Noriko Fujihara

B : b interactions are essential for polymerization of variant fibrinogens with impaired holes 'a'

Background: Fibrin polymerization is mediated by interactions between knobs ‘A’ and ‘B’ exposed by thrombin cleavage, and holes ‘a’ and ‘b’ always present in fibrinogen. The role of A:a interactions is well established, but the roles of knob:hole interactions A:b, B:b or B:a remain ambiguous.Objectives: To determine whether A:b or B:b interactions have a role in thrombin‐catalyzed polymerization, we examined a series of fibrinogen variants with substitutions altering holes ‘a’: γ364Ala, γ364His or γ364Val.Methods: We examined thrombin‐ and reptilase‐catalyzed fibrinopeptide release by high‐performance liquid chromatography, fibrin clot formation by turbidity, fibrin clot structure by scanning electron microscopy (SEM) and factor (F) XIIIa‐catalyzed crosslinking by sodium dodecylsulfate polyacrylamide gel electrophoresis.Results: Thrombin‐catalyzed fibrinopeptide A release was normal, but fibrinopeptide B release was delayed for all variants. The variant fibrinogens all showed markedly impaired thrombin‐catalyzed polymerization; polymerization of γ364Val and γ364His were more delayed than γ364Ala. There was absolutely no polymerization of any variant with reptilase, which exposed only knobs ‘A’. SEM showed that the variant clots formed after 24 h had uniform, ordered fibers that were thicker than normal. Polymerization of the variant fibrinogens was inhibited dose‐dependently by the addition of either Gly‐Pro‐Arg‐Pro (GPRP) or Gly‐His‐Arg‐Pro (GHRP), peptides that specifically block holes ‘a’ and ‘b’, respectively. FXIIIa‐catalyzed crosslinking between γ‐chains was markedly delayed for all the variants.Conclusion: These results demonstrate that B:b interactions are critical for polymerization of variant fibrinogens with impaired holes ‘a’. Based on these data, we propose a model wherein B:b interactions participate in protofibril formation.

Functional analysis of recombinant B beta 15C and B beta 15A fibrinogens demonstrates that B beta 15G residue plays important roles in FPB release and in lateral aggregation of protofibrils

Summary.  Background and objectives: Analysis of dysfibrinogens has improved our understanding of molecular defects and their effects on the function of intact fibrinogen. To eliminate the influence of plasma heterozygous molecules, we synthesized and analyzed recombinant‐variant fibrinogens. Methods: We synthesized two recombinant‐variant fibrinogens with a single amino acid substitution at the 15Gly residue in the Bβ‐chain: namely, Bβ15Cys and Bβ15Ala. Results: Western blotting analysis of purified fibrinogen revealed the existence of a small amount of a dimeric form only for Bβ15Cys fibrinogen. For Bβ15Cys fibrinogen, functional analysis indicated (a) no thrombin‐catalyzed fibrinopeptide B (FPB) release and (b) markedly impaired lateral aggregation in thrombin‐ and reptilase‐catalyzed fibrin polymerizations. For Bβ15Ala fibrinogen, such analysis indicated slight impairments of both thrombin‐catalyzed FPB release and lateral aggregation in thrombin‐catalyzed fibrin polymerization, but nearly normal lateral aggregation in reptilase‐catalyzed fibrin polymerization. These impaired lateral aggregations were accompanied by thinner fibrin fiber diameters (determined by scanning electron microscopy of the corresponding fibrin clots). Conclusion: We conclude that a region adjacent to Bβ15Gly plays important roles in lateral aggregation not only in desA fibrin polymerization, but also in desAB fibrin polymerization, and we speculate that the marked functional differences between Bβ15A and Bβ15C fibrinogens in FPB release and fibrin polymerization might not only be due to the presence of a substituted cysteine residue in Bβ15C fibrinogen, but also to the existence of disulfide‐bonded forms. Finally, our data indicate that the Bβ15Gly residue plays important roles in FPB release and lateral aggregation of protofibrils.

Factor H gene variants in Japanese: its relation to atypical hemolytic uremic syndrome.

Mutations and polymorphisms of factor H gene (FH1) are known to be closely involved in the development of atypical hemolytic uremic syndrome (aHUS). Several groups have identified disease risk mutations and polymorphisms of FH1 for the development of aHUS, and have investigated frequencies of aHUS in a number of ethnic groups. However, such studies on Japanese populations are limited. In the present study, we analyzed FH1 in Japanese aHUS patients and healthy volunteers, and examined whether those variants impacted on a tendency for the development of aHUS in Japanese populations. Similar to previous studies, we found that a high frequency of FH1 mutations, located in exon 23 of FH1, encodes short consensus repeat 20 in C-terminal end of factor H molecule in patients with aHUS (40%), but not in healthy volunteers. Interestingly, no significant differences in frequency of well-known disease risk polymorphisms for aHUS were observed between healthy volunteers and aHUS patients. Our results suggested that although FH1 mutations relates to the development of Japanese aHUS in accordance with other ethnic studies, other factor may be required for factor H polymorphism to be a risk factor of Japanese aHUS.

Evidence that heterodimers exist in the fibrinogen Matsumoto II (γ308N→K) proband and participate in fibrin fiber formation

INTRODUCTION In this report, we established the gamma308K/N fibrinogen-secreting Chinese hamster ovary (CHO) cell line, which is an artificially heterozygous, Matsumoto II (M-II; gamma308K-->N) type of dysfibrinogen, to indirectly demonstrate the existence of heterodimeric molecules in propositus plasma and the participation of these molecules in fibrin fiber formation. MATERIALS AND METHODS We co-transfected the gamma-chain of gamma308K- and gamma308N-coding vectors into CHO cells expressing Aalpha- and Bbeta-chains and selected the clones by utilizing the unique electrophoretic mobility of the variant gamma-chain of gamma308K. Although sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis under reducing conditions showed that the amount of the variant gamma-chain was slightly less than the amount of normal gamma-chain in recombinant gamma308K/N fibrinogen, we judged that our selected clone was still a useful model of the M-II individual. RESULTS AND CONCLUSION Functional analyses demonstrated that thrombin-catalyzed fibrin polymerization decreased in the following order: gamma308N, gamma308K/N, an equimolar mixture of gamma308K with gamma308N. The difference in the polymerization curves between gamma308N and gamma308K/N is highly similar to the difference between plasma fibrinogen from a normal control and the M-II proband. In addition, experimental results using the equimolar mixture indicated that gamma308K is able to polymerize into fibrin fibers and does not inhibit the gamma308N polymerization. In conclusion, our results indirectly demonstrated that gamma308K/N fibrinogen is the mixture of normal homodimers, heterodimers, and variant homodimers, and all of these can participate in the fibrin fiber formation.

In vitro transcription of compound heterozygous hypofibrinogenemia Matsumoto IX; first identification of FGB IVS6 deletion of 4 nucleotides and FGG IVS3-2A > G causing abnormal RNA splicing

BACKGROUND We reported a case of hypofibrinogenemia Matsumoto IX (M IX) caused by a novel compound heterozygous mutation involving an FGB IVS6 deletion of 4 nucleotides (Delta4b) (three T, one G; between FGB IVS6-10 and -16) and FGG IVS3-2A/G, which are both identified for the first time. To examine the transcription of mRNA from the M IX gene, we cloned the wild-type and mutant genes into expression vectors. METHODS The vectors were transfected into CHO cells and transiently produced wild-type, Bbeta- or gamma-mRNA in the cells. The mRNAs amplified with RT-PCR were analyzed by agarose gel electrophoresis and nucleotide sequencing. RESULTS The RT-PCR product from FGB IVS6Delta4b showed aberrant mRNA that included both introns 6 and 7, and that from FGG IVS3-2G showed two aberrant mRNAs, a major one including intron 3 and a minor in which intron 3 was spliced by a cryptic splice site in exon 4. We speculated that the aberrant mRNAs are degraded before translation into proteins, and/or translated variant chains are subjected to quality control and degraded in the cytoplasm. CONCLUSION The reduced plasma fibrinogen level of the M IX patient was caused by abnormal RNA splicing of one or both of the FGB and FGG genes.

Recombinant variant fibrinogens substituted at residues γ326Cys and γ339Cys demonstrated markedly impaired secretion of assembled fibrinogen

BACKGROUND To study the functions of residues gamma326Cys and gamma339Cys in the assembly and/or secretion of fibrinogen, recombinant fibrinogens were synthesized to replicate naturally occurring gamma326Tyr and gamma326Ser variants, along with gamma326Ala and gamma339Ala variants. METHODS A fibrinogen gamma-chain expression vector was altered and transfected into Chinese hamster ovary (CHO) cells. Cell lysates and culture media of the established cell lines were subjected to ELISA and immunoblotting analysis. In addition, pulse-chase analysis was performed. RESULTS The CHO cells synthesized mutant gamma-chains and assembled these into fibrinogen in the cells, although these variant fibrinogens were barely secreted into the culture media. Pulse-chase analysis indicated that the rates of both assembly and secretion of the variant fibrinogens were lower than that of normal fibrinogen. CONCLUSIONS The present study indicated that the 326-339 intrachain disulfide bond has a crucial role in maintaining the tertiary structure of the C-terminal domain of the gamma-module, which is necessary for fibrinogen assembly and specifically secretion. A combination of the present results and observations from naturally occurring heterozygous cases of gamma326Tyr and gamma326Ser suggest that heterozygous fibrinogen molecules containing variant gamma-chains might be secreted into plasma and show impaired fibrin polymerization, resulting in a phenotype of hypodysfibrinogenemia.

In vitro expression of β-thalassaemia gene (IVS1-1G>C) reveals complete inactivation of the normal 5' splice site and alternative aberrant RNA splicing

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 patients 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.