A new type 1 von Willebrand Disease (VWD) characterized by increased clearance of von Willebrand Factor (VWF) due to the heterozygous p.P1127S mutation: clinical phenotype and pathogenic mechanisms.

M. Tardugno¹, M. Sacco¹, R. De Cristofaro¹, S. Lancellotti², F. Bernardi³, M. Pinotti³, A. Branchini⁴, B. Giusti⁵, G. Castaman⁶

¹Catholic University of Sacred Heart, Translational Medicine and Surgery Department, Rome, Lazio, Italy, ²Fondazione Policlinico Universitario A. Gemelli, IRCCS, Rome, Rome, Lazio, Italy, ³Department of Life Sciences and Biotechnology, University of Ferrara, Italy., Ferrara, Emilia-Romagna, Italy, ⁴Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy, Ferrara, Emilia-Romagna, Italy, ⁵University of Florence, Department of Clinical and Applied Medicine, Florence, Toscana, Italy, ⁶“Careggi” University Hospital, Hemorrhagic and Coagulation Disease Unit, Department of Oncology., Florence, Toscana, Italy

Abstract Number: PB0804

Meeting: ISTH 2022 Congress

Theme: Platelet Disorders, von Willebrand Disease and Thrombotic Microangiopathies » von Willebrand Factor Biology

Background: A 21-year-old Italian woman with a large thigh hematoma, appeared after a minor trauma, showed a proportional decrease of both VWF antigen (VWF:Ag=34.3 IU/dL) and activity (VWF:GpIbR=32.8 IU/dL), and low levels of factor VIII (FVIII:C=55.3 IU/dL). She inherited, from her 51-year-old mother (VWF:Ag≈60 IU/dL), a new VWF heterozygous missense mutation c.C3379>T in the exon 25, causing the p.P1127S substitution in the D’D3 domain. Both patients signed an informed consent.

Aims: Because of VWD type 1 phenotype is extremely heterogeneous, low VWF levels are caused by different molecular mechanisms: alteration of secretion; intracellular retention; accelerated plasmatic clearance. This study focused on clinical/biochemical characterization of this new VWF gene mutation.

Methods: VWF:Ag and VWF:GpIbR were measured by chemiluminescence assays, FVIII:C by chromogenic assay; the VWF-FVIII binding (VWF:VIIIB) and pro-peptide levels (VWF:pp) by ELISA assays; ADAMTS-13:activity by FRET-based-assay; the VWF multimeric pattern (mVWF) by SDS-agarose-gel electrophoresis; ristocetin-induced platelet aggregation by Born-assay. Molecular modeling was performed by using I-TASSER software. Recombinant expression of VWF WT and p.P1127S-mutant variants was obtained by HEK-293 cells, while their interaction with the low-density lipoprotein receptor-related protein-1 (LRP1) was analyzed by fluorescence assay.

Results: The heterozygous p.P1127S mutation was clinically associated with a proportional mild decrease of both VWF:Ag and VWF:activity. Although 0.3 µg/Kg-BW desmopressin infusion normalized the patient’s VWF levels, the drug clearance resulted enhanced (t1/2=6.7h) than in normal subjects (t1/2=12±0.7h), (Figure 1). The VWF:VIIIB and ADAMTS-13:activity were normal, as well as the ristocetin-induced platelet aggregation and the mVWF pattern. The p.P1127S-mutant variant was normally synthesized and secreted by HEK-293 cells. Molecular modeling predicted p.P1127S-mutant conformational changes, showing an increased binding affinity for the VWF scavenging-receptor LRP1 (Figure 2), confirmed by in vitro studies.

Conclusion(s): The VWF p.P1127S mutation causes conformational rearrangements, due to multiple missing van der Waals contacts, accelerating the VWF clearance, but not its synthesis and secretion.

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Figure 1. Pharmacokinetics profile of VWF:Ag levels after i.v. infusion of 0.3 µg/Kg-BW desmopressin for the patient, her mother and three normal subjects. The calculated VWF t ½ clearance is listed in the inset table. The pharmacokinetic analysis was performed by using WinNonlin program, employing a non-compartmental and i.v. infusion model.

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Figure 2. Molecular modeling of VWF WT and p.P1127S mutant forms -in green- and their interaction -in yellow and red- with the LRP1 receptor -in blue-. The energetics is characterized by ∆G = -14.1 Kcal/mol -Kd at 25 °C=4.7×10-11 M- for the WT-LRP1 binding -Fig. 1A- and by ∆G = -15.2 Kcal/mol -Kd at 25 °C= 7.3×10-12 M- for the mutant-LRP1 binding -Fig. 1B-.

To cite this abstract in AMA style:

Tardugno M, Sacco M, De Cristofaro R, Lancellotti S, Bernardi F, Pinotti M, Branchini A, Giusti B, Castaman G. A new type 1 von Willebrand Disease (VWD) characterized by increased clearance of von Willebrand Factor (VWF) due to the heterozygous p.P1127S mutation: clinical phenotype and pathogenic mechanisms. [abstract]. https://abstracts.isth.org/abstract/a-new-type-1-von-willebrand-disease-vwd-characterized-by-increased-clearance-of-von-willebrand-factor-vwf-due-to-the-heterozygous-p-p1127s-mutation-clinical-phenotype-and-pathogenic-mechanisms/. Accessed September 24, 2023.

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