Thrombomodulin is a transmembrane proteoglycan with an ectodomain released in blood. Known as the thrombin-cofactor to inhibit coagulation, thrombomodulin has also a key role in fibrinolysis, development, cellular proliferation and inflammation. We previously demonstrated that the endothelial thrombomodulin has a specific signalling pathway and mediates Epidermal Growth Factor Receptor (EGFR) transactivation. Here, we explored the hypothesis that soluble thrombomodulin, which contains the N-terminal lectin C-like region and 6 EGF -like domains mediates the EGFR transactivation by thrombin.
Human endothelial cells were incubated with either thrombin to activate both thrombomodulin and PAR-1, or a mutant thrombin that is unable to activate PAR-1, or a recombinant soluble thrombomodulin. The EGFR tyrosine kinase was inhibited by AG1478 and metalloproteases by the pan inhibitor actinonin. Heparin-Binding EGF (HB-EGF), the metalloprotease TACE, and thrombomodulin were neutralized with monoclonal antibodies. Released soluble thrombomodulin was concentrated by ultrafiltration and quantified by ELISA. Static adhesion assays were performed with monocytic U937 cells labelled with calcein and incubated for 1h with endothelial cells.
Thrombin (20nM) activated the EGFR and its downstream effectors, the soluble tyrosine kinase Src and the Mitogen Activated Protein Kinases (MAPK), ERK1/2 and p38. Src phosphorylation was abolished by both actinonin and anti-thrombomodulin. Activation of ERK1/2 and p38 was also inhibited by actinonin and anti-thrombomodulin, but only by 50 %. Mutant thrombin and recombinant thrombomodulin stimulated the phosphorylation of EGFR and Src only, suggesting that PAR-1 signalling is required for MAPK activation. As expected, AG1478 suppressed thrombin-activated phosphorylation of Src and ERK1/2. However, neither anti-TACE nor anti-HB-EGF had any effect, indicating the participation of other metalloprotease and EGFR ligand. Interestingly, thrombin induced actinoninsensitive release of soluble thrombomodulin. Inhibition of the thrombomodulin pathway by incubating for 4h endothelial cells with both anti-thrombomodulin and thrombin twice increased the adherence of U937 monocytes compared to thrombin alone. Noteworthy, addition of soluble thrombomodulin during adhesion restored the adherence to the level of isotype-treated cells.
In conclusion, soluble thrombomodulin may negatively modulate the thrombin-induced monocyte adhesion through activation of EGFR signalling.Le texte complet de cet article est disponible en PDF.