Expression of ADAMTS13 in human vascular endothelial cells. (A) RT-PCR detects ADAMTS13 mRNA. The amplified cDNA fragments correspond to the signal peptide (lane 1), metalloprotease domain (lane 2), spacer domain (lane 3), and the second CUB domain (lane 4) of ADAMTS13. M indicates a 1-kb DNA maker. Double asterisks indicate nonspecific band. (B) Western blot detects full-length ADAMTS13 protein in both cell lysates and conditioned media of HAECs, HUVECs, and ECV304. MDCK cells transfected with (rFL-13) or without (–) ADAMTS13-V5-His were used as positive or negative control. The arrows indicate the intact full-length ADAMTS13 proteins. (C) ADAMTS13 protease in normal human plasma (NHP) or secreted from HUVECs, HAECs, and ECV304 cleaves FRETS-VWF73 specifically, which is completely inhibited by 10 mM EDTA (+ EDTA) and 40 μg/mL rabbit anti–ADAMTS13 IgG (+ anti–A13 IgG). The relative proteolytic activity (slope, fluorescent units/min) was shown as means ± SD from 3 independent experiments. (D) Metabolic labeling and detection of newly synthesized ADAMTS13 in the cell lysate (C) and conditioned medium (M) of HUVECs, HAECs, and ECV304 at 2 hours and 24 hours. The COS7 cells and LX-2 cells transfected with ADAMTS13-V5-His were used as negative and positive controls, respectively. The arrowheads indicate intracellular ADAMTS13 in cell lysate, whereas double asterisks indicate the secreted ADAMTS13 in the conditioned medium. The vertical white lines indicate areas of noncontiguous lanes assembled.