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LOCALIZATION OF A FACTOR VIII BINDING DOMAIN ON THE N- TERMINAL PORTION (FRAGMENT Spill) OF VON WILLEBRAND
| Content Provider | Semantic Scholar |
|---|---|
| Copyright Year | 2018 |
| Abstract | A new domain has been identified on the von Willebrand Factor (vWF) subunit. vWF binds to platelet glycoproteins GPib and GPIIb/IIIa as well as to collagen and corresponding domains have been isolated. vWF also binds to Factor VIII (F.VIII). We show here that the corresponding domain is located on the N-terminal portion of the vWF subunit (residues I to 1,365). For this purpose, F.VIII was tested for its ability to bind to purified vWF degradation fragments obtained by digestion with S.aureus V -8 protease, ie a dimeric N-terminal fragment of 320 kd (Spiii) and a dimeric C-terminal fragment of 220 kd (Spll). Human F.VIII was purified from cryoprecipitate by immunoadsorption of F.VIII/vWF onto a monoclonal antibody (MAb) to vWF coupled to Sepharose, followed by elution using 0.25 M CaCJ2: The F.VIII preparation contained 100 U/ml VIII:C and less than 0.001 U/ml vWFAg. The binding assay was performed using polystyrene tubes coated with 2 ug/ml of purified vWF, Spill or Spll. Coated albumin, fibrinogen or fibronectin were used as controls. Purified F.VIII (0.1 to 2 U/ml VIII:C) was incubated in the coated tubes for I hat 37•c. Following washing, bound F.VIII was estimated in situ by one-stage clotting and chromogenic assays. Immunoradiometric assay with 125 1MAbs to Spll or Spill demonstrated that the amount of coated protein remained constant throughout the experiments. F. VIII bound in a dosedependent manner to coated vWF and Spill but not to Spll. Binding was specific for F.VIII as demonstrated by inhibition experiments. Bound F.VIII could be removed with 0.25 M CaCI and its coagulant activity inhibited by a MAb or an oligoclonal (homotogous) antibody neutralising VIII:C. Binding of F.VIII to coated vWF or Spill was also inhibited in a dose-dependent way by vWF or Spill. In contrast, addition of Spll had no effect upon the binding. Binding of F. VIII to Spiii was confirmed using MAbs to vWF. Among 28 MAbs which bound Spill and had no anti VIII:C activity, 12 inhibited binding of F.VIII whereas the others had no effect. Among the latter, 3 MAbs blocked binding of vWF or Spill to GP!b and 6 MAbs inhibited binding of vWF or Spill to collagen. Ten Mf\bs to Spll had no inhibitory effect upon binding of F.VIII. These results indicate that a F.VIII binding domain of vWF is located in theN-terminal portion of vWF (residues I to I ,365) and that it is distinct from the GPib and collagen binding domains. The 12 MAbs to Spill which block binding of F. VIII to vWF or Spill should allow the precise localization of this new domain. 78 |
| File Format | PDF HTM / HTML |
| Alternate Webpage(s) | https://www.thieme-connect.de/products/ejournals/pdf/10.1055/s-0038-1642877.pdf |
| Language | English |
| Access Restriction | Open |
| Content Type | Text |
| Resource Type | Article |
