Abstract Number: PB1523
Meeting: ISTH 2020 Congress
Background: We recently identified in a 55-year-old man with hemorrhagic diathesis a compound heterozygous mutation in the VWF molecule, p.R924Q/ p.A2178S (DM-VWF), localized in the D’D3 and D4 domain of VWF. Although these mutations do not involve the A1 domain, commonly responsible for the type 2B VWD, the phenotype of this mutant clearly showed a type 2B-like phenotype.
Aims: Expression of the recombinant VWF with the observed mutations in cell cultures in order to study structural and functional features of this 2B-like VWD form.
Methods: WT and DM-VWF were expressed in HEK293T cells and purified by SE-FPLC. Transmission Electronic Microscopy (TEM) micrographs of all recVWF species, were collected with an acceleration voltage of 120 keV at a magnification of 30,000× (Libra 120; Carl Zeiss Meditec). Atomic Force Microscopy (AFM) was carried out with a silicon nitride cantilever of nominal force constant k =0.1 N/m (MikroMasch® NSC16). The region from S764 to V2191 was modeled on the I-TASSER threading modeling server (Iterative Threading ASSEmbly Refinement) to assess the putative structural effects of the compound heterozygous in cis mutation.
Results: TEM micrographs of the recombinant constructs in the absence of shear showed that WT-VWF has a globular shape, whereas the DM-VWF molecules are stretched and form reticular structures. AFM analysis obtained similar results. The molecular modeling agreed with these findings, as the S764-V2191 DM-VWF region showed a more “open” conformation than the WT form and less compact assembly of b5-b12 strands in the D3 domain.
Conclusions: The compound heterozygous p.R924Q/ p.A2178S mutation causes a less compact conformation of the VWF-monomer. The R924Q mutation eliminates several polar contacts with G913, H916 and K843, contributing to generate a less compact assembly of b5-b12 strands in the D3 domain of VWF. These conformational changes are compatible with the functional type 2B-like VWD features observed in the patient.
To cite this abstract in AMA style:Sacco M, Ferrarese M, Lancellotti S, Tardugno M, Basso M, Papi M, Bernardi F, Castaman G, De Cristofaro R. Structural Analysis of the VWF molecule with Compound in Cis Heterozygous p.R924Q and p.A2178S Mutations Causing a Type 2B-Like von Willebrand Disease [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/structural-analysis-of-the-vwf-molecule-with-compound-in-cis-heterozygous-p-r924q-and-p-a2178s-mutations-causing-a-type-2b-like-von-willebrand-disease/. Accessed September 27, 2023.
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