Antiphospholipid Antibodies with LAC Activity That Bind beta2-Glycoprotein I Cause Increased Resistance Against Activated Protein C.
Authors: de Laat Bas , Meijer Sander B. , Eckmann Carel M. , van Schagen M. , Mertens Koen , van Mourik Jan A. 
Published: April 2020

Background: The antiphospholipid syndrome is characterized by the occurrence of vascular thrombosis combined with the presence of antiphospholipid antibodies (aPL) in plasma of patients. Recently it was published that aPL with lupus anticoagulant activity (LAC), caused by anti-beta2-glycoprotein I (beta2GPI) antibodies, highly correlate with a history of thrombosis. aPL-related resistance against activated protein C (APC) is one of the proposed mechanism responsible for thrombosis. We investigated a possible correlation between a beta2GPI-dependent LAC and increased APC-resistance in a population of 22 plasma samples with LAC activity.

Methods: Twenty-two LAC-positive plasma samples were tested for beta2GPI-dependence (titration of cardiolipin into an APTT-based assay), increased APC-resistance, anti-beta2GPI IgG/IgM antibodies, anti-prothrombin IgG/IgM antibodies and anti-protein C IgG/IgM antibodies. In addition, a monoclonal anti-beta2GPI antibody and patient-purified IgG (both with LAC activity) were diluted in plasma with/without protein C and tested for occurrence of a beta2GPI-dependent LAC (normalization of clotting time by the addition of cardiolipin). To study aPL-induced APC-resistance in more detail, surface plasmon resonance analysis was used to investigate binding between APC and beta2GPI in the presence/absence of a mouse-derived monoclonal anti-beta2GPI antibody.

Results: Eleven plasma samples that displayed a beta2GPI-dependent LAC also showed increased APC resistance. In contrast, only 1 of the 11 plasma samples with a beta2GPI-independent LAC displayed increased APC-resistance. None of the other serological parameters (antibodies against beta2-glycoprotein I, prothrombin or protein C) displayed the same association with increased APC resistance as a beta2-glycoprotein I dependent LAC. Furthermore, we found a linear correlation between the potency of a beta2GPI-dependent LAC and the level of APC-resistance. When a monoclonal anti-beta2GPI antibody and a patient-purified IgG were tested for a beta2GPI-dependent LAC, both antibodies did not display a beta2GPI-dependent LAC when diluted in protein C deficient plasma. In literature it has been proposed that direct binding of beta2GPI to APC results in a decreased activity of APC. By using surface plasmon resonance analysis, we found that beta2GPI displayed a higher affinity for coated APC in the presence of the monoclonal anti-beta2GPI antibody (4 nM) compared to beta2GPI alone (400 nM).

Conclusion: The results of this study indicate that by adding cardiolipin into an APTT-based clotting assay, one can detect beta2GPI-dependent LAC based on increased resistance against APC. Increased resistance against activated protein C might result from direct binding of beta2GPI to activated protein C. In conclusion, our observations indicate a direct correlation between a major clinical symptom of APS (thrombosis), a diagnostic assay (beta2GPI-dependent LAC) and a potential mechanism responsible for thrombosis in the antiphospholipid syndrome (increased APC-resistance).