title: Functional analysis of recombinant Bbeta15C and Bbeta15A fibrinogens demonstrates that Bbeta15G residue plays important roles in FPB release and in lateral aggregation of protofibrils.
abstract: OBJECTIVE
  Analysis of dysfibrinogens has improved our understanding of molecular defects and their effects on the function of intact fibrinogen. To eliminate the influence of plasma heterozygous molecules, we synthesized and analyzed recombinant-variant fibrinogens.
  
  
  METHODS
  We synthesized two recombinant-variant fibrinogens with a single amino acid substitution at the 15Gly residue in the Bbeta-chain: namely, Bbeta15Cys and Bbeta15Ala.
  
  
  RESULTS
  Western blotting analysis of purified fibrinogen revealed the existence of a small amount of a dimeric form only for Bbeta15Cys fibrinogen. For Bbeta15Cys fibrinogen, functional analysis indicated (a) no thrombin-catalyzed fibrinopeptide B (FPB) release and (b) markedly impaired lateral aggregation in thrombin- and reptilase-catalyzed fibrin polymerizations. For Bbeta15Ala fibrinogen, such analysis indicated slight impairments of both thrombin-catalyzed FPB release and lateral aggregation in thrombin-catalyzed fibrin polymerization, but nearly normal lateral aggregation in reptilase-catalyzed fibrin polymerization. These impaired lateral aggregations were accompanied by thinner fibrin fiber diameters (determined by scanning electron microscopy of the corresponding fibrin clots).
  
  
  CONCLUSIONS
  We conclude that a region adjacent to Bbeta15Gly plays important roles in lateral aggregation not only in desA fibrin polymerization, but also in desAB fibrin polymerization, and we speculate that the marked functional differences between Bbeta15A and Bbeta15C fibrinogens in FPB release and fibrin polymerization might not only be due to the presence of a substituted cysteine residue in Bbeta15C fibrinogen, but also to the existence of disulfide-bonded forms. Finally, our data indicate that the Bbeta15Gly residue plays important roles in FPB release and lateral aggregation of protofibrils.
authors.affiliation: Department of Pathology, Shinshu University School of Medicine, Matsumoto, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Hirota-Kawadobora
authors.forename: S
authors.initials: S
authors.lastname: Kani
authors.forename: F
authors.initials: F
authors.lastname: Terasawa
authors.forename: N
authors.initials: N
authors.lastname: Fujihara
authors.forename: K
authors.initials: K
authors.lastname: Yamauchi
authors.forename: M
authors.initials: M
authors.lastname: Tozuka
authors.forename: N
authors.initials: N
authors.lastname: Okumura
mesh_terms: D000409:Alanine / Q000737:chemistry; D000818:Animals; D012103:Batroxobin / Q000737:chemistry; D015153:Blotting, Western; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005337:Fibrin / Q000737:chemistry / Q000648:ultrastructure; D005340:Fibrinogen / Q000737:chemistry*; D005345:Fibrinopeptide B / Q000737:chemistry*; D005998:Glycine / Q000737:chemistry; D006579:Heterozygote; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008855:Microscopy, Electron, Scanning; D016296:Mutagenesis; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry*; D012910:Snake Venoms; D013917:Thrombin / Q000737:chemistry; D013997:Time Factors
chemical_list: C115499:BBeta fibrinogen; D004220:Disulfides; D011994:Recombinant Proteins; D012910:Snake Venoms; D005345:Fibrinopeptide B; D005337:Fibrin; D005340:Fibrinogen; D012103:Batroxobin; D013917:Thrombin; D003545:Cysteine; D000409:Alanine; D005998:Glycine