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J. Biol. Chem., Vol. 280, Issue 43, 35999-36006, October 28, 2005

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Endothelial Thrombomodulin Induces Ca²⁺ Signals and Nitric Oxide Synthesis through Epidermal Growth Factor Receptor Kinase and Calmodulin Kinase II^*

From the Department of Signalisation Cellulaire et Atherosclerose Precoce, Universite Paris 6-CNRS, Paris 75014, France

Endothelial membrane-bound thrombomodulin is a high affinity receptor for thrombin to inhibit coagulation. We previously demonstrated that the thrombin-thrombomodulin complex restrains cell proliferation mediated through protease-activated receptor (PAR)-1. We have now tested the hypothesis that thrombomodulin transduces a signal to activate the endothelial nitric-oxide synthase (NOS3) and to modulate G protein-coupled receptor signaling. Cultured human umbilical vein endothelial cells were stimulated with thrombin or a mutant of thrombin that binds to thrombomodulin and has no catalytic activity on PAR-1. Thrombin and its mutant dose dependently activated NO release at cell surface. Pretreatment with anti-thrombomodulin antibody suppressed NO response to the mutant and to low thrombin concentration and reduced by half response to high concentration. Thrombin receptor-activating peptide that only activates PAR-1 and high thrombin concentration induced marked biphasic Ca²⁺ signals with rapid phosphorylation of PLC₃ and NOS3 at both serine 1177 and threonine 495. The mutant thrombin evoked a Ca²⁺ spark and progressive phosphorylation of Src family kinases at tyrosine 416 and NOS3 only at threonine 495. It activated rapid phosphatidylinositol-3 kinase-dependent NO synthesis and phosphorylation of epidermal growth factor receptor and calmodulin kinase II. Complete epidermal growth factor receptor inhibition only partly reduced the activation of phospholipase C₁ and NOS3. Prestimulation of thrombomodulin did not affect NO release but reduced Ca²⁺ responses to thrombin and histamine, suggesting cross-talks between thrombomodulin and G protein-coupled receptors. This is the first demonstration of an outside-in signal mediated by the cell surface thrombomodulin receptor to activate NOS3 through tyrosine kinase-dependent pathway. This signaling may contribute to thrombomodulin function in thrombosis, inflammation, and atherosclerosis.

Received for publication, June 10, 2005 , and in revised form, August 2, 2005.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

² Supported by INSERM.

³ Supported by the International Society of Thrombosis and Hemostasis.

¹ To whom correspondence should be addressed: Unite Mixte de Recherche, 7131 CNRS-Universite Paris 6, 102 Rue Didot, Paris 75014, France. Tel.: 33-1-43-95-92-97; Fax: 33-1-43-95-93-27; E-mail: monique.dufilho{at}brs.aphp.fr.

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