scholarly journals Crossveinless 2 contains cysteine-rich domains and is required for high levels of BMP-like activity during the formation of the cross veins in Drosophila

Development ◽  
2000 ◽  
Vol 127 (18) ◽  
pp. 3947-3959 ◽  
Author(s):  
C.A. Conley ◽  
R. Silburn ◽  
M.A. Singer ◽  
A. Ralston ◽  
D. Rohwer-Nutter ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Von Willebrand Factor ◽  
Secretory Pathway ◽  
Drosophila Wing ◽  
Transmembrane Sequence ◽  
The Cross ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Early Phases ◽  
Cross Vein

The BMP-like signaling mediated by the ligands Dpp and Gbb is required to reinforce the development of most veins in the Drosophila wing. However, the formation of the cross veins is especially sensitive to reductions in BMP-like signaling. We show here that the formation of the definitive cross veins occurs after the initial specification of the longitudinal veins in a process that requires localized BMP-like activity. Since Dpp and Gbb levels are not detectably higher in the early phases of cross vein development, other factors apparently account for this localized activity. Our evidence suggests that the product of the crossveinless 2 gene is a novel member of the BMP-like signaling pathway required to potentiate Gbb of Dpp signaling in the cross veins. crossveinless 2 is expressed at higher levels in the developing cross veins and is necessary for local BMP-like activity. The Crossveinless 2 protein contains a putative signal or transmembrane sequence, and a partial Von Willebrand Factor D domain similar to those known to regulate the formation of intramolecular and intermolecular bonds. It also contains five cysteine-rich domains, similar to the cysteine-rich domains found in Chordin, Short Gastrulation and Procollagen that are known to bind BMP-like ligands. These features strongly suggest that Crossveinless 2 acts extracelluarly or in the secretory pathway to directly potentiate Dpp or Gbb signaling.

VON WILLEBRAND FACTOR (vWF) PRO-POLYPEPTIDE IS REQUIRED FOR vWF MULTIMER FORMATION

1987 ◽  
Author(s):  
C L Verweij ◽  
M Hart ◽  
H Pannekoek
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Secretory Pathway ◽  
Vascular Endothelial Cells ◽  
Expression System ◽  
Proteolytic Processing ◽  
Information Encoding ◽  
Interchain Interactions ◽  
Von Willebrand ◽  
Willebrand Factor

The von Willebrand factor (vWF) is a multimeric plasma glycoprotein synthesized in vascular endothelial cells as a pre-pro-polypeptide with a highly repetitive domain structure, symbolized by the formula:(H)-D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-(0H).A heterologous expression system, consisting of a monkey kidney cell line (C0S-1), transfected with full-length vWF cDNA, is shown to mimic the constitutively, secretory pathway of vWF in endothelial cells. The assembly of pro-vWF into multimers and the proteolytic processing of these structures is found to oro-ceed along the following, consecutive steps. Pro-vWF subunits associate to form dimers, a process that does not involve the pro-polypeptide of pro-vWF. This observation is derived from transfection of C0S-1 cells with vWF cDNA, lacking the genetic information encoding the pro-polypeptide, composed of the domains D1 and D2. Pro-vWF dimers are linked intracellularly to form a regular series of multimeric structures that are secreted and cannot be distinguished from those released constitutively by endothelial cells. The presence of the pro-polypeptide, embedded in pro-vWF, is obligatory for multimerization since the deletion mutant lacking the D1 and D2 domains fails to assemble beyond the dimer stage. It is argued that the D domains are involved in interchain interactions.

scholarly journals The roles of protein kinase C and intracellular Ca2+ in the secretion of von Willebrand factor from human vascular endothelial cells

1992 ◽  
Vol 286 (2) ◽  
pp. 631-635 ◽  
Author(s):  
M A Carew ◽  
E M Paleolog ◽  
J D Pearson
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase ◽  
Von Willebrand Factor ◽  
Secretory Pathway ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Selective Inhibition ◽  
Von Willebrand ◽  
Willebrand Factor

Secretion of von Willebrand factor (vWf) glycoprotein from storage granules in human umbilical-vein endothelial cells was studied in vitro. Either elevation of intracellular Ca2+ concentration ([Ca2+]i) with a Ca2+ ionophore or activation of protein kinase (PK) C by phorbol 12-myristate 13-acetate caused vWf secretion, and together the agents acted synergistically. However, when vWf release was stimulated by receptor-mediated agonists, selective inhibition of PKC had no effect on histamine-induced secretion and significantly elevated thrombin-induced secretion. Furthermore, ATP, which efficiently elevates [Ca2+]i in these cells, was a very poor effector of vWf release. We conclude that elevation of [Ca2+]i by physiological agonists is necessary for vWf release, but other signalling mechanisms, as yet uncharacterized, but not due to PKC activation, are required for full induction of the secretory pathway.

Intracellular cotrafficking of factor VIII and von Willebrand factor type 2N variants to storage organelles

Blood ◽  
2009 ◽  
Vol 113 (13) ◽  
pp. 3102-3109 ◽  
Author(s):  
Maartje van den Biggelaar ◽  
Alexander B. Meijer ◽  
Jan Voorberg ◽  
Koen Mertens
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Secretory Pathway ◽  
Hek293 Cells ◽  
Binding Studies ◽  
Cargo Proteins ◽  
Storage Granules ◽  
Factor Type ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Weibel-Palade bodies (WPBs) are the endothelial storage organelles that are formed upon von Willebrand factor (VWF) expression. Apart from VWF, WPBs contain a variety of hemostatic and inflammatory proteins. Some of these are thought to be targeted to WPBs by directly interacting with VWF in the secretory pathway. Previous studies have demonstrated that coexpression of factor VIII (FVIII) with VWF results in costorage of both proteins. However, whether cotrafficking is driven by intracellular FVIII-VWF assembly has remained unclear. We now have addressed this issue using recombinant VWF type 2N variants that are known to display reduced FVIII binding in the circulation. Binding studies using purified fluorescent FVIII and VWF type 2N variants revealed FVIII binding defects varying from moderate (Arg854Gln, Cys1060Arg) to severe (Arg763Gly, Thr791Met, Arg816Trp). Upon expression in HEK293 cells, all VWF variants induced formation of WPB-like organelles that were able to recruit P-selectin, as well as FVIII. WPBs containing FVIII did not display their typical elongated shape, suggesting that FVIII affects the organization of VWF tubules therein. The finding that VWF type 2N variants are still capable of cotargeting FVIII to storage granules implies that trafficking of WPB cargo proteins does not necessarily require high-affinity assembly 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

Distinct Functions of pH-Sensing Histidine Residues in the Multimer Assembly and Storage of Von Willebrand Factor in Weibel-Palade Bodies

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3311-3311
Author(s):  
Jing Huang ◽  
Lisa A Westfield ◽  
J. Evan Sadler
Keyword(s):  
Von Willebrand Factor ◽  
Secretory Pathway ◽  
Conflicts Of Interest ◽  
Hek293 Cells ◽  
Ph Sensing ◽  
Wild Type ◽  
Length Width ◽  
Storage Granules ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 3311 The hemostatic function of von Willebrand factor (VWF) depends on the formation of disulfide-linked multimers. We have previously shown that several evolutionarily conserved His residues within the VWF propeptide are critical for pH-dependent VWF multimerization. For example, the charge-neutralization mutations H395A and H460A in the VWF D2 domain prevented multimer assembly, while the charge-stabilization mutation H460K preserved it, suggesting that protonation at these sites is essential. In this study, we investigated the role of these His residues in VWF targeting to storage granules and agonist-induced secretion. Full-length VWF was expressed in transiently-transfected HEK293 cells. If Weibel-Palade body-like storage granules (WPBs) were formed, their morphology was analyzed by immunofluorescence microscopy. A WPB was defined as “long” if length/width was > 2, or “round” if length/width was ≤ 2. To analyze the assembly of VWF multimers, we stimulated VWF secretion by incubating cells with 100 ng/ml of phorbol myristate acetate for 1 hour and collected cell lysates and supernatants for multimer gel electrophoresis. Expression of wild type VWF and VWF H460K induced the formation of approximately 60% long WPBs. Treatment of cells with monensin increases the pH of the Golgi, and as expected monensin reduced the storage of wild type VWF in long WPBs. However, monensin did not impair the storage of VWF H460K or reduce the fraction of long WPBs, indicating that a positive charge at residue 460 can sustain tubular storage of VWF despite neutralization of the late secretory pathway. Both WT VWF and VWF H460K were secreted with a normal distribution of multimers. VWF H395A and VWF H460A formed predominantly round pseudo-WPBs, with only 10–20% long granules, and these mutations also disrupted VWF multimerization. In contrast, VWF H395R and VWF H395A/H460R did not assemble multimers but nevertheless formed long WPBs. Insertion of an extra Gly between the vicinal cysteines in the D1 domain (159CGLC162 to 159CGGLC162) was reported to prevent multimerization but allow storage in long WPBs in AtT-20 cells (Mayadas & Wagner, PNAS 1992; 89:3531). We found that this mutation also inhibited VWF multimerization and resulted in the formation of long WPBs in HEK293 cells. These results indicate that VWF multimerization and packing in elongated WPBs both depend on the acidic pH of the late secretory pathway. However, these processes are independent, with distinct structural requirements, and they can be dissociated by mutations of specific His residues. VWF construct Multimers Granule Shape Granule Shape (with monensin) Wild type Yes Long Round H395A No Round H395R No Long H395A/H460R No Long H460A No Round H460K Yes Long Long 159CGGLC162 No Long Disclosures: No relevant conflicts of interest to declare.

Von Willebrand factor protects against acute CCl4-induced hepatotoxicity through phospho-p38 MAPK signaling pathway inhibition

2017 ◽  
Vol 65 (5) ◽  
pp. 1046-1058 ◽  
Author(s):  
Hai-Jian Sun ◽  
Jian Chen ◽  
Hao Zhang ◽  
Bing Ni ◽  
Jennifer C. van Velkinburgh ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
P38 Mapk ◽  
Von Willebrand Factor ◽  
Mapk Signaling ◽  
Mapk Signaling Pathway ◽  
Pathway Inhibition ◽  
Von Willebrand ◽  
Willebrand Factor

scholarly journals Differing polarity of the constitutive and regulated secretory pathways for von Willebrand factor in endothelial cells.

1989 ◽  
Vol 108 (4) ◽  
pp. 1283-1289 ◽  
Author(s):  
L A Sporn ◽  
V J Marder ◽  
D D Wagner
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Secretory Pathway ◽  
Molecular Form ◽  
Calcium Ionophore ◽  
Ionophore A23187 ◽  
Polycarbonate Membrane ◽  
Secretory Pathways ◽  
Von Willebrand ◽  
Willebrand Factor

von Willebrand factor (vWf) is secreted from endothelial cells by one of two pathways-a constitutive pathway and a regulated pathway originating from the Weibel-Palade bodies. The molecular form of vWf from each of these pathways differs, with the most biologically potent molecules being released from Weibel-Palade bodies (Loesberg, C., M. D. Gonsalves, J. Zandbergen, C. Willems, W. G. Van Aken, H. V. Stel, J. A. Van Mourik, and P. G. DeGroot. 1983. Biochim. Biophys. Acta. 763:160-168; Sporn, L. A., V. J. Marder, and D. D. Wagner. 1987. Cell. 46:185-190). We investigated the polarity of the two secretory pathways using human umbilical vein endothelial cells cultured on polycarbonate membrane filters which allowed sampling of media from both the apical and basolateral compartments. After metabolic labeling of cells, vWf (constitutively secreted during a 10-min period or released during a 10-min treatment with a secretagogue) was purified from the apical and basolateral chambers and subjected to gel analysis. Approximately equal amounts of vWf were constitutively secreted into both chambers, and therefore this secretory pathway appeared to be nonpolarized. On the contrary, an average of 90% of vWf released from Weibel-Palade bodies after treatment with the calcium ionophore A23187 or PMA appeared in the basolateral chamber, indicating that the regulated pathway of secretion is highly polarized. Thrombin, a secretagogue which promotes disruption of the endothelial monolayer, led to release of vWf from cells with no apparent polarity. The presence of microtubule-depolymerizing agents nocodazol and colchicine inhibited the polarized release of vWf. Ammonium chloride treatment did not disrupt the polarity of the regulated secretory pathway, indicating that maintenance of low pH in intracellular compartments was not required for the polarized delivery of preformed Weibel-Palade bodies to the plasma membrane.

scholarly journals A revised model for the secretion of tPA and cytokines from cultured endothelial cells

Blood ◽  
2010 ◽  
Vol 116 (12) ◽  
pp. 2183-2191 ◽  
Author(s):  
Laura Knipe ◽  
Athinoula Meli ◽  
Lindsay Hewlett ◽  
Ruben Bierings ◽  
John Dempster ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Secretory Pathway ◽  
Primary Source ◽  
Storage Efficiency ◽  
Monocyte Chemoattractant Protein 1 ◽  
Tissue Plasminogen ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Endothelial cells are reported to contain several distinct populations of regulated secretory organelles, including Weibel-Palade bodies (WPBs), the tissue plasminogen activator (tPA) organelle, and the type-2 chemokine-containing organelle. We show that the tPA and type-2 organelles in human endothelial cells represent a single compartment primarily responsible for unstimulated secretion of tPA or, in cells exposed to interleukin-1β (IL-1β), the cytokines IL-8, IL-6, monocyte chemoattractant protein-1 (MCP-1), and growth-regulated oncogene-α (GRO-α). This compartment was distinct from WPBs in that it lacked detectable von Willebrand factor, P-selectin, Rab27a, or CD63 immunoreactivity, displayed no time-dependent decrease in intragranule pH, underwent detectable unstimulated exocytosis, and was very poorly responsive to Ca2+-elevating secretagogues. WPBs could also contain tPA, and in IL-1β–treated cells, IL-8, IL-6, MCP-1, and GRO-α, and were the primary source for histamine or ionomycin-stimulated secretion of these molecules. However, analysis of the storage efficiency of cytokines and tPA revealed that all were very poorly stored compared with von Willebrand factor. The nonmammalian, nonsecretory protein EGFP, when expressed in the secretory pathway, also entered WPBs and had a storage efficiency similar to tPA and the cytokines tested. Based on these data, we proposed a revised model for storage and secretion of cytokines and tPA.

Basal secretion of von Willebrand factor from human endothelial cells

Blood ◽  
2008 ◽  
Vol 112 (4) ◽  
pp. 957-964 ◽  
Author(s):  
Jonathan P. Giblin ◽  
Lindsay J. Hewlett ◽  
Matthew J. Hannah
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Secretory Pathway ◽  
Significant Proportion ◽  
Brefeldin A ◽  
Human Endothelial Cells ◽  
Constitutive Secretion ◽  
Regulated Secretory Pathway ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Endothelial cells store the adhesive glycoprotein von Willebrand factor (VWF) in Weibel-Palade bodies (WPBs), distinctively shaped regulated secretory organelles that undergo exocytosis in response to secretagogue. A significant proportion of newly synthesized VWF is also secreted spontaneously from nonstimulated cells, through what is thought to be the constitutive secretory pathway. To learn more about VWF trafficking, we performed kinetic analyses of the storage and nonstimulated secretion of VWF in cultured human endothelial cells. We found that most VWF was secreted through a route that was significantly delayed compared with constitutive secretion, although this pathway was responsible for secretion of a small amount of uncleaved VWF precursor. Disruption of pH-dependent sorting processes with ammonium chloride converted the secretion kinetics of mature VWF to that of its precursor. Conversely, preventing constitutive secretion of nascent protein with brefeldin A had only a modest effect on the spontaneous release of VWF, showing that most VWF secreted by nonstimulated cells was not constitutive secretion but basal release of a post-Golgi storage organelle, presumably the WPB. These data suggest that VWF is sorted to the regulated secretory pathway in endothelial cells much more efficiently than previously reported.

Assembly, Storage, and Secretion of Von Willebrand Factor.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. sci-46-sci-46
Author(s):  
J. Evan Sadler
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Disulfide Bonds ◽  
Secretory Pathway ◽  
Three Dimensional ◽  
Von Willebrand Disease ◽  
Inflammatory Stress ◽  
Von Willebrand ◽  
Willebrand Factor

Von Willebrand factor (VWF) plays a central role in hemostasis, and dysregulation of VWF can cause either bleeding or thrombosis. Endothelial cells assemble VWF multimers in two stages that occur in distinct intracellular compartments: proVWF subunits dimerize in the endoplasmic reticulum through “tail-to-tail” disulfide bonds between C-terminal CK domains, and the proVWF dimers form enormous multimers in the Golgi through “head-to-head” disulfide bonds between N-terminal D3 domains. The finished multimers are packaged into ordered tubules within storage organelles called Weibel-Palade bodies (WPB), and tubular packing is necessary for the secretion of VWF filaments that have normal hemostatic function. We have recreated VWF tubule assembly in vitro, starting with pure VWF propeptide (domains D1D2) and disulfide-linked dimers of adjacent N-terminal D’D3 domains. No other cellular proteins or components of WPB are needed. Assembly requires low pH and calcium ions, similar to conditions in the Golgi. Quick-freeze deep-etch electron microscopy and three-dimensional reconstruction of negatively stained images show that tubules contain a repeating unit of one D’D3 dimer and two propeptides arranged in a right-handed helix with 4.2 units per turn. The symmetry and location of interdomain contacts suggest that decreasing pH along the secretory pathway coordinates the disulfide-linked assembly of VWF multimers with their tubular packaging. Secretion into the blood exposes VWF tubules to neutral pH conditions, releasing the constraints that maintain tubular packing and permitting the orderly unfurling of large VWF multimers. Some VWF multimers bind platelets and initiate the growth of platelet-rich thrombi. Under normal circumstances, these thrombi are limited in size by ADAMTS13, a metalloprotease that cleaves VWF multimers and releases the platelets. The absence of large VWF multimers causes bleeding that is typical of von Willebrand disease. Conversely, congenital or acquired deficiency of ADAMTS13 prevents the dissolution of VWF-platelet aggregates, which can cause the widespread microvascular thrombosis that characterizes thrombotic thrombocytopenic purpura (TTP). Interestingly, ADAMTS13 deficiency alone need not trigger TTP. Some patients persist for months or years without active disease but become ill whenever they suffer additional inflammatory stress associated with infection, surgery, or pregnancy. Thus, interactions between inflammatory mediators and endothelial cells can determine the course of VWF-dependent thrombosis. The mechanisms responsible for these phenomena remain poorly understood.

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