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

Abstract

SummaryThrombin interacts with its receptor and thrombomodulin on endothelial cells. We evaluated the respective roles of these two proteins on human umbilical vein endothelial cell (HUVEC) growth by comparing thrombin, S195A (a mutant thrombin in which the serine of the charge stabilizing system had been replaced by alanine), and the receptor activating peptide (TRAP). Thrombin and TRAP induced DNA synthesis (half maximal cell proliferation with 5 nM and 25 μM, respectively), whereas S195A thrombin was inactive, inferring that growth is mediated through the thrombin receptor. Surprisingly, cells stimulated by TRAP exhibited a maximal proliferation twice greater than that obtained with thrombin. Combination of thrombin and TRAP resulted in a mitogenic response higher than by thrombin alone, but lower than by TRAP alone. The role of thrombomodulin was evaluated by adding an anti-thrombomodulin antibody, which prevents formation of the thrombin-thrombomodulin complex. Antibody did not interfere with cell proliferation induced by TRAP, but enhanced that induced by thrombin. We conclude that formation of the thrombin-thrombomodulin complex restrains HUVEC proliferation mediated through the thrombin receptor.

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

“…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).…”

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

“…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.…”

“…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.…”

“…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.…”

“…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.…”