Figure 3
Figure 3. Cleavage of multimeric VWF by wild-type ADAMTS13 or mutants under various conditions. (A) Guanidine-HCl denatured VWF (37.5 μg/mL) was incubated at 37°C for 8 hours with wild-type ADAMTS13 (WT; lane 1) or various mutants (lanes 2-8; final concentration, 20nM) in the absence or presence of EDTA (20mM; lane 9) in 50mM Tris-HCl, 50mM NaCl, 5mM CaCl2, pH 8.0. (B) Native VWF (37.5 μg/mL) was incubated for 15 minutes with WT ADAMTS13 or various mutants (final concentration, 50nM) in the absence (−) or presence (+) of recombinant factor VIII (20nM) and lyophilized platelets (∼ 200 × 103/μL) in 50mM HEPES, pH 7.5, containing 1 mg/mL BSA, 150mM NaCl, and 5mM CaCl2. The reaction mixtures were subjected to vortexing at 2500 rpm for 5 minutes and then quenched by heating for 20 minutes at 60°C in the presence of SDS sample buffer. The proteolytic cleavage product (dimer of C-terminal fragments, ∼ 350 kDa) was determined by 1% agarose gel and Western blotting. HMW indicates the area with high-molecular-weight VWF multimers, whereas the arrowhead indicates the proteolytic cleavage product (∼ 350 kDa). (C-D) Human umbilical vascular endothelial cells cultured on 6-well plates or a gelatin-coated glass cover slides were stimulated with histamine (100μM) for 2 minutes and washed with PBS. The washed cells were treated for 5 minutes with a buffer alone, wild-type ADAMTS13, or mutants (10nM) in the absence of fluid shear stress (C) or in the presence of 2.5 dyne/cm2 of fluid shear stress generated (D). The conditioned media or perfusion buffers collected from the 6-well plate or flow chamber were concentrated by filtration. The UL-VWF antigen and degradation products in the conditioned media were determined by Western blot. The intact VWF polypeptide (∼ 250 kDa) is indicated by a solid line. The proteolytic cleavage products (176 kDa and 140 kDa) are marked by a closed and open arrowhead, respectively. Other bands between 176 kDa and 250 kDa, seen in panels C and D (lane 2 and lanes 7-9), may be the result of proteolysis by ADAMTS13 and/or other proteases in the conditioned medium.

Cleavage of multimeric VWF by wild-type ADAMTS13 or mutants under various conditions. (A) Guanidine-HCl denatured VWF (37.5 μg/mL) was incubated at 37°C for 8 hours with wild-type ADAMTS13 (WT; lane 1) or various mutants (lanes 2-8; final concentration, 20nM) in the absence or presence of EDTA (20mM; lane 9) in 50mM Tris-HCl, 50mM NaCl, 5mM CaCl2, pH 8.0. (B) Native VWF (37.5 μg/mL) was incubated for 15 minutes with WT ADAMTS13 or various mutants (final concentration, 50nM) in the absence (−) or presence (+) of recombinant factor VIII (20nM) and lyophilized platelets (∼ 200 × 103/μL) in 50mM HEPES, pH 7.5, containing 1 mg/mL BSA, 150mM NaCl, and 5mM CaCl2. The reaction mixtures were subjected to vortexing at 2500 rpm for 5 minutes and then quenched by heating for 20 minutes at 60°C in the presence of SDS sample buffer. The proteolytic cleavage product (dimer of C-terminal fragments, ∼ 350 kDa) was determined by 1% agarose gel and Western blotting. HMW indicates the area with high-molecular-weight VWF multimers, whereas the arrowhead indicates the proteolytic cleavage product (∼ 350 kDa). (C-D) Human umbilical vascular endothelial cells cultured on 6-well plates or a gelatin-coated glass cover slides were stimulated with histamine (100μM) for 2 minutes and washed with PBS. The washed cells were treated for 5 minutes with a buffer alone, wild-type ADAMTS13, or mutants (10nM) in the absence of fluid shear stress (C) or in the presence of 2.5 dyne/cm2 of fluid shear stress generated (D). The conditioned media or perfusion buffers collected from the 6-well plate or flow chamber were concentrated by filtration. The UL-VWF antigen and degradation products in the conditioned media were determined by Western blot. The intact VWF polypeptide (∼ 250 kDa) is indicated by a solid line. The proteolytic cleavage products (176 kDa and 140 kDa) are marked by a closed and open arrowhead, respectively. Other bands between 176 kDa and 250 kDa, seen in panels C and D (lane 2 and lanes 7-9), may be the result of proteolysis by ADAMTS13 and/or other proteases in the conditioned medium.

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