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 th e initial attachment of platelets to the wall of damaged arteries. In this study, an N-terminal fragment of human GP Ib(alpha) (residues 1-3 18), containing the ligand binding sites for von Willebrand factor (vW F) and thrombin, as well as the entire human GP Ib(alpha) were express ed in Chinese hamster ovary cells. The transfected cells secreted a 48 - and a 110-kDa protein, respectively, into the supernatant. Both reco mbinant proteins were purified by immunoaffinity chromatography. The p urified proteins bound soluble vWF in the presence of botrocetin as de monstrated in solid-phase binding assays. The dissociation constant (K (d)) for I-125-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 th rombin-Sepharose 4B. The 48-kDa protein contained two N-linked oligosa ccharide chains. A 125-kDa protein was identified in the lysate of cel ls transfected with the coding sequence for the entire GP Ib(alpha). T rypsin treatment of this protein generated a 110-kDa fragment, whereas the secreted 110-kDa protein remained unchanged. Post-translational r emoval of the C-terminal transmembrane domain of recombinant GP Ib(alp ha) might have facilitated the secretion of the soluble glycocalicin-l ike 110-kDa fragment. In addition, flow cytometry and immunofluorescen ce microscopy demonstrated that the expression of GP Ib(alpha) alone i s sufficient for its incorporation into the cell surface membrane. The se data indicate that two soluble fragments of human GP Ib(alpha) with binding activity for vWF and thrombin can be expressed in mammalian c ells and that the incorporation of GP Ib(alpha) into the surface membr ane does not depend on co-expression with GP Ib(beta) and/or GP IX.