OC 53.5 - Loss of Rab27A Targeting to Weibel-Palade Bodies in Endothelial Colony Forming Cells of Patients with Biallelic Mutations in the Guanine Nucleotide Exchange Factor MAP-Kinase Activating Death Domain

Tuesday, July 12, 2022

11:45 AM – 12:00 PM

Location: ExCel Center, ICC Capital Suite Room 7&12

Presentation Format: In-Person

Co-Author(s)

PB

Petra E. Bürgisser

Research Technician
Department of Hematology, Erasmus University Medical Center Rotterdam
Rotterdam, Zuid-Holland, Netherlands
MK

Marije Kat

PhD student
Molecular Hematology, Sanquin Research and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, The Netherlands
Amsterdam, Noord-Holland, Netherlands
Maurice Swinkels, MSc

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

Presenting Author(s)

Sophie Hordijk, Msc

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

Co-Author(s)

GK

Georg Christoph Korenke

Prof. Dr.
Klinik für Neuropädiatrie und angeborene Stoffwechselerkrankungen, Klinikum Oldenburg
Oldenburg, Niedersachsen, Germany
TH

Tomas Hónzik

Associate professor of Paediatrics, MD, PhD
Department of Pediatrics and Adolescent Medicine, First Faculty of Medicine, Charles University and General University Hospital
Prague, Hlavni mesto Praha, Czech Republic
Ruben Bierings, PhD

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

Background: Endothelial cells secrete hemostatic proteins such as Von Willebrand factor (VWF) from specific secretory organelles called Weibel-Palade bodies (WPBs). WPBs acquire secretion competence through recruitment of Rab GTPases such as Rab27A and Rab3 and their effectors. We have recently shown that activation and WPB recruitment of Rab27A and Rab3 depend on the guanine nucleotide exchange factor MAP-kinase activating death domain (MADD), which acts as a master regulator of WPB Rab recruitment and WPB exocytosis. Biallelic mutations in MADD have recently been linked to a rare, multisystem disorder that can also include bleeding abnormalities.

Aims: To determine the impact of disease-causing mutations in MADD on the recruitment of its Rab GTPases to WPBs using patient-derived endothelial cells.

Methods: Endothelial colony forming cells (ECFCs) and platelets were isolated from peripheral blood from 2 pediatric patients with biallelic mutations in MADD (patient 1:c.914G>T, c.(1862+1_1863-1)_(3759+1_3760-1)del: p.Gly305Val, p.?; patient 2:c.979C>T, c.4398delG: p.Arg327*, p.Leu1467Cysfs*20), unaffected heterozygous family members and matching controls. Intracellular localization of Rab27A in endothelial cells was determined using immunolocalization and counterstaining with VWF and VE-cadherin. Imaging was performed using a Leica SP8 confocal microscope. This study was performed according to the declaration of Helsinki with written informed consent from all participants and family members.

Results: ECFCs of patients with biallelic MADD mutations contained WPBs with typical, elongated morphology that were indistinguishable from those in ECFCs of healthy controls and unaffected heterozygous family members. Rab27A localized to WPBs in ECFCs of healthy controls and family members, but was lost from WPBs in ECFCs from the two patients with distinct biallelic MADD mutations.

Conclusion(s): These studies further emphasize the role of MADD in the recruitment of Rab27A to WPBs in endothelial cells. Future studies are needed to determine the impact of disease-causing mutations in MADD on the hemostatic response of the endothelium.