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| Formulation | 50%(vol/vol)glycerol/5mMCaCl2 |
| Storage | -20°C |
| Purity | >95%bySDS-PAGE |
| ActivityDetermination | Clottingassay |
| ShelfLife(properlystored) | 6months |
| Gel | Novex4-12%Bis-Tris |
|---|---|
| Load | HumanFactorVa,1µgperlane |
| Buffer | MOPS |
| Standard | SeeBluePlus2;Myosin(191kDa),PhosphorylaseB(97kDa),BSA(64kDa),GlutamicDehydrogenase(51kDa),AlcoholDehydrogenase(39kDa),CarbonicAnhydrase(28kDa),MyoglobinRed(19kDa),Lysozyme(14kDa) |
| SpecialNotes | FactorVisaverylABIleprotein.Somedegradedfragmentsareusuallynoticeableinpreparations. |
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.
| Localization | Plasmaandmembranesurfaces | |||||
|---|---|---|---|---|---|---|
| Modeofaction | cofactorforfactorXaintheprothrombinasecomplex | |||||
| Molecularweight | 168,000(baseduponthecombinedmolecularweightofsubunits) | |||||
| Extinctioncoefficient |
| |||||
| Structure | twosubunits,Mr~94,000(heavychain)and74,000(lightchain)(4) | |||||
| Percentcarbohydrate | approximately8%(baseduponthecalculatedmolecularweightofhumanfactorVa) |
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