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: Journal of Thrombosis and Haemostasis

Article Title: Cleavage by MMP‐13 renders VWF unable to bind to collagen but increases its platelet reactivity

doi: 10.1111/jth.14729

Figure Lengend Snippet: Cleavage of von Willebrand factor (VWF) by matrix metalloproteinase‐13 (MMP‐13). A, SDS‐PAGE of cleaved VWF samples. MMP‐13 but not ADAMTS13 at a concentration of 1.5 µmol/L was able to cleave purified human VWF (0.2 mg/mL) after 2 hours at 37°C. Degradation products were analyzed by (i) 12% reducing SDS‐PAGE and (ii) overnight separation of high molecular weight multimers on 15% acrylamide gels at 4°C. B, Schematic representation of MMP‐13 cleavage sites on VWF. Sequence analysis of MMP‐13 cleavage sites revealed two N‐terminal to the A1 domain and one within the C8‐4 domain. The ADAMTS13 (a disintegrin and metalloprotease with thrombospondin motif member 13) cleavage site within the A2 domain is also marked for reference

Article Snippet: Purified human VWF (ab88533; abcam) at 0.2 mg/mL (final concentration in Tris pH 7.4) was incubated with MMP‐13 or ADAMTS13 (6156‐AD‐020; R&D Systems) at 1.5 μmol/L final enzyme concentration for 2 hours at 37°C.

Techniques: SDS Page, Concentration Assay, Purification, High Molecular Weight, Sequencing

Journal: Circulation: Cardiovascular Imaging

Article Title: Molecular Imaging of VWF (von Willebrand Factor) and Platelet Adhesion in Postischemic Impaired Microvascular Reflow

doi: 10.1161/circimaging.118.007913

Figure Lengend Snippet: Figure 1. Mean (±SEM) microsphere- derived risk area from animals assigned to either (A) 30 min or (B) 45 min of coronary occlusion. C, Mean (±SEM) microvascular blood flow from myocardial contrast echocardiography (MCE) performed during coronary occlusion in the risk area and remote territories, as well as from sham-treated mice. D, Example of background-subtracted, color-coded MCE images during coronary occlusion, and time-intensity data after a destructive pulse sequence from the risk area and remote territory. For images, time after destructive pulse is at upper left and color-coded scale at bottom of the 5 s image. ADAMTS13 indicates a disintegrin and metalloprotein- ase with a thrombospondin type-1 motif member 13; LV, left ventricle; RA, risk area; and WT, wild-type. *P <0.05 vs remote territory.

Article Snippet: In Vivo Imaging Study Design Protocol 1 Closed-chest myocardial IR with 30 minutes of transient left anterior descending coronary artery (LAD) occlusion was performed in WT mice (n=40), WT mice treated with recombinant full-length human ADAMTS13, 5 μg IV (R&D Systems, Minneapolis, MN) just before IR (n=14), and ADAMTS13−/− mice (n=16).

Techniques: Derivative Assay, Sequencing

Journal: Circulation: Cardiovascular Imaging

Article Title: Molecular Imaging of VWF (von Willebrand Factor) and Platelet Adhesion in Postischemic Impaired Microvascular Reflow

doi: 10.1161/circimaging.118.007913

Figure Lengend Snippet: Figure 2. Postischemic myocardial perfusion by MCE in mice undergo- ing 30 min of ischemia and reperfusion measured in the remote region and the risk area. Data (mean±SEM) are shown for (A) myocardial microvascular blood flow (MBF), (B) microvascular blood volume (MBV), and (C) microvascular flux rate (β). ADAMTS13 indicates a disintegrin and metalloproteinase with a thrombo- spondin type-1 motif member 13. *P <0.05 vs remote territory; †P <0.05 vs WT (wild type); ‡P <0.05 only before correction for multiple comparison.

Article Snippet: In Vivo Imaging Study Design Protocol 1 Closed-chest myocardial IR with 30 minutes of transient left anterior descending coronary artery (LAD) occlusion was performed in WT mice (n=40), WT mice treated with recombinant full-length human ADAMTS13, 5 μg IV (R&D Systems, Minneapolis, MN) just before IR (n=14), and ADAMTS13−/− mice (n=16).

Techniques: Comparison

Journal: Circulation: Cardiovascular Imaging

Article Title: Molecular Imaging of VWF (von Willebrand Factor) and Platelet Adhesion in Postischemic Impaired Microvascular Reflow

doi: 10.1161/circimaging.118.007913

Figure Lengend Snippet: Figure 3. Mean (±SEM) signal enhance- ment on MCE molecular imaging after 30 min of ischemia and reperfusion for (A) VWF (von Willebrand factor) A-1 domain and (B) platelet GP (glycoprotein) Ibɑ. Data are expressed as signal difference between targeted and control microbubbles (MB). C, Examples of color-coded MCE molecular imaging in the short-axis plane for targeted and control MBs (color scale at bottom) and the microsphere-derived risk area in the same short-axis plane. *P <0.05 vs remote territory; †P <0.05 vs wild-type and ADAMTS13−/− (a disintegrin and metal- loproteinase with a thrombospondin type-1 motif member 13); ‡P <0.05 vs WT (wild type).

Article Snippet: In Vivo Imaging Study Design Protocol 1 Closed-chest myocardial IR with 30 minutes of transient left anterior descending coronary artery (LAD) occlusion was performed in WT mice (n=40), WT mice treated with recombinant full-length human ADAMTS13, 5 μg IV (R&D Systems, Minneapolis, MN) just before IR (n=14), and ADAMTS13−/− mice (n=16).

Techniques: Imaging, Control, Derivative Assay

Journal: Circulation: Cardiovascular Imaging

Article Title: Molecular Imaging of VWF (von Willebrand Factor) and Platelet Adhesion in Postischemic Impaired Microvascular Reflow

doi: 10.1161/circimaging.118.007913

Figure Lengend Snippet: Figure 5. Mean (±SEM) infarct size by triphenyltetrazolium chloride staining as a percentage of the total microsphere-de- rived risk area for mice undergoing either (A) 30 min of ischemia followed by 90 min of reperfusion or (B) 45 min of ischemia followed by 3 d of reperfusion. *P <0.05 vs WT (wild type) before Bonfer- roni correction; †P <0.05 vs WT+ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type-1 motif member 13) after Bonferroni correction.

Article Snippet: In Vivo Imaging Study Design Protocol 1 Closed-chest myocardial IR with 30 minutes of transient left anterior descending coronary artery (LAD) occlusion was performed in WT mice (n=40), WT mice treated with recombinant full-length human ADAMTS13, 5 μg IV (R&D Systems, Minneapolis, MN) just before IR (n=14), and ADAMTS13−/− mice (n=16).

Techniques: Staining

Journal: Circulation: Cardiovascular Imaging

Article Title: Molecular Imaging of VWF (von Willebrand Factor) and Platelet Adhesion in Postischemic Impaired Microvascular Reflow

doi: 10.1161/circimaging.118.007913

Figure Lengend Snippet: Figure 4. Examples of immunohistochemis- try from the sham-treated control WT (wild type) mice (anterior and posterior myocar- dial with anterior labeled as risk area) and from postischemic WT and ADAMTS13−/− (a disintegrin and metalloproteinase with a thrombospondin type-1 motif member 13) mice. The top and bottom rows show fused im- ages for platelet CD41 immunostaining (red), endothelial lectin staining (green), and nuclear counterstain (blue). The middle row shows corresponding images for only the red channel (platelet CD41) in the risk area. For the insets, arrowheads show single platelets, whereas the arrow in the ADAMTS13−/− mouse illustrates 3 platelets linearly arranged (scale bar=20 μm). MI indicates myocardial infarction.

Article Snippet: In Vivo Imaging Study Design Protocol 1 Closed-chest myocardial IR with 30 minutes of transient left anterior descending coronary artery (LAD) occlusion was performed in WT mice (n=40), WT mice treated with recombinant full-length human ADAMTS13, 5 μg IV (R&D Systems, Minneapolis, MN) just before IR (n=14), and ADAMTS13−/− mice (n=16).

Techniques: Control, Labeling, Immunostaining, Staining

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: 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: 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