Background/Purpose: Anti-cardiolipin/β₂ glycoprotein I (aCL/β₂GPI) antibodies are representative antiphospholipid antibodies(aPLs) that target the complex of cardiolipin (or anionic molecules) and β₂GPI. However, aCL is often regarded as an autoantibody with relatively lower specificity and the significance of its detection may considered to have secondary role to detection of aβ₂GPI. Recently, the antibodies to β₂GPI domain I (aβ₂GPI DI) are reported as more specific to the  manifestations of antiphospholipid syndrome (APS). In this study, the immunological and functional diversity of aCL/β₂GPI was examined in newly established monoclonal autoimmune aCL derived from a patient with APS. 

Methods: B cells from 40 APS patients were immortalized with EBV infection to clone aCL antibody-producing cells using limiting dilution and sorting techniques. A stable cell line containing an IgG-type monoclonal antibody (EV35102) was established by introducing genes in the IgG variable region into CHO-K1 cells. The presence of a homologous antigen of EV35102 was examined by ELISA. The anticoagulation function of EV35102 was evaluated in monocytes from healthy persons using quantification of induced tissue factor (TF) mRNA by real-time PCR after treatment of the cells with EV35102. The activation of intra-cellular signal protein was analyzed with the antibody array kit (Pathscan^TM : Cell Signaling Technology, USA). Regarding the result of ELISA tests for the monoclonal antibodies, the cohort of 182 APS patients (with and without systemic lupus erythematosus(SLE) and 141 patients with connective tissue diseases were analyzed of their aPL profiles. Five clotting assays for testing lupus anticoagulant and 7 enzyme-linked immunosorbent assays(IgG/IgM aCL, IgG/IgM aβ₂GPI and aβ₂GPI D1) were performed.

Results: EV35102 bound to cardiolipin only in the presence of β₂GPI, thus behaving as a typical β₂GPI-dependent aCL.  However, EV35102 did not recognize total β₂GPI molecule in any conditions (negative for aβ₂GPI).  Surprisingly EV35102 bound to domain I of β₂GPI (positive for aDI). EV35102 significantly induced monocyte TF mRNA expression compared with the IgG control (9.23+/-2.79 vs. 2.18+/-0.32, p<0.05). The p38 Mitogen-activated protein kinase(MAPK) significantly and specifically phosphorylated in the monocytes stimulated with EV35102 compared with the IgG control (OD value : 2.92+/-0.31 vs 1.23 +/-0.51 , p<0.05). Anti CL antibody positive with negative aβ₂GPI IgG was found in 5/141 in non-APS control and 20/182 in APS. Among 20 APS patients with aCL IgG positive and aβ₂GPI negative, 4/20 were aβ₂GPI DI positive. None of the non-APS with aCL positive, ab2GPI negative showed ab2GPI D1 positive.

Conclusion: EV35102 represented a new subset of aCL/β₂GPI in patients with APS; its epitope would locate on domain I of β₂GPI, but the antibodies are undetectable by anti-(total) β₂GPI assay. EV35102 had procoagulant property thus this subset of aCL/β₂GPI is possible to be pathogenic. The diversity in aCL/β₂GPI should be recognised and aCL assay is still useful for screening this subset.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/procoagulant-property-of-a-novel-patient-derived-autoimmune-igg-type-monoclonal-anticardiolipin-antibody-that-binds-to-beta-2-glycoprotein-domain-i-but-not-to-total-beta-2-glycoprotein-i-molecule/