Carbohydrate moiety of von Willebrand factor is not necessary for maintaining multimeric structure and ristocetin cofactor activity but protects from proteolytic degradation.
نویسندگان
چکیده
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.
منابع مشابه
von Willebrand disease "Vicenza" with larger-than-normal (supranormal) von Willebrand factor multimers.
When normal volunteers or patients with type I von Willebrand disease (VWD) are given desmopressin (DDAVP), a set of larger-than-normal (supranormal) von Willebrand factor (VWF) multimers, similar to those present in VWF-containing cells such as platelets megakaryocytes and endothelial cells, appear transiently in postinfusion plasma. In two kindreds with mild lifelong bleeding symptoms transmi...
متن کاملA new variant of type II von Willebrand disease with aberrant multimeric structure of plasma but not platelet von Willebrand factor (type IIF).
A patient with a lifelong bleeding disorder was diagnosed as having Type II von Willebrand disease. The larger multimers of von Willebrand factor were absent from her plasma but present in platelets. A high-resolution electrophoretic technique was used to study the complex structure of individual von Willebrand factor multimers. In normal plasma, each multimer could be resolved into five bands:...
متن کاملHigh - Resolution Analysis of von Willebrand Factor Multimeric Composition Defines
In Type I von Willebrand disease, the whole series of von Willebrand factor (vWF) multimers is present in plasma, but all are decreased in quantity. No structural abnormality of individual multimers has been demonstrated so far in these patients. We now describe five individuals, from two unrelated families. who had this form of the disease and in whom the complex banding pattern of each vWF mu...
متن کاملEnzymatic degradation of the factor-VIII-von-Willebrand protein: a unique tryptic fragment with ristocetin cofactor activity.
Human von Willebrand protein was purified from pooled cryoprecipitate, then degraded by trypsin and analyzed primarily for ristocetin cofactor activity in relation to the electrophoretic pattern in sodium dodecyl sulfate polyacrylamide gradient gels. Activity decreased rapidly at first. from 55 to 5.5 U/mI of activity after 20 mm of degradation. in association with the appearance and disappeara...
متن کاملResponse to desmopressin is influenced by the genotype and phenotype in type 1 von Willebrand disease (VWD): results from the European Study MCMDM-1VWD.
We have prospectively evaluated the biologic response to desmopressin in 77 patients with type 1 von Willebrand disease (VWD) enrolled within the Molecular and Clinical Markers for the Diagnosis and Management of type 1 VWD project. Complete response to desmopressin was defined as an increase of both ristocetin cofactor activity (VWF:RCo) and factor VIII coagulant activity (FVIII:C) to 50 IU/dL...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
عنوان ژورنال:
- The Journal of clinical investigation
دوره 74 6 شماره
صفحات -
تاریخ انتشار 1984