Journal: Blood Advances
Article Title: Development of a protease-resistant ADAMTS13 to improve stability against proteolytic degradation ∗
doi: 10.1182/bloodadvances.2024015212
Figure Lengend Snippet: 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.
Article Snippet: Recombinant (r)ADAMTS13 and human ADAMTS13 Quantikine enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN), kallikrein, FXIIa (Enzyme Research Laboratories, South Bend, IN), thrombin, FXa, FIXa, FXIa, FVIIa, activated protein C (APC), elastase, cathepsin G (Sigma-Aldrich, St Louis, MO), proteinase 3 (Enzo Life Sciences, Inc, Farmingdale, NY), FRETS-VWF73 (AnaSpec, Fremont, CA), AEBSF (4-[2-aminoethyl]benzenesulfonyl fluoride hydrochloride), Sivelestat (Tocris Bioscience, Bristol, United Kingdom), and cathepsin G inhibitor 1 (Cayman Chemical Company, Ann Arbor, MI).
Techniques: Purification, Clinical Proteomics, Incubation, SDS Page, Fluorescence, Generated, Software, Lysis, Polyacrylamide Gel Electrophoresis, Western Blot, Molecular Weight
![Characterizing the resistance of the ADAMTS13 mutants against proteolysis by purified proteases and in plasma fibrinolysis. (A) Four mutants of ADAMTS13 were developed: 2 with 1 of each C-terminal linker region mutated (T4L and T8L mutants), 1 with both linker regions mutated (T4L/T8L mutant), and a fourth with both linker regions and an additional elastase cleavage site mutation (T4L/T8L[I380G] mutant). (B) Using commercial recombinant ADAMTS13 (WT) as a control, each linker mutant (T4L, T8L, and/or T4L/T8L) was incubated at 50 nM with purified coagulation and fibrinolytic proteases at 50 nM (plasmin, thrombin, and kallikrein) or 100 nM (FXIa) for 0 to 180 minutes at 37°C. (C) The T4L/T8L mutant and 10 nM tPA were added to human platelet-poor plasma, followed by tissue factor and calcium at 37°C. Aliquots were removed at 0 and 45 minutes. (D) Mutants were incubated with 50 nM purified neutrophil-derived proteases (elastase, proteinase 3, and cathepsin G) at 37°C. The T4L/T8L(I380G) mutant was also incubated with 50 nM elastase. Western blotting was performed using an anti-ADAMTS13 metalloprotease domain antibody. The band corresponding to full-length protein (∼180 kDa) is indicated by black arrows.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9909/pmc12159909/pmc12159909__BLOODA_ADV-2024-015212-gr3.jpg)
