Figure 6
Figure 6. Analysis of VWF proteolysis under static and flow conditions. (A) VWFA1A2A3 and VWFA1CK (500 nM of each) were incubated with 10 nM ADAMTS13 in the absence of any denaturants. Subsamples were taken at various time points (0-3 hours) and analyzed by SDS-PAGE under reducing conditions, followed by Western blotting using an anti-His antibody. No cleavage was apparent and the uncleaved VWFA1A2A3 and VWFA1CK were visualized as bands of approximately 64 and 174 kDa, respectively. (B) VWFA1A2A3 and VWFA1CK were incubated with ADAMTS13, as in panel A, in the presence of 1.5 M urea. A band of approximately 42 kDa corresponding to the N-terminal cleavage product was detected for both VWFA1A2A3 and VWFA1CK, demonstrating proteolysis. No difference was evident in the rate of cleavage of the 2 fragments. (C) Increasing concentrations of VWFA3CK (0-500 nM) were preincubated with 20 nM ADAMTS13, before being added to 5 μg/mL VWF in the presence of 1.5 M urea. The reactions were stopped after 40 minutes and analyzed on a 1.5% agarose gel followed by Western blotting using an anti-D′D3 polyclonal antibody. In the absence of inhibitor, VWF was efficiently cleaved. No inhibition is observed in the presence of VWFA3CK. (D) Increasing concentrations of VWFA3CK (0-250 nM) were preincubated with 40 nM ADAMTS13, before being added to 5 μg/mL VWF and vortexing for 5 minutes. The reaction was analyzed on a 1.5% agarose gel. VWF was extensively cleaved after 5 minutes. In the presence of VWFA3CK, the amount of cleavage of high- and intermediate-molecular-weight multimers was reduced. (E) RU8 antibody (0-100 μg/mL) was preincubated with VWF, before being added to ADAMTS13 and vortexing as in panel D. The antibody significantly inhibited the cleavage of multimeric VWF. (F) A static experiment similar to the one described in panel C was prepared. The samples were analyzed on a 3% to 8% Tris-acetate gel and detected by an anti-D′D3 polyclonal antibody. The 140- to 140-kDa cleavage product was detected and no inhibition was observed in the presence of VWFA3CK. (G) A cleavage reaction under flow was prepared as in panel D, analyzed on a 3% to 8% Tris-acetate gel and detected by the anti-D′D3 polyclonal antibody. The intensity of the band corresponding to the 140- to 140-kDa homodimer gradually decreased when increasing concentrations of VWFA3CK were present in the reaction. (H) A cleavage reaction under flow was prepared as in panel E and analyzed on a 3% to 8% Tris-acetate gel, and the 176- to 176-kDa homodimer was detected by a polyclonal anti-VWF antibody HRP. RU8 antibody inhibited VWF proteolysis.

Analysis of VWF proteolysis under static and flow conditions. (A) VWFA1A2A3 and VWFA1CK (500 nM of each) were incubated with 10 nM ADAMTS13 in the absence of any denaturants. Subsamples were taken at various time points (0-3 hours) and analyzed by SDS-PAGE under reducing conditions, followed by Western blotting using an anti-His antibody. No cleavage was apparent and the uncleaved VWFA1A2A3 and VWFA1CK were visualized as bands of approximately 64 and 174 kDa, respectively. (B) VWFA1A2A3 and VWFA1CK were incubated with ADAMTS13, as in panel A, in the presence of 1.5 M urea. A band of approximately 42 kDa corresponding to the N-terminal cleavage product was detected for both VWFA1A2A3 and VWFA1CK, demonstrating proteolysis. No difference was evident in the rate of cleavage of the 2 fragments. (C) Increasing concentrations of VWFA3CK (0-500 nM) were preincubated with 20 nM ADAMTS13, before being added to 5 μg/mL VWF in the presence of 1.5 M urea. The reactions were stopped after 40 minutes and analyzed on a 1.5% agarose gel followed by Western blotting using an anti-D′D3 polyclonal antibody. In the absence of inhibitor, VWF was efficiently cleaved. No inhibition is observed in the presence of VWFA3CK. (D) Increasing concentrations of VWFA3CK (0-250 nM) were preincubated with 40 nM ADAMTS13, before being added to 5 μg/mL VWF and vortexing for 5 minutes. The reaction was analyzed on a 1.5% agarose gel. VWF was extensively cleaved after 5 minutes. In the presence of VWFA3CK, the amount of cleavage of high- and intermediate-molecular-weight multimers was reduced. (E) RU8 antibody (0-100 μg/mL) was preincubated with VWF, before being added to ADAMTS13 and vortexing as in panel D. The antibody significantly inhibited the cleavage of multimeric VWF. (F) A static experiment similar to the one described in panel C was prepared. The samples were analyzed on a 3% to 8% Tris-acetate gel and detected by an anti-D′D3 polyclonal antibody. The 140- to 140-kDa cleavage product was detected and no inhibition was observed in the presence of VWFA3CK. (G) A cleavage reaction under flow was prepared as in panel D, analyzed on a 3% to 8% Tris-acetate gel and detected by the anti-D′D3 polyclonal antibody. The intensity of the band corresponding to the 140- to 140-kDa homodimer gradually decreased when increasing concentrations of VWFA3CK were present in the reaction. (H) A cleavage reaction under flow was prepared as in panel E and analyzed on a 3% to 8% Tris-acetate gel, and the 176- to 176-kDa homodimer was detected by a polyclonal anti-VWF antibody HRP. RU8 antibody inhibited VWF proteolysis.

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