Isolation and characterization of two domains of human von Willebrand factor that interact with fibrillar collagen types I and III.
We have identified four discrete proteolytic fragments of von Willebrand factor (vWF) that define two collagen-binding domains. Two of the fragments tested, T 96 kDa and T 55 kDa, were generated by digestion with trypsin, and two, Fragments I and III, with Staphylococcus aureus V8 protease. The larger Fragment III, a disulfide-linked homodimer, extends between residues 1 and 1365 of the 2050-residue vWF subunit and comprises the sequence of all the others. T 96 kDa, also a disulfide-linked homodimer, extends between residues 449 and 728. T 55 kDa and Fragment I, both single-chain polypeptides, have a partial sequence overlap corresponding to residues 911-1114, and together extend from residue 730 to 1365. The ability of the fragments to interfere with the vWF-collagen interaction was evaluated by measuring inhibition of 125I-labeled vWF binding to fibrillar bovine collagen types I and III. All the four fragments tested inhibited binding. Native conformation was essential for expression of this function; denaturation with guanidine hydrochloride and reduction of disulfide bonds resulted in marked reduction or complete loss, respectively, of the inhibitory activity at all the concentrations tested. Two monoclonal antibodies were prepared, one directed against T 96 kDa and the other against Fragment I. Both antibodies partially inhibited vWF binding to collagen, and their inhibitory effect was enhanced when they were used together. 125I-Labeled Fragment I bound to collagen in a saturable manner, and the binding was completely blocked by both T 96 kDa and T 55 kDa. Thus, we have identified at least two distinct functional domains of vWF that concurrently mediate the vWF-collagen interaction. The two domains appear to share a common recognition site on collagen.
D000818:Animals; D002417:Cattle; D003094:Collagen / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D014357:Trypsin / Q000378:metabolism; D014841:von Willebrand Factor / Q000302:isolation & purification / Q000378:metabolism*
D010446:Peptide Fragments; D014841:von Willebrand Factor; D003094:Collagen; D014357:Trypsin