title: Recombinant γT305A fibrinogen indicates severely impaired fibrin polymerization due to the aberrant function of hole 'A' and calcium binding sites.
abstract: BACKGROUND
  We examined a 6-month-old girl with inherited fibrinogen abnormality and no history of bleeding or thrombosis. Routine coagulation screening tests showed a markedly low level of plasma fibrinogen determined by functional measurement and also a low level by antigenic measurement (functional/antigenic ratio=0.295), suggesting hypodysfibrinogenemia.
  
  
  METHODS
  DNA sequence analysis was performed, and γT305A fibrinogen was synthesized in Chinese hamster ovary cells based on the results. We then functionally analyzed and compared with that of nearby recombinant γN308K fibrinogen.
  
  
  RESULTS
  DNA sequence analysis revealed a heterozygous γT305A substitution (mature protein residue number). The γT305A fibrinogen indicated markedly impaired thrombin-catalyzed fibrin polymerization both in the presence or absence of 1mM calcium ion compared with that of γN308K fibrinogen. Protection of plasmin degradation in the presence of calcium ion or Gly-Pro-Arg-Pro peptide (analogue for so-called knob 'A') and factor XIIIa-catalyzed fibrinogen crosslinking demonstrated that the calcium binding sites, hole 'a' and D:D interaction sites were all markedly impaired, whereas γN308Kwas impaired at the latter two sites. Molecular modeling demonstrated that γT305 is localized at a shorter distance than γN308 from the high affinity calcium binding site and hole 'a'.
  
  
  CONCLUSIONS
  Our findings suggest that γT305 might be important for construction of the overall structure of the γ module of fibrinogen. Substitution of γT305A leads to both dysfibrinogenemic and hypofibrinogenemic characterization, namely hypodysfibrinogenemia. We have already reported that recombinant γT305A fibrinogen was synthesized normally and secreted slightly, but was significantly reduced.
authors: Department of Clinical Laboratory Investigation, Graduate School of Medicine, Shinshu University, Matsumoto, Japan.
authors: Minami
authors: M
authors: Ikeda
authors: Department of Clinical Laboratory Investigation, Graduate School of Medicine, Shinshu University, Matsumoto, Japan; Department of Clinical Laboratory, The University of Tokyo Hospital, Tokyo, Japan.
authors: Tamaki
authors: T
authors: Kobayashi
authors: Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan; Department of Laboratory Medicine, Graduate School of Medicine, Shinshu University, Matsumoto, Japan.
authors: Shinpei
authors: S
authors: Arai
authors: Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan; Department of Laboratory Medicine, Graduate School of Medicine, Shinshu University, Matsumoto, Japan.
authors: Saki
authors: S
authors: Mukai
authors: Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan; Department of Laboratory Medicine, Graduate School of Medicine, Shinshu University, Matsumoto, Japan.
authors: Yuka
authors: Y
authors: Takezawa
authors: Department of Clinical Laboratory Investigation, Graduate School of Medicine, Shinshu University, Matsumoto, Japan; Department of Health and Medical Sciences, Graduate School of Medicine, Shinshu University, Matsumoto, Japan.
authors: Fumiko
authors: F
authors: Terasawa
authors: Department of Clinical Laboratory Investigation, Graduate School of Medicine, Shinshu University, Matsumoto, Japan; Department of Health and Medical Sciences, Graduate School of Medicine, Shinshu University, Matsumoto, Japan. Electronic address: nobuoku@shinshu-u.ac.jp.
authors: Nobuo
authors: N
authors: Okumura
mesh_terms: D000347:Afibrinogenemia / Q000097:blood / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001777:Blood Coagulation; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D026122:Factor XIIIa / Q000378:metabolism; D005260:Female; D005337:Fibrin / Q000378:metabolism* / Q000648:ultrastructure; D015241:Fibrinogens, Abnormal / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000648:ultrastructure; D005341:Fibrinolysin / Q000378:metabolism; D006801:Humans; D007223:Infant; D058105:Polymerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D015241:Fibrinogens, Abnormal; D011994:Recombinant Proteins; D005337:Fibrin; D026122:Factor XIIIa; D005341:Fibrinolysin; D002118:Calcium
keywords: D:D interaction sites; fibrin polymerization; high affinity calcium binding sites; hole ‘a’; hypodysfibrinogenemia