PB0341 - Impaired secretion of Von Willebrand Factor in patients with biallelic mutations in the guanine nucleotide exchange factor MADD

Sunday, June 23, 2024

13:45 – 14:45 ICT

Room: Exhibition Hall

Presenting Author(s)

SH

Sophie Hordijk, Msc

PhD student
Hematology, Erasmus MC University Medical Center, Rotterdam, The Netherlands
Rotterdam, Zuid-Holland, Netherlands

Co-Author(s)

Stijn Groten, MSc

Phd student
Department of Molecular Hematology, Sanquin Research, 1066 CX Amsterdam, the Netherlands
Amsterdam, Noord-Holland, Netherlands
SL

Sebastiaan Laan

PhD student
Internal Medicine, Division of Thrombosis and Hemostasis, Leiden University Medical Center, Leiden, the Netherlands
Leiden, Zuid-Holland, Netherlands
PB

Petra Bürgisser

Research Technician
Hematology, Erasmus MC University Medical Center, Rotterdam, The Netherlands
Rotterdam, Zuid-Holland, Netherlands
GK

Georg Christoph Korenke

MD
Klinik für Neuropädiatrie und angeborene Stoffwechselerkrankungen, Klinikum Oldenburg, Oldenburg, Germany
Oldenburg, Niedersachsen, Germany
TH

Tomas Honzik

MD
Department of Pediatrics and Inherited Metabolic Disorders, First Faculty of Medicine, Charles University and General University Hospital, Prague, Czech Republic
Prague, Hlavni mesto Praha, Czech Republic
DB

Diane Beysen

MD
Division of Paediatric Neurology, Department of Paediatrics, Antwerp University Hospital/University of Antwerp, Edegem/Wilrijk, Belgium
Antwerp, Antwerpen, Belgium
Mv

Maartje van den biggelaar

Principal investigator
Molecular Hematology, Sanquin Research, Amsterdam, The Netherlands
Amsterdam, Noord-Holland, Netherlands
Ruben Bierings, PhD

Associate Professor
Hematology, Erasmus MC University Medical Center, Rotterdam, The Netherlands
Rotterdam, Zuid-Holland, Netherlands

Background: The release of von Willebrand factor (VWF) from Weibel-Palade bodies (WPBs) in endothelial cells is crucial in hemostasis. This process is dependent on the recruitment of Rab GTPases and their effectors to the WPB membrane. Key among them are Rab27A and Rab3, the activation and WPB recruitment of which depend on the guanine nucleotide exchange factor (GEF) MAP-kinase activating death domain (MADD). Patients with mutations in this protein suffer from a pleiotropic disorder that can include bleeding abnormalities.

Aims: To determine the impact of pathogenic MADD mutations on VWF secretion using patient-derived endothelial cells.

Methods: Endothelial colony forming cells (ECFCs) were isolated from peripheral blood from 3 pediatric patients with biallelic mutations in MADD (Table 1), unaffected heterozygous family members and matching controls. Their genotype was verified through Illumina Next Gen Sequencing (NGS) sequencing, focusing on a panel of VWF-related genes. Employing mass spectrometry, an unbiased proteomic analysis of ECFC protein levels was conducted. Rab27A and Rab3D GTPase activity were assessed through pull-down assays, and their localization to WPBs was studied using immunofluorescence. ECFCs were stimulated with 100 μM histamine to measure VWF release by ELISA.

Results: All patients exhibited low VWF plasma levels. NGS revealed no mutations or variants in the VWF gene that could explain their low VWF plasma levels. Proteomic analysis revealed the absence of MADD peptides in patient ECFCs, decreased levels of VWF and the reduced abundance of several other proteins in the exocytotic machinery, including Rab3D and the Rab3/27 effector Slp4-a. The activity of Rab27A and Rab3D was decreased and their localization to WPBs was absent in patient ECFCs. Stimulation with histamine resulted in impaired VWF and VWF propeptide release from patient ECFCs compared to controls.

Conclusion(s): Patients with mutations in the GEF MADD have impaired WPB exocytosis and low VWF plasma levels.