The von Willebrand factor-binding domain of platelet membrane glycoprotein Ib. Characterization by monoclonal antibodies and partial amino acid sequence analysis of proteolytic fragments.
The glycoprotein Ib (GPIb), a two-chain integral platelet membrane protein, acts as a receptor for von Willebrand factor. In order to obtain information on the domain involved in this function, as well as on the structural organization of GPIb, the protein has been purified and submitted to limited proteolysis using three different enzymes. The resulting fragments were topographically oriented by means of partial NH2-terminal sequence analysis and immunological identification using monoclonal antibodies. One of these antibodies (LJ-Ib1) inhibited the von Willebrand factor-GPIb interaction completely, one (LJ-P3) partially, and one (LJ-Ib10) had no inhibitory effect. Three distinct fragments, the 38-kDa fragment produced by Serratia marcescens protease as well as the 45- and 35-kDa fragments produced by trypsin, had the same NH2 terminus as the intact GPIb alpha-chain (apparent molecular mass = 140 kDa). These fragments and the alpha-chain reacted with the inhibitory antibodies. On the other hand, three fragments produced by Staphylococcus aureus V8 protease, one of 92 kDa similar to the previously described "macroglycopeptide" and two others of 52 and 45 kDa, had NH2-terminal sequences different from that of the GPIb alpha-chain and reacted only with the noninhibitor monoclonal antibody LJIb10. Thus, the binding domain for von Willebrand factor resides near the NH2 terminus of the GPIb alpha-chain, whereas the carbohydrate-rich region is part of the innermost portion of GPIb and does not appear to be involved in the von Willebrand factor binding function.
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D010450:Endopeptidases / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D007163:Immunosorbent Techniques; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins; D012697:Serine Endopeptidases; D014357:Trypsin / Q000378:metabolism; D014841:von Willebrand Factor / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D006023:Glycoproteins; D010446:Peptide Fragments; D010980:Platelet Membrane Glycoproteins; D014841:von Willebrand Factor; D010450:Endopeptidases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin