MOLECULAR CLONING OF HUMAN PLATELET GLYCOPROTEIN Ib

Glycoprotein Ib (GPIb) mediates von Willebrand factor-dependent platelet adhesion and participates in the resulting platelet activation process. In the present investigation, the primary structure of human platelet GPIb was studied. GPIb and its proteolytic fragment glycocalicin were purified to near homogeneity from human platelets by affinity chromatography using wheat germ agglutinin and anti-GPIb monoclonal antibody (D. Nugent, University of Washington) coupled to Sepharose. GPIba chain, β chain, and glycocalicin were isolated, reduced and carboxymethylated, and then fragmented by trypsin and S. aureus V8 protease. Peptides were isolated by HPLC and subjected to amino acid sequence analysis. Approximately 200 amino acid residues were identified. Affinity purified rabbit antibodies directed against the a chain, the ft chain, and glycocalicin were prepared and shown to be monospecific by Western blot analysis. Total RNA was prepared from human erythroleukemia cells grown in the presence of phorbol acetate. Poly(A)+ RNA was selected and used to prepare a cDNA library in λgt11. The library was screened with [125]I-labeled polyclonal antibody to glycocalicin. The clone with the largest cDNA insert was sequenced and shown to code for amino acid sequences corresponding to those determined by Edman degradation of glycocalicin. The predicted amino acid sequence contains at least six tandem repeats of 24 amino acids that are highly homologous with 13 repeats present in leucine rich α2 glycoprotein of human plasma. Another region in the protein contains a second repeat rich in threonine and serine, which shows some homology to a 9 amino acid repeat in the connecting region of human factor V. This region is probably the major site of attachment of clusters of O-linked carbohydrate in GPIbα. These results indicate that human platelet glycoprotein Ibα has a multi-domain structure composed of a number of repetitive sequences. Supported in part by grants from the American Heart Association, Robert Wood Johnson Foundation, Veterans Administration, and National Institutes of Health.