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Research and Practice in Thrombosis and... Jul 2023Variants of fibrinogen sequences that bind to thrombin's catalytic sites are mostly associated with bleeding phenotypes, while variants with fibrinogen...
BACKGROUND
Variants of fibrinogen sequences that bind to thrombin's catalytic sites are mostly associated with bleeding phenotypes, while variants with fibrinogen nonsubstrate-thrombin-binding sites are commonly believed to cause thrombosis. AαGlu39 and BβAla68 play important roles in fibrin(ogen)-thrombin-nonsubstrate binding. The BβAla68Thr variant has been described in several unrelated families with apparent thrombotic phenotypes.
OBJECTIVES
Homozygous AαGlu39Lys variant (fibrinogen BOE II) was identified in a boy with dysfibrinogenemia who had multiple cerebral hemorrhages. A series of analyses were performed to assess the variant's functions and elucidate underlying bleeding mechanisms.
METHODS
Abnormal fibrinogen was purified from plasma and subjected to Western blot, fibrinogen and fibrin monomer polymerization, clottability, fibrinopeptides release, activated factor (F)XIII (FXIIIa) cross-linking, fibrinolysis, and scanning electron microscopy analyses.
RESULTS
Fibrinogen BOE II weakened the binding capacity of thrombin to fibrinogen and delayed the formation of fibrin clots. The release of fibrinopeptides, polymerization of fibrinogen catalyzed by thrombin, and cross-linking of FXIIIa of fibrinogen BOE II were impaired. In contrast, batroxobin-catalyzed fibrinogen polymerization and desA/desAB fibrin monomer polymerization did not differ from those in normal controls. Fibrin clots formed by fibrinogen BOE II were composed of thicker fibrin fibers and showed a faster fibrinolysis rate.
CONCLUSION
Defective fibrin(ogen)-thrombin-nonsubstrate binding is not necessarily associated with thrombotic disorders. When the hypercoagulable state created by increased circulating free thrombin is insufficient to compensate for defective hemostasis caused by slowly formed but rapidly lysed clots, the primary concern of thrombin-binding deficiency dysfibrinogenemia appears to be hemorrhage rather than thrombosis.
PubMed: 37601017
DOI: 10.1016/j.rpth.2023.102145 -
Blood Advances Mar 2024Congenital fibrinogen deficiency (CFD) is a rare bleeding disorder caused by mutations in FGA, FGB, and FGG. We sought to comprehensively characterize patients with CFD...
Congenital fibrinogen deficiency (CFD) is a rare bleeding disorder caused by mutations in FGA, FGB, and FGG. We sought to comprehensively characterize patients with CFD using PRO-RBDD (Prospective Rare Bleeding Disorders Database). Clinical phenotypes, laboratory, and genetic features were investigated using retrospective data from the PRO-RBDD. Patients were classified from asymptomatic to grade 3 based on their bleeding severity. In addition, FGA, FGB, and FGG were sequenced to find causative variants. A total of 166 CFD cases from 16 countries were included, of whom 123 (30 afibrinogenemia, 33 hypofibrinogenemia, 55 dysfibrinogenemia, and 5 hypodysfibrinogenemia) were well characterized. Considering the previously established factor activity and antigen level thresholds, bleeding severity was correctly identified in 58% of the cases. The rates of thrombotic events among afibrinogenemic and hypofibrinogenemic patients were relatively similar (11% and 10%, respectively) and surprisingly higher than in dysfibrinogenemic cases. The rate of spontaneous abortions among 68 pregnancies was 31%, including 86% in dysfibrinogenemic women and 14% with hypofibrinogenemia. Eighty-six patients received treatment (69 on-demand and/or 17 on prophylaxis), with fibrinogen concentrates being the most frequently used product. Genetic analysis was available for 91 cases and 41 distinct variants were identified. Hotspot variants (FGG, p.Arg301Cys/His and FGA, p.Arg35Cys/His) were present in 51% of dysfibrinogenemia. Obstetric complications were commonly observed in dysfibrinogenemia. This large multicenter study provided a comprehensive insight into the clinical, laboratory, and genetic history of patients with CFDs. We conclude that bleeding severity grades were in agreement with the established factor activity threshold in nearly half of the cases with quantitative defects.
Topics: Humans; Female; Fibrinogen; Afibrinogenemia; Prospective Studies; Retrospective Studies; Hemorrhage; Hemostatics
PubMed: 38286442
DOI: 10.1182/bloodadvances.2023012186 -
Diagnostics (Basel, Switzerland) Nov 2021Congenital fibrinogen disorders are rare pathologies of the hemostasis, comprising quantitative (afibrinogenemia, hypofibrinogenemia) and qualitative (dysfibrinogenemia... (Review)
Review
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.
PubMed: 34829490
DOI: 10.3390/diagnostics11112140 -
Hamostaseologie Dec 2023Our study aimed to analyze the phenotype and genotype of a pedigree with inherited dysfibrinogenemia, and preliminarily elucidate the probable pathogenesis.
OBJECTIVE
Our study aimed to analyze the phenotype and genotype of a pedigree with inherited dysfibrinogenemia, and preliminarily elucidate the probable pathogenesis.
METHODS
The one-stage clotting method was used to test the fibrinogen activity (FIB:C), whereas immunoturbidimetry was performed to quantify the fibrinogen antigen (FIB:Ag). Furthermore, DNA sequence analysis was conducted to confirm the site of mutation. Conservation analysis and protein model analysis were performed using online bioinformatics software.
RESULTS
The FIB:C and FIB:Ag of the proband were 1.28 and 2.20 g/L, respectively. Gene analysis revealed a heterozygous c.293C > A (p.BβAla68Asp) mutation in . Bioinformatics and modeling analysis suggested that the missense mutation could potentially have a deleterious effect on fibrinogen.
CONCLUSION
The BβAla68Asp mutation in exon 2 of may account for the reduced FIB:C levels observed in the pedigree. To our knowledge, this point mutation is the first report in the world.
Topics: Humans; Fibrinogen; Afibrinogenemia; Genotype; Mutation, Missense; Mutation; Hemostatics; Pedigree
PubMed: 37516116
DOI: 10.1055/a-2116-8957 -
Frontiers in Pediatrics 2020Pediatric Acute Liver Failure (PALF) is a rapidly progressive clinical syndrome encountered in the pediatric ICU which may rapidly progress to multi-organ dysfunction,... (Review)
Review
Pediatric Acute Liver Failure (PALF) is a rapidly progressive clinical syndrome encountered in the pediatric ICU which may rapidly progress to multi-organ dysfunction, and on occasion to life threatening cerebral edema and hemorrhage. Pediatric Acute Liver Failure is defined as severe acute hepatic dysfunction accompanied by encephalopathy and liver-based coagulopathy defined as prolongation of International Normalized Ratio (INR) >1.5. However, coagulopathy in PALF is complex and warrants a deeper understanding of the hemostatic balance in acute liver failure. Although an INR value of >1.5 is accepted as the evidence of coagulopathy and has historically been viewed as a prognostic factor of PALF, it may not accurately reflect the bleeding risk in PALF since it only measures procoagulant factors. Paradoxically, despite the prolongation of INR, bleeding risk is lower than expected (around 5%). This is due to "rebalanced hemostasis" due to concurrent changes in procoagulant, anticoagulant and fibrinolytic systems. Since the liver is involved in both procoagulant (Factors II, V, IX, XI, and fibrinogen) and anticoagulant (Protein C, Protein S, and antithrombin) protein synthesis, PALF results in "rebalanced hemostasis" or even may shift toward a hypercoagulable state. In addition to rebalanced coagulation there is altered platelet production due to decreased thrombopoietin production by liver, increased von Willebrand factor from low grade endothelial cell activation, and hyperfibrinolysis and dysfibrinogenemia from altered synthetic liver dysfunction. All these alterations contribute to the multifactorial nature of coagulopathy in PALF. Over exuberant use of prophylactic blood products in patients with PALF may contribute to morbidities such as fluid overload, transfusion-associated lung injury, and increased thrombosis risk. It is essential to use caution when using INR values for plasma and factor administration. In this review we will summarize the complexity of coagulation in PALF, explore "rebalanced hemostasis," and discuss the limitations of current coagulation tests. We will also review strategies to accurately diagnose the coagulopathy of PALF and targeted therapies.
PubMed: 33425821
DOI: 10.3389/fped.2020.618119 -
Zhonghua Xue Ye Xue Za Zhi = Zhonghua... Nov 2023To analyze the phenotype and genotype of two pedigrees with inherited fibrinogen (Fg) deficiency caused by two heterozygous mutations. We also preliminarily probed the...
To analyze the phenotype and genotype of two pedigrees with inherited fibrinogen (Fg) deficiency caused by two heterozygous mutations. We also preliminarily probed the molecular pathogenesis. The prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and plasma fibrinogen activity (Fg∶C) of all family members (nine people across three generations and three people across two generations) were measured by the clotting method. Fibrinogen antigen (Fg:Ag) was measured by immunoturbidimetry. Direct DNA sequencing was performed to analyze all exons, flanking sequences, and mutated sites of FGA, FGB, and FGG for all members. Thrombin-catalyzed fibrinogen polymerization was performed. ClustalX 2.1 software was used to analyze the conservatism of the mutated sites. MutationTaster, PolyPhen-2, PROVEAN, SIFT, and LRT online bioinformatics software were applied to predict pathogenicity. Swiss PDB Viewer 4.0.1 was used to analyze the changes in protein spatial structure and molecular forces before and after mutation. The Fg∶C of two probands decreased (1.28 g/L and 0.98 g/L, respectively). The Fg∶Ag of proband 1 was in the normal range of 2.20 g/L, while it was decreased to 1.01 g/L in proband 2. Through genetic analysis, we identified a heterozygous missense mutation (c.293C>A; p.BβAla98Asp) in exon 2 of proband 1 and a heterozygous nonsense mutation (c.1418C>G; p.BβSer473*) in exon 8 of proband 2. The conservatism analysis revealed that Ala98 and Ser473 presented different conservative states among homologous species. Online bioinformatics software predicted that p.BβAla98Asp and p.BβSer473* were pathogenic. Protein models demonstrated that the p.BβAla98Asp mutation influenced hydrogen bonds between amino acids, and the p.BβSer473* mutation resulted in protein truncation. The dysfibrinogenemia of proband 1 and the hypofibrinogenemia of proband 2 appeared to be related to the p.BβAla98Asp heterozygous missense mutation and the p.BβSer473* heterozygous nonsense mutation, respectively. This is the first ever report of these mutations.
Topics: Humans; Afibrinogenemia; Codon, Nonsense; Pedigree; Phenotype; Fibrinogen; Genotype
PubMed: 38185523
DOI: 10.3760/cma.j.issn.0253-2727.2023.11.008 -
International Journal of Molecular... Jan 2021Venous thrombosis occurs in patients with quantitative and qualitative fibrinogen disorders. Injury-induced thrombosis in zebrafish larvae has been used to model human...
Venous thrombosis occurs in patients with quantitative and qualitative fibrinogen disorders. Injury-induced thrombosis in zebrafish larvae has been used to model human coagulopathies. We aimed to determine whether zebrafish models of afibrinogenemia and dysfibrinogenemia have different thrombotic phenotypes. Laser injuries were used to induce venous thrombosis and the time-to-occlusion (TTO) and the binding and aggregation of fluorescent thrombocytes measured. The larvae failed to support occlusive venous thrombosis and showed reduced thrombocyte binding and aggregation at injury sites. The larvae were largely unaffected. When genome editing zebrafish to produce fibrinogen Aα R28C, equivalent to the human Aα R35C dysfibrinogenemia mutation, we detected in-frame skipping of exon 2 in the fga mRNA, thereby encoding Aα. This mutation is similar to Fibrinogen Montpellier II which causes hypodysfibrinogenemia. Aα fish had prolonged TTO and reduced thrombocyte activity, a dominant effect of the mutation. Finally, we used transgenic expression of fga R28C cDNA in fga knock-down or mutants to model thrombosis in dysfibrinogenemia. Aα R28C expression had similar effects on TTO and thrombocyte activity as Aα. We conclude that thrombosis assays in larval zebrafish can distinguish between quantitative and qualitative fibrinogen disorder models and may assist in anticipating a thrombotic phenotype of novel fibrinogen mutations.
Topics: Animals; Base Sequence; Biomarkers; Blood Coagulation; Blood Platelets; Disease Models, Animal; Exons; Fibrinogen; Gene Editing; Gene Expression; Plasmids; Platelet Activation; Sequence Deletion; Venous Thrombosis; Zebrafish; RNA, Guide, CRISPR-Cas Systems
PubMed: 33440782
DOI: 10.3390/ijms22020655 -
Journal of Thrombosis and Haemostasis :... Jun 2023Fibrinogen is a soluble, multisubunit, and multidomain dimeric protein, which, upon its proteolytic cleavage by thrombin, is converted to insoluble fibrin, initiating...
BACKGROUND
Fibrinogen is a soluble, multisubunit, and multidomain dimeric protein, which, upon its proteolytic cleavage by thrombin, is converted to insoluble fibrin, initiating polymerization that substantially contributes to clot growth. Fibrinogen contains numerous, transiently accessible "cryptic" epitopes for hemostatic and immunologic proteins, suggesting that fibrinogen exhibits conformational flexibility, which may play functional roles in its temporal and spatial interactions. Hitherto, there have been limited integrative approaches characterizing the solution structure and internal flexibility of fibrinogen.
METHODS
Here, utilizing a multipronged, biophysical approach involving 2 solution-based techniques, temperature-dependent hydrogen-deuterium exchange mass spectrometry and small angle X-ray scattering, corroborated by negative stain electron microscopy, we present a holistic, conformationally dynamic model of human fibrinogen in solution.
RESULTS
Our data reveal 4 major and distinct conformations of fibrinogen accommodated by a high degree of internal protein flexibility along its central scaffold. We propose that the fibrinogen structure in the solution consists of a complex, conformational landscape with multiple local minima. This is further supported by the location of numerous point mutations that are linked to dysfibrinogenemia and posttranslational modifications, residing near the identified fibrinogen flexions.
CONCLUSION
This work provides a molecular basis for the structural "dynamism" of fibrinogen that is expected to influence the broad swath of its functionally diverse macromolecular interactions and fine-tune the structural and mechanical properties of blood clots.
Topics: Humans; Fibrin Fibrinogen Degradation Products; Fibrin; Fibrinogen; Thrombosis; Molecular Conformation
PubMed: 36746319
DOI: 10.1016/j.jtha.2023.01.034 -
Journal of Clinical Laboratory Analysis Sep 2022We reported a patient with congenital dysfibrinogenemia who was misdiagnosed and reviewed relevant literature, in order to discuss the methods to reduce misdiagnosis. (Review)
Review
BACKGROUND
We reported a patient with congenital dysfibrinogenemia who was misdiagnosed and reviewed relevant literature, in order to discuss the methods to reduce misdiagnosis.
METHODS
A 23-year-old pregnant woman was found to be with low fibrinogen in antenatal examination at another province teaching hospital, who was misdiagnosed to have hypofibrinogenemia. Fibrinogen infusion or cryoprecipitation was recommended if necessary. The patient came to our hospital for further diagnosis and treatment considering the safety of herself and the fetus. We examined the coagulation function and gene sequencing of the pregnant woman and her family members.
RESULTS
Fibrinogen (Clauss method) was significantly reduced in the patient and her mother, while the level of fibrinogen (PT-derived method) was normal. Thrombin time was prolonged. Heterozygous mutation site was found in exon 2 of the FGA gene, c.104G > A(p.Arg35His).
CONCLUSION
When the fibrinogen (Clauss method) is significantly reduced and the thrombin time is prolonged, PT-derived method and the investigation of family coagulation function should be added, which can be used to diagnose and distinguish congenital dysfibrinogenemia from hypofibrinogenemia.
Topics: Adult; Afibrinogenemia; Diagnostic Errors; Exons; Female; Fibrinogen; Humans; Pregnancy; Young Adult
PubMed: 35949040
DOI: 10.1002/jcla.24624 -
JRSM Cardiovascular Disease 2020Chronic thromboembolic pulmonary hypertension is characterized by incomplete thrombus resolution following acute pulmonary embolism, leading to pulmonary hypertension...
PURPOSE
Chronic thromboembolic pulmonary hypertension is characterized by incomplete thrombus resolution following acute pulmonary embolism, leading to pulmonary hypertension and right ventricular dysfunction. Conditions such as thrombophilias, dysfibrinogenemias, and inflammatory states have been associated with chronic thromboembolic pulmonary hypertension, but molecular mechanisms underlying this disease are poorly understood. We sought to characterize the molecular and functional features associated with chronic thromboembolic pulmonary hypertension using a multifaceted approach.
METHODS
We utilized functional assays to compare clot lysis times between chronic thromboembolic pulmonary hypertension patients and multiple controls. We then performed immunohistochemical characterization of tissue from chronic thromboembolic pulmonary hypertension, pulmonary arterial hypertension, and healthy controls, and examined RNA expression patterns of cultured lymphocytes and pulmonary arterial specimens. We then confirmed RNA expression changes using immunohistochemistry, immunofluorescence, and Western blotting in pulmonary arterial tissue.
RESULTS
Clot lysis times in chronic thromboembolic pulmonary hypertension patients are similar to multiple controls. Chronic thromboembolic pulmonary hypertension endarterectomized tissue has reduced expression of both smooth muscle and endothelial cell markers. RNA expression profiles in pulmonary arteries and peripheral blood lymphocytes identified differences in RNA transcript levels related to inflammation and growth factor signaling, which we confirmed using immunohistochemistry. Gene expression data also suggested significant alterations in metabolic pathways, and immunofluorescence and Western blot experiments confirmed that unglycosylated CD36 and adiponectin expression were increased in chronic thromboembolic pulmonary hypertension versus controls.
CONCLUSIONS
Our data do not support impaired clot lysis underlying chronic thromboembolic pulmonary hypertension, but did demonstrate distinct molecular patterns present both in peripheral blood and in pathologic specimens of chronic thromboembolic pulmonary hypertension patients suggesting that altered metabolism may play a role in chronic thromboembolic pulmonary hypertension pathogenesis.
PubMed: 32110389
DOI: 10.1177/2048004020906994