Binding and degradation of VWF by macrophages. THP-1 cells were differentiated into macrophages and incubated for 1 hour at 4°C with either radiolabeled pd-VWF (A-C) or VWF-GFP (D). (A) Binding of various concentrations of ¹²⁵I-labeled pd-VWF (0-135 μg/mL) was determined as described in “Cell-binding experiments.” (B) Radiolabeled VWF (13.5 μg/mL) was added to THP-1 cells in the presence of unlabeled VWF (0-250 μg/mL). (C) Radiolabeled pd-VWF was added to THP-1 cells for a 1-hour period at 4°C. Cells were then washed, and incubation was continued at 37°C to initiate endocytosis. At indicated time points, samples were taken to determine the amount of degraded material. Degraded material is defined as the radioactivity that is soluble in 10% trichloroacetic acid. In all experiments, controls were included to determine the amount of nonspecific degradation in the absence of cells, which routinely was less than 10% of degradation in the presence of cells. Data represent mean plus or minus SEM of 6 to 9 experiments. (D) THP-1 cells were incubated with VWF-GFP (65 μg/mL) for 1 hour at 4°C. After washing, cells were fixed and analyzed for the presence of bound VWF-GFP. (E-G) After incubation at 4°C and washing, cells were placed at 37°C to initiate endocytosis. After 2, 10, and 60 minutes, cells were fixed and processed for analysis (original magnification 630×).