Genetic alteration of the D2 domain abolishes von Willebrand factor multimerization and trafficking into storage.

BACKGROUND: The large von Willebrand factor (VWF) propeptide (VWFpp) plays a critical role in the multimerization and regulated storage of the mature VWF protein. Although our laboratory and others have identified mutations in von Willebrand disease patients that disrupt VWF multimerization, little is known about the affect of mutations on the regulated storage of VWF. PATIENTS/METHODS: We identified a heterozygous 18 base pair, in-frame deletion in exon 12 of the VWF gene in a patient with an unusual, dimer-intense multimer pattern. This deletion results in loss of amino acids 436-442 of VWFpp, which include one cysteine. RESULTS: Through expression studies, we demonstrate reduced secretion, loss of VWF multimerization, and defective regulated storage of the variant VWF. The loss of VWF storage is secondary to loss of propeptide storage resulting from an apparently defective sorting signal on VWFpp. Suprisingly, coexpressed wild-type VWF or VWFpp functioned in trans to partially restore multimerization of VWF from the variant allele. CONCLUSIONS: The deletion of six amino acids in VWFpp results in defects in VWF processing, regulated storage, and function. Although VWFpp may usually function in a homotypic fashion, acting on its own mature VWF subunit, VWFpp may retain the ability to function in trans on VWF expressed from the variant allele.

Type 2M von Willebrand disease: F606I and I662F mutations in the glycoprotein Ib binding domain selectively impair ristocetin- but not botrocetin-mediated binding of von Willebrand factor to platelets.

von Willebrand disease (vWD) is a common, autosomally inherited, bleeding disorder caused by quantitative and/or qualitative deficiency of von Willebrand factor (vWF). We describe two families with a variant form of vWD where affected members of both families have borderline or low vWF antigen levels, normal vWF multimer patterns, disproportionately low ristocetin cofactor activity, and significant bleeding symptoms. Whereas ristocetin-induced binding of plasma vWF from affected members of both families to fixed platelets was reduced, botrocetin-induced platelet binding was normal. The sequencing of genomic DNA identified unique missense mutations in each family in the vWF exon 28. In Family A, a missense mutation at nucleotide 4105T --> A resulted in a Phe606Ile amino acid substitution (F606I) and in Family B, a missense mutation at nucleotide 4273A --> T resulted in an Ile662Phe amino acid substitution (I662F). Both mutations are within the large disulfide loop between Cys509 and Cys695 in the A1 domain that mediates vWF interaction with platelet glycoprotein Ib. Expression of recombinant vWF containing either F606I or I662F mutations resulted in mutant recombinant vWF with decreased ristocetin-induced platelet binding, but normal multimer structure, botrocetin-induced platelet binding, collagen binding, and binding to the conformation-sensitive monoclonal antibody, AvW-3. Both mutations are phenotypically distinct from the previously reported variant type 2MMilwaukee-1 because of the presence of normal botrocetin-induced platelet binding, collagen binding, and AvW-3 binding, as well as the greater frequency and intensity of clinical bleeding. When the reported type 2M mutations are mapped on the predicted three-dimensional structure of the A1 loop of vWF, the mutations cluster in one region that is distinct from the region in which the type 2B mutations cluster.