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Background We encountered two patients with hypodysfibrinogenemia and designated them as Okayama II and Otsu I. Although the affected residue(s) in Okayama II and Otsu I overlapped, functionally determined fibrinogen levels and the ratio of functionally to immunologically determined plasma fibrinogen levels were markedly different. Methods DNA sequence and functional analyses were performed for purified plasma fibrinogen. A recombinant protein was synthesized in Chinese hamster ovary (CHO) cells to determine the secretion of variant fibrinogens. Results A heterozygous A>G in FGG , resulting in γ320Asp>Gly for Okayama II, and a heterozygous deletion of AATGAT in FGG , resulting in the deletion of γAsn319 and γAsp320 (γΔN319-ΔD320) for Otsu I, were obtained. SDS-PAGE and Coomassie staining revealed that the variant γ-chain was not clear in Okayama II, but was clearly present in Otsu I. The lag period for the fibrin polymerization of Okayama II was slightly slower than that of the normal control, whereas Otsu I fibrinogen indicated no polymerization within 30 min. Both variant γ-chains were synthesized in CHO cells and assembled into fibrinogen; however, the fibrinogen concentration ratio of the medium/cell lysate of γ320Gly was six-fold lower than that of γΔN319-ΔD320. Conclusions We concluded that the plasma fibrinogen of Okayama II, constituted by a lower ratio of the variant γ-chain, led to the almost normal functioning of fibrin polymerization. However, the plasma fibrinogen of Otsu I, with a higher ratio of the variant γ-chain, led to marked reductions in fibrin polymerization. [ABSTRACT FROM AUTHOR]

Abstract: Background: To study the functions of residues γ326Cys and γ339Cys in the assembly and/or secretion of fibrinogen, recombinant fibrinogens were synthesized to replicate naturally occurring γ326Tyr and γ326Ser variants, along with γ326Ala and γ339Ala variants. Methods: A fibrinogen γ-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 γ-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 γ-module, which is necessary for fibrinogen assembly and specifically secretion. A combination of the present results and observations from naturally occurring heterozygous cases of γ326Tyr and γ326Ser suggest that heterozygous fibrinogen molecules containing variant γ-chains might be secreted into plasma and show impaired fibrin polymerization, resulting in a phenotype of hypodysfibrinogenemia. [Copyright &y& Elsevier]

Abstract: Introduction: Emerging lines of evidence have suggested that certain dysfibrinogens present a significant risk of thrombosis. Patient/methods: The thrombophilic nature of a new-type of dysfibrinogen Kagoshima identified in a 36-year-old female with deep vein thrombosis during the postpartum period was studied. Results/discussion: Based on the analyses of the patient fibrinogen and the fibrinogen genes, fibrinogen Kagoshima was shown to have the amino acid substitution of γThr-314 to Ile that resulted in impaired function and hypofibrinogenemia. Polymerization of fibrin monomers derived from patient fibrinogen was severely impaired with a partial correction in the presence of calcium ions, causing very low clottability and delayed cross-linking of patient fibrin catalyzed by activated factor XIII. Because of the low clottability, a large amount of soluble fibrin was formed upon thrombin treatment, resulting in an increase of thrombin in the soluble fraction. Additionally, tPA-mediated plasmin generation on fibrin was impaired and calcium-ion-dependent integrity of the γ-chain D domain of Kagoshima fibrinogen was perturbed. The presence of many tapered-fiber ends inside the tangled fibrin networks, observed by scanning electron microscopy, suggested early termination of fibrin polymerization and the structural alteration. Conclusion: These data suggest that fibrinogen Kagoshima is dysfunctional, giving rise to formation of fibrinolysis-resistant soluble fibrin polymers and entrance of soluble fibrin associating with thrombin to the circulation, partly accounting for the thrombophilic nature of the affected fibrinogen and fibrin molecules. [Copyright &y& Elsevier]

This study describes a purification protocol of salmon fibrinogen that gives a consumable and highly clottable fibrinogen. Some characteristics of salmon and human fibrinogen are compared. Fibrinogen was purified from barium sulphate adsorbed plasma of Atlantic salmon, using two steps of 25% ammonium sulphate precipitation followed by ultrafiltration. The clottability of the purified salmon fibrinogen was 91%. The Aα chains of salmon fibrinogen were heterogeneous with a molecular mass of 90–110 kDa, compared to approximately 67 kDa of human fibrinogen Aα chains. The Bβ and γ chains of salmon and human fibrinogen had molecular masses of approximately 55 and 50 kDa, respectively. Western blotting revealed that polyclonal rabbit anti-human fibrinogen antibodies had affinity for the γ chains of salmon fibrinogen, making it possible to study factor XIII activity in purified salmon fibrinogen. Cross-linking of either γ–γ or γ–α chains was not detected upon incubation of the purified fibrinogen with thrombin and calcium alone, but was detected when clotting was performed in plasma indicating absence of factor XIII activity in the purified product. [Copyright &y& Elsevier]

Background: In the fibrin-forming process, thrombin cleaves fibrinogen to fibrin, which form fibrils and then fibers, producing a gel-like clot. Thrombin also activates coagulation factor XIII (FXIII), which crosslinks fibrin γ-chains and α-chains, stabilizing the clot. Many proteins bind to fibrin, including FXIII, an established regulation of clot structure, and platelet glycoprotein VI (GPVI), whose contribution to clot function is largely unknown. FXIII is present in plasma, but the abundant FXIII in platelet cytosol becomes exposed to the surface of strongly activated platelets., Objectives: We determined if GPVI interacts with FXIII and how this might modulate clot formation., Methods: We measured interactions between recombinant proteins of the GPVI extracellular domain: GPVI-dimer (GPVI-Fc 2 ) or monomer (GPVI ex ) and FXIII proteins (nonactivated and thrombin-activated FXIII, FXIII subunits A and B) by ELISA. Binding to fibrin clots and fibrin γ-chain crosslinking were analyzed by immunoblotting., Results: GPVI-dimer, but not GPVI-monomer, bound to FXIII. GPVI-dimer selectively bound to the FXIII A-subunit, but not to the B-subunit, an interaction that was decreased or abrogated by the GPVI-dimer-specific antibody mFab-F. The GPVI-dimer-FXIII interaction decreased the extent of γ-chain crosslinking, indicating a role in the regulation of clot formation., Conclusions: This is the first report of the specific interaction between GPVI-dimer and the A-subunit of FXIII, as determined in an in vitro system with defined components. GPVI-dimer-FXIII binding was inhibitory toward FXIII-catalyzed crosslinking of fibrin γ-chains in fibrin clots. This raises the possibility that GPVI-dimer may negatively modulate fibrin crosslinking induced by FXIII, lessening clot stability., (© 2022 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis (ISTH).)