Transient expression of recombinant glycoprotein Ib alpha polypeptides in COS cells that inhibit von Willebrand factor binding to the platelet glycoprotein Ib/IX complex.
The cDNA encoding the glycoprotein Ib alpha polypeptide has been expressed in COS cells. Transfection with full-length cDNA and a cDNA truncated at an internal XbaI site produced a recombinant polypeptide doublet with estimated molecular weights of 48 and 46 kDa (rGpIb alpha L318) which could be resolved into a single band of molecular weight 36 kDa following digestion with endoglycosidase F. A portion of the truncated polypeptide was retained in the endoplasmic reticulum of COS cells and slowly released. A second fraction was rapidly secreted into COS cell-conditioned medium and could be used for functional studies. Soluble rGpIb alpha L318 harvested from COS cell-conditioned medium inhibited ristocetin-dependent binding of [125I]-vWF to fixed washed human platelets (IC50 20 nM). Binding was inhibited by reduction and alkylation of "rGp1b alpha L318" suggesting the need for a critical disulfide bond to maintain biological activity of the recombinant polypeptide. We conclude that the recombinant polypeptides produced by transfection of GpIb alpha cDNA into heterologous cells are secreted, soluble, and can inhibit vWF binding to platelet GpIb/IX.
Petersen
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Hematology-Oncology Division, Brigham and Women's Hospital, Boston, MA 02115.
Handin
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D001665:Binding Sites; D002460:Cell Line; D004247:DNA / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D010980:Platelet Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014841:von Willebrand Factor / Q000378:metabolism*
D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D004247:DNA