Structural elements influencing von Willebrand factor (vWF) binding affinity for platelet glycoprotein Ib within a dispase-digested vWF fragment.
We investigated the structural elements in human von Willebrand factor (vWF) that influence binding affinity for platelet glycoprotein (GP) Ib using a dispase-digested vWF fragment as a prototype (residues Leu480/Val481-Gly718 of the vWF subunit; Andrews et al, Biochemistry 28:8326, 1989). The major structural features of this fragment are a large A1-loop formed by an intrachain disulfide bond between Cys509 and Cys695 and six O-linked sugar chains. The fragment was chemically modified by (1) reduction and S-carboxyamido-methylation (R/A), (2) desialylation (DS), or (3) a combination of both (R/A-DS). The GPIb binding affinity of these fragments was basically evaluated by competitive binding assay with anti-GPIb monoclonal antibody (LJ-Ib1), a receptor blocker for vWF (Sugimoto et al, Biochemistry 30:5202, 1991). Both the prototype and the R/A fragments were also assessed for their function in shear-induced platelet aggregation. Results unambiguously demonstrated that the presence of a disulfide bridge (Cys509-Cys695) within this domain downregulates the affinity of vWF to GPIb. In addition, it was also demonstrated that the terminal sialic acids attached to six o-linked sugar chains within this domain contribute to optimal functional modulation by the antibiotic ristocetin, but not by snake venom botrocetin.
Miura
S
S
Department of Pediatrics, Nara Medical College, Japan.
Fujimura
Y
Y
Sugimoto
M
M
Kawasaki
T
T
Ikeda
Y
Y
Titani
K
K
Yoshioka
A
A
D000818:Animals; D000911:Antibodies, Monoclonal; D001667:Binding, Competitive; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D007457:Iodine Radioisotopes; D007700:Kinetics; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D011869:Radioligand Assay; D014841:von Willebrand Factor / Q000302:isolation & purification / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D010980:Platelet Membrane Glycoproteins; D014841:von Willebrand Factor; D010450:Endopeptidases; C023139:dispase