Proteolysis of full-length VWF variants by ADAMTS13. (A) VWF, VWF-CC1, and VWF-CC2 with introduced disulfide bonds were predenatured in the presence of 2.5M urea for 1 hour at 37°C to induce unfolding. ADAMTS13 was preincubated with 5mM CaCl2. After preincubation, both mixtures were combined and contained 0.5 μg/mL VWF and 20nM ADAMTS13. After 0 and 16 hours of incubation at 37°C, samples were taken and reactions stopped by the addition of ethylenediaminetetraacetic acid. Samples were separated at 1.4% SDS-agarose gel and VWF multimers were detected using polyclonal anti-VWF antibody. (B) Same as in panel A, but samples were incubated for 0 to 24 hours, reduced with 5% β-mercaptoethanol for 30 minutes at 60°C, and separated on 3% to 8% acetate gel. (C) Proteolysis of VWF and VWF in which the vicinal cysteines C1669 and C1670 were changed to glycine (VWF-VicGG). Same as in panel A, but coincubation with 2nM ADAMTS13 for 0 to 16 hours in the absence (−) or presence (+) of 1.5M urea. (D) Same as in panel A, but using shorter incubation of 0 to 120 minutes in the presence of 2nM ADAMTS13 and 1.5M urea.
