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

Swinkels, M.; Hordijk, S.; Bürgisser, P. E.; Slotman, J. A.; Carter, T.; Leebeek, F. W. G.; Jansen, A. J. G.; Voorberg, J.; Bierings, R.

doi:10.1101/2022.10.25.513669

bioRxiv · 10.1101/2022.10.25.513669

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

Swinkels, M.·Hordijk, S.·Bürgisser, P. E.·Slotman, J. A.·Carter, T.·Leebeek, F. W. G.·Jansen, A. J. G.·Voorberg, J.·Bierings, R.

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Abstract

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

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Swinkels, M., Hordijk, S., Bürgisser, P. E., Slotman, J. A., Carter, T., Leebeek, F. W. G., Jansen, A. J. G., Voorberg, J., Bierings, R.. 2022-10-26. Quantitative super-resolution imaging of platelet degranulation reveals differential release of VWF and VWF propeptide from alpha-granules. https://doi.org/10.1101/2022.10.25.513669

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