β2-GPI regulates alternative and classic complement pathways. (A) NHS (20%) was preincubated with β2-GPI (0.1-0.4μM), and complement activation via the alternative pathway was assayed by determination of deposition of C5b-9. In the presence of β2-GPI, C5b-9 deposition was reduced (black columns). Factor H inhibited and bacterial inhibitor SCIN blocked complement activation (gray columns). BSA had no effect. (B) β2-GPI (black columns), C4BP (patterned columns), and bacterial inhibitor SCIN (gray columns) inhibited classic pathway activation in a dose-dependent manner. BSA had no effect. NHS (1%) was preincubated with equimolar amounts of β2-GPI, or C4BP, or SCIN or BSA (each 0.2-0.4μM) and added to IgM (10 μg/mL)–precoated wells. Complement activation was measured at 492 nm using a human C5b-9 monoclonal antibody. (A-B) Data are mean ± SD values of 3 independent experiments. *P < .05. **P < .01. ***P < .001. Activation of untreated NHS was set to 100%. (C) C3a is present in complement-activated NHS (lane 1). β2-GPI (0.4, 0.9, and 1.8μM; lanes 2-4) added to activated NHS inhibited complement activation on the level of C3a generation. Factor H/11–15 (1.8μM; lane 5) had minor effect and BSA (lane 1) had no effect. Purified C3a is shown in lane 7. (C) C3b (lane 1) is not cleaved by factor I in the presence of β2-GPI alone (lanes 2-4), but cleavage of C3b by factor I and cofactor factor H (lane 10) is enhanced in the presence of increasing amounts of β2-GPI (lanes 6-9), and additional cleavage products were generated. (C-D) Western blots show representative experiments of 3.
