The single-stranded DNA aptamer-binding site of human thrombin
A new class of thrombin inhibitors based on sequence-specific single-stranded DNA oligonucleotides (thrombin aptamer) has recently been identified. The aptamer-binding site on thrombin was examined by a solid-phase plate binding assay and by chemical modification. Binding assay results demonstrated...
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| Veröffentlicht in: | The Journal of biological chemistry 1993-10, Vol.268 (28), p.20808-20811 |
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| container_title | The Journal of biological chemistry |
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| creator | PABORSKY, L. R MCCURDY, S. N GRIFFIN, L. C TOOLE, J. J LEUNG, L. K |
| description | A new class of thrombin inhibitors based on sequence-specific single-stranded DNA oligonucleotides (thrombin aptamer) has
recently been identified. The aptamer-binding site on thrombin was examined by a solid-phase plate binding assay and by chemical
modification. Binding assay results demonstrated that the thrombin aptamer bound specifically to alpha-thrombin but not to
gamma-thrombin and that hirudin competed with aptamer binding, suggesting that thrombin's anion-binding exosite was important
for aptamer-thrombin interactions. To identify lysine residues of thrombin that participated in the binding of the thrombin
aptamer, thrombin was modified with fluorescein 5'-isothiocyanate in the presence or absence of the thrombin aptamer, reduced,
carboxymethylated, and digested with endoproteinase Arg-C. The digestion products were analyzed by reversed-phase high performance
liquid chromatography and the peptide maps compared. Four peptides with significantly decreased modification in the presence
of the aptamer were identified and subjected to N-terminal sequence analysis. Results indicated that B chain Lys-21 and Lys-65,
both located within the anion-binding exosite, are situated within or in close proximity to the aptamer-binding site of human
alpha-thrombin. The thrombin aptamer binds to the anion-binding exosite and inhibits thrombin's function by competing with
exosite binding substrates fibrinogen and the platelet thrombin receptor. |
| doi_str_mv | 10.1016/s0021-9258(19)36856-5 |
| format | Article |
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recently been identified. The aptamer-binding site on thrombin was examined by a solid-phase plate binding assay and by chemical
modification. Binding assay results demonstrated that the thrombin aptamer bound specifically to alpha-thrombin but not to
gamma-thrombin and that hirudin competed with aptamer binding, suggesting that thrombin's anion-binding exosite was important
for aptamer-thrombin interactions. To identify lysine residues of thrombin that participated in the binding of the thrombin
aptamer, thrombin was modified with fluorescein 5'-isothiocyanate in the presence or absence of the thrombin aptamer, reduced,
carboxymethylated, and digested with endoproteinase Arg-C. The digestion products were analyzed by reversed-phase high performance
liquid chromatography and the peptide maps compared. Four peptides with significantly decreased modification in the presence
of the aptamer were identified and subjected to N-terminal sequence analysis. Results indicated that B chain Lys-21 and Lys-65,
both located within the anion-binding exosite, are situated within or in close proximity to the aptamer-binding site of human
alpha-thrombin. The thrombin aptamer binds to the anion-binding exosite and inhibits thrombin's function by competing with
exosite binding substrates fibrinogen and the platelet thrombin receptor.</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>DOI: 10.1016/s0021-9258(19)36856-5</identifier><identifier>PMID: 8407909</identifier><identifier>CODEN: JBCHA3</identifier><language>eng</language><publisher>Bethesda, MD: American Society for Biochemistry and Molecular Biology</publisher><subject>Amino Acid Sequence ; Analytical, structural and metabolic biochemistry ; Base Sequence ; Binding Sites ; Biological and medical sciences ; DNA, Single-Stranded - chemistry ; DNA, Single-Stranded - metabolism ; Enzymes and enzyme inhibitors ; Fluorescein-5-isothiocyanate ; Fundamental and applied biological sciences. Psychology ; Hirudins - metabolism ; Humans ; Hydrolases ; Lysine - analysis ; Molecular Sequence Data ; Thrombin - metabolism</subject><ispartof>The Journal of biological chemistry, 1993-10, Vol.268 (28), p.20808-20811</ispartof><rights>1994 INIST-CNRS</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c506t-1348dc31e78b622c015006e3e149ca5526a6bed0c270a56750e5f5ca9a53f8923</citedby><cites>FETCH-LOGICAL-c506t-1348dc31e78b622c015006e3e149ca5526a6bed0c270a56750e5f5ca9a53f8923</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=3823937$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/8407909$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>PABORSKY, L. R</creatorcontrib><creatorcontrib>MCCURDY, S. N</creatorcontrib><creatorcontrib>GRIFFIN, L. C</creatorcontrib><creatorcontrib>TOOLE, J. J</creatorcontrib><creatorcontrib>LEUNG, L. K</creatorcontrib><title>The single-stranded DNA aptamer-binding site of human thrombin</title><title>The Journal of biological chemistry</title><addtitle>J Biol Chem</addtitle><description>A new class of thrombin inhibitors based on sequence-specific single-stranded DNA oligonucleotides (thrombin aptamer) has
recently been identified. The aptamer-binding site on thrombin was examined by a solid-phase plate binding assay and by chemical
modification. Binding assay results demonstrated that the thrombin aptamer bound specifically to alpha-thrombin but not to
gamma-thrombin and that hirudin competed with aptamer binding, suggesting that thrombin's anion-binding exosite was important
for aptamer-thrombin interactions. To identify lysine residues of thrombin that participated in the binding of the thrombin
aptamer, thrombin was modified with fluorescein 5'-isothiocyanate in the presence or absence of the thrombin aptamer, reduced,
carboxymethylated, and digested with endoproteinase Arg-C. The digestion products were analyzed by reversed-phase high performance
liquid chromatography and the peptide maps compared. Four peptides with significantly decreased modification in the presence
of the aptamer were identified and subjected to N-terminal sequence analysis. Results indicated that B chain Lys-21 and Lys-65,
both located within the anion-binding exosite, are situated within or in close proximity to the aptamer-binding site of human
alpha-thrombin. The thrombin aptamer binds to the anion-binding exosite and inhibits thrombin's function by competing with
exosite binding substrates fibrinogen and the platelet thrombin receptor.</description><subject>Amino Acid Sequence</subject><subject>Analytical, structural and metabolic biochemistry</subject><subject>Base Sequence</subject><subject>Binding Sites</subject><subject>Biological and medical sciences</subject><subject>DNA, Single-Stranded - chemistry</subject><subject>DNA, Single-Stranded - metabolism</subject><subject>Enzymes and enzyme inhibitors</subject><subject>Fluorescein-5-isothiocyanate</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Hirudins - metabolism</subject><subject>Humans</subject><subject>Hydrolases</subject><subject>Lysine - analysis</subject><subject>Molecular Sequence Data</subject><subject>Thrombin - metabolism</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1993</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkU1LJDEQhoO46Oj6E4Q-iOih3UoylU4ugvgNsh50wVtIp6vtlv4Ykx6W_feb0WE8GgpyeJ-qgqcYO-RwxoGrXxFA8NwI1CfcnEqlUeW4xWYctMwl8pdtNtsgu2wvxjdIb274DtvRcygMmBk7f24oi-3w2lEep-CGiqrs6vdF5haT6ynkZTtUKU7MRNlYZ82yd0M2NWHsU_ST_ahdF-lg_e-zPzfXz5d3-cPj7f3lxUPuEdSUcznXlZecCl0qITxwBFAkic-Nd4hCOVVSBV4U4FAVCIQ1emccylobIffZ8efcRRjflxQn27fRU9e5gcZltAUaIwrDvwW5QpN2FwnET9CHMcZAtV2Etnfhn-VgV4Lt08qeXdmz3NgPwRZT3-F6wbLsqdp0rY2m_Gidu-hdVyelvo0bTGohjSy-sKZ9bf62gWzZjr6h3gqlrUgFOh3yPzPJjPk</recordid><startdate>19931005</startdate><enddate>19931005</enddate><creator>PABORSKY, L. R</creator><creator>MCCURDY, S. N</creator><creator>GRIFFIN, L. C</creator><creator>TOOLE, J. J</creator><creator>LEUNG, L. K</creator><general>American Society for Biochemistry and Molecular Biology</general><scope>IQODW</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>7TM</scope><scope>7X8</scope></search><sort><creationdate>19931005</creationdate><title>The single-stranded DNA aptamer-binding site of human thrombin</title><author>PABORSKY, L. R ; MCCURDY, S. N ; GRIFFIN, L. C ; TOOLE, J. J ; LEUNG, L. K</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c506t-1348dc31e78b622c015006e3e149ca5526a6bed0c270a56750e5f5ca9a53f8923</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1993</creationdate><topic>Amino Acid Sequence</topic><topic>Analytical, structural and metabolic biochemistry</topic><topic>Base Sequence</topic><topic>Binding Sites</topic><topic>Biological and medical sciences</topic><topic>DNA, Single-Stranded - chemistry</topic><topic>DNA, Single-Stranded - metabolism</topic><topic>Enzymes and enzyme inhibitors</topic><topic>Fluorescein-5-isothiocyanate</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Hirudins - metabolism</topic><topic>Humans</topic><topic>Hydrolases</topic><topic>Lysine - analysis</topic><topic>Molecular Sequence Data</topic><topic>Thrombin - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>PABORSKY, L. R</creatorcontrib><creatorcontrib>MCCURDY, S. N</creatorcontrib><creatorcontrib>GRIFFIN, L. C</creatorcontrib><creatorcontrib>TOOLE, J. J</creatorcontrib><creatorcontrib>LEUNG, L. K</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Nucleic Acids Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>PABORSKY, L. R</au><au>MCCURDY, S. N</au><au>GRIFFIN, L. C</au><au>TOOLE, J. J</au><au>LEUNG, L. K</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>The single-stranded DNA aptamer-binding site of human thrombin</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>1993-10-05</date><risdate>1993</risdate><volume>268</volume><issue>28</issue><spage>20808</spage><epage>20811</epage><pages>20808-20811</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><coden>JBCHA3</coden><abstract>A new class of thrombin inhibitors based on sequence-specific single-stranded DNA oligonucleotides (thrombin aptamer) has
recently been identified. The aptamer-binding site on thrombin was examined by a solid-phase plate binding assay and by chemical
modification. Binding assay results demonstrated that the thrombin aptamer bound specifically to alpha-thrombin but not to
gamma-thrombin and that hirudin competed with aptamer binding, suggesting that thrombin's anion-binding exosite was important
for aptamer-thrombin interactions. To identify lysine residues of thrombin that participated in the binding of the thrombin
aptamer, thrombin was modified with fluorescein 5'-isothiocyanate in the presence or absence of the thrombin aptamer, reduced,
carboxymethylated, and digested with endoproteinase Arg-C. The digestion products were analyzed by reversed-phase high performance
liquid chromatography and the peptide maps compared. Four peptides with significantly decreased modification in the presence
of the aptamer were identified and subjected to N-terminal sequence analysis. Results indicated that B chain Lys-21 and Lys-65,
both located within the anion-binding exosite, are situated within or in close proximity to the aptamer-binding site of human
alpha-thrombin. The thrombin aptamer binds to the anion-binding exosite and inhibits thrombin's function by competing with
exosite binding substrates fibrinogen and the platelet thrombin receptor.</abstract><cop>Bethesda, MD</cop><pub>American Society for Biochemistry and Molecular Biology</pub><pmid>8407909</pmid><doi>10.1016/s0021-9258(19)36856-5</doi><tpages>4</tpages><oa>free_for_read</oa></addata></record> |
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| source | MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Alma/SFX Local Collection |
| subjects | Amino Acid Sequence Analytical, structural and metabolic biochemistry Base Sequence Binding Sites Biological and medical sciences DNA, Single-Stranded - chemistry DNA, Single-Stranded - metabolism Enzymes and enzyme inhibitors Fluorescein-5-isothiocyanate Fundamental and applied biological sciences. Psychology Hirudins - metabolism Humans Hydrolases Lysine - analysis Molecular Sequence Data Thrombin - metabolism |
| title | The single-stranded DNA aptamer-binding site of human thrombin |
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