| Formulation | 20mMHepes,150mMNaCl,pH7.4 |
| Storage | -80°C |
| Purity | >95%bySDS-PAGE |
| ActivityDetermination | ChromogenicAssay |
| ShelfLife(properlystored) | 12months |
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 |
| ||||||||
| 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 |
