β₂-GPI forms complexes with C3/C3b and factor H. β₂-GPI, β₂-GPI/I-IV, and β₂-GPI/IV-V together with increasing amounts of factor H were incubated with immobilized C3/C3b and binding of β₂-GPI and in parallel of factor H were analyzed by ELISA. (A) β₂-GPI/I-IV bound to immobilized C3, and binding was not increased with higher amounts of factor H present (patterned columns). β₂-GPI and β₂-GPI/IV-V showed no binding to C3 (black and light gray columns). Low binding of the ligands to the plate without C3 is shown (background). (B) β₂-GPI/I-IV bound to C3 enabled dose-dependent binding of factor H to C3 (patterned columns). Circular β₂-GPI had no effect on factor H binding to C3 (black columns) and fragment β₂-GPI/IV-V mediated minor binding of factor H to C3 (light gray columns). Factor H binding to C3 alone (small black column) or to the plate (background) was excluded. (C) Steady amounts of β₂-GPI/I-IV bound to C3b (patterned columns) in the presence of increasing concentrations of factor H. Circular β₂-GPI (black columns) and β₂-GPI/IV-V (light gray columns) did not bind to C3b. Binding of the proteins β₂-GPI, β₂-GPI/I-IV, and β₂-GPI/IV-V without C3b (background). (D) In the presence of β₂-GPI/I-IV, increasing amounts of factor H bound to C3b (patterned columns). Preincubation of C3b with circular β₂-GPI (black columns) or β₂-GPI/IV-V (light gray columns) did not enhance binding of factor H to C3b. (A-D) Data are mean ± SD of 3 independent experiments. **P < .01. ***P < .001. (E) β₂-GPI/C3 complexes were isolated from NHP as β₂-GPI (lanes 6 and 7 bottom panel) was bound to precipitated C3 (lanes 6 and 7 top panel) and showed similar mobility as plasma β₂-GPI (lane 1 bottom panel). β₂-GPI did not bind to the column alone (lane 2 bottom panel). NHP-derived C3 did not bind to immobilized β₂-GPI (lanes 3-5 top panel).