Proteolysis of glycan variants of the VWF A2 domain and FLVWF. (A) The VWF A2 variants (2 µM) WT, N1574Q, GnT^−/−, EndoH, and Y1544D/N1574Q and ADAMTS13 (20 nM) were separately preincubated with 20 mM Tris (pH7.9), 150 mM NaCl, 5 mM CaCl₂ and 2 M urea for at 37°C 45 minutes. Samples were combined and incubated at 37°C for proteolysis to occur, and reactions were stopped after 0.5 and 16 hours by the addition of EDTA. Proteolysis was assessed by SDS-PAGE on a 4% to 12% Bis-Tris gel followed by silver stain. (B) FLVWF containing mutation(s) to the stabilizing glycan (N1574Q) or hydrophobic substitution (Y1544D) or combination of (Y1544D/N1574Q) were expressed in HEK 293 cells. Multimer formation was analyzed on a 2% agarose gel and VWF bands detected on a western blot with a polyclonal anti-VWF antibody to determine multimer structure. (C) Proteolysis of FLVWF and its variants by ADAMTS13. FLVWF and its variants (1 µg/mL) and ADAMTS13 (20 nM) were separately preincubated with 20 mM Tris (pH 7.9), 150 mM NaCl, 5 mM CaCl₂ ± 2 M urea for at 37°C 45 minutes. Samples were combined and incubated at 37°C for proteolysis to occur, and reactions were stopped after 4 hours by the addition of EDTA. VWF cleavage products were resolved after the addition of reducing reagent. The samples were run on a 3% to 8% Tris-acetate gel and VWF and cleaved VWF bands detected by western blot using a cocktail of anti-VWF polyclonal and monoclonal antibody that detects the C-terminal cleavage product.