VWF-A2 FRET calcium dependence. (A-B) Schematic of VWF-A2 FRET and mVWF FRET proteins. Emission spectra of these FRET proteins were collected in the milieu of either 10 mM EDTA (0 mM CaCl₂) or 2 mM calcium, both in the presence of 1.6M urea. VWF-A2 FRET (C) and mVWF FRET (D) were incubated with different calcium concentrations. 1.6M urea was then added to trigger time-dependent protein unfolding. Changes in FRET ratio were measured. (E-F) 0.025 U/mL ADAMTS13 was added to samples at the end of the runs in panels C and D, respectively. The proteolysis reaction proceeded for 45 minutes (E) and 16 hours (F). Reaction products were analyzed using anti-His mAb to detect the cleavage band (arrow). Proteolysis is reduced at high calcium. Thus, VWF-A2 is predominantly unfolded/open at low calcium and folded/closed at high calcium. Unfolding and proteolysis of single domain VWF-A2 proceeds more efficiently compared with the full multimeric mVWF protein.