Calcium mobilization and protein kinase C activation downstream of protease activated receptor 4 (PAR4) is negatively regulated by PAR3 in mouse platelets
Thrombin activates platelets through protease activated receptors (PARs). Mouse platelets express PAR3 and PAR4. PAR3 does not signal in platelets. However, PAR4 is a relatively poor thrombin substrate and requires PAR3 as a cofactor at low thrombin concentrations. In this study we show that PAR3 al...
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| description | Thrombin activates platelets through protease activated receptors (PARs). Mouse platelets express PAR3 and PAR4. PAR3 does not signal in platelets. However, PAR4 is a relatively poor thrombin substrate and requires PAR3 as a cofactor at low thrombin concentrations. In this study we show that PAR3 also regulates PAR4 signaling. In response to thrombin (30-100 nM) or PAR4 activating peptide (AYPGKF), platelets from PAR3(-/-) mice had increased G(q) signaling compared to wild type mice as demonstrated by a 1.6-fold increase in the maximum intracellular calcium (Ca(2+)) mobilization, an increase in phosphorylation level of protein kinase C (PKC) substrates, and a 2-fold increase of Ca(2+) release from intracellular stores. Moreover, platelets from heterozygous mice (PAR3(+/-)) had an intermediate increase in maximum Ca(2+) mobilization. Treatment of PAR3(-/-) mice platelets with P2Y(12) antagonist (2MeSAMP) did not affect Ca(2+) mobilization from PAR4 in response to thrombin or AYPGKF. The activation of RhoA-GTP downstream G(12/13) signaling in response to thrombin was not significantly different between wild type and PAR3(-/-) mice. Since PAR3 influenced PAR4 signaling independent of agonist, we examined the direct interaction between PAR3 and PAR4 with bioluminescence resonance energy transfer (BRET). PAR3 and PAR4 form constitutive homodimers and heterodimers. In summary, our results demonstrate that in addition to enhancing PAR4 activation at low thrombin concentrations, PAR3 negatively regulates PAR4-mediated maximum Ca(2+) mobilization and PKC activation in mouse platelets by physical interaction. |
| doi_str_mv | 10.1371/journal.pone.0055740 |
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Mouse platelets express PAR3 and PAR4. PAR3 does not signal in platelets. However, PAR4 is a relatively poor thrombin substrate and requires PAR3 as a cofactor at low thrombin concentrations. In this study we show that PAR3 also regulates PAR4 signaling. In response to thrombin (30-100 nM) or PAR4 activating peptide (AYPGKF), platelets from PAR3(-/-) mice had increased G(q) signaling compared to wild type mice as demonstrated by a 1.6-fold increase in the maximum intracellular calcium (Ca(2+)) mobilization, an increase in phosphorylation level of protein kinase C (PKC) substrates, and a 2-fold increase of Ca(2+) release from intracellular stores. Moreover, platelets from heterozygous mice (PAR3(+/-)) had an intermediate increase in maximum Ca(2+) mobilization. Treatment of PAR3(-/-) mice platelets with P2Y(12) antagonist (2MeSAMP) did not affect Ca(2+) mobilization from PAR4 in response to thrombin or AYPGKF. The activation of RhoA-GTP downstream G(12/13) signaling in response to thrombin was not significantly different between wild type and PAR3(-/-) mice. Since PAR3 influenced PAR4 signaling independent of agonist, we examined the direct interaction between PAR3 and PAR4 with bioluminescence resonance energy transfer (BRET). PAR3 and PAR4 form constitutive homodimers and heterodimers. In summary, our results demonstrate that in addition to enhancing PAR4 activation at low thrombin concentrations, PAR3 negatively regulates PAR4-mediated maximum Ca(2+) mobilization and PKC activation in mouse platelets by physical interaction.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0055740</identifier><identifier>PMID: 23405206</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Activation ; Animals ; Biology ; Bioluminescence ; Bioluminescence Resonance Energy Transfer Techniques ; Blood clots ; Blood platelets ; Blood Platelets - metabolism ; Blotting, Western ; Calcium ; Calcium (intracellular) ; Calcium - metabolism ; Cell Adhesion Molecules - physiology ; Comparative analysis ; Energy transfer ; Enzyme Activation ; Enzyme-Linked Immunosorbent Assay ; GTP ; Guanosine triphosphate ; Intracellular ; Kinases ; Medicine ; Mice ; Mice, Knockout ; Pharmacology ; Phosphorylation ; Platelet Aggregation ; Platelets ; Protease ; Protein folding ; Protein kinase C ; Protein Kinase C - metabolism ; Protein kinases ; Protein Multimerization ; Proteinase ; Proteins ; Receptors ; Receptors, Purinergic P2Y12 - chemistry ; Receptors, Purinergic P2Y12 - metabolism ; Receptors, Thrombin - metabolism ; rhoA GTP-Binding Protein - metabolism ; RhoA protein ; Signal Transduction ; Signaling ; Studies ; Substrates ; Thrombin ; Thrombosis</subject><ispartof>PloS one, 2013-02, Vol.8 (2), p.e55740-e55740</ispartof><rights>COPYRIGHT 2013 Public Library of Science</rights><rights>2013 Arachiche et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License: https://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2013 Arachiche et al 2013 Arachiche et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c593t-8b0fe5e64fce4c9842b7a740290d8dccdc916e2c590d5ff1b509495dd92e39c03</citedby><cites>FETCH-LOGICAL-c593t-8b0fe5e64fce4c9842b7a740290d8dccdc916e2c590d5ff1b509495dd92e39c03</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3566007/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3566007/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,23847,27903,27904,53769,53771,79346,79347</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/23405206$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Lam, Wilbur</contributor><creatorcontrib>Arachiche, Amal</creatorcontrib><creatorcontrib>de la Fuente, María</creatorcontrib><creatorcontrib>Nieman, Marvin T</creatorcontrib><title>Calcium mobilization and protein kinase C activation downstream of protease activated receptor 4 (PAR4) is negatively regulated by PAR3 in mouse platelets</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Thrombin activates platelets through protease activated receptors (PARs). Mouse platelets express PAR3 and PAR4. PAR3 does not signal in platelets. However, PAR4 is a relatively poor thrombin substrate and requires PAR3 as a cofactor at low thrombin concentrations. In this study we show that PAR3 also regulates PAR4 signaling. In response to thrombin (30-100 nM) or PAR4 activating peptide (AYPGKF), platelets from PAR3(-/-) mice had increased G(q) signaling compared to wild type mice as demonstrated by a 1.6-fold increase in the maximum intracellular calcium (Ca(2+)) mobilization, an increase in phosphorylation level of protein kinase C (PKC) substrates, and a 2-fold increase of Ca(2+) release from intracellular stores. Moreover, platelets from heterozygous mice (PAR3(+/-)) had an intermediate increase in maximum Ca(2+) mobilization. Treatment of PAR3(-/-) mice platelets with P2Y(12) antagonist (2MeSAMP) did not affect Ca(2+) mobilization from PAR4 in response to thrombin or AYPGKF. The activation of RhoA-GTP downstream G(12/13) signaling in response to thrombin was not significantly different between wild type and PAR3(-/-) mice. Since PAR3 influenced PAR4 signaling independent of agonist, we examined the direct interaction between PAR3 and PAR4 with bioluminescence resonance energy transfer (BRET). PAR3 and PAR4 form constitutive homodimers and heterodimers. In summary, our results demonstrate that in addition to enhancing PAR4 activation at low thrombin concentrations, PAR3 negatively regulates PAR4-mediated maximum Ca(2+) mobilization and PKC activation in mouse platelets by physical interaction.</description><subject>Activation</subject><subject>Animals</subject><subject>Biology</subject><subject>Bioluminescence</subject><subject>Bioluminescence Resonance Energy Transfer Techniques</subject><subject>Blood clots</subject><subject>Blood platelets</subject><subject>Blood Platelets - metabolism</subject><subject>Blotting, Western</subject><subject>Calcium</subject><subject>Calcium (intracellular)</subject><subject>Calcium - metabolism</subject><subject>Cell Adhesion Molecules - physiology</subject><subject>Comparative analysis</subject><subject>Energy transfer</subject><subject>Enzyme Activation</subject><subject>Enzyme-Linked Immunosorbent Assay</subject><subject>GTP</subject><subject>Guanosine triphosphate</subject><subject>Intracellular</subject><subject>Kinases</subject><subject>Medicine</subject><subject>Mice</subject><subject>Mice, Knockout</subject><subject>Pharmacology</subject><subject>Phosphorylation</subject><subject>Platelet Aggregation</subject><subject>Platelets</subject><subject>Protease</subject><subject>Protein folding</subject><subject>Protein kinase C</subject><subject>Protein Kinase C - metabolism</subject><subject>Protein kinases</subject><subject>Protein Multimerization</subject><subject>Proteinase</subject><subject>Proteins</subject><subject>Receptors</subject><subject>Receptors, Purinergic P2Y12 - chemistry</subject><subject>Receptors, Purinergic P2Y12 - metabolism</subject><subject>Receptors, Thrombin - metabolism</subject><subject>rhoA GTP-Binding Protein - metabolism</subject><subject>RhoA protein</subject><subject>Signal Transduction</subject><subject>Signaling</subject><subject>Studies</subject><subject>Substrates</subject><subject>Thrombin</subject><subject>Thrombosis</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2013</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><sourceid>DOA</sourceid><recordid>eNptkt1u1DAQhSMEoqXwBggscVMudrFjO7FvKq1W_FSqBEJwbTn2ZPHixMFOipZH4WnxdtOqi6pcJJr55kzm6BTFS4KXhNbk3TZMsdd-OYQelhhzXjP8qDglkpaLqsT08b3vk-JZStsMUVFVT4uTkjLMS1ydFn_X2hs3dagLjfPujx5d6JHuLRpiGMH16KfrdQK0RtqM7vrQt-F3n8YIukOhPZB7ZibAoggGhjFExND5l9VX9ha5hHrY5PFr8Lvc30z-hmx2KAMU5U1dmLLIsK97GNPz4kmrfYIX8_us-P7h_bf1p8XV54-X69XVwnBJx4VocAscKtYaYEYKVja1zl6UElthjbFGkgrKDGPL25Y0HEsmubWyBCoNpmfF64Pu4ENSs61JEUqxqGsmZSYuD4QNequG6Doddypop24KIW6UjqMzHhTVIq-BSjbMsobUDROGGOCtbASvpclaF_O2qenAGujHqP2R6HGndz_UJlwryqsK4zoLnM8CMfyaII2qc8mA97qHbKAipaiFqLM3GX3zH_rwdTO10fkA17ch7zV7UbVitWCESi4ytXyAyo-Fzpmcwdbl-tEAOwyYGFKK0N7dSLDaJ_j2Z9Q-wWpOcB57dd-fu6HbyNJ_dV7wEg</recordid><startdate>20130206</startdate><enddate>20130206</enddate><creator>Arachiche, Amal</creator><creator>de la Fuente, María</creator><creator>Nieman, Marvin T</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7QO</scope><scope>7RV</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TG</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>KB0</scope><scope>KL.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>NAPCQ</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PATMY</scope><scope>PDBOC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PTHSS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope></search><sort><creationdate>20130206</creationdate><title>Calcium mobilization and protein kinase C activation downstream of protease activated receptor 4 (PAR4) is negatively regulated by PAR3 in mouse platelets</title><author>Arachiche, Amal ; de la Fuente, María ; Nieman, Marvin T</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c593t-8b0fe5e64fce4c9842b7a740290d8dccdc916e2c590d5ff1b509495dd92e39c03</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2013</creationdate><topic>Activation</topic><topic>Animals</topic><topic>Biology</topic><topic>Bioluminescence</topic><topic>Bioluminescence Resonance Energy Transfer Techniques</topic><topic>Blood clots</topic><topic>Blood platelets</topic><topic>Blood Platelets - metabolism</topic><topic>Blotting, Western</topic><topic>Calcium</topic><topic>Calcium (intracellular)</topic><topic>Calcium - metabolism</topic><topic>Cell Adhesion Molecules - physiology</topic><topic>Comparative analysis</topic><topic>Energy transfer</topic><topic>Enzyme Activation</topic><topic>Enzyme-Linked Immunosorbent Assay</topic><topic>GTP</topic><topic>Guanosine triphosphate</topic><topic>Intracellular</topic><topic>Kinases</topic><topic>Medicine</topic><topic>Mice</topic><topic>Mice, Knockout</topic><topic>Pharmacology</topic><topic>Phosphorylation</topic><topic>Platelet Aggregation</topic><topic>Platelets</topic><topic>Protease</topic><topic>Protein folding</topic><topic>Protein kinase C</topic><topic>Protein Kinase C - metabolism</topic><topic>Protein kinases</topic><topic>Protein Multimerization</topic><topic>Proteinase</topic><topic>Proteins</topic><topic>Receptors</topic><topic>Receptors, Purinergic P2Y12 - chemistry</topic><topic>Receptors, Purinergic P2Y12 - metabolism</topic><topic>Receptors, Thrombin - metabolism</topic><topic>rhoA GTP-Binding Protein - metabolism</topic><topic>RhoA protein</topic><topic>Signal Transduction</topic><topic>Signaling</topic><topic>Studies</topic><topic>Substrates</topic><topic>Thrombin</topic><topic>Thrombosis</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Arachiche, Amal</creatorcontrib><creatorcontrib>de la Fuente, María</creatorcontrib><creatorcontrib>Nieman, Marvin T</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Animal Behavior Abstracts</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Nursing & Allied Health Database</collection><collection>Ecology Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Immunology Abstracts</collection><collection>Meteorological & Geoastrophysical Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Agricultural Science Collection</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>ProQuest Public Health Database</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Technology Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>Materials Science & Engineering Collection</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest One Sustainability</collection><collection>ProQuest Central UK/Ireland</collection><collection>Advanced Technologies & Aerospace Collection</collection><collection>Agricultural & Environmental Science Collection</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Technology Collection</collection><collection>Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Materials Science Collection</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Materials Science Database</collection><collection>Nursing & Allied Health Database (Alumni Edition)</collection><collection>Meteorological & Geoastrophysical Abstracts - 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>TestCollectionTL3OpenAccess</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Arachiche, Amal</au><au>de la Fuente, María</au><au>Nieman, Marvin T</au><au>Lam, Wilbur</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Calcium mobilization and protein kinase C activation downstream of protease activated receptor 4 (PAR4) is negatively regulated by PAR3 in mouse platelets</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2013-02-06</date><risdate>2013</risdate><volume>8</volume><issue>2</issue><spage>e55740</spage><epage>e55740</epage><pages>e55740-e55740</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>Thrombin activates platelets through protease activated receptors (PARs). Mouse platelets express PAR3 and PAR4. PAR3 does not signal in platelets. However, PAR4 is a relatively poor thrombin substrate and requires PAR3 as a cofactor at low thrombin concentrations. In this study we show that PAR3 also regulates PAR4 signaling. In response to thrombin (30-100 nM) or PAR4 activating peptide (AYPGKF), platelets from PAR3(-/-) mice had increased G(q) signaling compared to wild type mice as demonstrated by a 1.6-fold increase in the maximum intracellular calcium (Ca(2+)) mobilization, an increase in phosphorylation level of protein kinase C (PKC) substrates, and a 2-fold increase of Ca(2+) release from intracellular stores. Moreover, platelets from heterozygous mice (PAR3(+/-)) had an intermediate increase in maximum Ca(2+) mobilization. Treatment of PAR3(-/-) mice platelets with P2Y(12) antagonist (2MeSAMP) did not affect Ca(2+) mobilization from PAR4 in response to thrombin or AYPGKF. The activation of RhoA-GTP downstream G(12/13) signaling in response to thrombin was not significantly different between wild type and PAR3(-/-) mice. Since PAR3 influenced PAR4 signaling independent of agonist, we examined the direct interaction between PAR3 and PAR4 with bioluminescence resonance energy transfer (BRET). PAR3 and PAR4 form constitutive homodimers and heterodimers. In summary, our results demonstrate that in addition to enhancing PAR4 activation at low thrombin concentrations, PAR3 negatively regulates PAR4-mediated maximum Ca(2+) mobilization and PKC activation in mouse platelets by physical interaction.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>23405206</pmid><doi>10.1371/journal.pone.0055740</doi><oa>free_for_read</oa></addata></record> |
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| subjects | Activation Animals Biology Bioluminescence Bioluminescence Resonance Energy Transfer Techniques Blood clots Blood platelets Blood Platelets - metabolism Blotting, Western Calcium Calcium (intracellular) Calcium - metabolism Cell Adhesion Molecules - physiology Comparative analysis Energy transfer Enzyme Activation Enzyme-Linked Immunosorbent Assay GTP Guanosine triphosphate Intracellular Kinases Medicine Mice Mice, Knockout Pharmacology Phosphorylation Platelet Aggregation Platelets Protease Protein folding Protein kinase C Protein Kinase C - metabolism Protein kinases Protein Multimerization Proteinase Proteins Receptors Receptors, Purinergic P2Y12 - chemistry Receptors, Purinergic P2Y12 - metabolism Receptors, Thrombin - metabolism rhoA GTP-Binding Protein - metabolism RhoA protein Signal Transduction Signaling Studies Substrates Thrombin Thrombosis |
| title | Calcium mobilization and protein kinase C activation downstream of protease activated receptor 4 (PAR4) is negatively regulated by PAR3 in mouse platelets |
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