Thrombomodulin Modulates the Mitogenic Response to Thrombin of Human Umbilical Vein Endothelial Cells

Lafay, Marina; Laguna, Roger; Bonniec, Bernard Le; Lasne, Dominique; Aiach, Martine; Rendu, Francine

doi:10.1055/s-0037-1615076

Cited by 25 publications

(25 citation statements)

References 28 publications

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“…Tem1/endosialin, however, lacks homology to thrombomodulin in the thrombin interaction domains (EGF repeats 5 and 6). An additional role for thrombomodulin is evident from its ability to inhibit arterial smooth muscle cell proliferation (32,33) and modulate mitogenic responses of endothelial cells to thrombin (34). Indeed, embryonic lethality in homozygous thrombomodulin knockout mice has been proposed to be due to thrombin-independent functions (35).…”

Section: Discussionmentioning

confidence: 99%

Molecular Characterization of the MouseTem1/endosialin Gene Regulated by Cell Density in Vitro and Expressed in Normal Tissues in Vivo

Opavský¹,

Haviernik²,

Jurkovičová³

et al. 2001

Journal of Biological Chemistry

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Human tumor endothelial marker 1/endosialin (TEM1/ endosialin) was recently identified as a novel tumor endothelial cell surface marker potentially involved in angiogenesis, although no specific function for this novel gene has been assigned so far. It was reported to be expressed in tumor endothelium but not in normal endothelium with the exception of perhaps the corpus luteum. Here we describe the cDNA and genomic sequences for the mouse Tem1/endosialin homolog, the identification and characterization of its promoter region, and an extensive characterization of its expression pattern in murine and human tissues and murine cell lines in vitro. The single copy gene that was mapped to chromosome 19 is intronless and encodes a 92-kDa protein that has 77.5% overall homology to the human protein. The remarkable findings are 1) this gene is ubiquitously expressed in normal human and mouse somatic tissues and during development, and 2) its expression at the mRNA level is density-dependent and up-regulated in serum-starved cells. In vitro, its expression is limited to cells of embryonic, endothelial, and preadipocyte origin, suggesting that the wide distribution of its expression in vivo is due to the presence of vascular endothelial cells in all the tissues. The ubiquitous expression in vivo is in contrast to previously reported expression limited to corpus luteum and highly angiogenic tissues such as tumors and wound tissue.

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Section: Discussionmentioning

confidence: 99%

Molecular Characterization of the MouseTem1/endosialin Gene Regulated by Cell Density in Vitro and Expressed in Normal Tissues in Vivo

Opavský¹,

Haviernik²,

Jurkovičová³

et al. 2001

Journal of Biological Chemistry

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“…All culture medium and material were purchased as previously described. 9 Texas Red streptavidin was from Jackson ImmunoResearch, Interchim. The nucleus marker 4,6-diamidino-2-phenylindol (DIAP) Vectashield was from Vector Laboratories.…”

Section: Methodsmentioning

confidence: 99%

“…22 As previously shown, HUVEC proliferation is more important in response to TRAP than to thrombin, at optimal concentrations, ie, 10 nmol/L and 100 mol/L for thrombin and TRAP, respectively (Figure 1). 9 Increasing concentrations of PD98059 resulted in significant decrease in DNA synthesis induced by thrombin or TRAP. The inhibitory effect of 10 mol/L PD98059 was Ͼ80% both with thrombin and TRAP.…”

Section: Effect Of Mek Inhibitor Pd98059 On 3 H-thymidine Incorporatimentioning

confidence: 96%

“…In view of the modulating effect of TM on PAR1 activation that we described on DNA synthesis in HUVECs, 9 we searched for a modulating effect of TM on ERK phosphorylation duration. For this purpose, we used 2 different tools: S195A, a mutant thrombin that binds to TM and has no proteolytic activity, 18 and a specific anti-TM antibody.…”

Section: Regulation Of the Par1-generated Activation Signal By Thrombmentioning

confidence: 99%

“…7 On endothelial cells, the inhibition of thrombin binding to TM by a monoclonal antibody 3E2, directed against the fourth EGF-like domain, 8 resulted in an increase of thrombin mitogenic effect. 9 In a wide range of tumoral cells, TM seems to exert an inhibitory effect on cell proliferation 10 independently of its thrombin cofactor activity. Thus, TM not only acts as a cofactor for protein C activation to inhibit thrombin generation but also seems to modulate the mitogenic signal triggered by PAR1 activation.…”

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confidence: 99%

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Thrombomodulin Prolongs Thrombin-Induced Extracellular Signal–Regulated Kinase Phosphorylation and Nuclear Retention in Endothelial Cells

Olivot

Estebanell

Lafay

et al. 2001

Circulation Research

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Abstract-On endothelial cells, thrombin binds to thrombomodulin (TM), an integral membrane-bound glycoprotein, and to protease-activated receptors (PARs). Thrombin binding to TM modulates endothelial cell and smooth muscle cell proliferation mediated through PAR1. We studied the phosphorylation and nuclear translocation of extracellular signal-regulated kinases (ERKs) 1 and 2 in human umbilical vein endothelial cells activated by thrombin. Thrombin and thrombin receptor-activating peptide (TRAP)-induced DNA synthesis were significantly inhibited by PD98059, an inhibitor of ERK phosphorylation. Immunoblots of phosphorylated ERKs (pERKs) and immunocytochemical studies of pERK localization revealed differences in the signal generated by thrombin and TRAP. After a short activation (15 minutes), the phosphorylation and the intracellular localization of pERKs were the same with the 2 agonists. After 4 hours, however, pERKs were visualized in the nuclei of thrombin-activated cells but barely detectable in TRAP-activated cells. Moreover, after 4 hours, the pERKs were visualized in the nuclei of cells stimulated by TRAP in the presence of a thrombin mutant that bound to TM, whereas they were around the nuclei in cells stimulated by thrombin in the presence of a monoclonal antibody preventing thrombin binding to TM. The results demonstrate that ERKs are involved in human umbilical vein endothelial cell DNA synthesis mediated by PAR agonists, that the duration of pERK nuclear retention is in inverse ratio to the mitogenic response, and that in addition to its role in the regulation of blood coagulation, TM acts as a thrombin receptor that modulates the duration of pERK nuclear retention and cell proliferation in response to thrombin. T hrombin is a multifunctional serine protease generated at sites of vascular injury. Thrombin plays a key role in blood coagulation and thrombotic disorders. It acts as the central enzyme of the coagulation cascade by cleaving fibrinogen into fibrin and favoring its own production by activating several coagulation factors by limited proteolysis. Thrombin also regulates its own formation after binding to thrombomodulin (TM), an integral membrane-bound glycoprotein expressed on endothelial cells. TM acts as a cofactor of thrombin to activate protein C, a serine protease ensuring proteolytic inactivation of 2 coagulation factors, factor Va and factor VIIIa. Thrombin also interacts with a variety of cells mediating inflammatory and proliferative responses to vascular injury. 1 For all protein and cellular interactions, thrombin has a recognition site and a catalytic active site. By the former, called the anion-binding exosite (ABE1), thrombin binds to specific negatively charged sequences. By the catalytic site, thrombin exerts its proteolytic activity. 2 On vascular endothelial cells, thrombin ABE1 binds to TM. This binding occurs through the epithelial growth factor (EGF)-like domains 4 and 5 of TM. 3 Thrombin ABE1 also binds to the typical heptahelical thrombin receptor, the first member of pr...

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Probing the activation of protein C by the thrombin-thrombomodulin complex using structural analysis, site-directed mutagenesis, and computer modeling

Knobe¹,

Berntsdotter²,

Shen³

et al. 1999

Proteins

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Thrombomodulin Modulates the Mitogenic Response to Thrombin of Human Umbilical Vein Endothelial Cells

Cited by 25 publications

References 28 publications

Molecular Characterization of the MouseTem1/endosialin Gene Regulated by Cell Density in Vitro and Expressed in Normal Tissues in Vivo

Molecular Characterization of the MouseTem1/endosialin Gene Regulated by Cell Density in Vitro and Expressed in Normal Tissues in Vivo

Thrombomodulin Prolongs Thrombin-Induced Extracellular Signal–Regulated Kinase Phosphorylation and Nuclear Retention in Endothelial Cells

Probing the activation of protein C by the thrombin-thrombomodulin complex using structural analysis, site-directed mutagenesis, and computer modeling

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