Background: Thrombin, the key clotting protease, acts on coagulation and inflammation via protease-activated receptors (PARs). PAR1 and PAR4 are major regulators of human platelet activation and vascular barrier integrity. Thrombin directly binds and cleaves PAR1 at very low nanomolar concentrations, while PAR4 lacks a thrombin binding site and therefore very high nanomolar concentrations of thrombin are required to cleave PAR4. We recently published that thrombomodulin, a co-factor expressed on both endothelial cells and leukocytes, improves PAR2 activation by thrombin, resulting in increased activation of inflammatory pathways. Thrombomodulin-dependent PAR4 activation was not addressed so far.

Aims: To test whether thrombomodulin recruitment of thrombin to the cell surface leads to a more efficient cleavage and activation of membrane-bound PAR4 compared to soluble thrombin.

Methods: To observe cleavage and activation of PAR4 by various thrombin concentrations, plasmids containing PAR4 tagged with an N-terminal alkaline phosphatase (AP) were transfected with a thrombomodulin plasmid in HEK 293T cells. The cleavage of the AP-PAR4 construct was detected by an established assay measuring activity of the cell bound and the released AP.

Results: We found that PAR4 was cleaved only at high thrombin concentrations of 30 nM in absence of thrombomodulin, whereas in presence of thrombomodulin PAR4 was significantly cleaved at low thrombin concentrations of 1 nM.

Conclusions: For the first time, we showed a switch of PAR4 from high- to low-dose thrombin receptor, which was caused by the thrombin binding to the endothelial receptor thrombomodulin. Whether the enhanced efficiency of PAR4 cleavage by thrombin in presence of thrombomodulin results in increased PAR4 activation will be studied by an in vitroNF-kB luciferase reporter system. The clinical relevance of PAR4 activation by low concentrations of thrombomodulin-bound thrombin will be tested on vascular barrier integrity as well as platelet activation.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/thrombomodulin-makes-the-difference-novel-role-of-protease-activated-receptor-4-as-low-dose-thrombin-receptor/