Fig 1: Western blot analysis of ADAMTS13 degradation by plasmin, with or without TXA. 100 nM ADAMTS13 was incubated with 10 nM plasmin in the absence (A) or presence (B) of 5 mM TXA. Aliquots were removed at various time points into reducing sample buffer and boiled immediately to stop the reaction. Samples were analyzed by Western Blot using an antibody targeting the metalloprotease domain of ADAMTS13. Bands at approximately 180 kDa correspond to full-length ADAMTS13, and the appearance of other bands indicates degradation of ADAMTS13 into smaller fragments. (C) The relative intensity of the intact ADAMTS13 band was determined at each time point and plotted as a function of time. Data were fit to a one phase exponential decay model using GraphPad Prism (V9). Values represent the mean ± standard deviation of 3 biological replicates.
Fig 2: Optimization of the stability of ATP in plasma. (A) Measurement of ATP concentration in citrated plasma in the presence or absence of various inhibitors (3 mM EDTA, 2 mM NaVO4, 1 µM IBMX, 5 nM NBTI or TBS (no inhibitor)) over the course of 4 h, expressed as a percentage of that at time 0 h. (B) Autobiotinylation activity of 100 nM ADAMTS13-BirA* in plasma in the absence of ATPase inhibitors (TBS) or presence of ATPase inhibitors (3 mM EDTA, or 2 mM NaVO4, 1 µM IBMX and 5 nM NBTI) over the course of 4 h. Samples were pulled down using streptavidin agarose and visualized by Western Blot using anti-FLAG antibody. (C) Labelling of plasma proteins by 100 nM ADAMTS13-BirA*, with serial supplementation of 2 mM ATP every hour, after the initial addition of 1 mM ATP at the beginning of the experiment, over the course of 4 h, and visualized by SYPRO-RUBY total protein stain.
Fig 3: Binding of various forms of plasminogen or apo(a) to immobilized ADAMTS13, MDTCS or fibrinogen. Various forms of plasminogen (A) (Glu-Pg, Lys-Pg, mini-Pg, µ-Pg, VFK-plasmin) or apo(a) (B) at varying concentrations 0–3.5 or 0–7 µM were injected onto immobilized ADAMTS13 (A—left), MDTCS (A—right), or fibrinogen. Change in response units was monitored over time, and corrected using a blank empty flow channel. Equilibrium value response units are plotted as a function of analyte concentration. Data points are presented as mean ± standard deviation or 3 biological replicates. Data points are fitted using a one-site binding model using GraphPad Prism.
Fig 4: Enzymatic activity of ADAMTS13-BirA*. Self-labelling of 250 nM ADAMTS13-BirA* in HBS in the presence or absence of 50 µM biotin, 1 mM ATP. Samples were subjected to pull-down by streptavidin-agarose and visualized by Western Blot with an anti-FLAG antibody.
Fig 5: Proteins identified from the in-vitro BioID screen of ADAMTS13 in plasma. Venn diagram of the number of proteins labelled from BioID assay of 100 nM ADAMTS13-BirA*, BirA* or PBS buffer (no protein) in citrated plasma, with 1 mM ATP added every hour, and 50 µM biotin, at 37 °C for 4 h. Labelled proteins were isolated using agarose streptavidin beads, digested with trypsin, and identified using LC–MS/MS. The numbers represent the number of unique labelled proteins present in each condition.
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