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

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.

Fibrin self-assembly is adapted to oxidation.

Fibrinogen is extremely susceptible to attack by reactive oxygen species (ROS). Having been suffered an oxidative modification, the fibrinogen molecules, now with altered spatial structure and function of fibrin network, affect hemostasis differently. However, the potential effects of the oxidative stress on the early stages of the fibrin self-assembly process remain unexplored. To clarify the damaging influence of ROS on the knob 'A': hole 'a' and the D:D interactions, the both are operating on the early stages of the fibrin polymerization, we have used a novel approach based on exploration of FXIIIa-mediated self-assembly of the cross-linked fibrin oligomers dissolved in the moderately concentrated urea solutions. The oligomers were composed of monomeric desA fibrin molecules created by cleaving the fibrinopeptides A off the fibrinogen molecules with a thrombin-like enzyme, reptilase. According to the UV-absorbance and fluorescence measurements data, the employed low ozone/fibrinogen ratios have induced only a slight fibrinogen oxidative modification that was accompanied by modest chemical transformations of the aromatic amino acid residues of the protein. Else, a slight consumption of the accessible tyrosine residues has been observed due to intermolecular dityrosine cross-links formation. The set of experimental data gathered with the aid of electrophoresis, elastic light scattering and analytical centrifugation has clearly witnessed that the oxidation can serve as an effective promoter for the observed enhanced self-assembly of the covalently cross-linked oligomers. At urea concentration of 1.20M, the pristine and oxidized fibrin oligomers were found to comprise a heterogeneous set of the double-stranded protofibrils that are cross-linked only by γ-γ dimers and the fibers consisting on average of four strands that are additionally linked by α polymers. The amounts of the oxidized protofibrils and the fibers accumulated in the system were higher than those of the non-oxidized counterparts. Moreover, the γ and α polypeptide chains of the oxidized molecules were more readily crosslinked by the FXIIIa. Upon increasing the urea solution concentration to 4.20M, the cross-linked double-stranded desA fibrin protofibrils have dissociated into the single-stranded fibrin oligomers, whereas the fibers dissociated into both the double-stranded desA fibrin oligomers, the structural integrity of the latter being maintained by means of the intermolecular α polymers, and the single-stranded fibrin oligomers cross-linked only by γ-γ dimers. The data we have obtained in this study indicate that the FXIIIa-mediated process of assembling the cross-linked protofibrils and the fibers constructed from the oxidized monomeric fibrin molecules was facilitated due to the strengthening of D:D interactions. The findings infer that the enhanced longitudinal D:D interactions become more essential in the assembly of soluble protofibrils when the interactions knobs 'A': holes 'a' are injured by oxidation. The new experimental findings presented here could be of help for elucidating the essential adaptive molecular mechanisms capable of mitigating the detrimental action of ROS in the oxidatively damaged fibrin self-assemblage processes.

Recombinant γT305A 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 γT305A fibrinogen was synthesized in Chinese hamster ovary cells based on the results. We then functionally analyzed and compared with that of nearby recombinant γN308K fibrinogen. RESULTS: DNA sequence analysis revealed a heterozygous γT305A substitution (mature protein residue number). The γT305A fibrinogen indicated markedly impaired thrombin-catalyzed fibrin polymerization both in the presence or absence of 1mM calcium ion compared with that of γN308K 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 γN308Kwas impaired at the latter two sites. Molecular modeling demonstrated that γT305 is localized at a shorter distance than γN308 from the high affinity calcium binding site and hole 'a'. CONCLUSION: Our findings suggest that γT305 might be important for construction of the overall structure of the γ module of fibrinogen. Substitution of γT305A leads to both dysfibrinogenemic and hypofibrinogenemic characterization, namely hypodysfibrinogenemia. We have already reported that recombinant γT305A fibrinogen was synthesized normally and secreted slightly, but was significantly reduced.

New aspects of π-d interactions in magnetic molecular conductors.

The 2 : 1 cation radical salts of bent donor molecules of ethylenedithio-tetrathiafulvalenoquinone-1,3-dithiolemethide (EDT-TTFVO), ethylenedithio-diselenadithiafulvalenoquinone-1,3-dithiolemethide (EDT-DSDTFVO), ethylenedithio-diselenadithiafulvalenothioquinone-1,3-diselenolemethide (EDT-DSDTFVSDS), ethylenedioxy-tetrathiafulvalenoquinone-1,3-dithiolemethide (EDO-TTFVO) and ethylenedioxy-tetrathiafulvalenoquinone-1,3-diselenolemethide (EDO-TTFVODS) with FeX4- (X = Cl, Br) ions are prepared by electrocrystallization. The crystal structures of these salts are composed of alternately stacked donor molecule and magnetic anion layers. The band structures of the donor molecule layers are calculated using the overlap integrals between neighboring donor molecules and are compared with the observed electronic transport properties. The magnetic ordering of the Fe(III) d spins of FeX4- ions is determined from magnetization and heat capacity measurements. The magnetic ordering temperatures are estimated by considering a combination of a direct d-d interaction between the d spins and an indirect π-d interaction between the conduction π electron and the d spins, whose magnitudes are separately calculated from the crystal structures with an extended Hückel molecular orbital method. The occurrence of a π-d interaction is proved by the negative magnetoresistance, and the magnitude of magnetoresistance reflects the strength of the π-d interaction. The effect of pressure on the magnetoresistance is studied, and the result indicates that the magnitude of magnetoresistance increases, namely, the π-d interaction is enhanced with increasing pressure. From these experimental results it is shown that (EDT-TTFVO)2•FeBr4 is a ferromagnetic semiconductor, (EDT-DSDTFVO)2•FeX4 (X = Cl, Br) and (EDT-DSDTFVSDS)2•FeBr4 are metals exhibiting antiferromagnetic ordering of the d spins, and (EDO-TTFVO)2•FeCl4 and (EDO-TTFVODS)2•FeBr4•(DCE)0.5 (DCE =-dichloroethane) are genuine antiferromagnetic metals. Among them, the (EDT-TTFVO)2•FeBr4 salt is the first π-d molecular system where the d spins of FeBr4- ions are ferromagnetically ordered through antiferromagnetic interaction with the conduction π electrons. Corresponding to this ferromagnetic ordering, an anomalous dielectric slow-down phenomenon toward the ordering temperature is observed. The π-d interaction in (EDT-DSDTFVSDS)2•FeBr4 is very large and comparable to that in λ-(BETS)2•FeCl4, which has the highest reported value so far, while the d-d interaction is fairly small. Concerning the ratio between the magnitudes of π-d and d-d interactions (Jπd/Jdd), this salt is currently the best π-d molecular system.