Interaction of the von Willebrand factor (vWF) with collagen. Localization of the primary collagen-binding site by analysis of recombinant vWF a domain polypeptides.
The von Willebrand factor (vWF) mediates platelet adhesion to the vascular subendothelium by binding to collagen, other matrix constituents, and the platelet receptor glycoproteins Ib/IX and IIb/IIIa. Although substantial progress has been made in defining vWF structure-function relationships, there are conflicting data regarding the location of its collagen-binding site(s). Possible collagen-binding sites have been localized in the A1 and A3 domains of vWF. To study the proposed binding sites, we have expressed cDNA sequences encoding the A1 and A3 domains of vWF in Escherichia coli and purified the resulting proteins from bacterial inclusion bodies. In addition, a chimeric molecule containing residues 465-598 of the vWF A1 domain polypeptide (vWF-A1) fused in frame to residues 1018-1114 of the vWF A3 domain polypeptide (vWF-A3) was also expressed. Each of the three recombinant proteins purified as a monomer and contained a single disulfide bond. As previously reported (Cruz, M. A., Handin, R. I., and Wise, R. J. (1993) J. Biol. Chem. 268, 21238-21245), recombinant vWF-A1 inhibited ristocetin-induced platelet agglutination, but did not compete with vWF multimers for collagen binding. In contrast, vWF-A3 inhibited the binding of multimeric vWF to immobilized collagen, but did not inhibit ristocetin-induced platelet agglutination. Metabolically labeled vWF-A3 bound to immobilized collagen in a saturable and reversible manner with a Kd of 1.8 x 10(-6) M. The vWF-A1/A3 chimera was bifunctional. It inhibited vWF binding to platelet glycoprotein Ib/IX with an IC50 of 0.6 x 10(-6) M and inhibited vWF binding to collagen with an IC50 of 0.5-1.0 x 10(-6) M. These results, taken together, provide firm evidence that the major collagen-binding site in vWF resides in the A3 domain.
Cruz
M A
MA
Brigham and Women's Hospital, Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA.
Yuan
H
H
Lee
J R
JR
Wise
R J
RJ
Handin
R I
RI
D001483:Base Sequence; D001665:Binding Sites; D003094:Collagen / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010974:Platelet Aggregation; D011994:Recombinant Proteins; D012310:Ristocetin / Q000494:pharmacology; D014841:von Willebrand Factor / Q000737:chemistry / Q000378:metabolism*
D017931:DNA Primers; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D012310:Ristocetin; D003094:Collagen