Formulation	Lyophilizedfrom20mMNaCit,20mMNaPO4,pH7.3
Storage	4°C
Purity	>95%bySDS-PAGE
ActivityDetermination	N/A
ShelfLife(properlystored)	12months

DomainStructureofFibrinogen
ThetrinodulararrangementoftheAα,Bβ,andγchains,theIIacleavagesites(IIa)releasingFPAandFPB,aswellastheplasmincleavagesites(Pm)yieldingFragmentDandFragmentEareshown.

SampleGelInformation:

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

Overview:

Thethrombin(IIa)catalyzedcleavageofsolublefibrinogen(Fbg)toformfibrin(Fbn)istheterminalproteolyticeventinthecoagulationcascade.ThesesolubleFbnmonomersspontaneouslypolymerizetoformaninsolubleFbnnetworkwhichisstABIlizedbythefactorXIIIacatalyzedcrosslinkingoflysandgluresiduesofαandγchains.ThisFbnnetworkisthemajorproteincomponentofthehemostaticplug.

Plasmafibrinogenislargeglycoprotein(Mr=340,000)synthesizedintheliverandcirculatingataconcentrationof2.6mg/ml.Itisadisulfidelinkeddimercomposedof3pairsofdisulfidelinkednon-identicalpolypeptidechains(Aα,Bβandγ).NotablefeaturesoftheAαchainaretheN-terminalpeptide(fibrinopeptideA(FPA,1-16)),factorXIIIacrosslinkingsitesand2phosphorylationsites.Whensynthesized,Fbgisfullyphosphorylated,butcirculatesatonly20-30%phosphorylation.TheBβchaincontainsfibrinopeptideB(FPB,1-14),oneofthe3N-linkedcarbohydratemoieties(Mr=2500)andanN-terminalpyroglutamicacid.TheγchaincontainstheotherN-linkedglycosylationsiteandafactorXIIIacrosslinkingsites.The2elongatedsubunits((AαBβγ)2)arealignedinanantiparallelmannerformingatrinodulararrangementofthesixchains.Thenodesareformedbydisulfideringsbetweenthe3parallelchains.Thecentralnode(n-disulfideknot,Edomain)isformedbytheN-terminiofallsixchainsheldtogetherby11disulfidebonds.Thisregioncontainsthe2IIa-sensitivesites.ThereleaseofFPAbycleavageatR16-G17generatesFbnI,exposingapolymerizationsite(17-20)ontheAαchain.TheseregionsbindtocomplimentaryregionsontheDdomainofFbntoformprotofibrils.SubsequentIIacleavageofFPB(R14-G15)fromtheBβchainexposesadditionalpolymerizationsitesandpromoteslateralgrowthoftheFbnnetwork.

Eachofthe2domainsbetweenthecentralnode(Edomain)andtheC-terminalnodes(Ddomain)iscomposedofparallelα-helicalregionsoftheAα,Bβandγchainscoiledaroundeachothertoforma"coiledcoil"withpolarresiduesdirectedoutwardandnonpolarresiduesformingahydrophobiccore.Inthisregion,all3chainspossessaprotease(plasmin)sensitivesite.Theothermajorplasminsensitivesiteisinthehydrophilicpreturbanceoftheα-chainfromtheC-terminalnode.ControlledplasmindegradationatthesesitesconvertsFbgintofragmentDandfragmentE.Theindividualfragmentsareisolatedbysaltfractionation,gelfiltrationandionexchangechromatography.Thefragmentsaresuppliedlyophilizedforstorageat4°C.Highlypurifiedresearchgradefibrinogen(>95%clottable)ispreparedbyacombinationofconventionalandaffinitytechniques.Itissuppliedasafrozensolutioninctirate-phosphateforstorageat-80°C.

Properties:

Localization	Plasma,platelets
Plasmaconcentration	2.6mg/ml
Modeofaction	Precursormoleculewhichiscleavedbythrombintoformfibrinclot.
Molecularweight	340,000
Extinctioncoefficient	E 1% 1cm,280nm =15.1
Isoelectricpoint	5.1-6.3
Structure	Dimerof3pairsofnon-identicalchainsAα(Mr=66,800),Bβ(Mr=52,000)andγ(Mr=46,500),Elongatedtrinodularmoleculewith2terminalDdomainsandonecentralEdomain.FactorXIIIacross-linkingsitesatE328,E366,K508,K584inAαchain;atE398andK406inBβchain.
Percentcarbohydrate	3%
Post-translationalmodifications	Aαchain:2phosphorylatedserines,S3,S346 Bβchain:N364glycosylationsiteMr=2500,N-terminalpyroglutamicacid γchain:glycosylatedN52

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