| Formulation | 20mMHepes,150mMNaCl,pH7.4 |
| MolecularWeight | |
| Storage | -80°C |
| Purity | >95%bySDS-PAGE |
| Compound | |
| Assay | Hippuryl-L-Argininehydrolysis |
| ShelfLife(properlystored) | 12months |
| ChemicalFormula | |
TheN-linkedglycosylationsites(N22,N51,N63.N86)arerepresentedbyN.TheactivesiteZn2+andresidues(S299,G336,D344)involvedinsubstratebindingareshown.
SampleGelInformation:
| Gel | Novex4-12%Bis-Tris |
|---|---|
| Load | HumanTAFI,1µgperlane |
| Buffer | MOPS |
| Standard | SeeBluePlus2;Myosin(191kDa),PhosphorylaseB(97kDa),BSA(64kDa),GlutamicDehydrogenase(51kDa),AlcoholDehydrogenase(39kDa),CarbonicAnhydrase(28kDa),MyoglobinRed(19kDa),Lysozyme(14kDa) |
Overview:
ThrombinActivatableFibrinolysisInhibitor(TAFI,Plasmapro-carboxypeptidaseB,carboxypeptidaseU)isasinglechainglycoproteinzymogen(Mr=60,000)synthesizedintheliverandcirculatingataplasmaconcentrationof50nM(1-4).Thrombin(plasmin,trypsin)cleavageofthezymogenreleasesa92aminoacidN-terminalactivationpeptidecontaining4N-linkedglycosylationsites(N22,N51,N63,N86)andtheproposedplasminogenrecognitionsite.TherateofthrombincatalyzedactivationofTAFIisincreased1250foldbyformationofaternarycomplexwiththrombomodulin(5).The309aminoacidC-terminal(Mr=35,783)catalyticdomain(TAFIa,pCPB)displaysthepropertiesofabasiccarboxypeptidase,hydrolyzinglysineandargininefromtheC-terminalpositionofpolypeptides.ThisportionofthemoleculeishomologoustotissuecarboxypeptidaseBandcontains7conservedcystineresidues(64,77,136,151,160,165,291),theactivesiteZn2+coordinationsite(H67,E69,H196)andthebasicC-terminalaminoacidsubstratebindingpocket(D257,G244,S207).
TAFIisproposedtoplayakeyroleintheinteractionbetweenprocoagulant,anticoagulantandfibrinolyticsystems(5-9).EffectivefibrinolysisresultsfromtheformationofaternarycomplexbetweentPA,plasminogenandC-terminallysineresiduesonfibrin.Plasminogenboundtofibrinismoreeffectivelyconvertedtoplasmin,therebylocalizingthelyticactivitytotheareaoftheclot.PlasmindegradationoffibringeneratesadditionalC-terminallysineresiduestherebyamplifyingthesystemlocally.TheABIlityofTAFItobindspecificallytoplasminogenandtocleaveC-terminallysinesonfibrin(andcellsurfaces)resultsindown-regulationoffibrinolysisbyreducingthenumberofplasminogenandtPAbindingsitesonfibrin.TheactivationofTAFIbythethrombin/thrombomodulincomplexcouplesboththephenomenonofcoagulationinducedinhibitionoffibrinolysisandtheprofibrinolyticeffectofactivatedproteinC.
TAFIispreparedfromfreshfrozenhumanplasmabyamodificationofthemethodofBajzar,et.al.(10),andsuppliedinHBSforstorageat-80°C.Activityisdeterminedmeasuringtherateofhydrolysisofhipuryl-L-Argfollowingactivationwiththethrombin/thrombomodulincomplex(11).
Properties:
| Localization | Plasma | |||||
|---|---|---|---|---|---|---|
| Plasmaconcentration: | 2.5µg/ml | |||||
| Modeofaction | Basiccarboxypeptidase,cleavesC-terminallysineandarginineresidues.Inhibitionoffibrinolysisbyremovalofplasminogenbindingsitesonfibrin. | |||||
| Molecularweight | 60,000 | |||||
| Extinctioncoefficient |
| |||||
| Isoelectricpoint | 5.0 | |||||
| Structure | Singlechainglycoprotein.92a.a.N-terminalactivationpeptide,309a.a.catalyticdomain,1molzinc, | |||||
| Percentcarbohydrate | 19% | |||||
| Post-translationalmodifications | 4N-linkedglycosylationsiteslocatedatresiduesN22,N51,N63,andN86oftheactivationpeptide |
