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

Abstract

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.

Physical Characteristics of von Willebrand Factor Binding with Platelet Glycoprotein Ibɑ Mutants at Residue 233 Causing Various Biological Functions

“…One important and specific characteristic of VWF binding with GPIbɑ is that their binding could be detected only under shear flow conditions or in the presence of specific modulators of ristocetin or botrocetin unless with a specific gain of function mutants. 4 15 18 45 46 Our MD simulation was conducted in the absence of any modulators. Thus, our results represent the conditions of transient VWF binding with GPIbɑ under shear flow conditions.…”

Von Willebrand Factor Multimer Analysis and Classification: A Comprehensive Review and Updates

“…Type 2M (platelet-binding defect) is the more common subtype and is characterized by decreased VWF plateletbinding activity with preservation of VWF multimers; the mutations are spread across the A1 domain, which results in decreased GPIB-V-IX binding affinity. 37,38 Type 2M (collagen-binding defect) mutations affect the ability of VWF to bind to type I, III, and/ or VI collagen via binding sites on the A1 or A3 domain, and are characterized by decreased VWF collagen-binding activity using a VWF:CB assay and preserved VWF platelet-binding activity. 39 Type 2N VWD is due to reduced VWF:FVIIIB activity.…”

Snake Venoms in Diagnostic Hemostasis and Thrombosis

“…45 46 47 Conversely, a normal VWF:BCof result in symptomatic patients with genuine type 2M VWD, including a reduced ristocetin-induced GPIb-binding based functional assay, is suggestive of specific mutations in the VWF GPIb-binding domain. 48 An assay for VWF inhibitors based on impaired ristocetin- or botrocetin-induced binding of biotin-labeled VWF to immobilized, washed platelets has been described, but is not commercially available. 49…”