Chapter 125: Hereditary Fibrinogen Abnormalities

Several detailed and thoroughly annotated reviews of mutations causing inherited fibrinogen disorders have been published^1,2,3 and the previous version of this chapter published in the eighth edition⁴ contained tables compiling causative mutations identified before 2009. In addition, a registry for hereditary fibrinogen abnormalities⁵ can be accessed at http://www.geht.org/databaseang/fibrinogen/ that lists variants reported in publications, conference abstracts, and submitted online, with original references. This chapter discusses the major molecular mechanisms leading to disease, as well as the laboratory and clinical aspects of fibrinogen disorders and their treatment, without listing all fibrinogen gene anomalies.

Fibrinogen plays a major role in hemostasis as the precursor molecule for the insoluble fibrin clot (Fig. 125–1). In addition fibrinogen participates in numerous other biologic processes, such as inflammation, wound healing, and angiogenesis. Fibrinogen binds plasminogen, α-antiplasmin, fibronectin, and factor XIII, among other proteins. It also binds to platelets and supports platelet aggregation. After fibrinogen is converted to fibrin by thrombin, it provides nonsubstrate binding sites for thrombin; consequently, fibrinogen is sometimes termed antithrombin I.⁶ Fibrinogen also binds to vascular endothelial and other cells, plasma or tissue matrix components such as fibronectin and glycosaminoglycans, and peptide growth factors. Fibrin provides a template for assembly and activation of the fibrinolytic system components and is the major substrate for the enzyme plasmin (Chap. 135). Both fibrinogen and fibrin serve as substrates for plasma factor XIIIa that catalyzes covalent crosslinking/ligation.

Fibrinogen is a 340-kDa glycoprotein synthesized in hepatocytes⁷ that circulates in plasma at a concentration of 1.5 to 3.5 mg/mL (~4 to 10 μM). Each fibrinogen molecule is approximately 45 nm in length. The core structure consists of two outer D regions (or D domains) and a central E region (or E domain) connected through coiled-coil connectors (Fig. 125–2).⁸ The molecule exhibits a twofold axis of symmetry perpendicular to the long axis, consisting of two sets of three polypeptide chains (Aα, Bβ, γ) that are joined in their aminoterminal regions by disulfide bridges to form the E region. The outer D regions contain the globular C terminal domains of the Bβ chain (βC) and γ chain (γC). The βC and γC domains, which are highly conserved in vertebrates, are members of the FreD (fibrinogen-related domain) family of proteins. Unlike the βC and γC domains, the C-terminal domains of the Aα chain (αC) are intrinsically unfolded and flexible and tend to be noncovalently tethered in the vicinity of the central E region (Fig. 125–2). The three genes encoding fibrinogen Bβ (FGB), Aα (FGA), and γ (FGG), ordered from centromere to telomere, are clustered in a region of approximately 50 kb on human chromosome 4.⁹ FGA and FGG are transcribed from the reverse strand, in the opposite direction to FGB. Alternative splicing¹⁰ results in two isoforms for the fibrinogen α chain: the common Aα chain, encoded by exons 1 to 5, and an extended Aα-E isoform, encoded by exons 1 to 6 which represents only 1 to 2 percent of transcripts. Alternative splicing for FGG also produces two transcripts: the major mRNA species contains all 10 exons and encodes the common γ chain (or γA), while the minor product (γ′) does not splice out intron 9 and the corresponding open reading frame replaces the four codons of exon 10 with 20 alternative codons. FGB encodes a single 1.9-kb transcript with a 1.5-kb coding sequence. Each gene is separately transcribed and translated to produce nascent polypeptides of 644 amino acids (Aα), 491 amino acids (Bβ), and 437 amino acids (γ).

During translocation of the single chains into the lumen of the endoplasmic reticulum (ER), a signal peptide is cotranslationally cleaved from each chain. The resulting chains have 625 amino acids (Aα), 461 amino acids (Bβ), and 411 amino acids (γ). Assembly proceeds in the ER with the formation of an Aα-γ or Bβ-γ intermediate. The addition of either a Bβ or Aα chain gives rise to a [AαBβγ] half-molecule, which dimerizes to form the functional hexamer.¹¹ The protein undergoes several posttranslational modifications in the Golgi complex, including maturation of N-linked oligosaccharides, phosphorylation, hydroxylation, and sulfation.¹²

Following assembly, which is completed within minutes, the mature molecule is constitutively secreted into the circulation, where it exhibits a half-life of approximately 4 days.¹³ In addition to plasma fibrinogen, blood contains an internalized intracellular fibrinogen pool that is stored within platelet α granules. Both megakaryocytes and platelets are capable of internalizing plasma fibrinogen via the fibrinogen integrin α_IIbβ₃ receptor,¹⁴ which binds to a C-terminal platelet recognition sequence that is present on γA chains but is absent from γ′ chains. Consequently, internalized platelet fibrinogen molecules contain only γA chains.¹⁵

Fibrin polymerization consists of several consecutive reactions, each affecting the ultimate structure and properties of the fibrin scaffold, which, in turn, determines the development and outcome of numerous diseases including coagulopathies and thrombosis.^16,17 Conversion of fibrinogen to a fibrin clot¹⁸ occurs in three distinct phases: (1) enzymatic cleavage by thrombin to produce fibrin monomers; (2) self-assembly of fibrin units to form an organized polymeric structure; and (3) covalent crosslinking of fibrin by factor XIIIa. In the first phase of conversion to fibrin, cleavage of fibrinogen at AαR35/G36 (R16/G17)* and later Bβ R44/G45 (R14/G15) results in release of fibrinopeptides A (FpA) and B (FpB), respectively, thus exposing “A” knobs and “B” knobs (Fig. 125–3). The “A” knob located at the new aminoterminal end of the fibrin α chain starts with the GPRV amino acid sequence. The “A” knob in fibrin interacts with the constitutive complementary association site known as hole “a” in another molecule to initiate the fibrin assembly process. Hole “a” is encompassed by residues 363 to 405 (337 to 379) of the γ chain.

A knob-hole a (A:a) interaction results in formation of double-stranded fibrils in which fibrin molecules become aligned in an end-to-middle, staggered overlapping arrangement (see Fig. 125–3).^16,17,18 Fibrils subsequently undergo branching by lateral fibril associations in which two fibrils converge to form a four-stranded “bilateral” fibril junction. Progressive lateral associations among fibrils result in larger fibril bundles or fibers. A second type of junction, termed equilateral branching, is formed by three fibrils converging to form a three-member junction.¹⁹ Both types of branch junctions provide scaffolding for the clot network, the ultimate structure of which is governed by several variables, including salt concentration, pH, fibrinogen concentration and thrombin concentration.^16,17,20

Fibrinopeptide B (FpB) release occurs more slowly than fibrinopeptide A (FpA) release and exposes another polymerization site known as the “B” knob beginning with the amino acid sequence GHRP. GHRP interacts with a constitutive hole “b” in the β chain encompassed by residues 427 to 462 (397 to 432). FpB cleavage is accelerated by fibrin polymerization, whereas FpA cleavage is independent of fibrin polymerization. B:b interactions are not required for lateral fibril associations, but they contribute to lateral association by inducing rearrangements in βC that allow βC:βC contacts to occur.^21,22

The flexible αC domains also participate in fibrin polymerization.²³ Fibrin clots made from plasma fibrinogen molecules lacking more than 100 C-terminal residues from the αC domain display prolonged thrombin times, reduced turbidity, and produce thinner fibers, indicating that αC domains participate in lateral fibril associations. In addition, αC domains become dissociated as a result of FpB cleavage. This allows αC domains to participate in noncovalent interactions with other αC domains, thereby promoting lateral fibril associations and fibrin network assembly. Finally, additional self-associating sites in the D region participate in fibrin assembly. These are the D:D sites and γ_XL sites that promote end-to-end alignment of assembling fibrin units and factor XIIIa crosslinking, respectively.^24,25

The clot formed by fibrin polymerization requires further stabilization to increase its mechanical strength and resist immediate degradation by the fibrinolytic pathway. Factor XIIIa (a heterotetramer FXIII-A2B2) is a transglutaminase that stabilizes the elongating protofibril by crosslinking adjacent γ chains through the formation of ε-(γ-glutamyl) lysine isopeptide bonds.²⁶ These occur between lysine 432 (406) of one γ chain and glutamine 424 (398) or 425 (399) of another chain. Crosslinking increases the resistance of the clot to deformation. The same process occurs, but at lower rate, between α chains and also between α chains and γ chains. In the presence of factor XIIIa, α-antiplasmin becomes covalently bound to the distal α chains of fibrin or fibrinogen.²⁶ The factor XIII binding site for fibrin has been characterized: residues in the Aα-C domain, that is, 408 to 421 (389 to 402) bind a cleft in FXIII-A2 that is exposed only after cleavage of the activation peptide by thrombin.²⁷ Fibronectin is also incorporated into the fibrin clot. This occurs by noncovalent interactions between the two proteins through specific binding sites, followed by their covalent crosslinking with factor XIIIa.²⁸ Fibronectin incorporation appears to affect the adhesion and migration of cells at sites of fibrin deposition, thereby contributing to wound healing and other cell-dependent processes.

Plasminogen and tissue-type plasminogen activator (t-PA) binding sites in the D regions (i.e., γ 337 to 350) (312 to 324), and αC domains (i.e., Aα 167 to 179) (148 to 160), are cryptic in fibrinogen and become exposed during fibrin assembly or during formation of crosslinked fibrinogen fibrils (Chap. 135).^29,30 Two phases can be distinguished in the t-PA induced lysis of a fibrin clot.³¹. In the first, slow, phase, t-PA activates plasminogen on the intact fibrin surface. The generation of C-terminal lysine residues in partially degraded fibrin (by plasmin) in the second phase of clot lysis may result in accumulation of plasminogen at the clot surface and a concomitant increase in lysis rate. Thrombin-activatable fibrinolysis inhibitor (TAFI) removes C-terminal lysine residues, resulting in a strongly reduced binding of plasminogen and in an inhibition of the second phase of clot lysis by a reduction of the activation of plasminogen on the fibrin surface. TAFI, as well as α-antiplasmin, lipoprotein(a), and histidine-rich glycoprotein, bind to fibrin and all have an inhibitory effect on fibrinolysis through various mechanisms.

Thrombin binds to its substrate, fibrinogen, through a fibrinogen recognition site in thrombin, referred to as exosite 1. The fibrin clot itself also exhibits significant thrombin-binding potential; this nonsubstrate binding potential of fibrin for thrombin is referred to as antithrombin activity I.⁶ This activity is defined by two classes of nonsubstrate thrombin-binding sites in fibrin, one of “low-affinity” in the E-region and the other of “high-affinity” in D regions of fibrin(ogen) molecules containing the variant γ′ chain. Altogether, heterodimeric γA/γ′ and homodimeric molecules γ′/γ′ chains make up 8 to 15 percent of the total γ-chain population.¹⁰ Low-affinity thrombin-binding activity reflects thrombin exosite 1 binding in the E region of fibrin, whereas high-affinity thrombin binding to γ′ chains takes place through exosite 2. The binding affinity of thrombin for γ′-containing fibrin molecules is increased by concomitant fibrin binding to thrombin exosite 1. Antithrombin I (fibrin) is an important inhibitor of thrombin generation that functions by sequestering thrombin in the forming fibrin clot, and also by reducing the catalytic activity of fibrin-bound thrombin. Vascular thrombosis may result from absence of antithrombin I (as in afibrinogenemia; see “Afibrinogenemia and Hypofibrinogenemia” below), reduced plasma γ′-chain content,³² or defective thrombin binding to fibrin as found in certain dysfibrinogenemias (see “Dysfibrinogenemia and Hypodysfibrinogenemia” below). In contrast, an increased susceptibility to arterial thrombosis has been reported when γ′-chain levels are significantly elevated. Moreover, thrombin bound to γ_A/γ′-fibrin is protected from inhibition by antithrombin to a greater extent than thrombin bound to γ_A/γ_A-fibrin. Thus, γ_A/γ′-fibrin serves as a reservoir of active thrombin, which may contribute to the prothrombotic nature of thrombi.³³

DEFINITION, HISTORY, AND EPIDEMIOLOGY

Type I disorders (afibrinogenemia and hypofibrinogenemia) affect the quantity of fibrinogen in circulation. Type II disorders (dysfibrinogenemia and hypodysfibrinogenemia) affect the quality of circulating fibrinogen.¹ While the first dysfibrinogenemia mutation was identified as early as 1968,³⁴ the molecular basis of afibrinogenemia was elucidated much later.³⁵ This disorder is characterized by autosomal recessive inheritance and the complete absence of fibrinogen in plasma.

The disease, originally described in 1920,³⁶ has an estimated prevalence of approximately one in 1,000,000. In populations where consanguineous marriages are common, the prevalence of afibrinogenemia, is increased.³⁷ Because hypofibrinogenemia (fibrinogen levels below 1.5 g L⁻¹) is often caused by heterozygosity for a fibrinogen gene mutation, this is much more frequent than afibrinogenemia. If one applies the Hardy Weinberg binomial distribution of alleles in the population to afibrinogenemia, carriers of fibrinogen deficiency causing mutations could be as frequent as 1 in 500.

ETIOLOGY AND PATHOGENESIS

Since the identification of the first causative mutation for congenital afibrinogenemia in 1999,³⁵ approximately 100 distinct mutations, the majority in FGA, have been identified in patients with afibrinogenemia (in homozygosity or in compound heterozygosity) or in hypofibrinogenemia. Causative mutations can be divided into two main classes: null mutations with no protein production at all and mutations producing abnormal protein chains which are retained inside the cell.¹

Large Deletions

The first causative mutation for afibrinogenemia was identified in a nonconsanguineous Swiss family with two pairs of afibrinogenemic brothers.³⁵ In a first step toward establishing whether or not the disease was linked to the fibrinogen gene cluster on chromosome 4, haplotype data were obtained for five microsatellite markers surrounding this locus. One of these, FGAi3, a (TCTT)n polymorphic marker located in intron 3 of the FGA gene, was found to be deleted in all four affected individuals and was hemizygous in the obligate carriers, implying that homozygous deletion of at least part of the FGA gene was responsible for the congenital afibrinogenemia in this family. Indeed, the genetic defect was found to be a recurrent deletion of approximately 11 kb of DNA, with breakpoints in FGA intron 1 and the FGA–FGB intergenic region, resulting in an absence of fibrinogen.

Three other large deletions in the fibrinogen gene cluster have been identified, all involving part of the FGA gene. These are: a deletion of 1.2 kb eliminating the entire FGA exon 4 in a Japanese patient³⁸; a deletion of 15 kb, with breakpoints situated in FGA intron 4 and in the FGA–FGB intergenic region in a Thai patient³⁹; and a 4.1-kb deletion encompassing FGA exon 1 in an Italian patient.⁴⁰ All patients were homozygous for the identified deletions except for the Thai patient, for whom complete maternal uniparental disomy was confirmed for the deleted chromosome 4.³⁹

Splice-Site Mutations

Several splice-site mutations have been identified in all three fibrinogen genes. In afibrinogenemic patients of European origin, the most common mutation is a donor splice mutation in intron 4, c.510+1G→T (previously described as IVS4+1 G→T).^1,41 Haplotype data suggest that this mutation, like the FGA 11-kb deletion, is also recurrent, or a very ancient mutation, because the c.510+1G→T mutation is found on multiple discrete haplotypes.

Frameshift Mutations

Frameshift mutations have been identified in all three fibrinogen genes. FGA exon 5, the largest fibrinogen-coding exon has the most frameshift mutations. Interestingly, seven single base-pair deletions in FGA exon 5 result in usage of the same new reading frame. All seven mutations are predicted to encode a long stretch of aberrant amino acids before terminating at the same premature stop codon, 69 to 158 codons downstream.⁴² The aberrant amino acid sequence (if the abnormal protein is synthesized and stable, which remains to be determined) may lead to abnormal folding of the Aα chain, thus affecting fibrinogen chain assembly or secretion.

Nonsense Mutations

Many nonsense mutations accounting for afibrinogenemia and hypofibrinogenemia have been identified. Of the nine nonsense mutations identified in FGB, four are located in FGB exon 8.⁴³ In particular, two FGB nonsense mutations—W467X (W437X) and W470X (W440X)—are localized very close to the β-chain C-terminus and are expected to cause the synthesis of βC chains truncated of only 25 and 22 residues, respectively.^44,45 Expression studies in transfected COS cells performed for both mutations showed that the mutations allowed individual chain synthesis and intracellular assembly of the hexamer but impaired secretion, suggesting that an intact FGB C-terminal domain is necessary for fibrinogen secretion into the circulation.⁴⁶

Missense Mutations

Null mutations, that is, large deletions, frameshift, early truncating nonsense, and splice-site mutations account for the majority of afibrinogenemia alleles, as expected. Missense mutations leading to complete fibrinogen deficiency are therefore particularly interesting, revealing the functional importance of individual residues or three-dimensional structures. Missense mutations are clustered in the highly conserved C-terminal globular domains of the γ and Bβ chains.^1,43 Expression studies in transfected cells for five FGB missense mutations, all identified in homozygosity or compound heterozygosity in afibrinogenemic patients, showed that these mutations, like the late-truncating nonsense mutations discussed previously, allowed individual chain synthesis and intracellular assembly of the hexamer but again impaired secretion.^47,48,49,50 Further characterization of the FGB G444S (G414S) mutant using immunostaining for fibrinogen and visualization by confocal microscopy revealed that the secretion-impaired mutant was retained in the ER proving the existence of an efficient quality control mechanism for fibrinogen secretion.⁴⁶

Several missense mutations have been identified in FGG in heterozygosity in patients with hypofibrinogenemia. For the majority of these mutations, analysis of patient plasma fibrinogen by mass spectrometry confirmed absence of the mutant γ chain in the circulation. Others have been studied at the functional level in transfected cells: fibrinogen Matsumoto IV C179R (C153R) was found to impair intracellular hexamer assembly,⁵¹ whereas fibrinogen Bratislava W253C (W227C) was found to impair fibrinogen secretion.⁵²

Mutations Causing Hepatic Endoplasmic Reticulum Retention and Hypofibrinogenemia

In the majority of patients with afibrinogenemia or hypofibrinogenemia there is no evidence of intracellular accumulation of the mutant fibrinogen chain. This implies the existence of an efficient degradation pathway for fibrinogen mutants that allow individual chain synthesis and assembly but not secretion. Four mutations, all in FGG, are known to cause hypofibrinogenemia accompanied by hepatic storage disease. These are three missense mutations (fibrinogen Brescia, Aguadilla, and Al duPont,^53,54,55 and a 15-bp deletion at the end of FGG exon 8 (fibrinogen Angers),⁵⁶ which creates a new FGG exon 8–intron 8 junction and donor splice site. All four mutations cause fibrinogen deficiency in the heterozygous state because of the absence of the mutant γ chain in patient plasma, but also progressive liver disease associated with hepatocellular cytoplasmic inclusions. The molecular mechanism by which these mutations, localized in the five-stranded β-sheet of γC and hole “a,” which are crucial for fibrin polymerization, leads to impaired secretion, retention in the ER, and formation of aggregates remains to be determined.

CLINICAL FEATURES

Afibrinogenemia

Bleeding because of afibrinogenemia usually manifests in the neonatal period, with 85 percent of cases presenting umbilical cord bleeding, but a later age of onset is not unusual. Bleeding may occur in the skin, gastrointestinal tract, genitourinary tract, or the central nervous system with intracranial hemorrhage being the major cause of death. Joint bleeding, which is common in patients with severe hemophilia, is less frequent: in a series of 72 patients with severe fibrinogen deficiency, hemarthrosis was observed in 25 percent of cases.⁵⁷ There is an intriguing susceptibility of spontaneous rupture of the spleen in afibrinogenemic patients. Bone cysts have also been described as a rare complication of afibrinogenemia and appear to benefit from prophylactic therapy with fibrinogen concentrate.⁵⁸

Menstruating women may experience menometrorrhagia but some have normal menses. First trimester abortion is usual in afibrinogenemic women. The importance of fibrinogen in pregnancy was demonstrated in studies with fibrinogen knockout mice that cannot carry fetuses to term.⁵⁹ Women may also have antepartum and postpartum hemorrhage. Hemoperitoneum after rupture of the corpus luteum has also been observed.

Paradoxically both arterial and venous thromboembolic complications are observed in afibrinogenemic patients. These complications can occur in the presence of concomitant risk factors such as a coinherited thrombophilic risk factor or after replacement therapy. However, in many patients, no known risk factors are present. Many hypotheses have been put forward to explain this predisposition to thrombosis. One explanation is that even in the absence of fibrinogen platelet aggregation is possible because of the action of von Willebrand factor and, in contrast to patients with severe hemophilia, afibrinogenemic patients are able to generate thrombin, both in the initial phase of limited production and also in the secondary burst of thrombin generation. In some patients, an increase of prothrombin activation fragments or thrombin–antithrombin complexes has been observed, which may reflect enhanced thrombin generation.⁶⁰ These abnormal levels can be normalized by fibrinogen infusions.

As previously mentioned, fibrin also acts as antithrombin I by both sequestering and downregulating thrombin activity.⁶ Thrombin which is not trapped by the clot is available for platelet activation and smooth muscle cell migration and proliferation, particularly in the arterial vessel wall. Thrombus formation is maintained in fibrinogen-deficient mice,⁶¹ and in fibrinogen-deficient zebrafish,⁶² but the thrombus is unstable and has a tendency to embolize. Similarly, the absence of fibrinogen in human plasma results in large but loosely packed thrombi under flow conditions.⁶³

Hypofibrinogenemia

Hypofibrinogenemia patients are very often heterozygous carriers of afibrinogenemia mutations.¹ These patients are usually asymptomatic with fibrinogen levels of approximately 1.0 g L⁻¹, levels which are in theory high enough to protect against bleeding and maintain pregnancy. However they can bleed when exposed to trauma, or if they have a second associated hemostatic abnormality. Hypofibrinogenemic women may also suffer from pregnancy loss.

LABORATORY FEATURES

The clinical diagnosis is established by functional and immunologic measurements of fibrinogen concentration backed by genetic analyses.

Phenotype Analysis

Absence of immunoreactive fibrinogen is essential for the diagnosis of congenital afibrinogenemia. All coagulation tests that depend on the formation of fibrin as the end point—that is, prothrombin time (PT), partial thromboplastin time (PTT), or thrombin time (TT)—are infinitely prolonged. Plasma activity of all other clotting factors is usually normal. Some abnormalities in platelet functions tests can be observed which can be reversed upon addition of fibrinogen. Because fibrinogen is one of the main determinants of erythrocyte sedimentation, it is not surprising that afibrinogenemic patients have very low erythrocyte sedimentation rates. When skin testing is performed for delayed hypersensitivity, there is no induration because of the lack of fibrin deposition.

Hypofibrinogenemia is defined as a proportional decrease of functional and immunoreactive fibrinogen. Coagulation tests depending on the formation of fibrin as well as the assays used are variably prolonged, the most sensitive assay being the TT.

Genotype Analysis

The large number of mutations identified in patients with afibrinogenemia allows the design of an efficient flow-chart for mutation detection in new cases.⁶⁴ Two common mutations are found in individuals of European origin, both in FGA: the c.510+1G→T intron 4 donor splice-site mutation and the FGA 11-kb deletion, both found on multiple haplotypes. In all new patients of European origin, the FGA c.510+1G→ T should be the first mutation to be screened. Southern blot or polymerase chain reaction (PCR) analysis of the FGA 11-kb deletion should also be performed, because it is the second most common mutation in patients of European origin and because of the risk of diagnostic error: a nonconsanguineous patient who appears to be homozygous for a mutation in FGA exons 2 to 6 may in reality be a heterozygous carrier of the large 11-kb deletion.⁶⁵ Given the high frequency of mutations in FGA, the other FGA exons (starting with exon 5) should then be studied for mutations before screening FGB (starting with exon 8) and FGG (starting with exons 7 and 8). The same strategy can also be applied to afibrinogenemic patients of non-European origin for whom recurrent mutations have yet to be identified. If the patient comes from a geographical region or population in which a mutation has already been identified, that mutation should be the first to be screened for. Screening of patients with hypofibrinogenemia can follow the same strategy apart from patients with ER fibrinogen-positive liver inclusions, for which four mutations in FGG are known so far to cause hepatic storage disease.

Prenatal diagnosis has been performed in a few cases.⁶⁶ This is important for families with afibrinogenemia and access to adequate treatment because the prenatal diagnosis of an affected infant allows initiation of treatment immediately after birth before the first bleeding manifestation.

Genotype–Phenotype Correlations: Potential Importance of Global Assays

Current diagnostic tests are appropriate for establishing the diagnosis but clearly additional tests are required for a more accurate prediction of the clinical phenotype of a patient and consequently the appropriate treatment. Indeed, although in afibrinogenemia all patients have unmeasurable functional fibrinogen, the severity of bleeding is highly variable amongst patients, even amongst those with the same genotype. Similarly, there is no clear relationship between the molecular defect and the risk of thrombosis.

One possible explanation for the observed variability of clinical manifestations is the existence of modifier genes/alleles: some variants may increase the severity of bleeding while others may ameliorate the phenotype. Such modifiers have yet to be identified. However, the common thrombophilias (e.g., factor V Leiden) most certainly play a role in decreasing the severity of bleeding. The existence of modifying genes/polymorphisms is also strongly suspected in the previously discussed cases of hypofibrinogenemia associated with fibrinogen inclusion bodies in hepatocytes. Indeed, all individuals heterozygous for one of the four FGG causative mutations have hypofibrinogenemia, but not all have fibrinogen aggregates and associated liver disease.

Global assays, such as thromboelastography and thrombin generation test, may provide a complementary and in some cases a better evaluation of an individual’s hemostatic state. Such global assays could be useful for the design of individual therapeutic strategies.⁶⁷

DIFFERENTIAL DIAGNOSIS

Inherited afibrinogenemia and hypofibrinogenemia have to be distinguished from acquired disorders. These include disseminated intravascular coagulation, primary fibrinolysis, liver disease, and can be caused by certain drugs (e.g., thrombolytic agents and l-asparaginase). In addition, one should be aware that artifactually low levels of fibrinogen can be observed with samples that have clotted as a result of improper collection. In most cases, the clinical context as well as the association with other laboratory abnormalities will allow differentiation of inherited from acquired disorders. Identification of a causative mutation in one of the three fibrinogen genes will confirm the diagnosis.

THERAPY

Available Treatments and Modalities

Replacement therapy is effective in treating bleeding episodes in congenital fibrinogen disorders. Depending on the country of residence, patients receive fresh-frozen plasma (FFP), cryoprecipitate, or fibrinogen concentrates.⁶⁴ Fibrinogen concentrate preparations include safety steps for inactivation or removal of viruses, which make them safer than cryoprecipitate or FFP. Furthermore, more precise dosing can be accomplished with fibrinogen concentrates because their potency is known, in contrast to FFP or cryoprecipitates.

The conventional treatment is on demand, in which fibrinogen is administered as soon as possible after onset of bleeding. Another approach is primary prophylaxis that includes administration of fibrinogen concentrates from an early age to prevent bleeding and, in the case of pregnancy, to prevent miscarriage. Effective long-term secondary prophylaxis with administration of fibrinogen every 7 to 14 days (particularly after central nervous system bleeds) has been advocated. The frequency and dose of fibrinogen concentrates should be adjusted to maintain a level above 0.5 g L⁻¹.⁶⁴

The United Kingdom guidelines on therapeutic products for coagulation disorders⁶⁸ provide recommendations about the best treatment options (dosage, management of bleeding, surgery and pregnancy as well as prophylaxis). According to these guidelines, in case of bleeding fibrinogen levels should be increased to 1.0 g L⁻¹ and maintained above this threshold until hemostasis is secured, and above 0.5 g L⁻¹ until wound healing is complete. To increase the fibrinogen concentration of 1 g L⁻¹, a dose of approximately 50 mg/kg is required. The doses and duration of treatment also vary depending on the type of injury or operative procedure and on the patient’s personal and familial history of bleeding and thrombosis.

Women with congenital afibrinogenemia are able to conceive and embryonic implantation is normal, but the pregnancy usually results in spontaneous abortion at 5 to 8 weeks of gestation unless fibrinogen replacement is given.⁶⁹ Maintaining the fibrinogen level above 0.6 g L⁻¹ and if possible higher than 1.0 g L⁻¹ is recommended. Lower fibrinogen concentrations (<0.4 g L⁻¹) have proven adequate to maintain pregnancy but not to avoid hemorrhagic complications. Continuous infusion of fibrinogen concentrate should be performed during labor to maintain fibrinogen higher than 1.5 g L⁻¹ (ideally greater than 2.0 g L⁻¹).⁷⁰ Thromboembolic events can occur, particularly with the use of cryoprecipitates that contain appreciable quantities of factor VIII and von Willebrand factor in addition to fibrinogen.

In addition to fibrinogen substitution, antifibrinolytic agents may be given, particularly to treat mucosal bleeding or to prevent bleeding following procedures such as dental extraction. Fibrin glue is useful to treat superficial wounds or following dental extractions. Oral contraceptive preparations are useful in case of menorrhagia. Oral iron preparations can be given in cases with associated iron-deficiency anemia. Routine vaccination against hepatitis, as well as a regular surveillance for both the disease and treatment-related complications in a comprehensive care setting, is highly recommended.⁶⁴

Finally, orthotopic liver transplantation is a possible rescue treatment for failure of fibrinogen replacement therapy. This procedure successfully restored normal hemostasis in an afibrinogenemic patient with severe Budd-Chiari syndrome and inferior cava vein thrombosis⁷¹ and in one of the four afibrinogenemic patients homozygous for the 11-kb FGA mutation.^35,72

Complications of Therapy

In many countries only FFP or cryoprecipitate are available, which is problematic because the viral inactivation process is in general not as efficient as it is for fibrinogen concentrates (although emerging nonviral pathogens such as the prion responsible for variant Creutzfeldt-Jacob disease must be considered, even for concentrates). Even if viral inactivation steps are performed, these preparations (particularly FFP) can induce volume overload. There is also a risk of transfusion-related acute lung injury, because of the presence of cytotoxic antibodies in the infused plasma.

Acquired inhibitors to fibrinogen after replacement therapy have been reported in only two cases. It is not clear why afibrinogenemic patients do not develop inhibitors more frequently. One explanation for some cases is that minute amounts of fibrinogen, which can only be detected by highly sensitive immunoassays, are present in the circulation.

One of the major complications in afibrinogenemic patients is thrombosis, which can occur spontaneously following blood component therapy. Some clinicians give small doses of heparin or low-molecular-weight heparin (LMWH) during administration of fibrinogen. Before surgery, patients with a thrombotic phenotype should be treated with compression stockings and LMWH. Successful use of lepirudin has been reported for an afibrinogenemic patient who suffered recurrent arterial thrombosis despite treatment with heparin and aspirin.⁷³ Thromboembolic complications are difficult to manage because both anticoagulants and fibrinogen preparations have to be administered.

New Preparations

The increasing need for fibrinogen preparations in congenital but also in acquired deficiencies has stimulated some companies to improve existing preparations or to develop new ones. A recombinant fibrinogen molecule is also under development.⁷⁴

DEFINITION, HISTORY, AND EPIDEMIOLOGY

The second class of hereditary fibrinogen abnormalities comprises the type II disorders, that is, dysfibrinogenemia and hypodysfibrinogenemia. Dysfibrinogenemia is defined by the presence of normal levels of functionally abnormal plasma fibrinogen. Hypodysfibrinogenemia is defined by low levels of a dysfunctional protein. As in afibrinogenemia and hypofibrinogenemia, both are heterogeneous disorders caused by many different mutations in the three fibrinogen-encoding genes. Dysfibrinogenemias and hypodysfibrinogenemias are autosomal dominant disorders. Most affected patients are heterozygous for missense mutations in the coding region of one of the three fibrinogen genes. Because the secreted fibrinogen hexamer contains two copies of each of the three fibrinogen chains, and the resulting fibrin network contains multiple copies of the molecule, heterozygosity for one mutant allele is sufficient to impair the structure and function of the fibrin clot (Fig. 125–4).

As of this writing, more than 100 distinct mutations have been identified in patients with dysfibrinogenemia and hypodysfibrinogenemia. The described mutants are very often named after the city of origin of the family or the city of the laboratory characterizing the mutation. Many cases are asymptomatic and are only identified as a result of routine coagulation screening. Indeed, a compilation of approximately 250 cases revealed that 55 percent of patients were asymptomatic, 25 percent had a history of bleeding, and 20 percent, a tendency toward thrombosis.⁷⁵ However, our retrospective multicentric study of the long-term outcomes of 101 genotyped patients suggests that bleeding and thrombotic events are more frequent.⁷⁶

ETIOLOGY AND PATHOGENESIS

Dysfibrinogenemic abnormalities usually are reflected in one or more phases of the fibrinogen-fibrin conversion and fibrin assembly process, notably impaired release of fibrinopeptides and defective fibrin polymerization or factor XIIIa–mediated crosslinking.^77,78 Other abnormalities involve abnormal tissue deposition such as in renal amyloidosis,⁷⁹ defective fibrinolysis,⁸⁰ abnormal interactions with platelets,^77,80 and defective calcium binding.⁸¹

Mutations Resulting in Abnormal “A” Knobs or Deficient Fibrinopeptide Release

Fibrinogen Detroit was the first abnormal fibrinogen in which the specific mutation was identified at the protein level.³⁴ This FGA R38S (R19S) mutation is located in the “A” knob (i.e., GPRV) resulting in impaired fibrin polymerization and a bleeding tendency. Other substitutions involving residue R38 (R19) have been found to be associated with bleeding in some cases, for example, Munich I, R38N (R19N), and Mannheim I, R38G (R19G), and with thrombosis in other cases, for example, Aarhus and Kumamoto, which are also a result of R38G (R19G). The mechanism for thrombophilia remains unclear, but coexisting risk factors may contribute to the clinical manifestations. Furthermore, the inability of a mutant fibrin to effectively bind and sequester thrombin may play a role in such a clinical presentation. Bleeding that occurs under conditions involving defective fibrinopeptide release or production of a defective “A” knob is most likely related to the reduced polymerization potential of the mutant fibrins that are produced, with resulting defective clot formation.⁸²

Missense mutations at residue FGA R35 (R16) which is part of the thrombin cleavage site in the fibrinogen α chain appear to be the most common causative mutations accounting for dysfibrinogenemia, based on information compiled in the GEHT registry for hereditary fibrinogen abnormalities.⁵ The R35 (R16) residue can be mutated to either H (CGT→CAT) or C (CGT→TGT) leading to delayed or absent FpA release, respectively, and subsequent delayed polymerization. A prolonged reptilase time is observed for both variants. Most patients do not have a bleeding tendency. Some patients have been found to be homozygous for these mutations or phenotypically homozygous,⁶⁵ as a result of compound heterozygosity for an R35 (R16) missense mutation and the large 11-kb FGA deletion first characterized in afibrinogenemia. In these cases, a mild bleeding tendency is observed.

Missense mutations in FGB affecting FpB release have been identified,⁸³ but are much less common than those affecting FpA release.

Mutations Leading to Polymerization Defects in the D Region

Sites in the D region important for fibrin polymerization are affected in many dysfibrinogenemias. Mutations affecting hole “a” in the γ chain are numerous, while no naturally occurring mutation involving hole “b” in the Bβ chain has been described, compatible with the view that A:a interactions are the driving force of fibrin polymerization.¹⁶ The interface for the end-to-end D:D site in the γ chain lies between R301 (R275) and S326 (S300), with T306 (T280) contacting R301 (R275) at the D:D interface. Mutations at the R301 (R275) residue to C (CGT→TGT) or H (CGT→CAT) are the second most common cause of dysfibrinogenemia, accounting for around 10 percent of fibrinogen variants.⁵ Impaired polymerization has been observed for all substitutions at this position. Most of these cases are asymptomatic, but some patients heterozygous for R301C (R275C) have thrombophilia, sometimes in association with an additional thrombotic risk factor such as factor V Leiden.⁸⁴

Mutations Accounting for Hypodysfibrinogenemia

Hypodysfibrinogenemia which is defined by low levels of a dysfunctional protein can be caused by different molecular mechanisms. One mechanism is heterozygosity for a single mutation that leads to synthesis of an abnormal fibrinogen chain which is secreted less efficiently than normal fibrinogen, for example, fibrinogen Kyoto IV.⁸⁵ Another mechanism is the presence of two different mutations with one mutation responsible for the fibrinogen deficiency (the “hypo phenotype”) and one mutation responsible for the abnormal function of the molecule (the “dys phenotype”). For example, in fibrinogen Keokuk,⁸⁶ there is compound heterozygosity for the common afibrinogenemia splice-site mutation c.510G→T and a premature truncating nonsense mutation in FGA Q347X (Q328X). Another example is fibrinogen Leipzig II in which the common hypofibrinogenemia mutation FGG A108G (A82G) and FGG G377S (G351S) are located on the same allele.⁸⁷ Homozygosity for a single mutation, which allows reduced secretion of a functionally impaired molecule, has been described in fibrinogens Otago⁸⁸ and Marburg.⁸⁹ Finally, maternal uniparental disomy for a nonsense mutation in FGB, W323X (W293X), was found to be the cause of severe hypodysfibrinogenemia in a Chinese patient.⁹⁰

CLINICAL FEATURES

Patients with inherited dysfibrinogenemia are frequently asymptomatic and can be discovered incidentally because of abnormal coagulation tests. A compilation of more than 260 cases of dysfibrinogenemia revealed that 55 percent of the patients had no clinical complications while 25 percent exhibited bleeding, and 20 percent had a tendency to thrombosis, mainly venous.⁷⁵ However, when 2376 patients with deep vein thrombosis were screened for thrombophilia, the prevalence of dysfibrinogenemia was very low (0.8 percent) and hence testing for dysfibrinogenemia in patients with deep vein thrombosis is not recommended.⁹¹ Patients with dysfibrinogenemia associated with hemorrhage bleed most often after trauma, surgery, or during the puerperium.⁷⁶ Thrombosis may also occur during pregnancy and in the postpartum period. Women with dysfibrinogenemia can also suffer from spontaneous abortions. The problems during and after pregnancy are not necessarily correlated to the fibrinogen concentration.

Some mutations in the Aα chain of fibrinogen are associated with a particular form of hereditary amyloidosis.^79,92 The E545V (E526V) amino acid substitution is the most common of these mutations.⁹² The abnormal fibrinogen fragments form amyloid fibrils and the extracellular deposition of these fibrils leads to renal failure. Chronic renal dialysis is performed for managing renal failure. Renal transplantation can be envisaged as an alternative to chronic dialysis. However, continuous fibrinogen-related amyloid deposition ultimately results in allograft destruction. Combined liver and kidney transplantation prevents further amyloid deposition in the renal allograft and elsewhere but is associated with additional perioperative and subsequent risks.

LABORATORY FEATURES

Phenotype Analysis

Initial screening tests for fibrinogen dysfunction should include fibrinogen concentration, measured functionally and immunochemically, TT, and reptilase time. Dysfibrinogenemia is diagnosed by a discrepancy between clottable and immunoreactive fibrinogen. However, even in specialized laboratories, this diagnosis can be difficult because the sensitivity of the tests depends on the specific mutation, reagents, and techniques.^93,94

In classical dysfibrinogenemias, the functional assay of fibrinogen yields low levels compared with the immunologic assays, but levels are sometimes concordant and the functional level may even be normal (as well as TT). The determination of the precise nature of a fibrinogen defect has to be performed in highly specialized laboratories since it involves purification of fibrinogen, measurement of the rate of fibrinopeptide cleavage, analysis of fibrin monomer polymerization, and fibrinolysis. Thromboelastography, commonly used for decision making for fibrinolytic and anticoagulant therapy, may be particularly useful for investigation of dysfibrinogenemia. The thromboelastography signal is fibrin dependant, its amplitude is enhanced by platelets and reflects the stretch and recovery of the clot during its formation.⁹⁵

Genotype Analysis

The gold standard for the diagnosis of dysfibrinogenemia is the characterization of the molecular defect. However, although advances in DNA analysis have made mutation detection easier, it is not always clear whether the identified mutation is the cause of the presenting phenotype. Family studies showing segregation of the mutation with the phenotype, exclusion that the DNA alteration is a common polymorphism in the general population, and structural correlations are necessary for establishing the link between the DNA alteration and the disorder. As previously mentioned, two mutations “hotspots” are of prime interest in screening for dysfibrinogenemia mutations: residue R35 (R16) situated in FGA exon 2, and residue R301 (R275) in FGG exon 8. Other causative mutations are common in the surrounding residues. Thus, it is recommended to initially screen FGA exon 2 and FGG exon 8 in cases of dysfibrinogenemia. In our recent study of 101 dysfibrinogenemia cases,⁷⁶ 87 percent of the causative mutations were located in these two exons. Here, mutations of FGG R301 (R275) were more common than mutations of FGA R35 (R16), 52 percent and 23 percent, respectively.

Genotype–Phenotype Correlations

As previously discussed, the clinical manifestations of dysfibrinogenemia are highly variable and may relate in some cases to differences in clot strength, structure and stability.^1,17 In a few cases, mutations are predictive of the clinical phenotype, such as the R573C (R554C) substitution in the Aα chain (e.g., fibrinogens Chapel Hill III, Paris V, and Dusart) that predisposes patients to thrombosis. Impaired fibrinolysis exhibited by this dysfibrinogen appears to be responsible for the thrombotic complications. Other examples associated with thrombosis include dysfibrinogens Barcelona III, Haifa I, or Bergamo II as a result of the common γ R301H (R275H) mutation and Cedar Rapids I caused by γ R301C (R275C). However, in fibrinogen Cedar Rapids I, only patients heterozygous for both factor V Leiden and the FGG R301C (R275C) substitutions were symptomatic, suggesting that this mutation causes thrombosis when associated with another defect. On the other hand, several mutations in the aminoterminal region of the Aα chain, such as fibrinogen Detroit R38S (R19S) and Mannheim I R38G (R19G), are associated with bleeding.

DIFFERENTIAL DIAGNOSIS

Inherited dysfibrinogenemia has to be distinguished from acquired dysfibrinogenemia. Liver diseases (e.g., cirrhosis, chronic active liver disease, hepatoma, liver failure) are the main causes of acquired dysfibrinogenemia. l-Asparaginase treatment also may result in the production of abnormal fibrinogen. In addition, there are a few case reports of acquired dysfibrinogenemia secondary to pancreatitis, paraneoplastic syndrome, and renal carcinoma. The acquired dysfibrinogenemias represent a heterogeneous group of disorders with multiple pathogenetic mechanisms, the most clearly defined fibrinogen abnormalities being an increase in carbohydrate content in patients with liver disease. These abnormal fibrinogens are usually characterized by prolonged thrombin and reptilase times, by abnormal fibrin monomer polymerization but with normal fibrinopeptide release. Fibrinogen concentration is variable.

In some cases no underlying disease is found, and to determine whether a fibrinogen abnormality is congenital or acquired may be difficult. The demonstration of the same fibrinogen abnormality in another family member is a strong argument for a congenital disorder. When measured in newborns, fibrinogen levels should be interpreted with caution because neonatal fibrinogen has an altered content of carbohydrate that can mimic dysfibrinogenemia in certain laboratory tests.

Rare cases of circulating autoantibodies to fibrinogen, for example in systemic lupus erythematosus and in patients receiving surgical sealants containing bovine fibrinogen, have also been reported.

THERAPY

Any treatment considered in patients with dysfibrinogenemia should be based on the personal and family history. Indeed, as already discussed, subjects with hereditary dysfibrinogenemias may be asymptomatic throughout their whole life or may suffer from bleeding and/or thrombotic complications.^1,75,76 In patients who bleed, functional levels of fibrinogen should be raised above 1.0 g L⁻¹ and maintained above this threshold until hemostasis is secured and above 0.5 g L⁻¹ until wound healing is complete. Topical fibrin glue or antifibrinolytic agents may be used for superficial bleeds. In pregnant women with a bleeding phenotype, the recommendations for afibrinogenemia and hypofibrinogenemia can be followed. With a personal or familial history of thrombosis, thromboprophylaxis and antithrombotic treatments may be proposed after a careful analysis of each particular situation. Long-term management strategies for thrombophilic dysfibrinogenemia are the same as the strategies for patients with recurrent thromboembolism and may include long-term anticoagulant therapy.