Recombinant γT305A fibrinogen indicates severely impaired fibrin polymerization due to the aberrant function of hole ‘a’ and calcium binding sites

• Published in: Thrombosis research Vol. 134; no. 2; pp. 518 - 525
• Main Authors: Ikeda, Minami, Kobayashi, Tamaki, Arai, Shinpei, Mukai, Saki, Takezawa, Yuka, Terasawa, Fumiko, Okumura, Nobuo
• Format: Journal Article
• Language: English
• Published: United States Elsevier Ltd 01-08-2014
• Subjects: Afibrinogenemia - blood; Afibrinogenemia - genetics; Afibrinogenemia - metabolism; Animals; Binding Sites; Blood Coagulation; Calcium - metabolism; CHO Cells; Cricetinae; Cricetulus; D:D interaction sites; Factor XIIIa - metabolism; Female; Fibrin - metabolism; Fibrin - ultrastructure; fibrin polymerization; Fibrinogens, Abnormal - chemistry; Fibrinogens, Abnormal - genetics; Fibrinogens, Abnormal - metabolism; Fibrinogens, Abnormal - ultrastructure; Fibrinolysin - metabolism; Hematology, Oncology and Palliative Medicine; high affinity calcium binding sites; hole ‘a’; Humans; hypodysfibrinogenemia; Infant; Polymerization; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Recombinant Proteins - ultrastructure; Gly-Pro-Arg-Pro; PT; hypodysfibrinogenemia; GPRP; prothrombin time; N-[2-hydroxyethyl] piperazine-N′-[2-ethanesulfonic acid], pH 7.4, 0.12 M NaCl; CHO; FpA; ethylenediaminetetraacetic acid; HBS; FpB; polyacrylamide gel electrophoresis; sodium dodecyl sulfate; fibrinopeptide A; fibrinopeptide B; high affinity calcium binding sites; fibrin polymerization; D:D interaction sites; scanning electron microscopy; SDS; N-[2-hydroxyethyl] piperazine-N′-[2-ethanesulfonic acid]; PAGE; EDTA; APTT; Chinese hamster ovary; activated partial thromboplastin time; HEPES; SEM; hole ‘a’
• ISSN: 0049-3848; 1879-2472
• DOI: 10.1016/j.thromres.2014.06.002

Abstract Introduction 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. Materials and 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 1 mM 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’. Conclusion 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.