scholarly journals Differential regulation of endothelial exocytosis of P-selectin and von Willebrand factor by protease-activated receptors and cAMP

Blood ◽  
2006 ◽  
Vol 107 (7) ◽  
pp. 2736-2744 ◽  
Author(s):  
John H. Cleator ◽  
Wen Qin Zhu ◽  
Douglas E. Vaughan ◽  
Heidi E. Hamm
Keyword(s):  
Von Willebrand Factor ◽  
Umbilical Vein ◽  
Surface Expression ◽  
Inhibitory Effect ◽  
Differential Regulation ◽  
Cell Surface Expression ◽  
Protease Activated Receptors ◽  
Umbilical Vein Endothelial Cells ◽  
Von Willebrand ◽  
Willebrand Factor

AbstractThrombin-mediated endothelial-cell release of von Willebrand factor (VWF) and P-selectin functionally links protease-activated receptors (PARs) to thrombosis and inflammation. VWF release can be stimulated by both Ca2+ and cAMP, and, although both VWF and P-selectin are found in Weibel-Palade bodies (WPBs), we found that their release could be differentially regulated. In these studies, human umbilical vein endothelial cells stimulated with cAMP or PAR2-AP led to a delayed release of VWF and significantly less P-selectin release compared with histamine, thrombin, or PAR1-AP. Dose-response studies revealed that PAR2-AP was significantly less efficacious in promoting the release of P-selectin compared with VWF. PAR2-AP–induced robust stimulation of intracellular Ca2+ coupled with a significantly greater inhibitory effect of calcium chelation on release of VWF compared with cell-surface expression of P-selectin, suggests an additional Ca2+-independent pathway involved in release of P-selectin. PAR2-AP failed to increase global cAMP levels; however, inhibition of protein kinase A led to a significant attenuation of PAR2-AP–mediated release of VWF. Confocal microscopy studies revealed that PAR2 and forskolin caused preferential release of a population of Weibel-Palade bodies (WPBs) consisting of only VWF. Thus, WPBs are pharmacologically and morphologically heterogeneous, and distinct granule populations are susceptible to differential regulation.

Glycoprotein Ib Can Mediate Endothelial Cell Attachment to a von Willebrand Factor Substratum

1995 ◽  
Vol 73 (02) ◽  
pp. 309-317 ◽  
Author(s):  
Dorothy A Beacham ◽  
Miguel A Cruz ◽  
Robert I Handin
Keyword(s):  
Endothelial Cell ◽  
Von Willebrand Factor ◽  
Cell Attachment ◽  
Umbilical Vein ◽  
Single Amino Acid ◽  
Cell Surface Expression ◽  
Platelet Glycoprotein ◽  
Glycoprotein Ib ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryIntroduction of single amino acid substitutions into the C-terminal Arg-Gly-Asp-Ser (RGDS) site of von Willebrand Factor, referred to as RGD mutant vWF, selectively abrogated vWF binding to platelet glycoprotein IIb/IIIa (GpIIb/IIIa, αIIbβ3 and abolished human umbilical vein endothelial cell (HUVEC) spreading, but not attachment, to RGD mutant vWF (Beacham, D. A., Wise, R. J., Turci, S. M. and Handin, R. I. 1992. J. Biol. Chem. 167, 3409-3415). These results suggested that in addition to the vitronectin receptor (VNR, αvβ3), a second endothelial membrane glycoprotein can mediate HUVEC adhesion to vWF. HUVEC attachment to wild-type (WT) and RGD-mutant vWF was reduced by two proteins known to block the vWF-platelet glycoprotein Ib/IX (GpIb/IX) interaction, the monoclonal antibody AS-7 and the recombinant polypeptide, vWF-A1. The addition of cytochalasin B or DNase I to disrupt potential GPIbα-cytoskeletal interactions enhanced the immunoprecipitation of endothelial GPIbα, caused HUVEC to round up, and increased HUVEC adhesion to RGD mutant vWF. These results indicate that while the VNR is the primary adhesion receptor for vWF, endothelial GPIbα can mediate HUVEC attachment to vWF. GpIb-dependent attachment could contribute to HUVEC adhesion under conditions when cell surface expression of the VNR is downregulated, and VNR-dependent adhesion is reduced.

Hydrogen peroxide stimulates exocytosis of von Willebrand factor in human umbilical vein endothelial cells

2017 ◽  
Vol 44 (5) ◽  
pp. 531-537 ◽  
Author(s):  
P. V. Avdonin ◽  
A. A. Tsitrina ◽  
G. Y. Mironova ◽  
P. P. Avdonin ◽  
I. L. Zharkikh ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Umbilical Vein ◽  
Human Umbilical Vein ◽  
Umbilical Vein Endothelial Cells ◽  
Von Willebrand ◽  
Willebrand Factor

FACTOR VIII/VON WILLEBRAND FACTOR COMPLEX: ONLY THE 440 000 SUBUNIT OF ENDOTHELIAL CELL-DERIVED VON WILLEBRAND FACTOR FORMS A COMPLEX WITH PURIFIED PLASMA FACTOR VIIIC

1987 ◽  
Author(s):  
B Pötzsch ◽  
U Delvos ◽  
E Anders ◽  
N Heimburger ◽  
G Müller-Berghaus
Keyword(s):  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Umbilical Vein ◽  
Sds Page ◽  
Plasma Factor ◽  
Umbilical Vein Endothelial Cells ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Recombination Experiments ◽  
Monoclonal Mouse

Von Willebrand Factor (vWF) circulates in plasma as a series of multimers with moleculag weight ranging from M = 0.44 x 106 up to more than 20 x 106 . Besides the mediation of platelet adhesion to exposed subendothelium, the protein plays an important role in the stabilization and the transport of Factor VIIIC (FVIIIC). In the present study the interaction between FVIIIC and vWF was studied by recombination experiments. vWF was isolated from cultured human umbilical vein endothelial cells by immunoprécipitation. This source of vWF ascertained, that it was free of FVIIIC as indicated by the absence of FVIIIC activity as well as FVIIIC antigen. FVIIIC was prepared by immunoabsorption from human plasma yielding an activity of 1600 IJ/mg. SDS-PAGE analysis showed two main bands at Mr= 0.28 x 106 and 0.18 x 106 , respectively. vWF-multimers were separated by SDS agarose gel electrophoresis and were electrophoretically transferred onto nitrocellulose sheets. After extensive washing, the sheets were incubated for 12 h with 20 U/ml FVIIIC in PBS, pH 7.4, containing 2.5 mM calcium chloride. Subsequently, associated FVIIIC was detected by autoradiography with a 125-I-labelled monoclonal mouse anti-(human FVIIIC) antibody. The results of recombination experiments exclusively showed prominent staining of the Mr= 0.44 x 106 vWF band in the autoradiography. However, proteolytically degraded FVIIIC with partly retained procoagulant activity did not show a positive stain. The results indicate that an intact FVIIIC molecule and the smallest multimer of vWF are required for the formation of a stable FVIII/vWF complex.

THE EFFECT OF PHORBOL DIESTERS AND INTERLEUKINS ON THE SECRETION OF VON WILLEBRAND FACTOR BY HUMAN ENDOTHELIAL CELLS IN CULTURE

1987 ◽  
Author(s):  
J C Giddings ◽  
L Shall
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Umbilical Vein ◽  
Interleukin 1 ◽  
Interleukin 2 ◽  
Morphological Evidence ◽  
Adhesive Properties ◽  
Umbilical Vein Endothelial Cells ◽  
Von Willebrand ◽  
Willebrand Factor

Human umbilical vein endothelial cells (EC) were cultured in the presence of 4p-phorbol 12-myristate 13-acetate (PMA, 10ug/l), interleukin 1 (IL-1, 1 unit/ml) and interleukin 2 (IL-2, 1 unit/ml), and secretion of von Willebrand factor activity (vWF, Ristocetin co-factor) and von Willebrand factor antigen (vWFAG, ELISA Technique) measured at intervals. Confluent control EC were treated with PMA, IL-1 and IL-2, and the supernatant medium assayed for release of vWF and vWFAg. Treated cells were also examined for vWFAg by immuno-fluorescence. The levels of both vWF and vWFAg in cultures containing IL-1 were significantly higher than those in control cultures after 5-6 days growth. Moreover, vWF and vWFAg increased significantly in the supernatant of confluent control EC incubated further in the presence of IL-1. Furthermore, the characteristic fluorescence pattern of endothelial vWFAg was markedly reduced in EC treated with IL-1. The levels of vWF and vWFAg in cultures containing PMA were also significantly higher than those of control cultures. In these conditions, however, the growth of cells appeared to be enhanced, and confluence was observed after about 6 days in the presence of PMA compared to 9 - 10 days in control cultures. The mean levels of vWF and vWFAg in the supernatant of EC incubated with PMA were higher than the control values but the differences were not statistically significant. Immunofluorescence of PMA-treated cells suggested that vWFAg might be less granular than in control cells but the differences were not as marked as those seen with IL-1. The results of all assays in the presence of IL-2 were not significantly different from those of control cells. In all instances no morphological evidence of endothelial injury was observed and more than 90% of cells remained viable at the termination of cultures. The results indicated that the synthesis and release of vWF were increased in the presence of PMA, and secretion of vWF was stimulated by IL-1. The data suggest that secreted vWF might contribute to the previously reported enhanced procoagulant and adhesive properties of EC treated with these substances.

scholarly journals Sulfation of von Willebrand factor

Blood ◽  
1990 ◽  
Vol 76 (12) ◽  
pp. 2530-2539 ◽  
Author(s):  
JA Carew ◽  
PJ Browning ◽  
DC Lynch
Keyword(s):  
Amino Acids ◽  
Von Willebrand Factor ◽  
Time Course ◽  
Umbilical Vein ◽  
Cell Monolayer ◽  
Sodium Chlorate ◽  
Normal Hemostasis ◽  
Umbilical Vein Endothelial Cells ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract von Willebrand factor (vWF) is a multimeric adhesive glycoprotein essential for normal hemostasis. We have discovered that cultured human umbilical vein endothelial cells incorporate inorganic sulfate into vWF. Following immunoisolation and analysis by polyacrylamide or agarose gel electrophoresis, metabolically labeled vWF was found to have incorporated [35S]-sulfate into all secreted multimer species. The time course of incorporation shows that sulfation occurs late in the biosynthesis of vWF, near the point at which multimerization occurs. Quantitative analysis suggests the presence, on average, of one molecule of sulfate per mature vWF subunit. Virtually all the detectable sulfate is released from the mature vWF subunit by treatment with endoglycosidases that remove asparagine-linked carbohydrates. Sulfated carbohydrate was localized first to the N-terminal half of the mature subunit (amino acids 1 through 1,365) by partial proteolytic digestion with protease V8; and subsequently to a smaller fragment within this region (amino acids 273 through 511) by sequential digestions with protease V8 and trypsin. Thus, the carbohydrate at asparagine 384 and/or 468 appears to be the site of sulfate modification. Sodium chlorate, an inhibitor of adenosine triphosphate- sulfurylase, blocks sulfation of vWF without affecting either the ability of vWF to assemble into high molecular weight multimers or the ability of vWF multimers to enter Weible-Palade bodies. The stability of vWF multimers in the presence of an endothelial cell monolayer also was unaffected by the sulfation state. Additionally, we have found that the cleaved propeptide of vWF is sulfated on asparagine-linked carbohydrate.

scholarly journals Molecular studies of von Willebrand disease: reduced von Willebrand factor biosynthesis, storage, and release in endothelial cells derived from patients with type I von Willebrand disease [published erratum appears in Blood 1990 Nov 1;76(9):1901]

Blood ◽  
1990 ◽  
Vol 75 (7) ◽  
pp. 1466-1472 ◽  
Author(s):  
BM Ewenstein ◽  
A Inbal ◽  
JS Pober ◽  
RI Handin
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Messenger Rna ◽  
Umbilical Vein ◽  
Von Willebrand Disease ◽  
Type I ◽  
Molecular Defects ◽  
Umbilical Vein Endothelial Cells ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Endothelial cells were cultured from the umbilical veins of two neonates with type I von Willebrand disease (vWD) and compared with cells cultured in parallel from normal control umbilical veins. In both cases, cultured vWD endothelial cells contained less messenger RNA (mRNA) encoding von Willebrand factor (vWF), and constitutively secreted two- to fourfold less vWF protein than their matched controls. Regulated secretion of stored vWF induced by thrombin or phorbol-12- myristate-13-acetate (PMA) was also diminished in vWD cells. Both the mRNA and protein produced by each of these type I vWD cells appeared to be of normal size. However, despite the diminished size of the vWF storage pool, electron microscopy of endothelial cells in situ showed normal appearing vWF storage organelles (Weibel-Palade bodies). These studies show that cultured umbilical vein endothelial cells can be used to explore the molecular defects in type I and perhaps other forms of vWD, and suggest that at least some forms of type I vWD are caused by diminished mRNA transcription or subsequent translation due to a defective vWF allele.

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