Mapping the glycoprotein Ib-binding site in the von willebrand factor A1 domain.
The von Willebrand factor (vWF) mediates platelet adhesion to exposed subendothelium at sites of vascular injury. It does this by forming a bridge between subendothelial collagen and the platelet glycoprotein Ib-IX-V complex (GPIb). The GPIb-binding site within vWF has been localized to the vWF-A1 domain. Based on the crystal structure of the vWF-A1 domain (Emsley, J., Cruz, M., Handin, R., and Liddington, R. (1998) J. Biol. Chem. 273, 10396-10401), we introduced point mutations into 16 candidate residues that might form all or part of the GPIb interaction site. We also introduced two mutations previously reported to impair vWF function yielding a total of 18 mutations. The recombinant vWF-A1 mutant proteins were then expressed in Escherichia coli, and the activity of the purified proteins was assessed by their ability to support flow-dependent platelet adhesion and their ability to inhibit ristocetin-induced platelet agglutination. Six mutations located on the front and upper anterior face of the folded vWF-A1 domain, R524S, G561S, H563T, T594S/E596A, Q604R, and S607R, showed reduced activity in all the assays, and we suggest that these residues form part of the GPIb interaction site. One mutation, G561S, with impaired activity occurs in the naturally occurring variant form of von Willebrand's disease-type 2M underscoring the physiologic relevance of the mutations described here.
Cruz
M A
MA
Hematology Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
Diacovo
T G
TG
Emsley
J
J
Liddington
R
R
Handin
R I
RI
D001665:Binding Sites; D001792:Blood Platelets / Q000166:cytology / Q000378:metabolism; D002448:Cell Adhesion; D018360:Crystallography, X-Ray; D006801:Humans; D008958:Models, Molecular; D016296:Mutagenesis; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014841:von Willebrand Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D019038:Platelet Glycoprotein GPIb-IX Complex; D011994:Recombinant Proteins; D014841:von Willebrand Factor