Clinical, Laboratory, and Molecular Aspects of Congenital Fibrinogen Disorders.

Congenital fibrinogen disorders (CFDs) include afibrinogenemia, hypofibrinogenemia, dysfibrinogenemia, and hypodysfibrinogenemia. The fibrinogen levels, the clinical features, and the genotype define several sub-types, each with specific biological and clinical issues. The diagnosis of CFDs is based on the measurement of activity and antigen fibrinogen levels as well as on the genotype. While relatively easy in quantitative fibrinogen disorders, the diagnosis can be more challenging in qualitative fibrinogen disorders depending on the reagents and methods used, and the underlying fibrinogen variants. Overall, quantitative and qualitative fibrinogen defects lead to a decrease in clottability, and usually in a bleeding tendency. The severity of the bleeding phenotype is moreover related to the concentration of fibrinogen. Paradoxically, patients with CFDs are also at risk of thrombotic events. The impact of the causative mutation on the structure and the fibrinogen level is one of the determinants of the thrombotic profile. Given the major role of fibrinogen in pregnancy, women with CFDs are particularly at risk of obstetrical adverse outcomes. The study of the fibrin clot properties can help to define the impact of fibrinogen disorders on the fibrin network. The development of next generation sequencing now allows the identification of genetic modifiers able to influence the global hemostasis balance in CFDs. Their integration in the assessment of the patient risk on an individual scale is an important step toward precision medicine in patients with such a heterogeneous clinical course.

Highly thrombogenic phenotype and impaired wound healing in a patient with congenital dysfibrinogenemia: case report.

Background: Congenital fibrinogen disorders are classified based on both fibrinogen levels and the clinical phenotype. For dysfibrinogenemia, normal fibrinogen levels are typical. Key Clinical Question: We highlight the importance of comprehensive thrombotic risk assessment, including lipoprotein a (Lp[a]) and hypertriglyceridemia in association with severe thrombosis and poor wound healing in dysfibrinogenemia. Clinical Approach: We report the case of a 42-year-old male patient with a rare congenital thrombotic-related dysfibrinogenemia (fibrinogen Naples) and multiple thrombotic episodes throughout his life and an unhealing ankle wound. Despite all thrombotic episodes and surgery, the patient had undetectable D-dimer, suggestive of fibrinolytic defect, further supported by over 4-fold elevated Lp(a) levels. The last arterial thrombosis was preoperatively managed by plasma exchange, antithrombotics, and thereafter continued fibrinogen replacement therapy, under which the chronic wound has healed. Conclusion: The combination of thrombogenesis, abnormal fibrinogen, and high Lp(a) levels is a clinical and research topic deserving more attention.

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.

Congenital fibrinogen disorders: a retrospective clinical and genetic analysis of the Prospective Rare Bleeding Disorders Database.

ABSTRACT: 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.

Composition of thrombi in zebrafish: similarities and distinctions with mammals.

BACKGROUND: Blood clots are primarily composed of red blood cells (RBCs), platelets/thrombocytes, and fibrin. Despite the similarities observed between mammals and zebrafish, the composition of fish thrombi is not as well known. OBJECTIVES: To analyze the formation of zebrafish blood clots ex vivo and arterial and venous thrombi in vivo. METHODS: Transgenic zebrafish lines and laser-mediated endothelial injury were used to determine the relative ratio of RBCs and thrombocytes in clots. Scanning electron and confocal microscopy provided high-resolution images of the structure of adult and larval clots. Adult and larval thrombocyte spreading on fibrinogen was evaluated ex vivo. RESULTS: RBCs were present in arterial and venous thrombi, making up the majority of cells in both circulations. However, bloodless mutant fish demonstrated that fibrin clots can form in vivo in the absence of blood cells. Scanning electron and confocal microscopy showed that larval and adult zebrafish thrombi and mammalian thrombi look surprisingly similar externally and internally, even though the former have nucleated RBCs and thrombocytes. Although adult thrombocytes spread on fibrinogen, we found that larval cells do not fully activate without the addition of plasma from adult fish, suggesting a developmental deficiency of a plasma activating factor. Finally, mutants lacking αIIbß3 demonstrated that this integrin mediates thrombocyte spreading on fibrinogen. CONCLUSION: Our data showed strong conservation of arterial and venous and clot/thrombus formation across species, including developmental regulation of thrombocyte function. This correlation supports the possibility that mammals also do not absolutely require circulating cells to form fibrin clots in vivo.

Coexpression of factor VIII and factor von Willebrand variants in a woman with heavy menstrual bleeding.

Heavy menstrual bleeding is one of the most common causes of consultation in haematology. We present the clinical case of a 20-year-old woman referred by her gynaecologist due to heavy menstrual bleeding since menarche, complicated by iron deficiency anaemia. Haemostasis work-up was initially suggestive of a von Willebrand disease type 1. Genetic analyses by whole exome sequencing lead to a fortuitous discovery of haemophilia by identifying a heterozygous missense mutation in F8 , exon 8 c.1127T>G:p.Val376Gly, previously reported in a patient with mild haemophilia A. The bleeding phenotype worsened by concomitant low von Willebrand factor (VWF) due to VWF variants influencing VWF levels. Our case highlights how whole exome sequencing can help to correct an erroneous diagnosis and identify polymorphisms that eventually contribute to the overall haemostatic balance.

Physiological correction of hereditary mild hypofibrinogenemia during pregnancy.

INTRODUCTION: Hereditary hypofibrinogenemia is a rare fibrinogen disorder characterised by decreased levels of fibrinogen. Pregnant women with hypofibrinogenemia are at risk of adverse obstetrical outcomes, depending on the fibrinogen level. AIM: We investigated how the physiological changes of hemostasis throughout the pregnancy impact the hemostatic balance in a woman with hereditary mild hypofibrinogenemia. METHODS: Fibrin clot properties were analyzed by turbidimetry and scanning electron microscopy, clot weight and red blood cells retention were measured by whole clot contraction, and in vitro thrombin generation was assessed by calibrated automated thrombogram and ex vivo by TAT. RESULTS: Throughout the pregnancy, the fibrinogen levels increased reaching normal values in the third trimester (activity 3.1 g/L, antigen 3.2 g/L). In parallel, the fibrin polymerisation increased, the fibrinolysis decreased, the fibrin clot network became denser with thicker fibrin fibers, and the fibrin clot weight and red blood cells retention increased, reaching control's value at the third trimester. Similarly, in vitro and ex vitro thrombin generation increased, reaching maximum values at the delivery. CONCLUSION: In this case of hereditary mild hypofibrinogenemia we observed a physiological increase of fibrinogen and thrombin generation. Future studies should focus on moderate and severe hypofibrinogenemia, to assess fibrinogen variation and the overall impact of increased TG on the hemostasis balance.

One Hundred Years of Congenital Fibrinogen Disorders.

Congenital fibrinogen disorders encompass a broad range of fibrinogen defects characterized by a wide molecular and clinical spectrum. From the first clinical description of afibrinogenemia in 1920, many major achievements have contributed to a better understanding of these complex disorders. The finding of causative mutations in all three fibrinogen genes has contributed to reveal the molecular mechanisms involved in biosynthesis of the fibrinogen molecule and to clarify the basic processes of fibrin polymerization and fibrinolysis. The compilation of abundant cases with detailed genetic, biological, and clinical features has enabled the classification of congenital fibrinogen disorders into several types and subtypes. Thus, the recent classification of congenital fibrinogen disorder is based not only on the clottable and antigenic fibrinogen levels but also on the patient's clinical phenotype and genotype. Fibrinogen supplementation is the cornerstone of bleeding management in fibrinogen disorders. Since the discovery of blood fractionation, the method of production of fibrinogen concentrate has been progressively modified to significantly improve purity and safety. Nevertheless, the availability of such products is still limited to a few countries and the optimal threshold of fibrinogen to target is still not established. In this review, we describe the major advances that have characterized 100 years of congenital fibrinogen disorders, focusing on afibrinogenemia and dysfibrinogenemia.

Impact of Fibrinogen Infusion on Thrombin Generation and Fibrin Clot Structure in Patients with Inherited Afibrinogenemia.

INTRODUCTION: Inherited afibrinogenemia is a very rare disease characterized by complete absence of fibrinogen in the circulation and an increased risk in both thrombosis and bleeding. Infusion of fibrinogen concentrate (FC) is the main approach for prevention and management of bleeding; however, it has been reported to carry a thrombotic risk. METHODS: We investigated the impact of a standard dose (40-100 mg/kg) of FC infusion on the thrombin generation (TG) parameters and the fibrin clot structure formed in plasma samples of patients with afibrinogenemia. Blood samples were collected from 20 patients before (T0) and 1 hour after infusion of FC (T1). TG was studied with calibrated automated thrombography. Fibrin clot structure was assessed with turbidimetry and scanning electron microscopy. RESULTS: FC infusions (mean Clauss fibrinogen plasma level: 1.21 g/L at T1) led to a statistically significant increase in endogenous thrombin potential (ETP) (p < 0.0001) and thrombin peaks (p = 0.02). Nevertheless, when compared with healthy controls, patients' T1 lag times were longer (p = 0.002), ETP values were lower (p = 0.0003), and thrombin peaks were lower (p < 00001). All fibrin polymerization parameters (turbidimetry) obtained at T1 were comparable to those of patients with inherited hypofibrinogenemia matched for fibrinogen plasma levels. CONCLUSION: In summary, fibrinogen infusion with a standard dose of FC increased but did not correct TG and led to formation of fibrin clots similar to those of patients with hypofibrinogenemia. All in all, our results do not support the biological evidence of hypercoagulability induced by FC in patients with afibrinogenemia.

Mutations Accounting for Congenital Fibrinogen Disorders: An Update.

Fibrinogen is a complex protein that plays a key role in the blood clotting process. It is a hexamer composed of two copies of three distinct chains: Aα, Bß, and γ encoded by three genes, FGA, FGB, and FGG, clustered on the long arm of chromosome 4. Congenital fibrinogen disorders (CFDs) are divided into qualitative deficiencies (dysfibrinogenemia, hypodysfibrinogenemia) in which the mutant fibrinogen molecule is present in the circulation and quantitative deficiencies (afibrinogenemia, hypofibrinogenemia) with no mutant molecule present in the bloodstream. Phenotypic manifestations are variable, patients may be asymptomatic, or suffer from bleeding or thrombosis. Causative mutations can occur in any of the three fibrinogen genes and can affect one or both alleles. Given the large number of studies reporting on novel causative mutations for CFDs since the review on the same topic published in 2016, we performed an extensive search of the literature and list here 120 additional mutations described in both quantitative and qualitative disorders. The visualization of causative single nucleotide variations placed on the coding sequences of FGA, FGB, and FGG reveals important structure function insight for several domains of the fibrinogen molecule.

A homozygous duplication of the FGG exon 8-intron 8 junction causes congenital afibrinogenemia. Lessons learned from the study of a large consanguineous Turkish family.

Congenital afibrinogenemia is the most severe congenital fibrinogen disorder, characterized by undetectable fibrinogen in circulation. Causative mutations can be divided into two main classes: null mutations with no protein production at all and missense mutations producing abnormal protein chains that are retained inside the cell. The vast majority of cases are due to single base pair mutations or small insertions or deletions in the coding regions or intron-exon junctions of FGB, FGA and FGG. Only a few large rearrangements have been described, all deletions involving FGA. Here we report the characterization of a 403 bp duplication of the FGG exon 8-intron 8 junction accounting for congenital afibrinogenemia in a large consanguineous family from Turkey. This mutation, which had escaped detection by Sanger sequencing of short polymerase chain reaction (PCR) amplicons of coding sequences and splice sites, was identified by studying multiple alignments of reads obtained from whole exome sequencing of a heterozygous individual followed by PCR amplification and sequencing of a larger portion of FGG. Because the mutation duplicates the donor splice site of intron 8, we predicted that the impact of the mutation would be on FGG transcript splicing. Analysis of mRNA produced by cells transiently transfected with normal or mutant minigene constructs showed that the duplication causes production of several aberrant FGG transcripts generating premature truncating codons.

Novel missense mutations affecting the structure of the conserved fibrinogen Bß C-terminal domain cause congenital hypofibrinogenemia.

This study describes the identification of two new mutations of the fibrinogen beta-chain in patients with inherited fibrinogen deficiency. Modelling of the impact of the mutations predict that these single amino acid substitutions are sufficient to abolish secretion of the mutant chains into the circulation, resulting in low fibrinogen levels in the patients. In addition, whole exome sequencing identified genetic modifiers for both patients which could contribute to the patients' global hemostatic function. Our results yield clinically relevant information for the personalised management of patients and eventually precision medicine for fibrinogen disorders.

Illustrated State-of-the-Art Capsules of the ISTH 2020 Congress.

This year's Congress of the International Society of Thrombosis and Haemostasis (ISTH) was hosted virtually from Philadelphia July 17-21, 2021. The conference, now held annually, highlighted cutting-edge advances in basic, population and clinical sciences of relevance to the Society. Despite being held virtually, the 2021 congress was of the same scope and quality as an annual meeting held in person. An added feature of the program is that talks streamed at the designated times will then be available on-line for asynchronous viewing. The program included 77 State of the Art (SOA) talks, thematically grouped in 28 sessions, given by internationally recognized leaders in the field. The SOA speakers were invited to prepare brief illustrated reviews of their talks that were peer reviewed and are included in this article. The topics, across the main scientific themes of the congress, include Arterial Thromboembolism, Coagulation and Natural Anticoagulants, COVID-19 and Coagulation, Diagnostics and Omics, Fibrinogen, Fibrinolysis and Proteolysis, Hemophilia and Rare Bleeding Disorders, Hemostasis in Cancer, Inflammation and Immunity, Pediatrics, Platelet Disorders, von Willebrand Disease and Thrombotic Angiopathies, Platelets and Megakaryocytes, Vascular Biology, Venous Thromboembolism and Women's Health. These illustrated capsules highlight the major scientific advances with potential to impact clinical practice. Readers are invited to take advantage of the excellent educational resource provided by these illustrated capsules. They are also encouraged to use the image in social media to draw attention to the high quality and impact of the science presented at the congress.

Whole Blood Thromboelastometry by ROTEM and Thrombin Generation by Genesia According to the Genotype and Clinical Phenotype in Congenital Fibrinogen Disorders.

The outcome of congenital fibrinogen defects (CFD) is often unpredictable. Standard coagulation assays fail to predict the clinical phenotype. We aimed to assess the pheno- and genotypic associations of thrombin generation (TG) and ROTEM in CFD. We measured fibrinogen (Fg) activity and antigen, prothrombin fragments F1+2, and TG by ST Genesia® with both Bleed- and ThromboScreen in 22 patients. ROTEM was available for 11 patients. All patients were genotyped for fibrinogen mutations. Ten patients were diagnosed with hypofibrinogenemia, nine with dysfibrinogenemia, and three with hypodysfibrinogenemia. Among the 17 mutations, eight were affecting the Fg γ chain, four the Fg Bß chain, and five the Fg Aα chain. No statistical difference according to the clinical phenotypes was observed among FGG and FGA mutations. Median F1+2 and TG levels were normal among the different groups. Fg levels correlated negatively with F1+2 and peak height, and positively with lag time and time to peak. The pheno- and genotypes of the patients did not associate with TG. FIBTEM by ROTEM detected hypofibrinogenemia. Our study suggests an inverse link between low fibrinogen activity levels and enhanced TG, which could modify the structure-function relationship of fibrin to support hemostasis.