Diagnostic value of clot formation parameters determined by rotational thromboelastometry in 63 patients with congenital dysfibrinogenemia.

Rotational thromboelastometry (ROTEM) is a global hemostasis assay. The diagnosis added value of ROTEM in congenital dysfibrinogenemia remains to be established. The aim of this study was to analyze clot formation by ROTEM in a cohort of dysfibrinogenemic patients and to establish correlations with genotype, clinical features, and coagulation parameters. The study included genetically confirmed congenital dysfibrinogenemia cases (n = 63) and healthy controls ( n  = 50). EXTEM, INTEM, FIBTEM tests were used to measure ROTEM parameters, that is, clotting time (CT), clot formation time (CFT), maximal clot firmness (MCF) and amplitude 10 min after CT (A10). The ISTH bleeding assessment tool was used to determine bleeding episodes. CT (INTEM) was statistically significantly shorter in congenital dysfibrinogenemia patients compared to controls while CFT (EXTEM) was prolonged. Patients's MCF in EXTEM, INTEM, and FIBTEM were similar to controls while A10 (FIBTEM) was statistically significantly lower. Fibrinogen activity was positively correlated with fibrinogen antigen, A10 and MCF in all three assays. Bleeding phenotypes were observed in 23 (36.5%) patients. Only CFT in EXTEM and CT in INTEM were statistically different in patients with bleeding phenotype versus controls. Carriers of the FGA mutation p.Arg35His had a CT (EXTEM) slightly prolonged and a reduced A10 (FIBTEM) compared to controls. Some ROTEM parameters were able to distinguish congenital dysfibrinogenemia patients from controls, and patients with a bleeding phenotype. Prolonged CFT in EXTEM were associated with congenital dysfibrinogenemia and bleeding phenotype. Bleeding episodes in most patients were generally mild and prevalence of thrombosis was very low.

Basic Principles of Rotational Thromboelastometry (ROTEM®) and the Role of ROTEM-Guided Fibrinogen Replacement Therapy in the Management of Coagulopathies.

Rotational thromboelastometry (ROTEM) is a viscoelastic method, which provides a graphical and numerical representation of induced hemostasis in whole blood samples. Its ability to quickly assess the state of hemostasis is used in the management of bleeding from a variety of causes. The separate activation of particular parts of hemocoagulation in INTEM, EXTEM, and FIBTEM tests allows for a more comprehensive and faster evaluation of the missing component of hemostasis followed by targeted therapy. One of the most common cause of coagulopathy is trauma-induced coagulopathy. Fibrinogen replacement therapy by ROTEM allows for the use of a standard dosage of fibrinogen, which has been shown to be successful in preventing dilutional coagulopathy following colloid and crystalloid replacement and excessive amount of allogeneic blood transfusions. The best reflection of fibrinogen activity is observed in the FIBTEM assay, where fibrinogen replacement therapy is recommended at an MCF (maximum clot firmness) of FIBTEM < 10 mm and FIBTEM A10 < 7 mm. ROTEM also plays an important role in the diagnostic and management of inherited fibrinogen disorders. These can be manifested by bleeding complications, where changes in the MCF parameter are the most useful tool for assessing the effectiveness of fibrinogen replacement therapy. ROTEM-guided bleeding management algorithms effectively reduce the number of transfusions, healthcare costs, and complications, leading to the improvement of patient safety and overall health.

SARS-CoV-2 Infection-Associated Aortic Thrombosis Treated with Oral Factor Xa Inhibition.

Coronavirus disease 2019 (COVID-19) is an acute complex systemic disorder caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).While SARS-CoV-2 is known to cause significant pulmonary disease, various extrapulmonary manifestations of COVID-19 have also been reported. Growing evidence suggests that COVID-19 is associated with coagulopathy leading to micro and macrovascular complications. Although in patients with COVID-19, venous thromboembolic events are more frequent, arterial thrombosis also occurs at an increased rate. These often lead to acute life-threatening ischemia, which requires urgent diagnosis and treatment. We present case reports of two patients with an abnormal thrombus formation in the thoracic aorta who recently overcame COVID-19, which led to systemic embolism and splenic infarction. Ambulatory oral factor Xa inhibitor therapy led to aortic thrombosis resolution in both patients.

Heterogeneity of Genotype-Phenotype in Congenital Hypofibrinogenemia-A Review of Case Reports Associated with Bleeding and Thrombosis.

Congenital fibrinogen disorders are diseases associated with a bleeding tendency; however, there are also reports of thrombotic events. Fibrinogen plays a role in the pathogenesis of thrombosis due to altered plasma concentrations or modifications to fibrinogen's structural properties, which affect clot permeability, resistance to lysis, and its stiffness. Several distinct types of genetic change and pathogenetic mechanism have been described in patients with bleeding and a thrombotic phenotype, including mutations affecting synthesis or processing in three fibrinogen genes. In this paper, we focused on familial hypofibrinogenemia, a rare inherited quantitative fibrinogen disorder characterized by decreased fibrinogen levels with a high phenotypic heterogeneity. To begin, we briefly review the basic information regarding fibrinogen's structure, its function, and the clinical consequences of low fibrinogen levels. Thereafter, we introduce 15 case reports with various gene mutations derived from the fibrinogen mutation database GFHT (French Study Group on Hemostasis and Thrombosis), which are associated with congenital hypofibrinogenemia with both bleeding and thrombosis. Predicting clinical presentations based on genotype data is difficult. Genotype-phenotype correlations would be of help to better understand the pathologic properties of this rare disease and to provide a valuable tool for the identification of patients who are not only at risk of bleeding, but also at risk of a thrombotic event.

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.

Multimer Analysis of Von Willebrand Factor in Von Willebrand Disease with a Hydrasys Semi-Automatic Analyzer-Single-Center Experience.

von Willebrand disease (VWD) is reportedly the most common inherited bleeding disorder. This disorder develops as a result of defects and/or deficiency of the plasma protein von Willebrand factor (VWF). Laboratory testing for VWF-related disorders requires the assessment of both VWF level and VWF activity, the latter requiring multiple assays. As an additional step, an evaluation of VWF structural features by multimer analysis is useful in selective investigations. Multimer analysis is also important for the selection of a suitable VWF therapy preparation (desmopressin, VWF/FVIII concentrate, recombinant VWF) and the determination of the correct dose for the patient. Based on clinical and laboratory findings, including the analysis of VWF multimers, we classified our patients into individual types of VWD. Our study group included 58 patients. The study group consisted of 66% (38 patients) with VWD type 1, 5% (3 patients) with VWD type 2, 7% (4 patients) with VWD type 3, 5% (3 patients) with mixed type 1/2A VWD, and 17% (10 patients) comprising an unclassified group. In this article, we provide an overview of our practical experience using a new complementary method-the analysis of von Willebrand factor multimers with a semi-automatic analyzer Hydrasys 2 scan. We explain the principle, procedure, advantages, and pitfalls associated with the introduction of the VWF multimer analysis methodology into standard VWD diagnostics.

Use of Fibrinogen Determination Methods in Differential Diagnosis of Hypofibrinogenemia and Dysfibrinogenemia.

BACKGROUND: Fibrinogen plays an important role in hemostasis. The normal concentration of fibrinogen in blood plasma is between 1.8 - 4.2 g/L. Decreased fibrinogen levels are observed in congenital afibrinogenemia, hypofibrinogenemia, dysfibrinogenemia, disseminated intravascular coagulation, fibrinolytic therapy, some more severe hepatic parenchymal disorders, and increased blood loss. Elevated fibrinogen levels occur in inflammatory diseases and neoplastic diseases, in pregnancy, and postoperative conditions. Functional fibrinogen measurement is also one of the basic coagulation screening tests. The fibrinogen antigen assay is used to distinguish between qualitative and quantitative fibrinogen disorders. METHODS: The aim of the study was the use of fibrinogen determination methods in differential diagnosis of hypofibrinogenemia and dysfibrinogenemia, statistical evaluation and determine the relationship of fibrinogen Clauss assay, prothrombin time (PT) derived fibrinogen assay, and fibrinogen antigen in the group of 60 patients with congenital fibrinogen disorders (n = 40 dysfibrinogenemia; n = 20 hypofibrinogenemia). RESULTS: The results measured by the PT-derived fibrinogen assay were approximately four times higher compared to the fibrinogen Clauss assay in the group of patients with dysfibrinogenemia. In patients with hypofibrinogenemia, there is a correlation (r = 0.9016) between the fibrinogen Clauss assay and PT-derived fibrinogen assay with a statistical significance of p < 0.0001. Using a linear or quadratic interpolation function, we were able to determine the fibrinogen Clauss assay and the fibrinogen antigen assay before analysis. CONCLUSIONS: The higher level of the PT-derived fibrinogen assay compared to the fibrinogen Clauss assay in the group of patients with dysfibrinogenemia may pose a greater risk to asymptomatic patients who require diagnosis and treatment in case of bleeding. The fibrinogen value using the PT-derived fibrinogen assay could erroneously give a normal level. The use of the interpolation function is important to estimate the value of fibrinogen activity and antigen before the analysis itself by the Clauss assay or analysis by the fibrinogen antigen assay.

A Novel Nonsense Mutation in FGB (c.1421G>A; p.Trp474Ter) in the Beta Chain of Fibrinogen Causing Hypofibrinogenemia with Bleeding Phenotype.

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 Bß 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 ß 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 Bß 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.

Genetic Variants in the FGB and FGG Genes Mapping in the Beta and Gamma Nodules of the Fibrinogen Molecule in Congenital Quantitative Fibrinogen Disorders Associated with a Thrombotic Phenotype.

Fibrinogen is a hexameric plasmatic glycoprotein composed of pairs of three chains (Aα, Bß, and γ), which play an essential role in hemostasis. Conversion of fibrinogen to insoluble polymer fibrin gives structural stability, strength, and adhesive surfaces for growing blood clots. Equally important, the exposure of its non-substrate thrombin-binding sites after fibrin clot formation promotes antithrombotic properties. Fibrinogen and fibrin have a major role in multiple biological processes in addition to hemostasis and thrombosis, i.e., fibrinolysis (during which the fibrin clot is broken down), matrix physiology (by interacting with factor XIII, plasminogen, vitronectin, and fibronectin), wound healing, inflammation, infection, cell interaction, angiogenesis, tumour growth, and metastasis. Congenital fibrinogen deficiencies are rare bleeding disorders, characterized by extensive genetic heterogeneity in all the three genes: FGA, FGB, and FGG (enconding the Aα, Bß, and γ chain, respectively). Depending on the type and site of mutations, congenital defects of fibrinogen can result in variable clinical manifestations, which range from asymptomatic conditions to the life-threatening bleeds or even thromboembolic events. In this manuscript, we will briefly review the main pathogenic mechanisms and risk factors leading to thrombosis, and we will specifically focus on molecular mechanisms associated with mutations in the C-terminal end of the beta and gamma chains, which are often responsible for cases of congenital afibrinogenemia and hypofibrinogenemia associated with thrombotic manifestations.

Comparison of clinical phenotype with genetic and laboratory results in 31 patients with congenital dysfibrinogenemia in northern Slovakia.

Congenital dysfibrinogenemia (CD) is a rare disorder of hemostasis. The majority of cases are caused by heterozygous missense mutations in one of the three fibrinogen genes. Patients with CD may experience bleeding and thrombosis, but many are asymptomatic. To better describe the clinical, laboratory, and genotypic picture of CD, we evaluated 31 patients from seven unrelated families using standard coagulation tests and genetic analysis. The clinical phenotype consisted of bleeding in 13/31 (42%) patients; other patients (18/31; 58%) were asymptomatic. Among patients with bleeding, symptoms were mostly in single anatomical sites, with variable intensity of bleeding. Compared to results from a previous large systematic survey, our results showed a similar mean bleeding score, but a higher incidence of bleeding episodes without thrombotic complications. In the present study, we identified three known pathogenic point mutations in the FGA (c.95G > A, c.104G > A) and FGB (c.586C > T) genes. The variants of CD identified in this cross-sectional study were either asymptomatic or had bleeding manifestations and showed similar laboratory features, irrespective of genotype. Results from genetic and clinical studies will continue to yield valuable information on the structure and function of the fibrinogen molecule.