Characterization of neuraminidase-treated VWF. (A) Purified human VWF was incubated overnight with either α2-3,6,8,9 neuraminidase or PNGase F, and residual expression of α2-3,6–linked sialic acid on VWF was analyzed by modified S nigra lectin binding ELISA as described in “Methods.” All experiments were performed in triplicate, and results described represent the means ± SEM (**P < .001 and ***P < .001 in comparison to WT-VWF, respectively). (B) VWF was treated with either α2-3 neuraminidase or PNGase F, and residual expression of α2-3–linked sialic acid was analyzed by modified Maackia amurensis lectin ELISA. After treatment with either PNGase F, α2-3,6,8,9 neuraminidase, β1-3,4 galactosidase, or both neuraminidase and β-galactosidase, the multimeric structure of VWF was assessed by agarose gel electrophoresis in 1.8% gels under nonreducing conditions (C) or collagen binding activity (D).