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