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

Maurice Swinkels; Sophie Hordijk; Petra E Bürgisser; Johan A Slotman; Tom Carter; Frank W G Leebeek; A J Gerard Jansen; Jan Voorberg; Ruben Bierings

doi:10.1016/j.jtha.2023.03.041

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

J Thromb Haemost. 2023 Jul;21(7):1967-1980. doi: 10.1016/j.jtha.2023.03.041. Epub 2023 Apr 13.

Authors

Maurice Swinkels¹, Sophie Hordijk², Petra E Bürgisser³, Johan A Slotman⁴, Tom Carter⁵, Frank W G Leebeek³, A J Gerard Jansen³, Jan Voorberg⁶, Ruben Bierings⁷

Affiliations

¹ Department of Hematology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands. Electronic address: https://twitter.com/MauriceSwinkels.
² Department of Hematology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands. Electronic address: https://twitter.com/sophiehordijk.
³ Department of Hematology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands.
⁴ Optical Imaging Center, Department of Pathology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands.
⁵ Molecular and Clinical Sciences Research Institute, St George's University of London, London, United Kingdom.
⁶ Molecular Hematology, Sanquin Research and Landsteiner Laboratory, Amsterdam University Medical Center, University of Amsterdam, Amsterdam, The Netherlands; Experimental Vascular Medicine, Amsterdam University Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
⁷ Department of Hematology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands. Electronic address: r.bierings@erasmusmc.nl.

PMID: 37061132
DOI: 10.1016/j.jtha.2023.03.041

Abstract

Background: Von Willebrand factor (VWF) and VWF propeptide (VWFpp) are stored in eccentric nanodomains within platelet alpha-granules. VWF and VWFpp can undergo differential secretion following Weibel-Palade body exocytosis in endothelial cells; however, it is unclear if the same process occurs during platelet alpha-granule exocytosis. Using a high-throughput 3-dimensional super-resolution imaging workflow for quantification of individual platelet alpha-granule cargo, we studied alpha-granule cargo release in response to different physiological stimuli.

Objectives: To investigate how VWF and VWFpp are released from alpha-granules in response to physiological stimuli.

Methods: Platelets were activated with protease-activated receptor 1 (PAR-1) activating peptide (PAR-1 ap) or collagen-related peptide (CRP-XL). Alpha-tubulin, VWF, VWFpp, secreted protein acidic and cysteine rich (SPARC), and fibrinogen were imaged using 3-dimensional structured illumination microscopy, followed by semiautomated analysis in FIJI. Uptake of anti-VWF nanobody during degranulation was used to identify alpha-granules that partially released content.

Results: VWFpp overlapped with VWF in eccentric alpha-granule subdomains in resting platelets and showed a higher degree of overlap with VWF than SPARC or fibrinogen. Activation of PAR-1 (0.6-20 μM PAR-1 ap) or glycoprotein VI (GPVI) (0.25-1 μg/mL CRP-XL) signaling pathways caused a dose-dependent increase in alpha-granule exocytosis. More than 80% of alpha-granules remained positive for VWF, even at the highest agonist concentrations. In contrast, the residual fraction of alpha-granules containing VWFpp decreased in a dose-dependent manner to 23%, whereas SPARC and fibrinogen were detected in 60% to 70% of alpha-granules when stimulated with 20 μM PAR-1 ap. Similar results were obtained using CRP-XL. Using an extracellular anti-VWF nanobody, we identified VWF in postexocytotic alpha-granules.

Conclusion: We provide evidence for differential secretion of VWF and VWFpp from individual alpha-granules.

Keywords: blood platelets; exocytosis; hemostasis; secretory vesicles; von Willebrand factor.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Blood Platelets / metabolism
Endothelial Cells* / metabolism
Exocytosis
Fibrinogen / metabolism
Humans
Weibel-Palade Bodies / metabolism
von Willebrand Factor* / metabolism

Substances

von Willebrand Factor
Fibrinogen