Comparative analysis of VWF A2-ΔCC and A2-VicCC. (A) Purified VWF A2-VicCC was detected on a Coomassie-stained SDS-PAGE gel (lane 1). VWF A2-ΔCC was incubated with maleimide-PEG₂-biotin (MBP) without (NR, lane 2), and with prior reduction by incubation with tris(2-carboxyethyl)phosphine (TCEP) agarose beads (lane 3, R). Afterward, reactions were quenched with reduced L-glutathione. Biotin-labeled sulfhydryl groups were detected on Western blot using peroxidase-labeled streptavidin. The same samples were also detected on Western blot using c-myc monoclonal antibody to control for equal loading (lanes 4 and 5). (B) Recombinant ADAMTS13 (2nM) was preincubated with 5mM CaCl₂ for 1 hour at 37°C. Then, 2μM VWF A2-ΔCC or A2-VicCC (NR) variant and 1.5M urea were added and samples were taken at the indicated time points (0-3 hours). Reactions were stopped by the addition of 10mM ethylenediaminetetraacetic acid. We simultaneously also performed this assay using A2-VicCC that was reduced with 10mM DTT, then carboxymethylated with 20mM iodoacetic acid, and afterward quenched with 40mM DTT and extensively dialyzed before use in the activity assay (A2-VicCC R). Samples were separated on SDS-PAGE and visualized by silver staining. On proteolysis by ADAMTS13, the N- and C-terminal cleavage fragments became apparent. The experiment was performed 3 times; a representative result is shown. (C) Binding of serial dilutions of purified ADAMTS13 to 50nM immobilized VWF A2-ΔCC, A2-VicCC (NR) and reduced and carboxymethylated VWF A2-VicCC (R). The assay was performed in the presence of 10mM ethylenediaminetetraacetic acid to prevent proteolysis. Bound ADAMTS13 was detected using biotinylated anti–TSR2-4 polyclonal antibodies, followed by streptavidin-peroxidase. Binding curves were generated using GraphPad Prism software, fitting the data to the one-binding site equation. (D) Purified ADAMTS13 (30nM) was preincubated for 1 hour with 0 to 600nM soluble A2-ΔCC, A2-VicCC (NR), and reduced and carboxymethylated A2-VicCC (R). After preincubation, the mixture was added to wells with 50nM immobilized VWF A2-ΔCC, and ADAMTS13 was allowed to bind for 1 hour in the presence of the soluble VWF A2 variants. Bound ADAMTS13 was then detected as in panel C. Curves were fitted to a one-site competition model using GraphPad Prism software. (E) CD spectra of VWF A2-ΔCC, A2-VicCC, A2-CC1, and A2-CC2 at 20°C. Measurements were taken over 190 to 260 nm, at a 0.5-nm interval, bandwidth 1 nm, 1 second per data point. Data were converted to mean residue molar ellipticity, and averages of 6 to 12 measurements are shown. (F) Change in mean residue molar ellipticity on increase of temperature at 5°C intervals from 20°C to 80°C, as determined at 222 nm. Averages of 5 measurements are shown. Curves were plotted using GraphPad Prism software.