A simple method to discriminate between beta2-glycoprotein I- and prothrombin-dependent lupus anticoagulants.

Lupus anticoagulants (LAC) are a heterogeneous group of autoantibodies that prolong phospholipid-dependent clotting assays. The autoantibodies that cause LAC activity are predominantly directed against beta2-glycoprotein I (beta2GPI) or prothrombin. In the present study, we describe a method to differentiate between LAC caused by antibodies directed against beta2GPI or prothrombin. Monoclonal antibodies, affinity purified patient antibodies, and selected patient samples were used to show that in an aPTT-based clotting assay (PTT-LA; Diagnostica Stago), the use of cardiolipin vesicles in the neutralization procedure discriminates between beta2GPI- or prothrombin-dependent LAC activities. Addition of cardiolipin vesicles shortened the prolonged clotting time caused by anti-beta2GPI antibodies with LAC activity, whereas this procedure further prolonged clotting times caused by antiprothrombin antibodies with LAC activity. In contrast, addition of phosphatidylcholine/phosphatidylserine vesicles corrected prolonged clotting times caused by either anti-beta2GPI or antiprothrombin antibodies with LAC activity. The effects of cardiolipin (CL) on beta2GPI-induced LAC activity were specific for contact activation mediated clotting assays. Possible explanations for these findings are the relatively high affinity of beta2GPI for cardiolipin, as determined by surface plasmon resonance analysis, and inhibition by anti-beta2GPI antibodies of the CL-induced prolongation of the PTT-LA.

Quantification of lupus anticoagulants in clinical samples using anti-beta2GP1 and anti-prothrombin monoclonal antibodies.

Quantification of lupus anticoagulant (LA) in clinical samples is hampered by the lack of a suitable standard of activity. We evaluated the use of mAbs displaying LA activity for this purpose. As most patient samples contain both beta2Glycoprotein I (beta2GP1) and prothrombin dependent LA, a combination of two mAbs, one of each specificity, was added to normal plasma in a concentration from 0 to 60 microg/ml. Eight assay systems using different reagents and instruments were used. The calibration curves were linear for all but one, with marked differences between the responsiveness to each mAb. A panel of plasmas from 69 patients with persistent LA diagnosed using the SSC-ISTH criteria was tested. An antiphospholipid syndrome (APS) was present in 40, whereas 29 were asymptomatic. LA activities of individual plasmas varied between assays (p < 10(-4)), but homogeneous subgroups were identified. In a majority of samples, LA activity displayed a prothrombin-dependent profile, with a variable contribution of beta2GP1-dependent activity. The latter was associated to beta2GP1 antibodies detected by solid-phase immunoassay. By using 3 dilute Russell viper venom time assays, higher LA titers were found in APS, compared to asymptomatic patients (p <0.05).

Lack of association between antiphospholipid antibody and WHO classification in lupus nephritis.

OBJECTIVE: To study the prevalence of antiphospholipid antibody (aPL) in patients with biopsy proven lupus nephritis (LN) and to investigate if there is any association between the presence of serum aPL and WHO classes. METHODS: Seventy-one patients (68 female and 3 male, mean age 31 years, range 10-67) meeting ACR criteria for the classification of SLE and with biopsy proven LN were included. For every patient, we evaluated anticardiolipin antibodies, lupus anticoagulant and renal biopsy classified according to the WHO classification criteria (activity and chronicity scores were included). Twenty-four hour urinary protein at the time of biopsy was considered. RESULTS: Twenty-nine patients had class V LN, 27 had class IV, 11 had class III, 3 had class II and 1 had class I. Twenty-seven (40.2%) patients were aPL positive. The prevalence of aPL positive patients was 45% in class V, 33.3% in class IV and 45.6% in class III. We did not find any significant association between the presence of aPL and the WHO class (p = 0.61 with class V, p = 0.31 with class IV and p = 0.73 with class III). There was no association between the presence of aPL and activity (p = 0.52) or chronicity scores (p = 0.42). We also did not find any association between proteinuria and the presence of aPL (p = 0.3). CONCLUSIONS: Our results suggest that there is no association between the presence of aPL and the different WHO classes. The presence of these antibodies does not seem to be related to histological activity or the chronicity of lupus nephritis nor proteinuria.

Methods for subtyping lupus anticoagulants.

Two simple procedures were tested for their potential to identify beta2-glycoprotein 1 (beta2GP1) or prothrombin cofactor dependence among lupus anticoagulants (LA). The first comprised mixing test plasma 1:4 with beta2GP1-deficient plasma instead of with normal plasma. beta2GP1 deficiency decreased, but did not abolish most LA detectable in KCT, DRVVT and APTT clotting tests. Mixing 1:4 with bovine plasma was evaluated in a second test based on the KCT in the expectation that prothrombin-dependent LA would be preferentially shortened. Bovine plasma had a similar correcting effect on LA in all three tests considered here. Conversely, a prothrombin antibody was found to have similar prolonging effect on all three of these tests. LA patient plasmas displayed considerable heterogeneity when analysed using a combination of these two tests. The clinical significance of these tests remains uncertain. DRVVT and KCT tests do not appear to discriminate beta2GP1-dependent from prothrombin-dependent LA.