Functional modulation of the isolated glycoprotein Ib binding domain of von Willebrand factor expressed in Escherichia coli.
We have expressed in Escherichia coli the domain of von Willebrand factor (vWF) containing the binding site for platelet glycoprotein (GP) Ib and used it to study the regulation of vWF-platelet interaction. The recombinant fragment, comprising residues 445-733 of the mature vWF subunit and designated rvWF445-733, did not have the native conformation of the corresponding domain in the intact molecule because, in order to prevent formation of random aggregates, the seven cysteine residues in the sequence were reduced and alkylated. Unlike native vWF, rvWF445-733 bound to GP Ib in the absence of any modulator, suggesting that the lack of disulfide bonds and/or carbohydrate side chains within this domain may expose platelet interaction sites. In the presence of two modulators, the glycopeptide ristocetin and the snake protein botrocetin, rvWF445-733 inhibited native vWF binding to GP Ib as well as platelet aggregation mediated by vWF, suggesting that both the fragment and the native molecule interact with the same site on platelets. This conclusion was also supported by the observation that the recombinant fragment competed with the binding to platelets of an anti-GP Ib monoclonal antibody known to inhibit vWF binding. Botrocetin formed a complex with rvWF445-733, but the affinity of this interaction was approximately 25-fold lower than with native vWF. However, the complexes of botrocetin with either rvWF445-733 or multimeric native vWF bound to GP Ib with similar dissociation constant. Therefore, conformational attributes of vWF regulate its affinity for botrocetin, but once the complex is formed, interaction with GP Ib is independent of native vWF conformation. These findings provide insights into the regulation of vWF-platelet interaction.
Sugimoto
M
M
Roon Research Laboratory for Arteriosclerosis and Thrombosis, Division of Experimental Hemostasis and Thrombosis, Scripps Clinic and Research Foundation, La Jolla, California 92037.
Ricca
G
G
Hrinda
M E
ME
Schreiber
A B
AB
Searfoss
G H
GH
Bottini
E
E
Ruggeri
Z M
ZM
D001665:Binding Sites; D003001:Cloning, Molecular; D003435:Crotalid Venoms / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000378:metabolism; D010974:Platelet Aggregation; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D012310:Ristocetin / Q000378:metabolism; D013329:Structure-Activity Relationship; D014841:von Willebrand Factor / Q000378:metabolism*
D003435:Crotalid Venoms; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D012310:Ristocetin; C030961:botrocetin