Formulation	20mMTris,150mMNaCl,0.05%PDOC,pH7.4
Storage	-80°C
Purity	>95%bySDS-PAGE
ActivityDetermination	ProteinCactivationassay
ShelfLife(properlystored)	12months

DomainStructureofThrombomodulin
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	E 1% 1cm,280nm =8.8(5)
Isoelectricpoint	2.5(2)
Structure	Singlechain,NH2-terminalhydrophobicdomain,sixEGFdomains,oneO-glycosylationrichdomain,onetransmembranedomain,COOH-terminalcytoplasmicdomain
Post-translationalmodifications	β-hydroxyasparagine(0.5mol/molofprotein)(6)

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