Changes in the pattern of distribution of von Willebrand factor in rat aortic endothelial cells following thrombin generation in vivo

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.

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Multi-Cellular Activation In Vivo by Endotoxin in Humans – Limited Protection by Adenosine Infusion

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614032 ◽

2000 ◽

Vol 84 (09) ◽

pp. 381-387 ◽

Cited By ~ 19

Author(s):

Nailin Li ◽

Anne Soop ◽

Alf Sollevi ◽

Paul Hjemdahl

Keyword(s):

Von Willebrand Factor ◽

Thrombin Generation ◽

Platelet Reactivity ◽

Adenosine Infusion ◽

Cellular Activation ◽

Soluble P ◽

Von Willebrand ◽

Willebrand Factor ◽

Factor Secretion

SummaryThe influence of adenosine infusion (40 µg/kg/min for 4 h) on inflammatory and hemostatic parameters was investigated in healthy males without (n = 10) or with (n = 11) intravenous endotoxin injection (4 ng/kg). Without endotoxin, adenosine elevated circulating leukocytes and circulating platelet-leukocyte aggregates. Endotoxin activated platelets and leukocytes in vivo. Platelet activation was seen as slightly increased platelet P-selectin expression, decreased platelet counts, and elevated plasma soluble P-selectin (from 39.6 ± 3.4 to 68.9 ± 6.6 ng/ml; P <0.01). Leukocyte activation was evidenced by increased CD11b expression (from MFI of 0.54 ± 0.02 to 2.21 ± 0.17; P <0.01) and plasma elastase levels (from 25.3 ± 2.5 to 169.3 ± 22.5 ng/ml; P <0.01). Endotoxin also enhanced platelet and leukocyte responsiveness to in vitro stimulation. Endotoxin induced von Willebrand factor secretion (from 92 ± 8 units to 265 ± 19 units at 4 h; P <0.001) and enhanced thrombin generation in vivo. Endotoxin induced leukocytosis and thus increased circulating platelet-leukocyte, mainly platelet-neutrophil, aggregates. Adenosine caused slight attenuation of platelet reactivity to agonist stimulation, enhanced the endotoxin-induced leukocytosis, and detained more platelet-leukocyte aggregates in circulation, but did not attenuate endotoxin-induced neutrophil elastase secretion, von Willebrand factor secretion, or thrombin generation. Thus, endotoxemia induces multi-cellular activation in vivo. Adenosine inhibits leukocyte adhesion and extravasation, and mildly attenuates platelet responsiveness and soluble P-selectin release. Adenosine has the potential of becoming a therapeutic antiinflammatory drug, but an optimal treatment strategy needs to be developed.

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The Interaction of the Platelet and von Willebrand Factor

10.1055/s-0039-1682472 ◽

1977 ◽

Author(s):

D.N. Fass ◽

F. Booyse ◽

J.C. Lewis ◽

E. J. W. Bowie

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Cell Cultures ◽

Von Willebrand Factor ◽

Sandwich Technique ◽

Aortic Endothelial Cells ◽

Confluent Culture ◽

Endothelial Cell Cultures ◽

Von Willebrand ◽

Willebrand Factor

A culture of pig aortic endothelial cells was used for experiments to investigate the interaction between the platelet and von Willebrand factor. An antibody was raised in rabbits to purified porcine von Willebrand factor. A semi-confluent culture of pig endothelial cells was stained immunofluorescently by the sandwich technique using anti-Willebrand factor IgG. An extensive extracellular meshwork of microfilaments was revealed. In endothelial cell cultures from von Willebrand pigs, no immunoreactive microfilaments were found. Immunoelectronmicro-scopy with peroxidase linked antibody has been used to identify similar filaments in normal pig endothelial cells. Washed platelets were shown to adhere to semiconfluent or damaged normal endothelial cell cultures. If the cultures had been previously incubated with anti-Willebrand factor IgG, the washed platelets did not adhere. There was no adherence of platelets when they were added to semiconfluent or damaged von Willebrand endothelial cells.

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Expression of von Willebrand Factor by Human Pulmonary Endothelial Cells in vivo1

Respiration ◽

10.1159/000066471 ◽

2002 ◽

Vol 69 (6) ◽

pp. 526-533 ◽

Cited By ~ 21

Author(s):

Annette M. Müller ◽

Carmen Skrzynski ◽

Guido Skipka ◽

Klaus-Michael Müller

Keyword(s):

Endothelial Cells ◽

Von Willebrand Factor ◽

Pulmonary Endothelial Cells ◽

Von Willebrand ◽

Willebrand Factor

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DIVERGENT PATES OF VON WILLEBRAND FACTOR AND ITS PROPOLYPEPTIDE (VON WILLEBRAND ANTIGEN II) AFTER SECRETION FROM ENDOTHELIAL CELLS

10.1055/s-0038-1642832 ◽

1987 ◽

Author(s):

D D Wagner ◽

P J Fay ◽

L A Sporn ◽

S Sinha ◽

S O Lawrence ◽

...

Keyword(s):

Endothelial Cells ◽

Von Willebrand Factor ◽

Culture Medium ◽

Platelet Adhesion ◽

Human Foreskin Fibroblasts ◽

Covalently Linked ◽

Von Willebrand ◽

Willebrand Factor

The intracellular site of cleavage of pro-von Willebrand factor subunit and the subsequent fate of the propolypeptide (von Willebrand antigen II) and of the mature von Willebrand factor (vWf) were investigated. Both the propolypeptide, which was found to be a homodimer of non-covalently linked subunits, and mature vWf were released from Weibel-Palade bodies of endothelial cells following stimulation with secretagogues. The stoichiometry of the two proteins in the releasate was essentially equimolar. This indicates that vWf and the propolypeptide were packaged into the Weibel-Palade bodies as one unit, pro-vWf, and that the proteolytic cleavage of pro-vWf is likely to be a post-Golgi event. The association of prosequences into dimers provides support for their hypothetical role in the multimerization process. After secretion, the two proteins were distributed differently, as based on the following observations. The propolypeptide did not associate with vWf in the culture medium, did not co-distribute with vWf in the extracellular "patches of release" on stimulated endothelial cells, and was not detected in the endothelial cell extracellular matrix, which did contain vWf. Additionally, in contrast to vWf, the propolypeptide did not bind to matrix of human foreskin fibroblasts. Since the propolypeptide does not associate with vWf and does not interact witji extracellular matrices in vitro, it is highly unlikely that it would promote platelet adhesion to subendothelium in vivo.

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Unwinding the von Willebrand factor strings puzzle

Blood ◽

10.1182/blood-2012-07-442285 ◽

2013 ◽

Vol 121 (2) ◽

pp. 270-277 ◽

Cited By ~ 84

Author(s):

Karen De Ceunynck ◽

Simon F. De Meyer ◽

Karen Vanhoorelbeke

Keyword(s):

Endothelial Cells ◽

Von Willebrand Factor ◽

Cleavage Sites ◽

Normal Functional ◽

Von Willebrand ◽

Willebrand Factor

Abstract von Willebrand factor (VWF) is amongst others synthesized by endothelial cells and stored as ultra-large (UL) VWF multimers in Weibel-Palade bodies. Although UL-VWF is proteolysed by ADAMTS13 (a disintegrin-like and metalloprotease domain with thrombospondin type-1 motif, number 13) on secretion from endothelial cells, in vitro experiments in the absence of ADAMTS13 have demonstrated that a proportion of these UL-VWF multimers remain anchored to the activated endothelium. These multimers unravel, bind platelets, and wave in the direction of the flow. These so-called VWF “strings” have also been visualized in vivo, lining the lumen of activated mesenteric veins of Adamts13−/− mice. Various studies have demonstrated the extraordinary length of these VWF strings, the availability of their platelet binding and ADAMTS13 cleavage sites, and the possible nature of their endothelial attachment. VWF strings are also capable of tethering leukocytes and parasite-infected red blood cells. However, the majority of studies have been performed in the absence of ADAMTS13, a condition only experienced in thrombotic thrombocytopenic purpura. A normal functional role of VWF strings in healthy persons or in other disease pathologies remains unclear. In this review, we discuss some of the puzzling characteristics of VWF strings, and we debate whether the properties of VWF strings in the absence of ADAMTS13 might be relevant for understanding (patho)physiologic mechanisms.

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EPAC regulates von Willebrand factor secretion from endothelial cells in a PI3K/eNOS-dependent manner during inflammation

10.1101/2020.09.04.282806 ◽

2020 ◽

Author(s):

Jie Xiao ◽

Ben Zhang ◽

Zhengchen Su ◽

Yakun Liu ◽

Thomas R. Shelite ◽

...

Keyword(s):

Endothelial Cells ◽

Von Willebrand Factor ◽

Umbilical Vein ◽

Null Mouse ◽

Intracellular Camp ◽

Dependent Manner ◽

Von Willebrand ◽

Willebrand Factor

AbstractCoagulopathy is associated with both inflammation and infection, including infection with the novel SARS-CoV-2 (COVID-19). Endothelial cells (ECs) fine tune hemostasis via cAMP-mediated secretion of von Willebrand factor (vWF), which promote the process of clot formation. The exchange protein directly activated by cAMP (EPAC) is a ubiquitously expressed intracellular cAMP receptor that plays a key role in stabilizing ECs and suppressing inflammation. To assess whether EPAC could regulate vWF release during inflammation, we utilized our EPAC1-null mouse model and revealed an increased secretion of vWF in endotoxemic mice in the absence of the EPAC1 gene. Pharmacological inhibition of EPAC1 in vitro mimicked the EPAC1−/− phenotype. EPAC1 regulated TNFα-triggered vWF secretion from human umbilical vein endothelial cells (HUVECs) in a phosphoinositide 3-kinases (PI3K)/endothelial nitric oxide synthase (eNOS)-dependent manner. Furthermore, EPAC1 activation reduced inflammation-triggered vWF release, both in vivo and in vitro. Our data delineate a novel regulatory role of EPAC1 in vWF secretion and shed light on potential development of new strategies to controlling thrombosis during inflammation.Key PointPI3K/eNOS pathway-mediated, inflammation-triggered vWF secretion is the target of the pharmacological manipulation of the cAMP-EPAC system.

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Quantitative analysis of von Willebrand factor propeptide release in vivo: effect of experimental endotoxemia and administration of 1- deamino-8-D-arginine vasopressin in humans

Blood ◽

10.1182/blood.v88.8.2951.bloodjournal8882951 ◽

1996 ◽

Vol 88 (8) ◽

pp. 2951-2958 ◽

Cited By ~ 137

Author(s):

A Borchiellini ◽

K Fijnvandraat ◽

JW ten Cate ◽

D Pajkrt ◽

SJ van Deventer ◽

...

Keyword(s):

Endothelial Cells ◽

Von Willebrand Factor ◽

Arginine Vasopressin ◽

Molar Ratio ◽

Cultured Endothelial Cells ◽

Baseline Values ◽

Von Willebrand ◽

Willebrand Factor ◽

Von Willebrand Factor Propeptide

The results of studies with cultured endothelial cells have shown that most von Willebrand factor (vWF) synthesized is directly secreted (constitutive pathway) and consists of both mature vWF, its precursor molecule pro-vWF, and the cleaved vWF prosequence. Only fully processed, functionally mature vWF is stored within the cell, together with the propeptide, and leaves the cell only on stimulation (regulated secretion). Both in resting and stimulated cultured endothelial cells, the stoichiometry of the released propeptide to the released mature vWF is essentially equimolar. In the present study, we have measured the molar ratio of propeptide to mature vWF in vivo, both under resting conditions and conditions that reflect activation of the endothelium. To this end, we devised a method that allows the measurement of the propeptide (vW antigen II) on a quantitative, is, molar basis, using purified recombinant propeptide as a standard. Our results show that the molar concentration of the propeptide in normal plasma is about one tenth of the concentration of mature vWF (expressed as half-dimer concentration). This ratio is approximately 1:1 in the medium of cultured endothelial cells. On administration in healthy subjects of either 1-deamino-8-D-arginine vasopressin or endotoxin, both agents being known to elicit an intravascular increase of vWF, the molar ratio of propeptide to mature vWF increased fourfold to fivefold. The propeptide concentration returned to baseline values after about 6 to 7 hours of injection of each stimulus, whereas the increase of mature vWF was much more sustained. Because the respective half-lives of mature vWF and its propeptide clearly differ, measurement of the concentration of these proteins could provide a means to assess the extent of activation of the endothelium under physiological and pathophysiological conditions.

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