title: Effect of deletion of the A1 domain of von Willebrand factor on its binding to heparin, collagen and platelets in the presence of ristocetin.
abstract: In order to study the functional importance of the collagen, heparin and glycoprotein-Ib-binding domain, we deleted the A1 domain of von Willebrand factor (vWF), corresponding to residues 478-716, by oligonucleotide-directed mutagenesis. The resulting delta A1-vWF cDNA was expressed in COS-1 monkey kidney cells and compared to wild-type vWF. The higher-molecular-mass multimers were decreased in delta A1 recombinant von Willebrand factor (delta A1-rvWF) compared to plasma vWF and rvWF. The reactivity of delta A1-rvWF and rvWF with monoclonal antibodies directed against the collagen-binding domain (residues 969-992), the vessel-wall-binding domain, and the binding site for glycoprotein IIb-IIIa on platelets was identical. The interaction with vWF of the monoclonal antibody directed against the glycoprotein Ib binding domain was abolished for delta A1-rvWF, and similar to plasma vWF for rvWF. The binding of factor VIII to delta A1-rvWF and rvWF was similar. delta A1-rvWF and rvWF bound similarly to collagen, but the binding of delta A1-rvWF to heparin and to platelets in the presence of ristocetin were abolished. These data indicate that the heparin-binding site in the A1 domain is essential. There is no second binding domain for glycoprotein Ib outside the A1 domain. The collagen-binding domain in the A1 domain is either not active or its action can be compensated by the second collagen-binding domain.
authors.affiliation: Department of Haematology, University Hospital Utrecht, The Netherlands.
authors.forename: J J
authors.initials: JJ
authors.lastname: Sixma
authors.forename: M E
authors.initials: ME
authors.lastname: Schiphorst
authors.forename: C L
authors.initials: CL
authors.lastname: Verweij
authors.forename: H
authors.initials: H
authors.lastname: Pannekoek
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002460:Cell Line; D002872:Chromosome Deletion; D003094:Collagen / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005169:Factor VIII / Q000378:metabolism; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010974:Platelet Aggregation; D011994:Recombinant Proteins / Q000378:metabolism; D012310:Ristocetin / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D014162:Transfection; D014841:von Willebrand Factor / Q000235:genetics / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D012310:Ristocetin; D005169:Factor VIII; D006493:Heparin; D003094:Collagen; D004247:DNA