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Golgi SNARE STX5 Regulates Golgi Morphology and WPB Biogenesis

1Sanquin Research, Molecular and Cellular Hemostasis, Amsterdam, the Netherlands, 2Leiden University Medical Center, Molecular Cell Biology, Leiden, the Netherlands, 3Amsterdam UMC, University of Amsterdam, Medical Biology, Amsterdam, the Netherlands, 4Amsterdam UMC, University of Amsterdam, Experimental Vascular Medicine, Amsterdam, the Netherlands, 5Erasmus University Medical Center, Hematology, Rotterdam, the Netherlands

Abstract Number: PB1520

Meeting: ISTH 2020 Congress

Theme: Platelet Disorders and von Willebrand Disease » von Willebrand Factor Biology

Background: Von Willebrand factor (VWF), a multimeric hemostatic protein primarily synthesized in endothelial cells (ECs), facilitates the arrest of platelets from the circulation at the site of vascular damage and initiates the primary hemostatic plug. VWF is temporarily stored in endothelial storage organelles, the Weibel-Palade bodies (WPBs), whose biogenesis and morphology are strongly related to VWF anterograde trafficking and Golgi morphology. We have previously identified Sec22b, an ER-to-Golgi SNARE, as a novel regulator of WPB elongation though its involvement in Golgi integrity and VWF anterograde transport.

Aims: In this study we aimed to further elucidate the molecular determinants of WPB morphology by looking into the role of endothelial Sec22b interaction partners, focusing on cognate SNAREs.

Methods: Using a mass spectrometry-based approach we identified a vast array of proteins within the Sec22b interactome that potentially regulate WPB biogenesis and morphology, including the Golgi Qa-SNARE STX5. To investigate the role of STX5 in WPB biogenesis we depleted STX5 in endothelial cells using shRNA silencing approach and followed WPB biogenesis and ER and Golgi morphology using confocal and electron microscopy. Additionally, we explored the effect of STX5 depletion on the exocytotic behavior of WPB by quantifying the VWF release under basal and stimulated conditions.

Results: STX5 depleted ECs exhibited decreased WPB length, ER dilation and extensive Golgi fragmentation. This subsequently led to reduced intracellular VWF levels and reduced stimulus-induced VWF secretion. Finally, we examined the whole proteome changes in shSTX5 and shSec22b KD ECs and found that STX5 downregulation resulted in a drastic alteration of the proteome, that negatively affected several intracellular compartments.

Conclusions: Taken together our study has identified STX5 as a novel SNARE protein that plays a major role in WPB formation and VWF release.

To cite this abstract in AMA style:

Karampini E, Olins J, Mulder A, van Alphen F, Geerts D, Jost C, van den Biggelaar M, Voorberg J, Bierings R. Golgi SNARE STX5 Regulates Golgi Morphology and WPB Biogenesis [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/golgi-snare-stx5-regulates-golgi-morphology-and-wpb-biogenesis/. Accessed September 22, 2023.

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