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Abstract
Purpose Serine protease (SP) enzymes play a critical role in both the coagulation and complement cascades. Anti-SP antibodies are found in approximately 40% of patients systemic lupus erythematosus (SLE) and antiphospholipid Syndrome (APS). Complement activation is important in SLE and APS. However, little is known about the effects of anti-SP antibodies on complement activation in these diseases. We sought to investigate whether affinity-purified antibodies to the SP Factor(F)Xa and Thrombin (Thr) alter the effects of these SP on complement cleavage in the presence or absence of the physiological inhibitor anti-thrombin (AT).
Methods Serum was obtained by informed consent from patients with SLE (n=10) and/or APS (n=2) under long-term follow-up at University College London Hospital. A novel method was developed to affinity purify anti-FXa and anti-Thr IgG separately. We incubated FXa (2 µM) and Thr (2.7 µM) separately with complement component C3 to determine baseline C3 cleavage. We then repeated the experiment in the presence of AT3, the appropriate anti-SP (anti-FXa or anti-Thr) or both. Finally we reviewed medical records of 40 patients with SLE to determine whether low C3 was associated with seropositivity for anti-FXa and/or anti-Thr in these patients.
Results Both FXa and Thr cleaved C3 into C3a/C3 b. This was inhibited by AT, though both cleavage and inhibition by AT were stronger for Thr than FXa. Conversely anti-SP antibodies enhanced C3 cleavage by SP. Anti-FXa IgG (n=3) increased FXa-mediated cleavage of C3 by 1.3-fold. Anti-Thr IgG (n=8) increased Thr-mediated C3 cleavage by 1.8-fold. AT mediated inhibition was prevented by addition of anti-FXa IgG (n=2) but not anti-Thr IgG. Analysis of clinical serology records for the last 10 consecutive clinic visits of 28 anti-SP-positive patients showed a lower level of C3 (0.92 g/L) in the patients double positive for anti-FXa and anti-Thr than for anti-Thr alone (1.12 g/L) or anti-FXa alone (1.16 g/L).
Conclusions Anti-FXa and anti-Thr enhance cleavage of C3 by FXa and Thr respectively. Presence of these antibodies in-vivo in patients with SLE and/or APS may promote increased complement activation and disease activity. This finding may have potential translational implications for future treatment of these diseases.
Data showing relative C3 cleavage of Thr and FXa in the presence and absence of antibodies and inhibitors