PB0804 - 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.

Monday, July 11, 2022

6:30 PM – 7:30 PM

Location: Exhibition

Presentation Format: In-Person

Presenting Author(s)

Maira Tardugno, Dr

PhD Student
Catholic University, Translational Medicine and Surgery Department Rome, Italy
Roma, Lazio, Italy

Co-Author(s)

MS

Monica Sacco

Post-doc
Catholic University, Translational Medicine and Surgery Department Rome, Italy
Roma, Lazio, Italy
RD

Raimondo De Cristofaro, MD, PhD

Professor, Haemostasis and Thrombosis Center Director
1.Fondazione Policlinico Universitario A. Gemelli, IRCCS, Center for Hemorrhagic and Thrombotic Diseases and Haemophilia Center, Rome, Italy; 2. Catholic University, Translational Medicine and Surgery Department Rome, Italy
Roma, Lazio, Italy
Stefano Lancellotti, PhD

Biologist Health Manager
Fondazione Policlinico Universitario A. Gemelli, IRCCS, Center for Hemorrhagic and Thrombotic Diseases and Haemophilia Center, Rome, Italy
Roma, Lazio, Italy
FB

Francesco Bernardi

Professor of Biochemistry
Department of Life Sciences and Biotechnology, Section of Biochemistry and Molecular Biology, Ferrara University, Ferrara (Italy)
Verona, Veneto, Italy
MP

Mirko Pinotti

Professor
University of Ferrara, Department of Life Sciences and Biotechnology
Ferrara, Emilia-Romagna, Italy
AB

Alessio Branchini

Assistant Professor
Department of Life Sciences and Biotechnology and LTTA Center, University of Ferrara, Ferrara 44121, Italy
Ferrara, Emilia-Romagna, Italy
BG

Betti Giusti

Professor
University of Florence, Department of Clinical and Applied Medicine
Florence, Toscana, Italy
Giancarlo Castaman, MD

Medical Director
Center for Bleeding Disorders and Coagulation, Careggi University Hospital, Florence, Italy
Florence, Toscana, Italy

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.