A role for von Willebrand factor proline residues 702-704 in ristocetin-mediated binding to platelet glycoprotein Ib.
Mutant domains of von Willebrand factor (vWF) were constructed to determine the effects of altering net charge, and presumably conformation, within a peptide sequence (residues 694-708) previously shown to be involved in the platelet receptor glycoprotein (GP) Ib binding function of vWF. Non-conservative substitutions replaced a triplet of proline residues (proline702-704) with either a triplet of arginine (positively-charged) or aspartic acid (negatively-charged) residues. After establishing stable CHO cell transformants, we observed the secretion of covalently-linked dimeric molecules analogous to a domain with native sequence. Functional assays using immunopurified molecules revealed that the ristocetin-dependent binding to GP Ib was abolished with both charge mutants. However, in the absence of disulfide-bond dependent conformation both mutant molecules and the molecule with native sequence interacted with GP Ib. The results demonstrate that vWF proline702-704 are important for the ristocetin-mediated interaction between vWF and GP Ib, but are not essential residues of the GP Ib binding site within vWF.
Azuma
H
H
Roon Research Laboratory for Arteriosclerosis and Thrombosis, Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, CA 92037.
Sugimoto
M
M
Ruggeri
Z M
ZM
Ware
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010974:Platelet Aggregation / Q000235:genetics / Q000502:physiology; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D011392:Proline / Q000235:genetics / Q000378:metabolism; D012310:Ristocetin / Q000378:metabolism; D014162:Transfection; D014841:von Willebrand Factor / Q000235:genetics* / Q000378:metabolism
D010980:Platelet Membrane Glycoproteins; D014841:von Willebrand Factor; D012310:Ristocetin; D011392:Proline