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A cluster of aspartic residues in the extracellular loop II of PAR 4 is important for thrombin interaction and activation of platelets
Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences. Nanyang Technology University, Singapore.
Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.ORCID iD: 0000-0002-0256-1958
Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences.ORCID iD: 0000-0002-1920-3962
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2017 (English)In: Thrombosis Research, ISSN 0049-3848, E-ISSN 1879-2472, Vol. 154, 98-106 p.Article in journal (Refereed) Published
Abstract [en]

Thrombin activates platelets via proteolytic cleavage of protease-activated receptors (PARs) 1 and 4. The two PARs have distinct but complementary roles. The mechanisms responsible for PAR1 activation by thrombin have been extensively studied. However, much less is known regarding thrombin activation of PAR4, especially the potential involvement of regions of PAR4 other than the N-terminal, which is bound to the catalytic site of thrombin. We have studied PAR4 in S. cerevisiae strainMMY12, an expression system in which the GPCR receptors are connected to a Lac Z reporter gene resulting in increased beta-galactosidase activity. This approach was used to assess PAR4 mutants to evaluate the contribution of different aspartic residues in facilitating PAR4 activation. Furthermore, peptides mimicking parts of the PAR4 N-terminal and the second extracellular loop (ECLII) were tested for their ability to inhibit platelet activation by thrombin. Binding of these peptides to gamma-thrombin was studied by monitoring the decrease in tryptophan fluorescence intensity of thrombin. We conclude that not only the N-terminal but also the electronegative aspartic residues D224, D230 and D235 (located in ECLII) are be important for PAR4 binding to thrombin. We further suggest that they play a role for the tethered ligand binding to the receptor, as mutations also affected activation in response to a PAR4-activating peptide mimicking the new N-terminal formed after cleavage. This agrees with previous results on PAR1 and thrombin binding. We suggest that the ECLII of PAR4 could be a potential target for antithrombotic drug development. (C) 2017 Elsevier Ltd. All rights reserved.

Place, publisher, year, edition, pages
PERGAMON-ELSEVIER SCIENCE LTD , 2017. Vol. 154, 98-106 p.
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Biochemistry and Molecular Biology
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URN: urn:nbn:se:liu:diva-138473DOI: 10.1016/j.thromres.2017.04.006ISI: 000402491600016OAI: oai:DiVA.org:liu-138473DiVA: diva2:1111754
Note

Funding Agencies|Swedish Research Council [K2013-65x-15060-10-3]; Swedish Heart-Lung foundation [2014-0410]

Available from: 2017-06-19 Created: 2017-06-19 Last updated: 2017-06-19

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Sanchez Centellas, DanielGudlur, SushanthVicente Carrillo, AlejandroRamström, SofiaLindahl, Tomas
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