Identification of functional domains on von Willebrand factor by binding of tryptic fragments to collagen and to platelets in the presence of ristocetin

With the use of monoclonal antibodies that inhibit the ristocetin- induced binding of von Willebrand factor (VWF) to platelets and the binding to collagen, we have previously identified two distinct tryptic fragments. To prove that these fragments contain the platelet binding or the collagen binding domain, we investigated the direct binding of tryptic fragments of 125I-VWF to platelets in the presence of ristocetin and to collagen fibrils. During the course of the tryptic digestion, there was a rapid and parallel decrease in binding to platelets and collagen. In the first ten minutes, binding decreased greater than 50%; a further decrease to 19% and 29%, respectively, was noted at 90 minutes, but no further decrease was observed thereafter. The bound fragments were eluted from platelets and collagen and analyzed on polyacrylamide gradient gels. The fragments bound to the platelets appeared to be reduced, probably by endogenous reducing substances from the platelets. This was prevented by addition of N- ethylmaleimide during the incubation. After 24 hours of digestion, platelets predominantly bound fragments of 116 kd and collagen bound a single fragment of 48 kd. These fragments are similar to those previously identified with the monoclonal antibodies.