THE INTERACTION OF THE VON-WILLEBRAND FACTOR-A1 DOMAIN WITH PLATELET GLYCOPROTEIN-IB-IX - THE ROLE OF GLYCOSYLATION AND DISULFIDE BONDING IN A MONOMERIC RECOMBINANT A1 DOMAIN PROTEIN

The interaction of von Willebrand factor (vWF) with platelet glycoprot ein Ib/IX plays an important role in primary hemostasis. Previous stud ies have localized the GpIbalpha binding domain of vWF to amino acid r esidues 449-728, a region containing the vWF-A1 domain. In order to as sess the role of A1 domain structure in vWF binding functions, a cDNA encoding residues 475-709 of vWF was expressed in Escherichia coli (no n-glycosylated) and in Chinese hamster ovary (CHO) cells (glycosylated ). These recombinant proteins contain a single intrachain disulfide bo nd between C509 and C695 and were purified as monomers with apparent m olecular weights of 36,000 (E. coli) and 39,000 (CHO). S-35-Labeled-vW F-A1 proteins bound directly to GpIb/IX receptors on platelets. The no n-glycosylated form had a slightly higher affinity (K(d) = 1.4 +/- 0.4 muM) than the glycosylated vWF-A1 protein (K(d) = 4.5 +/- 0.9 muM) bu t had similar binding capacity of 28,000 GpIb/IX-specific binding site s per platelet. Additionally, both recombinant vWF-A1 proteins bound t o heparin but neither bound to immobilized type I and III collagen. Bo th E. coli- and CHO-derived vWF-A1 proteins inhibited ristocetin-induc ed platelet agglutination with IC50 values of 300 and 700 nM, respecti vely. Reduction of the only disulfide bond between C509 and C695 aboli shed platelet binding activity at concentration up to 2 muM of protein . Confirmation of the importance of the 509-695 disulfide bond was obt ained from a full-length vWF mutant containing substitutions at C509 a nd C695 (C509/695S) which failed to bind to the platelet GpIb/IX recep tor. These studies document that vWF-A1 domain can bind to GpIb/IX and heparin but not collagen, and that binding to GpIb/IX requires an int act disulfide bond between C509 and C695. Furthermore, glycosylation i ncreases the solubility but reduces binding affinity of recombinant vW F A1.