Thrombin, a multifunctional serine protease generated at sites of vascular injury, induces multiple phenotypic changes of blood and vascular cells to affect vascular tone, cell permeability and growth, and leukocyte trafficking. Thrombin mediates cellular events by signal transduction through two different types of thrombin receptors: protease-activated receptors (PARs) and thrombomodulin (TM). Comparison of thrombin-PAR1 as to their ability to induce proliferation, thrombin-TM complex seems to restrains cell proliferation. These regulatory processes may rely on cross-talks between TM and PARs. We investigated the TM-modulating role on
thrombin-induced signal transduction pathways in PAR1-expressed human embryonic kidney (HEK293) cells transfected with TM. In the present works, TM-expressing cells (HEK293TM) cells grew in culture as close clustered colonies, while HEK293 cells dispersed in culture. TM expression resulted in a significant decrease of cell proliferation of HEK293 cells.In vitro analysis of cell migration by wound assay showed 1.5 fold accelerated migration of the HEK293TM compared to HEK293 cells. The activation of signaling was analyzed by Western blotting using an antibody specific for the phosphorylated forms of extracellular signal–regulated kinases (ERKs), protein kinase C (PKC), and protein kinase B (PKB), key signaling events central to the action of thrombin have been identified. 7.5 nM thrombin induced transient phosphorylation of ERKs in HEK293 cells with a peak at 20 min, whereas HEK293TM cells enhanced and sustained the phosphorylation of ERKs. Increased level of ERKs phosphorylation remained on activation state at 4 hours after thrombin stimulation. Thrombin also induced a higher phosphorylation of PKC or PKB/Akt in HEK293TM compared with control HEK293 cells. Add PKC inhibitor (3 μM Ro318220) after thrombin did not alter ERK phosphrylation prolonged .The results suggested that TM could modulate signaling through PAR-1 and the regulatory function of TM might in involved in sustained ERKs phosphorylation.
