A Role for Thrombin Receptor Signaling in Endothelial Cells During Embryonic Development

Science ◽  
2001 ◽  
Vol 293 (5535) ◽  
pp. 1666-1670 ◽  
Author(s):  
C. T. Griffin
Keyword(s):  
Endothelial Cells ◽  
Embryonic Development ◽  
Thrombin Receptor ◽  
Receptor Signaling

Two molecules related to the VEGF receptor are expressed in early endothelial cells during avian embryonic development

1993 ◽  
Vol 42 (1-2) ◽  
pp. 33-48 ◽  
Author(s):  
Anne Eichmann ◽  
Christophe Marcelle ◽  
Christiane Bréant ◽  
Nicole M. Le Douarin
Keyword(s):  
Endothelial Cells ◽  
Embryonic Development ◽  
Vegf Receptor

scholarly journals An autoantibody directed against human thrombin anion-binding exosite in a patient with arterial thrombosis: effects on platelets, endothelial cells, and protein C activation

Blood ◽  
1994 ◽  
Vol 84 (6) ◽  
pp. 1843-1850 ◽  
Author(s):  
E Arnaud ◽  
M Lafay ◽  
P Gaussem ◽  
V Picard ◽  
M Jandrot-Perrus ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Arterial Thrombosis ◽  
Protein C ◽  
Receptor Activation ◽  
Anion Binding ◽  
Thrombin Receptor ◽  
Cell Functions ◽  
Human Thrombin

Abstract An autoantibody, developed by a patient with severe and recurrent arterial thrombosis, was characterized to be directed against the anion- binding exosite of thrombin, and inhibited all thrombin interactions requiring this secondary binding site without interfering with the catalytic site. The effect of the antibody was studied on thrombin interactions with platelets and endothelial cells from human umbilical veins (HUVEC). The autoantibody specifically and concentration- dependently inhibited alpha-thrombin-induced platelet activation and prostacyclin (PGI2) synthesis from HUVEC. It had no effect when gamma- thrombin or the thrombin receptor activation peptide SFLLR were the inducers. The effect of the antibody on protein C activation has been studied. The antibody blocked the thrombin-thrombomodulin activation of protein C. The inhibition of the activation was maximal with a low concentration of thrombomodulin. The fact that the autoantibody inhibited concentration-dependent alpha-thrombin-induced platelet and endothelial cell functions emphasizes the crucial role of the anion- binding exosite of thrombin to activate its receptor. In regard to the pathology, the antibody inhibited two vascular processes implicated in thrombin-antithrombotic functions, PGI2 secretion, and protein C activation, which could be implicated in this arterial thrombotic disease.

The functional thrombin receptor is associated with the plasmalemma and a large endosomal network in cultured human umbilical vein endothelial cells

1995 ◽  
Vol 108 (3) ◽  
pp. 1155-1164 ◽  
Author(s):  
R. Horvat ◽  
G.E. Palade
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Morphological Evidence ◽  
Thrombin Receptor ◽  
Human Umbilical Vein ◽  
Cultured Endothelial Cells ◽  
Human Thrombin ◽  
Membrane Spanning ◽  
Umbilical Vein Endothelial Cells ◽  
G Protein Coupled

The functional thrombin receptor, normally expressed by endothelial cells and platelets, is a member of the G protein-coupled, seven membrane-spanning-domain receptor family and is thought to be responsible for most, if not all, the cell stimulatory effects of thrombin. Upon binding, thrombin cleaves the receptor's N-terminal ectodomain, unmasking a new N terminus, which by itself activates the receptor. Using antibodies to different domains of the human thrombin receptor, we have localized the receptor in cultured human umbilical vein endothelial cells by indirect immunofluorescence and immunoelectron microscopy. We found the receptor expressed on the plasmalemma of cultured endothelial cells in individual units rather than in clusters, at lower concentration than, and at different sites from, thrombomodulin. We also found the receptor associated with a distinct, intracellular, transferrin receptor-containing, tubulovesicular network. The thrombin receptor-positive structure spread from the perinuclear region to the periphery of the cells, exhibiting a number of varicosities interconnected by branching tubular elements, strikingly similar to an image recently described for a continuous endosomal reticulum. Our results provide morphological evidence for the presence of the functional thrombin receptor at relative low density on the surface of cultured endothelial cells (compared to thrombomodulin) and in relatively large quantities inside the cells, associated with an endosomal compartment.

Thrombin receptor-activating peptide sensitizes the human endothelial thrombin receptor

1995 ◽  
Vol 268 (1) ◽  
pp. C36-C44 ◽  
Author(s):  
H. J. Kruse ◽  
C. Mayerhofer ◽  
W. Siess ◽  
P. C. Weber
Keyword(s):  
Endothelial Cells ◽  
Ligand Binding ◽  
Umbilical Vein ◽  
Thrombin Receptor ◽  
Receptor Desensitization ◽  
Short Term ◽  
Tethered Ligand ◽  
Continuous Presence ◽  
Sensitizing Effect ◽  
Umbilical Vein Endothelial Cells

Receptor-operated effects of alpha-thrombin and of the thrombin receptor-activating peptide TRAP14 on cytoplasmic Ca2+ concentration ([Ca2+]i) were examined in fura 2-loaded endothelial cells. Experiments with hirudin showed that alpha-thrombin-induced Ca2+ influx requires the continuous presence of active alpha-thrombin. YFLLRNP, known to antagonize alpha-thrombin- and TRAP7-induced [Ca2+]i transients in platelets, did not antagonize [Ca2+]i transients in response to alpha-thrombin and TRAP14 in human umbilical vein endothelial cells (HUVEC). Repetitive short-term stimulations with alpha-thrombin desensitized [Ca2+]i transients to subsequent stimulations with either alpha-thrombin or TRAP14. In contrast, repeated short-term stimulations with TRAP14 sensitized [Ca2+]i transients to subsequent stimulations with either agonist. Blockade of Ca2+ influx by SKF-96365 abolished the sensitizing effect of TRAP14. The results indicate distinct characteristics of platelet and endothelial thrombin receptors and suggest that alpha-thrombin and TRAP14 activate the receptor differently. It appears that receptor desensitization occurs independently of TRAP14 binding and, hence, tethered ligand binding to and activation of the receptor. Persistent receptor desensitization after alpha-thrombin seems to depend on both alpha-thrombin binding to the hirudin-like receptor domain and the irreversible proteolytic cleavage of the receptor. It does not involve the TRAP14/tethered ligand binding site of the receptor. TRAP14 primes the receptor by a mechanism mediated by Ca2+ influx.

Thrombin receptor 14-amino acid peptide binds to endothelial cells and stimulates calcium transients

1992 ◽  
Vol 263 (5) ◽  
pp. L595-L601 ◽  
Author(s):  
C. Tiruppathi ◽  
H. Lum ◽  
T. T. Andersen ◽  
J. W. Fenton ◽  
A. B. Malik
Keyword(s):  
Endothelial Cells ◽  
Intracellular Calcium ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Calcium Transients ◽  
Cell Protein ◽  
Affinity Constant ◽  
Thrombin Receptor ◽  
Vascular Endothelial ◽  
Amino Terminal

We examined the binding characteristics of the recently described thrombin receptor amino-terminal peptide, SFLLRNPNDKYEPF (T. K. H. Vu, D. T. Hung, V. I. Wheaton, and S. R. Coughlin. Cell 64: 1057-1068, 1991), termed TRP-14, and its effect in activating intracellular calcium transients in pulmonary vascular endothelial cells. Binding of 125I-labeled TRP-14 was found to be saturable with a affinity constant of 2 microM and maximum binding of 41 pmol/mg of cell protein. The 125I-labeled TRP-14 also interacted with bovine pulmonary microvessel endothelial cells, human umbilical vein endothelial cells, and porcine pulmonary artery smooth muscle cells. Binding of 125I-labeled diisopropylphosphoryl (DIP)-alpha-thrombin, which is catalytically inactive but binds to thrombin receptors, was not inhibited by TRP-14 or vice versa, indicating that TRP-14 did not compete for the alpha-thrombin binding site(s) on the endothelial cell surface. TRP-14 (> 1 microM) increased the concentration of intracellular calcium ([Ca2+]i) in endothelial cells with kinetics similar to the increase in [Ca2+]i triggered by alpha-thrombin. In contrast, DIP-alpha-thrombin did not increase [Ca2+]i and also did not prevent the rise in [Ca2+]i induced by the subsequent challenge with either TRP-14 or alpha-thrombin. Because the generation of TRP-14 by the proteolytically active forms of thrombin stimulated a rise in endothelial [Ca2+]i, TRP-14 may be the agonist responsible for the activation of the alpha-thrombin receptor in pulmonary vascular endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Differential transcriptional regulation of the human thrombin receptor gene by the Sp family of transcription factors in human endothelial cells

1998 ◽  
Vol 330 (3) ◽  
pp. 1469-1474 ◽  
Author(s):  
Yaxu WU ◽  
Johannes RUEF ◽  
N. Gadiparthi RAO ◽  
Cam PATTERSON ◽  
S. Marschall RUNGE
Keyword(s):  
Endothelial Cells ◽  
Promoter Activity ◽  
Receptor Gene ◽  
Thrombin Receptor ◽  
Wild Type ◽  
Mobility Shift ◽  
Human Thrombin ◽  
Gc Box

The mitogenic effects of thrombin are mediated by a G-protein-coupled receptor. Because the effects of thrombin are strongly influenced by the expression of its receptor, an understanding of its regulatory mechanisms is essential. To identify mechanisms of human thrombin receptor (HTR) gene regulation, a series of HTR-promoter-luciferase constructs were made and transfected into human microvascular endothelial cells for analysis. Deletion from bp -303 to -164 abolished reporter gene expression. Dimethyl sulphate treatment in vivo and DNase I footprinting in vitro demonstrated that a cluster of three GC box consensus sites was occupied, and electrophoretic mobility-shift assays established that Sp1 and Sp3 both bind to this 3ʹ GC box cluster. We mutated each of the three GC boxes individually and all three collectively within this 3ʹ cluster. Basal promoter activity was decreased to 46%, 78% and 29% of control for each of the GC boxes mutated individually, and to 6% when the three were mutated collectively. To test the individual abilities of Sp1 and Sp3 to activate or repress HTR transcription, we conducted co-transfection experiments with wild-type or mutated HTR-promoter-luciferase constructs. Co-transfection with Sp1 significantly augmented wild-type HTR promoter activity. Sp3 alone did not affect activity, and inhibited Sp1-mediated activation. Competition for shared binding sites by Sp1 and Sp3 might differentially regulate HTR expression in vascular endothelial cells.

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