Header

UZH-Logo

Maintenance Infos

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


Heuberger, Dorothea M; Franchini, Alessandro G; Madon, Jerzy; Schuepbach, Reto A (2019). Thrombin cleaves and activates the protease-activated receptor 2 dependent on thrombomodulin co-receptor availability. Thrombosis research, 177:91-101.

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 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-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.

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 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-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.

Statistics

Citations

Dimensions.ai Metrics
6 citations in Web of Science®
5 citations in Scopus®
Google Scholar™

Altmetrics

Downloads

12 downloads since deposited on 17 Jan 2020
7 downloads since 12 months
Detailed statistics

Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > University Hospital Zurich > Institute of Intensive Care Medicine
Dewey Decimal Classification:610 Medicine & health
Scopus Subject Areas:Health Sciences > Hematology
Uncontrolled Keywords:Hematology
Language:English
Date:1 May 2019
Deposited On:17 Jan 2020 12:47
Last Modified:29 Jul 2020 12:50
Publisher:Elsevier
ISSN:0049-3848
OA Status:Hybrid
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1016/j.thromres.2019.02.032
PubMed ID:30861432
Project Information:
  • : FunderSNSF
  • : Grant IDPZ00P3_136639
  • : Project TitleCytoprotection through non Anticoagulant Engineered Chimeric Activated Protein C

Download

Hybrid Open Access

Download PDF  'Thrombin cleaves and activates the protease-activated receptor 2 dependent on thrombomodulin co-receptor availability'.
Preview
Content: Published Version
Filetype: PDF
Size: 1MB
View at publisher
Licence: Creative Commons: Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)