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.

Homocysteine inhibits von Willebrand factor processing and secretion by preventing transport from the endoplasmic reticulum

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 683-689 ◽  
Author(s):  
SR Lentz ◽  
JE Sadler
Keyword(s):  
Endothelial Cells ◽  
Endoplasmic Reticulum ◽  
Endothelial Cell ◽  
Von Willebrand Factor ◽  
Protein Transport ◽  
Cell Protein ◽  
Carboxyl Terminal ◽  
Insulinoma Cells ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Intracellular protein transport in endothelial cells is selectively inhibited by homocysteine, a thiol amino acid associated with both thrombosis and atherosclerosis. In a previous study, homocysteine decreased cell surface expression of the surface transmembrane glycoprotein thrombomodulin without decreasing secretion of another endothelial cell protein, plasminogen activator inhibitor-1. To define further the effects of homocysteine on protein transport, we examined the processing and secretion of the multimeric glycoprotein von Willebrand factor (vWF) in human umbilical vein endothelial cells. Incubation with 2 mmol/L homocysteine resulted in complete loss of vWF multimers and prevented asparagine-linked oligosaccharide maturation, propeptide cleavage, and secretion; these effects are consistent with impaired exit from the endoplasmic reticulum (ER). Dimerization was only partially inhibited, suggesting that homocysteine causes retention of provWF in the ER without preventing dimer formation. In pulse-chase incubations, intracellular provWF was degraded before exiting the ER in homocysteine-treated cells. Homocysteine also inhibited the processing and secretion of a carboxyl-terminal truncation mutant of human provWF expressed in rat insulinoma cells, indicating that retention in the endoplasmic reticulum can be mediated by regions of provWF apart from the carboxyl-terminal 20-Kd segment. These results suggest that retention of secretory proteins in the ER is regulated by redox mechanisms and imply that the intracellular transport of multiple endothelial cell proteins may be altered in patients with homocystinuria.

NAADP links histamine H1 receptors to secretion of von Willebrand factor in human endothelial cells

Blood ◽  
2011 ◽  
Vol 117 (18) ◽  
pp. 4968-4977 ◽  
Author(s):  
Bianca Esposito ◽  
Guido Gambara ◽  
Alexander M. Lewis ◽  
Fioretta Palombi ◽  
Alessio D'Alessio ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endoplasmic Reticulum ◽  
Von Willebrand Factor ◽  
Calcium Release ◽  
Umbilical Vein ◽  
Pore Channel ◽  
Human Endothelial Cells ◽  
Signaling Mechanism ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract A variety of endothelial agonist–induced responses are mediated by rises in intracellular Ca2+, suggesting that different Ca2+ signatures could fine-tune specific inflammatory and thrombotic activities. In search of new intracellular mechanisms modulating endothelial effector functions, we identified nicotinic acid adenine dinucleotide phosphate (NAADP) as a crucial second messenger in histamine-induced Ca2+ release via H1 receptors (H1R). NAADP is a potent intracellular messenger mobilizing Ca2+ from lysosome-like acidic compartments, functionally coupled to the endoplasmic reticulum. Using the human EA.hy926 endothelial cell line and primary human umbilical vein endothelial cells, we show that selective H1R activation increases intracellular NAADP levels and that H1R-induced calcium release involves both acidic organelles and the endoplasmic reticulum. To assess that NAADP links H1R to Ca2+-signaling we used both microinjection of self-inactivating concentrations of NAADP and the specific NAADP receptor antagonist, Ned-19, both of which completely abolished H1R-induced but not thrombin-induced Ca2+ mobilization. Interestingly, H1R-mediated von Willebrand factor (VWF) secretion was completely inhibited by treatment with Ned-19 and by siRNA knockdown of 2-pore channel NAADP receptors, whereas thrombin-induced VWF secretion failed to be affected. These findings demonstrate a novel and specific Ca2+-signaling mechanism activated through H1R in human endothelial cells, which reveals an obligatory role of NAADP in the control of VWF secretion.

scholarly journals Homocysteine inhibits von Willebrand factor processing and secretion by preventing transport from the endoplasmic reticulum

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 683-689 ◽  
Author(s):  
SR Lentz ◽  
JE Sadler
Keyword(s):  
Endothelial Cells ◽  
Endoplasmic Reticulum ◽  
Endothelial Cell ◽  
Von Willebrand Factor ◽  
Protein Transport ◽  
Cell Protein ◽  
Carboxyl Terminal ◽  
Insulinoma Cells ◽  
Von Willebrand ◽  
Willebrand Factor

Intracellular protein transport in endothelial cells is selectively inhibited by homocysteine, a thiol amino acid associated with both thrombosis and atherosclerosis. In a previous study, homocysteine decreased cell surface expression of the surface transmembrane glycoprotein thrombomodulin without decreasing secretion of another endothelial cell protein, plasminogen activator inhibitor-1. To define further the effects of homocysteine on protein transport, we examined the processing and secretion of the multimeric glycoprotein von Willebrand factor (vWF) in human umbilical vein endothelial cells. Incubation with 2 mmol/L homocysteine resulted in complete loss of vWF multimers and prevented asparagine-linked oligosaccharide maturation, propeptide cleavage, and secretion; these effects are consistent with impaired exit from the endoplasmic reticulum (ER). Dimerization was only partially inhibited, suggesting that homocysteine causes retention of provWF in the ER without preventing dimer formation. In pulse-chase incubations, intracellular provWF was degraded before exiting the ER in homocysteine-treated cells. Homocysteine also inhibited the processing and secretion of a carboxyl-terminal truncation mutant of human provWF expressed in rat insulinoma cells, indicating that retention in the endoplasmic reticulum can be mediated by regions of provWF apart from the carboxyl-terminal 20-Kd segment. These results suggest that retention of secretory proteins in the ER is regulated by redox mechanisms and imply that the intracellular transport of multiple endothelial cell proteins may be altered in patients with homocystinuria.

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.

Lack of Stability of Aggregates after Thrombin-Induced Reaggregation of Thrombin-Degranulated Platelets

1992 ◽  
Vol 67 (04) ◽  
pp. 453-457 ◽  
Author(s):  
Raelene L Kinlough-Rathbone ◽  
Marian A Packham ◽  
Dennis W Perry ◽  
J Fraser Mustard ◽  
Marco Cattaneo
Keyword(s):  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Aggregate Stability ◽  
Relative Importance ◽  
Platelet Aggregates ◽  
The Stability ◽  
Von Willebrand ◽  
Induced Aggregation ◽  
Willebrand Factor

SummaryThe stability of platelet aggregates is influenced by the extent of the release of granule contents; if release is extensive and aggregation is prolonged, deaggregation is difficult to achieve. The relative importance of the contributions of released substances to aggregate stability are not known, although stable thrombin-induced aggregates form in platelet-rich plasma from patients with barely detectable plasma or platelet fibrinogen, and ADP stabilizes thrombin-induced aggregates of platelets from patients with delta storage pool deficiency which otherwise deaggregate more readily than normal platelets. We degranulated platelets with thrombin (0.9 U/ml caused greater than 90% loss of delta and alpha granule contents) and recovered them as individual platelets in fresh medium. The degranulated platelets were reaggregated by thrombin (2 U/ml). To prevent continuing effects of thrombin, FPRCH2C1 was added when thrombin-induced aggregation of thrombin-degranulated platelets reached its maximum. EDTA (5 mM) or EGTA (5 mM) added at maximum aggregation did not deaggregate these platelets, indicating that the stability of these aggregates does not depend on Ca2+ in the medium. Whereas with control platelets a combination of PGE1 (10 μM) and chymotrypsin(10 U/ml) was required for deaggregation, with thrombin-degranulated platelets either PGE1 or chymo-trypsin alone caused extensive deaggregation. The rate and extent of deaggregation of thrombin-degranulated platelets by a combination of PGE1 and chymotrypsin was greater than with control platelets.Electron microscope gold immunocytochemistry using antihuman fibrinogen IgG, anti-von Willebrand factor and anti-fibronectin showed a) that fibrinogen in the vacuoles of degranulated platelets was visible at focal points of platelet contact in the aggregates, but that large areas of platelet contact had no fibrinogen detectable between them; and b) in comparison to fibrinogen, little fibronectin or von Willebrand factor (vWf) was detectable in the platelets.Since the linkages between thrombin-degranulated platelets reaggregated by thrombin can be disrupted either by raising cAMP (thus making glycoprotein IIb/IIIa unavailable) or by proteolysis, these linkages are less stable than those formed between normal platelets. It might therefore be expected that platelets that take part in thrombus formation and then recirculate are likely to form less stable thrombi than platelets that have not released their granule contents.

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.

Epinephrine Induces von Willebrand Factor Release from Cultured Endothelial Cells: Involvement of Cyclic AMP-dependent Signalling in Exocytosis

1997 ◽  
Vol 77 (06) ◽  
pp. 1182-1188 ◽  
Author(s):  
Ulrich M Vischer ◽  
Claes B Wollheinn
Keyword(s):  
Endothelial Cells ◽  
Adenylate Cyclase ◽  
Von Willebrand Factor ◽  
Umbilical Vein ◽  
Free Calcium ◽  
Camp Content ◽  
Von Willebrand ◽  
Willebrand Factor

Summaryvon Willebrand factor (vWf) is released from endothelial cell storage granules after stimulation with thrombin, histamine and several other agents that induce an increase in cytosolic free calcium ([Ca2+]i). In vivo, epinephrine and the vasopressin analog DDAVP increase vWf plasma levels, although they are thought not to induce vWf release from endothelial cells in vitro. Since these agents act via a cAMP-dependent pathway in responsive cells, we examined the role of cAMP in vWf secretion from cultured human umbilical vein endothelial cells. vWf release increased by 50% in response to forskolin, which activates adenylate cyclase. The response to forskolin was much stronger when cAMP degradation was blocked with IBMX, an inhibitor of phosphodiesterases (+200%), whereas IBMX alone had no effect. vWf release could also be induced by the cAMP analogs dibutyryl-cAMP (+40%) and 8-bromo-cAMP (+25%); although their effect was weak, they clearly potentiated the response to thrombin. Epinephrine (together with IBMX) caused a small, dose-dependent increase in vWf release, maximal at 10-6 M (+50%), and also potentiated the response to thrombin. This effect is mediated by adenylate cyclase-coupled β-adrenergic receptors, since it is inhibited by propranolol and mimicked by isoproterenol. In contrast to thrombin, neither forskolin nor epinephrine caused an increase in [Ca2+]j as measured by fura-2 fluorescence. In addition, the effects of forskolin and thrombin were additive, suggesting that they act through distinct signaling pathways. We found a close correlation between cellular cAMP content and vWf release after stimulation with epinephrine and forskolin. These results demonstrate that cAMP-dependent signaling events are involved in the control of exocytosis from endothelial cells (an effect not mediated by an increase in [Ca2+]i) and provide an explanation for epinephrine-induced vWf release.

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.

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