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Blood. 2019 Jul 1. pii: blood.2019000786. doi: 10.1182/blood.2019000786. [Epub ahead of print]

Proximity proteomics of endothelial Weibel-Palade bodies identifies novel regulator of von-Willebrand factor secretion.

Author information

1
Institute of Medical Biochemistry, University of Muenster, Germany.
2
Bioanalytical Mass Spectrometry, Max Planck Institute for Molecular Biomedicine, Germany.
3
University of Muenster, Germany.
4
Institute of Medical Biochemistry, University of Muenster, Germany gerke@uni-muenster.de.

Abstract

Weibel-Palade bodies (WPB) are unique secretory organelles of endothelial cells that store factors regulating vascular haemostasis and local inflammation. Endothelial activation triggers rapid exocytosis of WPB, leading to the surface presentation of adhesion molecules relevant for leukocyte rolling (P-selectin) and platelet capture (von-Willebrand factor, VWF). Despite its role as an important secretory organelle, a comprehensive compilation of factors associated with WPB has not been carried out. We addressed this by a proximity proteomics approach employing the peroxidase APEX2 coupled to two known WPB-associated proteins, the Rab GTPases Rab3b and Rab27a. We show that APEX2-Rab3b/27a fusion constructs are correctly targeted to WPB of primary endothelial cells and that proteins in their close proximity can be biotinylated via the WPB-recruited APEX2. Mass spectrometry analysis of the biotinylated proteins identified 183 WPB-associated proteins. While these include factors reported before to localize to WPB, the majority comprises proteins not previously associated with WPB biology. Among them, the SNARE-interacting protein Munc13-2 was shown here to specifically localize to WPB and to serve as a novel factor promoting histamine-evoked WPB exocytosis and VWF secretion. Thus, APEX2-based proximity proteomics can be used to specifically identify novel organelle-associated factors in primary endothelial cells.

PMID:
31262780
DOI:
10.1182/blood.2019000786

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