Intracellular cleavage of recombinant VWF expressed in HUVECs. (A) Schematic of Myc-tagged proteins VWF-Myc/wild-type (W), VWF-Lock (L), and VWF-FLAG-His. (B) 2 µg/mL recombinant VWF-Myc and VWF-Lock from HEK293Ts was incubated with 1.6 M urea with or without 4 U/mL recombinant ADAMTS13 (AD13) for 4 hours. Anti c-Myc mAb detected the ∼176 kDa C-terminal fragment in the case of VWF-Myc but not VWF-Lock (arrow). (C) HUVECs and HEK293Ts were transfected with VWF-Myc and VWF-Lock for 72 hours. Culture supernatant was concentrated 20-fold before western blotting with anti-c-Myc mAb. VWF-Myc and VWF-Lock were equally cleaved in HUVECs only. (D) VWF-Myc or VWF-Lock expressed in HEK293Ts either upon cotransfection with ADAMTS13 (lanes 1, 2) or control plasmid pLKO.1-PGK-DsRed (DsR, lanes 3, 4). Equal cleavage of VWF-Myc and VWF-Lock is observed upon ADAMTS13 cotransfection. (E) Both HUVECs and HEK293Ts were transiently transfected with VWF-FLAG-His. Transfection media were replaced the next day, and recombinant c-Myc tagged VWF-Myc or VWF-Lock protein produced in HEK293Ts was added to culture media to monitor extracellular cleavage in culture medium. Western blot of culture supernatant collected at 48 hours shows that only VWF-His produced in HUVECs was cleaved. Exogenously added c-Myc tagged proteins in culture medium remained intact.