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

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

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

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

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

Schematic domain structure of full-length VWF and recombinant VWF fragments.

<p>The domain structure of human preproVWF is shown above the structures of recombinant VWF fragments designed in this study. ADAMTS-13 cleaves the Y1605-M1606 peptidyl bond in the A2 domain (D1459-L1668). Five different recombinant VWF domains with His-tags (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022157#pone-0022157-g005" target="_blank">Figure 5A</a>) and 5 different recombinant proteins derived from the VWF A2 domain flanked with GST- and His-tags (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022157#pone-0022157-g005" target="_blank">Figure 5B</a>).</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

Summary of 8 mAbs to VWF and their effects on VWF proteolysis by ADAMTS13.

<p>Summary of 8 mAbs to VWF and their effects on VWF proteolysis by ADAMTS13.</p

Comparison of the binding activity of SZ34 to native and denatured pVWF using Western blot in combination with ELISA based on polystyrene microspheres.

<p>SZ34 (20 µg/ml) was coated on polystyrene microspheres, then incubated with native pVWF or denatured pVWF (100 nM). The denatured pVWF was obtained by thermal treatment (20 min at 80°C) or treatment with 1.5 M guanidine-HCl (2 h at 37°C) of native pVWF. Bound VWF was separated on 6% SDS-PAGE in reducing conditions, followed by Western blotting with SZ34. SZ129 (anti-VWF A1) was a control as a mAb with a linear epitope. The figure is representative of four separate experiments.</p