SummaryWe report two novel hypofibrinogenemias, Shizuoka III and Kanazawa II, which are caused by heterozygous mutations in FGG. Shizuoka III showed c.147delT and 147_149insACA in FGG exon 3 and a subsequent frameshift mutation, resulting in γ23X (stop codon), and Kanazawa II showed c.1205G>A in FGG exon 9, resulting in γ376X.To determine whether the truncated γ-chains, γ23X and γ376X, were synthesized and participated in the assembly of fibrinogen, mutant-type cDNA vectors were transfected into Chinese hamster ovary (CHO) cells. Significant levels of mutant fibrinogen were not detected by ELISA in the culture media and cell lysates. Immunoblot analysis of cell lysates revealed that the mutant γ-chain of γ376X was observed but intact fibrinogen was not. On the other hand, mutant γ-chain was not observed in γ23X-expressing cells.To demonstrate the involvement of the mechanisms of nonsense-mediated mRNA decay (NMD), we cloned wild-and mutant-type mini-genes containing γ23-or γ376-codon and transfected these into CHO cell lines in the absence or presence of cycloheximide (CHX) as an NMD inhibitor. mRNA levels were determined using real-time quantitative RT-PCR in CHO cells. In the absence of CHX, levels of mRNAs transcribed from the mutant gene were lower than from the wild-type gene whereas, in the presence of CHX, levels of mRNAs transcribed from the mutant gene increased dose-dependently. 3Finally, these results demonstrated that aberrant mRNAs containing γ23X or γ376X are degraded by the NMD system and translation decrease in hepatocytes, resulting in hypofibrinogenemias.4

Word count: manuscript, 3711(excluding table, figure legends and references) ; summary, 230 2 Summary Fibrin polymerization is mediated by interactions between knobs 'A' and 'B' exposed by thrombin cleavage, and holes 'a' and 'b'. We demonstrated markedly delayed thrombin-catalyzed fibrin polymerization, through B:b interactions alone, of recombinant γD364H-fibrinogen with impaired hole 'a'. To determine whether recombinant variant fibrinogens with no release of fibrinopeptide A (FpA) polymerize similarly to γD364H-fibrinogen, we examined two variant fibrinogens with substitutions altering knob 'A', Aα17A-and Aα17C-fibrinogen. We examined thrombin-or batroxobin-catalyzed fibrinopeptide release by HPLC, fibrin clot formation by turbidity and fibrin clot structure by scanning electron microscopy (SEM) and compared the results of the variants with those for γD364H-fibrinogen. Thrombin-catalyzed FpA release of Aα17A-fibrinogen was substantially delayed and none observed for Aα17C-fibrinogen; fibrinopeptide B (FpB) release was delayed for all variants. All variant fibrinogens showed substantially impaired thrombin-catalyzed polymerization; for Aα17A-fibrinogen it was delayed less, and for Aα17C more than for γD364H-fibrinogen. No variants polymerized with batroxobin, which exposed only knob 'A'. The inhibition of variant fibrinogens' polymerization was dose-dependent on the concentration of either GPRP or GHRP, and both peptides that block holes 'b'. SEM showed that the variant clots from Aα17A-and γD364H-fibrinogen had uniform, ordered fibers, thicker than normal, whereas Aα17C-fibrinogen formed less 3 organized clots with shorter, thinner, and tapered ends. These results demonstrate that FpA release per se is necessary for effective B:b interactions during polymerization of variant fibrinogens with impaired A:a interactions.

A 48-year-old man presented with a sustained fever. Abdominal computed tomography revealed multilocular liver abscesses. He underwent percutaneous needle aspiration, yielding straw-colored pus. Gram staining revealed Gram-negative coccobacilli. The organism grew only on chocolate II agar in a 7% carbon dioxide atmosphere. Identification of Aggregatibacter aphrophilus was confirmed using mass spectrometry and 16S rRNA gene sequencing. He was successfully treated with antibiotics. Liver abscess caused by A. aphrophilus is extremely rare. We herein report the first such case in Japan. Even fastidious organisms, such as A. aphrophilus, should be correctly identified using mass spectrometry or 16S rRNA gene sequencing for adequate treatment.