GTH 2017, 61st Annual Meeting of the Society of Thrombosis and Hemostasis Research

Novel insights in the molecular etiology of recessive von Willebrand disease 2N due to D’D3 Factor VIII-binding site mutations of von Willebrand factor

J. J. Michiels^1,2, F. Hansen³, R. Dingle³, A. Batorova⁴, T. Prigancova⁴, P. Smejkal⁵, M. Penka⁵, I. Vangenechten¹, A. Gadisseur¹ (¹Edegem, Belgium, ²Rotterdam, Netherlands, ³London, United Kingdom, ⁴Bratislava, Slovakia, ⁵Brno, Czech Republic)

Bleeding disorders, coagulation and fibrinolytic factors
Date: 17.02.2017,
Time: 17:15 - 18:15

Objective: Introduction. Von Willebrand disease (VWD) type 2N is caused by a Factor VIII(FVIII) binding site on von Willebrand factor (VWF) located in the D’ region of VWF (766-864). The D’ region consists of 2 domains: trypsin-inhibitor-like (TIL’) and E’. The trypsin-inhibitor like (TIL’) E’ domains form a protrusion from the D3 domain, thereby presenting the TIL’E’ domains to the physiological binding partner FVIII. A detailed characteristic of the VWF-FVIII binding site has remained intractable until recently when the two distinct and conserved domains TIL’(residues 766-827)E’(residues 829-863) were shown to contain a majority of the known type 2N mutations. This has led to better insights in the structure of the FVIII binding domain in the VWF protein and the VWF binding domain in FVIII protein, which prompted to look into the molecular etiology and the clinical and laboratory manifestation on autosomal recessive von Willebrand disease (VWD) type 2N.

Methods: FVIII binding D’ domain of VWF. The TIL’E’ domains are located immediately after the Furin cleavage site and form independently folding domains separating D’ into two distinct domains and all cysteine residues of these domains form disulfide bridges (Cys767-Cys808;Cys776-Cys804;Cys810-Cys821) (Figure 2). The TIL’ and E’ domains tumble as a single entity with only limited inter-domain motion [16]. TIL’ is formed of short beta sheets 12 (residues 772-775;806-809) and 3:4 (residues 814-817;820-823). The 1-to-2 loop encompass an eight residue insertion between the second and third conserved TIL' cysteine residues. The second antiparallel  sheet, formed of strands 3 and 4, is connected by a reversed turn forming a small hairpin structure. TIL’ and E’ are connected at 827-829. The greater proportion of E’ is formed of a triple-stranded antiparallel -sheet formed of strands 3 (residues 839-844), 4 (residues 847-852) and 5 (residues 855-858). The N-terminus of E’ contains a short double strand 1:2 sheet (residues 829-831:834-836). The relative positions of these two  sheets are restrained by the E’ Cys829-Cys851 disulfide bond. The dynamic characteristics of TIL’E’ is of great importance for elucidating the mechanism by which FVIII is stabilized in plasma

Results: Results. Correct pairing of the 16 cysteine residues and hence correct folding of the Til’E’ is of critically importance for the binding between Til’E’ and FVIII. The homozygous Factor VIII binding defects (FVIII-BD) R854W/R854W and R816W/R816W in the D’ domain (VWD 2N in its purity) result in a clinical phenotype of mild/moderate hemophilia A with normal bleeding time and VWF function. The E787K, T791M and R816W mutations have pronounced type 2N VWD with decreased FVIII-BD and normal VWF functions. Type 2N mutations that involve a cysteine (C788R/Y, Y795C, and C804F in TiL’, C858S/F in E’) are associated with aberrant multimerization and poor secretion on top of reduced FVIII-BD. Two mutations, T791M and R816W together with T789P, M800V and H817Q are clustered around a region of positive charged density of TiL’. VWD type 2N patients with the heterozygous mutation near to or at the Furin cleavage site R763G present with VWD type 2 in early childhood featured by a smeary pattern and ultralarge VWF multimers caused by the mixture of circulating R763G mutant Pro-VWF and mature wild type VWF on top of a FVIII-BD of about 30%.

Conclusion: Discussion. Recessive VWD type 2N in its purity due to homozygous non-cystein mutations in the D’domain of the VWF gene (eg ) is different from all other VWD subtypes in that the primary defect, a decreased FVIII-BD, results in a clinical mild to moderate hemophilia A, featured by FVIII:C deficiency and low FVIII:C/VWF:Ag ratio, normal VWF:RCo/Ag and VWF:CB/Ag ratios with normal bleeding time, functional VWF parameters and VWF multimers. Heterozygous mutations in the C-domain of the FVIII gene impairing VWF binding produced a similar mild to moderate hemophilia A and completely normal VWF functions as seen in “VWD type 2N” in its purity. VWD type 2N mutations that involve a cysteine residue (C788R/Y; Y795C and C804F in TIL’; C858C/F in E’) are associated with aberrant multimerization, poor secretion on top of reduced FVIII binding by the cystein mutation itself or when associated with a non-cystein mutation in the D’ domain. Double heterozygous VWD 2N non-cystein and a cysteine 2N mutation in the FVIII binding site of VWF produce a hybrid phenotype of FVIII-BD defect with abnormal multimers. VWD type 2N mutations that involve a cysteine residue (C788R/Y; Y795C and C804F in TIL’; C858C/F in E’) are associated with aberrant multimerization, poor secretion on top of reduced FVIII binding by the cystein mutation itself or when associated with a non-cystein mutation in the D’ domain.