Publications by authors named "Nobuo Okumura"

85 Publications

A novel variant fibrinogen, AαE11del, demonstrating the importance of AαE11 residue in thrombin binding.

Int J Hematol 2021 Jul 31. Epub 2021 Jul 31.

Department of Clinical Laboratory Investigation, Graduate School of Medicine, Shinshu University, Matsumoto, Japan.

Introduction: 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.

Materials And Methods: 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.

Results: 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.

Conclusions: 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.
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http://dx.doi.org/10.1007/s12185-021-03200-zDOI Listing
July 2021

Automated screening procedure for the phenotypes of congenital fibrinogen disorders using novel parameters, |min1|c and Ac/|min1|c, obtained from clot waveform analysis using the Clauss method.

Clin Chim Acta 2021 Jul 15;521:170-176. Epub 2021 Jul 15.

Department of Clinical Laboratory Investigation, Graduate School of Medicine, Shinshu University, Matsumoto, Japan; Laboratory of Clinical Chemistry and Immunology, Department of Biomedical Laboratory Sciences, School of Health Sciences, Shinshu University, Matsumoto, Japan.

Introduction: Fibrinogen activity (Ac) is widely measured, but fibrinogen antigen (Ag) is measured only in specialized laboratories, so it is difficult to discriminate congenital fibrinogen disorders (CFDs) from acquired hypofibrinogenemia (aHypo). In this study, to screen for CFD phenotypes we adopted novel parameters, |min1|c and Ac/ |min1|c, and compared these with validated Ac, Ag, and Ac/Ag, and previously proposed Ac/dH and Ac/|min1|.

Materials And Methods: We calibrated |min1| using a CN-6000 instrument and investigated the correlation between Ag and |min1|c for aHypo (n = 131) and CFD [18 dysfibrinogenemia (Dys), two hypodysfibrinogenemia (Hypodys) and four hypofibrinpogenemia (Hypo)]. Furthermore, we proposed a schema for screening CFD phenotypes using |min1|c and Ac/|min1|c.

Results: The |min1|c correlated well with Ag in aHypo, and Ac/|min1|c was a better parameter for screening Dys and Hypodys than Ac/dH and Ac/|min1|. With the combination of |min1|c and Ac/|min1|c parameters, 15 Dys, 2 Hypodys and four Hypo were categorized in agreement with the phenotype determined using Ag and Ac/Ag; conversely three Dys were classified as one Hypodys (AαR16C) and two Hypo (BβG15C).

Conclusion: We demonstrated that |min1|c and Ac/|min1|c are valuable parameters for screening CFD patients and phenotypes in laboratories that do not measure Ag or perform genetic analysis.
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http://dx.doi.org/10.1016/j.cca.2021.07.012DOI Listing
July 2021

Novel variant fibrinogen γp.C352R produced hypodysfibrinogenemia leading to a bleeding episode and failure of infertility treatment.

Int J Hematol 2021 Jun 12. Epub 2021 Jun 12.

Department of Clinical Laboratory Investigation, Graduate School of Medicine, Shinshu University, 3-1-1 Asahi, Matsumoto, 390-8621, Japan.

Introduction: 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.

Materials And Methods: 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.

Results: 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.

Conclusion: 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.
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http://dx.doi.org/10.1007/s12185-021-03174-yDOI Listing
June 2021

Recombinant γY278H Fibrinogen Showed Normal Secretion from CHO Cells, but a Corresponding Heterozygous Patient Showed Hypofibrinogenemia.

Int J Mol Sci 2021 May 14;22(10). Epub 2021 May 14.

Department of Clinical Laboratory Investigation, Graduate School of Medicine, Shinshu University, Matsumoto 390-8621, Japan.

We identified a novel heterozygous hypofibrinogenemia, γY278H (Hiroshima). To demonstrate the cause of reduced plasma fibrinogen levels (functional level: 1.12 g/L and antigenic level: 1.16 g/L), we established γY278H fibrinogen-producing Chinese hamster ovary (CHO) cells. An enzyme-linked immunosorbent assay demonstrated that synthesis of γY278H fibrinogen inside CHO cells and secretion into the culture media were not reduced. Then, we established an additional five variant fibrinogen-producing CHO cell lines (γL276P, γT277P, γT277R, γA279D, and γY280C) and conducted further investigations. We have already established 33 γ-module variant fibrinogen-producing CHO cell lines, including 6 cell lines in this study, but only the γY278H and γT277R cell lines showed disagreement, namely, recombinant fibrinogen production was not reduced but the patients' plasma fibrinogen level was reduced. Finally, we performed fibrinogen degradation assays and demonstrated that the γY278H and γT277R fibrinogens were easily cleaved by plasmin whereas their polymerization in the presence of Ca and "D:D" interaction was normal. In conclusion, our investigation suggested that patient γY278H showed hypofibrinogenemia because γY278H fibrinogen was secreted normally from the patient's hepatocytes but then underwent accelerated degradation by plasmin in the circulation.
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http://dx.doi.org/10.3390/ijms22105218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8156302PMC
May 2021

Congenital Hypofibrinogenemia in a Neonate with a Novel Mutation in the Gene.

Pediatr Rep 2021 Mar 1;13(1):113-117. Epub 2021 Mar 1.

Uji-Tokushukai Medical Center, Department of Pediatrics, Uji, Kyoto 611-0041, Japan.

Detection of severe hypofibrinogenemia (<50 mg/dL) in a neonate soon after birth is alarming because of the risk of hemorrhage. A female neonate was noted to be hypofibrinogenemic (<50 mg/dL) on day 0 of birth; she showed no thrombocytopenia/coagulopathy or hemorrhagic symptoms. Considering the possibility of afibrinogenemia, which may cause bleeding, fresh frozen plasma (FFP) was initiated twice a week to maintain her plasma fibrinogen level at 50-100 mg/dL. Thereafter, we found hypofibrinogenemia in her father and elder sister and plasma fibrinogen levels, determined by clot formation and immunological methods, showed similarly reduced values in both the neonate (proband) and her father. Based on a presumed diagnosis of congenital hypofibrinogenemia, sequencing of the fibrinogen genes was performed, revealing a novel heterozygous mutation of (Genbank NG008833); a p.403Try>Stop. The neonate was treated with repeat FFP infusions until two months of age, when treatment was stopped because she remained asymptomatic.
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http://dx.doi.org/10.3390/pediatric13010016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7930968PMC
March 2021

A Novel Amino Acid Substitution, Fibrinogen Bβp.Pro234Leu, Associated with Hypofibrinogenemia Causing Impairment of Fibrinogen Assembly and Secretion.

Int J Mol Sci 2020 Dec 10;21(24). Epub 2020 Dec 10.

Department of Clinical Laboratory Investigation, Graduate School of Medicine, Shinshu University, Matsumoto 390-8621, Japan.

We identified a novel heterozygous variant, Bβp.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 Bβp.P234L and γp.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 Bβp.P234L and γp.G242E impaired the secretion and synthesis of fibrinogen. Moreover, immunoblotting analysis elucidated the mobility migration of the Bβγ complex in Bβp.P234L. On the other hand, the fibrin polymerization of fibrinogen Tokorozawa was similar to that of normal fibrinogen. In-silico analysis revealed that the Bβp.P234 residue is located in the contact region between the Bβ and γ chains and contacts γp.G242 residue. The present study demonstrated that the Bβp.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 Bβ and γ chains impact the assembly and secretion of fibrinogen.
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http://dx.doi.org/10.3390/ijms21249422DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7764081PMC
December 2020

Changes in serum citrullinated fibrinogen concentration associated with the phase of bacteremia patients.

Clin Chim Acta 2021 Jan 4;512:127-134. Epub 2020 Nov 4.

Department of Health and Medical Sciences, Graduate School of Medicine, Shinshu University, Matsumoto, Japan; Department of Biomedical Laboratory Sciences, Shinshu University School of Medicine, Matsumoto, Japan.

Background: 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.

Methods: 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.

Result: 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).

Conclusion: 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.
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http://dx.doi.org/10.1016/j.cca.2020.10.038DOI Listing
January 2021

Screening method for congenital dysfibrinogenemia using clot waveform analysis with the Clauss method.

Int J Lab Hematol 2021 Apr 8;43(2):281-289. Epub 2020 Oct 8.

Department of Clinical Laboratory Investigation, Graduate School of Medicine, Shinshu University, Matsumoto, Japan.

Introduction: Congenital fibrinogen disorders (CFDs) are classified as afibrinogenemia or hypofibrinogenemia (Hypo), dysfibrinogenemia (Dys), or hypodysfibrinogenemia (Hypodys), according to functional and antigenic fibrinogen concentrations. However, in routine laboratory tests, plasma fibrinogen levels are mostly measured using the functional Clauss method and not as an antigenic level. Therefore, it is difficult to discriminate CFD from acquired hypofibrinogenemia (aHypo). To establish a screening method for CFD, we investigated the parameters of clot waveform analysis (CWA) from the Clauss method.

Methods: We compared fibrinogen concentrations determined using Clauss and prothrombin time (PT)-derived methods for 67 aHypo and CFD cases (19 Dys, 4 Hypodys, and 1 Hypo determined using antigen levels and DNA sequence analysis) with a CS-2400 instrument, and the CWA parameters, dH and Min1, were analyzed automatically with an on-board algorithm. dH and Min1 are the maximum change in transmittance at the end of coagulation and the maximum velocity of transmittance change during coagulation, respectively.

Results: Clauss/PT-derived ratios detected 18 cases of Dys and Hypodys but no Hypo cases, whereas Clauss/dH plus Clauss/Min1 ratios were calculated from fibrinogen concentration using the Clauss method and CWA parameters detected 21 cases of Dys and Hypodys and one Hypo case. Moreover, the Clauss/PT-derived ratio and Clauss/dH plus Clauss/Min1 ratio detected 22 cases of Dys and Hypodys cases and one Hypo case.

Conclusion: This report demonstrates that CWA parameters of the Clauss method, Clauss/dH plus Clauss/Min1 ratio, screened Dys patients with a higher rate, whereas Clauss/PT-derived ratios did not.
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http://dx.doi.org/10.1111/ijlh.13358DOI Listing
April 2021

Congenital fibrinogen disorder with a compound heterozygote possessing two novel FGB mutations, one qualitative and the other quantitative.

Thromb Res 2020 12 20;196:152-158. Epub 2020 Aug 20.

Department of Clinical Laboratory Investigation, Graduate School of Medicine, Shinshu University, Matsumoto, Japan; Department of Biomedical Laboratory Sciences, School of Health Sciences, Shinshu University, Matsumoto, Japan.

Introduction: Congenital fibrinogen disorders result from genetic mutations in FGA, FGB, or FGG resulting in quantitative fibrinogen deficiencies (afibrinogenemia or hypofibrinogenemia) or qualitative fibrinogen deficiencies (dysfibrinogenemia). Hypodysfibrinogenemia sharing features with hypo- and dysfibrinogenemia is rare. We performed genetic and functional analyses of a 31-year-old woman with suspected hypodysfibrinogenemia.

Materials And Methods: Functional and antigenic fibrinogen values of patient were 1.05 and 1.24 g/L, respectively. DNA sequence and western blotting analyses for plasma fibrinogen were performed. A minigene incorporating the mutational region was transfected into a Chinese hamster ovary cell line (CHO), and reverse transcription products were analyzed. Assembly and secretion were examined using the recombinant variant fibrinogen. We purified the patient's plasma fibrinogen and analyzed thrombin-catalyzed fibrin polymerization (TCFP).

Results And Conclusions: DNA sequencing revealed compound heterozygous nucleotide mutations with FGB 35 bp c.1245-17_1262 or -16_1263 del and FGB c.510T>A (resulting in Bβp.N170K substitution) on different alleles. We did not detect shortened Bβ-chain peptides in the plasma using western blotting analysis. A minigene incorporating the deletion DNA showed two aberrant mRNA products. The secretion of Bβp.N170K-fibrinogen-CHO was almost same as normal Bβ-fibrinogen-CHO. TCFP of plasma Bβp.N170K fibrinogen was slightly lower than that of normal plasma fibrinogen. Aberrant splicing products derived from the 35 bp deletion caused hypofibrinogenemia due to nonsense-mediated mRNA decay and suggested the presence of only Bβp.N170K fibrinogen in patient's plasma. Bβp.N170K caused dysfibrinogenemia due to a delay in lateral aggregation. These findings demonstrated that these mutations respectively affected the fibrinogen quality and quantity, resulting in hypodysfibrinogenemia.
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http://dx.doi.org/10.1016/j.thromres.2020.08.031DOI Listing
December 2020

Comparison of molecular structure and fibrin polymerization between two Bβ-chain N-terminal region fibrinogen variants, Bβp.G45C and Bβp.R74C.

Int J Hematol 2020 Sep 19;112(3):331-340. Epub 2020 Jun 19.

Department of Clinical Laboratory Investigation, Graduate School of Medicine, Shinshu University, Matsumoto, Japan.

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.
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http://dx.doi.org/10.1007/s12185-020-02919-5DOI Listing
September 2020

Acquired dysfibrinogenemia: monoclonal λ-type IgA binding to fibrinogen caused lower functional plasma fibrinogen level and abnormal clot formation.

Int J Hematol 2020 Jul 6;112(1):96-104. Epub 2020 Apr 6.

Department of Biomedical Laboratory Sciences, School of Health Sciences, Shinshu University, Matsumoto, Japan.

We report a case of acquired dysfibrinogenemia with monoclonal gammopathy of undetermined significance presenting λ-type IgA M protein. The patient showed lower functional (0.4 g/dL) and normal immunological fibrinogen (2.9 g/dL). To examine the cause of the false lower value of fibrinogen, we performed experiments using the patient's purified fibrinogen and IgA. Fibrinogen was purified from the patient's plasma; IgA was purified from plasma or serum by immunoaffinity chromatography. We performed thrombin-catalyzed fibrin polymerization, scanning electron microscopy (SEM), immunoblotting analysis, and enzyme-linked immunosorbent assays (ELISAs). Fibrin polymerization in the patient's plasma was markedly reduced and SEM showed no fiber bundles or sponge-like structures. Purified IgA did not influence polymerization, whereas immunoprecipitated plasma with an anti-IgA (α-chain) antibody indicated normalization of polymerization and clot structure. Western blotting analysis revealed the presence of monoclonal λ-type IgA-bound fibrinogen, the proportion of which was significantly higher than normal control plasma using ELISA. Our results suggest that IgA M protein-bound fibrinogen is not normally converted into fibrin, but rather leads to formation of an aberrantly structured fragile clot. The patient's reduced plasma fibrinogen level was caused by the presence of IgA M protein-bound fibrinogen, not by IgA M protein alone.
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http://dx.doi.org/10.1007/s12185-020-02874-1DOI Listing
July 2020

Heterozygous variant fibrinogen γA289V (Kanazawa III) was confirmed as hypodysfibrinogenemia by plasma and recombinant fibrinogens.

Int J Lab Hematol 2020 Apr 20;42(2):190-197. Epub 2020 Jan 20.

Department of Clinical Laboratory Investigation, Graduate School of Medicine, Shinshu University, Matsumoto, Japan.

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.
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http://dx.doi.org/10.1111/ijlh.13152DOI Listing
April 2020

γD318Y fibrinogen shows no fibrin polymerization due to defective "A-a" and "B-b" interactions, whereas that of γK321E fibrinogen is nearly normal.

Thromb Res 2019 Oct 20;182:150-158. Epub 2019 Aug 20.

Department of Clinical Laboratory Investigation, Graduate School of Medicine, Shinshu University, Matsumoto, Japan. Electronic address:

Background: The fibrinogen γ-module has several functional sites and plays a role in dysfibrinogenemia, which is characterized by impaired fibrin polymerization. Variants, including γD318Y and γΔN319D320, have been reported at the high affinity Ca-binding site, and analyses using recombinant fibrinogen revealed the importance of this site for fibrinogen functions and secretion. We examined the polymerization abilities of the recombinant fibrinogen variants, γD318Y and γK321E.

Materials And Methods: γD318Y and γK321E were produced using CHO cells and fibrinogen functions were examined using thrombin- or batroxobin-catalyzed polymerization, gel chromatography, protection against plasmin degradation, and factor XIIIa cross-linking.

Results: γD318Y did not show any polymerization by thrombin or batroxobin, similar to γΔN319D320, whereas γK321E had slightly impaired polymerization. The functions of Ca binding, hole 'a', and the "D-D" interaction were markedly reduced in γD318Y, and gel chromatography suggested altered protofibril formation. In silico analyses revealed that structural changes in the γ-module of these variants were inconsistent with polymerization results. The degree of structural changes in γD318Y was moderate relative to those in γD318A and γD320A, which had markedly impaired polymerization, and γK321E, which showed slightly impaired polymerization.

Conclusion: Our results suggest that no polymerization of γD318Y or γΔN319D320 was due to the loss of both "A-a" and "B-b" interactions. Previous studies demonstrated that "B-b" interaction alone causes polymerization of neighboring γD318A and γD320A fibrinogen, which is subsequently decreased. Marked changes in the tertiary structure of the γD318Y γ-module influenced the location and/or orientation of the adjacent β-module, which led to impaired "B-b" interactions.
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http://dx.doi.org/10.1016/j.thromres.2019.08.017DOI Listing
October 2019

Fibrin monomers derived from thrombogenic dysfibrinogenemia, Naples-type variant (BβAla68Thr), showed almost entirely normal polymerization.

Thromb Res 2018 12 4;172:1-3. Epub 2018 Oct 4.

Department of Clinical Laboratory Investigation, Graduate School of Medicine, Shinshu University, Matsumoto, Japan.

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http://dx.doi.org/10.1016/j.thromres.2018.10.004DOI Listing
December 2018

Hereditary Fibrinogen Aα-Chain Amyloidosis in Asia: Clinical and Molecular Characteristics.

Int J Mol Sci 2018 Jan 22;19(1). Epub 2018 Jan 22.

Department of Clinical Laboratory Medicine, Shinshu University School of Health Sciences, Matsumoto 390-8621, Japan.

Hereditary fibrinogen Aα-chain amyloidosis (Aα-chain amyloidosis) is a type of autosomal dominant systemic amyloidosis caused by mutations in α gene (). 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.
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http://dx.doi.org/10.3390/ijms19010320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5796263PMC
January 2018

A Novel Mutation in the Fibrinogen Bβ Chain (c.490G>A; End of Exon 3) Causes a Splicing Abnormality and Ultimately Leads to Congenital Hypofibrinogenemia.

Int J Mol Sci 2017 Nov 20;18(11). Epub 2017 Nov 20.

Department of Health and Medical Sciences, Graduate School of Medicine, Shinshu University, 3-1-1 Asahi, Matsumoto 390-8621, Japan.

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 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. 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.
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http://dx.doi.org/10.3390/ijms18112470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5713436PMC
November 2017

A novel frameshift mutation in the fibrinogen γC terminal region, FGG c.1169_1170 del AT, leading to hypofibrinogenemia.

Thromb Res 2017 11 2;159:82-85. Epub 2017 Oct 2.

Department of Health and Medical Sciences, Graduate School of Medicine, Shinshu University, Matsumoto, Japan.

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http://dx.doi.org/10.1016/j.thromres.2017.10.002DOI Listing
November 2017

Congenital dysfibrinogenemia in a Japanese family with fibrinogen Naples (BβAla68Thr) manifesting as superior sagittal sinus thrombosis.

Blood Coagul Fibrinolysis 2017 Oct;28(7):580-584

aDepartment of Pediatrics bDepartment of Neurosurgery cDepartment of Hematology and Internal medicine, Seirei-Mikatahara General Hospital, Hamamatsu, Shizuoka dDepartment of Biomedical Laboratory Sciences, School of Health Sciences, Shinshu University, Matsumoto, Nagano, Japan.

: 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.
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http://dx.doi.org/10.1097/MBC.0000000000000641DOI Listing
October 2017

Hypodysfibrinogenemia with a Heterozygous Mutation of γCys326Ser by the Novel Transversion of TGT to TCT in a Patient with Pulmonary Thromboembolism and Right Ventricular Thrombus.

Cardiology 2017;137(3):167-172. Epub 2017 Apr 19.

Division of Cardiology, Department of Medicine, Tokai University Hachioji Hospital, Tokyo, Japan.

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.
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http://dx.doi.org/10.1159/000457899DOI Listing
August 2018

Causal mechanisms of seismo-EM phenomena during the 1965-1967 Matsushiro earthquake swarm.

Sci Rep 2017 03 21;7:44774. Epub 2017 Mar 21.

Genesis Research Institute, Inc., 4-1-35 Noritake-shinmachi, Nishi-ku, Nagoya, Aichi, 451-0051, Japan.

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 CO/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 CO gas and the dielectric-barrier discharge of the modelled fields in laboratory experiments. We found that CO 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.
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http://dx.doi.org/10.1038/srep44774DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5359576PMC
March 2017

Rapid ABO genotyping by high-speed droplet allele-specific PCR using crude samples.

J Clin Lab Anal 2018 Jan 13;32(1). Epub 2017 Mar 13.

Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan.

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.
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http://dx.doi.org/10.1002/jcla.22196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6816865PMC
January 2018

The fibrous form of intracellular inclusion bodies in recombinant variant fibrinogen-producing cells is specific to the hepatic fibrinogen storage disease-inducible variant fibrinogen.

Int J Hematol 2017 Jun 4;105(6):758-768. Epub 2017 Feb 4.

Department of Health and Medical Sciences, Graduate School of Medicine, Shinshu University, Matsumoto, Japan.

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.
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http://dx.doi.org/10.1007/s12185-017-2185-5DOI Listing
June 2017

Genetic analyses of novel compound heterozygous hypodysfibrinogenemia, Tsukuba I: FGG c.1129+62_65 del AATA and FGG c.1299+4 del A.

Thromb Res 2016 Dec 5;148:111-117. Epub 2016 Nov 5.

Department of Health and Medical Sciences, Graduate School of Medicine, Shinshu University, Matsumoto, Japan. Electronic address:

Introduction: We found a novel hypodysfibrinogenemia designated Tsukuba I caused by compound heterozygous nucleotide deletions with 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 γ'-chain, but not the γA-chain. A Western blot analysis of plasma fibrinogen from our patient revealed an aberrant γ-chain that migrated slightly faster than the normal Bβ-chain.

Materials And Methods: To clarify the complex genetic mechanism underlying 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, γ'409ΔA (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 γ'409ΔA-CHO cells secreted variant fibrinogen more 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 γA- and γ'-chains; moreover, the Ex-9 deletion causes hypodysfibrinogenemia due to the absence of normal γA- and γ'-chain production (hypofibrinogenemia) and augmented aberrant γ'-chain production (dysfibrinogenemia).
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http://dx.doi.org/10.1016/j.thromres.2016.11.002DOI Listing
December 2016

Familial discrepancy of clinical outcomes associated with fibrinogen Dorfen: A case of huge genital hematoma after episiotomy.

J Obstet Gynaecol Res 2016 Jun 23;42(6):722-725. Epub 2016 Mar 23.

Departments of Obstetrics and Gynecology, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan.

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.
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http://dx.doi.org/10.1111/jog.12972DOI Listing
June 2016

Differences in the function and secretion of congenital aberrant fibrinogenemia between heterozygous γD320G (Okayama II) and γΔN319-ΔD320 (Otsu I).

Thromb Res 2015 Dec 10;136(6):1318-24. Epub 2015 Nov 10.

Department of Health and Medical Sciences, Graduate School of Medicine, Shinshu University, Matsumoto, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.thromres.2015.11.011DOI Listing
December 2015

[A Case of Secondary Cryofibrinogenemia with Cholangiocarcinoma and Deep Venous Thrombosis].

Rinsho Byori 2015 Apr;63(4):421-6

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.
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April 2015

[Polymorphism frequency of fibrinogen Bβ-chain 448Arg and Lys, and the differences of plasma fibrinogen level and clotting function with three genotypes in Japanese].

Rinsho Byori 2014 Dec;62(12):1185-90

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).
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December 2014

Rapid single nucleotide polymorphism based method for hematopoietic chimerism analysis and monitoring using high-speed droplet allele-specific PCR and allele-specific quantitative PCR.

Clin Chim Acta 2015 May 20;445:101-6. Epub 2015 Mar 20.

Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan.

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 8min. 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.
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http://dx.doi.org/10.1016/j.cca.2015.03.018DOI Listing
May 2015

Novel heterozygous dysfibrinogenemia, Sumida (AαC472S), showed markedly impaired lateral aggregation of protofibrils and mildly lower functional fibrinogen levels.

Thromb Res 2015 Apr 15;135(4):710-7. Epub 2015 Jan 15.

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:

Introduction: We encountered a 6-year-old girl with systemic lupus erythematosus. Although no bleeding or thrombotic tendency was detected, routine coagulation screening tests revealed slightly lower plasma fibrinogen levels, as determined by functional and antigenic measurements (functional/antigenic ratio=0.857), suggesting hypodysfibrinogenemia.

Materials And Methods: DNA sequence and functional analyses were performed on purified plasma fibrinogen, and recombinant variant fibrinogen was synthesized in Chinese hamster ovary cells based on the results obtained.

Results: DNA sequencing revealed a heterozygous AαC472S substitution (mature protein residue number) in the αC-domain. AαC472S fibrinogen indicated the presence of additional disulfide-bonded molecules, and markedly impaired lateral aggregation of protofibrils in spite of slightly lower functional plasma fibrinogen levels. Scanning electron microscopic observations showed a thin fiber fibrin clot, and t-PA and plasminogen-mediated clot lysis was similar to that of a normal control. Recombinant variant fibrinogen-producing cells demonstrated that destruction of the Aα442C-472C disulfide bond did not prevent the synthesis or secretion of fibrinogen, whereas the variant Aα chain of the secreted protein was degraded faster than that of the normal control.

Conclusion: Our results suggest that AαC472S fibrinogen may cause dysfibrinogenemia, but not hypofibrinogenemia. The destruction and steric hindrance of the αC-domain of variant fibrinogen led to the impaired lateral aggregation of protofibrils and t-PA and plasminogen-mediated fibrinolysis, as well as several previously reported variants located in the αC-domain, and demonstrated the presence of disulfide-bonded molecules.
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http://dx.doi.org/10.1016/j.thromres.2015.01.013DOI Listing
April 2015

[College education for medical technologists of the next generation].

Authors:
Nobuo Okumura

Rinsho Byori 2014 May;62(5):487-92

Medical Technologists (MTs) of the next generation will be expected to: 1) perform clinical tests in clinical laboratories as so-called Clinical Laboratory Scientists(CLS), 2)research and develop highly advanced reagents, devices, or procedures for clinical laboratories, and 3) educate MTs and research in the college or university. CLS are required to develop and maintain highly advanced medical skills as follows: (1) explaining medical tests and those results to patients, (2) evaluating and explaining test results to medical doctors, (3) advising medical doctors of laboratory diagnoses, (4) analyzing the patients' pathophysiology based on samples with aberrant results, (5) evaluating newly developed reagents, devices, or procedures, and (6) promoting the total medical cure of patients with specialized skills. In the MT course at Shinshu University, to develop the skills necessary to become a CLS before graduation, students participate in a number of programs, i.e., freshman seminars, observing the clinical laboratory, and basic training for medical tests (first grade), special lectures from MTs working in the clinical laboratory (second and third grades), examination for clinical practice, 12-week clinical practice, and 15-week laboratory research (fourth grade). Several academic members working in a clinical laboratory and collaboration with the Department of Clinical Laboratory at Shinshu University Hospital are essential to realize the above-mentioned course.
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May 2014
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