Thrombomodulin prolongs thrombin-induced extracellular signal-regulated kinase phosphorylation and nuclear retention in endothelial cells

On endothelial cells, thrombin binds to thrombomodulin (TM), an integral me mbrane-bound glycoprotein, and to protease-activated receptors (PARs). Thro mbin binding to TM modulates endothelial cell and smooth muscle cell prolif eration mediated through PAR1. We studied the phosphorylation and nuclear t ranslocation of extracellular signal-regulated kinases (ERKs) 1 and 2 in hu man umbilical vein endothelial cells activated by thrombin. Thrombin and th rombin receptor-activating peptide (TRAP)-induced DNA synthesis were signif icantly inhibited by PD98059, an inhibitor of ERK phosphorylation. Immunobl ots of phosphorylated ERKs (pERKs) and immunocytochemical studies of pERK l ocalization revealed differences in the signal generated by thrombin and TR AP. After a short activation (15 minutes), the phosphorylation and the intr acellular localization of pERKs were the same with the 2 agonists. After 4 hours, however, pERKs were visualized in the nuclei of thrombin-activated c ells but barely detectable in TRAP-activated cells. Moreover, after 4 hours , the pERKs were visualized in the nuclei of cells stimulated by TRAP in th e 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 ER Ks are involved in human umbilical vein endothelial cell DNA synthesis medi ated by PAR agonists, that the duration of pERK nuclear retention is in inv erse ratio to the mitogenic response, and that in addition to its role in t he regulation of blood coagulation, TM acts as a thrombin receptor that mod ulates the duration of pERK nuclear retention and cell proliferation in res ponse to thrombin.