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Article, Blood Advances. 5(19): 3830-3838 (2021)

We identified a novel heterozygous AαE11del variant in a patient with congenital dysfibrinogenemia. This mutation is located in fibrinopeptide A (FpA). We analyzed the effect of AαE11del on the catalyzation of thrombin and batroxobin and simulated the stability of the complex structure between the FpA fragment (AαG6-V20) peptide and thrombin. We performed fibrin polymerization and examined the kinetics of FpA release catalyzed by thrombin and batroxobin using purified plasma fibrinogen. To clarify the association between the AαE11 residue and thrombin, we calculated binding free energy using molecular dynamics simulation trajectories. Increasing the thrombin concentration improved release of FpA from the patient’s fibrinogen to approximately 90%, compared to the previous 50% of that of normal fibrinogen. Fibrin polymerization of variant fibrinogen also improved. In addition, greater impairment of variant FpA release from the patient’s fibrinogen was observed with thrombin than with batroxobin. Moreover, the calculated binding free energy showed that the FpA fragment–thrombin complex became unstable due to the missing AαE11 residue. Our findings indicate that the AαE11 residue is involved in FpA release in thrombin catalyzation more than in batroxobin catalyzation, and that the AαE11 residue stabilizes FpA fragment–thrombin complex formation.

We identified a patient with a novel heterozygous variant fibrinogen, γp.C352R (Niigata II; N-II), who had a bleeding episode and failed infertility treatment and was suspected to have hypodysfibrinogenemia based on low and discordant fibrinogen levels (functional assay 0.33 g/L, immunological assay 0.91 g/L). We analyzed the mechanism of this rare phenotype of a congenital fibrinogen disorder. Patient plasma fibrinogen was purified and protein characterization and thrombin-catalyzed fibrin polymerization performed. Recombinant fibrinogen-producing Chinese hamster ovary (CHO) cells were established and the assembly and secretion of variant fibrinogen analyzed by ELISA and western blotting. Purified N-II plasma fibrinogen had a small lower molecular weight band below the normal γ-chain and slightly reduced fibrin polymerization. A limited proportion of p.C352R fibrinogen was secreted into the culture medium of established CHO cell lines, but the γ-chain of p.C352R was synthesized and variant fibrinogen was assembled inside the cells. We demonstrated that fibrinogen N-II, γp.C352R was associated with markedly reduced secretion of variant fibrinogen from CHO cells, that fibrin polymerization of purified plasma fibrinogen was only slightly affected, and that fibrinogen N-II produces hypodysfibrinogenemia in plasma.

Article, Case Reports in Clinical Medicine. 10: 108-116 (2021)

Article, International journal of laboratory hematology. 43(2): 281-289(2021)

Article, Pediatric reports. 13(1): 113-117(2021)

Citrullinated fibrinogen (C-Fbg) has been detected in rheumatoid arthritis; however, few studies have reported the role of C-Fbg in other inflammatory diseases. This study aimed to clarify the changes in serum C-Fbg associated with the bacteremia phase.We measured serum C-Fbg concentration in bacteremia patients. C-Fbg levels at each phase of bacteremia, classified by white blood cell (WBC) count and neutrophil left shift change, were compared with those of healthy control (HC). The correlation between C-Fbg concentration and certain inflammatory markers, or citrullinated histone H3 concentration was assessed. Multiple linear regression (MLR) analysis was used to examine the association of log C-Fbg with certain inflammatory markers.Serum C-Fbg levels were significantly higher in bacteremia patients than in HC (p 0.001) and positively correlated with WBC and neutrophil count. Further, C-Fbg levels were significantly higher in phases III and IV of bacteremia than in HC (p 0.001). MLR analysis indicated that log C-Fbg had a stronger relationship with log neutrophil counts than other certain inflammatory markers (p 0.01).Serum C-Fbg levels increased in bacteremia patients, and this was consistent with an influx of neutrophils into the blood stream in accordance with the bacteremia phase.

Article, Thrombosis research. 196: 152-158. (2020)

Article, International journal of hematology. 112(1): 96-104. (2020)

Article, Thrombosis research. 182: 150-158. (2019)

We identified a novel heterozygous AαE11del variant in a patient with congenital dysfibrinogenemia. This mutation is located in fibrinopeptide A (FpA). We analyzed the effect of AαE11del on the catalyzation of thrombin and batroxobin and simulated the stability of the complex structure between the FpA fragment (AαG6-V20) peptide and thrombin.We performed fibrin polymerization and examined the kinetics of FpA release catalyzed by thrombin and batroxobin using purified plasma fibrinogen. To clarify the association between the AαE11 residue and thrombin, we calculated binding free energy using molecular dynamics simulation trajectories.Increasing the thrombin concentration improved release of FpA from the patient's fibrinogen to approximately 90%, compared to the previous 50% of that of normal fibrinogen. Fibrin polymerization of variant fibrinogen also improved. In addition, greater impairment of variant FpA release from the patient's fibrinogen was observed with thrombin than with batroxobin. Moreover, the calculated binding free energy showed that the FpA fragment-thrombin complex became unstable due to the missing AαE11 residue.Our findings indicate that the AαE11 residue is involved in FpA release in thrombin catalyzation more than in batroxobin catalyzation, and that the AαE11 residue stabilizes FpA fragment-thrombin complex formation.

Article, Clinica chimica acta. 521: 170-176(2021)

Article, International journal of molecular sciences. 22(10): 5218(2021)

We identified a patient with a novel heterozygous variant fibrinogen, γp.C352R (Niigata II; N-II), who had a bleeding episode and failed infertility treatment and was suspected to have hypodysfibrinogenemia based on low and discordant fibrinogen levels (functional assay 0.33 g/L, immunological assay 0.91 g/L). We analyzed the mechanism of this rare phenotype of a congenital fibrinogen disorder.Patient plasma fibrinogen was purified and protein characterization and thrombin-catalyzed fibrin polymerization performed. Recombinant fibrinogen-producing Chinese hamster ovary (CHO) cells were established and the assembly and secretion of variant fibrinogen analyzed by ELISA and western blotting.Purified N-II plasma fibrinogen had a small lower molecular weight band below the normal γ-chain and slightly reduced fibrin polymerization. A limited proportion of p.C352R fibrinogen was secreted into the culture medium of established CHO cell lines, but the γ-chain of p.C352R was synthesized and variant fibrinogen was assembled inside the cells.We demonstrated that fibrinogen N-II, γp.C352R was associated with markedly reduced secretion of variant fibrinogen from CHO cells, that fibrin polymerization of purified plasma fibrinogen was only slightly affected, and that fibrinogen N-II produces hypodysfibrinogenemia in plasma.

We identified a novel heterozygous variant, B&beta, p.Pro234Leu (fibrinogen Tokorozawa), which was suspected to be associated with hypofibrinogenemia. Therefore, we analyzed the assembly and secretion of this fibrinogen using Chinese hamster ovary (CHO) cells. To determine the impact on the synthesis and secretion of fibrinogen of the B&beta, p.P234L and &gamma, p.G242E substitutions, we established recombinant variant fibrinogen-producing CHO cell lines. Synthesis and secretion analyses were performed using an enzyme-linked immunosorbent assay (ELISA) and immunoblotting analysis with the established cell lines. In addition, we performed fibrin polymerization using purified plasma fibrinogen and in-silico analysis. Both B&beta, p.G242E impaired the secretion and synthesis of fibrinogen. Moreover, immunoblotting analysis elucidated the mobility migration of the B&beta, &gamma, complex in B&beta, p.P234L. On the other hand, the fibrin polymerization of fibrinogen Tokorozawa was similar to that of normal fibrinogen. In-silico analysis revealed that the B&beta, p.P234 residue is located in the contact region between the B&beta, and &gamma, chains and contacts &gamma, p.G242 residue. The present study demonstrated that the B&beta, p.P234L substitution resulted in hypofibrinogenemia by decreasing the assembly and secretion of fibrinogen. Therefore, there is a possibility that substitutions in the contact region between the B&beta, chains impact the assembly and secretion of fibrinogen.

We identified two heterozygous dysfibrinogenemias, Bβp.Gly45Cys (Kyoto VII; K-VII) and Bβp.Arg74Cys (Iida II; I-II). The impairment of polymerization of Bβp.G45C has been well analyzed; however, that of Bβp.R74C has not. Thus, we compared fibrin polymerization between these variants. To determine the structural and functional characterization of purified fibrinogens, we performed immunoblotting analysis, kinetic analyses of fibrinopeptide A and B release, and thrombin- or batroxobin-catalyzed fibrin or fibrin monomer polymerization. Immunoblotting analysis showed that both variant fibrinogens had variant fibrinogen-albumin complexes and variant fibrinogen multimers, and the amounts of fibrinogen-albumin complexes with fibrinogen K-VII was more than with fibrinogen I-II. Moreover, fibrinopeptide B release from fibrinogen K-VII was about 50% of the control, whereas the others were normal. The maximum slopes of polymerization for variant fibrinogens were reduced, but fibrinogen K-VII was reduced more than fibrinogen I-II. The present study demonstrated that both Bβp.G45C and Bβp.R74C variants showed the presence of variant fibrinogen-albumin complexes and variant fibrinogen multimers, and polymerization of Bβp.G45C was impaired more than Bβp.R74C. Our study and several previous reports concerning the clinical phenotype of both variants suggested the risks of bleeding for patients with Bβp.G45C and thrombosis for patients with Bβp.R74C.

Article, THROMBOSIS RESEARCH.159:82-85(2017)

Fibrinogen storage disease (FSD) is a rare disorder that is characterized by the accumulation of fibrinogen in hepatocytes and induces liver injury. Six mutations in the γC domain (γG284R, γT314P, γD316N, the deletion of γG346-Q350, γG366S, and γR375W) have been identified for FSD. Our group previously established γ375W fibrinogen-producing Chinese hamster ovary (CHO) cells and observed aberrant large granular and fibrous forms of intracellular inclusion bodies. The aim of this study was to investigate whether fibrous intracellular inclusion bodies are specific to FSD-inducible variant fibrinogen. Thirteen expression vectors encoding the variant γ-chain were stably or transiently transfected into CHO cells expressing normal fibrinogen Aα- and Bβ-chains or HuH-7 cells, which were then immunofluorescently stained. Six CHO and HuH-7 cell lines that transiently produced FSD-inducible variant fibrinogen presented the fibrous (3.2?22.7 and 2.1?24.5%, respectively) and large granular (5.4?25.5 and 7.7?23.9%) forms of intracellular inclusion bodies. Seven CHO and HuH-7 cell lines that transiently produced FSD-non-inducible variant fibrinogen only exhibit the large granular form. These results demonstrate that transiently transfected variant fibrinogen-producing CHO cells and inclusion bodies of the fibrous form may be useful in non-invasive screening for FSD risk factors for FSD before its onset., Article, INTERNATIONAL JOURNAL OF HEMATOLOGY.105:758-768(2017)

We encountered a 45-year-old Japanese man who suffered from pulmonary thromboembolism and huge right ventricular thrombus after inferior vena cava (IVC) filter implantation without apparent thrombus in either the deep veins or inside the IVC filter. The biochemical data showed a discrepancy in the level of fibrinogen between the immunological and thrombin time methods, suggesting hypodysfibrinogenemia. The sequencing of the fibrinogen γ-chain gene (FGG) revealed a novel heterozygous missense mutation in exon 8 - a TGT to TCT transversion in codon 326 - resulting in an amino acid substitution of serine for cysteine (γCys326Ser). The characterization of the protein did not show known mechanisms for thrombosis in dysfibrinogenemia, such as dimer or albumin-binding complex formation. In summary, the current case with a life-threatening thrombotic event was found to have a novel heterozygous missense mutation resulting in γCys326Ser, which was suggested as a predisposing factor of the thrombosis. Known mechanisms responsible for thrombosis in the current case were not demonstrated, suggesting other mechanisms including superimposing inherited and/or acquired risk factors. When a patient presents with unusual thrombosis such as breakthrough pulmonary embolism and huge thrombus in the right ventricle, as in the current case, the laboratory process for heritable thrombophilia should be considered.

Introduction: Congenital fibrinogen disorders are classified as afibrinogenemia, hypofibrinogenemia, dysfibrinogenemia, and hypodysfibrinogenemia. However, difficulties are associated with discriminating between dysfibrinogenemia, hypofibrinogenemia, and hypodysfibrinogenemia using routine analyses. We previously reported a heterozygous variant fibrinogen (γA289V; Kanazawa III) as hypodysfibrinogenemia; however, the same variant had previously been described as hypofibrinogenemia. To clarify the production of γA289V fibrinogen, we expressed recombinant γA289V (r-γA289V) fibrinogen and compared it with wild-type (WT) and adjacent recombinant variant fibrinogens. Methods: Target mutations were introduced into a fibrinogen γ-chain expression vector by site-directed mutagenesis, and the vector was then transfected into Chinese hamster ovary cells to produce recombinant fibrinogen. Fibrinogen was purified from the plasma of the proposita, and culture media and fibrinogen functions were analyzed using fibrin polymerization, plasmin protection, and FXIIIa-catalyzed fibrinogen cross-linking. Results: The fibrinogen concentration ratio of the culture media to cell lysates was markedly lower for r-γA289V fibrinogen than for WT. Because the secretion of recombinant γF290L (r-γF290L) fibrinogen was similar to WT, we compared r-γF290L fibrinogen functions with WT. The fibrin polymerization of Kanazawa III plasma (K-III) fibrinogen was significantly weaker than normal plasma fibrinogen. Moreover, K-III fibrinogen showed a markedly reduced “D:D” interaction. However, all functions of r-γF290L fibrinogen were similar to WT. An in silico analysis confirmed the above results. Conclusion: The present results demonstrated that γA289 is crucial for the γ-module structure, and the γA289V substitution markedly reduced fibrinogen secretion. Moreover, K-III fibrinogen showed markedly reduced fibrin polymerization and “D:D” interactions. γA289V fibrinogen was confirmed as hypodysfibrinogenemia., Article, INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY.42(2):190-197(2020)

Epub 2016 Nov 5, Introduction: Wefound a novel hypodysfibrinogenemia designated Tsukuba I caused by compound heterozygous nucleotide deletionswith FGG c. 1129+ 62_ 65 del AATA and FGG c. 1299+ 4 del A on different alleles. The former was deep in intron 8 of FGG (IVS-8 deletion) and the latter in exon 9 of FGG (Ex-9 deletion), which is translated for the gamma'-chain, but not the.A-chain. AWestern blot analysis of plasma fibrinogen from our patient revealed an aberrant gamma-chain that migrated slightly faster than the normal B beta-chain. Materials andmethods: To clarify the complex genetic mechanismunderlying Tsukuba I's hypodysfibrinogenemia induced by nucleotide deletions in two regions, we generated two minigenes incorporating each deletion region, transfected them into Chinese Hamster Ovary (CHO) cells, and analyzed RT-PCR products. We also established CHO cells producing the recombinant variant fibrinogen,gamma' 409.A (Ex-9 deletion). Results and conclusions: Minigene I incorporating the IVS-8 deletion showed two products: a normal splicing product and the unspliced product. Minigene II incorporating the Ex-9 deletion only produced the unspliced product. The established gamma' 409.A-CHOcells secreted variant fibrinogenmore effectively than normal fibrinogen. Therefore, the aberrant splicing products derived from the IVS-8 deletion cause hypofibrinogenemia most likely due to nonsense-mediated mRNA decay and the partial production of normal.A-and gamma'-chains; moreover, the Ex-9 deletion causes hypodysfibrinogenemia due to the absence of normal.A-and gamma'-chain production (hypofibrinogenemia) and augmented aberrant.'-chain production (dysfibrinogenemia). (C) 2016 Elsevier Ltd. All rights reserved., Article, THROMBOSIS RESEARCH. 148:111-117 (2016)

This study demonstrates a case of a huge genital hematoma after delivery, associated with fibrinogen Dorfen. Fibrinogen Dorfen is the mutation of a fibrinogen-coded exon gene, which has a single heterozygous GCC → GTC transition at codon 289 of the γ gene, predicting an Ala → Val substitution. Because Ala289 plays a crucial role in maintaining the structure of the polymerization site of hole 'a' via a hydrogen bond, it is speculated that the γ 289Ala → Val substitution can change not only the fibrinogen structure, but also the function of polymerization. In our case, although the patient's gene mutation was the same as that of her mother, there was a discrepancy in the clinical outcomes. Although the precise mechanism regarding this discrepancy remains unknown, it may cause different perinatal outcomes in terms of vaginal delivery, such as the severe bleeding in this patient and the absence of clinical symptoms in her mother. This is the first report suggesting the heterogeneity of fibrinogen functions of fibrinogen Dorfen, which may be critical to the clinical outcome.

Background Chimerism analysis is important for the evaluation of engraftment and predicting relapse following hematopoietic stem cell transplantation (HSCT). We developed a chimerism analysis for single nucleotide polymorphisms (SNPs), including rapid screening of the discriminable donor/recipient alleles using droplet allele-specific PCR (droplet-AS-PCR) pre-HSCT and quantitation of recipient DNA using AS-quantitative PCR (AS-qPCR) following HSCT. Methods SNP genotyping of 20 donor/recipient pairs via droplet-AS-PCR and the evaluation of the informativity of 5 SNP markers for chimerism analysis were performed. Samples from six follow-up patients were analyzed to assess the chimerism via AS-qPCR. These results were compared with that determined by short tandem repeat PCR (STR-PCR). Results Droplet-AS-PCR could determine genotypes within 8 min. The total informativity using all 5 loci was 95% (19/20). AS-qPCR provided the percentage of recipient DNA in all 6 follow-up patients without influence of the stutter peak or the amplification efficacy, which affected the STR-PCR results. Conclusion The droplet-AS-PCR had an advantage over STR-PCR in terms of rapidity and simplicity for screening before HSCT. Furthermore, AS-qPCR had better accuracy than STR-PCR for quantification of recipient DNA following HSCT. The present chimerism assay compensates for the disadvantages of STR-PCR and is readily performable in clinical laboratories.

Hereditary fibrinogen Aα-chain amyloidosis (Aα-chain amyloidosis) is a type of autosomal dominant systemic amyloidosis caused by mutations in fibrinogen Aα-chain gene (FGA). Patients with Aα-chain amyloidosis have been mainly reported in Western countries but have been rarely reported in Asia, with only five patients with Aα-chain amyloidosis being reported in Korea, China, and Japan. Clinically, the most prominent manifestation in Asian patients with Aα-chain amyloidosis is progressive nephropathy caused by excessive amyloid deposition in the glomeruli, which is similar to that observed in patients with Aα-chain amyloidosis in Western countries. In molecular features in Asian Aα-chain amyloidosis, the most common variant, E526V, was found in only one Chinese kindred, and other four kindred each had a different variant, which have not been identified in other countries. These variants are located in the C-terminal region (amino acid residues 517?555) of mature Aα-chain, which was similar to that observed in patients with Aα-chain amyloidosis in other countries. The precise number of Asian patients with Aα-chain amyloidosis is unclear. However, patients with Aα-chain amyloidosis do exist in Asian countries, and the majority of these patients may be diagnosed with other types of systemic amyloidosis., Article, INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES.19:320(2018)

We found a novel heterozygous mutation in the fibrinogen Bβ chain (c.490G>A) of a 3-year-old girl with congenital hypofibrinogenemia. To clarify the complex genetic mechanism, we made a mini-gene including a FGB c.490G>A mutation region, transfected it into a Chinese Hamster Ovary (CHO) cell line, and analyzed reverse transcription (RT) products. The assembly process and secretion were examined using recombinant mutant fibrinogen. Direct sequencing demonstrated that the mutant RT product was 99 bp longer than the wild-type product, and an extra 99 bases were derived from intron 3. In recombinant expression, a mutant Bβ-chain was weakly detected in the transfected CHO cell line, and aberrant fibrinogen was secreted into culture media; however, an aberrant Bβ-chain was not detected in plasma. Since the aberrant Bβ-chain was catabolized faster in cells, the aberrant Bβ-chain in a small amount of secreted fibrinogen may catabolize in the bloodstream. FGB c.490G>A indicated the activation of a cryptic splice site causing the insertion of 99 bp in intron 3. This splicing abnormality led to the production of a Bβ-chain possessing 33 aberrant amino acids, including two Cys residues in the coiled-coil domain. Therefore, a splicing abnormality may cause impaired fibrinogen assembly and secretion., Article, INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES.18:2470(2017)

Congenital dysfibrinogenemia refers to the presence of a dysfunctional fibrinogen molecule, typically because of mutations in the fibrinogen gene. About 20% of fibrinogen gene mutations are responsible for thrombosis. Here, we described the case of a 17-year-old Japanese boy, who had a sudden stroke because of superior sagittal sinus thrombosis associated with dysfibrinogenemia. Genetic testing confirmed the presence of homozygous fibrinogen Naples (BβAla68Thr) mutation, which was previously reported as a causative mutation for thrombotic dysfibrinogenemia only in an Italian family. In this Japanese family, the patient's 12-year-old asymptomatic sister was also homozygous for this mutation. She, like her brother, was started on warfarin therapy. This report highlights the occurrence of fibrinogen Naples that has caused severe thrombotic complications in a young member of a Japanese family.

Background ABO genotyping has common tools for personal identification of forensic and transplantation field. We developed a new method based on a droplet allele-specific PCR (droplet-AS-PCR) that enabled rapid PCR amplification. We attempted rapid ABO genotyping using crude DNA isolated from dried blood and buccal cells. Methods We designed allele-specific primers for three SNPs (at nucleotides 261, 526, and 803) in exons 6 and 7 of the ABO gene. We pretreated dried blood and buccal cells with proteinase K, and obtained crude DNAs without DNA purification. Results Droplet-AS-PCR allowed specific amplification of the SNPs at the three loci using crude DNA, with results similar to those for DNA extracted from fresh peripheral blood. The sensitivity of the methods was 5%-10%. The genotyping of extracted DNA and crude DNA were completed within 8 and 9 minutes, respectively. The genotypes determined by the droplet-AS-PCR method were always consistent with those obtained by direct sequencing. Conclusion The droplet-AS-PCR method enabled rapid and specific amplification of three SNPs of the ABO gene from crude DNA treated with proteinase K. ABO genotyping by the droplet-AS-PCR has the potential to be applied to various fields including a forensic medicine and transplantation medical care.

We encountered a 45-year-old Japanese man who suffered from pulmonary thromboembolism and huge right ventricular thrombus after inferior vena cava (IVC) filter implantation without apparent thrombus in either the deep veins or inside the IVC filter. The biochemical data showed a discrepancy in the level of fibrinogen between the immunological and thrombin time methods, suggesting hypodysfibrinogenemia. The sequencing of the fibrinogen γ-chain gene (FGG) revealed a novel heterozygous missense mutation in exon 8 - a TGT to TCT transversion in codon 326 - resulting in an amino acid substitution of serine for cysteine (γCys326Ser). The characterization of the protein did not show known mechanisms for thrombosis in dysfibrinogenemia, such as dimer or albumin-binding complex formation. In summary, the current case with a life-threatening thrombotic event was found to have a novel heterozygous missense mutation resulting in γCys326Ser, which was suggested as a predisposing factor of the thrombosis. Known mechanisms responsible for thrombosis in the current case were not demonstrated, suggesting other mechanisms including superimposing inherited and/or acquired risk factors. When a patient presents with unusual thrombosis such as breakthrough pulmonary embolism and huge thrombus in the right ventricle, as in the current case, the laboratory process for heritable thrombophilia should be considered.

Background Hepatic endoplasmic reticulum (ER) storage disease (HERSD) associated with hypofibrinogenemia has been reported in patients with four types of heterozygous γ-chain variant fibrinogen in the C terminal region. Of interest, substitution of γR375W induced hypofibrinogenemia and HERSD, whereas γR375G induced dysfibrinogenemia. Objectives To analyze the synthesis of variant fibrinogen and morphological characteristics, we established variant fibrinogen-producing cells and compared them with wild-type fibrinogen-synthesizing cells. Methods The fibrinogen γ-chain expression vectors coding γ375W and γ375G were altered by oligonucleotide-directed mutagenesis and transfected into Chinese hamster ovary (CHO) cells. Synthesis of fibrinogen (media and cell lysates) was measured by ELISA for each cloned cell line and morphological characteristics were observed by immunofluorescence and transmission electron microscopy. Results The medium/cell lysate fibrinogen ratio of γ375W-CHO cells was markedly lower than that of the normal cells and γ375G-CHO cells. Immunostaining with anti-fibrinogen antibody showed only γ375W-CHO cells, but revealed two types of cells containing cytoplasmic inclusion bodies, scattered large-granular bodies and fibrous forms. Observation by confocal microscopy indicated that both inclusion bodies were colocalized with fibrinogen and ER-membrane protein; furthermore, transmission electron microscopic observation demonstrated dilatation of the ER by large-granular inclusion bodies and fibrous forms filled with regularly structured fibular materials within the dilated ER. Conclusion These results demonstrated that assembled and non-secreted γ375W fibrinogen was accumulated in the dilated ER and aggregated variant fibrinogen was seen as regularly structured fibular materials, which was similar to the fingerprint-like pattern observed at inclusion bodies in patients’ hepatocytes affected with HERSD.

We 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 mature protein γ23X (native protein: γ49X), and Kanazawa II showed c.1205G>A in FGG exon 9, resulting in γ376X (native protein: γ402X). 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 as an NMD inhibitor. mRNA levels were determined using real-time quantitative RT-PCR in CHO cells. In the absence of cycloheximide, levels of mRNAs transcribed from the mutant gene were lower than from the wild-type gene whereas, in the presence of cycloheximide, levels of mRNAs transcribed from the mutant gene increased dose-dependently. Finally, these results demonstrated that mRNAs containing γ23X or γ376X are degraded by the NMD system and translation of the truncated γ-chain polypeptide decrease in patients' hepatocytes, resulting in hypofibrinogenemias., Article, THROMBOSIS RESEARCH. 132(4):465-470 (2013)

Fibrin polymerisation is mediated by interactions between knobs 'A' and 'B' exposed by thrombin cleavage, and holes 'a' and 'b'. We demonstrated markedly delayed thrombin-catalysed fibrin polymerisation, through B:b interactions alone, of recombinant gamma D364H-fibrinogen with impaired hole 'a'. To determine whether recombinant variant fibrinogens with no release of fibrinopeptide A (FpA) polymerise similarly to gamma D364H-fibrinoge, we examined two variant fibrinogens with substitutions altering knob 'A', A alpha 17A- and A alpha 17C-fibrinogen. We examined thrombin- or batroxobin-catalysed 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 gamma D364H-fibrinogen. Thrombin-catalysed FpA release of A alpha 17A-fibrinogen was substantially delayed and none observed for A alpha 17C-fibrinogen; fibrinopeptide B (FpB) release was delayed for all variants. All variant fibrinogens showed substantially impaired thrombin-catalysed polymerisation; for A alpha 17A-fibrinogen it was delayed less, and for A alpha 17C more than for gamma D364H-fibrinogen. No variants polymerised with batroxobin, which exposed only knob 'A'. The inhibition of variant fibrinogens' polymerisation 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 A alpha 17A- and gamma D364H-fibrinogen had uniform, ordered fibres, thicker than normal, whereas A alpha 17C-fibrinogen formed less organised clots with shorter, thinner, and tapered ends. These results demonstrate that FpA release per se is necessary for effective B:b interactions during polymerisation of variant fibrinogens with impaired A:a interactions., Article, THROMBOSIS AND HAEMOSTASIS. 109(2):221-228 (2013)

The 1965–1967 Matsushiro earthquake swarm in central Japan exhibited two unique characteristics. The first was a hydro-mechanical crust rupture resulting from degassing, volume expansion of CO2/water, and a crack opening within the critically stressed crust under a strike-slip stress. The other was, despite the lower total seismic energy, the occurrence of complexed seismo-electromagnetic (seismo-EM) phenomena of the geomagnetic intensity increase, unusual earthquake lights (EQLs) and atmospheric electric field (AEF) variations. Although the basic rupture process of this swarm of earthquakes is reasonably understood in terms of hydro-mechanical crust rupture, the associated seismo-EM processes remain largely unexplained. Here, we describe a series of seismo-EM mechanisms involved in the hydro-mechanical rupture process, as observed by coupling the electric interaction of rock rupture with CO2 gas and the dielectric-barrier discharge of the modelled fields in laboratory experiments. We found that CO2 gases passing through the newly created fracture surface of the rock were electrified to generate pressure-impressed current/electric dipoles, which could induce a magnetic field following Biot-Savart’s law, decrease the atmospheric electric field and generate dielectric-barrier discharge lightning affected by the coupling effect between the seismic and meteorological activities.

A 9-year-old boy presented with sudden onset of nausea, vomiting and unsteady gait after a bread-eating game, which possibly caused neck hyperextension. Neurological examination revealed hemisensory loss of pain and temperature sensation in the right trunk and limbs along with left Horner’s syndrome, suggesting lateral medullary syndrome (LMS). Magnetic resonance (MR) imaging of the brain revealed infarction at the left lateral medulla. MR angiography showed no sign of arterial dissection and no occlusion or stenosis of the intracranial, basilar or vertebral arteries or their branches. No evidence of cardioemboli or systemic inflammation was apparent. Repeated blood examination revealed low activity of fibrinogen. Genetic testing confirmed the presence of hereditary dysfibrinogenemia with a mutation in the FGB gene (BβGly15Cys). This fibrinogen variant has previously been found in Japanese patients with atherosclerosis obliterans or no symptoms. Under conservative treatment without anticoagulation and aspirin, the patient made a good recovery within a few months. We presume that microthrombosis may have been deposited within the vertebral system as a result of extension and rotation of the neck during sports activity, with a contribution from hereditary dysfibrinogenemia.

Background Quantitative evaluation of minimal residual disease (MRD) following hematopoietic stem cell transplantation (HSCT) is indispensable for patients with hematological malignancies. In addition to established MRD markers such as immunoglobulin and T-cell receptor gene rearrangements, fusion genes, or aberrantly expressed genes, single nucleotide mutations are considered one of the MRD markers that reflect the malignant cell clone. Methods We compared the quantity of mutant genes by allele-specific quantitative polymerase chain reaction (AS-qPCR) for single nucleotide mutations ( TP53 410T > A and PTPN11 1508G > A) with the percentage of autologous DNA by short tandem repeat (STR)-PCR. Results Following HSCT, the quantity of mutant genes detected by AS-qPCR correlated with the percentage of autologous DNA assessed by the STR-PCR. Moreover, mutant DNAs were detected at a quantifiable level before relapse, whereas the percentage of autologous DNA was less than 5%, that is, complete chimerism. Conclusions The AS-qPCR approach for single nucleotide mutations was accurate and highly sensitive for monitoring pre-transplantation as well as post-transplantation MRD. AS-qPCR for single nucleotide mutation is suitable for monitoring MRD in patients who lack previously established MRD markers.

A heterozygous patient with dysfibrinogenemia with slight bleeding and no thrombotic complications was diagnosed with fibrinogen Kyoto VI (K-VI). To elucidate the genetic mutation(s) and characterize the variant protein, we performed the following experiments and compared with identical and similar variants that have already been reported. The proposita's PCR-amplified DNA was analyzed by sequencing and her purified plasma fibrinogen underwent SDS-PAGE followed by immunoblotting, fibrin polymerization, and scanning electron microscopic observation of fibrin clot and fibers. Sequence analyses showed that K-VI fibrinogen substituted W (TGG) for terminal codon (TAG), resulting in 12 amino acid elongation 462-473 (WSPIRRFLLFCM) in the Bβ-chain. Protein analyses indicated that the presence of some albumin-binding variant fibrinogens and a dimeric molecule of variant fibrinogens reduced fibrin polymerization, with a thinner fiber and aberrant fibrin network. These results are almost the same as for the identical variant of Magdeburg, however, different from the similar variant of Osaka VI [12 amino acid elongation 462-473 (KSPIRRFLLFCM) in the Bβ-chain] in the presence of variant forms and clot structure. We speculate the side-chain difference at 462 residues, W in K-VI, K in Osaka VI, and/or the difference in the presence of disulfide bridged forms of variant fibrinogens, led to the notable difference in the fibrin bundle network. Although a strong evolutional and structural association between Bβ-chain and γ-chain molecules is established, the corresponding recombinant 15 residue elongation variants of the fibrinogen γ-chain showed reduced assembly and secretion.

Background Monitoring of minimal residual disease (MRD) in patients with hematological malignancies is important for evaluating the patients' therapeutic response and risk of relapse. Single nucleotide mutations associated with leukemogenesis can be considered as applicable MRD markers. Methods We developed an allele-specific quantitative polymerase chain reaction (AS-qPCR) for FLT3 2503G > T, KIT 2446G > T, and KIT 2447A > T and compared the change in the expression levels of the FLT3 or KIT mutations assessed by AS-qPCR to those of the RUNX1–RUNX1T1 fusion gene and WT1 by conventional quantitative PCR. Results The AS-qPCR using primers including template-mismatched nucleotide or template-mismatched nucleotide plus locked nucleic acid substituted nucleotide provided higher selectivity for mutant nucleotides. The change in the expression levels of the FLT3 or KIT mutations at the time of relapse and just after hematopoietic stem cell transplantation correlated well with that of the RUNX1–RUNX1T1 fusion gene and WT1 . Moreover, during complete remission, only AS-qPCR could detect low-level expression of residual mutations. Conclusions The AS-qPCR for analyzing single nucleotide mutations contributes to the monitoring of MRD in patients without recurrent fusion gene throughout the clinical course and thus broadens the spectrum of patients in whom MRD can be monitored.

SummaryWe found a novel hypofibrinogenemia designated as Matsumoto VII (M-VII), which is caused by a heterozygous nucleotide deletion at position g.7651 in FGG and a subsequent frameshift mutation in codon 387 of the γ-chain. This frameshift results in 25 amino acid substitutions, late termination of translation with elongation by 15 amino acids, and the introduction of a canonical glycosylation site. Western blot analysis of the patient’s plasma fibrinogen visualised with anti-γ-chain antibody revealed the presence of two extra bands. To identify the extra bands and determine which of the above-mentioned alterations caused the assembly and/or secretion defects in the patient, 11 variant vectors that introduced mutations into the cDNA of the γ-chain or γ′-chain were transfected into Chinese hamster ovary cells. In vitro expression of transfectants containing γΔ7651A and γΔ7651A/399T (γΔ7651A with an amino acid substitution of 399Asn by Thr and a variant lacking the canonical glycosylation site) demonstrated a reduction in secretion to approximately 20% of the level seen in the transfectants carrying the normal γ-chain. Furthermore, results from other transfectants demonstrated that eight aberrant residues between 391 and 398 of the M-VII variant, rather than the 15 amino acid extension or the additional glycosylation, are responsible for the reduced levels of assembly and secretion of M-VII variant fibrinogen. Finally, the results of this study and our previous reports demonstrate that the fibrinogen γ-chain C-terminal tail (388–411) is not necessary for protein assembly or secretion, but the aberrant amino acid sequence observed in the M-VII variant (especially 391–398) disturbs these functions.

We found a heterozygous dysfibrinogenemia caused by the substitution of BbetaGly15Cys and designated it fibrinogen Hamamatsu II (H-II). Although the propositus suffered an infarction of the medulla oblongata, other thrombotic risk factors, paradoxical cerebral infarction, and arterial dissection were not found. To determine whether the delayed lysis of fibrin clots or not in the context of the BbetaGly15Cys substitution, we examined the clot lysis and plasmin generation of propositus' fibrinogen. Fibrinogen was purified from the propositus' and normal control plasma by immunoaffinity chromatography and was used for the following experiments: sodium dodecyl sulfate-polyacrylamide gel electrophoresis, fibrin polymerization, scanning electron microscopic observation of fibrin clot and fibers, clot lysis, and tissue-type plasminogen activator-mediated plasminogen activation. The H-II plasma fibrinogen showed the presence of albumin-binding variant forms, a dimeric molecule of variant fibrinogen, and impairment of lateral aggregation during fibrin polymerization. The H-II fibrin clot showed lower density of bundles and thinner diameters of fibers than in the normal fibrin clot. In the clot lysis experiments with overlaid plasmin, H-II fibrin showed a similar lysis period and lysis rate to the normal control. Moreover, plasmin generation from a mixture of thrombin, tissue-type plasminogen activator, plasminogen, and H-II fibrinogen also showed a similar rate to normal fibrinogen. Although the propositus suffered an infarction, the present study did not observe delayed clot lysis, that is, the clot was not resistant to plasmin degradation. Therefore, we did not clarify an association between the BbetaGly15Cys dysfibrinogenemia and arterial thrombosis.

Background Cysteine-bound transthyretin (TTR) in serum is believed to be associated with deposition of amyloid in familial amyloidotic polyneuropathy (FAP). We examined the binding of cysteine and homocysteine with TTR, and the effect of such binding on TTR structure. Methods The concentration of serum cysteine and homocysteine was measured using HPLC and fluorescence detection. TTR was analyzed using isoelectric focusing (IEF), SDS-polyacrylamide gel electrophoresis (PAGE) and mass spectrometry. Results Incubation of serum at 4 °C resulted in an approximate doubling of the amount of both cysteine and homocysteine bound to TTR. Incubation of serum TTR with cysteine–HCl at 37 °C markedly altered the IEF and SDS-PAGE profiles of TTR. The main ion peaks of TTR were observed at m / z 13,761 and 13,881, and assigned as free TTR and TTR + cysteine respectively. Incubation of purified TTR with dithiothreitol followed by cysteine–HCl resulted in loss of the latter peak and an increase in the m / z 13,761 peak, while incubation with cysteine–HCl alone did not cause such a change. Conclusion Cysteine- and homocysteine-bound TTR was more easily denatured by cysteine–HCl than free TTR. Binding of cysteine and homocysteine may alter the structure and characteristics of serum TTR, and may facilitate TTR denaturation and deposition.

Background Apolipoprotein (apo) E in the cerebrospinal fluid (CSF) is abundant with sialic acid (SA), and sialylation of certain proteins is known to modulate biological function. The aim of the present study was to quantify the SA content in CSF apoE and carry out the more detailed characterization of the CSF apoE-containing lipoproteins. Methods The method for the determination of the SA in CSF apoE was based on the conversion of SA into p-aminobenzoic acid ethyl ester-derivatized N-acetylmannosamine, followed by HPLC analysis. Results The levels of CSF SA and serum SA were 25.9 ± 1.5 and 2209 ± 196 μmol/l, respectively; however, when the SA values were corrected by the total protein concentrations, CSF SA values were approximately 3.5-fold of those in the serum. The SA levels in the CSF apoE-containing lipoprotein fractions were 5.3 ± 1.3% of total CSF SA, and were correlated with the CSF apoE concentrations. However, the ratios of SA to apoE were inversely proportional to the CSF lipid concentrations. The lipoprotein particle sizes were larger when the ratios of SA to CSF apoE were greater. Conclusion The SA moiety of the CSF apoE molecules may affect the formation of the apoE-containing lipoprotein particles and the regulation of lipid delivery in CNS.

Article, Clinica Chimica Acta. 401(1-2):119-123 (2009)

The interaction between apolipoprotein (apo) E and beta-amyloid (Abeta) is associated with the development of Alzheimer's disease (AD); however, the details remain unknown. ApoE in cerebrospinal fluid is extensively sialylated, and sialylation of certain proteins are known to modulate biological function. We investigated the effects of a sialic acid moiety of apoE on the apoE-Abeta interaction.We prepared normal apoE3 and its mutant (Thr(194) --Ala) and analyzed their interactions with Abeta(1-42) by using the surface plasmon resonance (SPR) assay. In addition, we performed the SPR assay by using apoE-containing lipoproteins treated with neuraminidase. We also assessed the effect of the mutation on the interaction of apoE3 with liposomes.The binding avidity of the mutant apoE3(#) was approximately 50% that of normal apoE3 (p0.0001). The binding avidity of the apoE-containing lipoproteins for Abeta(1-42) reduced after neuraminidase treatment.We suggest that AD development is controlled not only by the apoE isoforms but also by the posttranslational modifications in apoE, such as those in the sialic acid moieties, which are abundant in apoE derived from the brain.

This is an electronic version of an Article published in Journal of Thrombosis and Haemostasis 2007; 5(12): 2352-2359., Article, JOURNAL OF THROMBOSIS AND HAEMOSTASIS. 5(12): 2352-2359 (2007)

We previously reported a case of heterozygous β-thalassaemia with IVS1-1G > C substitution in the β-globin gene and a non-detectable level of mutant mRNA in the patient's reticulocytes. The purpose of this study was to determine whether the transcription and RNA splicing and processing of the mutant gene occurred. We analysed the expression of the mRNA encoded by the cloned mutant gene in COS-1 cells by reverse transcription-polymerase chain reaction followed by agarose gel electrophoresis and nucleotide sequencing. The G > C mutation completely inactivated the normal 5- splice site and resulted in the activation of two cryptic 5- splice sites, located 16 and 38 nt upstream of the normal site. The usage of these two cryptic sites accords with the findings of reports on IVS1-1G > A or IVS1-1G > C substitution of exon 1 of the β-globin gene. Additional experiments that involved transfection of equal amounts of both normal and mutant vectors into COS-1 cells indicated the presence of mutant mRNAs. In conclusion, the β-thalassaemia gene (IVS1-1G > C) was expressed in transfected cells, but showed aberrant RNA splicing. Further studies will be required to clarify the molecular mechanism that results in severe reduction in the mutant mRNA level in vivo.

Cryofibrinogen (CF) is a type of cryoprotein (CP) that can precipitate in cooled plasma but not in serum, and resolves upon warming. We identified a case of secondary cryofibrinogenemia with cholangiocarcinoma and deep venous thrombosis. The patient's cryocrit measured using a Wintrobe tube was 19% in sodium citrate plasma stored for 7 days at 4 degrees C. We performed quantitative analysis of plasma proteins (fibrinogen, IgG, IgA, IgM, C3, C4, α1-antitrypsin, and C-reactive protein) before and after precipitation for 12 hours at 4 degrees C. The plasma fibrinogen concentration decreased by 16.7% (120 mg/dL --100 mg/dL), whereas the others were unaffected by precipitation. The CP purified from the patient's plasma was washed three times with saline and subjected to Western blot and Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) analyses. Western blot analysis indicated that the purified CP was composed of not only fibrinogen but also fibronectin, α1-antitrypsin, α2-macroglobulin, coagulation factor VIII, and IgG, IgA, and IgM. Interestingly, SDS-PAGE analysis showed that the molecular weight of the patient's CF differed from that of purified normal fibrinogen (340 KDa) and consisted of several low-molecular-weight bands (50-250 KDa). From these results, we speculated that CF found in this case was a mixture of degradated fibrinogen and some plasma proteins. In summary, cryofibrinogenemia is a rare and under-recognized disease. Sample information in routine clinical practice is valuable to diagnose this disease.

Background We encountered two patients with hypodysfibrinogenemia and designated them as Okayama II and Otsu I. Although the affected residue(s) in Okayama II and Otsu I overlapped, functionally determined fibrinogen levels and the ratio of functionally to immunologically determined plasma fibrinogen levels were markedly different. Methods DNA sequence and functional analyses were performed for purified plasma fibrinogen. A recombinant protein was synthesized in Chinese hamster ovary (CHO) cells to determine the secretion of variant fibrinogens. Results A heterozygous A>G in FGG, resulting in γ320Asp>Gly for Okayama II, and a heterozygous deletion of AATGAT in FGG, resulting in the deletion of γAsn319 and γAsp320 (γΔN319-ΔD320) for Otsu I, were obtained. SDS-PAGE and Coomassie staining revealed that the variant γ-chain was not clear in Okayama II, but was clearly present in Otsu I. The lag period for the fibrin polymerization of Okayama II was slightly slower than that of the normal control, whereas Otsu I fibrinogen indicated no polymerization within 30 min. Both variant γ-chains were synthesized in CHO cells and assembled into fibrinogen; however, the fibrinogen concentration ratio of the medium/cell lysate of γ320Gly was six-fold lower than that of γΔN319-ΔD320. Conclusions We concluded that the plasma fibrinogen of Okayama II, constituted by a lower ratio of the variant γ-chain, led to the almost normal functioning of fibrin polymerization. However, the plasma fibrinogen of Otsu I, with a higher ratio of the variant γ-chain, led to marked reductions in fibrin polymerization.

The 448th residue in the fibrinogen Bβ-chain molecule is known to be polymorphic (Arg/Lys, R/K), and its allele frequency was previously estimated to be R: 0.85 and K: 0.15 in the US. In the present study, we collected blood samples from 64 healthy individuals and examined the frequency of the fibrinogen Bβ-chain 448 polymorphism in the Japanese population as well as the relationship between polymorphic types and the function and levels of fibrinogen. The polymorphic site was confirmed by MnlI restriction analysis and direct sequencing analysis for amplified 860 bp PCR products containing the Bβ 448 residue. Fibrinogen plasma levels were estimated based on functional and immunological methods. Functional analyses were performed on the R/R, R/K, and K/K types using thrombin-catalyzed fibrin polymerization. The R/R type was detected in 48 out of 64 subjects, R/K in 15, and K/K in one. Therefore, the allele frequency was found to be R: 0.87 and K: 0.13 for the Bβ 448 site, which was similar to that reported previously in the US. The polymorphism did not affect fibrinogen plasma levels. The results of the analysis on fibrin polymerization of the three types suggested that lateral aggregation may be significantly slower in the fibrinogen Bβ-chain 448R/K and K/K types than in the R/R type. (Original).