Fibrinopeptide A release is necessary for effective B:b interactions in polymerisation of variant fibrinogens with impaired A:a interactions

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.ArticleTHROMBOSIS AND HAEMOSTASIS. 109(2):221-228 (2013)journal articl

Nonsense-mediated mRNA decay was demonstrated in two hypofibrinogenemias caused by heterozygous nonsense mutations of FGG, Shizuoka III and Kanazawa II

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.ArticleTHROMBOSIS RESEARCH. 132(4):465-470 (2013)journal articl

siRNA down-regulation of FGA mRNA in HepG2 cells demonstrated that heterozygous abnormality of the A alpha-chain gene does not affect the plasma fibrinogen level

Introduction: We encountered two afibrinogenemia patients with homozygous and compound heterozygous FGA mutation. Of interest, the patients' parents, who are heterozygous, had normal levels of plasma fibrinogen; thus, we hypothesized that liver FGA mRNA levels were higher than those of FGB and/or FGG mRNA. Materials and Methods: To test the hypothesis, we quantitated mRNA levels of a normal liver and a human hepatocyte cell line, HepG2 cells, and performed siRNA-mediated down-regulation of the fibrinogen gene in HepG2 cells. mRNA levels were determined using real-time quantitative RT-PCR for three normal livers and HepG2 cells. Down-regulation of FGA, FGB, or FGG in HepG2 cells was performed by the addition of siRNA corresponding to each of the three genes, and the mRNA levels determined in the cells and the secreted fibrinogen concentration in media. Results: The mRNA level of normal human liver was FGA=FGB>FGG and the FGG mRNA level was about 2-fold lower than the others, that of HepG2 cells was FGA>FGG>FGB and FGA mRNA was approximately 2- or 4-fold higher than FGG mRNA and FGB mRNA. When FGA, FGB, or FGG mRNA expression levels were down-regulated by nearby 50%, fibrinogen concentrations in media were 78%, 49%, or 57% of the control, respectively. Conclusions: Our results suggest that FGG mRNA levels limit fibrinogen expression in normal liver and HepG2 cells and that 50% reduction of FGA mRNA levels would not limit fibrinogen expression in normal liver and HepG2 cells.ArticleTHROMBOSIS RESEARCH. 131(4):342-348 (2013)journal articl

Novel heterozygous dysfibrinogenemia, Sumida (A alpha C472S), showed markedly impaired lateral aggregation of protofibrils and mildly lower functional fibrinogen levels

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 A alpha C472S substitution (mature protein residue number) in the alpha C-domain. A alpha C472S 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 A alpha 442C-472C disulfide bond did not prevent the synthesis or secretion of fibrinogen, whereas the variant A alpha chain of the secreted protein was degraded faster than that of the normal control. Conclusion: Our results suggest that A alpha C472S fibrinogen may cause dysfibrinogenemia, but not hypofibrinogenemia. The destruction and steric hindrance of the alpha C-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 alpha C-domain, and demonstrated the presence of disulfide-bonded molecules.ArticleTHROMBOSIS RESEARCH. 135(4):710-717 (2015)journal articl

Nonsense-mediated mRNA decay was demonstrated in two hypofibrinogenemias caused by heterozygous nonsense mutations of FGG, Shizuoka III and Kanazawa II

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.ArticleTHROMBOSIS RESEARCH. 132(4):465-470 (2013)journal articl

Analysis of plasmin generation and clot lysis of plasma fibrinogen purified from a heterozygous dysfibrinogenemia, B beta Gly15Cys (Hamamatsu II)

This is a non-final version of an article published in final form in Blood Coagulation & Fibrinolysis. 20(8):726-732, December 2009.We found a heterozygous dysfibrinogenemia caused by the substitution of B beta Gly15Cys 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 B beta Gly15Cys 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 B beta Gly15Cys dysfibrinogenemia and arterial thrombosis.ArticleBLOOD COAGULATION & FIBRINOLYSIS. 20(8):726-732 (2009)journal articl

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

Introduction: We examined a 6-month-old girl with inherited fibrinogen abnormality and no history of bleeding or thrombosis. Routine coagulation screening tests showed a markedly low level of plasma fibrinogen determined by functional measurement and also a low level by antigenic measurement (functional/antigenic ratio = 0.295), suggesting hypodysfibrinogenemia. Materials and methods: DNA sequence analysis was performed, and gamma T305A fibrinogen was synthesized in Chinese hamster ovary cells based on the results. We then functionally analyzed and compared with that of nearby recombinant gamma N308K fibrinogen. Results: DNA sequence analysis revealed a heterozygous gamma T305A substitution (mature protein residue number). The gamma T305A fibrinogen indicated markedly impaired thrombin-catalyzed fibrin polymerization both in the presence or absence of 1 mM calcium ion compared with that of gamma N308K fibrinogen. Protection of plasmin degradation in the presence of calcium ion or Gly-Pro-Arg-Pro peptide (analogue for so-called knob 'A') and factor XIIIa-catalyzed fibrinogen crosslinking demonstrated that the calcium binding sites, hole 'a' and D:D interaction sites were all markedly impaired, whereas gamma N308K was impaired at the latter two sites. Molecular modeling demonstrated that gamma T305 is localized at a shorter distance than gamma N308 from the high affinity calcium binding site and hole 'a'. Conclusion: Our findings suggest that gamma T305 might be important for construction of the overall structure of the. module of fibrinogen. Substitution of gamma T305A leads to both dysfibrinogenemic and hypofibrinogenemic characterization, namely hypodysfibrinogenemia. We have already reported that recombinant gamma T305A fibrinogen was synthesized normally and secreted slightly, but was significantly reduced.ArticleTHROMBOSIS RESEARCH. 134(2):518-525 (2014)journal articl

gamma 375W fibrinogen-synthesizing CHO cells indicate the accumulation of variant fibrinogen within endoplasmic reticulum

Background: Hepatic endoplasmic reticulum (ER) storage disease (HERSD) associated with hypofibrinogenemia has been reported in patients with four types of heterozygous gamma-chain variant fibrinogen in the C terminal region. Of interest, substitution of gamma R375W induced hypofibrinogenemia and HERSD, whereas gamma R375G 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 gamma-chain expression vectors coding gamma 375W and gamma 375G 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 gamma 375W-CHO cells was markedly lower than that of the normal cells and gamma 375G-CHO cells. Immunostaining with anti-fibrinogen antibody showed only gamma 375W-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 gamma 375W 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.ArticleTHROMBOSIS RESEARCH. 133(1):101-107 (2014)journal articl

A C-terminal amino acid substitution in the gamma-chain caused by a novel heterozygous frameshift mutation (Fibrinogen Matsumoto VII) results in hypofibrinogenemia

This article is not an exact copy of the original published article in THROMBOSIS AND HAEMOSTASIS. The definitive publisher-authenticated version of THROMBOSIS AND HAEMOSTASIS. 104(2):213-223 (2010) is available online at: https://doi.org/10.1160/TH09-08-0540 .We 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 visualized 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 orγ’-chain were transfected into CHO cells. In vitro expression of transfectants containingγΔ7651A 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 8 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.ArticleTHROMBOSIS AND HAEMOSTASIS. 104(2):213-223 (2010)journal articl

Molecular analysis of afibrinogenemic mutations caused by a homozygous FGA1238 bp deletion, and a compound heterozygous FGA1238 bp deletion and novel FGA c.54+3A > C substitution

We identified two afibrinogenemic girls in two Japanese families and performed molecular analysis to clarify the mechanisms of fibrinogen defects. Genetic analyses were performed by PCR amplification of the fibrinogen gene and DNA sequence analysis. To analyze the mechanisms of mature fibrinogen defects in plasma, we cloned minigenes from the proposita's PCR-amplified DNA, transfected them into CHO cells, and sequenced the cDNA amplified with the RT reaction followed by PCR. Sequence analyses indicated that one was caused by a homozygous 1238 bp deletion of the fibrinogen A alpha-chain gene (FGA Delta 1238) and the other was a compound heterozygous FGA Delta 1238 and novel FGA c.54+3A > C substitution. The minigene corresponding to FGA Delta 1238 generates two aberrant mRNAs, both of which may induce a frameshift and terminate prematurely. In contrast, the minigene corresponding to FGA c.54+3A > C generates two aberrant mRNAs, one of which may induce a frameshift and terminate prematurely, and the other uses a cryptic 5' splice site in exon 1, resulting in the deletion of six amino acids in signal peptides. Molecular analyses of both genetic variants suggest that the lack of a mature A alpha-chain, impaired assembly, and/or secretion of the fibrinogen molecule may lead to afibrinogenemia.ArticleINTERNATIONAL JOURNAL OF HEMATOLOGY. 96(1):39-46 (2012)journal articl