Carbohydrate moiety of von Willebrand factor is not necessary for maintaining multimeric structure and ristocetin...

To better define the role of carbohydrate in the structure and ristocetin cofactor activity of von Willebrand factor, we have removed up to 83% of total hexose by sequential treatment of the molecule with endo-beta-N-acetyl-glucosaminidase F (endo F), neuraminidase, and beta-galactosidase. Endo F alone removed 69% of total hexose and D-galactose, and 71% of sialic acid. However, there was no discernible loss of large multimers and the ristocetin cofactor activity was decreased by only 11%. The reduced von Willebrand factor subunit migrated more rapidly in polyacrylamide gels containing SDS, consistent with a 10% decrease of molecular mass. All multimers of unreduced carbohydrate-modified von Willebrand factor migrated more rapidly in SDS-agarose, but the triplet pattern of individual multimers was unchanged. This alteration in multimer migration rate did not resemble alterations found so far in von Willebrand disease variants. Further treatment of von Willebrand factor with neuraminidase and beta-galactosidase reduced the D-galactose to 15% and ristocetin cofactor activity to 57%. A similar decrease in ristocetin cofactor activity was seen if von Willebrand factor was treated only with neuraminidase and beta-galactosidase. In contrast, treating von Willebrand factor with neuraminidase and beta-galactosidase in the presence of protease inhibitors (20 mM benzamidine, 20 U/ml aprotonin, 15 micrograms/ml leupeptin) resulted in a comparable removal of carbohydrate with no change in ristocetin cofactor activity. Moreover, the multimeric structure remained intact in spite of 80% removal of D-galactose. This suggested that carbohydrate was protecting von Willebrand factor against traces of one or more protease contaminants. Evidence in support of this hypothesis was obtained by exposing von Willebrand factor to plasmin after pretreatment with neuraminidase alone or with neuraminidase and beta-galactosidase. A loss of large multimers was observed from von Willebrand factor that had been pretreated with neuraminidase, but this was even greater if pretreatment was also with beta-galactosidase. In contrast, the multimeric structure of von Willebrand factor with intact carbohydrate was not affected by plasmin under similar conditions. These studies suggest that carbohydrate protects von Willebrand factor from disaggregation occurring secondarily to proteolytic attack but does not play a direct role in maintaining its multimeric structure or ristocetin cofactor activity.