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

Abstract

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-κB) 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-κB 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 EGFlike 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-κB 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.

Staphylococcus aureus and Neutrophil Extracellular Traps: The Master Manipulator Meets Its Match in Immunothrombosis

“…FXII induces NET formation via uPAR (urokinase plasminogen activator receptor)induced pAkt2 signaling, 53 while thrombin activates protease activated receptors (PAR1/2/4) on platelets and potentiates platelet-induced NETosis. 71,72 NETs and VWF VWF is a multimeric protein released by activated platelets and by exocytosis of Weibel-Palade bodies from activated endothelial cells. 73 Once released in the vasculature, ultra-large VWF multimers elongate in response to shear stress from the flowing bloodstream and expose the A1 domain, which can recruit neutrophils by binding to their PSGL-1 and αmβ2 receptors 74 (Figure 3) but also directly binds to platelets and S. aureus.…”

Piezo1 activation attenuates thrombin-induced blebbing in breast cancer cells

“…2). Thrombin has been reported to act directly on PAR2, but may also transactivate PAR2 via the PAR1 tethered ligand [14][15][16] . Notably, we observed Ca 2+ oscillations following agonist-activation of PAR2, that were not observed in thrombin treated cells (Fig.…”

“…2e, f (Supplementary Video 2). Thrombin has the capacity to directly and indirectly activate PAR2 [14][15][16] and PAR2 expression is elevated in breast tumor specimens and in breast cancer cell lines, including MDA-MB-231 cells 34 . Exposure of MDA-MB-231 cells to the PAR2 agonist SLIGRL mimicked the effects of thrombin with respect to bleb induction and increased cytosolic Ca 2+ (Fig.…”

“…Consequently, cancer cells may exist within, and contribute to, a milieu enriched in various components of the coagulation cascade, including thrombin 12 (reviewed in 13 ). Thrombin acts on the G protein-coupled protease activated receptors (PARs) 1-4 [14][15][16][17][18] to irreversibly sever a part of their N-terminal extracellular domain exposing a tethered ligand, which activates the receptor through an intramolecular interaction. Thrombin activation of PARs results in phospholipase-C b (PLCb)-mediated release of inositol 1,4,5-trisphosphate (IP3) from phosphatidylinositol 4,5-bisphosphate (PIP2), leaving diacylglycerol (DAG) in the PM, which can bind protein kinase C (PKC).…”

The development of proteinase-activated receptor-2 modulators and the challenges involved

“…Whilst trypsin could be thought of as the endogenous activator of PAR2 in the intestine, trypsin was unlikely to fulfil this role for PAR2 in other systems. Indeed, over the years the list of proteases capable of PAR2 activation and/or disarming the receptor has grown, and now includes tryptase [9], tissue factor (TF)/factor VIIa (FVIIa) complex [10,11], matriptase [12], thrombin [13,14], cathepsins [15], kallikreins and human leukocyte elastase [16,17], plus, many more exogenous nonmammalian proteases [4,18] which contribute towards PAR2-mediated inflammatory activity (for a comprehensive review see [4,19]). These proteases presumably bind with different affinities and display different enzymatic activities depending on the physiological context.…”