Expression and characterization of functionally active fragments of the platelet glycoprotein (GP) Ib-IX complex in mammalian cells. Incorporation of GP Ib alpha into the cell surface membrane.
The platelet glycoprotein Ib-IX complex (GP Ib-IX) is essential for the initial attachment of platelets to the wall of damaged arteries. In this study, an N-terminal fragment of human GP Ib alpha (residues 1-318), containing the ligand binding sites for von Willebrand factor (vWF) and thrombin, as well as the entire human GP Ib alpha were expressed in Chinese hamster ovary cells. The transfected cells secreted a 48- and a 110-kDa protein, respectively, into the supernatant. Both recombinant proteins were purified by immunoaffinity chromatography. The purified proteins bound soluble vWF in the presence of botrocetin as demonstrated in solid-phase binding assays. The dissociation constant (Kd) for 125I-vWF binding to the recombinant 110-kDa protein was 1.2 +/- 0.2 nM as compared with 1.0 +/- 0.3 nM for vWF binding to purified platelet GP Ib-IX. Both recombinant proteins were also retained on thrombin-Sepharose 4B. The 48-kDa protein contained two N-linked oligosaccharide chains. A 125-kDa protein was identified in the lysate of cells transfected with the coding sequence for the entire GP Ib alpha. Trypsin treatment of this protein generated a 110-kDa fragment, whereas the secreted 110-kDa protein remained unchanged. Post-translational removal of the C-terminal transmembrane domain of recombinant GP Ib alpha might have facilitated the secretion of the soluble glycocalicin-like 110-kDa fragment. In addition, flow cytometry and immunofluorescence microscopy demonstrated that the expression of GP Ib alpha alone is sufficient for its incorporation into the cell surface membrane. These data indicate that two soluble fragments of human GP Ib alpha with binding activity for vWF and thrombin can be expressed in mammalian cells and that the incorporation of GP Ib alpha into the surface membrane does not depend on co-expression with GP Ib beta and/or GP IX.
Pharma Division, F. Hoffmann-La Roche Ltd., Basel, Switzerland.
S
S
Meyer
G
G
Kresbach
P
P
Häring
B
B
Schumpp-Vonach
K J
KJ
Clemetson
P
P
Hadváry
B
B
Steiner
D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D015153:Blotting, Western; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism*; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D005784:Gene Amplification; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008727:Methotrexate / Q000494:pharmacology; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins / Q000032:analysis / Q000096:biosynthesis* / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D014162:Transfection; D014841:von Willebrand Factor / Q000378:metabolism
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D008727:Methotrexate