Bürgisser, P. E. | bioRxiv Science

bioRxiv2022

Quantitative super-resolution imaging of platelet degranulation reveals differential release of VWF and VWF propeptide from alpha-granules

BackgroundPlatelet alpha-granules contain Von Willebrand factor (VWF), which is stored in eccentric alpha-granule nanodomains, and VWF propeptide (VWFpp). Differential release of VWF and VWFpp has been reported from endothelial cells. It is unclear if this also occurs during platelet alpha-granule exocytosis. We have recently developed a 3D super-resolution imaging workflow for quantification of platelet alpha-granule content based on Structured Illumination Microscopy (SIM). With this we can study alpha-granule cargo release following platelet activation in hundreds of platelets simultaneously. AimsTo study release of VWF and VWFpp from alpha-granules using quantitative super-resolution microscopy. MethodsPlatelets were activated with PAR-1 activating peptide (PAR-1 ap) or collagen-related peptide (CRP-XL). Alpha-tubulin, VWF, VWFpp, SPARC and fibrinogen were imaged using 3D-SIM, followed by semi-automated analysis in FIJI. Uptake of anti-VWF nanobody during degranulation was used to identify alpha-granules that partially released content. ResultsVWF+ and VWFpp+ structures overlapped nearly completely ([~]90%) in resting platelets, implying they are stored in similar eccentric alpha-granule nanodomains. A subset of VWF+/VWFpp+-structures was released completely at 0.6 {micro}M PAR-1 ap, but at higher concentration (20 {micro}M) significantly more VWFpp (85.3{+/-}1.6%) was released than VWF (37.6{+/-}1.4%). Release of other cargo was intermediate at 20 {micro}M (SPARC: 62.2{+/-}1.4%; fibrinogen: 51.9{+/-}2.9%), providing further evidence for differential cargo release. Similar results were obtained using CRP-XL. Anti-VWF nanobody was taken up by VWF+/VWFpp-structures and increased with stimulus strength, demonstrating these were post-exocytotic structures. ConclusionsVWF and VWFpp are differentially released from alpha-granules. This may affect how platelet-derived VWF and VWFpp contribute to formation and stabilization of hemostatic clots. Key pointsO_LIVWFpp and VWF are localized in the same, eccentric alpha-granule subdomain in resting platelets and do not overlap with other alpha-granule cargo proteins such as fibrinogen C_LIO_LIVWFpp and VWF are differentially secreted from individual alpha-granules upon activation with platelet agonists PAR-1 activating peptide and collagen-related peptide C_LI

cell biology↗

bioRxiv2021

Syntaxin 5 determines Weibel-Palade body size and Von Willebrand factor secretion by controlling Golgi architecture

Von Willebrand factor (VWF) is a multimeric hemostatic protein primarily synthesized in endothelial cells (ECs). VWF is stored in endothelial storage organelles, the Weibel-Palade bodies (WPBs), whose biogenesis strongly depends on VWF anterograde trafficking and Golgi architecture. Elongated WPB morphology is correlated to longer VWF strings with better adhesive properties. We previously identified the SNARE SEC22B, which is involved in anterograde ER-to-Golgi transport, as a novel regulator of WPB elongation. To elucidate novel determinants of WPB morphology we explored endothelial SEC22B interaction partners in a mass spectrometrybased approach, identifying the Golgi SNARE Syntaxin 5 (STX5). We established STX5 knockdown in ECs using shRNA-dependent silencing and analyzed WPB and Golgi morphology, using confocal and electron microscopy. STX5-depleted ECs exhibited extensive Golgi fragmentation and decreased WPB length, which was associated with reduced intracellular VWF levels, and impaired stimulated VWF secretion. However, the secretion-incompetent organelles in shSTX5 cells maintained WPB markers such as Angiopoietin 2, P-selectin, Rab27A, and CD63. Taken together, our study has identified SNARE protein STX5 as a novel regulator of WPB biogenesis.

cell biology↗