Effect of deletion of the A1 domain of von Willebrand factor on its binding to heparin, collagen and platelets in the presence of ristocetin.
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.
Sixma
J J
JJ
Department of Haematology, University Hospital Utrecht, The Netherlands.
Schiphorst
M E
ME
Verweij
C L
CL
Pannekoek
H
H
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*
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D012310:Ristocetin; D005169:Factor VIII; D006493:Heparin; D003094:Collagen; D004247:DNA