PB0816 - Phenotypic characterization of Von Willebrand disease type 3 patient-derived ECFCs with a homozygous p.M771V mutation

Monday, June 26, 2023

18:30 – 19:30 ET

Room: Exhibition

Presenting Author(s)

Isabel Bär, MSc

PhD candidate
Department of Hematology, Erasmus University Medical Centre, Rotterdam, the Netherlands
Rotterdam, Zuid-Holland, Netherlands

Co-Author(s)

PB

Petra E. Bürgisser

Research Technician
Department of Hematology, Erasmus University Medical Center Rotterdam
Rotterdam, Zuid-Holland, Netherlands
Calvin B. van Kwawegen, MD (he/him/his)

PhD-candidate
Department of Hematology, Erasmus MC, Erasmus University Medical Center Rotterdam, Rotterdam, the Netherlands
Rotterdam, Zuid-Holland, Netherlands
JV

Jan Voorberg

Professor, Principal investigator
Department of Molecular Hematology, Sanquin Research and Landsteiner Laboratory
Amsterdam, Noord-Holland, Netherlands
Jeroen C.J Eikenboom, MD. PhD

Professor, Head of Division of Thrombosis and Hemostasis
Leiden University Medical Center
Leiden, Zuid-Holland, Netherlands
Frank W.G. Leebeek, MD, PhD

Professor and Head of Department of Hematology
Erasmus MC, University Medical Center Rotterdam, The Netherlands
Rotterdam, Zuid-Holland, Netherlands
Ruben Bierings, PhD

Principal investigator
Department of Hematology, Erasmus MC, University Medical Center Rotterdam, the Netherlands
Rotterdam, Zuid-Holland, Netherlands

Background: Von Willebrand disease (VWD) is the most common inherited bleeding disorder. Most patients carry missense mutations in the Von Willebrand Factor (VWF) gene. The severity and type of VWD can however vary drastically between patients due to heterogeneity of the disease genotype. A family with p.M771V mutations has been diagnosed with type 2N and type 3 VWD.

Aims: Characterization of patient endothelial colony forming cells (ECFCs) with a p.M771V mutation to gain insight in the molecular mechanism that underpins the severe VWD phenotype.

Methods: ECFCs are isolated from venous blood of two patients selected from the Willebrand in the Netherlands (WiN) cohort that carry the p.M771V mutation. Biosynthesis, storage, and secretion of VWF was studied using biochemical methods (VWF ELISA, immunoblotting, VWF multimer gel electrophoresis), as well as confocal microscopy.

Results: VWF activity < 0.07 U/ml and VWF antigen levels < 0.1 U/ml were measured in both patients’ plasma. Proteolytic processing of proVWF into mature VWF and VWF propeptide (VWFpp) was impaired, judging from high ratio of proVWF:VWF and the low abundance of VWFpp in p.M771V ECFC lysates. Intracellular VWF content and secreted VWF were severely reduced in p.M771V ECFCs compared to ECFCs of healthy control donors. Multimer analysis of intracellular and secreted VWF showed a deficiency of high molecular weight (HMW) VWF multimers. Immunofluorescent staining of VWF revealed the absence of Weibel-Palade bodies in p.M771V ECFCs. The remaining intracellular VWF was found retained in the endoplasmic reticulum (ER).

Conclusion(s): The severe endothelial disease phenotype observed in p.M771V ECFCs suggests that ER retention of VWF and failure to undergo subsequent processing steps is the basis of the severe bleeding phenotype of VWD3 patients with homozygous mutations at M771.

Funding was received from the Netherlands Organization for Scientific Research (NWO), Domain Applied and Engineering Sciences (TTW), ‘Connecting Innovators’ Open Technology Program, Project#18712