Formulation	50%glycerol/water(v/v)
Storage	-20°C
Purity	>95%bySDS-PAGE
ActivityDetermination	Clottingassay
ShelfLife(properlystored)	6months

ActivationofFactorVbyAlpha-Thrombin
TheactivationofsinglechainfactorV(330,000)byalpha-thrombinisrepresented.ArrowsindicatethesiteatwhichfactorViscleavedbythrombintoyieldtwoactivationpeptides(71,000and120,000),inadditiontotheheavy(94,000)andlight(74,000)chainswhichformVa.TheheavyandlightchainsoffactorVaremainnon-covalentlyassociatedinthepresenceofcalciumions.

SampleGelInformation:

Gel	Novex4-12%Bis-Tris
Load	HumanFactorV,1µgperlane
Buffer	MOPS
Standard	SeeBluePlus2;Myosin(191kDa),PhosphorylaseB(97kDa),BSA(64kDa),GlutamicDehydrogenase(51kDa),AlcoholDehydrogenase(39kDa),CarbonicAnhydrase(28kDa),MyoglobinRed(19kDa),Lysozyme(14kDa)
SpecialNotes	FactorVisaverylABIleprotein.Somedegradedfragmentsareusuallynoticeableinpreparations.

Overview:

FactorVisalarge,singlechain,plasmaglycoproteinwhichisanessentialcomponentinthebloodcoagulationcascade(1).Duringcoagulation,theprocofactor,factorV,isconvertedtotheactivecofactor,factorVa,vialimitedproteolysisbytheserineproteasealpha-thrombin(illustratedabove),andlessefficientlybyfactorXa.TheactivecofactoriscomposedofanNH2-terminalderivedheavychain(Mr=94,000)andaCOOH-terminalderivedlightchain(Mr=74,000)whichremainnon-covalentlyassociatedinthepresenceofcalciumions.FactorVaservesasacofactorfortheserineproteasefactorXa.FactorVaandXaassembleonaphospholipidsurfaceinanon-covalentandcalciumion-dependentmanner,toformtheprothrombinasecomplex.TheprothrombinasecomplexisresponsIBLefortherapidconversionofthezymogenprothrombintotheactiveserineprotease,alpha-thrombin.Assemblyoftheprothrombinasecomplexincreasestherateatwhichprothrombinisconvertedtothrombinbynearly300,000-foldrelativetotheratewithfactorXaalone.DownregulationoftheprothrombinasecomplexisaccomplishedpartlythroughtheinactivationoffactorVabyactivatedproteinC.

HumanfactorVispreparedfromfreshfrozenhumanplasmausingimmunoaffinitychromatographyasdescribedbyKatzmannandcoworkers(2).BovinefactorVispreparedfromfreshbovineplasmausingconventionalchromatographictechniquesasdescribedbyNesheimandcoworkers(3).PurifiedfactorVissuppliedin50%glycerol(vol/vol),andshouldbestoredat-20°C.PurityisdeterminedbySDS-PAGEanalysisandactivityismeasuredinafactorVclottingassay.

Properties:

Localization	Plasmaandplatelets
Plasmaconcentration	7.0µg/ml(human)(4,5) 35µg/ml(bovine)(5)
Modeofaction	Procofactor;activatedbythrombintoformtheactivecofactor,factorVa
Molecularweight	330,000(3)-(determinedforbovinefactorV)
Extinctioncoefficient	E 1% 1cm,280nm =9.6(3)-(determinedforbovinefactorV)
Structure	onesubunit,2196aminoacids(1,6)-(determinedforhumanfactorV)
Percentcarbohydrate	approximately25%(baseduponthecalculatedmolecularweightofhumanfactorV)

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