CHARACTERIZATION OF THE VON WILLEBRAND FACTOR-BINDING DOMAIN OF PLATELET MEMBRANE GLYCOPROTEIN Ib

Abstract

We have previously obtained immunochemical evidence that the von Willebrand factor (vWF)-binding domain of the platelet membrane glycoprotein (GP) Ib is located near the amino terminus of the a subunit (Journal of Biological Chemistry 261: 12579-12585, 1986). We have now determined the complete amino acid sequence of the 45 kDa tryptic fragment of glycocalicin that contains this domain. Purified glycocalicin was subjected to limited digestion with trypsin and the proteolytic fragments were separated by size-exclusion high-pressure liquid chromatography. Two fragments of 45 kDa and 84 kDa, respectively, were obtained under nonreducing conditions. After reduction and S-carboxymethylation, the 84 kDa fragment was unchanged, while the 45 kDa fragment yielded two new fragments, one of 35 kDa and the other of 7 kDa. This finding proves the existence of a trypsin cleavage site within a disulfide loop. Two primary sets of overlapping fragments were obtained by cleavage of the carboxymethylated protein at methionyl and lysyl bonds following treatment with cyanogen bromide and Achromobacter protease I, respectively. Additional fragments were obtained by treatment of glycocalicin with Staphylococcus aureus V8 protease and Serratia marcescens protease. Analysis of all these fragments provided data that allowed determination of the sequence of the amino terminal 299 residues of the GP Ib a-chain. This includes the 45 kDa tryptic fragment containing the vWF-binding domain. This 299-residue sequence, corresponding approximately to two thirds of the α-chain polypeptide, is largely hydrophobic and contains only two N-linked and one O-linked carbohydrate chains. A hydrophilic region exists between residues 215-299, with a cluster of ten negatively charged residues at 269-287. This area is likely to attract positively charged molecules. The hydrophilic, highly glycosylated (at Ser/Thr residues) region corresponding to the previously described "macroglycopeptide" begins at residue 292. The determined sequence of glycocalicin contains a region with seven repeats, indicative of gene duplication, and is highly homologous to human leucine-rich α2-glycoprotein.