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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 -
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 -
Journal of Thrombosis and Haemostasis :... Sep 2011This review of published studies was conducted to derive data on patients with congenital fibrinogen deficiency (CFD), including dosing of fibrinogen replacement... (Review)
Review
This review of published studies was conducted to derive data on patients with congenital fibrinogen deficiency (CFD), including dosing of fibrinogen replacement therapy, outcome, and adverse events, either temporally related or distant to fibrinogen replacement, in order to assist clinicians in developing treatment plans for patients with CFD. A systematic review was performed of case reports identified by a MEDLINE search between 1961 and 2010. Eligible studies included subjects with a diagnosis of CFD who received fibrinogen replacement. An attempt was made to extract dose, frequency, duration, hemostatic efficacy and adverse events such as thrombosis or allergic reactions. Reported thrombotic events distant from fibrinogen replacement were also recorded. From 104 papers reviewed, a total of 50 cases were identified: afibrinogenemia (35), hypofibrinogenemia (6), and dysfibrinogenemia (9). Fibrinogen replacement therapy was generally effective in preventing or treating bleeding in doses adequate to achieve and maintain fibrinogen activity above 50-100 mg dL(-1) (non-surgical and obstetric use) or 100-200 mg dL(-1) (surgical prophylaxis). Increased fibrinogen clearance was observed with massive hemorrhage, major surgery, and advanced pregnancy. Obstetric outcomes were optimized when fibrinogen replacement was initiated prior to conception. Uncontrolled hemorrhage, allergic reactions and antibody formation were rare events. However, thromboses, both related and unrelated to fibrinogen replacement, occurred in 15 of 50 (30%) patients overall, and in eight of 12 (67%) adult non-obstetric patients with afibrinogenemia. Published fibrinogen replacement regimens are presented for 50 CFD patients. Fibrinogen replacement therapy requires careful monitoring of fibrinogen levels. Afibrinogenemia is associated with thromboembolic complications with or without treatment.
Topics: Adolescent; Adult; Afibrinogenemia; Child; Child, Preschool; Female; Fibrinogen; Hemorrhage; Hemostasis, Surgical; Humans; Infant; Infant, Newborn; Male; Pregnancy; Pregnancy Complications, Hematologic; Thrombosis; Young Adult
PubMed: 21711446
DOI: 10.1111/j.1538-7836.2011.04424.x -
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 -
Journal of Clinical Laboratory Analysis May 2018In this study, the significance of fibrinogen concentration assessed by a combination of Clauss and prothrombin time (PT)-derived methods for screening for congenital...
BACKGROUND
In this study, the significance of fibrinogen concentration assessed by a combination of Clauss and prothrombin time (PT)-derived methods for screening for congenital dysfibrinogenemia were investigated, and the screening efficiency of fibrinogen PT-derived/Clauss ratio on congenital dysfibrinogenemia was analyzed.
METHODS
We compared fibrinogen concentrations determined by the Clauss, PT-derived, and enzyme-linked immunosorbent assay (ELISA) methods in 73 patients with congenital dysfibrinogenemia and 81 normal controls. Receiver operating characteristic (ROC) curves were utilized to evaluate the efficacy of fibrinogen PT-derived/Clauss ratio in screening for congenital dysfibrinogenemia.
RESULTS
Fibrinogen concentrations determined by the Clauss method were dramatically lower than by the PT-derived method and ELISA, and correlated poorly with the latter two methods in patients with congenital dysfibrinogenemia. Fibrinogen concentrations in normal controls were slightly lower according to the Clauss method than to the PT-derived method and ELISA; however, each method yielded results within the normal range and the correlation was good. The area under the ROC curve of fibrinogen PT-derived/Clauss ratio for diagnosis of congenital dysfibrinogenemia was 1 with a standard error of 0, 95% confidence interval of 0.976-1.00, and optimal critical diagnosis point of 1.43. When fibrinogen PT-derived/Clauss ratio was >1.43, the sensitivity and specificity for diagnosis of congenital dysfibrinogenemia were both 100%.
CONCLUSIONS
The combined use of Clauss and PT-derived methods for determining fibrinogen concentrations improves the efficiency of screening for congenital dysfibrinogenemia, as the fibrinogen PT-derived/Clauss ratio has high sensitivity and specificity in diagnosis of congenital dysfibrinogenemia. This ratio could serve an important screening tool for this disease.
Topics: Adolescent; Adult; Afibrinogenemia; Aged; Aged, 80 and over; Blood Coagulation Tests; Case-Control Studies; Child; Enzyme-Linked Immunosorbent Assay; Female; Fibrinogen; Humans; Male; Middle Aged; Sensitivity and Specificity; Young Adult
PubMed: 28922493
DOI: 10.1002/jcla.22322 -
Indian Journal of Hematology & Blood... Sep 2017Congenital dysfibrinogenemia is a rare autosomal recessive bleeding disorder, which is characterized by the absence of functional fibrinogen. Patients may have bleeding...
Congenital dysfibrinogenemia is a rare autosomal recessive bleeding disorder, which is characterized by the absence of functional fibrinogen. Patients may have bleeding and paradoxical arterial and venous thrombotic problems from early childhood. The optimal antithrombotic therapy in these patients hasn't been determined yet. In this report we present a dysfibrogenemic patient, who has suffered recurrent arterial thrombosis under aspirin treatment. Intravenous fibrinogen concentrates (fc) along with reduced doses of rivaroxaban (10 mg daily), cilostazol (50 mg bid) and aspirin (100 mg daily) were given as antithrombotic treatment. The pain and the cyanosis clinically recovered within 6 weeks. This is, to our knowledge, the first time in which a new oral anticoagulant, rivaroxaban and cilostazol combination was used in a dysfibrinogenemic patient with thrombotic episodes. We determined the type, the dosage and the duration of antithrombotic treatment according to the clinical progress of the symptoms. Rivaroxaban, cilostazol and fibrinogen concentrate replacement; combination may represent a useful alternative for the antithrombotic treatment in dysfibrinogenemic patients.
PubMed: 28824252
DOI: 10.1007/s12288-016-0751-2 -
Pathophysiology of Haemostasis and... 2002Until recently the laboratory diagnosis of thrombophilia consisted on investigation of the plasmatic anticoagulant pathways and the search for dysfibrinogenemia and... (Review)
Review
Until recently the laboratory diagnosis of thrombophilia consisted on investigation of the plasmatic anticoagulant pathways and the search for dysfibrinogenemia and antiphospholipid antibodies/lupus anticoagulants. More recently, the laboratory investigation has been expanded by including activated protein C (APC) resistance, due or not to the presence of the factor V Leiden mutation; hyperprothrombinemia, due to the presence of the prothrombin mutation G20210A and hyperhomocysteinemia, due to impairment of the relevant metabolic pathway because of enzymatic and/or vitamin deficiency. Testing for thrombophilia may be useful for many reasons. First, the results of testing may provide valuable information to assess the risk of recurrence in the proband. Second, testing family members is useful for prophylactic and diagnostic purposes. Third, the identification of patients bearing combined defect helps to identify those at increased risk for thrombosis. Testing is recommended for patients with a past history of thrombosis and should be extended to their first-degree family members. Since most of the tests are not reliable during anticoagulation, it is preferable to postpone laboratory testing until after discontinuation of the treatment. Whenever possible testing should be performed by means of functional assays. DNA analysis is required for the prothrombin mutation G20210A. Laboratory diagnosis for antiphospholipid antibodies/lupus anticoagulant should be performed by a combination of tests including phospholipid-dependent clotting assays and solid phase anticardiolipin antibodies. Hyperhomocysteinemia may be assessed by high-pressure liquid chromatography methods, or by fluorescence polarization immunoassays.
Topics: Hematology; Humans; Laboratories; Thrombophilia
PubMed: 13679650
DOI: 10.1159/000073574 -
World Journal of Clinical Cases Dec 2022The purpose of this study was to report the rare case of a pregnant woman with congenital dysfibrinogenemia (CD) misdiagnosed as acute fatty liver. She was treated...
BACKGROUND
The purpose of this study was to report the rare case of a pregnant woman with congenital dysfibrinogenemia (CD) misdiagnosed as acute fatty liver. She was treated according to the principles of acute fatty liver but achieved good clinical results.
CASE SUMMARY
A 30-year-old woman presented with 39 (6/7) wk of menopause and 6 h of irregular abdominal pain and attended our hospital. Emergency surgery was performed due to fetal distress. Postoperative management followed the treatment principle of acute fatty liver. DNA sequencing was carried out on the pregnant woman and her pedigree. Coagulation values of the patient on admission were prothrombin time 33.7 s, activated partial thromboplastin time 60.4 s, thrombin time 45.2 s, and fibrinogen 0.60 g/L. DNA sequencing results showed that the woman carried a pathogenic heterozygous variation of the fibrinogen alpha chain gene (FGA), which is closely related to hereditary fibrinogen abnormality, and the mutation site was located in . After a follow-up period of 12 mo, the mother and her newborn had a good prognosis without bleeding or thrombosis.
CONCLUSION
Pregnant women with CD may have atypical symptoms, which can easily lead to misdiagnosis. In addition, treatment can be attempted according to the principles of acute fatty liver management. This rare pregnant patient with CD was caused by a novel FGA () gene mutation.
PubMed: 36569010
DOI: 10.12998/wjcc.v10.i35.12996 -
Blood Jan 2015We conducted a multicenter study of 101 patients with congenital dysfibrinogenemia (CD) to characterize the incidence of hemorrhagic and thrombotic events as well as...
We conducted a multicenter study of 101 patients with congenital dysfibrinogenemia (CD) to characterize the incidence of hemorrhagic and thrombotic events as well as complications of pregnancy and surgery. At the time of diagnosis, 10.9% and 13.9% had experienced major bleeding and thrombotic events, respectively. During a mean follow-up of 8.8 years after CD diagnosis, the incidence of major bleeding and thrombotic events was 2.5 and 18.7 per 1000 patient-years, respectively, with estimated cumulative incidences at age 50 years of 19.2% and 30.1%. We identified 111 pregnancies with an overall incidence of spontaneous abortions and postpartum hemorrhage of 19.8% and 21.4%, respectively. The risk of postpartum hemorrhage was associated with a previously identified bleeding phenotype (odds ratio, 5.8; 95% CI, 1.2 to 28.0). Among 137 surgical procedures analyzed, 9 (6.5%) were complicated by abnormal bleeding. Propositi vs relatives, sex, mutation hotspots, fibrinogen levels, and activity:antigen ratios were not associated with the risk of thrombotic or bleeding outcomes. In conclusion, the results of our study, the largest in genotyped CD and the first including long-term history, indicate that propositi with CD and their relatives carry not only a high risk of major bleeding, including postpartum hemorrhage, but also of thrombotic event.
Topics: Abortion, Spontaneous; Adolescent; Adult; Afibrinogenemia; Europe; Female; Follow-Up Studies; Hemorrhage; Humans; Incidence; Male; Postoperative Complications; Pregnancy; Pregnancy Complications, Hematologic; Prognosis; Retrospective Studies; Risk Factors; Thrombosis; Young Adult
PubMed: 25320241
DOI: 10.1182/blood-2014-06-582866