Congenital hypofibrinogenemia is a rare bleeding disorder characterized by a proportional decrease of functional and antigenic fibrinogen levels. Hypofibrinogenemia can be considered the phenotypic expression of heterozygous loss of function mutations occurring within one of the three fibrinogen genes (FGA, FGB, and FGG). Clinical manifestations are highly variable; most patients are usually asymptomatic, but may appear with mild to severe bleeding or thrombotic complications. We have sequenced all exons of the FGA, FGB, and FGG genes using the DNA isolated from the peripheral blood in two unrelated probands with mild hypofibrinogenemia. Coagulation screening, global hemostasis, and functional analysis tests were performed. Molecular modeling was used to predict the defect of synthesis and structural changes of the identified mutation. DNA sequencing revealed a novel heterozygous variant c.1421G>A in exon 8 of the FGB gene encoding a Bβ chain (p.Trp474Ter) in both patients. Clinical data from patients showed bleeding episodes. Protein modelling confirmed changes in the secondary structure of the molecule, with the loss of three β sheet arrangements. As expected by the low fibrinogen levels, turbidity analyses showed a reduced fibrin polymerisation and imaging difference in thickness fibrin fibers. We have to emphasize that our patients have a quantitative fibrinogen disorder; therefore, the reduced function is due to the reduced concentration of fibrinogen, since the Bβ chains carrying the mutation predicted to be retained inside the cell. The study of fibrinogen molecules using protein modelling may help us to understand causality and effect of novel genetic mutations.

Congenital fibrinogen disorders are caused by mutations in genes coding for fibrinogen and may lead to various clinical phenotypes. Here, we present a functional and structural analysis of 4 novel variants located in the FGB gene coding for fibrinogen Bβ chain-heterozygous missense BβY416C and BβA68S, homozygous nonsense BβY345*, and heterozygous nonsense BβW403* mutations. The cases were identified by coagulation screening tests and further investigated by various methods. Fibrin polymerization had abnormal development with decreased maximal absorbance in all patients. Plasmin-induced fibrin degradation revealed different lytic phases of BβY416C and BβW403* than those of the control. Fibrinopeptide cleavage measured by reverse phase high pressure liquid chromatography of BβA68S showed impaired release of fibrinopeptide B. Morphological properties, studied through scanning electron microscopy, differed significantly in the fiber thickness of BβY416C, BβA68S, and BβW403*, and in the fiber density of BβY416C and BβW403*. Finally, homology modeling of BβA68S showed that mutation caused negligible alternations in the protein structure. In conclusion, all mutations altered the correct fibrinogen function or structure that led to congenital fibrinogen disorders.

Background Cardiovascular diseases are linked with oxidative stress which is the source of reactive oxidative and nitrative species, contributors of post-translational modification. Fibrinogen due to its high concentration in blood is considered as one of the most sought of targets of oxidative stress substances. Post-translational modifications of fibrinogen might influence its physiological function, thus affect hemostasis in the terms of fibrin nets forming and architecture or interaction with platelets. The aim of this study was to observe influence of in vivo fibrinogen modifications on formation of fibrin net and to identify amino acid residues prone to changes related to oxidative stress. Methods Plasma samples were collected from patients of The Military University Hospital Prague in the agreement with ethical committees of participating institutions and with informed consents from all subjects. Samples were divided into 4 groups: patients with acute coronary syndrome (A), patients with stroke (B), patients with thrombus localized in carotid vein (C) and control group (patients without coronary atherosclerosis; D). Fibrin net architecture was studied by scanning electron microscopy (Mira 3 LMH, Tescan Orsay Holding, a.s., Brno, Czech Republic). For identification of modified amino acids residue mass spectroscopy was used (Triple TOF 6600, Sciex). Molecular dynamics simulations of hydrated protein were performed in Gromacs software with Gromos force fields. Crystal structure 3GHG was used as a reference structure to which post-translational modifications were introduced manually in Yasara View. Results We found extensive both qualitative and quantitative changes in the structure of fibrinogen molecule in all groups of patients. Oxidative stress level differed among patient groups and between the control group. Different oxidative changes caused by in vivo modifications of fibrinogen affected quite distinctly the architecture of fibrin net. Modified amino acids were detected in all three fibrinogen chains. In gamma chain the localisation of modified amino acid residues correlated with the part of fibrinogen important for fibrin polymerisation. The impact of the most pronounced post-translational modifications on the secondary structure of fibrinogen was described by molecular dynamics simulations. Conclusions The results show that the degree of impairment of fibrinogen functions in the cardiovascular diseases is related to the level of oxidative stress. Characterization of oxidative fibrinogen modification and its precise meaning to the function of fibrinogen in hemostasis appears to be extremely helpful to better understanding of thrombotic/bleeding complications linked with various cardiovascular diseases. Acknowledgments This work was supported by the Ministry of Health, Czech Republic, no. 00023736, by the Academy of Sciences, Czech Republic no. P205/12/G118 and NV18-08-00149, by ERDF OPPK CZ.2.16/3.1.00/24001 and by the European Regional Development Fund and the state budget of the Czech Republic (project AIIHHP: CZ.02.1.01/0.0/0.0/16_025/0007428, OP RDE, Ministry of Education, Youth and Sports). Disclosures No relevant conflicts of interest to declare.

A single-center study was conducted on 120 patients with inherited disorders of primary hemostasis followed at our hematological center. These patients presented a variety of bleeding symptoms; however, they had no definitive diagnosis. Establishing a diagnosis has consequences for the investigation of probands in families and for treatment management; therefore, we aimed to improve the diagnosis rate in these patients by implementing advanced diagnostic methods. According to the accepted international guidelines at the time of study, we investigated platelet morphology, platelet function assay, light-transmission aggregometry, and flow cytometry. Using only these methods, we were unable to make a definitive diagnosis for most of our patients. However, next-generation sequencing (NGS), which was applied in 31 patients, allowed us to establish definitive diagnoses in six cases (variants in ANKRD26, ITGA2B, and F8) and helped us to identify suspected variants (NBEAL2, F2, BLOC1S6, AP3D1, GP1BB, ANO6, CD36, and ITGB3) and new suspected variants (GFI1B, FGA, GP1BA, and ITGA2B) in 11 patients. The role of NGS in patients with suspicious bleeding symptoms is growing and it changes the diagnostic algorithm. The greatest disadvantage of NGS, aside from the cost, is the occurrence of gene variants of uncertain significance.