title: Promotion of binding of von Willebrand factor to platelet glycoprotein Ib by dimers of ristocetin.
abstract: In the absence of high shear forces, the in vitro binding of human von Willebrand factor (vWF) to its platelet receptor glycoprotein Ib (GPIb) can be promoted by two well-characterized mediators, botrocetin and ristocetin. Using purified vWF and GPIb, we have investigated the mechanisms by which ristocetin mediates this binding. Specific binding of vWF monomers to GPIb occurred with a 1:1 stoichiometry, but high-affinity binding required the participation of two ristocetin dimers. Binding was strongly dependent on pH and inhibited by low poly-L-lysine concentrations, indicating ristocetin-dependent charge neutralization during the interaction. With increasing ristocetin concentrations, vWF binding depended progressively less on the involvement of its A1 loop, which is compatible with a model in which the two ristocetin dimers bridge the vWF-GPIb complex on secondary sites. In agreement with this model, the ristocetin-dimer-promoted stabilization of vWF on GPIb was abolished by low concentrations of poly(Pro-Gly-Pro), which is known to complex ristocetin dimers. Mechanistic analysis of the inhibition of vWF binding by the recombinant vWF fragment Leu504-Ser728 (VCL), which covers the entire A1 loop, revealed an affinity of VCL for GPIb comparable with that of the botrocetin-vWF complex for GPIb, and identified a specific but 20-fold lower affinity of VCL in the presence of ristocetin. The proline-rich peptides flanking the vWF A1 loop, Cys474-Val489 and Leu694-Asp709, inhibited vWF binding semispecifically by competitively interfering with the formation of the GPIb-vWF complex rather than by complexation of free ristocetin dimers. In conclusion, ristocetin-promoted binding of vWF to its GPIb receptor results from charge neutralization and interactions involving proline residues in the vicinity of the natural interaction sites present on both GPIb and the A1 domain of vWF.
authors.affiliation: Center for Molecular and Vascular Biology, University of Leuven, Belgium.
authors.forename: M F
authors.initials: MF
authors.lastname: Hoylaerts
authors.forename: K
authors.initials: K
authors.lastname: Nuyts
authors.forename: K
authors.initials: K
authors.lastname: Peerlinck
authors.forename: H
authors.initials: H
authors.lastname: Deckmyn
authors.forename: J
authors.initials: J
authors.lastname: Vermylen
mesh_terms: D000595:Amino Acid Sequence; D001667:Binding, Competitive; D006801:Humans; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D008433:Mathematics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D011107:Polylysine / Q000494:pharmacology; D012310:Ristocetin / Q000737:chemistry / Q000494:pharmacology*; D014841:von Willebrand Factor
chemical_list: D046911:Macromolecular Substances; D010446:Peptide Fragments; D010980:Platelet Membrane Glycoproteins; D014841:von Willebrand Factor; D012310:Ristocetin; D011107:Polylysine