Masako Hirota-Kawadobora

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.

Fibrinogens Kosai and Ogasa: Bβ15Gly→Cys (GGT→TGT) substitution associated with impairment of fibrinopeptide B release and lateral aggregation

Summary.  We found two heterozygous dysfibrinogenemias, designated fibrinogen Kosai and fibrinogen Ogasa. Kosai was associated with arteriosclerosis obliterans but Ogasa showed no bleeding or thrombotic tendencies. The plasma fibrinogen concentrations from the two propositi (Ogasa and Kosai) were much lower when determined by the thrombin‐time method (0.94 and 1.06 g L−1, respectively) than when determined by the immunological method (2.87 and 2.72 g L−1, respectively). We performed DNA sequencing and functional analyses to clarify the relationship between the structural and functional abnormalities. Genetic analysis of PCR‐amplified DNA from the propositi identified the heterozygous substitution Bβ15Gly→Cys (GGT→TGT). Western blotting analysis of purified fibrinogen revealed the existence of albumin–fibrinogen complexes. Functional analyses indicated that compared with the normal control, the propositis fibrinogen released only half the normal amount of fibrinopeptide B and showed markedly impaired polymerization. In addition, the observation of thinner fibers in fibrin clots (by scanning electron microscopy) indicated markedly defective lateral aggregation in the variant fibrinogens. The impaired functions may be due to the substitution of Cys for Bβo15Gly plus the existence of some additional disulfide‐bonded forms.

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.

Recombinant fibrinogen, γ275Arg → Cys, exhibits formation of disulfide bond with cysteine and severely impaired D:D interactions

Summary.  Background and objectives: Analysis of dysfibrinogens has provided useful information aiding our understanding of molecular defects in fibrin polymerization. We have already reported impaired fibrin polymerization in a variant fibrinogen (γArg275Cys), the Cys being located in the D:D interface. Since this substitution occurred in a heterozygous individual, interpretation of the functional analysis was complicated. We tried to resolve this complication by synthesizing a recombinant variant fibrinogen. Methods: A variant γ‐chain expression plasmid was transfected into Chinese hamster ovary cells expressing normal human fibrinogen Aα‐ and Bβ‐chains. The recombinant variant fibrinogen (γ275C) was purified using an immunoaffinity column, and we compared its structure and functions with those of normal recombinant fibrinogen (γ275R) and plasma variant fibrinogen. Results: Mass analyses showed the existence of disulfide‐linked Cys in both patient and recombinant variant fibrinogens. Functional analyses indicated that both fibrin polymerization and γ–γ dimer formation were markedly impaired in the variant fibrinogen. The impairments were much more pronounced in γ275C than in plasma variant fibrinogen. In addition, scanning electron microscopic observation of fibrin clots made from γ275C revealed less dense fibrin fiber bundles and larger fiber diameter than in those made from γ275R, and also the existence of many aberrant fibrin fibers with tapered ends. Conclusion: These results indicate that γArg275 has an important residue affecting the structure and function of the γ‐chain C‐terminal domain. However, the variant D:D interface can interact with that of the normal fibrinogen existing in a heterozygous patient with dysfibrinogenemia.

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.

Quantitative RT-PCR analysis demonstrates that synthesis of the recombinant fibrinogen is dependent on the transcription and synthesis of γ-chain

Background: The purpose of this study was to examine the relationship between the production of secreted fibrinogen and the synthesis of γ-chain mRNA. Methods: We transfected a γ-chain expression vector into Chinese hamster ovary cells already expressing both Aα- and Bβ-chains of fibrinogen and measured fibrinogen output concentrations by ELISA. We quantified both γ-chain and Bβ-chain mRNA concentrations using the recently developed TaqMan fluorogenic detection system. Results: The concentration of secreted fibrinogen into the media positively correlated with the amount of fibrinogen contained in the cell lysates. Additionally, quantitative mRNA assays revealed that the fibrinogen concentration in the cell lysates correlated well with the concentration of γ-chain mRNA (r=0.7077, p<0.01) but not with the concentration of Bβ-chain mRNA (r=0.0224, NS). Conclusions: These results demonstrate that the amount of recombinant fibrinogen produced in cells transfected with the γ-chain vector, also expressing normal Aα- and Bβ-chains, is dependent on the transcription of γ-chain mRNA. Namely, in this recombinant expression system using a two-step transfection procedure, γ-chain synthesis is the rate-limiting factor for fibrinogen production. This quantitative method to measure mRNA may prove very useful for further in vivo analysis of fibrinogen gene transcription.

Heterozygous Bβ-chain C-terminal 12 amino acid elongation variant, BβX462W (Kyoto VI), showed dysfibrinogenemia.

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

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.

Comparison of thrombin-catalyzed fibrin polymerization and factor XIIIa-catalyzed cross-linking of fibrin among three recombinant variant fibrinogens, gamma 275C, gamma 275H, and gamma 275A.

Summary.  Background and objectives: We have previously reported that recombinant γ275Cys fibrinogen exhibits a marked impairment of functions as well as aberrant fibrin clot and bundle structures, as compared with wild‐type, γ275Arg, and plasma fibrinogen from a heterozygous proband. Since γArg275His mutations have also been reported in 10 families, we synthesized recombinant γ275His fibrinogen and γ275Ala fibrinogen (as a control) and analyzed and compared them with γ275Cys and γ275Arg. Methods: A variant γ‐chain expression plasmid was transfected into Chinese hamster ovary cells expressing normal human fibrinogen Aα‐ and Bβ‐chains. After purification of the recombinant variant fibrinogens, we performed functional analyzes for thrombin‐catalyzed fibrin polymerization and factor XIIIa (FXIIIa)‐catalyzed γ‐γ dimer formation from fibrin or fibrinogen and also ultrastructural analysis of fibrin clots and bundles. Results: By comparison with both γ275His and γ275Ala fibrinogens, recombinant γ275Cys fibrinogen exhibited a more impaired γ‐γ dimer formation from fibrin or fibrinogen, a more aberrant fibrin clot structure, and thicker fibers in fibrin bundles. In 1 : 1 mixtures of γ275Arg and γ275Cys fibrinogens or γ275Arg and γ275His fibrinogens, thrombin‐catalyzed fibrin polymerization and both fibrin clot and fiber structures showed some compensation (as compared with γ275Cys or γ275His alone). Conclusion: These results strongly suggest that an amino acid substitution of γ275Arg alone disrupts D:D interactions in thrombin‐catalyzed fibrin polymerization and the formation of fibrin bundles and fibrin clots. Moreover, the existence of a subsequent disulfide‐linked Cys in γ275C fibrinogen augments the impairment caused by a His or Ala substitution.