Specificity of PNA binding to VWF. (A) Microtiter wells coated with polyclonal anti-VWF antibodies (3 μg/mL) were incubated with plasma that was diluted in PBS to reach a VWF antigen concentration of 50 mU/mL. After catching VWF, wells were incubated with neuraminidase (5 mU/mL) in PBS/1 mM CaCl2 overnight at 37°C. Subsequently, wells were incubated for 1 hour at 37°C with btPNA (5 μg/mL) in the presence or absence of various carbohydrate structures (0-1 mM): □ indicate glucose; ○, galactose; ▵, GalNAc; ⋄, GlcNAc; and •, Gal-(β1-3)-GalNAc. After washing, HRP-conjugated streptavidin was added to the wells, and bound btPNA was detected by measuring HRP activity using OPD as a substrate. Plotted is residual PNA binding versus sugar concentration. One-hundred percent refers to PNA binding in the absence of competitors. (B) Microtiter wells coated with polyclonal anti-VWF antibodies (3 μg/mL) were incubated with equimolar concentrations of pd-VWF, wt-VWF, VWF/delta-pro, or VWF/D′-D3 (7.5 nM based on monomer concentration). Where indicated, pd-VWF was incubated with recombinant O-glycosidase (12.5 mU/mL) for 3 hours at 37°C following neuraminidase incubation. PNA binding was determined as described for panel A. Plotted is relative PNA binding using wt-VWF as a reference (100%). (C) Microtiter wells coated with a recombinant anti-VWF A1 antibody were incubated with equimolar concentrations of A1/1260-1481 or A1/1238-1494 (7.5 nM). PNA binding was determined as described for panel A. Plotted is relative PNA binding using A1/1238-1494 as a reference (100%).
