component to their disease processes.     

Autoantibodies to plasminogen and tissue plasminogen activator in women with recurrent pregnancy loss

Reduced fibrinolytic activity has been described in primary anti-phospholipid syndrome (PAPS), and may be responsible for thrombotic events. Antibodies to tissue type plasminogen activator (t-PA) or plasminogen (PLG) might contribute to the hypofibrinolytic state in autoimmune diseases, but the clinical significance of these antibodies is still unclear in recurrent pregnancy loss (RPL). The aim of this study is to evaluate the prevalence and clinical significance of anti-PLG and anti-t-PA antibodies in 87 patients with a history of RPL: 54 women with well-defined PAPS (mean age 32.5 years; range 26-38) and 33 women with unexplained RPL (mean age 30 years; range 24-39). IgG anti-PLG antibodies were found in 20 and four patients from the group with RPL/PAPS and unexplained RPL, respectively; IgG anti-t-PA antibodies were found in 11 and two patients from the above two groups, respectively. IgG anti-PLG antibodies were associated with the high risk of RPL (OR 7.2, P = 0.004), especially with RPL/PAPS (OR 11.2, P < 0.001) evaluated by Fisher's exact test, while IgG anti-t-PA were associated with RPL/PAPS (OR 10.0, P = 0.01) but not with RPL (OR 6.8, P = 0.06). A significant inhibition of exogenous fibrinolysis was observed by IgG fractions from patients with anti-PLG or anti-t-PA antibodies on microplates and on the human umbilical vein endothelial cells, compared with those from healthy controls. The prevalence of IgG anti-PLG antibodies was high in RPL patients, especially in RPL/PAPS, while the prevalence of IgG anti-t-PA antibodies was high in RPL/PAPS but not in RPL, and some of them might inhibit fibrinolysis in patients.     

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.     

Serum amyloid A in autoimmune thrombosis

The objectives of this study were (1) to determine how levels of serum amyloid A (SAA), high sensitivity C-reactive protein (CRP) and interleukin-6 (IL-6) correlate to autoimmune diseases in patients with or without thrombosis, and (2) to discuss the parameters that influence the relative SAA values. SAA, CRP and IL-6 concentrations were determined by enzyme linked immunosorbent assay (ELISA). 84 patients with secondary antiphospholipid syndrome (SAPS), primary antiphospholipid syndrome (PAPS), systemic lupus erythematosus with antiphospholipid antibodies (SLE+aPL), SLE, venous thrombosis (VT), arterial thrombosis (AT) were compared to healthy donors (n=60). The percentages of patients above cut-off were highest in the SAPS, SLE and SLE+aPL groups. Significant differences were observed between healthy donors and inflammatory groups of patients (SAPS and SLE+aPL) in all three measured parameters. SAA and CRP were shown to be correlated to a greater extent in SAPS patients than SLE+aPL patients. In summary, this cross-sectional, retrospective, small study and accompanying clinical considerations limit the ability to make definite conclusions. SAA would not serve as a useful marker for venous, arterial thrombosis or PAPS (pro-coagulant events). It could however, be a good predictor of progression from a non-inflammatory thrombotic condition to an inflammatory one.     

Induction of phospholipid-binding antibodies in mice and rabbits by immunization with human beta 2 glycoprotein 1 or anticardiolipin antibodies alone.

Anticardiolipin (aCL) antibodies are autoantibodies present in high concentrations in patients with the antiphospholipid syndrome (APS), a disorder of recurrent thrombosis and pregnancy loss. What induces aCL antibodies is uncertain, but a recent report suggested that immunization of mice with beta 2 glycoprotein 1 (beta 2 GP1) in Freund's complete adjuvant (FCA) resulted in aCL antibody production in the recipient mice. Since this observation might explain how autoantibodies might be induced by poor immunogens, such as phospholipids, we decided to explore the question further. In our first series of experiments, we found that aCL antibodies were induced in mice by beta 2GP1 mixed with adjuvants that did not contain lipids (Adju-Prime or aluminium hydroxide). This excluded the possibility that antibody induction occurred because beta 2GP1 formed complexes with lipids in FCA. We also found that aCL antibodies always appeared before anti-beta 2GP1 antibodies, excluding the possibility that aCL antibodies were directed to beta 2GP1 or were induced by formation of anti-idiotypic antibodies (to anti-beta 2GP1). In experiments, we found that immunization of mice with human IgG antibodies from patients with the APS (IgG-APS), also induced aCL antibodies. Immunization with pure bovine serum albumin (BSA) did not induce aCL antibodies. We propose that aCL antibodies are induced by proteins with high avidity for phospholipids. These proteins may be bound to phospholipids when introduced, or may bind circulating phospholipids, so transforming phospholipid molecules into immunogens. Similar mechanisms might explain autoantibody induction to other poor immunogens.     

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.     

Specificity of anti-phospholipid antibodies in infectious mononucleosis: a role for anti-cofactor protein antibodies

The antigen specificity of anti-phospholipid antibodies in infectious mononucleosis (IM) was studied using ELISA for the detection of anti-beta2-glycoprotein I (beta2-GPI), anti-annexin V, anti-protein S and anti-prothrombin antibodies and TLC immunostaining for the detection of anti-phospholipid antibodies. This technique enabled us to look at antibodies reacting to 'pure' phospholipid antigens in the absence of protein contamination. Sera from 46 patients with IM, 18 with systemic lupus erythematosus (SLE), 21 with primary anti-phospholipid antibody syndrome (PAPS), 50 with Helicobacter pylori infection and 30 healthy blood donors were tested. This study highlights anti-phospholipid antibodies in patients with IM as specific 'pure' anti-cardiolipin antibodies, while in PAPS and SLE patients anti-phosphatidylserine and anti-phosphatidylethanolamine antibodies were also found. This investigation also shows that the anti-cardiolipin antibodies found in IM can be present with anti-cofactor protein antibodies. The higher prevalence of anti-cofactor antibodies found in IM sera than in Helicobacter pylori sera may be due to the immunostimulatory effect and/or the polyclonal activation often observed in course of Epstein-Barr virus infection. However, anti-beta2-GPI and, to a lesser extent, anti-prothrombin antibodies occur with a significantly lower prevalence in IM than in PAPS patients. This finding suggests that these antibodies should be regarded as the expression of the broad autoimmune syndrome involving the phospholipid-binding plasma proteins.     

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).     

Which are the best biological markers of the antiphospholipid syndrome?

The diagnosis of antiphospholipid syndrome (APS) requires the presence of both clinical and biological features. Due to the heterogeneity of anti-phospholipid antibodies (aPL) the laboratory approach for their detection includes clotting-based tests for lupus anticoagulant (LA) as well as solid-phase assays for anticardiolipin antibodies (aCL). In addition, as it has been shown that autoimmune aPL recognize epitopes on phospholipid (PL)-binding plasma proteins, assays detecting antibodies to beta 2-glycoprotein I (beta 2-GPI) or prothrombin have been developed. The association between venous or arterial thrombosis and recurrent fetal loss with the presence of conventional aPL (LA and/or aCL) has been confirmed by many studies. The LA and IgG aCL at moderate/high titre seem to exhibit the strongest association with clinical manifestations of the APS. Several reports indicate that LA is less sensitive but more specific than aCL for the APS. Assays against PLs other than CL as well as the use of mixtures of PLs have been proposed to improve the detection of APS-related aPL. Concerning antibodies to PL-binding proteins (detected in the absence of PLs), there is evidence that anti-beta 2-GPI are closely associated with thrombosis and other clinical features of the APS. Moreover, these antibodies may be more specific in the recognition of the APS and in some cases may be present in the absence of aPL detected by standard tests. Many issues are still under debate and are discussed in this review, such as the problems of standardization of anti-beta 2-GPI assays, detection of the IgA isotype of aCL and anti-beta 2-GPI, the coagulation profiles of LA in the recognition of the thrombotic risk and the association of particular markers with subsets of patients with APS.     

The combination of different antiphospholipid antibody subgroups in the sera of patients with autoimmune diseases is a strong predictor for thrombosis. A retrospective study from a single center

OBJECTITVE: To determine the distribution of different antiphospholipid antibodies (APL-Ab) and their association with thrombosis in patients with autoimmune diseases. METHODS: Clinical data and laboratory features of 30 patients with different autoimmune diseases with positive APL-Ab were retrospectively studied for a period of more than two years. Anti-cardiolipin (aCL), anti-phosphatidylserine (aPS) and anti-beta2-glycoprotein I (abeta2-GPI) antibodies were determined by ELISA. RESULTS: Autoantibodies that target only PS were detected in 53.3% (n = 16) patients, aCL antibodies only were found in one patient (3,3%). In 43.3% (n = 13), aPS were associated with elevated levels of aCL and/or abeta2-GPI antibodies. No thrombotic event occurred in patients with aPS antibodies only compared to 6 patients from the group with different APL-Ab during 808 +/- 92 days of observation. CONCLUSION: The combination of different antiphospholipid antibody subgroups seems to be a predictor for thrombosis. The presence of aPS antibodies without additional aCL or abeta2-GPI is not associated with thrombosis. The measurement of the APL specificities in addition to the aCL antibodies may be important to develop predictive markers for the risk to develop thrombotic events.