Weibel-Palade Body Localized Syntaxin-3 Modulates Von Willebrand Factor Secretion From Endothelial Cells

Schillemans, Maaike; Karampini, Ellie; Eshof, Bart L. van den; Gangaev, Anastasia; Hofman, Menno; Breevoort, Dorothee van; Meems, Henriet; Janssen, Hans; Mulder, Aat A.; Jost, Carolina R.; Escher, Johanna C.; Adam, Rüdiger; Carter, Tom; Koster, Abraham J.; Biggelaar, Maartje van den; Voorberg, Jan; Bierings, Ruben

doi:10.1161/atvbaha.117.310701

Cited by 37 publications

(39 citation statements)

References 66 publications

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“…A different localization pattern was observed for syntaxin‐3 and CD63, 2 proteins that are normally found both on WPBs and on endosomes . It has previously been shown that CD63 is trafficked to the WPBs via the endosomal system in an annexin A8‐ and AP‐3–dependent manner .…”

Section: Resultsmentioning

confidence: 85%

“…EGM-18 was replaced every 2 to 3 days, and after 7 days colonies started to form in some wells. Conditioned media of wells that were >50% confluent were assayed using VWF ELISA 40 to identify candidates no longer secreting VWF. VWF-deficient wells were passaged and expanded for further analysis including immunoblotting of cell lysates and Sanger sequencing and NGS of the genomic DNA isolated with the DNeasy Blood & Tissue kit (Qiagen, Venlo, The Netherlands).…”

Section: Generation Of Crispr-edited Vwf −/− Cbboecsmentioning

confidence: 99%

“…A different localization pattern was observed for syntaxin-3 and CD63, 2 proteins that are normally found both on WPBs and on endosomes. 40,49 It has previously been shown that CD63 is trafficked to the WPBs via the endosomal system in an annexin A8-and AP-3dependent manner. 21,[50][51][52] As expected, whereas CD63 localization to the elongated WPBs is lost in VWF −/− clones, its localization to spherical vesicles that are most likely endosomes remains similar ( Figure 7).…”

Section: Alternative Routing Of Weibel-palade Body Cargo Proteinsmentioning

confidence: 99%

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Alternative trafficking of Weibel‐Palade body proteins in CRISPR/Cas9‐engineered von Willebrand factor–deficient blood outgrowth endothelial cells

Schillemans

Kat

Westeneng

et al. 2019

Research and Practice in Thrombosis and Haemostasis

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BackgroundSynthesis of the hemostatic protein von Willebrand factor (VWF) drives formation of endothelial storage organelles called Weibel‐Palade bodies (WPBs). In the absence of VWF, angiogenic and inflammatory mediators that are costored in WPBs are subject to alternative trafficking routes. In patients with von Willebrand disease (VWD), partial or complete absence of VWF/WPBs may lead to additional bleeding complications, such as angiodysplasia. Studies addressing the role of VWF using VWD patient–derived blood outgrowth endothelial cells (BOECs) have reported conflicting results due to the intrinsic heterogeneity of patient‐derived BOECs.ObjectiveTo generate a VWF‐deficient endothelial cell model using clustered regularly interspaced short palindromic repeats (CRISPR) genome engineering of blood outgrowth endothelial cells.MethodsWe used CRISPR/CRISPR‐associated protein 9 editing in single‐donor cord blood–derived BOECs (cbBOECs) to generate clonal VWF −/− cbBOECs. Clones were selected using high‐throughput screening, VWF mutations were validated by sequencing, and cells were phenotypically characterized.ResultsTwo VWF −/− BOEC clones were obtained and were entirely devoid of WPBs, while their overall cell morphology was unaltered. Several WPB proteins, including CD63, syntaxin‐3 and the cargo proteins angiopoietin (Ang)‐2, interleukin (IL)‐6, and IL‐8 showed alternative trafficking and secretion in the absence of VWF. Interestingly, Ang‐2 was relocated to the cell periphery and colocalized with Tie‐2.ConclusionsCRISPR editing of VWF provides a robust method to create VWF‐ deficient BOECs that can be directly compared to their wild‐type counterparts. Results obtained with our model system confirmed alternative trafficking of several WPB proteins in the absence of VWF and support the theory that increased Ang‐2/Tie‐2 interaction contributes to angiogenic abnormalities in VWD patients.

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Section: Resultsmentioning

confidence: 85%

Section: Generation Of Crispr-edited Vwf −/− Cbboecsmentioning

confidence: 99%

Section: Alternative Routing Of Weibel-palade Body Cargo Proteinsmentioning

confidence: 99%

See 1 more Smart Citation

Alternative trafficking of Weibel‐Palade body proteins in CRISPR/Cas9‐engineered von Willebrand factor–deficient blood outgrowth endothelial cells

Schillemans

Kat

Westeneng

et al. 2019

Research and Practice in Thrombosis and Haemostasis

Self Cite

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“…WPBs contain two v‐SNAREs, VAMP3 and VAMP8, of which only VAMP3 has so far been shown to support stimulus‐induced WPB release . t‐SNAREs that take part in WPB release include the SNAP25 homolog SNAP23, syntaxin‐3, syntaxin‐4, and most likely also syntaxin‐2 . Owing to their promiscuous nature, various combinations of these SNAREs exist: in endothelial cells, syntaxin‐4 engages with SNAP23 and VAMP3 or VAMP8 , and syntaxin‐3 primarily interacts with VAMP8 and SNAP23 .…”

Section: Exocytotic Machinery Of Wpbsmentioning

confidence: 99%

“…This suggests that endothelial cells can employ at least three (syntaxin‐4–SNAP23–VAMP3, syntaxin‐4–SNAP23–VAMP8, and syntaxin‐3–SNAP23–VAMP8) and possibly more distinct SNARE complexes for release of WPBs. Interestingly, whereas syntaxin‐4 is localized on the plasma membrane, syntaxin‐3 is found on WPBs , suggesting that, in certain cases, WPBs can also function as the acceptor compartment during fusion.…”

Section: Exocytotic Machinery Of Wpbsmentioning

confidence: 99%

Exocytosis of Weibel–Palade bodies: how to unpack a vascular emergency kit

Schillemans

Karampini

Kat

et al. 2019

Journal of Thrombosis and Haemostasis

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Summary The blood vessel wall has a number of self‐healing properties, enabling it to minimize blood loss and prevent or overcome infections in the event of vascular trauma. Endothelial cells prepackage a cocktail of hemostatic, inflammatory and angiogenic mediators in their unique secretory organelles, the Weibel–Palade bodies (WPBs), which can be immediately released on demand. Secretion of their contents into the vascular lumen through a process called exocytosis enables the endothelium to actively participate in the arrest of bleeding and to slow down and direct leukocytes to areas of inflammation. Owing to their remarkable elongated morphology and their secretory contents, which span the entire size spectrum of small chemokines all the way up to ultralarge von Willebrand factor multimers, WPBs constitute an ideal model system for studying the molecular mechanisms of secretory organelle biogenesis, exocytosis, and content expulsion. Recent studies have now shown that, during exocytosis, WPBs can undergo several distinct modes of fusion, and can utilize fundamentally different mechanisms to expel their contents. In this article, we discuss recent advances in our understanding of the composition of the WPB exocytotic machinery and how, because of its configuration, it is able to support WPB release in its various forms.

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Riociguat inhibits ultra‐large VWF string formation on pulmonary artery endothelial cells from chronic thromboembolic pulmonary hypertension patients

et al. 2022

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Chronic thromboembolic pulmonary hypertension (CTEPH) is characterized by elevated pulmonary arterial pressure and organized thrombi within pulmonary arteries. Riociguat is a soluble guanylate cyclase stimulator and is approved for patients with inoperable CTEPH or residual pulmonary hypertension after pulmonary endarterectomy (PEA). Previous work suggested that riociguat treatment is associated with an increased risk of bleeding, although the mechanism is unclear. The aim of this study is to assess how

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Weibel-Palade Body Localized Syntaxin-3 Modulates Von Willebrand Factor Secretion From Endothelial Cells

Abstract: Supplemental Digital Content is available in the text.

Cited by 37 publications

References 66 publications

Alternative trafficking of Weibel‐Palade body proteins in CRISPR/Cas9‐engineered von Willebrand factor–deficient blood outgrowth endothelial cells

Alternative trafficking of Weibel‐Palade body proteins in CRISPR/Cas9‐engineered von Willebrand factor–deficient blood outgrowth endothelial cells

Exocytosis of Weibel–Palade bodies: how to unpack a vascular emergency kit

Riociguat inhibits ultra‐large VWF string formation on pulmonary artery endothelial cells from chronic thromboembolic pulmonary hypertension patients

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