PAR-1-Stimulated Factor IXa Binding to a Small Platelet Subpopulation Requires a Pronounced and Sustained Increase of Cytoplasmic Calcium
We previously reported that only a subpopulation of PAR-1-stimulated platelets binds coagulation factor IXa, since confirmed by other laboratories. Since calcium changes have been implicated in exposure of procoagulant aminophospholipids, we have now examined calcium fluxes in this subpopulation by...
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| Veröffentlicht in: | Biochemistry (Easton) 2006-06, Vol.45 (23), p.7289-7298 |
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| creator | London, Fredda S Marcinkiewicz, Mariola Walsh, Peter N |
| description | We previously reported that only a subpopulation of PAR-1-stimulated platelets binds coagulation factor IXa, since confirmed by other laboratories. Since calcium changes have been implicated in exposure of procoagulant aminophospholipids, we have now examined calcium fluxes in this subpopulation by measuring fluorescence changes in Fura Red/AM-loaded platelets following PAR-1 stimulation. While fluorescence changes in all platelets indicated calcium release from internal stores and influx of external calcium, a subpopulation of platelets displayed a pronounced increase in calcium transients by 15 s and positive factor IXa binding by 2 min, with calcium transients sustained for 45 min. Pretreatment of platelets with Xestospongin C to inhibit IP3-mediated dense tubule calcium release, and the presence of impermeable calcium channel blockers nifedipine, SKF96365, or LaCl3, inhibited PAR-1-induced development of a subpopulation with pronounced calcium transients, factor IXa binding, and platelet support of FXa generation, suggesting the importance of both release of calcium from internal stores and influx of extracellular calcium. When platelets were stimulated in EDTA for 5−20 min before addition of calcium, factor IXa binding sites developed on a smaller subpopulation but with unchanged rate, indicating sustained opening of calcium channels and continued availability of signaling elements required for binding site exposure. While pretreatment of platelets with 100 μM BAPTA/AM (K d 160 nM) had minimal effects, 100 μM 5,5‘-dimethylBAPTA/AM (K d 40 nM) completely inhibited the appearance and function of the platelet subpopulation, indicating the importance of minor increases of cytoplasmic calcium. We conclude that PAR-1-stimulated development of factor IXa binding sites in a subpopulation of platelets is dependent upon release of calcium from internal stores leading to sustained and pronounced calcium transients. |
| doi_str_mv | 10.1021/bi060294m |
| format | Article |
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Since calcium changes have been implicated in exposure of procoagulant aminophospholipids, we have now examined calcium fluxes in this subpopulation by measuring fluorescence changes in Fura Red/AM-loaded platelets following PAR-1 stimulation. While fluorescence changes in all platelets indicated calcium release from internal stores and influx of external calcium, a subpopulation of platelets displayed a pronounced increase in calcium transients by 15 s and positive factor IXa binding by 2 min, with calcium transients sustained for 45 min. Pretreatment of platelets with Xestospongin C to inhibit IP3-mediated dense tubule calcium release, and the presence of impermeable calcium channel blockers nifedipine, SKF96365, or LaCl3, inhibited PAR-1-induced development of a subpopulation with pronounced calcium transients, factor IXa binding, and platelet support of FXa generation, suggesting the importance of both release of calcium from internal stores and influx of extracellular calcium. When platelets were stimulated in EDTA for 5−20 min before addition of calcium, factor IXa binding sites developed on a smaller subpopulation but with unchanged rate, indicating sustained opening of calcium channels and continued availability of signaling elements required for binding site exposure. While pretreatment of platelets with 100 μM BAPTA/AM (K d 160 nM) had minimal effects, 100 μM 5,5‘-dimethylBAPTA/AM (K d 40 nM) completely inhibited the appearance and function of the platelet subpopulation, indicating the importance of minor increases of cytoplasmic calcium. We conclude that PAR-1-stimulated development of factor IXa binding sites in a subpopulation of platelets is dependent upon release of calcium from internal stores leading to sustained and pronounced calcium transients.</description><identifier>ISSN: 0006-2960</identifier><identifier>EISSN: 1520-4995</identifier><identifier>DOI: 10.1021/bi060294m</identifier><identifier>PMID: 16752917</identifier><language>eng</language><publisher>United States: American Chemical Society</publisher><subject>Blood Platelets - drug effects ; Blood Platelets - metabolism ; Calcium - metabolism ; Cytoplasm - metabolism ; Factor IXa - metabolism ; Humans ; Imidazoles - pharmacology ; Ion Transport ; Lanthanum - pharmacology ; Macrocyclic Compounds ; Oxazoles - pharmacology ; Protein Binding ; Receptor, PAR-1 - physiology ; Thapsigargin - pharmacology</subject><ispartof>Biochemistry (Easton), 2006-06, Vol.45 (23), p.7289-7298</ispartof><rights>Copyright © 2006 American Chemical Society</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a538t-32ab29dcc60d5bcf86c9cefb657dff0a637c96d0af7c86d62fe536276f9de0313</citedby><cites>FETCH-LOGICAL-a538t-32ab29dcc60d5bcf86c9cefb657dff0a637c96d0af7c86d62fe536276f9de0313</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://pubs.acs.org/doi/pdf/10.1021/bi060294m$$EPDF$$P50$$Gacs$$H</linktopdf><linktohtml>$$Uhttps://pubs.acs.org/doi/10.1021/bi060294m$$EHTML$$P50$$Gacs$$H</linktohtml><link.rule.ids>230,314,776,780,881,2752,27053,27901,27902,56713,56763</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/16752917$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>London, Fredda S</creatorcontrib><creatorcontrib>Marcinkiewicz, Mariola</creatorcontrib><creatorcontrib>Walsh, Peter N</creatorcontrib><title>PAR-1-Stimulated Factor IXa Binding to a Small Platelet Subpopulation Requires a Pronounced and Sustained Increase of Cytoplasmic Calcium</title><title>Biochemistry (Easton)</title><addtitle>Biochemistry</addtitle><description>We previously reported that only a subpopulation of PAR-1-stimulated platelets binds coagulation factor IXa, since confirmed by other laboratories. Since calcium changes have been implicated in exposure of procoagulant aminophospholipids, we have now examined calcium fluxes in this subpopulation by measuring fluorescence changes in Fura Red/AM-loaded platelets following PAR-1 stimulation. While fluorescence changes in all platelets indicated calcium release from internal stores and influx of external calcium, a subpopulation of platelets displayed a pronounced increase in calcium transients by 15 s and positive factor IXa binding by 2 min, with calcium transients sustained for 45 min. Pretreatment of platelets with Xestospongin C to inhibit IP3-mediated dense tubule calcium release, and the presence of impermeable calcium channel blockers nifedipine, SKF96365, or LaCl3, inhibited PAR-1-induced development of a subpopulation with pronounced calcium transients, factor IXa binding, and platelet support of FXa generation, suggesting the importance of both release of calcium from internal stores and influx of extracellular calcium. When platelets were stimulated in EDTA for 5−20 min before addition of calcium, factor IXa binding sites developed on a smaller subpopulation but with unchanged rate, indicating sustained opening of calcium channels and continued availability of signaling elements required for binding site exposure. While pretreatment of platelets with 100 μM BAPTA/AM (K d 160 nM) had minimal effects, 100 μM 5,5‘-dimethylBAPTA/AM (K d 40 nM) completely inhibited the appearance and function of the platelet subpopulation, indicating the importance of minor increases of cytoplasmic calcium. We conclude that PAR-1-stimulated development of factor IXa binding sites in a subpopulation of platelets is dependent upon release of calcium from internal stores leading to sustained and pronounced calcium transients.</description><subject>Blood Platelets - drug effects</subject><subject>Blood Platelets - metabolism</subject><subject>Calcium - metabolism</subject><subject>Cytoplasm - metabolism</subject><subject>Factor IXa - metabolism</subject><subject>Humans</subject><subject>Imidazoles - pharmacology</subject><subject>Ion Transport</subject><subject>Lanthanum - pharmacology</subject><subject>Macrocyclic Compounds</subject><subject>Oxazoles - pharmacology</subject><subject>Protein Binding</subject><subject>Receptor, PAR-1 - physiology</subject><subject>Thapsigargin - pharmacology</subject><issn>0006-2960</issn><issn>1520-4995</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2006</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqF0d2KEzEUB_BBFLeuXvgCkhsFL0bzMZNpboTd4mqXoqVdxbtwJpOsWWeS2SQj7iP41qa0VAXBq3A4v_xzyCmKpwS_IpiS163FHFNRDfeKGakpLish6vvFDGPMSyo4PikexXiTywo31cPihPCmpoI0s-Ln-mxTknKb7DD1kHSHLkAlH9DyC6Bz6zrrrlHyCNB2gL5H6x3qdULbqR39uLtjvUMbfTvZoGN26-Cdn5zKUeC67GIC63K1dCpoiBp5gxZ3yY89xMEqtIBe2Wl4XDww0Ef95HCeFp8u3l4t3perj--Wi7NVCTWbp5JRaKnolOK4q1tl5lwJpU3L66YzBgNnjRK8w2AaNecdp0bXjNOGG9FpzAg7Ld7sc8epHXSntEsBejkGO0C4kx6s_Lvj7Fd57b9LWjPWsDoHvDgEBH876ZjkYKPSfQ9O-ylKPsdVJQj7LySCCcZoleHLPVTBxxi0OU5DsNxtWB43nO2zP8f_LQ8rzaDcAxuT_nHsQ_gmecOaWl6tt7K6_LD6fH65kSL753sPKsobPwWXf_8fD_8CGbO_NQ</recordid><startdate>20060613</startdate><enddate>20060613</enddate><creator>London, Fredda S</creator><creator>Marcinkiewicz, Mariola</creator><creator>Walsh, Peter N</creator><general>American Chemical Society</general><scope>BSCLL</scope><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>7QP</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20060613</creationdate><title>PAR-1-Stimulated Factor IXa Binding to a Small Platelet Subpopulation Requires a Pronounced and Sustained Increase of Cytoplasmic Calcium</title><author>London, Fredda S ; Marcinkiewicz, Mariola ; Walsh, Peter N</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a538t-32ab29dcc60d5bcf86c9cefb657dff0a637c96d0af7c86d62fe536276f9de0313</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2006</creationdate><topic>Blood Platelets - drug effects</topic><topic>Blood Platelets - metabolism</topic><topic>Calcium - metabolism</topic><topic>Cytoplasm - metabolism</topic><topic>Factor IXa - metabolism</topic><topic>Humans</topic><topic>Imidazoles - pharmacology</topic><topic>Ion Transport</topic><topic>Lanthanum - pharmacology</topic><topic>Macrocyclic Compounds</topic><topic>Oxazoles - pharmacology</topic><topic>Protein Binding</topic><topic>Receptor, PAR-1 - physiology</topic><topic>Thapsigargin - pharmacology</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>London, Fredda S</creatorcontrib><creatorcontrib>Marcinkiewicz, Mariola</creatorcontrib><creatorcontrib>Walsh, Peter N</creatorcontrib><collection>Istex</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Biochemistry (Easton)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>London, Fredda S</au><au>Marcinkiewicz, Mariola</au><au>Walsh, Peter N</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>PAR-1-Stimulated Factor IXa Binding to a Small Platelet Subpopulation Requires a Pronounced and Sustained Increase of Cytoplasmic Calcium</atitle><jtitle>Biochemistry (Easton)</jtitle><addtitle>Biochemistry</addtitle><date>2006-06-13</date><risdate>2006</risdate><volume>45</volume><issue>23</issue><spage>7289</spage><epage>7298</epage><pages>7289-7298</pages><issn>0006-2960</issn><eissn>1520-4995</eissn><abstract>We previously reported that only a subpopulation of PAR-1-stimulated platelets binds coagulation factor IXa, since confirmed by other laboratories. Since calcium changes have been implicated in exposure of procoagulant aminophospholipids, we have now examined calcium fluxes in this subpopulation by measuring fluorescence changes in Fura Red/AM-loaded platelets following PAR-1 stimulation. While fluorescence changes in all platelets indicated calcium release from internal stores and influx of external calcium, a subpopulation of platelets displayed a pronounced increase in calcium transients by 15 s and positive factor IXa binding by 2 min, with calcium transients sustained for 45 min. Pretreatment of platelets with Xestospongin C to inhibit IP3-mediated dense tubule calcium release, and the presence of impermeable calcium channel blockers nifedipine, SKF96365, or LaCl3, inhibited PAR-1-induced development of a subpopulation with pronounced calcium transients, factor IXa binding, and platelet support of FXa generation, suggesting the importance of both release of calcium from internal stores and influx of extracellular calcium. When platelets were stimulated in EDTA for 5−20 min before addition of calcium, factor IXa binding sites developed on a smaller subpopulation but with unchanged rate, indicating sustained opening of calcium channels and continued availability of signaling elements required for binding site exposure. While pretreatment of platelets with 100 μM BAPTA/AM (K d 160 nM) had minimal effects, 100 μM 5,5‘-dimethylBAPTA/AM (K d 40 nM) completely inhibited the appearance and function of the platelet subpopulation, indicating the importance of minor increases of cytoplasmic calcium. We conclude that PAR-1-stimulated development of factor IXa binding sites in a subpopulation of platelets is dependent upon release of calcium from internal stores leading to sustained and pronounced calcium transients.</abstract><cop>United States</cop><pub>American Chemical Society</pub><pmid>16752917</pmid><doi>10.1021/bi060294m</doi><tpages>10</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | Biochemistry (Easton), 2006-06, Vol.45 (23), p.7289-7298 |
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| source | ACS Publications; MEDLINE |
| subjects | Blood Platelets - drug effects Blood Platelets - metabolism Calcium - metabolism Cytoplasm - metabolism Factor IXa - metabolism Humans Imidazoles - pharmacology Ion Transport Lanthanum - pharmacology Macrocyclic Compounds Oxazoles - pharmacology Protein Binding Receptor, PAR-1 - physiology Thapsigargin - pharmacology |
| title | PAR-1-Stimulated Factor IXa Binding to a Small Platelet Subpopulation Requires a Pronounced and Sustained Increase of Cytoplasmic Calcium |
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