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.

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 Hypoxia Results in Upregulation and De Novo Activation of Von Willebrand Factor Expression in Lung Endothelial Cells

2013 ◽  
Vol 33 (6) ◽  
pp. 1329-1338 ◽  
Author(s):  
Anahita Mojiri ◽  
Maryam Nakhaii-Nejad ◽  
Wei-Lee Phan ◽  
Stephen Kulak ◽  
Aneta Radziwon-Balicka ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
De Novo ◽  
Factor Expression ◽  
Lung Endothelial Cells ◽  
Von Willebrand ◽  
Willebrand Factor

P-selectin binds to the D′-D3 domains of von Willebrand factor in Weibel-Palade bodies

Blood ◽  
2006 ◽  
Vol 107 (10) ◽  
pp. 3922-3924 ◽  
Author(s):  
Grégoire Michaux ◽  
Timothy J. Pullen ◽  
Sandra L. Haberichter ◽  
Daniel F. Cutler
Keyword(s):  
Endothelial Cells ◽  
Membrane Protein ◽  
Cell Surface ◽  
Von Willebrand Factor ◽  
Integral Membrane Protein ◽  
Hek293 Cells ◽  
Binding Partner ◽  
Lumenal Domain ◽  
Von Willebrand ◽  
Willebrand Factor

It has recently been shown that the ultralarge platelet–recruiting von Willebrand factor (VWF) strings formed immediately at exocytosis from endothelial cells may be anchored to the cell surface by interaction with the integral membrane protein P-selectin. This finding of a new binding partner for VWF immediately prompts the question which domains of VWF bind to P-selectin. We have exploited the fact that VWF expression in HEK293 cells triggers the formation of Weibel-Palade body–like structures that can recruit P-selectin. A suitably modified version of this assay using coexpressed truncations of VWF, together with P-selectin variants in HEK293 cells, allowed us to determine which domains of VWF would recruit P-selectin within a physiologically appropriate intracellular environment. Confirming the results of such a cellular assay by conventional coimmunoprecipitation, we concluded that the lumenal domain of P-selectin interacts with the D′-D3 domains of VWF.

scholarly journals Perturbation of human endothelial cells by thrombin or PMA changes the reactivity of their extracellular matrix towards platelets.

1987 ◽  
Vol 104 (3) ◽  
pp. 697-704 ◽  
Author(s):  
P G de Groot ◽  
J H Reinders ◽  
J J Sixma
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
De Novo ◽  
The Matrix ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Adhesion Of Platelets ◽  
De Novo Protein Synthesis

In this study we have examined the influence of perturbation of endothelial cells on the amounts of fibronectin and von Willebrand factor in their extracellular matrix and the consequences of a changed composition of the matrix on platelet adhesion. For this purpose, we have used an in vitro perfusion system with which we can investigate the interactions of platelets in flowing blood with cultured endothelial cells and their extracellular matrix (Sakariassen, K. S., P. A. M. M. Aarts, P. G. de Groot, W. P. M. Houdgk, and J. J. Sixma, 1983, J. Lab. Clin Med. 102:522-535). Treatment of endothelial cells with 0.1-1.0 U/ml thrombin for 2 h increased the reactivity of the extracellular matrix, isolated after the thrombin treatment, towards platelets by approximately 50%. The increased reactivity did not depend on de novo protein synthesis but was inhibited by 3-deazaadenosine, an inhibitor of phospholipid methylation, which also inhibits the stimulus-induced instantaneous release of von Willebrand factor from endothelial cells. However, no changes in the amounts of von Willebrand factor and fibronectin in the matrix were detected. Thrombin may change the organization of the matrix proteins, not the composition. When endothelial cells were perturbed with the phorbol ester PMA or thrombin for 3 d, the adhesion of platelets to the extracellular matrix of treated cells was strongly impaired. This impairment coincided with a decrease in the amounts of von Willebrand factor and fibronectin present in the matrix. These results indicate that, after perturbation, endothelial cells regulate the composition of their matrix, and that this regulation has consequences for the adhesion of platelets.

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 von Willebrand factor biosynthesis and partitioning between constitutive and regulated pathways of secretion after thrombin stimulation

Blood ◽  
1989 ◽  
Vol 73 (3) ◽  
pp. 706-711 ◽  
Author(s):  
T Mayadas ◽  
DD Wagner ◽  
PJ Simpson
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
De Novo ◽  
Umbilical Vein ◽  
Protein Levels ◽  
Storage Pool ◽  
Umbilical Vein Endothelial Cells ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Specific Mrna

Abstract The major part of von Willebrand factor (vWf) synthesized in cultured endothelial cells is secreted constitutively without stimulation and consists of all multimeric forms of vWf. In contrast, stimulation with secretagogues such as thrombin results in the release of vWf from the storage pool, the Weibel-Palade bodies which contain only the largest, most biologically potent multimeric forms of vWf. We wished to determine whether the signal for release of vWf might also function as a signal for replenishment of the vWf by enhancing de novo biosynthesis and if replenishment of the vWf storage pool involved a diversion of newly synthesized vWf from the constitutive pathway to the regulated pathway. vWf mRNA and protein levels in unstimulated human umbilical vein endothelial cells were compared with cells that were briefly stimulated with 1 U/mL thrombin for 15 minutes and then incubated without thrombin for periods up to 72 hours. A comparison was also made between unstimulated cells and cells continuously exposed to thrombin for up to 48 hours. Thrombin stimulation, brief or continuous, had no significant effect on subsequent biosynthesis of vWf protein or vWf- specific mRNA. Since thrombin releases vWf only from the storage pool, we examined the possibility of diversion of newly synthesized vWf from the constitutive pathway to the regulated pathway. Cells were pulse- labeled, incubated for 15 minutes with and without thrombin, chased for various periods in unlabeled media, and briefly restimulated with thrombin. No significant redistribution of vWf between the two pathways was observed as a result of thrombin stimulation for the time periods tested.

scholarly journals von Willebrand factor biosynthesis and partitioning between constitutive and regulated pathways of secretion after thrombin stimulation

Blood ◽  
1989 ◽  
Vol 73 (3) ◽  
pp. 706-711 ◽  
Author(s):  
T Mayadas ◽  
DD Wagner ◽  
PJ Simpson
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
De Novo ◽  
Umbilical Vein ◽  
Protein Levels ◽  
Storage Pool ◽  
Umbilical Vein Endothelial Cells ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Specific Mrna

The major part of von Willebrand factor (vWf) synthesized in cultured endothelial cells is secreted constitutively without stimulation and consists of all multimeric forms of vWf. In contrast, stimulation with secretagogues such as thrombin results in the release of vWf from the storage pool, the Weibel-Palade bodies which contain only the largest, most biologically potent multimeric forms of vWf. We wished to determine whether the signal for release of vWf might also function as a signal for replenishment of the vWf by enhancing de novo biosynthesis and if replenishment of the vWf storage pool involved a diversion of newly synthesized vWf from the constitutive pathway to the regulated pathway. vWf mRNA and protein levels in unstimulated human umbilical vein endothelial cells were compared with cells that were briefly stimulated with 1 U/mL thrombin for 15 minutes and then incubated without thrombin for periods up to 72 hours. A comparison was also made between unstimulated cells and cells continuously exposed to thrombin for up to 48 hours. Thrombin stimulation, brief or continuous, had no significant effect on subsequent biosynthesis of vWf protein or vWf- specific mRNA. Since thrombin releases vWf only from the storage pool, we examined the possibility of diversion of newly synthesized vWf from the constitutive pathway to the regulated pathway. Cells were pulse- labeled, incubated for 15 minutes with and without thrombin, chased for various periods in unlabeled media, and briefly restimulated with thrombin. No significant redistribution of vWf between the two pathways was observed as a result of thrombin stimulation for the time periods tested.

scholarly journals Differential regulation of von Willebrand factor exocytosis and prostacyclin synthesis in electropermeabilized endothelial cell monolayers

1995 ◽  
Vol 309 (2) ◽  
pp. 473-479 ◽  
Author(s):  
J A Frearson ◽  
P Harrison ◽  
M C Scrutton ◽  
J D Pearson
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Umbilical Vein ◽  
Secretory Granules ◽  
Guanine Nucleotide Binding Protein ◽  
Intact Cells ◽  
Nucleoside Triphosphates ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Prostacyclin Synthesis

We have developed a system to permeabilize human umbilical vein endothelial cells in monolayer culture by application of a high-voltage electric field. The permeabilized preparation allows access of small molecules (M(r) < 1000) without loss of large cytosolic proteins. Electropermeabilized cells exocytose highly multimeric von Willebrand factor from secretory granules in response to added Ca2+ (EC50 = 0.8 +/- 0.02 microM), with levels comparable with those observed on stimulation of intact endothelial cells by physiological agonists. MgATP2- potentiates Ca(2+)-driven von Willebrand factor secretion. Other nucleoside triphosphates, but not non-hydrolysable analogues, can replace ATP. Electropermeabilized cells also synthesize and release prostacyclin in response to added Ca2+ (EC50 = 0.3 +/- 0.08 microM), but nucleoside triphosphates markedly inhibit, whereas nonhydrolysable GTP analogues increase, Ca(2+)-driven prostacyclin synthesis. We conclude that elevation of the intracellular [Ca2+] is sufficient to cause efficient exocytosis of von Willebrand factor from permeabilized cells, despite evidence that additional second messengers are needed in intact cells. We find no evidence in endothelial cells for a guanine nucleotide-binding protein promoting exocytosis, although one is clearly involved in stimulating Ca(2+)-driven prostacyclin synthesis.

Requirement for Both D Domains of the Propolypeptide in von Willebrand Factor Muitimerization and Storage

1993 ◽  
Vol 70 (06) ◽  
pp. 1053-1057 ◽  
Author(s):  
Agnès M Journet ◽  
Simin Saffaripour ◽  
Denisa D Wagner
Keyword(s):  
Endothelial Cells ◽  
Endoplasmic Reticulum ◽  
Von Willebrand Factor ◽  
Deletion Mutants ◽  
Relative Importance ◽  
D2 Domain ◽  
Constitutive Secretion ◽  
Von Willebrand ◽  
And Storage ◽  
Willebrand Factor

SummaryBiosynthesis of the adhesive glycoprotein von Willebrand factor (vWf) by endothelial cells results in constitutive secretion of small multimers and storage of the largest multimers in rodshaped granules called Weibel-Palade bodies. This pattern is reproduced by expression of pro-vWf in heterologous cells with a regulated pathway of secretion, that store the recombinant protein in similar elongated granules. In these cells, deletion of the vWf prosequence prevents vWf storage. The prosequence, composed of two homologous domains (D1 and D2), actively participates in vWf multimer formation as well. We expressed deletion mutants lacking either the D1 domain (D2vWf) or the D2 domain (D1vWf) in various cell lines to analyze the relative importance of each domain in vWf muitimerization and storage. Both proteins were secreted efficiently without being retained in the endoplasmic reticulum. Despite this, neither multimerized past the dimer stage and they were not stored. We conclude that several segments of the prosequence are jointly involved in vWf muitimerization and storage.

Adverse Influence of Cigarette Smoking on the Endothelium

1993 ◽  
Vol 70 (04) ◽  
pp. 707-711 ◽  
Author(s):  
Andrew D Blann ◽  
Charles N McCollum
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Tobacco Smoke ◽  
Lipid Peroxides ◽  
Short Exposure ◽  
Acute Effects ◽  
Adverse Influence ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryThe effect of smoking on the blood vessel intima was examined by comparing indices of endothelial activity in serum from smokers with that from non-smokers. Serum from smokers contained higher levels of von Willebrand factor (p <0.01), the smoking markers cotinine (p <0.02) and thiocyanate (p <0.01), and was more cytotoxic to endothelial cells in vitro (p <0.02) than serum from non-smokers. The acute effects of smoking two unfiltered medium tar cigarettes was to briefly increase von Willebrand factor (p <0.001) and cytotoxicity of serum to endothelial cells in vitro (p <0.005), but lipid peroxides or thiocyanate were not increased by this short exposure to tobacco smoke. Although there were correlations between von Willebrand factor and smokers consumption of cigarettes (r = 0.28, p <0.02), number of years smoking (r = 0.41, p <0.001) and cotinine (r = 0.45, p <0.01), the tissue culture of endothelial cells with physiological levels of thiocyanate or nicotine suggested that these two smoking markers were not cytotoxic. They are therefore unlikely to be directly responsible for increased von Willebrand factor in the serum of smokers. We suggest that smoking exerts a deleterious influence on the endothelium and that the mechanism is complex.

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