Figure 1.
SZ34 inhibits the proteolytic cleavage of pVWF by rADAMTS13 under shear stress.
(A) 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, the 350 kDa cleavage products were visualized by 5% SDS-PAGE under non-reducing conditions and Western blot analysis. 1C1E7 (an anti-VWF D'D3 mAb), SZ129 (an anti-VWF A1 mAb) and SZ123 (an anti-VWF A3 mAb) were used as negative controls. (B) Changes in the cleavage products detected relative to that observed in the absence of mAbs were determined under shear stress by densitometry. The extent of cleavage was analyzed by detection of the intensity of the 350 kDa cleavage products. Results represent the mean ± standard deviation of four independent experiments.
Table 1.
Summary of 8 mAbs to VWF and their effects on VWF proteolysis by ADAMTS13.
Figure 2.
SZ34 inhibits the proteolysis of VWF multimers by ADAMTS13 under shear stress.
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.
Figure 3.
SZ34 has no effects on the proteolysis of denatured pVWF by rADAMTS13 under static conditions.
(A) Purified pVWF (150 nM) pretreated with guanidine-HCl was incubated with SZ34 (0–200 µg/ml), and then incubated with 25 nM rADAMTS13. After 1.5 h, the reaction was quenched by adding 20 mM EDTA. The 350 kDa cleavage products were analyzed by Western blot as above. 1C1E7, SZ129 and SZ123 were used as controls. (B) Changes in the cleavage product detected relative to that observed in the absence of SZ34 were determined under denatured conditions by densitometry. The extent of cleavage was analyzed by detection of the intensity of the 350 kDa cleavage products. Results represent the mean ± standard deviation of four independent experiments.
Figure 4.
SZ34 had no effect on proteolysis of VWF-R1597W mutant by ADAMTS13.
Recombinant VWF-R1597W (150 nM) was incubated with 0 or 100 µg/ml SZ34 at 37°C for 30 min and then for 18 h with 25 nM rADAMTS13 at 37°C. VWF multimers were separated by 1.5% agarose gel electrophoresis and immunologic analysis. A representative image of 4 independent experiments is shown.
Figure 5.
Schematic domain structure of full-length VWF and recombinant VWF fragments.
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 (Figure 5A) and 5 different recombinant proteins derived from the VWF A2 domain flanked with GST- and His-tags (Figure 5B).
Table 2.
Kinetics of SZ34 and SZ29 interactions with native or denatured pVWF and various recombinant VWF fragments.
Figure 6.
Comparison of the binding activity of SZ34 to native and denatured pVWF using Western blot in combination with ELISA based on polystyrene microspheres.
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.