Abstract
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 PLC(beta3) 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 Cgamma1 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.
Highlights
Thrombin plays a key role in vessel wound healing and revascularization
We report that endothelial TM mediates Ca2ϩ spark and nitric oxide (NO) synthesis through the epidermal growth factor (EGF) receptor (EGFR) kinase and calmodulin kinase II (CaMKII)
The dose-response curve for thrombin was biphasic with a first plateau between 0.5 and 2 nM and a second one at 20 nM, suggesting that the two receptors TM and protease-activated receptor (PAR)-1 are involved in NOS3 activation (Fig. 1A)
Summary
Thrombin plays a key role in vessel wound healing and revascularization It induces multiple phenotypic changes of blood and vascular cells to affect vascular tone, cell permeability and growth, and leukocyte trafficking [5]. Thrombin mediates cellular events by signal transduction through protease-activated receptors (PARs) [6]. The purpose of the present study was to investigate whether thrombin binding to endothelial membrane-bound TM induced signaling events to activate NOS3 and modulate G protein-coupled receptor (GPCR) signals. Tel.: 33-1-43-95-92-97; Fax: EGF receptor; PAR, protease-activated receptor; TRAP, thrombin receptor-activating peptide; ERK, extracellular signal-regulated kinase; NO, nitric oxide; NOS, NO synthase; HUVEC, human umbilical vein endothelial cell; GPCR, G protein-coupled receptor; CaMKII, calmodulin kinase II; PKC, protein kinase C; PLC, phospholipase C
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
