Formulation | 20mMHepes,150mMNaCl,pH7.4 |
MolecularWeight | |
Storage | -80°C |
Purity | >95%bySDS-PAGE |
Compound | |
Assay | Hippuryl-L-Argininehydrolysis |
ShelfLife(properlystored) | 12months |
ChemicalFormula | |
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) |
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).
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 |
SouthernBiotech/Goat Anti-Human Lambda-Alexa Fluor® 555/2070-32/1.0 mg
vectorlabs/Biotinylated Aleuria Aurantia Lectin (AAL)/B-1395/1 mg
vectorlabs/VECTASTAIN® Elite® ABC-HRP Kit (Peroxidase, Mouse IgG)/PK-6102/1 kit
vectorlabs/VECTASTAIN® Elite® ABC-HRP Kit (Peroxidase, Universal)/PK-6200/1 kit
vectorlabs/Unconjugated Musa Paradisiaca (Banana) Lectin (BanLec)/L-1410/5 mg
GiottoBiotech/Calmodulin N60D/5 mg/G02CLM60cn