title: Isolation and functional characterization of the von Willebrand factor-binding domain located between residues His1-Arg293 of the alpha-chain of glycoprotein Ib.
abstract: In the present report we describe the isolation of a functional domain of platelet membrane glycoprotein (GP) Ib which retains von Willebrand factor (vWF)-binding activity. Glycocalicin, a proteolytic fragment of the alpha-chain of GP Ib generated by an endogenous calcium-activated protease, was submitted to digestion with trypsin. The two resulting fragments, one of 45 kDa extending between residues His1 and Arg293 and representing the amino terminus of the alpha-chain, the other of 84 kDa corresponding to the previously described macroglycopeptide, were purified to homogeneity. Glycocalicin, as well as the 45- and 84-kDa fragments, inhibited the ristocetin-dependent binding of native vWF to platelet GP Ib. The concentration inhibiting 50% of binding (IC50) was between 1 and 5 microM with all these molecules. In contrast, the binding of asialo-vWF to platelet GP Ib, measured directly in the absence of ristocetin, was blocked by glycocalicin and the 45-kDa fragment with a similar IC50, but not by the 84-kDa fragment. Both glycocalicin and the 45-kDa fragment bound to purified surface-bound vWF in a ristocetin-dependent manner and with similar affinities. Monoclonal antibodies against vWF or GP Ib inhibited this interaction in a way consistent with their inhibition of vWF binding to platelet GP Ib. These studies demonstrate that the amino-terminal extracytoplasmic region of the alpha-chain, extending between residues 1 and 293, contains a functional domain that interacts with vWF in the absence of any other structure of the GP Ib complex or any other platelet membrane component. Whereas the ristocetin-dependent binding of vWF may involve also other domains in the macroglycopeptide region, the direct vWF-GP Ib interaction appears to be mediated only by a domain in the amino-terminal region of GP Ib alpha.
authors.affiliation: Department of Basic and Clinical Research, Research Institute of Scripps Clinic, La Jolla, California 92037.
authors.forename: V
authors.initials: V
authors.lastname: Vicente
authors.forename: P J
authors.initials: PJ
authors.lastname: Kostel
authors.forename: Z M
authors.initials: ZM
authors.lastname: Ruggeri
mesh_terms: D001120:Arginine; D001665:Binding Sites; D006639:Histidine; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D011485:Protein Binding; D014841:von Willebrand Factor / Q000378:metabolism*
chemical_list: D046911:Macromolecular Substances; D010446:Peptide Fragments; D010980:Platelet Membrane Glycoproteins; D014841:von Willebrand Factor; D006639:Histidine; D001120:Arginine