Functional analysis of a recombinant glycoprotein Ib alpha polypeptide which inhibits von Willebrand factor binding to the platelet glycoprotein Ib-IX complex and to collagen.
By deletion mutagenesis and transient expression in COS cells, a 96-amino acid hydrophilic sequence in the glycoprotein Ib alpha polypeptide located between L220 and L318 was identified which appeared to contain its von Willebrand factor- (vWF) binding site. The cDNA encoding this fragment was then expressed in Escherichia coli and purified from the bacterial cell lysate. The recombinant polypeptide, rGpIb alpha Q221-L318, was monomeric and had an apparent molecular weight of 14,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It inhibited both ristocetin-induced binding of 125I-vWF to fixed washed platelets and ristocetin-induced platelet agglutination. The recombinant polypeptide also inhibited the binding of 125I-vWF to immobilized type I and III collagen. Inhibition of 125I-vWF binding to platelets and collagen was dose-dependent, with IC50 values of 500 and 200 nM rGpIb alpha Q221-L318, respectively. Fifty % inhibition of ristocetin-induced platelet agglutination required 500 nM rGpIb alpha Q221-L318. Although rGpIb alpha Q221-L318 inhibited vWF binding to collagen it did not, itself, bind to collagen-coated surfaces. Reduction of the disulfide bond between C248 and C264 abolished activity. 125I-rGpIb alpha Q221-L318 bound directly to GpIb/IX sites on multimeric vWF. These studies document that a portion of the sequence between Q221 and L318 is needed for recognition and binding to vWF and that binding requires an intact disulfide bond between C248 and C264. The binding of this recombinant polypeptide to vWF multimers inhibits vWF interaction with two important substrates, platelet GpIb/IX and collagen.
Cruz
M A
MA
Hematology-Oncology Division, Brigham and Women's Hospital, Boston, Massachusetts 02115.
Petersen
E
E
Turci
S M
SM
Handin
R I
RI
D000244:Adenosine Diphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003094:Collagen / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010980:Platelet Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D011233:Precipitin Tests; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012310:Ristocetin / Q000494:pharmacology; D014841:von Willebrand Factor / Q000037:antagonists & inhibitors / Q000378:metabolism*
D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D012310:Ristocetin; D000244:Adenosine Diphosphate; D003094:Collagen; D004247:DNA