Journal: ASAIO journal (American Society for Artificial Internal Organs : 1992)

Article Title: Benchtop von Willebrand Factor Testing: Comparison of Commercially Available VADs and Evaluation of Variables for a Standardized Test Method

doi: 10.1097/MAT.0000000000000849

Figure Lengend Snippet: Normal FFP, FFP+rhADAMTS13 (1 or 4μg/mL) and FFP+EDTA (10mM) were assayed for ADAMTS13 antigen level(A) and activity(B). The double solid line (=) denotes normal levels. Error bars are +/− one standard deviation.

Article Snippet: Aliquots supplemented with 1 or 4μg/mL recombinant human ADAMTS13 (rhADAMTS13, R&D Systems, Minneapolis, MN), or 10mM ethylenediaminetetraacetic acid (EDTA) were rocked on a VariMix platform rocker (Model {"type":"entrez-nucleotide","attrs":{"text":"M79735","term_id":"271754","term_text":"M79735"}} M79735 , Thermo Scientific) at 1Hz, with samples collected into low protein retention microcentrifuge (LPRM) tubes (Fisher Scientific, Pittsburgh, PA) at 30 and 60 minutes, then hourly until 6h.

Techniques: Activity Assay, Standard Deviation

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: Fig. 1. 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, Clinical Proteomics, 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: Fig. 4. 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: Clinical Proteomics, 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: Fig. 5. 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) (R2 = 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: 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: Fig. 6. 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, Drug discovery, Activity Assay, Clinical Proteomics

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: Fig. 7. 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, Drug discovery

Journal: PLoS ONE

Article Title: Structural Basis of Type 2A von Willebrand Disease Investigated by Molecular Dynamics Simulations and Experiments

doi: 10.1371/journal.pone.0045207

Figure Lengend Snippet: Qualitatively similar unfolding pathways were observed in all runs with the wild-type and mutants (see , , , , , , , , , , ). ( A ) Applied tensile force. Events observed during the simulations corresponding to force peaks (i.e., sharp increases followed by drops) are indicated. ( B ) Formation of secondary structure elements. The colors are explained in the legend on the right. The position of the Tyr -Met cleavage site is indicated by a red line and labeled on the right. ( C ) C RMSD of the two C-terminus proximal helices 5 and 6. ( D ) Solvent accessible surface area of the Tyr -Met cleavage site. ( E ) C RMSD from the native state for the N-terminal part of the (residues 1497 to 1605) and the C-terminal part of the protein (residues 1606 to 1668). ( F ) Solvent accessible surface area of the minimum docking unit for ADAMTS13 (residues 1645–1668) identified in a previous experimental study .

Article Snippet: Recombinant ADAMTS13 was quantified in western blots probed with streptavidin-HRP by comparison to serial dilutions of a reference preparation of biotinylated albumin, which contains 4 moles of biotin per mole of albumin, prepared by the chemical biotinylating agent ChromaLink (SoluLink, San Diego, CA).

Techniques: Labeling

Journal: PLoS ONE

Article Title: The Interaction between Factor H and Von Willebrand Factor

doi: 10.1371/journal.pone.0073715

Figure Lengend Snippet: ( A ) Cleavage of VWF-A2 (100 nM) by ADAMTS-13 (10 nM) after 1 hr incubation in the presence or absence of fH (0.6 µM) was detected by immunoblotting (IB) with polyclonal anti-VWF antibody. A representative blot is shown (n=5). ( B ) The results of three separate experiments with ADAMTS-13-mediated VWF-A2 cleavage were summarized as bar graphs. The VWF-A2 cleavage (%) was calculated by measuring the ratio of band density of cleaved to uncleaved + cleaved VWF-A2. ( C ) Recombinant GST VWF-A2 (100 nM) was incubated with ADAMTS-13 (10 nM), in the presence or absence of fH (0.6 µM) for different time intervals. The cleavage products were detected by immunoblotting with anti-GST antibody. A representative blot is shown (n=3). ( D ) The half maximal cleavage time of GST VWF-A2 by ADAMTS-13 was calculated using the data obtained from different incubation intervals (20 minutes to 2 hours), in the presence or absence of fH. The results of these experiments were summarized as a linear graph (n=3, t-test, ** p<0.01, * p<0.05).

Article Snippet: To quantify the effect of fH on ADAMTS-13-mediated VWF cleavage, we measured the activity of recombinant ADAMTS-13 in the presence or absence of plasma-purified fH using FRETS-VWF73 as substrate in a commercial kit (ATS-13, Gen-Probe).

Techniques: Incubation, Western Blot, Recombinant

Journal: PLoS ONE

Article Title: The Interaction between Factor H and Von Willebrand Factor

doi: 10.1371/journal.pone.0073715

Figure Lengend Snippet: ( A ) Cleavage of ADAMTS-13 substrate (FRETS-VWF73) at different concentrations of fH was quantified using a commercial kit (ATS-13, Gen-Probe). Polyclonal anti-fH antibody (100 nM) was used to block the effect of fH on ADAMTS-13-mediated cleavage of the substrate (n=3). ( B ) Factor H was removed from normal serum by immunoadsorption using sepharose beads coated with polyclonal anti-fH antibody. Immunoblotting of 1 µl of fH-depleted serum with an anti-fH antibody showed the absence of fH band in comparison to normal serum. ( C ) The number of ULVWF strings anchored to the surface of histamine-stimulated-HUVECs (with adherent platelets) in the presence of recombinant ADAMTS-13 (control); ADAMTS-13 and factor H; or ADAMTS-13, factor H and a polyclonal anti-factor H antibody was counted starting at 10 sec or 2 min after perfusion in 20 review fields (×200 magnifications; n=4, *p<0.05).

Article Snippet: To quantify the effect of fH on ADAMTS-13-mediated VWF cleavage, we measured the activity of recombinant ADAMTS-13 in the presence or absence of plasma-purified fH using FRETS-VWF73 as substrate in a commercial kit (ATS-13, Gen-Probe).

Techniques: Blocking Assay, Western Blot, Recombinant

Journal: PLoS ONE

Article Title: The Interaction between Factor H and Von Willebrand Factor

doi: 10.1371/journal.pone.0073715

Figure Lengend Snippet: ( A ) Cleavage of recombinant VWF-A2 (100 nM) after 30 minutes of incubation with ADAMTS-13 (10 nM) in the presence or absence of full-length or truncated fH (100 nM) was studied by Western-blotting using anti-GST antibody. A representative blot is shown (n=7). ( B ) The band intensity of the VWF-A2 cleavage products was compared between samples with full-length and truncated fH (n=7, t-test).

Article Snippet: To quantify the effect of fH on ADAMTS-13-mediated VWF cleavage, we measured the activity of recombinant ADAMTS-13 in the presence or absence of plasma-purified fH using FRETS-VWF73 as substrate in a commercial kit (ATS-13, Gen-Probe).

Techniques: Recombinant, Incubation, Western Blot

Journal: Clinical Pharmacology and Therapeutics

Article Title: Use of PopPK and E‐R Analyses toward Explaining Causal Link Between ADAMTS13 in Recombinant vs. Plasma‐Based Therapies and Clinical Effects in cTTP

doi: 10.1002/cpt.3720

Figure Lengend Snippet: General framework for repeated time‐to‐event manifestations. ADAMTS13, a disintegrin and metalloproteinase with thrombospondin motifs 13; C ave , average ADAMTS13 activity; C trough , trough ADAMTS13 activity; E max , maximum effect; FOCE, first‐order conditional estimation; LAPLACE, Laplacian method; LDH, lactate dehydrogenase; NONMEM, nonlinear mixed‐effects modeling; PK, pharmacokinetic. a Two patients included in the exploratory data analysis and repeated time‐to‐event modeling were not considered part of the prophylaxis cohort in the count exposure–response analysis.

Article Snippet: We therefore conducted integrated population pharmacokinetics (PopPK) analysis and exposure–response modeling based on three clinical trials of recombinant ADAMTS13 (rADAMTS13, Takeda Pharmaceuticals U.S.A., Inc.) in patients with cTTP.

Techniques: Activity Assay

Journal: Clinical Pharmacology and Therapeutics

Article Title: Use of PopPK and E‐R Analyses toward Explaining Causal Link Between ADAMTS13 in Recombinant vs. Plasma‐Based Therapies and Clinical Effects in cTTP

doi: 10.1002/cpt.3720

Figure Lengend Snippet: Prediction‐corrected visual predictive checks: ( a ) PBT; ( b ) rADAMTS13 (phase III study). The dashed black lines represent the 5 th and 95 th percentiles of ADAMTS13 activity, and the solid black line represents the median (50 th percentile) of ADAMTS13 activity. The red solid lines are the model‐predicted 5 th and 95 th percentiles, and the shaded red area represents their respective 95% PI. The blue solid line is the model‐predicted median 50 th percentile, and the shaded blue area represents the 95% PI. “Other” is the remaining PBT types in the PopPK methods dataset. ADAMTS13, a disintegrin and metalloproteinase with thrombospondin motifs 13; FFP, fresh frozen plasma; PBT, plasma‐based therapy; pd FVIII/VWF, plasma‐derived factor VIII/ von Willebrand factor; PI, percentile interval; PK‐I, pharmacokinetic infusion I; PopPK, population pharmacokinetics; rADAMTS13, recombinant ADAMTS13; S/D, solvent/detergent.

Article Snippet: We therefore conducted integrated population pharmacokinetics (PopPK) analysis and exposure–response modeling based on three clinical trials of recombinant ADAMTS13 (rADAMTS13, Takeda Pharmaceuticals U.S.A., Inc.) in patients with cTTP.

Techniques: Activity Assay, Clinical Proteomics, Derivative Assay, Drug discovery, Recombinant, Solvent

Journal: Clinical Pharmacology and Therapeutics

Article Title: Use of PopPK and E‐R Analyses toward Explaining Causal Link Between ADAMTS13 in Recombinant vs. Plasma‐Based Therapies and Clinical Effects in cTTP

doi: 10.1002/cpt.3720

Figure Lengend Snippet: Exposure (average ADAMTS13 activity)‐response (hazard for thrombocytopenia count) relationship for thrombocytopenia count. The final Poisson model with random effect and sigmoidal E max drug effect adequately described the observed thrombocytopenia counts. The x‐axis represents the distribution (histogram) of the average ADAMTS13 activity values from Periods 1 and 2 of the phase III study. ¹⁰ The left y‐axis represents the model‐predicted hazard (solid blue line) or risk of thrombocytopenia. The right y‐axis represents the total number of patients falling under each bin of exposure (highlighted on the x‐axis). Data are from patients of all ages. ADAMTS13, a disintegrin and metalloproteinase with thrombospondin motifs 13; C ave , average ADAMTS13 activity; E max , maximum effect; PBT, plasma‐based therapy; Q1W, once every week; Q2W, once every 2 weeks; rADAMTS13, recombinant ADAMTS13.

Article Snippet: We therefore conducted integrated population pharmacokinetics (PopPK) analysis and exposure–response modeling based on three clinical trials of recombinant ADAMTS13 (rADAMTS13, Takeda Pharmaceuticals U.S.A., Inc.) in patients with cTTP.

Techniques: Activity Assay, Clinical Proteomics, Recombinant

Journal: Clinical Pharmacology and Therapeutics

Article Title: Use of PopPK and E‐R Analyses toward Explaining Causal Link Between ADAMTS13 in Recombinant vs. Plasma‐Based Therapies and Clinical Effects in cTTP

doi: 10.1002/cpt.3720

Figure Lengend Snippet: C ave exposure distribution and model‐predicted probability of zero thrombocytopenia events by percentiles of C ave ( N = 41). ADAMTS13, a disintegrin and metalloproteinase with thrombospondin motifs 13; C ave , average ADAMTS13 activity; P, percentile; Q1W, once every week; Q2W, once every 2 weeks; rADAMTS13, recombinant ADAMTS13.

Article Snippet: We therefore conducted integrated population pharmacokinetics (PopPK) analysis and exposure–response modeling based on three clinical trials of recombinant ADAMTS13 (rADAMTS13, Takeda Pharmaceuticals U.S.A., Inc.) in patients with cTTP.

Techniques: Activity Assay, Recombinant