OC 09.3 - Differential fate of Von Willebrand factor and Von Willebrand factor propeptide following release from platelet alpha-granules

Saturday, July 9, 2022

1:30 PM – 1:45 PM

Location: ExCel Center, ICC Capital Suite Room 1

Presentation Format: In-Person

Presenting Author(s)

Maurice Swinkels, MSc

PhD student
Hematology, Erasmus MC, University Medical Center Rotterdam
Rotterdam, Zuid-Holland, Netherlands

Co-Author(s)

TL

Titus Lemmens

PhD candidate
Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University
Maastricht, Limburg, Netherlands
PB

Petra E. Bürgisser

Research Technician
Department of Hematology, Erasmus University Medical Center Rotterdam
Rotterdam, Zuid-Holland, Netherlands
JS

Johan A. Slotman

Scientist
Optical Imaging Center, Department of Pathology, Erasmus MC, University Medical Center Rotterdam
Rotterdam, Zuid-Holland, Netherlands
AH

Adriaan B. Houtsmuller

Professor
Optical Imaging Center, Department of Pathology, Erasmus MC, University Medical Center Rotterdam
Rotterdam, Zuid-Holland, Netherlands
JC

Judith Cosemans, PhD

Professor
Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University
Maastricht, Limburg, Netherlands
Frank W.G. Leebeek, MD, PhD

Professor and Head of Department of Hematology
Erasmus MC, University Medical Center Rotterdam, The Netherlands
Rotterdam, Zuid-Holland, Netherlands
JV

Jan Voorberg

Professor, Principal investigator
Department of Molecular Hematology, Sanquin Research and Landsteiner Laboratory
Amsterdam, Noord-Holland, Netherlands
GA

Gerard A. J. Jansen

Internist hematologist
Department of Hematology, Erasmus MC, University Medical Center
Rotterdam, Zuid-Holland, Netherlands
Ruben Bierings, PhD

Principal investigator
Department of Hematology, Erasmus MC, University Medical Center Rotterdam, the Netherlands
Rotterdam, Zuid-Holland, Netherlands

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 and what happens post-release. We have recently developed a quantitative 3D super-resolution imaging workflow for 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 ImageJ. Uptake of anti-VWF nanobody during degranulation was used to identify alpha-granules that partially released content. In vitro thrombi were generated by perfusion of plasma-depleted blood, reconstituted with thrombin and fibrinogen, over collagen surfaces at 1000 s-1 shear rate.

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 0.6µM PAR-1-ap, but at higher concentration (20µM) significantly more VWFpp (85.3±1.6%) was released than VWF (37.6±1.4%). Release of other cargo was intermediate at 20µM (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 incorporated in VWF+/VWFpp--structures and increased with stimulus strength, demonstrating these were post-exocytotic structures. In thrombi formed under flow, VWFpp was primarily localized in the core while VWF was more uniformly present in the thrombus shell.

Conclusion(s): VWF and VWFpp are differentially released from alpha-granules and associate with distinct regions within the thrombus post-release. This may affect how platelet-derived VWF and VWFpp contribute to formation and stabilization of hemostatic clots.