Journal: Scientific Reports

Article Title: GC1126A, a novel ADAMTS13 mutein, evades autoantibodies in immune-mediated thrombotic thrombocytopenic purpura

doi: 10.1038/s41598-024-80674-x

Figure Lengend Snippet: Comparison of therapeutic efficacy of GC1126A, rh WT-ADAMTS13, and caplacizumab in mitigating platelet count reduction in the iTTP-mimic mouse model. ( a ) Study design for therapeutic efficacy comparison. ( b ) Platelet count results. ( c ) Residual activity of ADAMTS13 in the plasma of an iTTP-mimic mouse model. Values below the limit (0.03 IU/mL) are also not present on the graph. ( d ) Inhibitory antibody levels (BU levels). Values below the limit (0.42 BU/mL) are also not present on the graph. ( b )–( d ) Outliers were identified using the Grubbs’ test (α = 0.05) and excluded from each group. Each bar represents the mean and standard error of the mean ( n = 4 mice per group).

Article Snippet: The recombinant human ADAMTS13 (full-length) protein (rhADAMTS13 FL; R&D Systems, Minnesota, USA) served as the antigen.

Techniques: Comparison, Activity Assay

Journal: Scientific Reports

Article Title: GC1126A, a novel ADAMTS13 mutein, evades autoantibodies in immune-mediated thrombotic thrombocytopenic purpura

doi: 10.1038/s41598-024-80674-x

Figure Lengend Snippet: Comparison of therapeutic efficacy of GC1126A, rh WT-ADAMTS13, and caplacizumab in restoring severely reduced platelet counts in the iTTP-mimic mouse model. ( a ) Study design for therapeutic efficacy comparison. ( b ) Platelet count results. Outliers were identified using the Grubbs’ test (α = 0.05) and excluded from each group. Each bar represents the mean and standard error of the mean ( n = 3–10 mice per group).

Article Snippet: The recombinant human ADAMTS13 (full-length) protein (rhADAMTS13 FL; R&D Systems, Minnesota, USA) served as the antigen.

Techniques: Comparison

Journal: Scientific Reports

Article Title: GC1126A, a novel ADAMTS13 mutein, evades autoantibodies in immune-mediated thrombotic thrombocytopenic purpura

doi: 10.1038/s41598-024-80674-x

Figure Lengend Snippet: The process and outcomes of candidate screening and the pharmacokinetic profile of candidates. ( a ) The diagram shows the binding domains of anti-ADAMTS13 neutralizing antibodies (Nabs). ( b ) The strategy and process of candidate screening. ( c ) The specific activity of selected candidates in the media following transient expression ( n = 1 or 2). ( d ) The relative residual activity of selected candidates in the presence of 9 Nabs ( n = 1 or 2). ( e ) The plasma concentration profiles after the intravenous administration of the selected candidates or controls (MDTCS or MDTCS-Fc) at the dose level of 160 IU/kg. Each point represents the mean and standard error of the mean ( n = 4 mice per time point).

Article Snippet: The recombinant human ADAMTS13 (full-length) protein (rhADAMTS13 FL; R&D Systems, Minnesota, USA) served as the antigen.

Techniques: Binding Assay, Activity Assay, Expressing, Concentration Assay

Journal: Scientific Reports

Article Title: GC1126A, a novel ADAMTS13 mutein, evades autoantibodies in immune-mediated thrombotic thrombocytopenic purpura

doi: 10.1038/s41598-024-80674-x

Figure Lengend Snippet: Autoantibody escaping ability of GC1126A using the iTTP patient’s plasma. ( a ) Relative residual activity of ADAMTS13 and GC1126A in the presence of autoantibodies from iTTP patient’s plasma ( n = 2). Each patient’s plasma was diluted to inhibitory antibody concentrations of 0.5, 1, 2, and 3 BU/ml. Each diluted plasma was mixed with the same molar concentration (3 nM) of ADAMTS13 or GC1126A. Relative activity is the ratio of activity remaining after neutralizing antibodies in the patient’s plasma to activity when each substance is free of neutralizing antibodies. ( b ) Binding level of autoantibodies from patients with iTTP to ADAMTS13 and GC1126A. Statistical significance was determined using the one-way analysis of variance followed by Tukey’s test (α = 0.05). Each bar represents the mean and standard error of the mean ( n = 3).

Article Snippet: The recombinant human ADAMTS13 (full-length) protein (rhADAMTS13 FL; R&D Systems, Minnesota, USA) served as the antigen.

Techniques: Activity Assay, Concentration Assay, Binding Assay

Journal: Scientific Reports

Article Title: GC1126A, a novel ADAMTS13 mutein, evades autoantibodies in immune-mediated thrombotic thrombocytopenic purpura

doi: 10.1038/s41598-024-80674-x

Figure Lengend Snippet: Relationship between the anti-ADAMTS13 inhibitor titer and GC1126A EC50. The distribution of the amount of GC1126A required to achieve 0.5 IU/ml activity (EC50) for different patient samples at inhibitor titers of 0.6–0.9 BU/ml ( n = 4), 1 BU/ml ( n = 23), 3 BU/ml ( n = 15), 6 BU/ml ( n = 9), and 9 BU/ml ( n = 5) (the observed data are shown with closed circles). The estimated regression line and the 95% confidence interval are depicted in solid and dotted lines, respectively. The linear regression analysis yielded the following relationship: EC50 (µg/mL) = 0.0347 + 0.0396 × anti-ADAMTS13 inhibitor titer (BU/mL) (R 2 = 0.5842).

Article Snippet: The recombinant human ADAMTS13 (full-length) protein (rhADAMTS13 FL; R&D Systems, Minnesota, USA) served as the antigen.

Techniques: Activity Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: The effect of ADAMTS13 on graft‐versus‐host disease

doi: 10.1111/jcmm.18457

Figure Lengend Snippet: Role of VWF in the binding of T cells. (A) One hundred thousand Jurkat cells (immortalize malignant T lymphocytes) were incubated with HUVECs cells plated on 6‐well plates. After 15 min incubation at 37°C, the number of adhered Jurkat cells in each well was counted using pictures taken with an inverted microscope and compared between histamine‐stimulated and non‐stimulated HUVECs. The effect of recombinant ADAMTS13 (1 mg/mL), recombinant VWF‐A2 (1 mg/mL) and anti‐α L antibodies (1:1000 dilution) on the number of adhered Jurkat cells to histamine‐stimulated HUVECs were compared ( n = 4, each experiment in triplicates). p‐ values were calculated using a one‐way ANOVA test with Dunnett's multiple comparison correction compared to the number of adhered Jurkat cells to histamine‐stimulated HUVECs. * p < 0.05. (B) One hundred thousand Jurkat cells were incubated in VWF‐coated 96‐wells plates for 15 min at 37°C. Jurkat cells, either pre‐stimulated with 200 ng/mL of CCL21 or resting, were added to each well. The effect of recombinant ADAMTS13, recombinant VWF‐A2 and anti‐αL antibodies on the number of adhered CCL21‐stimulated Jurkat cells to VWF was compared ( n = 8, each experiment in triplicates). p ‐values were calculated using a one‐way ANOVA test with Dunnett's multiple comparison correction compared to the number of adhered CCL21‐stimulated Jurkat cells VWF. * p < 0.05.

Article Snippet: In some experiments, prior to adding Jurkat cells, histamine‐stimulated HUVEC cells were treated with 1 μg/mL of recombinant human ADAMTS13 (6156‐AD; R&D Systems) for 10 min at room temperature.

Techniques: Binding Assay, Incubation, Inverted Microscopy, Recombinant, Comparison

Journal: Scientific reports

Article Title: Optimization of plasma-based BioID identifies plasminogen as a ligand of ADAMTS13.

doi: 10.1038/s41598-024-59672-6

Figure Lengend Snippet: Figure 1. 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.

Article Snippet: ADAMTS13 (2.5 nM) as wild-type (R&D Systems: 6156-AD) or as ADAMTS13-BirA* was reacted with FRETS-VWF73 (1 μM) in FRETS buffer (20 mM Tris–HCl, 25 mM CaCl2, 0.05% Tween-20, pH 7.4) and the activity was measured using SpectraMax M3 (Molecular Devices) fluorescence mode at ex = 340 nm and em = 450 nm.

Techniques: Activity Assay, Western Blot

Journal: Scientific reports

Article Title: Optimization of plasma-based BioID identifies plasminogen as a ligand of ADAMTS13.

doi: 10.1038/s41598-024-59672-6

Figure Lengend Snippet: Figure 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.

Article Snippet: ADAMTS13 (2.5 nM) as wild-type (R&D Systems: 6156-AD) or as ADAMTS13-BirA* was reacted with FRETS-VWF73 (1 μM) in FRETS buffer (20 mM Tris–HCl, 25 mM CaCl2, 0.05% Tween-20, pH 7.4) and the activity was measured using SpectraMax M3 (Molecular Devices) fluorescence mode at ex = 340 nm and em = 450 nm.

Techniques: Clinical Proteomics, Concentration Assay, Activity Assay, Western Blot, Staining

Journal: Scientific reports

Article Title: Optimization of plasma-based BioID identifies plasminogen as a ligand of ADAMTS13.

doi: 10.1038/s41598-024-59672-6

Figure Lengend Snippet: Figure 4. Binding of various analytes to immobilized ADAMTS13. Analytes Glu-Plasminogen (Glu-Pg), vitronectin (Vn), stanniocalcin-2 (STC2) and bovine serum albumin (BSA) were passed over immobilized ADAMTS13 at varying concentrations (0–7 µM). 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.

Article Snippet: ADAMTS13 (2.5 nM) as wild-type (R&D Systems: 6156-AD) or as ADAMTS13-BirA* was reacted with FRETS-VWF73 (1 μM) in FRETS buffer (20 mM Tris–HCl, 25 mM CaCl2, 0.05% Tween-20, pH 7.4) and the activity was measured using SpectraMax M3 (Molecular Devices) fluorescence mode at ex = 340 nm and em = 450 nm.

Techniques: Binding Assay, Concentration Assay, Standard Deviation

Journal: Scientific reports

Article Title: Optimization of plasma-based BioID identifies plasminogen as a ligand of ADAMTS13.

doi: 10.1038/s41598-024-59672-6

Figure Lengend Snippet: Figure 3. 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.

Article Snippet: ADAMTS13 (2.5 nM) as wild-type (R&D Systems: 6156-AD) or as ADAMTS13-BirA* was reacted with FRETS-VWF73 (1 μM) in FRETS buffer (20 mM Tris–HCl, 25 mM CaCl2, 0.05% Tween-20, pH 7.4) and the activity was measured using SpectraMax M3 (Molecular Devices) fluorescence mode at ex = 340 nm and em = 450 nm.

Techniques: In Vitro, Clinical Proteomics, Isolation, Liquid Chromatography with Mass Spectroscopy

Journal: Scientific reports

Article Title: Optimization of plasma-based BioID identifies plasminogen as a ligand of ADAMTS13.

doi: 10.1038/s41598-024-59672-6

Figure Lengend Snippet: Figure 6. Effect of EACA or TXA onto Glu-Pg and Lys-Pg binding to immobilized ADAMTS13. Increasing concentration (0–5 mM) of EACA or TXA were injected along with 2 µM Glu-Pg or Lys-Pg onto immobilized ADAMTS13. 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 a competitive inhibition model ([inhibitor] vs normalized response—variable slope) using GraphPad Prism (V9).

Article Snippet: ADAMTS13 (2.5 nM) as wild-type (R&D Systems: 6156-AD) or as ADAMTS13-BirA* was reacted with FRETS-VWF73 (1 μM) in FRETS buffer (20 mM Tris–HCl, 25 mM CaCl2, 0.05% Tween-20, pH 7.4) and the activity was measured using SpectraMax M3 (Molecular Devices) fluorescence mode at ex = 340 nm and em = 450 nm.

Techniques: Binding Assay, Concentration Assay, Injection, Standard Deviation, Inhibition

Journal: Scientific reports

Article Title: Optimization of plasma-based BioID identifies plasminogen as a ligand of ADAMTS13.

doi: 10.1038/s41598-024-59672-6

Figure Lengend Snippet: Figure 5. 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.

Article Snippet: ADAMTS13 (2.5 nM) as wild-type (R&D Systems: 6156-AD) or as ADAMTS13-BirA* was reacted with FRETS-VWF73 (1 μM) in FRETS buffer (20 mM Tris–HCl, 25 mM CaCl2, 0.05% Tween-20, pH 7.4) and the activity was measured using SpectraMax M3 (Molecular Devices) fluorescence mode at ex = 340 nm and em = 450 nm.

Techniques: Binding Assay, Injection, Concentration Assay, Standard Deviation

Journal: Scientific reports

Article Title: Optimization of plasma-based BioID identifies plasminogen as a ligand of ADAMTS13.

doi: 10.1038/s41598-024-59672-6

Figure Lengend Snippet: Figure 7. 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.

Article Snippet: ADAMTS13 (2.5 nM) as wild-type (R&D Systems: 6156-AD) or as ADAMTS13-BirA* was reacted with FRETS-VWF73 (1 μM) in FRETS buffer (20 mM Tris–HCl, 25 mM CaCl2, 0.05% Tween-20, pH 7.4) and the activity was measured using SpectraMax M3 (Molecular Devices) fluorescence mode at ex = 340 nm and em = 450 nm.

Techniques: Western Blot, Incubation, Standard Deviation