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Active Site-labeled Prothrombin Inhibits Prothrombinase in Vitro and Thrombosis in Vivo


Metadata FieldValueLanguage
dc.contributorPeter Panizzi, prp0003@auburn.eduen_US
dc.creatorKroh, Heather K.
dc.creatorPanizzi, Peter
dc.creatorTchaikovski, Svetlana
dc.creatorBaird, T. Regan
dc.creatorWei, Nancy
dc.creatorKrishnaswam, Sriram
dc.creatorTans, Guido
dc.creatorRosing, Jan
dc.creatorFurie, Bruce
dc.creatorFurie, Barbara C.
dc.creatorBock, Paul E.
dc.date.accessioned2020-09-16T15:40:32Z
dc.date.available2020-09-16T15:40:32Z
dc.date.created2011
dc.identifier10.1074/jbc.M111.230292en_US
dc.identifier.urihttps://www.jbc.org/content/286/26/23345.full.pdfen_US
dc.identifier.urihttps://aurora.auburn.edu/handle/11200/49929
dc.identifier.urihttp://dx.doi.org/10.35099/aurora-17
dc.description.abstractMouse and human prothrombin (ProT) active site specifically labeled with D-Phe-Pro-Arg-CH2Cl (FPR-ProT) inhibited tissue factor-initiated thrombin generation in platelet-rich and platelet-poor mouse and human plasmas. FPR-prethrombin 1 (Pre 1), fragment 1 (F1), fragment 1.2 (F1.2), and FPR-thrombin produced no significant inhibition, demonstrating the requirement for all three ProT domains. Kinetics of inhibition of ProT activation by the inactive ProTS195A mutant were compatible with competitive inhibition as an alternate nonproductive substrate, although FPR-ProT deviated from this mechanism, implicating a more complex process. FPR-ProT exhibited 10- fold more potent anticoagulant activity compared with ProTS195A as a result of conformational changes in the ProT catalytic domain that induce a more proteinase-like conformation upon FPR labeling. Unlike ProT and ProTS195A, the pathway of FPR-ProT cleavage by prothrombinase was redirected from meizothrombin toward formation of the FPR-prethrombin 2 (Pre 2)F1.2 inhibitory intermediate. Localization of ProT labeled with Alexa Fluor 660 tethered through FPR-CH2Cl ([AF660]FPR-ProT) during laser-induced thrombus formation in vivo in murine arterioles was examined in real time wide-field and confocal fluorescence microscopy. [AF660]FPR-ProT bound rapidly to the vessel wall at the site of injury, preceding platelet accumulation, and subsequently to the thrombus proximal, but not distal, to the vessel wall. [AF660]FPR-ProT inhibited thrombus growth, whereas [AF660]FPR-Pre 1, lacking the F1 membrane-binding domain did not bind or inhibit. Labeled F1.2 localized similarly to [AF660]FPR-ProT, indicating binding to phosphatidylserine-rich membranes, but did not inhibit thrombosis. The studies provide new insight into the mechanism of ProT activation in vivo and in vitro, and the properties of a unique exosite-directed prothrombinase inhibitor.en_US
dc.formatPDFen_US
dc.publisherThe American Society for Biochemistry and Molecular Biology, Incen_US
dc.relation.ispartofJournal of Biological Chemistryen_US
dc.relation.ispartofseries0021-9258en_US
dc.rights© 2011. This is the version of record published by ASBMB PUBLISHER and is made available under the CC-BY-NC-ND 4.0 license. Item should be cited as: Active Site-labeled Prothrombin Inhibits Prothrombinase in Vitro and Thrombosis in Vivo By: Heather K. Kroh, Peter Panizzi, Svetlana Tchaikovski, T. Regan Baird, Nancy Wei, Sriram Krishnaswamy, Guido Tans, Jan Rosing, Bruce Furie, Barbara C. Furie and Paul E. Bock- J. Biol. Chem. 2011, 286:23345-23356- ASBMBen_US
dc.subjectCoagulation Factors Enzyme Kinetics Enzyme Mechanisms Fluorescence Protein-Protein Interactions Proteolytic Enzymes Prothrombin Serine Protease Thrombosisen_US
dc.titleActive Site-labeled Prothrombin Inhibits Prothrombinase in Vitro and Thrombosis in Vivoen_US
dc.typeTexten_US
dc.type.genreJournal Article, Academic Journalen_US
dc.citation.volume286en_US
dc.citation.issue26en_US
dc.citation.spage23345en_US
dc.citation.epage23356en_US
dc.description.statusPublisheden_US
dc.description.peerreviewyesen_US

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