Abstract Number: OC 09.3
Meeting: ISTH 2021 Congress
Background: A 55-year-old Italian man showed mucocutaneous bleeding with low levels of VWF, mild thrombocytopenia, increased ristocetin-induced platelet aggregation and a deficiency of high molecular weight multimers. The gene sequence analysis revealed a compound heterozygous mutation in the VWF molecule, p.R924Q/ p.A2178S (DM-VWF), localized in the D’D3 and D4 domains 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 VWD 2B-like characteristics .
Aims: This study is aimed to express of the recombinant VWF with the observed mutations in order to study structural and functional features of this 2B-like VWD form.
Methods: Wild Type (WT) and DM-VWF were expressed in HEK293T cells and purified by SE-FPLC. Transmission Electronic Microscopy (TEM) micrographs of all rec-VWF species were collected at a magnification of 30,000× (Libra 120). Atomic Force Microscopy (AFM) was carried out with a silicon nitride cantilever of nominal force constant k=0.1 N/m. The region from S764 to V2191 was modeled on the I-TASSER threading modeling server to assess the structural effect of the double mutation.
Results: TEM micrographs of the recombinant constructs in the absence of shear showed that recombinant 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 β5-β12 strands in the D3-domain.
Conclusions: The DM-VWF 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 β5-β12 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, Lancellotti S, Ferrarese M, Bernardi F, Pinotti M, Tardugno M, De Candia E, Di Gennaro L, Basso M, Giusti B, Papi M, Perini G, Castaman G, De Cristofaro R. Expression and Functional Studies of a Compound Heterozygous Mutation in Cis Position in the D’D3 (p.R924Q ) and D4 (p.A2178S) Domains of von Willebrand Factor (VWF) [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 1). https://abstracts.isth.org/abstract/expression-and-functional-studies-of-a-compound-heterozygous-mutation-in-cis-position-in-the-dd3-p-r924q-and-d4-p-a2178s-domains-of-von-willebrand-factor-vwf/. Accessed September 25, 2021.
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