Identification of discontinuous von Willebrand factor sequences involved in complex formation with botrocetin. A model for the regulation of von Willebrand factor binding to platelet glycoprotein Ib.
We have used proteolytic fragments and overlapping synthetic peptides to define the domain of von Willebrand factor (vWF) that forms a complex with botrocetin and modulates binding to platelet glycoprotein (GP) Ib. Both functions were inhibited by the dimeric 116-kDa tryptic fragment and by its constituent 52/48-kDa subunit, comprising residues 449-728 of mature vWF, but not by the dimeric fragment III-T2 which lacks amino acid residues 512-673. Three synthetic peptides, representing discrete discontinuous sequences within the region lacking in fragment III-T2, inhibited vWF-botrocetin complex formation; they corresponded to residues 539-553, 569-583, and 629-643. The 116-kDa domain, with intact disulfide bonds, exhibited greater affinity for botrocetin than did the reduced and alkylated 52/48-kDa molecule, and both fragments had significantly greater affinity than any of the inhibitory peptides. Thus, conformational attributes, though not strictly required for the interaction, contribute to the optimal functional assembly of the botrocetin-binding site. Accordingly, 125I-labeled botrocetin bound to vWF and to the 116-kDa fragment immobilized onto nitrocellulose but not to equivalent amounts of the reduced and alkylated 52/48-kDa fragment; it also bound to the peptide 539-553, but only when the peptide was immobilized onto nitrocellulose at a much greater concentration than vWF or the proteolytic fragments. These studies demonstrate that vWF interaction with GP Ib may be modulated by botrocetin binding to a discontinuous site located within residues 539-643. The finding that single point mutations in Type IIB von Willebrand disease are located in the same region of the molecule supports the concept that this domain may contain regulatory elements that modulate vWF affinity for platelets at sites of vascular injury.
Roon Research Center for Arteriosclerosis and Thrombosis, Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, CA 92037.
M
M
Sugimoto
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H
Mohri
R A
RA
McClintock
Z M
ZM
Ruggeri
D000595:Amino Acid Sequence; D001345:Autoradiography; D001665:Binding Sites; D015153:Blotting, Western; D003435:Crotalid Venoms / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010974:Platelet Aggregation; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D014357:Trypsin; D014841:von Willebrand Factor / Q000235:genetics* / Q000378:metabolism
D003435:Crotalid Venoms; D010980:Platelet Membrane Glycoproteins; D014841:von Willebrand Factor; C030961:botrocetin; D014357:Trypsin