Azzudin E. Gharavi

Thrombosis in systemic lupus erythematosus: striking association with the presence of circulating lupus anticoagulant

The lupus anticoagulant was found in the plasma of 31 of 60 patients with systemic lupus erythematosus and other connective tissue disorders (mixed connective tissue disease, systemic vasculitis, polyarteritis nodosa, primary sicca syndrome, discoid lupus, Behcets syndrome, and systemic sclerosis). Strong associations were found with biological false positive seroreaction for syphilis and thrombocytopenia. The most striking association, however, was with the high prevalence of thrombosis. This tendency to thrombosis was independent of disease activity of systemic lupus erythematosus. The lupus anticoagulant appears to be a useful marker for a subset of patients with systemic lupus erythematosus at risk for the development of thromboembolic complications.

Activation of cultured vascular endothelial cells by antiphospholipid antibodies.

Circulating antiphospholipid antibodies (aPL) are associated with a syndrome of thrombosis, recurrent fetal loss, and thrombocytopenia. We have demonstrated the activation of cultured human umbilical vein endothelial cells (HUVEC) by IgG from patients with anticardiolipin antibodies (aCL). Incubation of HUVEC for 4 h with purified IgG (100 micrograms/ml) from patients with high-titer aCL induced a 2.3-fold increase in monocyte adhesion over that seen in HUVEC incubated with IgGs from normal subjects. The effect of aCL was not attributable to LPS contamination, Fc receptors, or immune complexes. Monocyte adhesion was not induced when the aCL were added in serum-free media but was restored by the addition of purified beta 2GP1, previously described as a necessary cofactor for aCL reactivity. Purified rabbit polyclonal IgG raised against beta 2GP1 also induced monocyte adhesion when incubated with HUVEC. Preadsorption of patient serum with cardiolipin reduced monocyte adhesion by 60%. Immunofluorescent microscopy demonstrated that endothelial cells incubated with patient IgG expressed cell adhesion molecules, including E-selectin, vascular cell adhesion molecule-1, and intracellular adhesion molecule-1. These data support the hypothesis that aPL activate vascular endothelial cells, thereby leading to a pro-thrombotic state.

Immune mediated mechanism for thrombosis: antiphospholipid antibody binding to platelet membranes.

Because thrombocytopenia occurs frequently in patients with anticardiolipin (aCL) antibodies and thrombosis, some investigators have proposed that aCL antibodies may play a direct part in thrombosis by binding and activating platelets. To test this proposal experiments were performed to determine whether aCL antibodies can bind platelets. Preincubation of aCL positive sera with freeze-thawed platelets caused significant inhibition of aCL activity in four serum samples tested. Antibodies with cardiolipin binding activity were subsequently eluted from these platelets. Total phospholipids extracted from platelets inhibited aCL activity, and the specific phospholipids bound were shown to be phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol. It is concluded that aCL antibodies can bind phospholipids in platelet membranes but pertubation of the membrane must first occur.

Prophylaxis of the antiphospholipid syndrome: a consensus report

Hypothetical circumstances that may require prophylaxis for a potential antiphospholipid syndrome (primary prophylaxis), or in some instances when there already had been some manifestations of the syndrome (secondary prophylaxis), were presented to a panel of experts for their consideration on potential prophylactic intervention. These were subsequently presented to the participants in the First InternationalConsensuson Treatment of the AntiphospholipidSyndrome. In most instances there was consensus in adding low dose aspirin, an exception being aspirin allergy when other antiaggregants could be used in nonpregnant subjects. General measures to prevent thrombosis and other vaso-protective actions should also be provided. Higher risk of fetal loss or thrombosis called for anticoagulation with coumadin in nonpregnant subjects or subcutaneous low molecular weight heparin in pregnant ones. When indicated, prophylaxis of the antiphospholipid syndrome should be provided in systemic lupus erythematosus patients who are being treated for their disease. In no instanceshould corticosteroidsor immunosuppresantsbe given as prophylacticof an antiphospholipid syndrome.

A role for the polymorphism at position 247 of the β2‐glycoprotein I gene in the generation of anti–β2‐glycoprotein I antibodies in the antiphospholipid syndrome

OBJECTIVE To determine the frequencies at which either a valine or leucine occurs at position 247 in the beta2-glycoprotein I (beta2GPI) gene of normal individuals of the Caucasian, African American, and Asian ethnic groups and to compare these data with those in patients with the antiphospholipid syndrome (APS), with and without anti-beta2GPI antibodies. METHODS The DNA segment containing the position-247 polymorphism was amplified by seminested polymerase chain reaction, and the polymorphism was detected by restriction endonuclease digestion. DNA samples from 370 healthy controls of different racial backgrounds were analyzed, and the results were compared with those from 149 APS patients (66 primary; 83 secondary). Allele and genotype frequencies were compared using Fishers exact test. When significant differences were detected, pairwise comparisons were made using Fishers exact test with a Bonferroni adjustment. RESULTS Allele and genotype expression was significantly different (P < 0.0001 for both) among the 3 races, with the V allele and the VV genotype occurring most often among Caucasians, less among African Americans, and least among Asians. Conversely, the V allele and the VV genotype were found more frequently among Asian APS patients than among controls (P = 0.0028 and P = 0.0023, respectively). No significant differences in allele or genotype frequencies were seen in comparisons of the Caucasian or the African American patients with appropriate controls. The differences in allele and genotype frequencies seen in the Asian APS patients were restricted to the anti-beta2GPI-positive patients (P = 0.0018 and P = 0.0005, respectively). CONCLUSION In Asian patients with APS, expression of a V at position 247, especially in the homozygous state, is significantly associated with the presence of anti-beta2GPI antibodies and, therefore, can be viewed as a major risk factor in this ethnic group (odds ratio 9.19 and 16.33, respectively).

Antiphospholipid Antibodies Differ in aPL Cofactor Requirement

Although autoimmune antiphospholipid antibodies (aPL) may require a serum cofactor, beta2-glycoprotein I (β2GPI), for maximal binding in aPL ELISA, it is not known whether cofactor is absolutely required or is merely an enhancing factor for binding, nor is it clear whether aPL bind to cofactor itself, a cofactor-lipid complex, or a phospholipid modified in some way by cofactor. We therefore isolated and purified β2GPI and evaluated its relationship to both IgG and IgM aPL binding. aPL derived from different sera appear to have differing requirements for cofactor; the proportion of total binding attributable to cofactor varies from 46% to 95%. aPL do not bind to β 2GPI in the absence of phospholipid. Enhanced binding to phospholipid is seen if β2GPI is provided either before or with the test antibody. Autoimmune aPL bind phospholipid better with human rather than bovine cofactor. The requirement for cofactor is greater for low-avidity aPL as measured in an IgG-human cofactor system. Cofactor requirement alone does not predict the presence or absence of associated clinical complications.

Lupoid sclerosis: a possible pathogenetic role for antiphospholipid antibodies.

The case of a 45-year-old woman is described who developed transverse myelitis over a one-year period. Serological tests suggested a lupus-like illness. Antibodies to cardiolipin of the IgM class were detected in high titres in her serum. These may have played a part in the pathogenesis of her disease.

IgG subclass and light chain distribution of anticardiolipin and anti-DNA antibodies in systemic lupus erythematosus.

The IgG subclass and light chain distribution of anticardiolipin and anti-DNA antibodies were determined in serum samples from patients with systemic lupus erythematosus. With an enzyme linked immunosorbent assay (ELISA) and mouse monoclonal antibodies to individual subclasses, significant differences in the distributions of IgG2, IgG3, and IgG4 subclasses were observed between anticardiolipin and anti-DNA antibodies. Whereas anti-DNA antibodies were predominantly IgG1 and IgG3, all subclasses of anticardiolipin were detected with a prevalence ranging from 34% (IgG3) to 57% (IgG1). Clinical complications were found slightly more frequently (83%) in patients with sera containing the non or weak complement fixing subclasses (IgG2 and IgG4) than in patients with sera containing complement fixing (IgG1 and IgG3) subclasses (62%). Light chain analysis by ELISA showed a trend towards use of kappa chains for anti-DNA and lambda chains for anticardiolipin antibodies. These findings further emphasise the differences between anti-DNA and anticardiolipin antibodies in terms of their origins and potential mechanisms for producing tissue injury.

Autoantibodies in the sicca syndrome (primary Sjögren's syndrome).

IgA and IgM rheumatoid factors as well as antibodies to Ro and La were measured in 37 patients with the sicca syndrome and 9 patients with Sjögrens syndrome secondary to rheumatoid arthritis. Positive results were found in 84% (IgA rheumatoid factor), 76% (IgM rheumatoid factor), 62% (Ro), and 50% (La) of patients with the sicca syndrome. There was no significant difference in the frequency of positive results in patients with glandular versus extraglandular disease. Antibodies to La invariably occurred in patients with antibodies to Ro. In addition, patients who were anti-Ro positive also had significantly higher IgA (p less than 0.05) and IgM (p less than 0.01) rheumatoid factor activity. Since 81% of the sicca patients were concordant for 3 of the 4 antibodies tested, production of these autoantibodies appears to be related.

Diagnosis of the antiphospholipid syndrome: A proposal for use of laboratory tests

The presence of antiphospholipid (aPL) antibodies has been associated with thrombosis, pregnancy loss and thrombocytopenia in the antiphospholipid syndrome (APS). The anticardiolipin and the lupus anticoagulant tests are frequently used to detect aPL antibodies. The anticardiolipin ELISA utilizes cardiolipin coated on polystyrene plates as antigen and is a very sensitive test but lacks specificity, since it can be positive in a number of infectious (such as syphilis, HIV) and autoimmune diseases other than APS. In an effort to improve specificity, new ELISA techniques that employ alternative antigens (such as β2-glycoprotein 1, particularly when coated onto oxidized microtiter plates or mixture of phospholipids) have been developed. Several investigators have reported that these new assays enable more specific determination of aPL antibodies and thus can be used more reliably for the diagnosis and confirmation of APS. This article examines the results of those studies, including data that shows correlations of these assays with clinical manifestations of APS, and proposes a new protocol for the use of laboratory tests in the diagnosis of APS.