ADAMTS13 endocytosis by MDMs is receptor mediated. (A) ADAMTS13-AF488 was incubated with MDMs for 1 hour at 37°C or 4°C. Subsequently, cells were analyzed by flow cytometry. (A) Left panel shows ADAMTS13-AF488 uptake at 37°C (solid red line). The sample incubated without ADAMTS13-AF488 is shown by the blue area. Right panel displays reduced uptake of ADAMTS13-AF488 at 4°C. (B) ADAMTS13-AF488 (green) was added to MDMs for 30 minutes at 37°C. MDMs were stained for EEA1 (Alexa Fluor 568; red) and nuclei (DAPI; blue). Scale bars are 10 μm. (C) Addition of EDTA reduces the uptake of ADAMTS13-AF488 (dotted line), control cells incubated without ADAMTS13-AF488 are shown by the blue area, and ADAMTS13-AF488 uptake is shown by the solid red line. Right panel quantifies the reduced uptake of ADAMTS13-AF488 in the presence of 5 mM EDTA. (D) Preincubation with dynasore for 20 minutes reduces the uptake of ADAMTS13-AF488 by 90% (dotted line). Control cells incubated without ADAMTS13-AF488 are shown by the blue area, and ADAMTS13 uptake is shown by the red solid line. Right panel quantifies the reduced uptake of ADAMTS13-AF488 upon incubation with dynasore. Bar graphs represent data from 3 independent experiments ± standard deviation (SD). Data are expressed as percentage of mean fluorescent intensity (MFI) at 37°C; 100% corresponds to the highest MFI observed for individual experiments. ***P < .001 (Student t test).