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

Ikeda, Minami; Tamaki, Kunihiko; Arai, Shinpei; Mukai, Saki; Takezawa, Yuka; Terasawa, Fumiko; Okumura, Nobuo

doi:10.1016/j.thromres.2014.06.002

Cited by 20 publications

(45 citation statements)

References 27 publications

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“…In order to analyze all exons and exon-intron boundaries of the fibrinogen genes, long-range polymerase chain reaction (PCR) for FGA, FGB, and FGG and direct sequencing were performed as described elsewhere [19].…”

Section: Dna Sequence Analysismentioning

confidence: 99%

“…Purification was performed by immunoaffinity chromatography, utilizing a calcium-dependent monoclonal antibody (IF-1; Iatron Laboratories, Tokyo, Japan), as described [19]. Eluted fractions were pooled and dialyzed at 4°C against 20 mM N- [2-hydroxyethyl] piperazine-N′-[2-ethanesulfonic acid] (HEPES), pH 7.4, 0.12 M NaCl (polymerization buffer).…”

Section: Purification and Characterization Of Plasma Fibrinogenmentioning

confidence: 99%

“…Thrombin-catalyzed fibrin polymerization was followed by measuring changes in turbidity at 350 nm at ambient temperature using an UVmini-1240 spectrophotometer (Shimazu, Tokyo, Japan), as described elsewhere [19]. Three parameters: the lag time, maximum slope, and absorbance change (ΔAbs) for 30 min, were obtained from the turbidity curves, as previously described [19].…”

Section: Thrombin-catalyzed Fibrin Polymerization and Clottabilitymentioning

confidence: 99%

“…Three parameters: the lag time, maximum slope, and absorbance change (ΔAbs) for 30 min, were obtained from the turbidity curves, as previously described [19]. The reactions were performed in triplicate for each sample.…”

Section: Thrombin-catalyzed Fibrin Polymerization and Clottabilitymentioning

confidence: 99%

“…The protection of the plasmin digestion of fibrinogen in the presence of 1 or 5 mM CaCl 2 , and GPRP (the synthetic peptides Gly-Pro-Arg-Pro acetate salt, purity N 97%; Sigma-Aldrich, St. Louis, MO), was analyzed by SDS-PAGE under non-reduced conditions (10% polyacrylamide gel) and followed by Coomassie brilliant blue R-250 staining, as described before [19].…”

Section: Protection Assay Of the Plasmin Digestion Of Fibrinogenmentioning

confidence: 99%

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

Mukai

Ikeda

Takezawa

et al. 2015

Thrombosis Research

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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 ANG in FGG, resulting in γ320AspNGly 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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Section: Dna Sequence Analysismentioning

confidence: 99%

Section: Purification and Characterization Of Plasma Fibrinogenmentioning

confidence: 99%

Section: Thrombin-catalyzed Fibrin Polymerization and Clottabilitymentioning

confidence: 99%

Section: Thrombin-catalyzed Fibrin Polymerization and Clottabilitymentioning

confidence: 99%

Section: Protection Assay Of the Plasmin Digestion Of Fibrinogenmentioning

confidence: 99%

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

Mukai

Ikeda

Takezawa

et al. 2015

Thrombosis Research

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

et al. 2020

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Background: We reported a case of acquired dysfibrinogenemia with monoclonal gammopathy of undetermined significance presenting λ-type IgA M protein. The patient showed lower functional (0.38 g/dL) and normal immunological fibrinogen (3.24 g/dL).To examine the cause of the false lower value of fibrinogen, we performed experiments using the patient's purified fibrinogen and IgA. Methods: Fibrinogen was purified from the patient's plasma, and IgA was purified from patient's plasma or serum by immunoaffinity chromatography. We performed thrombincatalyzed fibrin polymerization, scanning electron microscopy (SEM), immunoblotting analysis, and enzyme-linked immunosorbent assays (ELISAs).Results: 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, and the proportion was significantly higher than normal control plasma using ELISA. Conclusion: Our results suggested that IgA M protein-bound fibrinogen is not normally converted into fibrin, and instead finally forms an aberrantly structured fragile clot. The 3 patient's reduced plasma fibrinogen level was caused by the presence of IgA M proteinbound fibrinogen and not by IgA M protein alone.

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

et al. 2020

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

Abstract: Introduction: We examined a 6-month-old girl with inherited fibrinogen abnormality

Cited by 20 publications

References 27 publications

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

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

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

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

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