| Formulation | 50%(vol/vol)glycerol/5mMCaCl2 |
| Storage | -20°C |
| Purity | >95%bySDS-PAGE |
| ActivityDetermination | Clottingassay |
| ShelfLife(properlystored) | 6months |
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ParticipationofFactorVainProthrombinaseAssemblyandexpressionoftheprothrombinasecomplexisillustrated.FactorVa,composedofaheavy(VaH)andlight(VaL)chain,bindstonegativelychargedphospholipidmembranesandeffectivelyservesasareceptorformembraneboundfactorXa(Xa).Theenzymecomplexservestoconvertthezymogenprothrombin(II)totheactiveserineproteasethrombin(IIa),inaproteolyticeventwhichremovesthefragment1.2(F1.2)portionofprothrombin.
SampleGelInformation:
| Gel | Novex4-12%Bis-Tris |
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| Load | HumanFactorVa,1µgperlane |
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| Buffer | MOPS |
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| Standard | SeeBluePlus2;Myosin(191kDa),PhosphorylaseB(97kDa),BSA(64kDa),GlutamicDehydrogenase(51kDa),AlcoholDehydrogenase(39kDa),CarbonicAnhydrase(28kDa),MyoglobinRed(19kDa),Lysozyme(14kDa) |
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| SpecialNotes | FactorVisaverylABIleprotein.Somedegradedfragmentsareusuallynoticeableinpreparations. |
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Overview:
FactorVaisacofactorfortheserineproteasefactorXa,andinthepresenceofcalciumionstheycollectivelyassembleonaphospholipidsurfacetoformtheprothrombinasecomplex(1).TheprothrombinasecomplexisresponsIBLefortherapidconversionofprothrombintothrombin.FactorVaisderivedfromthepro-cofactor,factorV,uponlimitedproteolysisbyalpha-thrombin.ThethrombincleavageoffactorVliberatestwoheavilyglycosylatedactivationpeptidesfromthecentralportionofthemoleculewhichhavenocofactorfunction.FactorVaiscomprisedofanNH2-terminalderivedheavychain(Mr=94,000)andaCOOH-terminalderivedlightchain(Mr=74,000)whichremainassociatedinthepresenceofcalciumions.Thecofactorbindstophospholipid(cellmembrane)surfacesandeffectivelyservesasareceptorformembraneboundfactorXa.Completeassemblyoftheprothrombinasecomplex(factorXa,factorVa,phospholipid,andcalcium)resultsina300,000-foldincreaseintherateofprothrombinconversionrelativetotherateobservedwithfactorXaalone.TheinteractionbetweenfactorVaandfactorXaismediatedbyboththeheavyandlightchainoffactorVa,whilethebindingofprothrombintofactorVaismediatedsolelybytheheavychain.
FactorVaispreparedbyactivatingpurifiedfactorVwiththrombinandissubsequentlypurifiedbyimmunoaffinitychromatography(2).Thisprocessresultsincofactorpreparationswhicharefreeofbothactivationpeptidesandthrombin.PurifiedfactorVaissuppliedin50%glycerol(vol/vol),5.0mMCaCl2,andshouldbestoredat-20°C.PurityisdeterminedbySDS-PAGEanalysisandactivityismeasuredinafactorVaclottingassay.
Properties:
| Localization | Plasmaandmembranesurfaces |
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| Modeofaction | cofactorforfactorXaintheprothrombinasecomplex |
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| Molecularweight | 168,000(baseduponthecombinedmolecularweightofsubunits) |
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| Extinctioncoefficient | |
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| Structure | twosubunits,Mr~94,000(heavychain)and74,000(lightchain)(4) |
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| Percentcarbohydrate | approximately8%(baseduponthecalculatedmolecularweightofhumanfactorVa) |
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