Defective AP-3-dependent VAMP8 trafficking impairs Weibel-Palade body exocytosis in Hermansky-Pudlak Syndrome type 2 blood outgrowth endothelial cells.

ABSTRACT: Weibel-Palade bodies (WPBs) are endothelial secretory organelles that contain VWF, P-selectin and CD63. Release of VWF from WPBs is crucial for platelet adhesion during primary hemostasis. Endosomal trafficking of proteins like CD63 to WPBs during maturation is dependent on the AP-3 complex. Mutations in the AP3B1 gene, which encodes the AP-3 β1 subunit, result in Hermansky-Pudlak syndrome 2 (HPS-2), a rare genetic disorder that leads to neutropenia and a mild bleeding diathesis. This is caused by abnormal granule formation in neutrophils and platelets due to defects in trafficking of cargo to secretory organelles. The impact of these defects on the secretory pathway of the endothelium is largely unknown. In this study we have investigated the role of AP-3-dependent mechanisms in trafficking of proteins during WPB maturation in endothelial cells. An ex vivo patient-derived endothelial model of HPS-2 was established using blood outgrowth endothelial cells (BOECs) that were isolated from an HPS-2 patient with compound heterozygous mutations in AP3B1. HPS-2 BOECs and CRISPR/Cas9-engineered AP3B1-/- BOECs contain WPBs that are entirely devoid of CD63, indicative of disrupted endosomal trafficking to the WPB membrane. HPS-2 BOECs have impaired Ca2+- and cAMP-mediated WPB exocytosis. Whole proteome analysis of HPS-2 BOECs revealed that apart from AP-3β1 also the AP-3µ1 subunit and the v-SNARE VAMP8 were depleted. Stimulus-induced VWF secretion was impaired in CRISPR/Cas9-engineered VAMP8-/- BOECs. Our data show that defects in AP-3 dependent maturation of WPBs impairs WPB exocytosis by affecting the recruitment of the WPB-localized member of the SNARE fusion machinery VAMP8.

INSTRUMENT(S): Orbitrap Fusion ETD

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Cell, Endothelial Cell

DISEASE(S): Hermansky-pudlak Syndrome 2

SUBMITTER: Maartje van den Biggelaar  

LAB HEAD: M. van den Biggelaar

PROVIDER: PXD011294 | Pride | 2019-01-31

REPOSITORIES: Pride