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:https://doi.org/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

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

Research output: Contribution to journal › Article › Academic › peer-review

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.

Original language	English
Pages (from-to)	1967-1980
Number of pages	14
Journal	Journal of thrombosis and haemostasis : JTH
Volume	21
Issue number	7
Early online date	13 Apr 2023
DOIs	https://doi.org/10.1016/j.jtha.2023.03.041
Publication status	Published - Jul 2023

Keywords

blood platelets
exocytosis
hemostasis
secretory vesicles
von Willebrand factor

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https://doi.org/10.1016/j.jtha.2023.03.041

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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. (2023). Quantitative super-resolution imaging of platelet degranulation reveals differential release of von Willebrand factor and von Willebrand factor propeptide from alpha-granules. Journal of thrombosis and haemostasis : JTH, 21(7), 1967-1980. https://doi.org/10.1016/j.jtha.2023.03.041

@article{0ad0368afb1c4eb691ca1708daf08ff8,

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

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",

author = "Maurice Swinkels and Sophie Hordijk and B{\"u}rgisser, {Petra E} and Slotman, {Johan A} and Tom Carter and Leebeek, {Frank W G} and Jansen, {A J Gerard} and Jan Voorberg and Ruben Bierings",

note = "Funding Information: We thank Dr Coen Maas (University Medical Center Utrecht, The Netherlands) for the generous supply of Alpaca anti-VWF C-terminal cystine knot nanobodies. We thank Titus Lemmens (Maastricht University Medical Center+, The Netherlands) for stimulating discussion and experimental assistance. M.S. S.H. P.E.B. J.A.S. and R.B. performed experiments and analyzed the data. T.C. and F.W.G.L. provided essential reagents and expertise. M.S. S.H. A.J.G.J. J.V. and R.B. designed the research and wrote the paper. All authors critically revised and approved the final version of the manuscript. F.W.G.L. received research support from CSL Behring, Takeda, uniQure, and Sobi and is a consultant for uniQure, Biomarin, CSL Behring, and Takeda, of which the fees go to the institute. He was a DSMB member for a study sponsored by Roche. A.J.G.J. received speaker fees and travel cost payments from 3SBio, Amgen, and Novartis, is on the international advisory board at Novartis, and received research support from CSL Behring, Principia, and Argenx. None of the other authors have conflicts of interest to declare. Funding information This study was supported by grants from the Landsteiner Stichting voor Bloedtransfusie Research (LSBR-1707 and LSBR-2005) and an European Hematology Association (EHA) Clinical Research Fellowship (to A.J.G.J.). Funding Information: Funding information This study was supported by grants from the Landsteiner Stichting voor Bloedtransfusie Research (LSBR-1707 and LSBR-2005) and an European Hematology Association (EHA) Clinical Research Fellowship (to A.J.G.J.). Publisher Copyright: {\textcopyright} 2023 The Author(s)",

year = "2023",

month = jul,

doi = "https://doi.org/10.1016/j.jtha.2023.03.041",

language = "English",

volume = "21",

pages = "1967--1980",

journal = "Journal of thrombosis and haemostasis : JTH",

issn = "1538-7933",

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}

Swinkels, M, Hordijk, S, Bürgisser, PE, Slotman, JA, Carter, T, Leebeek, FWG, Jansen, AJG, Voorberg, J & Bierings, R 2023, 'Quantitative super-resolution imaging of platelet degranulation reveals differential release of von Willebrand factor and von Willebrand factor propeptide from alpha-granules', Journal of thrombosis and haemostasis : JTH, vol. 21, no. 7, pp. 1967-1980. https://doi.org/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. / Swinkels, Maurice; Hordijk, Sophie; Bürgisser, Petra E et al.
In: Journal of thrombosis and haemostasis : JTH, Vol. 21, No. 7, 07.2023, p. 1967-1980.

Research output: Contribution to journal › Article › Academic › peer-review

TY - JOUR

T1 - Quantitative super-resolution imaging of platelet degranulation reveals differential release of von Willebrand factor and von Willebrand factor propeptide from alpha-granules

AU - Swinkels, Maurice

AU - Hordijk, Sophie

AU - Bürgisser, Petra E

AU - Slotman, Johan A

AU - Carter, Tom

AU - Leebeek, Frank W G

AU - Jansen, A J Gerard

AU - Voorberg, Jan

AU - Bierings, Ruben

N1 - Funding Information: We thank Dr Coen Maas (University Medical Center Utrecht, The Netherlands) for the generous supply of Alpaca anti-VWF C-terminal cystine knot nanobodies. We thank Titus Lemmens (Maastricht University Medical Center+, The Netherlands) for stimulating discussion and experimental assistance. M.S. S.H. P.E.B. J.A.S. and R.B. performed experiments and analyzed the data. T.C. and F.W.G.L. provided essential reagents and expertise. M.S. S.H. A.J.G.J. J.V. and R.B. designed the research and wrote the paper. All authors critically revised and approved the final version of the manuscript. F.W.G.L. received research support from CSL Behring, Takeda, uniQure, and Sobi and is a consultant for uniQure, Biomarin, CSL Behring, and Takeda, of which the fees go to the institute. He was a DSMB member for a study sponsored by Roche. A.J.G.J. received speaker fees and travel cost payments from 3SBio, Amgen, and Novartis, is on the international advisory board at Novartis, and received research support from CSL Behring, Principia, and Argenx. None of the other authors have conflicts of interest to declare. Funding information This study was supported by grants from the Landsteiner Stichting voor Bloedtransfusie Research (LSBR-1707 and LSBR-2005) and an European Hematology Association (EHA) Clinical Research Fellowship (to A.J.G.J.). Funding Information: Funding information This study was supported by grants from the Landsteiner Stichting voor Bloedtransfusie Research (LSBR-1707 and LSBR-2005) and an European Hematology Association (EHA) Clinical Research Fellowship (to A.J.G.J.). Publisher Copyright: © 2023 The Author(s)

PY - 2023/7

Y1 - 2023/7

N2 - 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.

AB - 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.

KW - blood platelets

KW - exocytosis

KW - hemostasis

KW - secretory vesicles

KW - von Willebrand factor

UR - http://www.scopus.com/inward/record.url?scp=85159195815&partnerID=8YFLogxK

U2 - https://doi.org/10.1016/j.jtha.2023.03.041

DO - https://doi.org/10.1016/j.jtha.2023.03.041

M3 - Article

C2 - 37061132

SN - 1538-7933

VL - 21

SP - 1967

EP - 1980

JO - Journal of thrombosis and haemostasis : JTH

JF - Journal of thrombosis and haemostasis : JTH

IS - 7

ER -

Swinkels M, Hordijk S, Bürgisser PE, Slotman JA, Carter T, Leebeek FWG et al. Quantitative super-resolution imaging of platelet degranulation reveals differential release of von Willebrand factor and von Willebrand factor propeptide from alpha-granules. Journal of thrombosis and haemostasis : JTH. 2023 Jul;21(7):1967-1980. Epub 2023 Apr 13. doi: https://doi.org/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

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