Recombinant gamma T305A fibrinogen indicates severely impaired fibrin polymerization due to the aberrant function of hole 'a' and calcium binding sites

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 gamma T305A fibrinogen was synthesized in Chinese hamster ovary cells based on the results. We then functionally analyzed and compared with that of nearby recombinant gamma N308K fibrinogen. Results: DNA sequence analysis revealed a heterozygous gamma T305A substitution (mature protein residue number). The gamma T305A fibrinogen indicated markedly impaired thrombin-catalyzed fibrin polymerization both in the presence or absence of 1 mM calcium ion compared with that of gamma N308K 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 gamma N308K was impaired at the latter two sites. Molecular modeling demonstrated that gamma T305 is localized at a shorter distance than gamma N308 from the high affinity calcium binding site and hole 'a'. Conclusion: Our findings suggest that gamma T305 might be important for construction of the overall structure of the. module of fibrinogen. Substitution of gamma T305A leads to both dysfibrinogenemic and hypofibrinogenemic characterization, namely hypodysfibrinogenemia. We have already reported that recombinant gamma T305A fibrinogen was synthesized normally and secreted slightly, but was significantly reduced.ArticleTHROMBOSIS RESEARCH. 134(2):518-525 (2014)journal articl