Isolated recombinant domain of von Willebrand factor displaying increased sensitivity to ristocetin

Type 2B von Willebrand disease is characterized by an abnormal von Willebrand factor molecule with increased affinity for the platelet glycoprotein (GP) Ib-IX receptor. A diagnostic feature of type 2B von Willebrand disease is a characteristic loss of von Willebrand factor high molecular weight multimers. In vitro, the soluble interaction of normal von Willebrand factor with platelets can be initiated with exogenous modulators, the most common being the antibiotic ristocetin. The variant molecules resulting in type 2B von Willebrand disease can sustain binding to platelets at subnormal levels of ristocetin. We characterized the von Willebrand factor gene of an individual with type 2B von Willebrand disease and identified a nucleotide transition resulting in an Arg543-->Trp amino-acid substitution within the GP Ib-IX binding domain of von Willebrand factor. In this study we demonstrate that a recombinant plasmid capable of expressing the isolated GP Ib-IX binding domain of von Willebrand factor, and containing the Arg543-->Trp amino-acid substitution, secretes a dimeric molecule that supports platelet agglutination using subnormal levels of ristocetin. These results demonstrate that the mutation at position 543 increases the affinity between the variant molecule and platelet GP Ib-IX as an intrinsic feature of the isolated von Willebrand factor domain. Thus, structural perturbations within the GP Ib-IX binding domain that are independent of the von Willebrand factor multimeric structure can sufficiently increase the affinity of von Willebrand factor to sustain platelet aggregation, using subnormal levels of ristocetin.