Self-association of VWD type 2A and calcium-binding–deficient mutants. (A) Multimer distribution of WT- and mutant-VWF produced in HEK293T-furin cells. (B) A total of 1 U/mL ADAMTS13 was added to various proteins for 4 hours in the absence or presence of 1.6 M urea. VWF proteolysis was greater for the mutant molecules, and this was evident even in the absence of urea. (C-D) PRP from normal human donors was diluted 50-fold and shear mixed with 10 μg/mL ΔA1-488 at 9600/s in a viscometer in the absence of supplemented calcium, along with either 5 μg/mL WT-VWF, D1498A, or R1597W. VWF self-association (C) and platelet activation (D) were measured identically to Figure 1. (E) Thrombus formation on collagen type I was measured at 1000/s following methods in Figure 2D-E. Greater thrombus formation was noted upon supplementing washed blood with D1498A and R1597W (either 2 or 4 µg/mL) compared with WT-VWF. (F) Representative images of thrombus formation at 3 minutes. Assays were performed 3 to 4 times, each with 2 to 4 repeats. *P < .05 with respect to all other treatments. Scale bars in panel F, 100 µm; BCECF platelet stain.