A3 DOMAIN IS ESSENTIAL FOR INTERACTION OF VON-WILLEBRAND-FACTOR WITH COLLAGEN TYPE-III

von Willebrand factor (vWF) mediates platelet adhesion at sites of vas cular damage. It acts as a bridge between receptors on platelets and c ollagens present in the connective tissue. Two collagen binding sites have been identified on the A1 and A3 domain of the vWF subunit. To st udy the functional importance of these binding sites, we have made two deletion mutants that lack the Al domain (residues 478-716; Delta A1- vWF; Sixma et al. fur. J. Biochem. 196, 369, 1991 [1]) or the A3 domai n (residues 910-1113; Delta A3-vWF). After transfection in baby hamste r kidney cells overexpressing furin, the mutants were processed and se creted efficiently. Ristocetin or botrocetin induced platelet binding was normal. for Delta A3-vWF as was binding to heparin and factor vm. As reported by Sixma et al. (1) Delta A1-vWF still binds to collagen t ype III, indicating that the A3 domain is sufficient for the interacti on. In the current study, we investigated the binding of Delta A3-vWF to collagen type m. When preincubated on collagen type III it did not support platelet adhesion under flow conditions, whereas it was able t o support platelet adhesion when coated directly to a glass surface. T he binding of I-125-Delta A3-vWF to collagen was specific but maximal binding was about 40 times less compared to I-125-vWF. When added at 2 5 times excess, Delta A3-vWF did not compete with I-125-vWF for bindin g to collagen type III, whereas Delta A1-vWF did. The binding of I-125 -Delta A3-vWF could be blocked by excess unlabeled VWF but not by Delt a A1-vWF. In conclusion, we demonstrate that the A3 domain in VWF cont ains the major collagen binding site. The major binding site present o n the A3 domain and the minor site present on A1 bind to different sit es on collagen.