Intracellular cleavage of VWF calcium mutants in HUVECs. VWF-A2 FRET proteins were collected and concentrated from HUVEC culture supernatant. These proteins were either incubated in buffer with 1.6M urea alone or buffer with urea and 8 U/mL ADAMTS13 at 37°C for 4 hours. Monoclonal anti-VWF-A2-C-domain antibody (MAB2764, R&D Systems) was used for probing the western blot. Results show that R1597W is completely cleaved and D1498M is mostly cleaved, even in the absence of exogenous ADAMTS13 addition.