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Article: Kinetic analysis of a unique direct prothrombinase, fgl2, and identification of a serine residue critical for the prothrombinase activity

TitleKinetic analysis of a unique direct prothrombinase, fgl2, and identification of a serine residue critical for the prothrombinase activity
Authors
Issue Date2002
PublisherAmerican Association of Immunologists. The Journal's web site is located at http://www.jimmunol.org
Citation
Journal Of Immunology, 2002, v. 168 n. 10, p. 5170-5177 How to Cite?
Abstractfgl2 prothrombinase, by its ability to generate thrombin, has been shown to be pivotal to the pathogenesis of viral-induced hepatitis, cytokine-induced fetal loss syndrome, and xeno- and allograft rejection. In this study, the molecular basis of fgl2 prothrombinase activity was examined in detail. Purified fgl2 protein generated in a baculovirus expression system had no measurable prothrombinase activity, whereas the activity was restored when the purified protein was reconstituted into phosphatidyl-L-serine-containing vesicles. Reconstituted fgl2 catalyzed the cleavage of human prothrombin to thrombin with kinetics consistent with a first order reaction, with an apparent Vmax value of 6 mol/min/mol fgl2 and an apparent Km value for prothrombin of 8.3 μM. The catalytic activity was totally dependent on calcium, and factor Va (500 nM) enhanced the catalytic efficiency of fgl2 by increasing the apparent Vmax value to 3670 mol/min/mol fgl2 and decreasing the apparent Km value for prothrombin to 7.2 μM. By a combination of site-directed mutagenesis and production of truncated proteins, it was clearly shown that residue Ser89 was critical for the prothrombinase activity of fgl2. Furthermore, fgl2 prothrombinase activity was not inhibited by antithrombin III, soybean trypsin inhibitor, 4-aminobenzamidine, aprotinin, or phenylmethylsulfonyl fluoride, whereas diisopropylfluorophosphate completely abrogated the activity. In this work we provide direct evidence that fgl2 cleaves prothrombin to thrombin consistent with serine protease activity and requires calcium, phospholipids, and factor Va for its full activity.
Persistent Identifierhttp://hdl.handle.net/10722/149610
ISSN
2015 Impact Factor: 4.985
2015 SCImago Journal Rankings: 3.549
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorChan, CWYen_US
dc.contributor.authorChan, MWCen_US
dc.contributor.authorLiu, Men_US
dc.contributor.authorFung, Len_US
dc.contributor.authorCole, EHen_US
dc.contributor.authorLeibowitz, JLen_US
dc.contributor.authorMarsden, PAen_US
dc.contributor.authorClark, DAen_US
dc.contributor.authorLevy, GAen_US
dc.date.accessioned2012-06-26T05:55:57Z-
dc.date.available2012-06-26T05:55:57Z-
dc.date.issued2002en_US
dc.identifier.citationJournal Of Immunology, 2002, v. 168 n. 10, p. 5170-5177en_US
dc.identifier.issn0022-1767en_US
dc.identifier.urihttp://hdl.handle.net/10722/149610-
dc.description.abstractfgl2 prothrombinase, by its ability to generate thrombin, has been shown to be pivotal to the pathogenesis of viral-induced hepatitis, cytokine-induced fetal loss syndrome, and xeno- and allograft rejection. In this study, the molecular basis of fgl2 prothrombinase activity was examined in detail. Purified fgl2 protein generated in a baculovirus expression system had no measurable prothrombinase activity, whereas the activity was restored when the purified protein was reconstituted into phosphatidyl-L-serine-containing vesicles. Reconstituted fgl2 catalyzed the cleavage of human prothrombin to thrombin with kinetics consistent with a first order reaction, with an apparent Vmax value of 6 mol/min/mol fgl2 and an apparent Km value for prothrombin of 8.3 μM. The catalytic activity was totally dependent on calcium, and factor Va (500 nM) enhanced the catalytic efficiency of fgl2 by increasing the apparent Vmax value to 3670 mol/min/mol fgl2 and decreasing the apparent Km value for prothrombin to 7.2 μM. By a combination of site-directed mutagenesis and production of truncated proteins, it was clearly shown that residue Ser89 was critical for the prothrombinase activity of fgl2. Furthermore, fgl2 prothrombinase activity was not inhibited by antithrombin III, soybean trypsin inhibitor, 4-aminobenzamidine, aprotinin, or phenylmethylsulfonyl fluoride, whereas diisopropylfluorophosphate completely abrogated the activity. In this work we provide direct evidence that fgl2 cleaves prothrombin to thrombin consistent with serine protease activity and requires calcium, phospholipids, and factor Va for its full activity.en_US
dc.languageengen_US
dc.publisherAmerican Association of Immunologists. The Journal's web site is located at http://www.jimmunol.orgen_US
dc.relation.ispartofJournal of Immunologyen_US
dc.subject.meshAnimalsen_US
dc.subject.meshBaculoviridae - Geneticsen_US
dc.subject.meshBlood Coagulation - Geneticsen_US
dc.subject.meshCho Cellsen_US
dc.subject.meshCalcium - Physiologyen_US
dc.subject.meshCricetinaeen_US
dc.subject.meshEnzyme Activation - Geneticsen_US
dc.subject.meshFemaleen_US
dc.subject.meshFibrinogen - Biosynthesis - Genetics - Metabolism - Physiologyen_US
dc.subject.meshKineticsen_US
dc.subject.meshMacrophages, Peritoneal - Enzymology - Metabolism - Virologyen_US
dc.subject.meshMiceen_US
dc.subject.meshMice, Inbred Balb Cen_US
dc.subject.meshMurine Hepatitis Virus - Physiologyen_US
dc.subject.meshMutagenesis, Site-Directeden_US
dc.subject.meshPhospholipids - Physiologyen_US
dc.subject.meshRecombinant Proteins - Biosynthesis - Metabolismen_US
dc.subject.meshSerine - Genetics - Metabolismen_US
dc.subject.meshSpodoptera - Genetics - Virologyen_US
dc.subject.meshThrombin - Metabolismen_US
dc.subject.meshThromboplastin - Biosynthesis - Genetics - Metabolism - Physiologyen_US
dc.titleKinetic analysis of a unique direct prothrombinase, fgl2, and identification of a serine residue critical for the prothrombinase activityen_US
dc.typeArticleen_US
dc.identifier.emailChan, CWY:camchan@hku.hken_US
dc.identifier.authorityChan, CWY=rp01311en_US
dc.description.naturelink_to_subscribed_fulltexten_US
dc.identifier.pmid11994472-
dc.identifier.scopuseid_2-s2.0-0037094135en_US
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-0037094135&selection=ref&src=s&origin=recordpageen_US
dc.identifier.volume168en_US
dc.identifier.issue10en_US
dc.identifier.spage5170en_US
dc.identifier.epage5177en_US
dc.identifier.isiWOS:000175480100043-
dc.publisher.placeUnited Statesen_US
dc.identifier.scopusauthoridChan, CWY=12240386600en_US
dc.identifier.scopusauthoridChan, MWC=36941295800en_US
dc.identifier.scopusauthoridLiu, M=7406299782en_US
dc.identifier.scopusauthoridFung, L=7101721367en_US
dc.identifier.scopusauthoridCole, EH=7202331632en_US
dc.identifier.scopusauthoridLeibowitz, JL=7006843902en_US
dc.identifier.scopusauthoridMarsden, PA=7201783899en_US
dc.identifier.scopusauthoridClark, DA=7404788893en_US
dc.identifier.scopusauthoridLevy, GA=35391580500en_US

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