The interaction of von Willebrand factor (vWF) with platelet glycoprotein Ib/IX plays an important role in primary hemostasis. Previous studies have localized the GpIb alpha binding domain of vWF to amino acid residues 449-728, a region containing the vWF-A1 domain. In order to assess the role of A1 domain structure in vWF binding functions, a cDNA encoding residues 475-709 of vWF was expressed in Escherichia coli (non-glycosylated) and in Chinese hamster ovary (CHO) cells (glycosylated). These recombinant proteins contain a single intrachain disulfide bond between C509 and C695 and were purified as monomers with apparent molecular weights of 36,000 (E. coli) and 39,000 (CHO). 35S-Labeled-vWF-A1 proteins bound directly to GpIb/IX receptors on platelets. The non-glycosylated form had a slightly higher affinity (Kd = 1.4 +/- 0.4 microM) than the glycosylated vWF-A1 protein (Kd = 4.5 +/- 0.9 microM) but had similar binding capacity of 28,000 GpIb/IX-specific binding sites per platelet. Additionally, both recombinant vWF-A1 proteins bound to heparin but neither bound to immobilized type I and III collagen. Both E. coli- and CHO-derived vWF-A1 proteins inhibited ristocetin-induced platelet agglutination with IC50 values of 300 and 700 nM, respectively. Reduction of the only disulfide bond between C509 and C695 abolished platelet binding activity at concentration up to 2 microM of protein. Confirmation of the importance of the 509-695 disulfide bond was obtained from a full-length vWF mutant containing substitutions at C509 and C695 (C509/695S) which failed to bind to the platelet GpIb/IX receptor. 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 intact disulfide bond between C509 and C695. Furthermore, glycosylation increases the solubility but reduces binding affinity of recombinant vWF A1.