Identification of a site in the alpha chain of platelet glycoprotein Ib that participates in von Willebrand factor binding.
The binding of von Willebrand factor (vWF) to the platelet receptor glycoprotein (GP) Ib-IX complex is a key event in hemostasis and may participate in the development of thrombotic vascular occlusion. We present here evidence that residues Ser251-Tyr279 in the GP Ib alpha-chain participate in this function. Initial studies suggested that the modality of vWF interaction with GP Ib depended on the conditions used for induction of binding, either in the presence of ristocetin, or botrocetin, or with asialo-vWF. In fact, only the 45-kDa amino-terminal fragment of GP Ib alpha inhibited the vWF-GP Ib interaction under all conditions tested, while the 84-kDa macroglycopeptide was significantly effective only in the presence of ristocetin. Moreover, the 45-kDa fragment with reduced disulfide bonds still inhibited ristocetin-induced binding but had no effect, at the concentrations tested, on botrocetin-mediated or direct asialo-vWF binding. In order to localize in more detail the functional site, the entire sequence of the 45-kDa fragment was reproduced in 27 overlapping synthetic peptides that were then used in inhibition of binding assays. This led to the identification of a linear GP Ib alpha sequence (residues Ser251-Tyr279) that effectively inhibited platelet interaction with vWF mediated by ristocetin and, at higher concentration, also by botrocetin. A shorter peptide overlapping with the longer one (residues Gly271-Glu285) was the second most active inhibitory species. This region of the molecule contains several residues with a high surface probability index, as expected for a site involved in ligand binding. Thus, while native conformation of GP Ib alpha appears to be important for optimal interaction with vWF, the results obtained with short synthetic peptides may help in defining the amino acid residues participating in this essential function.
Vicente
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Department of Molecular and Experimental Medicine, Scripps Clinic and Research Foundation, La Jolla, California 92037.
Houghten
R A
RA
Ruggeri
Z M
ZM
D000595:Amino Acid Sequence; D001212:Asialoglycoproteins; D001665:Binding Sites; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D003435:Crotalid Venoms / Q000494:pharmacology; D004220:Disulfides / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000494:pharmacology; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism* / Q000494:pharmacology; D012310:Ristocetin / Q000494:pharmacology; D012694:Serine; D014443:Tyrosine; D014841:von Willebrand Factor / Q000031:analogs & derivatives / Q000378:metabolism*
D001212:Asialoglycoproteins; D003435:Crotalid Venoms; D004220:Disulfides; D010446:Peptide Fragments; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins; C044131:asialo-von Willebrand Factor; C023592:glycocalicin; D014841:von Willebrand Factor; D012310:Ristocetin; D014443:Tyrosine; D012694:Serine; C030961:botrocetin