A more recent version of this article appeared on April 13, 2001

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(Circulation Research. 2001;0:hh0701.088769.)
© 2001 American Heart Association, Inc.

Online First Article

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

Jean-Marc Olivot, Eva Estebanell, Monique Lafay, Brigitte Brohard, Martine Aiach Francine Rendu

From the Unité INSERM 428, Faculté de Pharmacie, Université René Descartes, Paris, France. Present address for J.-M.O. is Service de Neurologie, Hôpital Tenon, Paris, France. Present address for E.E. is Servicio de hemoterapia y hemostasia, Hospital Clinic, Barcelona, Spain.

Correspondence to Dr Francine Rendu, U428 INSERM, Faculté de Pharmacie, 4 avenue de l’Observatoire, F-75270, Paris, Cedex, France. E-mail rendu{at}pharmacie.univ-paris5.fr

Abstract

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.

Key Words: thrombin • thrombomodulin • human endothelial cells • mitogen-activated protein kinase

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