ADAMTS13 degradation by purified proteases and in plasma-based assays. (A) Commercial rADAMTS13 (200 nM) was incubated with various proteases (100 nM) for 2 hours at 37°C. Samples were separated under reducing conditions using SDS-PAGE, and cleavage was visualized using SYPRO Ruby. (B) rADAMTS13, varying dilutions of tissue factor (TF), and 1 mg/mL GPRP-amide were incubated in human platelet-poor plasma for 15 minutes. Thrombin generation was then initiated with calcium and quantified over time in a parallel plate by measuring fluorescence (λ_ex = 360 nm; λ_em = 460) at 37°C. Curves were generated using the Technothrombin TGA Software in triplicate, and representative images are shown. (C) rADAMTS13 and varying concentrations of tPA were added to human platelet-poor plasma, followed by TF and calcium. Clot formation and lysis was quantified by measuring OD at 405 nm every 30 seconds in triplicate, and representative images are shown. Samples were separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, and ADAMTS13 degradation was visualized via western blot using an anti-ADAMTS13 metalloprotease domain antibody. Molecular weight references are indicated on the left of blots (kDa), and full-length bands are indicated by the black arrows. APC, activated protein C; GPRP-amide, Gly-Pro-Arg-Pro-amide; OD, optical density.