Effect of intact/nicked β2GPI on the VEGF-dependent tube formation of HUVECs cocultured with fibroblasts in the presence or in the absence of AS4.5. VEGF-dependent tube formation of HUVECs cocultured with primary human fibroblasts was evaluated in the presence or absence of AS4.5_. (A) HUVECs were visualized by immunostaining with anti–human CD31 antibodies. In the top 2 lines of the panels, the assay was done without AS4.5_. AS4.5 was added in the bottom 2 lines of the panels. Serial concentrations of intact β2GPI were added in lines 1 and 3, whereas nicked β2GPI was added in lines 2 and 4. Similar results were obtained in the second and third experiments (original magnification, ×40). (B) Capillary tube formation was quantified using KURABO Angiogenesis Image Analyzer, Version 2. Obtained data (pixels) were plotted onto the graph. Concentrations of intact/nicked β2GPI were as follows: 0.025, 0.1, 0.4, 1.0, or 4.0 μM. Error bars represent SE. Tube areas in the presence of AS4.5 alone (50 nM)* were compared with those in the presence of both AS4.5 (50 nM) and nicked β2GPI (0.4 μM; **P = .044; Student t test).