IDENTIFICATION OF A SECOND COLLAGEN-BINDING DOMAIN IN HUMAN VON WILLEBRAND FACTOR

Abstract

We have recently reported (Journal of Biological Chemistry 261: 15310-15315, 1986) that von Willebrand factor (vWF) possesses a collagen-binding domain localized in a reduced and alkylated tryptic fragment of apparent 52/48 kDa molecular weight extending between residues Val (449) and Lys (728) of the constituent subunit. This proteolytic fragment of vWF also contains a glycoprotein lb-binding domain and a heparin-binding domain. We have now identified a second collagen-binding domain in the Staphylococcus aureus V8 protease-generated fragment I that extends from residue Gly (911) to Glu (1365). The two binding domains exhibit different interaction with collagens of different origin. The reduced and alkylated 52/48 kDa tryptic fragment was a potent inhibitor of vWF binding to equine collagen type I, but had no effect on the binding to bovine collagen type I and III. In contrast, a purified fraction containing the unreduced 52/48 kDa domain inhibited vWF binding to all types of collagen, as did anti-52/48 kDa monoclonal antibodies. Some of these antibodies, however, were more effective in inhibiting binding to equine collagen. On the other hand, fragment I markedly inhibited the binding of vWF to bovine collagen type I and III, but was less effective with equine collagen type I. Direct binding studies using 425j_qabeled fragment I demonstrated that the association constant was 5 to 10 times greater with the bovine collagens than with the equine collagen. The Staphylococcus aureus V8 protease-generated fragment III, which extends from residue Ser (1) to Glu (1365) and contains both collagen-binding domains, was the most potent inhibitor of vWF binding to all types of collagen tested. Thus, vWF has at least two collagen-binding domains. Native conformation appears to be necessary for binding of the 52/48 kDa domain to bovine collagen type I and III, but not to the equine collagen type I tested. The two domains appear to function concurrently in mediating vWF binding to collagen.