Fibrinogen Secretion Research Articles

Regulation of hepatic inclusions and fibrinogen biogenesis by SEL1L-HRD1 ERAD

Impaired secretion of an essential blood coagulation factor fibrinogen leads to hepatic fibrinogen storage disease (HFSD), characterized by the presence of fibrinogen-positive inclusion bodies and hypofibrinogenemia. However, the molecular mechanisms underlying the biogenesis of fibrinogen in the endoplasmic reticulum (ER) remain unexplored. Here we uncover a key role of SEL1L-HRD1 complex of ER-associated degradation (ERAD) in the formation of aberrant inclusion bodies, and the biogenesis of nascent fibrinogen protein complex in hepatocytes. Acute or chronic deficiency of SEL1L-HRD1 ERAD in the hepatocytes leads to the formation of hepatocellular inclusion bodies. Proteomics studies followed by biochemical assays reveal fibrinogen as a major component of the inclusion bodies. Mechanistically, we show that the degradation of misfolded endogenous fibrinogen Aα, Bβ, and γ chains by SEL1L-HRD1 ERAD is indispensable for the formation of a functional fibrinogen complex in the ER. Providing clinical relevance of these findings, SEL1L-HRD1 ERAD indeed degrades and thereby attenuates the pathogenicity of two disease-causing fibrinogen γ mutants. Together, this study demonstrates an essential role of SEL1L-HRD1 ERAD in fibrinogen biogenesis and provides insight into the pathogenesis of protein-misfolding diseases.

Genomewide Association Study Identifies Copy Number Variants Associated With Warfarin Dose Response and Risk of Venous Thromboembolism in African Americans.

The anticoagulant warfarin is commonly used to control and prevent thrombotic disorders, such as venous thromboembolism (VTE), which disproportionately afflicts African Americans. Despite the importance of copy number variants (CNVs), few studies have focused on characterizing and understanding their role in drug response and disease risk among African Americans. In this study, we conduct the first genome-wide analysis of CNVs to more comprehensively account for the contribution of genetic variation in warfarin dose requirement and VTE risk among African Americans. We used hidden Markov models to detect CNVs from high-throughput single-nucleotide polymorphism arrays for 340 African American participants in the International Warfarin Pharmacogenetics Consortium. We identified 11,570 CNVs resulting in 2,038 copy number variable regions (CNVRs) and found 3 CNVRs associated with warfarin dose requirement and 3 CNVRs associated with VTE risk in African Americans. CNVRs 1q31.2del and 6q14.1del were associated with increased warfarin dose requirement (β=11.18 and 4.94, respectively; Pemp =< 0.002); CNVR 19p13.31del was associated with decreased warfarin dose requirement (β=-1.41, Pemp =0.0004); CNVRs (2p22.1del and 5q35.1-q35.2del) were found to be associated with increased risk of VTE (odds ratios (ORs)=1.88 and 14.9, respectively; Pemp ≤0.02); and CNVR 10q26.12del was associated with a decreased risk of VTE (OR=0.6; Pemp =0.05). Modeling of the 10q26.12del in HepG2 cells revealed that this deletion results in decreased fibrinogen gene expression, decreased fibrinogen and WDR11 protein levels, and decreased secretion of fibrinogen into the extracellular matrix. We found robust evidence that CNVRs could contribute to warfarin dose requirement and risk of VTE in African Americans and for 10q26.3del describe a possible pathogenic mechanism.

222 Maternal Recognition of Pregnancy in the Mare: What is Needed to Make Progress in our Understanding of This Complex Issue

Abstract Maternal recognition of pregnancy (MRP) resulting in prolonged luteal lifespan is crucial to maintenance of pregnancy. Mechanisms underlying MRP differ in between species, whereat the precise mechanism by which luteolysis is prevented has yet to be described for equine pregnancy. Transcriptional profiling studies entailing analysis of endometrial and embryonic tissues have shed light into the wider context of maternal recognition of pregnancy. Our own studies revealed the secretion of fibrinogen by preimplantation equine embryos, which is a peculiar finding as fibrinogen synthesis almost exclusively occurs in the liver. Conceptus-derived fibrinogen likely contributes to cell adhesion during fixation and underlines the helpfulness of transcriptome studies in identifying species-specific aspects of early pregnancy. Despite all our advances, we yet have to decipher the signaling pathways leading to reduced endometrial production of prostaglandin F2alpha during MRP. We recently analyzed samples from mares undergoing spontaneous pregnancy loss in the face of high progesterone in comparison to samples collected from mares at Day 20 of intact pregnancy. No differentially expressed transcripts could be detected. It appears that after maternal recognition of pregnancy and consequent suppression of luteolysis, progesterone, not the presence of a viable conceptus, is the driving force of gene expression. This finding underlines the importance of understanding the events surrounding luteostasis. We propose that a standardized approach in vitro cell culture model is needed to address mechanistic questions pertaining to MRP. Approaches currently underway such as CRISPR/Cas9-mediated targeted integration of TERT and TERC to immortalize equine endometrial stromal fibroblasts will be discussed.

SARS-CoV-2 Spike Proteins and Cell-Cell Communication Inhibits TFPI and Induces Thrombogenic Factors in Human Lung Microvascular Endothelial Cells and Neutrophils: Implications for COVID-19 Coagulopathy Pathogenesis.

In SARS-CoV-2-infected humans, disease progression is often associated with acute respiratory distress syndrome involving severe lung injury, coagulopathy, and thrombosis of the alveolar capillaries. The pathogenesis of these pulmonary complications in COVID-19 patients has not been elucidated. Autopsy study of these patients showed SARS-CoV-2 virions in pulmonary vessels and sequestrated leukocytes infiltrates associated with endotheliopathy and microvascular thrombosis. Since SARS-CoV-2 enters and infects target cells by binding its spike (S) protein to cellular angiotensin-converting enzyme 2 (ACE2), and there is evidence that vascular endothelial cells and neutrophils express ACE2, we investigated the effect of S-proteins and cell–cell communication on primary human lung microvascular endothelial cells (HLMEC) and neutrophils expression of thrombogenic factors and the potential mechanisms. Using S-proteins of two different SARS-CoV-2 variants (Wuhan and Delta), we demonstrate that exposure of HLMEC or neutrophils to S-proteins, co-culture of HLMEC exposed to S-proteins with non-exposed neutrophils, or co-culture of neutrophils exposed to S-proteins with non-exposed HLMEC induced transcriptional upregulation of tissue factor (TF), significantly increased the expression and secretion of factor (F)-V, thrombin, and fibrinogen and inhibited tissue factor pathway inhibitor (TFPI), the primary regulator of the extrinsic pathway of blood coagulation, in both cell types. Recombinant (r)TFPI and a thiol blocker (5,5′-dithio-bis-(2-nitrobenzoic acid)) prevented S-protein-induced expression and secretion of Factor-V, thrombin, and fibrinogen. Thrombomodulin blocked S-protein-induced expression and secretion of fibrinogen but had no effect on S-protein-induced expression of Factor-V or thrombin. These results suggests that following SARS-CoV-2 contact with the pulmonary endothelium or neutrophils and endothelial–neutrophil interactions, viral S-proteins induce coagulopathy via the TF pathway and mechanisms involving functional thiol groups. These findings suggest that using rTFPI and/or thiol-based drugs could be a viable therapeutic strategy against SARS-CoV-2-induced coagulopathy and thrombosis.

Fibrinogen mediates cadmium-induced macrophage activation and serves as a predictor of cadmium exposure in chronic obstructive pulmonary disease.

The etiologies of chronic obstructive pulmonary disease (COPD) remain unclear. Cadmium (Cd) causes both pulmonary fibrosis and emphysema; however, the predictors for Cd exposure and the mechanisms by which Cd causes COPD remain unknown. We demonstrated that Cd burden was increased in lung tissue from subjects with COPD and this was associated with cigarette smoking. Fibrinogen levels increased markedly in lung tissue of patients with smoked COPD compared with never-smokers and control subjects. Fibrinogen concentration also correlated positively with lung Cd load, but inversely with the predicted % of FEV1 and FEV1/FVC. Cd enhanced the secretion of fibrinogen in a cdc2-dependent manner, whereas fibrinogen further mediated Cd-induced peptidylarginine deiminase 2 (PAD2)-dependent macrophage activation. Using lung fibroblasts from CdCl2-treated Toll-like receptor 4 (TLR4) wild-type and mutant mice, we demonstrated that fibrinogen enhanced Cd-induced TLR4-dependent collagen synthesis and cytokine/chemokine production. We further showed that fibrinogen complexed with connective tissue growth factor (CTGF), which in turn promoted the synthesis of plasminogen activator inhibitor-2 (PAI-2) and fibrinogen and inhibited fibrinolysis in Cd-treated mice. The amounts of fibrinogen were increased in the bronchoalveolar lavage fluid (BALF) of Cd-exposed mice. Positive correlations were observed between fibrinogen with hydroxyproline. Our data suggest that fibrinogen is involved in Cd-induced macrophage activation and increases in fibrinogen in patients with COPD may be used as a marker of Cd exposure and predict disease progression.

Congenital Afibrinogenemia and Hypofibrinogenemia: Laboratory and Genetic Testing in Rare Bleeding Disorders with Life-Threatening Clinical Manifestations and Challenging Management.

Congenital fibrinogen disorders are rare pathologies of the hemostasis, comprising quantitative (afibrinogenemia, hypofibrinogenemia) and qualitative (dysfibrinogenemia and hypodysfibrinogenemia) disorders. The clinical phenotype is highly heterogeneous, being associated with bleeding, thrombosis, or absence of symptoms. Afibrinogenemia and hypofibrinogenemia are the consequence of mutations in the homozygous, heterozygous, or compound heterozygous state in one of three genes encoding the fibrinogen chains, which can affect the synthesis, assembly, intracellular processing, stability, or secretion of fibrinogen. In addition to standard coagulation tests depending on the formation of fibrin, diagnostics also includes global coagulation assays, which are effective in monitoring the management of replacement therapy. Genetic testing is a key point for confirming the clinical diagnosis. The identification of the precise genetic mutations of congenital fibrinogen disorders is of value to permit early testing of other at risk persons and better understand the correlation between clinical phenotype and genotype. Management of patients with afibrinogenemia is particularly challenging since there are no data from evidence-based medicine studies. Fibrinogen concentrate is used to treat bleeding, whereas for the treatment of thrombotic complications, administered low-molecular-weight heparin is most often. This review deals with updated information about afibrinogenemia and hypofibrinogenemia, contributing to the early diagnosis and effective treatment of these disorders.

A novel fibrinogen γ-chain frameshift mutation, p. Cys365Phefs*41, causing hypofibrinogenemia with bleeding phenotype in a Chinese family.

BackgroundCongenital hypofibrinogenemia is a rare bleeding disease that is classified as the quantitative deficient type. In the present study, investigated the relationship between the genotype and phenotype in a family with hypofibrinogenemia.MethodsThe proband was aware of a predisposition to bleeding. Functional analysis was performed for her all family members, including coagulation function tests, thrombus molecular markers, thromboelastography, scanning electron microscopy, DNA sequencing, and high-performance liquid chromatography-mass spectrometry (HPLC-MS). Pathogenicity analysis and protein modeling of mutant amino acids were also performed.ResultsA novel heterozygous mutation in c.1094delG was detected in FGG exon 8, which resulted in p. Cys365Phefs*41 (containing the signal peptide) in the proband and her mother, who showed a corresponding decrease in fibrinogen function and levels. Thromboelastography indicated that the strength of their blood clots decreased and they had an increased risk of bleeding. The proband fibrin network structure was looser than healthy controls, with large pores in the network, which increased the permeability of lytic enzymes. Results of HPLC-MS showed a lack of mutant peptide chain expression in their plasma, indicating that the family had congenital hypofibrinogenemia, with a clinical phenotype that is related to the degree of fibrinogen deficiency. The mutation truncated the γ-peptide chain and destroyed the functional structure of fibrinogen, including the γ352Cys-γ365Cys disulfide bond. The truncated peptide chains may also lead to nonsense-mediated decay.ConclusionsThe mutation induced a structural change at the carboxyl-terminal of the fibrinogen molecule, leading to fibrinogen secretion dysfunction.

Novel variant fibrinogen γp.C352R produced hypodysfibrinogenemia leading to a bleeding episode and failure of infertility treatment.

We identified a patient with a novel heterozygous variant fibrinogen, γp.C352R (Niigata II; N-II), who had a bleeding episode and failed infertility treatment and was suspected to have hypodysfibrinogenemia based on low and discordant fibrinogen levels (functional assay 0.33g/L, immunological assay 0.91g/L). We analyzed the mechanism of this rare phenotype of a congenital fibrinogen disorder. Patient plasma fibrinogen was purified and protein characterization and thrombin-catalyzed fibrin polymerization performed. Recombinant fibrinogen-producing Chinese hamster ovary (CHO) cells were established and the assembly and secretion of variant fibrinogen analyzed by ELISA and western blotting. Purified N-II plasma fibrinogen had a small lower molecular weight band below the normal γ-chain and slightly reduced fibrin polymerization. A limited proportion of p.C352R fibrinogen was secreted into the culture medium of established CHO cell lines, but the γ-chain of p.C352R was synthesized and variant fibrinogen was assembled inside the cells. We demonstrated that fibrinogen N-II, γp.C352R was associated with markedly reduced secretion of variant fibrinogen from CHO cells, that fibrin polymerization of purified plasma fibrinogen was only slightly affected, and that fibrinogen N-II produces hypodysfibrinogenemia in plasma.

Head-to-head comparison of cytokines storm-coagulopathy in septic shock and COVID-19

IntroductionHost immune response to the coronavirus disease 2019 (COVID-19) is variable and can induce a dysregulated inflammatory response associated with venous and arterial thrombosis called COVID-19 associated coagulopathy (CAC). During septic shock, inflammatory reaction generates endothelial activation and procoagulant state with microvascular thrombi inducing disseminated intravascular coagulation (DIC). Although CAC and DIC induce altered coagulation responses, their clinical outcomes are different.ObjectiveWe investigated and compared coagulopathy between septic shock and critical COVID-19 patients.MethodSeptic shock patients were diagnosed following the Survival Sepsis Campaign guidelines. COVID-19 patients were admitted in intensive care unit (ICU) for severe acute respiratory distress syndrome. Both were included in the study within 2 days after admission. Biomarkers were measured by ELISA from patient's plasma.ResultsWe observed an increase in vWF and TFPI in both septic and COVID-19 patients compared to controls, highlighting endothelial damage. Interestingly, circulating TF was only elevated in COVID-19 patients. Platelet activation differed between the two cohorts of patients. P-selectin and TLT-1 were specifically heightened in septic shock whereas CD40L was only augmented in COVID-19. Coagulation markers were increased in a disease-dependent way, with PAI-1, tPA and D-Dimers higher in septic shock and fibrinogen level, higher in COVID-19.DiscussionCOVID-19 patients had longer length-of-stay with more pronounced respiratory failure. This strong lung disruption overtime induced plasmatic TF release with sustained inflammatory response characterized by sCD40L and fibrinogen secretion. Given the similarities between COVID-19 and septic shock regarding fibrinolysis and coagulation, but not platelet activation, endothelium seems to play a central role in COVID-19 and might explain the differences between CAC and DIC.

Mechanism of Human Platelet Activation by Endotoxic Glycolipid-Bearing Mutant Re595 of Salmonella minnesota

The mechanism through which human blood platelets interact with gram-negative bacteria with well-defined structural variations in endotoxic lipopolysaccharide was studied. Secretion of 14C-serotonin and aggregation of platelets separated from plasma proteins were observed on challenge with rough mutant Re595 of Salmonella minnesota possessing a glycolipid outer layer composed of Lipid A and 2-keto-3-deoxyoctonate (KDO) but lacking heptose phosphate in the core and O-polysaccharide in its outer portion. Both 14C-serotonin secretion and platelet aggregation were concentration-dependent, with a half-maximum response at the ratio of one bacterial colony-forming unit (CFU) to two platelets. The aggregation of human platelets induced by mutant Re595 was divalent cation-dependent and required secretion of ADP and fibrinogen from platelet storage granules because it was inhibited by chelators, by the ADP-splitting enzyme apyrase, and by monospecific antifibrinogen Fab fragments. The synthetic peptide analog of the platelet receptor recognition site on the gamma chain of fibrinogen, gamma 400-411, inhibited platelet aggregation induced by mutant Re595 (IC50 160 mumol/L), whereas serotonin secretion was unaffected. Tetrapeptide, RGDS, analogous to human fibrinogen alpha chain (alpha 572-575) and to the cell adhesion site of fibronectin, also inhibited aggregation induced by mutant Re595 (IC50 60 mumol/L). Secretion of 14C-serotonin was preceded by a very rapid phosphorylation of a platelet protein of mol wt 47,000, which is associated with protein kinase C activation. Myosin light chain (mol wt 20,000) was also phosphorylated. Both phosphoproteins were dephosphorylated while secretion was reaching maximum. Furthermore, release of 3H-arachidonic acid from platelet phospholipids and generation of thromboxane B2 via the cyclooxygenase pathway were observed. Inhibition of this pathway with acetylsalicylic acid (10(-4) mol/L) or indomethacin (5 X 10(-4) mol/L) reduced 14C-serotonin secretion and platelet aggregation. The role of Lipid A in the interaction of mutant Re595 with human platelets was deduced from the inhibitory effect of the Lipid A-binding protein present in Limulus amebocyte lysate. Likewise, polymyxin B, known to complex with Lipid A, was inhibitory. The reactivity of mutant Re595 toward platelets was attenuated by mild acid hydrolysis, during which KDO was dissociated from the glycolipid, and by alkaline hydrolysis, which breaks ester-linked fatty acids in Lipid A. In contrast to mutant Re595, strain S218 of S minnesota bearing "complete" endotoxic lipopolysaccharide did not induce secretion and aggregation of human platelets.(ABSTRACT TRUNCATED AT 400 WORDS)

A Novel Amino Acid Substitution, Fibrinogen Bβp.Pro234Leu, Associated with Hypofibrinogenemia Causing Impairment of Fibrinogen Assembly and Secretion.

We identified a novel heterozygous variant, Bβp.Pro234Leu (fibrinogen Tokorozawa), which was suspected to be associated with hypofibrinogenemia. Therefore, we analyzed the assembly and secretion of this fibrinogen using Chinese hamster ovary (CHO) cells. To determine the impact on the synthesis and secretion of fibrinogen of the Bβp.P234L and γp.G242E substitutions, we established recombinant variant fibrinogen-producing CHO cell lines. Synthesis and secretion analyses were performed using an enzyme-linked immunosorbent assay (ELISA) and immunoblotting analysis with the established cell lines. In addition, we performed fibrin polymerization using purified plasma fibrinogen and in-silico analysis. Both Bβp.P234L and γp.G242E impaired the secretion and synthesis of fibrinogen. Moreover, immunoblotting analysis elucidated the mobility migration of the Bβγ complex in Bβp.P234L. On the other hand, the fibrin polymerization of fibrinogen Tokorozawa was similar to that of normal fibrinogen. In-silico analysis revealed that the Bβp.P234 residue is located in the contact region between the Bβ and γ chains and contacts γp.G242 residue. The present study demonstrated that the Bβp.P234L substitution resulted in hypofibrinogenemia by decreasing the assembly and secretion of fibrinogen. Therefore, there is a possibility that substitutions in the contact region between the Bβ and γ chains impact the assembly and secretion of fibrinogen.

Abstract LB-105: Plasma exposure-target engagement biomarker analysis for danvatirsen after multiple dosing schedules and route of administrations in patients with advanced solid tumors

Abstract Background: Danvatirsen (Da) is a generation 2.5 antisense oligonucleotide targeting STAT3 mRNA under clinical development in combination with Durvalumab (NCT03421353). The recommended Phase II dose is intravenous (IV) 200 mg weekly (QW, A). However, alternative 400 mg IV every two weeks (Q2W, B) and subcutaneous 200 mg QW (C) are also under investigation. The JAK-STAT signaling pathway is involved in immunity, cell division, cell death, and tumor formation. Additionally, activation of hepatic STAT3 results in increased secretion of C-reactive protein (CRP) and fibrinogen into blood. Therefore, target engagement in patients may be better understood by analyzing Da exposure and related biomarkers in the blood/plasma. We investigated Da exposure and subsequent impact on target engagement biomarkers such as CRP, platelets, fibrinogen, neutrophils and STAT3 mRNA levels. Methods: Extensive pharmacokinetics (PK) samples were collected at lead-in monotherapy and at steady state (SS) in combination. Plasma biomarker samples were collected at baseline and every two weeks thereafter. Whole blood samples for the analysis of STAT3 mRNA were collected at baseline, after lead-in monotherapy, and every four weeks thereafter. The PK parameters were estimated by non-compartmental analysis by WinNonlin. The biomarkers were analyzed by standard clinical methods and STAT3 mRNA level was measured by NanoString technology. Results: The area under plasma concentration-time curve of Da at SS within dosing interval (AUC0-τ, ng.h/ml) for A, B, and C were 69110 (24.64%), 101600 (22.11%), and 44600 (30.25%), respectively. Additionally, SS Ctrough (ng/ml) levels for A, B, and C were 28.53 (48.86%), 14.56 (36.14%), and 27.66 (49.22%), respectively. Maximum median % decrease from baseline in CRP levels was observed at week 4; A (-91%), B (-94%), C (-82%). Additionally, maximum median % changes from baseline for fibrinogen were -60%, -40%, -53%, in A, B, and C at week 4, respectively. Maximum % decrease from baseline in platelets was observed at week 8 in A (-42%), B (-41%), and C (-69%). In contrast, maximum % change from baseline in neutrophil count was observed at week 6; -37%, -31%, and -32% in A, B, and C, respectively. Additionally, the median maximum change from baseline for STAT3 mRNA was -42.4% and -33.6% for A and C, respectively. Conclusion: Cumulative AUC in A was very similar to B, whereas, AUC in C was 30% lower as compared to A or B. The SS Ctrough values in A and C were similar, although Ctrough value in B was 50% lower. Maximum % decreases in CRP levels were similar across all the doses. Moreover, route of administration did not appear to impact level of STAT3 knockdown. In summary, a similar degree of changes in pharmacodynamic biomarkers were achieved at all three dose levels regardless of route of administration, despite differences in plasma exposure. Citation Format: Kowser Miah, Patricia McCoon, Deanna Russell, Patrick Mitchell, Martin Scott, Amy Rosenfeld, Li Zhang, Pablo Martinez Rodriguez, Alicia Savage, Ganesh Mugundu. Plasma exposure-target engagement biomarker analysis for danvatirsen after multiple dosing schedules and route of administrations in patients with advanced solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-105.

Generation of Transplantable Three-Dimensional Hepatic-Patch to Improve the Functionality of Hepatic Cells In Vitro and In Vivo.

Cell therapy and tissue engineering (TE) are considered alternative therapeutic approaches to organ transplantation. Since cell therapy approaches achieved little success for liver failure treatment, liver TE is considered a more promising alternative. In this study, we produced a liver tissue equivalent (called "liver-derived extracellular matrix scaffold [LEMS]-Patch") by co-culture of human bone marrow stromal cells, human umbilical vein endothelial cells, and a hepatoma cell line, Huh7, within an artificial three-dimensional liver-extracellular matrix scaffold. The results showed significant increase in the liver-specific gene expression and hepatic functions, in terms of albumin (ALB) and fibrinogen secretion, urea production, and cytochrome inducibility in the LEMS-Patch compared to controls. In addition, transplanted LEMS-Patch was successfully incorporated into the recipient liver of acute liver failure mice and produced human ALB. Consequently, our data demonstrated that the generated LEMS-Patch could be used as a good platform for functional improvement of hepatic cells in vitro and in vivo.

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

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. 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. 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. 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.

A novel fibrinogen gamma-chain mutation, p.Cys165Arg, causes disruption of the γ165Cys–Bβ227Cys disulfide bond and ultimately leads to hypofibrinogenemia

BackgroundCongenital hypofibrinogenemia is a type of hereditary disease characterized by impaired fibrinogen synthesis and/or secretion induced by mutations in the fibrinogen gene. ObjectivesWe investigated the phenotypes, genotypes, and pathogenesis of congenital hypofibrinogenemia in an affected family. Patients/methodsThe proband had a risk of bleeding; therefore, conventional coagulation screening was performed for the proband and her family members. Mutation sites in all exons and flanking sequences of FGA, FGB, and FGG were identified, with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) performed to indicate the expression of abnormal chains. The effect of the mutation sites on fibrinogen structure and function was predicted by molecular modeling, and purified plasma fibrinogen from the proband was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and scanning electron microscopy. Thromboelastography was applied to assess the risk of bleeding and clotting in the proband. ResultsFibrinogen levels in the proband were 1.21 g/L, 1.31 g/L, and 1.38 g/L according to Clauss assay, the prothrombin time method, and enzyme-linked immunosorbent assay, respectively. A novel heterozygous mutation (γCys165Arg), a heterozygous mutation (AαIle6Val), and two genetic polymorphisms (AαThr331Ala and BβArg478Lys) in fibrinogen were found in the proband, and MALDI-TOF MS indicated absence of the mutated chain in patient plasma. Additionally, the heterozygous mutation (γCys165Arg) displayed substitution of a nonpolar γ165Cys (low mass) with a positively charged Arg (high mass) along with a small fiber diameter and loose network structure. ConclusionsFibrinogen γCys165Arg mutations cause damage to the interchain disulfide bonds of fibrinogen and hinder fibrinogen secretion, possibly explaining the pathological mechanism associated with congenital hypofibrinogenemia.

Congenital hypofibrinogenemia associated with γK232T

SummaryWe have previously reported a case of congenital hypofibrinogenemia caused by a novel heterozygous A→C transition at nucleotide 5864 of FGG, leading to the K232T substitution in the fibrinogen γ-chain. However, the pathogenic mechanism is still unclear. To further reveal its molecular basis, we examined the effects of γK232T substitution on fibrinogen synthesis, stability, and secretion through in vitro expression of mutant γ232T in Chinese hamster ovary (CHO) cells. Quantitative RT-PCR of the variant γ-chain mRNA showed that the γ232T transcribed the variant cDNA. Enzyme-linked immunosorbent assay and Western blot analysis of the cell lysates and culture media showed that the CHO cells transfected with successfully synthesized the variant fibrinogen, but failed to secrete it into the culture medium. Furthermore, fibrinogen purified from the plasma of patient showed a normal thrombin-catalyzed fibrin polymerization, also indicating the impeding secretion of variant γ232T fibrinogen. In conclusion, our data reveal that the γK232T is responsible for the congenital hypofibrinogenaemia through interfering with the correct secretion of fibrinogen.

Identification of Two Novel Fibrinogen Bβ Chain Mutations in Two Slovak Families with Quantitative Fibrinogen Disorders.

Congenital fibrinogen disorders are caused by mutations in one of the three fibrinogen genes that affect the synthesis, assembly, intracellular processing, stability or secretion of fibrinogen. Functional studies of mutant Bβ-chains revealed the importance of individual residues as well as three-dimensional structures for fibrinogen assembly and secretion. This study describes two novel homozygous fibrinogen Bβ chain mutations in two Slovak families with afibrinogenemia and hypofibrinogenemia. Peripheral blood samples were collected from all subjects with the aim of identifying the causative mutation. Coagulation-related tests and rotational thromboelastometry were performed. All exons and exon–intron boundaries of the fibrinogen genes (FGA, FGB and FGG) were amplified by PCR followed by direct sequencing. Sequence analysis of the three fibrinogen genes allowed us to identify two novel homozygous mutations in the FGB gene. A novel Bβ chain truncation (BβGln180Stop) was detected in a 28-year-old afibrinogenemic man with bleeding episodes including repeated haemorrhaging into muscles, joints, and soft tissues, and mucocutaneous bleeding and a novel Bβ missense mutation (BβTyr368His) was found in a 62-year-old hypofibrinogenemic man with recurrent deep and superficial venous thromboses of the lower extremities. The novel missense mutation was confirmed by molecular modelling. Both studying the molecular anomalies and the modelling of fibrinogenic mutants help us to understand the extremely complex machinery of fibrinogen biosynthesis and finally better assess its correlation with the patient’s clinical course.

Dysfibrinogenemia-associated novel heterozygous mutation, Shanghai (FGA c.169_180+2 del), leads to N-terminal truncation of fibrinogen Aα chain and impairs fibrin polymerization

AimsA novel heterozygous variant, FGA c.169_180+2 del (designated fibrinogen Shanghai), was identified in a patient with dysfibrinogenemia with antiphospholipid antibody syndrome (APS) and recurrent venous thrombosis, and in his asymptomatic...

Molecular characterization of 7 patients affected by dys- or hypo-dysfibrinogenemia: Identification of a novel mutation in the fibrinogen Bbeta chain causing a gain of glycosylation

Fibrinogen is a hexameric glycoprotein consisting of two sets of three polypeptides (the Aα, Bβ, and γ chains, encoded by the three genes FGA, FGB, and FGG). It is involved in the final phase of the coagulation process, being the precursor of the fibrin monomers necessary for the formation of the hemostatic plug. Rare inherited fibrinogen disorders can manifest as quantitative deficiencies, qualitative defects, or both. In particular, dysfibrinogenemia and hypo-dysfibrinogenemia are characterized by reduced functional activity associated with normal or reduced antigen levels, and are usually determined by heterozygous mutations affecting any of the three fibrinogen genes.In this study, we investigated the genetic basis of dys- and hypo-dysfibrinogenemia in seven unrelated patients. Mutational screening disclosed six different variants, two of which novel (FGB-p.Asp185Asn and FGG-p.Asn230Lys). The molecular characterization of the FGG-p.Asn230Lys mutation, performed by transient expression experiments of the recombinant mutant protein, demonstrated that it induces an almost complete impairment in fibrinogen secretion, according to a molecular mechanism often associated with quantitative fibrinogen disorders. Conversely, the FGB-p.Asp185Asn variant was demonstrated to be a gain-of-glycosylation mutation leading to a hyperglycosylation of the Bβ chain, not affecting fibrinogen assembly and secretion. To our knowledge, this is the second gain-of-glycosylation mutation involving the FGB gene.

Novel heterozygous dysfibrinogenemia, Sumida (AαC472S), showed markedly impaired lateral aggregation of protofibrils and mildly lower functional fibrinogen levels

IntroductionWe encountered a 6-year-old girl with systemic lupus erythematosus. Although no bleeding or thrombotic tendency was detected, routine coagulation screening tests revealed slightly lower plasma fibrinogen levels, as determined by functional and antigenic measurements (functional/antigenic ratio=0.857), suggesting hypodysfibrinogenemia. Materials and methodsDNA sequence and functional analyses were performed on purified plasma fibrinogen, and recombinant variant fibrinogen was synthesized in Chinese hamster ovary cells based on the results obtained. ResultsDNA sequencing revealed a heterozygous AαC472S substitution (mature protein residue number) in the αC-domain. AαC472S fibrinogen indicated the presence of additional disulfide-bonded molecules, and markedly impaired lateral aggregation of protofibrils in spite of slightly lower functional plasma fibrinogen levels. Scanning electron microscopic observations showed a thin fiber fibrin clot, and t-PA and plasminogen-mediated clot lysis was similar to that of a normal control. Recombinant variant fibrinogen-producing cells demonstrated that destruction of the Aα442C-472C disulfide bond did not prevent the synthesis or secretion of fibrinogen, whereas the variant Aα chain of the secreted protein was degraded faster than that of the normal control. ConclusionOur results suggest that AαC472S fibrinogen may cause dysfibrinogenemia, but not hypofibrinogenemia. The destruction and steric hindrance of the αC-domain of variant fibrinogen led to the impaired lateral aggregation of protofibrils and t-PA and plasminogen-mediated fibrinolysis, as well as several previously reported variants located in the αC-domain, and demonstrated the presence of disulfide-bonded molecules.