scholarly journals Arf GTPase-Activating proteins SMAP1 and AGFG2 regulate the size of Weibel-Palade bodies and exocytosis of von Willebrand factor

2021 ◽  
Author(s):  
Asano Watanabe ◽  
Hikari Hataida ◽  
Naoya Inoue ◽  
Kosuke Kamon ◽  
Keigo Baba ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Intracellular Transport ◽  
Secretory Granules ◽  
Secretory Proteins ◽  
Adp Ribosylation ◽  
Gtpase Activating Proteins ◽  
Summary Statement ◽  
Golgi Network ◽  
Von Willebrand ◽  
Willebrand Factor

AbstractArf GTPase-Activating proteins (ArfGAPs) mediate the hydrolysis of GTP bound to ADP-ribosylation factors, which are important for intracellular transport. ArfGAPs have been shown to be critical for cargo sorting in the Golgi-to-ER and post-Golgi traffic. However, their roles in the sorting of secretory proteins remains unclear. Weibel-Palade bodies (WPBs) are cigar-shaped secretory granules in endothelial cells that contain von Willebrand factor (vWF) as their main cargo. WPBs are formed at the trans-Golgi Network, and this process is thought to be coupled with the sorting of vWF. WPB biogenesis was reported to be regulated by ADP-ribosylation factors and their regulators, but the role of ArfGAPs has been unknown. In this study, we performed siRNA screening of ArfGAPs to investigate the biogenesis of WPBs. We found two ArfGAPs, SMAP1 and AGFG2, to be involved in WPB size and vWF exocytosis, respectively. SMAP1 depletion resulted in small-sized WPBs, and the lysosomal inhibitor leupeptin recovered the size of WPBs. These results indicate that SMAP1 functions in preventing the degradation of cigar-shaped WPBs. However, AGFG2 downregulation resulted in the inhibition of vWF secretion upon Phorbol 12-myristate 13-acetate (PMA)-stimulation, suggesting that AGFG2 plays a role in vWF exocytosis. Our study revealed unexpected processes regulated by ArfGAPs for vWF transport.Summary StatementThe ArfGAP proteins SMAP1 and AGFG2 were identified as regulating WPB size and vWF exocytosis.

scholarly journals Arf GTPase-Activating proteins SMAP1 and AGFG2 regulate the size of Weibel-Palade bodies and exocytosis of von Willebrand factor

Biology Open ◽  
2021 ◽  
Author(s):  
Asano Watanabe ◽  
Hikari Hataida ◽  
Naoya Inoue ◽  
Kosuke Kamon ◽  
Keigo Baba ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Secretory Granules ◽  
The Other ◽  
Negative Regulators ◽  
Histamine Stimulation ◽  
Gtpase Activating Proteins ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Hydrolysis Of

Arf GTPase-Activating proteins (ArfGAPs) mediate the hydrolysis of GTP bound to ADP-ribosylation factors (Arfs), that are critical to form transport intermediates. ArfGAPs have been thought to be negative regulators of Arfs, however, accumulating evidence indicates that ArfGAPs are important for cargo sorting and promote membrane traffic. Weibel-Palade bodies (WPBs) are cigar-shaped secretory granules in endothelial cells that contain von Willebrand factor (vWF) as their main cargo. WPB biogenesis at the Golgi was reported to be regulated by Arf and their regulators, but the role of ArfGAPs has been unknown. In this study, we performed siRNA screening of ArfGAPs to investigate the role of ArfGAPs in the biogenesis of WPBs. We found two ArfGAPs, SMAP1 and AGFG2, to be involved in WPB size and vWF exocytosis, respectively. SMAP1 depletion resulted in small-sized WPBs, and the lysosomal inhibitor leupeptin recovered the size of WPBs. The results indicate that SMAP1 functions in preventing the degradation of cigar-shaped WPBs. On the other hand, AGFG2 downregulation resulted in the inhibition of vWF secretion upon Phorbol 12-myristate 13-acetate (PMA) or histamine stimulation, suggesting that AGFG2 plays a role in vWF exocytosis. Our study revealed unexpected roles of ArfGAPs in vWF transport.

scholarly journals Actomyosin II contractility expels von Willebrand factor from Weibel–Palade bodies during exocytosis

2011 ◽  
Vol 194 (4) ◽  
pp. 613-629 ◽  
Author(s):  
Thomas D. Nightingale ◽  
Ian J. White ◽  
Emily L. Doyle ◽  
Mark Turmaine ◽  
Kimberly J. Harrison-Lavoie ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Secretory Granules ◽  
Myosin Ii ◽  
Human Endothelial Cells ◽  
Spatiotemporal Resolution ◽  
Regulated Secretion ◽  
Experimental Systems ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Extracellular Environment

The study of actin in regulated exocytosis has a long history with many different results in numerous systems. A major limitation on identifying precise mechanisms has been the paucity of experimental systems in which actin function has been directly assessed alongside granule content release at distinct steps of exocytosis of a single secretory organelle with sufficient spatiotemporal resolution. Using dual-color confocal microscopy and correlative electron microscopy in human endothelial cells, we visually distinguished two sequential steps of secretagogue-stimulated exocytosis: fusion of individual secretory granules (Weibel–Palade bodies [WPBs]) and subsequent expulsion of von Willebrand factor (VWF) content. Based on our observations, we conclude that for fusion, WPBs are released from cellular sites of actin anchorage. However, once fused, a dynamic ring of actin filaments and myosin II forms around the granule, and actomyosin II contractility squeezes VWF content out into the extracellular environment. This study therefore demonstrates how discrete actin cytoskeleton functions within a single cellular system explain actin filament–based prevention and promotion of specific exocytic steps during regulated secretion.

scholarly journals Weibel-Palade body size modulates the adhesive activity of its von Willebrand Factor cargo in cultured endothelial cells

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Francesco Ferraro ◽  
Mafalda Lopes da Silva ◽  
William Grimes ◽  
Hwee Kuan Lee ◽  
Robin Ketteler ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Secretory Granules ◽  
Vascular Wall ◽  
Endothelial Cell Surface ◽  
Endothelial Surface ◽  
Wide Range ◽  
Storage Organelle ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Changes in the size of cellular organelles are often linked to modifications in their function. Endothelial cells store von Willebrand Factor (vWF), a glycoprotein essential to haemostasis in Weibel-Palade bodies (WPBs), cigar-shaped secretory granules that are generated in a wide range of sizes. We recently showed that forcing changes in the size of WPBs modifies the activity of this cargo. We now find that endothelial cells treated with statins produce shorter WPBs and that the vWF they release at exocytosis displays a reduced capability to recruit platelets to the endothelial cell surface. Investigating other functional consequences of size changes of WPBs, we also report that the endothelial surface-associated vWF formed at exocytosis recruits soluble plasma vWF and that this process is reduced by treatments that shorten WPBs, statins included. These results indicate that the post-exocytic adhesive activity of vWF towards platelets and plasma vWF at the endothelial surface reflects the size of their storage organelle. Our findings therefore show that changes in WPB size, by influencing the adhesive activity of its vWF cargo, may represent a novel mode of regulation of platelet aggregation at the vascular wall.

scholarly journals von Willebrand factor proteolytic processing and multimerization precede the formation of Weibel-Palade bodies

Blood ◽  
1994 ◽  
Vol 83 (12) ◽  
pp. 3536-3544 ◽  
Author(s):  
UM Vischer ◽  
DD Wagner
Keyword(s):  
Von Willebrand Factor ◽  
Healing Process ◽  
Proteolytic Processing ◽  
Wound Healing Process ◽  
Endothelial Surface ◽  
High Calcium ◽  
High Calcium Concentration ◽  
Golgi Network ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract We investigated the intracellular site of pro-von Willebrand factor (pro-vWF) cleavage and multimerization, as well as the fate of the propolypeptide (von Willebrand antigen II) after cleavage. Analysis of subcellular fractions of endothelial cells metabolically labeled with sulfate showed that both cleavage and covalent multimerization occur after sulfation and precede the formation of Weibel-Palade bodies. Because sulfation is a processing step localized to the trans-Golgi network (TGN), our results indicate that multimerization and prosequence cleavage also occur in this organelle. After cleavage, the propolypeptide remains noncovalently associated with the mature vWF subunit. This association is promoted by a high calcium concentration and an acidic pH (conditions thought to prevail in the TGN) and explains the 1:1 stoichiometry of the propolypeptide and mature vWF found in Weibel-Palade bodies. The propolypeptide remains an integral part of the large multimeric vWF aggregates in the Weibel-Palade body until secretion. When secretion occurs under slightly acidic conditions, such as may be found in poorly perfused wounds, the propolypeptide remains associated with the endothelial surface-bound vWF, and may thus participate in the wound healing process.

scholarly journals Selective and Signal-dependent Recruitment of Membrane Proteins to Secretory Granules Formed by Heterologously Expressed von Willebrand Factor

2002 ◽  
Vol 13 (5) ◽  
pp. 1582-1593 ◽  
Author(s):  
Anastasia D. Blagoveshchenskaya ◽  
Matthew J. Hannah ◽  
Simon Allen ◽  
Daniel F. Cutler
Keyword(s):  
Endothelial Cells ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Von Willebrand Factor ◽  
De Novo ◽  
Secretory Granules ◽  
Synaptotagmin I ◽  
Organelle Membrane ◽  
Von Willebrand ◽  
Willebrand Factor

von Willebrand factor (vWF) is a large, multimeric protein secreted by endothelial cells and involved in hemostasis. When expressed in AtT-20 cells, vWF leads to the de novo formation of cigar-shaped organelles similar in appearance to the Weibel-Palade bodies of endothelial cells in which vWF is normally stored before regulated secretion. The membranes of this vWF-induced organelle, termed the pseudogranule, are uncharacterized. We have examined the ability of these pseudogranules, which we show are secretagogue responsive, to recruit membrane proteins. Coexpression experiments show that the Weibel-Palade body proteins P-selectin and CD63, as well as the secretory organelle membrane proteins vesicle-associated membrane protein-2 and synaptotagmin I are diverted away from the endogenous adrenocorticotropic hormone-containing secretory granules to the vWF-containing pseudogranules. However, transferrin receptor, lysosomal-associated membrane protein 1, and sialyl transferase are not recruited. The recruitment of P-selectin is dependent on a tyrosine-based motif within its cytoplasmic domain. Our data show that vWF pseudogranules specifically recruit a subset of membrane proteins, and that in a process explicitly driven by the pseudogranule content (i.e., vWF), the active recruitment of at least one component of the pseudogranule membrane (i.e., P-selectin) is dependent on residues of P-selectin that are cytosolic and therefore unable to directly interact with vWF.

Reversible Self-Assembly of Weibel-Palade Body-Like Tubules from Recombinant N-Terminal Domains of von Willebrand Factor.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 291-291 ◽  
Author(s):  
Ren-Huai Huang ◽  
Ying Wang ◽  
Robyn Roth ◽  
Xiong Yu ◽  
Angie R. Purvis ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Building Blocks ◽  
Reconstruction Method ◽  
Thin Sections ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Weibel-Palade bodies (WPBs) are elongated secretory granules of endothelial cells that are packed with tubules composed of von Willebrand factor (VWF), a multimeric protein required for hemostasis. Disruption of tubular packing prevents the orderly secretion of VWF multimers and blocks the subsequent binding of platelets. The cigar-like shape and tubular cross section of WPBs are conserved in all vertebrates, but little is known about how VWF specifies this packing arrangement. Starting from recombinant 82 kDa VWF propeptide (domains D1D2) and 114 kDa disulfide-bonded D’D3 dimer, we now have assembled tubules reversibly in vitro with the same dimensions as VWF tubules in WPBs. Assembly was induced at pH 6.2, reversed at pH 7.4, and required Ca2+. Recombinant D’D3 dimers did not self-associate at pH 7.4 or pH 6.2, with or without Ca2+. Without Ca2+, VWF propeptide did not bind to D’D3 dimers. At pH 7.4, with Ca2+, VWF propeptide formed noncovalent 160 kDa dimers and, when mixed with D’D3 dimers, assembled a 280 kDa complex of two propeptides and one D’D3 dimer as shown by gel filtration chromatography and multi-angle light scattering. Lowering the pH to 6.2 caused the formation of >3 MDa aggregates with the same stoichiometry, which dissociated upon adding EDTA or raising the pH to 7.4. Quick-freeze deep-etch EM showed that the large aggregates are hollow right-handed tubular helices. The iterative helical real space reconstruction method was used to make 3D reconstructions of the tubules at 22 Å resolution from negative stain EM images (Figure, left). Tubules consist of a right-handed helix with axial rise of 26.2 Å and twist of 85.6 degrees per subunit, or 4.2 subunits per 11 nm turn. The dimensions (outside diameter 25 nm, inside diameter 12 nm) are similar to those of tubules in WPBs in thin sections of endothelial cells by transmission EM (Figure, right and its insert). Each subunit contains one D’D3 dimer flanked by two D1D2 propeptides (Figure, center). Each D’D3 dimer makes a total of six contacts with D1D2 domains. Each D1D2 propeptide makes three contacts with D’D3 and just one end-to-end homotypic contact. The spatial arrangement of these building blocks and inter-domain contacts in tubules suggest a model by which decreasing pH along the secretory pathway coordinates the formation of intersubunit disulfide bonds with the tubular packaging of VWF multimers. Within the WPB, Ca2+-dependent and pH-dependent binding of D1D2 to D’D3 domains stabilizes the packing of VWF multimers into tubules, which behave as constrained springs. Upon secretion, the increased pH weakens these constraints and permits the helical tubules to unfurl into flowing blood without tangling. Figure Figure

scholarly journals Modulation of endothelial organelle size as an antithrombotic strategy

2020 ◽  
Author(s):  
Francesco Ferraro ◽  
Joana R. Costa ◽  
Robin Ketteler ◽  
Janos Kriston-Vizi ◽  
Daniel F. Cutler
Keyword(s):  
Von Willebrand Factor ◽  
Pharmacological Treatment ◽  
Antithrombotic Therapy ◽  
Secretory Granules ◽  
Dramatic Reduction ◽  
Endothelial Surface ◽  
Wide Range ◽  
Novel Target ◽  
Von Willebrand ◽  
Willebrand Factor

AbstractIt is long-established that Von Willebrand Factor (VWF) is central to haemostasis and thrombosis. Endothelial VWF is stored in cell-specific secretory granules, Weibel-Palade bodies (WPBs), uniquely rod-like exocytic organelles generated in a wide range of lengths (0.5 to 5.0 µm). It has been shown that WPB size responds to physiological cues and pharmacological treatment and that, under flow, VWF secretion from shortened WPBs produces a dramatic reduction of platelet and plasma VWF adhesion to an endothelial surface. WPB-shortening therefore represents a novel target for antithrombotic therapy acting via modulation of VWF adhesive activity. To this aim, we screened a library of licenced drugs and identified several that prompt WPB size reduction. These compounds therefore constitute a novel set of potentially antithrombotic compounds.SummaryThe size of the endothelial secretory granules that store Von Willebrand Factor correlates with its activity, central to haemostasis and thrombosis. Here, human-licenced drugs that reduce the size of these secretory granules are identified, providing a set of novel potential anti-thrombotic compounds.

von Willebrand Factor (vWf) as a Plasma Marker of Endothelial Activation in Diabetes: Improved Reliability with Parallel Determination of the vWf Propeptide (vWf:AgII)

1998 ◽  
Vol 80 (12) ◽  
pp. 1002-1007 ◽  
Author(s):  
Jef Emeis ◽  
Henk Bilo ◽  
Coen Stehouwer ◽  
Claus Thomsen ◽  
Ole Rasmussen ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Secretory Granules ◽  
Control Diet ◽  
Close Correlation ◽  
Endothelial Activation ◽  
Diabetic Patients ◽  
Monounsaturated Fat ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryElevated plasma von Willebrand factor (vWf) levels are found in diabetes and other vasculopathies, and predict cardiovascular mortality. vWf is stored and released from endothelial cell secretory granules, along with equimolar amounts of its propeptide (vWf:AgII). In the present study, we examined plasma propeptide levels as a marker of endothelial secretion in vivo, using an ELISA based on monoclonal antibodies. vWf but not propeptide levels are influenced by blood groups, explaining in part the smaller variation in plasma propeptide levels among normal individuals. In both controls and insulin-dependent diabetic patients, we found a close correlation between propeptide and immunoreactive vWf levels (r2 = 0.54, p <0.0001). vWf and propeptide were elevated in patient subgroups with microalbuminuria or overt diabetic nephropathy, whereas only the propeptide was significantly elevated in the normoalbuminuric subgroup. This observation suggests that in conjunction with vWf, propeptide measurements may improve the identification of endothelial activation, which occurs frequently even without increased urinary albumin excretion. In 12 NIDDM patients, a 3-week diet enriched in monounsaturated fat (MUFA) resulted in parallel decreases in vWf (-22%, p <0.05) and propeptide (-17%, p <0.05) levels, indicating that the experimental diet affected endothelial secretion rather than vWf catabolism. A carbohydrate-enriched control diet did not significantly influence either marker.Our results suggest that concomittant determinations of plasma vWf and propeptide are useful tools to assess endothelial activation in vivo, and reinforce our previous conclusion that a diet rich in MUFA can improve endothelial function in NIDDM.

scholarly journals von Willebrand factor proteolytic processing and multimerization precede the formation of Weibel-Palade bodies

Blood ◽  
1994 ◽  
Vol 83 (12) ◽  
pp. 3536-3544 ◽  
Author(s):  
UM Vischer ◽  
DD Wagner
Keyword(s):  
Von Willebrand Factor ◽  
Healing Process ◽  
Proteolytic Processing ◽  
Wound Healing Process ◽  
Endothelial Surface ◽  
High Calcium ◽  
High Calcium Concentration ◽  
Golgi Network ◽  
Von Willebrand ◽  
Willebrand Factor

We investigated the intracellular site of pro-von Willebrand factor (pro-vWF) cleavage and multimerization, as well as the fate of the propolypeptide (von Willebrand antigen II) after cleavage. Analysis of subcellular fractions of endothelial cells metabolically labeled with sulfate showed that both cleavage and covalent multimerization occur after sulfation and precede the formation of Weibel-Palade bodies. Because sulfation is a processing step localized to the trans-Golgi network (TGN), our results indicate that multimerization and prosequence cleavage also occur in this organelle. After cleavage, the propolypeptide remains noncovalently associated with the mature vWF subunit. This association is promoted by a high calcium concentration and an acidic pH (conditions thought to prevail in the TGN) and explains the 1:1 stoichiometry of the propolypeptide and mature vWF found in Weibel-Palade bodies. The propolypeptide remains an integral part of the large multimeric vWF aggregates in the Weibel-Palade body until secretion. When secretion occurs under slightly acidic conditions, such as may be found in poorly perfused wounds, the propolypeptide remains associated with the endothelial surface-bound vWF, and may thus participate in the wound healing process.

Subcellular Localization of Von Willebrand Factor Mutants Correlates with Particular VWF Multimer Patterns

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 98-98
Author(s):  
Maria A. Brehm ◽  
Tobias Obser ◽  
Ulrich Budde ◽  
Sonja Schneppenheim ◽  
Reinhard Schneppenheim
Keyword(s):  
Subcellular Localization ◽  
Von Willebrand Factor ◽  
Intracellular Transport ◽  
Conflicts Of Interest ◽  
Intracellular Localization ◽  
Hek293 Cells ◽  
Missense Mutations ◽  
Type 3 ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 98 Von Willebrand disease (VWD) is a bleeding disorder caused by mutations of von Willebrand factor (VWF), a huge multimeric glycoprotein that is essential for platelet-dependent primary hemostasis. VWF undergoes a highly complex biosynthesis that starts with the production of the 250 kD monomers that are further post-translationally modified within the ER. Here, N-linked glycosylation occurs and dimerization is facilitated by the formation of interchain disulfide bonds. These dimers are being released from the ER to the Golgi apparatus where the high mannose glycans are processed to the complex form and O-linked oligosaccharides are added. The pH-dependent assembly of VWF High Molecular Weight Multimers (HMWM), up to 80 monomers in size, occurs in the trans-Golgi where they become organized in tubules that are subsequently stored in Weibel-Palade bodies (WPB) until stimulated release into the circulation. This process may partly or completely be disrupted, in particular in several VWD type 2A subtypes (IIC, IID, IIE) but also in some VWD type 3 mutants with missense mutations. Mutant VWF in such patients is characterized by lack or a significant decrease of VWF HMWM which can be due to impaired dimerization and multimerization or poor secretion. The phenotypes are ranging from a relative decrease of HMWM (IIE, IID) over a severe reduction (IIC) to their complete absence beyond the size of a dimer in some patients with VWD type 3. To identify the cause of VWF HMWM deficiency in type 2A (subtypes IIC, IID and IIE) and type 3 VWD patients with known or novel missense mutations, we overexpressed representative VWF mutants in HEK293 cells. Confocal immunofluorescence was employed to investigate their intracellular localization by parallel visualization of cellular sub-compartments using antibodies against marker proteins for the ER, Golgi, endosomes and pseudo-WPB. The type IIC mutants (I94N, L526S, and G550R) as well as the IID mutants (C2771R, S2775C and G2752D) did not form any cigar-shaped pseudo-WPB and were almost completely localized within the ER where small granules were formed. The type 2A/IIE mutants W1144G, Y1146C and C1169W in contrast exhibited a massive increase in pseudo-WPB formation with most of their contents remaining intracellularly as a result of poor secretion. VWF Type 3 mutants (W377C, W1120S and C2304Y) are retained in the ER in a similar pattern as type IIC and IID mutants. In conclusion, we show that type 2A (IIC, IID, IIE), and type 3 VWF mutations correspond to a discrete disruption in the VWF intracellular transport, resulting in a characteristic subcellular localization of the particular VWF mutants and correlating with a particular VWF multimer pattern. The respective localization is indicative for the processing and secretion defect of VWF which is ultimately responsible for the manifestation of a specific VWD phenotype. Disclosures: No relevant conflicts of interest to declare.

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