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The Aa chain composition of plasma fibrinogen catabolites
THROMBOSIS Pergamon
RESEARCH 15; 287-289 Printed Press Ltd.1979.
in Great Britain
LEXTER'IOTHEEDITORS-INXHIEF
'IHEAa CH?ZN CCMP%XITION OF PL&%A FIBRINXEN CATABOLIX'ES
D.K. Galanakis and M.W. msesson SUNY, DownstateMedical Center, Departzents ofPathologyand%dicine, 450 Clarkson Avenue, Brooklyn, New York 11203, USA (Received
14.3.1979.
Accepted
by A.L. Copley)
Weinstein and Deykin (1) recently reported their studies concerningthe Aa chain ccnposition of plasma fibrinogenmolecules that are distinguishable as discrete bands (tenredbands I and II, respectively)in dodecyl sulfatecontaining gels. Their conclusion regarding the subunit ccmpositionof materialwithinthenrxtcathodal electrophoreticband (band I) wnfirmedprevious studies on this subject (2). They also concluded that the Aa chain population of the predaninant molecular species accounting for their band II fibrinogenmolecules wnsistedofone intactA~ chainorAu/;! remnant (mol.wt. 70,900 and 67,300, respectively)plus an AU rermantchainhavingaml.wt. that was-30-40,000less than that of the parent& chain (i.e.,AU/~-10). Our recently reported investigations (3) are at variance with this reclusion (Table 1). We foundthatthepmtband II fibrinogenn&xules~re ccqrisedoftw0Aarenma.n ts termed Aa/4 (nrol. wt. 57,700),which migrate close to the Bf3chain position (nol.wt. 60,400) in dodecyl sulfate gel electrophoreticexper~~,arsd~ch~tobeobscuredwhensignificant munts of the B8 chain are also present. Furthenrrxe,we found that species of fibrinogenwntaini.ngAa or&/2 chains Were presentalnustexclusivelyin bandIrolecules,whereas specieswntaining& remnants smaller than Au/4 (i.e.,Aa/6 -Aa/ll) migratedina sarewhatnxxe ancdalbandthat*have M band III (2,3). We believe that the plasma fractions studied ard the analytical techniques employedbyWeinstei_nand~eykindid notpermitth~to accurately evaluate the& chainpopulationofplasma fibrinogen,andwnsequently led to the discrepanciesbetween their wnclusions and ours. Weinstein and Deykin employed a gel slicing technique (electrophowsis of reduced material sliced fnxn gels of unreduced SarrpleS) for aSSeSSi.% the Subunit c-ition of the fibrinogenbands; their conclusionthat rrostband II molecules contained an Aa or Aa/ chain was based upon results UShg this Our previous experiencewith this analyticalprocedure (2) made us techniw. keenly aware of an inportantlimitation- there is likely to be significant overlap of nuterial frrrmthe anodal region of band I and the cathodal region 287
288
FIBRINOGEN Ad CHAINS
Vo1.15,No.l/2
TABlIE
Ca'qarisonof Pqmsed Aa Chain Compositionof FibrinogenSpecies Migratingin Electrophoretic Bands I !!Jx III Electmphoretic Weinstein& Deykin (ref.1) Au+Aa/z W7 to 10
Galanakiset al. (ref.3) Aa+&/ AC'/4h/6 to9
++ ++ I ++ 11 + + III (nZtanalyzedseparaLy) ____---__-_____-____-~--~~-~~-~~~~~~~~~~-~~-~-~-~ Each horizontalpair of crossesrepresentsthe Aa chain coqomts of the diuric fibrinogenmlecules withineach band. In order to sinplifythe cm parison,only the predanimnt speciesare depicted:the readeris referredto the originalarticlesfor additionaldetails. The mlecular weightsof these chains,as reportedelsewhere(4)are: Au, 70,900;Au/Z, 67,300;Au/g, 57,700; h/6, 46,500;Aa/7, 37,600;Au/a, 33,900;An/g, 31,800;Acr/lOr 29,200;h/11, 25,000;&x/12, 22,600. of band II. This situationresultedinourde~loping armreincisive approachtothepmblem (3). Anotheriqortant featureof the Weinsteinand Deykinstudythat seer& to contributeto thediscrepantconclusionswas theirunwarrantedassurrption was esthat the fibrinogenfractionthey preparedby B-alanineprecipitation sentiallyequivalentin conposition to fractionI-4. FractionI-4 is usually prepzed francohn fractionI, or an equivalentplasma fraction. This fraction displaysa praninentelectmplxmtic band II (2,3),contains significant quantitiesof Au/4 (2-4),but has relativelyminor atmuntsof Au renmants of plasm fibrinogen with f3mller than Aci/4.In contrast,precipitation alanine (5)or glycine (6)can be e to includein addition,significant amunts of the relativelyhigh solubilityfibrinogencatabolitespecies that tend to be excludedfmn Cohn fractionI preparations(i.e.,rmlecules toAa/4 or smaller). Impection of ContainingAarmmantchains corresponding WeinsteinandDeykin'sgels indicatesthatthiswas indeedthe case since such renmantchainsare clearlypresent(c.f.Figs. 2 & 3, ref.1). Furthermre, the relativewidth& stainingintensityoftheB6 Win theirband II sanplesaqaredwith thatfranband I suggeststhat considerable armmts ofAa/4 chainswerepresentinband11, althoughtheydidnotinterprettheir resultsin thisway. Consideringthe B-alaninefractionthat Weinsteinand Deykinanalyzed, plus the fact that bands II and III fibrinogenare not clearlyresolvedfrcxn oneanother inmixtuxes (3),it seemsevidentthattheytreatedmterialmigratingin the band II and III positionsas 9 singlespecies(i.e.,their band II). Incontrasttotheirpropxal thatAa/4 chainsplayedonlyaminor role in formingthe~l~ulesmigratinginthatregion,ourresults provided clear evidenceof the importanceof Au/4 ohainsin -rising the molecules migratingin eitherband II or band III (3). ?hesefind.ingsamconsistent with our earlierwork (2,4)and with the data of Semram et al. (7),fran which itcanbecalculatedthatAa/4 chainsaccountfor - 35%ofallAa chain remnantsinplam fibrinogen.
Vo1.15,No.1/2
FIBRINOGEN
Ad CHAINS
289
1.
WEINSTEIN, M.J. tm-dimmsional tric focusing.
and DEYKIN, D. Low solubilityfibrinogenexmined by sczdiundodecylsulfate gelelectrophoresis andisoelecThron33.Res. 13, 361, 1978. --
2.
MXESSON, M.W., CACANAKIS,D.K. and FINIAYSON,J.S. Caqarison of human fibrinogensubfractionsandearlyplasmic fibrinogenderivatives. 2. -Biol. Chm. 249, 4656, 1974.
3.
@LANAKIS, D-K., MXESSCN, M.W. and S!PAlXAKIS, N.E. Hm fibrinogen heterogeneities:Distributionand charge characteristicsof chains of Aa origin. IL.Lab. Clin. Med. 92, 376, 1978. --
4.
MXESSCN, M.W., FINlAY=, J.S., UMETBXT, R.A. and GALANAKIS,D.K. Humn fibrinogm heterogeneitiesI. Structuraland related studies of plasm fibrincqenswhich are high solubilitycatabolic intermediates. J. Biol. Chm. 247, 5210, 1972. ---
5.
STRAUGHN,W. III and WAGNER, R.H. A smle method for preparing fibrinogen. lkrcmh. Diath. Haemrrh. 16, 198, 1966. --
6.
m, M.W. and SHERRY, S. l%e preparationand properties of human fibrinogenof relativelyhigh solubility. Bicchemistry 5, 2829, 1966.
RESEARCH 15; 287-289 Printed Press Ltd.1979.
in Great Britain
LEXTER'IOTHEEDITORS-INXHIEF
'IHEAa CH?ZN CCMP%XITION OF PL&%A FIBRINXEN CATABOLIX'ES
D.K. Galanakis and M.W. msesson SUNY, DownstateMedical Center, Departzents ofPathologyand%dicine, 450 Clarkson Avenue, Brooklyn, New York 11203, USA (Received
14.3.1979.
Accepted
by A.L. Copley)
Weinstein and Deykin (1) recently reported their studies concerningthe Aa chain ccnposition of plasma fibrinogenmolecules that are distinguishable as discrete bands (tenredbands I and II, respectively)in dodecyl sulfatecontaining gels. Their conclusion regarding the subunit ccmpositionof materialwithinthenrxtcathodal electrophoreticband (band I) wnfirmedprevious studies on this subject (2). They also concluded that the Aa chain population of the predaninant molecular species accounting for their band II fibrinogenmolecules wnsistedofone intactA~ chainorAu/;! remnant (mol.wt. 70,900 and 67,300, respectively)plus an AU rermantchainhavingaml.wt. that was-30-40,000less than that of the parent& chain (i.e.,AU/~-10). Our recently reported investigations (3) are at variance with this reclusion (Table 1). We foundthatthepmtband II fibrinogenn&xules~re ccqrisedoftw0Aarenma.n ts termed Aa/4 (nrol. wt. 57,700),which migrate close to the Bf3chain position (nol.wt. 60,400) in dodecyl sulfate gel electrophoreticexper~~,arsd~ch~tobeobscuredwhensignificant munts of the B8 chain are also present. Furthenrrxe,we found that species of fibrinogenwntaini.ngAa or&/2 chains Were presentalnustexclusivelyin bandIrolecules,whereas specieswntaining& remnants smaller than Au/4 (i.e.,Aa/6 -Aa/ll) migratedina sarewhatnxxe ancdalbandthat*have M band III (2,3). We believe that the plasma fractions studied ard the analytical techniques employedbyWeinstei_nand~eykindid notpermitth~to accurately evaluate the& chainpopulationofplasma fibrinogen,andwnsequently led to the discrepanciesbetween their wnclusions and ours. Weinstein and Deykin employed a gel slicing technique (electrophowsis of reduced material sliced fnxn gels of unreduced SarrpleS) for aSSeSSi.% the Subunit c-ition of the fibrinogenbands; their conclusionthat rrostband II molecules contained an Aa or Aa/ chain was based upon results UShg this Our previous experiencewith this analyticalprocedure (2) made us techniw. keenly aware of an inportantlimitation- there is likely to be significant overlap of nuterial frrrmthe anodal region of band I and the cathodal region 287
288
FIBRINOGEN Ad CHAINS
Vo1.15,No.l/2
TABlIE
Ca'qarisonof Pqmsed Aa Chain Compositionof FibrinogenSpecies Migratingin Electrophoretic Bands I !!Jx III Electmphoretic Weinstein& Deykin (ref.1) Au+Aa/z W7 to 10
Galanakiset al. (ref.3) Aa+&/ AC'/4h/6 to9
++ ++ I ++ 11 + + III (nZtanalyzedseparaLy) ____---__-_____-____-~--~~-~~-~~~~~~~~~~-~~-~-~-~ Each horizontalpair of crossesrepresentsthe Aa chain coqomts of the diuric fibrinogenmlecules withineach band. In order to sinplifythe cm parison,only the predanimnt speciesare depicted:the readeris referredto the originalarticlesfor additionaldetails. The mlecular weightsof these chains,as reportedelsewhere(4)are: Au, 70,900;Au/Z, 67,300;Au/g, 57,700; h/6, 46,500;Aa/7, 37,600;Au/a, 33,900;An/g, 31,800;Acr/lOr 29,200;h/11, 25,000;&x/12, 22,600. of band II. This situationresultedinourde~loping armreincisive approachtothepmblem (3). Anotheriqortant featureof the Weinsteinand Deykinstudythat seer& to contributeto thediscrepantconclusionswas theirunwarrantedassurrption was esthat the fibrinogenfractionthey preparedby B-alanineprecipitation sentiallyequivalentin conposition to fractionI-4. FractionI-4 is usually prepzed francohn fractionI, or an equivalentplasma fraction. This fraction displaysa praninentelectmplxmtic band II (2,3),contains significant quantitiesof Au/4 (2-4),but has relativelyminor atmuntsof Au renmants of plasm fibrinogen with f3mller than Aci/4.In contrast,precipitation alanine (5)or glycine (6)can be e to includein addition,significant amunts of the relativelyhigh solubilityfibrinogencatabolitespecies that tend to be excludedfmn Cohn fractionI preparations(i.e.,rmlecules toAa/4 or smaller). Impection of ContainingAarmmantchains corresponding WeinsteinandDeykin'sgels indicatesthatthiswas indeedthe case since such renmantchainsare clearlypresent(c.f.Figs. 2 & 3, ref.1). Furthermre, the relativewidth& stainingintensityoftheB6 Win theirband II sanplesaqaredwith thatfranband I suggeststhat considerable armmts ofAa/4 chainswerepresentinband11, althoughtheydidnotinterprettheir resultsin thisway. Consideringthe B-alaninefractionthat Weinsteinand Deykinanalyzed, plus the fact that bands II and III fibrinogenare not clearlyresolvedfrcxn oneanother inmixtuxes (3),it seemsevidentthattheytreatedmterialmigratingin the band II and III positionsas 9 singlespecies(i.e.,their band II). Incontrasttotheirpropxal thatAa/4 chainsplayedonlyaminor role in formingthe~l~ulesmigratinginthatregion,ourresults provided clear evidenceof the importanceof Au/4 ohainsin -rising the molecules migratingin eitherband II or band III (3). ?hesefind.ingsamconsistent with our earlierwork (2,4)and with the data of Semram et al. (7),fran which itcanbecalculatedthatAa/4 chainsaccountfor - 35%ofallAa chain remnantsinplam fibrinogen.
Vo1.15,No.1/2
FIBRINOGEN
Ad CHAINS
289
1.
WEINSTEIN, M.J. tm-dimmsional tric focusing.
and DEYKIN, D. Low solubilityfibrinogenexmined by sczdiundodecylsulfate gelelectrophoresis andisoelecThron33.Res. 13, 361, 1978. --
2.
MXESSON, M.W., CACANAKIS,D.K. and FINIAYSON,J.S. Caqarison of human fibrinogensubfractionsandearlyplasmic fibrinogenderivatives. 2. -Biol. Chm. 249, 4656, 1974.
3.
@LANAKIS, D-K., MXESSCN, M.W. and S!PAlXAKIS, N.E. Hm fibrinogen heterogeneities:Distributionand charge characteristicsof chains of Aa origin. IL.Lab. Clin. Med. 92, 376, 1978. --
4.
MXESSCN, M.W., FINlAY=, J.S., UMETBXT, R.A. and GALANAKIS,D.K. Humn fibrinogm heterogeneitiesI. Structuraland related studies of plasm fibrincqenswhich are high solubilitycatabolic intermediates. J. Biol. Chm. 247, 5210, 1972. ---
5.
STRAUGHN,W. III and WAGNER, R.H. A smle method for preparing fibrinogen. lkrcmh. Diath. Haemrrh. 16, 198, 1966. --
6.
m, M.W. and SHERRY, S. l%e preparationand properties of human fibrinogenof relativelyhigh solubility. Bicchemistry 5, 2829, 1966.