Formulation	50%(vol/vol)Glycerol/H2O
Storage	-20°C
Purity	>95%bySDS-PAGE
ActivityDetermination	ChromogenicAssay
ShelfLife(properlystored)	12months

InactivationofFactorVabyActivatedProteinC(APC)
ProteolyticinactivationoffactorVabyAPCisrepresented,where:PS=proteinS,PCPS=phospholipidvesicles(orcellularsurface)andCa++=calciumions.TheinactivationoffactorVatoformfactorVairesultsfromproteolysisofthefactorVaheavychain(94K)atthreespecificsitesbyAPC(solidarrows)(1,2).ThelocationofthesecleavagesitesinthefactorVaheavychainareasfollows:(human:R306,R506,&R679)and(bovine:R306,R505,&R662).CompleteinactivationofthecofactormoleculerequirescleavageattheArginine-306position.CleavageatArginine-306byactivatedproteinCoccursonlyinthepresenceofmembrane,andrequirespriorcleavageoftheheavychainatArginine-505.ProteolysisofthefactorValightchainbyAPCoccursonlyinthebovinemoleculeandisnotrequiredforinactivation(dashedarrow).

SampleGelInformation:

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

Overview:

ActivatedproteinC(APC)isananticoagulantserineproteasederivedfromthetwochain,vitaminK-dependentzymogen,proteinC(3-7).Acomplexbetweenalpha-thrombinandthrombomodulincatalyzesasinglecleavageatArg-12(Arg-14inbovine)intheheavychainofproteinC,togenerateactivatedProteinC.Severalnon-physiologicallyrelevantproteasessuchasRVV-Xactivator,trypsin,andPROTACarealsocapableofactivatingproteinC.

APCfunctionsasananticoagulantwhichcatalyzestheproteolyticinactivationofthecofactors,factorsVaandVIIIa,leADIngtoinhibitionoftheprothrombinaseandfactorXasecomplexes.TheinactivationoffactorsVaandVIIIaisbothCa2+andphospholipiddependent.ThevitaminKdependentcofactor,proteinS,moderatelyincreasesthisrateofinactivationbyforminga1:1complexwithAPC(Kd=6x10-9M)(8).

SeveralfactorsattenuatetheanticoagulantactivityofAPC.FactorXaprotectsfactorVafromproteolysisbyAPCbycompetingforasimilarbindingsiteonfactorVa.ThrombinhasalsobeenproposedasaregulatorofAPCbyproteolyticinactivationofproteinS.Inaddition,APCisregulatedbyacirculatingheparin-dependentproteinCinhibitor(PAI-3),acirculatingheparin-independentproteinCinhibitor,aplatelet-derivedproteinCinhibitor,andPAI-1.ThecomplexesformedbetweenAPCandbothtypesofPAIhavebeenreportedtoaccountforincreasedfibrinolysisobserveduponinfusionofAPCorthegenerationofAPCinvivo.

InadditiontoourstandardAPCpreparation,anactivesite-blockedformcontainingDansyl-EGR-chloromethlyketoneisalsoavailable.

ActivatedproteinCispreparedfrompurifiedproteinCbyactivationwiththrombinfollowedbyionexchangechromatography(4).APCissuppliedin50%(vol/vol)glycerol/H2Oandshouldbestoredat-20oC.PurityisdeterminedbySDS-PAGEanalysisandactivityismeasuredusingachromogenicsubstrateassay.AllproductionlotsofAPCarealsotestedfortheirABIlitytoprolongtheaPTTofnormalhumanplasma,asrequiredfortheAPCresistanceassay(10,11).Theresultsofthistestareprovidedforeachlot,asanaPTT(+/-APC)ratio(10nMAPC).

 

Properties:

Localization	Plasma
Modeofaction	Anticoagulant,inactivatesfactorsVaandVIIIa
Molecularweight	56,200(human)(5) 52,650(bovine)(5)
Extinctioncoefficient	E 1% 1cm,280nm =14.5(human)(9)     =13.7(bovine)(9)
IsoelectricPoint	4.4-4.8(human)(9) 4.2-4.5(bovine)(9)
Structure	twochains,Mr=35,000and21,000,disulfidelinked,NH2-terminalgladomaintwoEGFdomains
Percentcarbohydrate	23%(human)(5) 14%(bovine)(5)
Post-translationmodifications	elevenglaresidues(bovine),nineglaresidues(human),oneβ-hydroxyaspartate

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