Additional file 2: Figure S1. of A novel monoclonal antibody against the von Willebrand Factor A2 domain reduces its cleavage by ADAMTS13

Characterization of mAb SZ-179. (A) Quantification of ELISA analyses detecting SZ-179 binding to IgG1, IgG2a, IgG2a, IgG3, or IgM. (B) Quantification of ELISA analyses for SZ-179 or murine IgG1 binding to VWFα5. Dose–response curves are shown. (C) Quantification of ELISA analyses for SZ-179 or murine IgG1 binding to plasma-derived VWF. Dose–response curves are shown. Data are mean ± SD of four independent experiments. (DOCX 974 kb

Additional file 3: Figure S2. of A novel monoclonal antibody against the von Willebrand Factor A2 domain reduces its cleavage by ADAMTS13

SZ-179 inhibits cleavage of VWF by ADAMTS13 in plasma under denaturing conditions. (A, B) Pooled normal human plasma was pre-incubated with SZ-179 or isotype control IgG1 for 2 h at 37°C, and then incubated with 1.5M urea for 16 h. The proteolytic products were separated by electrophoresis in a 1.3% agarose gel and detected by anti-VWF. (C) Dose–response curve for inhibition of plasma ADAMTS13-mediated cleavage of plasma-VWF. (D) Dose–response curve for inhibition of rADAMTS13-mediated GST-VWF73-H cleavage. Results represented as mean ± SD of four independent experiments. (DOCX 1519 kb

Additional file 1: of A novel monoclonal antibody against the von Willebrand Factor A2 domain reduces its cleavage by ADAMTS13

Supplemental data. Detailed methods and materials are shown. (DOC 115 kb

Analysis of plasma and platelet VWF multimers.

<p>Plasma (A) and platelet (B) VWF multimers were assessed by 1.6% SDS-agarose gel electrophoresis and Western blotting. Normal plasma samples were diluted 1∶20 and patient plasma samples were diluted 1∶5. Samples from family members and a normal control (N) were indicated. Platelet samples were not available from IV-4.</p

Identification of a VWF gene mutation.

<p>Sequence analysis of the VWF gene in the proband detected a 6-bp nucleotides deletion in exon 28. The mutation caused D1529V1530 deletion (ΔD1529V1530) in VWF A2 domain. The ADAMTS13 cleavage site is indicated by an arrow.</p

Pedigree of the family with bleeding disorder.

<p>Squares and circles indicate males and females, respectively, and arrow indicates the proband. Black color denotes affected individuals. A slash through the symbol indicates decreased individuals.</p

Expression of WT and mutant VWF in transfected HEK293 cells.

<p>VWF:Ag, VWF antigen; VWF:CB, VWF collagen-binding activity; WT, wild-type VWF; WT (1/2), one-half amount of WT plasmid; Data represent mean ± SD,</p>**<p><i>p</i><0.01 <i>vs.</i> WT,</p>*<p><i>p</i><0.05 <i>vs.</i> WT. Data were from three independent experiments.</p

Multimer analysis of recombinant VWF expressed in HEK293 cells.

<p>Multimeric analysis of recombinant VWF was performed with the conditioned medium from HEK293 cells transfected with plasmids for WT and the mutant, individually or in combination. Samples from the vector-transfected cells were used as a negative control.</p

Detection of WT and mutant VWF in transfected HEK293 cells by immunostaining.

<p>HEK293 cells were transfected with a control vector or plasmids expressing WT or mutant VWF. The cells were stained for VWF (red), PDI (green) or DAPI (nucleus, blue). In the right column panels, merged pictures of green, red and blue channels are shown. The images were obtained with a confocal microscope. Scale bars: 10 µm.</p

SZ34 inhibits the proteolysis of VWF multimers by ADAMTS13 under shear stress.

<p>Purified pVWF (150 nM) was pre-incubated with SZ34 (0–200 µg/ml) for 30 min at 37°C, and then incubated with 50 nM rADAMTS13. After 5 min of vortexing at 2,500 rpm on a mini vortexer, VWF multimers were separated by 1.5% agarose gel electrophoresis and immunologic analysis. A representative image of 4 independent experiments is shown.</p