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

Abstract

Background:Platelet 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.Aims:To study release of VWF and VWFpp from alpha-granules using quantitative super-resolution microscopy.Methods:Platelets 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.Results:VWF+ 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 μM PAR-1-ap, but at higher concentration (20 μM) significantly more VWFpp (85.3±1.6%) was released than VWF (37.6±1.4%). Release of other cargo was intermediate at 20 μM (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.Conclusions:VWF 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.