Thrombin cleaves and activates the protease-activated receptor 2 dependent on thrombomodulin co-receptor availability

Introduction: Protease-activated receptors (PARs) evolved to react to extracellular proteolytic activity. In mammals, three of the four PARs (PAR1, PAR3, and PAR4) that are expressed respond to the prototypical procoagulant enzyme thrombin, whereas PAR2 was assumed to resist activation by thrombin. To date, involvement of cell surface thrombin-recruiting co-receptors such as thrombomodulin (TM), which potentially facilitates PAR2 cleavage, has not been addressed. Thus, we examined whether TM-bound thrombin cleaved PAR2 and tested biological responses such as nuclear factor kappa B (NF-kappa B) DNA binding activity and cytokine release. Materials and methods: We examined 293T cells overexpressing PAR2 and TM for thrombin recruitment by TM promoting PAR2 cleavage. To test for the TM-thrombin interactions required for PAR2 cleavage and to map cleavage sites on PAR2, mutant constructs of TM or PAR2 were engineered. Biological effects because of PAR2 activation were investigated using an NF-kappa B reporter system and cytokine release. Results and conclusions: We identified that, at low to moderate concentrations, thrombin cleaved PAR2 in a TM co-receptor-dependent manner with cleavage efficiency comparable to that of trypsin. In TM's presence, thrombin efficiently cleaved both, PAR1 and PAR2, albeit kinetics differed. Whereas the majority of surface expressed PAR1 was immediately cleaved off, prolonged exposure to thrombin resulted in few additional cleavage. In contrast, PAR2 cleavage was sustained upon prolonged exposure to thrombin. However, TM EGF-like domain 5 was required and TM chondroitin sulfate (CS) proteoglycan sites serine 490 and serine 492 assisted in PAR2 cleavage, while thrombin preferentially cleaved at arginine 36 on PAR2's N-terminus. Note that thrombin-induced activation of NF-kappa B via PAR2 resulted in release of interleukin-8. Thus, we provide a novel concept of how thrombin efficiently cleaves PAR2 in a TM-dependent manner, resulting in pro-inflammatory interleukin-8 release. This unexpected pro-inflammatory role of TM, promoting cleavage and activation of PAR2 by thrombin, may lead to novel therapeutic options for treating inflammatory and malignant diseases.