von Willebrand factor remodeling during exocytosis from vascular endothelial cells

Abstract

Summary. Background: In vascular endothelial cells, high molecular weight multimers of von Willebrand factor (VWF) are folded into tubular structures for storage in Weibel-Palade bodies. On stimulation, VWF is secreted and forms strings to induce primary hemostasis. The structural changes composing the transition of stored tubular VWF into secreted unfurled VWF strings are still unresolved even though they are vital for normal hemostasis. The secretory pod is a novel structure that we previously described in endothelial cells. It is formed on stimulation and has been postulated to function as a VWF release site. In this study, we investigated the actual formation of secretory pods and the subsequent remodeling of VWF into strings. Methods: Human umbilical vein endothelial cells were stimulated and studied using various imaging techniques such as live-cell imaging and correlative light and electron microscopy. Results: We found by using live-cell imaging that secretory pods are formed through the coalescence of multiple Weibel-Palade bodies without involvement of other large structures. Secreted VWF expelled from secretory pods was found to adopt a globular conformation. We visualized that VWF strings derive from those globular masses of VWF. Flow experiments showed that, on secretion, the globular masses of VWF move to the edge of the cell, where they anchor and generate VWF strings. Conclusion: On secretion, VWF adopts a globular conformation that remodels into strings after translocation and anchoring at the edge of the cell. This finding reveals new pathophysiological mechanisms that could be affected in patients with von Willebrand disease.

Neutralizing capacity of inhibitors on FVIII is lower for natural FVIII/VWF complex than for isolated FVIII: in vitro comparative study in eleven different therapeutic FVIII concentrates

“…Both FVIII and ultra-large multimers of VWF (ULVWF) are packaged in the WeibelPalade bodies of select types of primary endothelial cells [5,6]. Upon secretion of the granules, strings of ULVWF anchored on the cell surface are cleaved by the metalloprotease ADAMTS13 into smaller FVIII/ VWF multimers [6,7], while pH-induced dissociation of VWF propeptide allows both FVIII to bind and FVIII/VWF multimers to adopt a globular conformation [8]. Given the different sizes of VWF multimers (up to 20 000 kDa) vs. FVIII (approximately 300 kDa) and the different stoichiometric proportion (50-100 VWF molecules per 1 FVIII molecule) [9], FVIII represents only 1-2% of the total protein composition within the natural FVIII/VWF complex.…”

Correlative Light Microscopy and Electron Tomography to Study Von Willebrand Factor Exocytosis from Vascular Endothelial Cells

“…By LM, we identified a novel structure involved in VWF exocytosis, the secretory pod. This structure is only present in stimulated endothelial cells and appears as a large VWF positive spherical-shaped structure that associates with WPBs and VWF strings (Mourik et al, 2013;Valentijn et al, 2010). Transmission electron microscopy (TEM) revealed that stimulated endothelial cells contained large membrane-enclosed organelles of similar dimensions.…”

“…The membrane-enclosed organelles contained lacy material and were often in close proximity with WPBs. To show that the structures observed by TEM contained VWF and represented, the VWF-positive secretory pods seen by LM, CLEM was performed (Mourik et al, 2013;Valentijn et al, 2010). We combined confocal laser scanning microscopy with conventional TEM and electron tomography to reveal the structures in three dimensions using serial sections.…”

“…The solid line highlights the borders of the imaged square. Scale bar panel (C and D), 100 mm.This figure was partially reproduced fromMourik et al (2013).…”

Development of a Hyperactive Primary Hemostatic System During Off-Pump Lung Transplantation Resulting From an Unbalance Between von Willebrand Factor and Its Cleaving Protease ADAMTS13

“…We recently demonstrated a marked unbalance between the platelet binding protein von Willebrand factor (VWF) and its cleaving protease: A disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), in plasma of brain dead organ donors (1). This unbalance is indicative of an increased activity of the endothelial cells after brain death (1,2). Continuous extensive VWF release by activated endothelial cells may lead to the consumption of ADAMTS13 until the levels of the molecule are insufficient to process VWF (3).…”