Weibel-Palade bodies are endothelial secretory organelles that contain
von Willebrand factor,
P-selectin and CD63. Release of
von Willebrand factor from Weibel-Palade bodies is crucial for platelet adhesion during primary hemostasis. Endosomal trafficking of
proteins like CD63 to Weibel-Palade bodies during maturation is dependent on the
adaptor protein complex 3 complex. Mutations in the AP3B1 gene, which encodes the
adaptor protein complex 3 β1 subunit, result in
Hermansky-Pudlak syndrome 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 investigated the role of adaptor
protein complex 3-dependent mechanisms in trafficking of
proteins during Weibel-Palade body maturation in endothelial cells. An ex vivo patient-derived endothelial model of
Hermansky-Pudlak syndrome type 2 was established using blood outgrowth endothelial cells that were isolated from a patient with compound heterozygous mutations in AP3B1
Hermansky-Pudlak syndrome type 2 endothelial cells and CRISPR-Cas9-engineered AP3B1-/- endothelial cells contain Weibel-Palade bodies that are entirely devoid of CD63, indicative of disrupted endosomal trafficking.
Hermansky-Pudlak syndrome type 2 endothelial cells have impaired Ca2+-mediated and cAMP-mediated exocytosis. Whole
proteome analysis revealed that, apart from
adaptor protein complex 3 β1, also the μ1 subunit and the
v-SNARE VAMP8 were depleted. Stimulus-induced
von Willebrand factor secretion was impaired in CRISPR-Cas9-engineered VAMP8-/-endothelial cells. Our data show that defects in adaptor
protein complex 3-dependent maturation of Weibel-Palade bodies impairs exocytosis by affecting the recruitment of VAMP8.