β₂-GPI binds to apoptotic particles, recruits C3/C3b, and mediates inactivation by factor H and factor I. (A) β₂-GPI (i, green fluorescence) and also annexin V (ii, red fluorescence) bound to apoptotic particles from human umbilical vein endothelial cells. Overlay demonstrates colocalization of β₂-GPI and annexin V to particles (iii), and unstained particles are shown by differential interference contrast (DIC; iv) by laser scanning microscopy (LSM 510 META, Zeiss, oil immersion objective 63×/1.4 NA, LSM519 Version 3.2 software). Bars represent 5μm. (B) β₂-GPI (0.05-5 μg) bound to the particles in a dose-dependent manner as measured by flow cytometry. (C) β₂-GPI and β₂-GPI/IV-V bound to apoptotic particles, in contrast to fragment β₂-GPI/I-IV, which has the C-terminal SCR5 deleted. (D) C3/C3b from NHP is deposited on apoptotic particles and addition of β₂-GPI to the plasma enhanced C3/C3b binding to the particles. (E) Purified C3b is recruited by β₂-GPI (black columns) but not by β₂-GPI/IV-V (gray columns) to the surface of apoptotic particles. Data are mean ± SD of 3 independent experiments. ***P < .001. (F) Apoptotic particles were loaded with increasing amounts of β₂-GPI and constant amounts of factor H. C3 and factor I were added and C3 cleavage followed by immunoblotting. β₂-GPI enhanced degradation of C3 (lanes 3-5). Novel cleavage products are indicated. Factor I alone (lane 2) or β₂-GPI/IV-V (lane 6) had no enhancing effect. (A-C,E) Data are representative results from 3 experiments.