| Formulation | 20mMTris,150mMNaCl,0.05%PDOC,pH7.4 |
| Storage | -80°C |
| Purity | >95%bySDS-PAGE |
| ActivityDetermination | ProteinCactivationassay |
| ShelfLife(properlystored) | 12months |
Thedomainstructureofthrombomodulinisrepresented,where:EGF=regionscontainingsequencehomologoustohumanepidermalgrowthfactor,O-GLYCOS=regionrichinO-glycosylationsites,MEM=membranespanningregion,CYTO=cytoplasmicdomaincontainingafreecycteine.
Overview:
Thrombomodulin(TM)isanintegralmembraneglycoproteinexpressedonthesurfaceofendothelialcells.ItsdiscoverybyEsmonandOwenhasfocusedattentionontheimportanceoftheproteinCanticoagulantpathway(1,2).TMservesasacofactorforproteinCactivationbyforminga1:1stoichiometriccomplexwiththrombin(Kd=10-10M),whichacceleratestherateofproteinCactivationby1000-foldrelativetotheratewiththrombinalone.InadditiontofacilitatingproteinCactivation,thebindingofthrombintoTMdrasticallyalterstheprocoagulantactivityofthrombin.WhenboundtoTM,thrombinnolongerclotsfibrinogen,activatesfactorV,inactivatesproteinSortriggersplateletaggregation.
TMisasinglechainproteincomposedof5distinctdomains.ThedomainstructureofTMissimilartothelowdensitylipoprotein(LDL)receptor.AshortcytoplasmicdomaincontainingafreecysteineislocatedattheCOOH-terminalendandisjoinedbyamembranespanningregiontoanO-glycosylationrichdomain.Thelatterisfollowedbyanepidermalgrowthfactor(EGF)homologyregionandtheNH2-terminalhydrophobicdomain.TheEGFhomologyregioncontains6EGF-likedomainsandcontainsthebindingsitesforboththrombinandproteinC.
Thrombomodulinisisolatedfromrabbitlungbyamodification(3)oftheproceduredescribedbyGalvinetal.(4)andissuppliedasanaqueoussolutionin0.02MTris,0.15MNaCl,pH7.4,containing0.05%polidocanol,andcanbestoredat-80°Cforuptoayear.PurityisdeterminedbySDS-PAGEanalysisandactivityisdeterminedusingachromogenicassayforproteinCactivation.
Properties:
| Localization | Endothelialcellmembrane,tracesofdegraded,yetfunctional,TMdetectedinhumanurineandplasma(7,8) | |||||
|---|---|---|---|---|---|---|
| ModeofAction | Cofactor;enhancestheproteolyticactivationofproteinCbythrombin | |||||
| Molecularweight | 74,000(5) | |||||
| Extinctioncoefficient |
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| Isoelectricpoint | 2.5(2) | |||||
| Structure | Singlechain,NH2-terminalhydrophobicdomain,sixEGFdomains,oneO-glycosylationrichdomain,onetransmembranedomain,COOH-terminalcytoplasmicdomain | |||||
| Post-translationalmodifications | β-hydroxyasparagine(0.5mol/molofprotein)(6) |
