A novel fibrinogen variant--Liberec: dysfibrinogenaemia associated with gamma Tyr262Cys substitution

Objective:  A 22-yr-old woman had abnormal preoperative coagulation test results and congenital dysfibrinogenaemia was suspected.
Patients and methods:  The patient from Liberec (Czech Republic) had a low fibrinogen plasma level as determined by Clauss method, normal fibrinogen level as determined by immunoturbidimetrical method, and prolonged thrombin time. To identify the genetic mutation responsible for this dysfibrinogen, genomic DNA extracted from the blood was analysed. Fibrin polymerisation measurement, kinetics of fibrinopeptide release, fibrinogen clottability measurement and scanning electron microscopy were performed.
Results:  DNA sequencing showed the heterozygous fibrinogen γ Y262C mutation. Kinetics of fibrinopeptide release was normal, however fibrin polymerisation was impaired. Fibrinogen clottability measurement showed that only about 45% molecules of fibrinogen are involved in the clot formation. Scanning electron microscopy revealed thicker fibres, which were significantly different from the normal control.
Conclusion:  A case of dysfibrinogenaemia, found by routine coagulation testing, was genetically identified as a novel fibrinogen variant (γ Y262C) that has been named Liberec.     

Two novel fibrinogen variants in the C-terminus of the Bβ-chain: fibrinogen Rokycany and fibrinogen Znojmo

Hereditary dysfibrinogenemia is a rare disorder wherein an inherited abnormality in fibrinogen structure may result in defective fibrin function and/or structure. Congenital hypofibrinogenemia is a rare autosomal bleeding disorder, either recessive or dominant, characterized by a low fibrinogen plasma level. A 28-year-old asymptomatic woman (fibrinogen Rokycany) and a 54-year-old man with thrombosis and pulmonary embolism (fibrinogen Znojmo) were investigated for a suspected fibrinogen mutation after abnormal coagulation tests results were obtained. DNA sequencing showed the heterozygous point mutation Bβ Asn351Lys in fibrinogen Rokycany and the heterozygous point mutation Bβ Arg237Ser in fibrinogen Znojmo, respectively. The kinetics of fibrinopeptide release was found to be normal in both cases. Fibrinolysis was impaired in the Znojmo variant. The average fibril diameters of Znojmo fibrin was slightly increased, but not differing significantly from normal; formed by less fibrils with abrupt fibril terminations. Rheological studies revealed a softer clot. Rokycany fibrin was formed by significantly narrower fibrils than normal fibrin; and the clot was denser than the control clot. Rheological studies revealed a stiffer clot. Impaired fibrinolysis and abnormal clot morphology may be the cause of thrombotic episodes in the patient with Znojmo mutation. New cases of hypofibrinogenemia and dysfibrinogenemia, found by routine coagulation testing, were genetically identified as a novel fibrinogen variants Bβ Asn351Lys (fibrinogen Rokycany) and Bβ Arg237Ser (fibrinogen Znojmo), respectively.     

Fibrinogen kaiserslautern III: a new case of congenital dysfibrinogenemia with aalpha 16 arg-->cys substitution.

An abnormal fibrinogen was identified in a man with suspicious prolonged prothrombin time and a mild bleeding tendency. Coagulation studies showed marked prolonged thrombin and reptilase clotting times and a discrepancy between functional fibrinogen test and fibrinogen antigen. The rate of fibrinopeptide B release by thrombin was slightly delayed while the release of fibrinopeptide A was only half the normal amount. DNA sequencing revealed a heterozygous C to T point mutation in position 1202 of exon 2 of the Aalpha chain, resulting in the substitution of Arg-->Cys at position 16, the thrombin cleavage site. This mutation was found also in his 2 children. Both had a mild bleeding tendency too.     

Fibrinogen Baltimore II: congenital hypodysfibrinogenemia with delayed release of fibrinopeptide B and decreased rate of fibrinogen synthesis.

A congenital hypodysfibrinogenemia, fibrinogen Baltimore II, was found in a young asymptomatic Caucasian female. Prothrombin, partial thromboplastin, and euglobulin lysis times were normal, as were platelet function and coagulation factor assays. Subnormal plasma fibrinogen levels were found using chronometric, rate-independent, and immunologic assay methods. Kinetic analysis of fibrinopeptide release revealed a delay in the thrombin-catalyzed release of fibrinopeptide B from the abnormal protein. Proteolysis of fibrinopeptide A by thrombin or Arvin, fibrin monomer polymerization, and fibrin polymer ligation occurred at normal rates. Catabolism of radiolabeled autologous and homologous fibrinogen was also normal, but the fibrinogen synthetic rate was less than half the normal value. Comparison of the coagulation characteristics of fibrinogen Baltimore II with those of other abnormal fibrinogens indicates that it represents a unique example of hypodysfibrinogenemia.     

Fibrinogen Saint-Germain I: a case of the heterozygous Aalpha GLY 12 --> VAL fibrinogen variant.

A fibrinogen variant was suspected based on the results of routine coagulation tests in a 2-year-old asymptomatic child. Coagulation studies showed marked prolongation of both the thrombin and reptilase times, and discrepancy was noted between the level of plasma fibrinogen as measured by a kinetic versus immunological determination. Family studies revealed that the father beared the same abnormality. Studies of purified fibrinogen revealed an impaired release of both fibrinopeptides by thrombin. Fibrin monomer polymerization and fibrin stabilization were normal. DNA sequencing revealed a heterozygous G --> T point mutation in exon 2 of the gene coding for the Aalpha chain, which substituted a Gly for Val at position 12. Although the mutation is the same as in fibrinogen Rouen, fibrinogen Saint-Germain I shows a different fibrinopeptide release pattern and a mild factor V deficiency.     

Novel fibrinogen mutation (gamma 313 Ser-->Asn) associated with hypofibrinogenemia in two unrelated families.

Congenital hypofibrinogenemia is a rare disorder caused by a number of different mutations in the fibrinogen genes. The aim of the study was the elucidation of molecular defects in two unrelated families with hypofibrinogenemia. DNA samples from the patients were screened for mutations in the fibrinogen genes by direct sequencing of polymerase chain reaction-amplified gene segments. Isolated plasma fibrinogen was studied by sodium dodecyl sulfate electrophoresis and electrospray ionization mass spectrometry in order to detect variant polypeptides. Fibrin polymerization was analyzed both in plasma and using purified fibrinogen samples. A novel mutation in the FGG gene (G7590A) was found in all patients from the two families with hypofibrinogenemia. This mutation causes the amino acid exchange 313 Ser-->Asn in the gamma chain. When plasma fibrinogen from a heterozygous individual was analyzed for the presence of variant gamma chains by reverse-phase high-performance liquid chromatography and electrospray ionization mass spectrometry, only normal gamma chains could be detected. The molecular defect affecting an evolutionary highly conserved amino acid residue in human fibrinogen interferes with plasma expression of the variant molecules and is causative for the observed hypofibrinogenemic phenotype.     

Fibrinogen Columbus: a novel gamma Gly200Val mutation causing hypofibrinogenemia in a family with associated thrombophilia

Fibrinogen is an essential component of the coagulation cascade and the acute phase response. The native 340 kDa molecule has a symmetrical trinodular structure composed of a central E-domain connected to outer D-domains by triple helical coiled-coils.1 Several mutations known to cause hypofibrinogenemia occur within the C-terminal gammaD-domain and have helped to elucidate the structurally and functionally important areas of this domain.2-5 Here we report the identification of a novel point mutation gammaG200V (fibrinogen Columbus) causing hypofibrinogenemia and co-segregating with three genetic thrombophilia risk factors.     

Novel fibrinogen gamma375 Arg-->Trp mutation (fibrinogen aguadilla) causes hepatic endoplasmic reticulum storage and hypofibrinogenemia

The proposita and her sister had chronically elevated liver function test results, and needle biopsy specimens showed scattered eosinophilic inclusions within the hepatocytes. On immunoperoxidase staining, the inclusions reacted strongly with anti-fibrinogen antisera; on electron-microscopic (EM) examination, the material appeared confined to the endoplasmic reticulum (ER) and was densely packed into tubular structures with a swirling fingerprint appearance. Coagulation investigations showed low functional and antigenic fibrinogen concentrations that were indicative of hypofibrinogenemia. Amplification and DNA sequencing showed a heterozygous CGG-->TGG mutation at codon 375 of the fibrinogen gamma chain gene. This novel gamma375 Arg-->Trp substitution segregated with hypofibrinogenemia in 3 family members and was absent from 50 normal controls. When purified plasma fibrinogen chains were examined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, reverse-phase chromatography, electrospray ionization mass spectrometry, and isoelectric focusing, only normal gamma chains were detected. In conclusion, we propose that this nonconservative mutation causes a conformational change in newly synthesized molecules and that this provokes aggregation within the ER and in turn causes the observed hypofibrinogenemia. Whereas the mutation site, gamma375, is located in the gammaD domain at the jaws of the primary E-to-D polymerization site, purified plasma fibrinogen showed normal polymerization, supporting our contention that molecules with variant chains never reach the circulation but accumulate in the ER.     

Fibrinogen Šumperk II: dysfibrinogenemia in an individual with two coding mutations

Fibrinogen—a 340-kDa glycoprotein—plays a crucial role in blood coagulation, platelet aggregation, wound healing, and other physiological processes. A mutation in fibrinogen may lead to congenital dysfibrinogenemia,a rare disease characterized by the functional deficiency of fibrinogen. About 580 cases of abnormal fibrinogens have been reported worldwide; thereof 335 cases in the fibrinogen Aa chain[1]. To our knowledge, only five cases of abnormal fibrinogens with two mutations [2–6] and one case of two different mutations in the same family [7] have been described earlier. A 52-year-old female was examined for bleeding. Routine hemostasis screening resulted in a diagnosis of dysfibrinogenemia. Functional testing revealed prolonged fibrin polymerization, prolonged lysis of the clot, abnormal fibrin morphology,and fibrinopeptides release. Genetic analysis showed two heterozygous nonsense mutations—previously described mutation AaGly13Glu and a novel mutation Aa Ser314Cys. The mutation Aa Gly13-Glu was found in her brother and niece, but there was no evidence in either of the mutation Aa Ser314Cys. While mutation Aa Gly13Glu is responsible for abnormal fibrinopeptide release and prolonged thrombin time, the novel mutation Aa Ser314Cys seems to affect fibrin morphology and fibrinolysis.     

Fibrinogen "New York"--an abnormal fibrinogen associated with thromboembolism: functional evaluation.

A 54-yr-old woman presented with a 23-yr history of repeated life-threatening thromboembolism. The presence of a qualitatively abnormal fibrinogen was suggested by the demonstration of delayed and incomplete coagulation of plasma or partially purified fibrinogen by thrombin or Reptilase. Two brothers showed a similar in vitro defect but were clinically not affected. The plasma fibrinogen concentration was 0.50-1.64 mg/ml when estimated by heat turbidity, clottability, or immunologic techniques. The serum contained 80-820 mug/ml of unclottable fibrinogen-related materials even after 24 hr exposure to thrombin. The fibrinogen-related material in the serum showed faster anodal mobility an immunoelectrophoresis than that of normal plasma. Immunodiffusion studies with rabbit antihuman fibrinogen antiserum showed lines of identity between control plasma and the patient's plasma and serum. Studies of the kinetics of thrombin action on fibrinogen demonstrated impaired release of fibrinopeptide A and B and defective polymerization of preformed fibrin monomers. The maximum amount of fibrinopeptide A released by exhaustive treatment with thrombin was similar (per milligram protein) for both the patient's and control fibrinogen. This abnormal fibrinogen varient is tentatively designated fibrinogen "New York"; its possible identity with one of the previously described abnormal fibrinogens has not been excluded.     

Fibrinogen Milano IV, another case of congenital dysfibrinogenemia with an abnormal fibrinopeptide A release (A alpha 16 Arg----His).

An abnormal fibrinogen, denoted as 'fibrinogen Milano IV', has been found in a 36-year-old woman without any bleeding manifestations or thrombotic tendency. Routine coagulation studies revealed prolonged thrombin and reptilase clotting times, very low plasma fibrinogen concentration determined by the functional assay but a normal fibrinogen concentration measured by the immunologic assay. Turbidity curves, measured following addition of thrombin to purified fibrinogen Milano IV, both in presence of calcium or EDTA, were markedly delayed. Release of fibrinopeptide B by thrombin was normal, whereas only half the normal amount of fibrinopeptide A was cleaved. The fibrinopeptide A peak of fibrinogen was preceded by an abnormal fibrinopeptide A*. Both peaks were collected for amino acid analysis which showed an exchange of arginine by histidine in position 16 of the A alpha chain of the fibrinopeptide A*.     

Dysfibrinogenemia and thrombosis.

Congenital abnormal fibrinogen molecules (dysfibrinogenemias) are due to structural defects in the molecule. The molecular structure of the fibrinogen molecule is to a great extent known and this has allowed identification of the abnormalities at a molecular level. While most patients with dysfibrinogenemia are clinically asymptomatic, some present with a bleeding diathesis, others with thrombophilia, and occasionally with both, bleeding and thromboembolism. In principle, the dysfibrinogenemias are due to either impaired release of the fibrinopeptides, defective fibrin polymerization, or abnormal cross-linking by factor XIIIa. Dysfibrinogenemias associated with thrombophilia have been reported in those related to abnormal fibrinopeptide release or defective polymerization. In addition, abnormal interactions with platelets, defective fibrinolysis, defective assembly of the fibrinolytic system, and abnormal calcium binding have been described. The presently identified dysfibrinogenemias associated with thrombosis and their molecular defects are described in this review. It must also be recognized that some patients with abnormal fibrinogen molecules have additional hemostasis defects, such as abnormalities of antithrombin, protein C, protein S, factor V Leiden, and others. On rare occasions, dysfibrinogenemias can be associated with hypofibrinogenemia.     

Acquired dysfibrinogenemia caused by monoclonal production of immunoglobulin lambda light chain

Disorders of fibrinogen are usually caused by genetic mutations that result in low protein levels (hypofibrinogenemia) or an abnormal molecule (dysfibrinogenemia). However, environmental and plasma factors can have an acquired effect on its expression or function. For example, antibodies can bind fibrinogen and/or fibrin to interfere with polymerization and inhibit coagulation. The objective here was to determine the cause of dysfibrinogenemia in a 63-year-old man. Despite a low functional fibrinogen concentration and prolonged thrombin time, no inherited fibrinogen abnormality could be detected after extensive protein analysis and gene sequencing. Thus, electrophoresis methods and fibrinogen binding studies were used to establish the cause of the acquired dysfibrinogenemia. An immunoglobulin lambda light chain was found to bind fibrinogen as a monomer. It had no significant effect on fibrinopeptide release, but caused substantial defects in all other stages of thrombin-catalyzed fibrin polymerization. Binding to fibrinogen also seemed to prevent the light chain from being filtered through the kidneys, causing only low levels of it in the urine. Once in the urine, the lambda chain lost its anti-fibrinogen activity, apparently due to dimerization. The 63-year-old patient acquired dysfibrinogenemia from a monoclonal production of lambda light chain that bound and inhibited the function of fibrinogen. At age 64.5 he was diagnosed with monoclonal gammopathy of undetermined significance, explaining the abnormal immunoglobulin chain production. This case was particularly unusual in that the inhibition of fibrin polymerization was caused by a single immunoglobulin light chain, rather than by a whole antibody molecule.     

Fibrinogen Poissy II (gammaN361K): a novel dysfibrinogenemia associated with defective polymerization and peptide B release.

A fibrinogen variant was identified in a pregnant patient with disseminated intravascular coagulation and abruptio placentae. This dysfibrinogen was also found in four asymptomatic members of the patient's family. Coagulation studies showed prolongation of both the thrombin and reptilase times, and discrepancy was noted between the levels of plasma fibrinogen as determined by a kinetic versus an immunological determination or light-scattering assay. Studies on purified fibrinogen revealed an impaired release of fibrinopeptide B by thrombin related to a delayed thrombin-induced fibrin polymerization. DNA sequencing revealed a heterozygous T <-- A point mutation in position 9373 of the gamma-chain gene (exon 9), which substituted a K for an N at position 361.     

Idiopathic autoantibody that inhibits fibrin monomer polymerization

S ummary . A 73-year-old female was found to have prolonged thrombin and reptilase times in the immediate post-operative period. These abnormalities were not corrected by the addition of normal plasma. They were subsequently shown to be due to an IgG immunoglobulin which inhibited fibrin monomer polymerization. The IgG immunoglobulin activity could be neutralized completely by prior incubation with either patient or normal fibrinogen, uncrosslinked fibrin monomers or IgG antisera. No inhibitory effect on thrombin activity, fibrinopeptide A release or on the fibrin cross-linking reaction of factor XIIIa could be detected. Purified patient fibrinogen was functionally normal as demonstrated by normal fibrinogen–fibrin polymerization and fibrinopeptide A release. No underlying cause for this phenomenon was found. The presence of the inhibitor was associated with excessive blood loss during the post-operative period.     

Fibrinogen Philadelphia, a hypodysfibrinogenemia characterized by abnormal polymerization and fibrinogen hypercatabolism due to gamma S378P mutation

Fibrinogen Philadelphia, a hypodysfibrinogenemia described in a family with a history of bleeding, is characterized by prolonged thrombin time, abnormal fibrin polymerization, and increased catabolism of the abnormal fibrinogen. Turbidity studies of polymerization of purified fibrinogen under different ionic conditions reveal a reduced lag period and lower final turbidity, indicating more rapid initial polymerization and impaired lateral aggregation. Consistent with this, scanning and transmission electron microscopy show fibers with substantially lower average fiber diameters. DNA sequence analysis of the fibrinogen genes A, B, and G revealed a T>C transition in exon 9 resulting in a serine-to-proline substitution near the gamma chain C-terminus (S378P). The S378P mutation is associated with fibrinogen Philadelphia in this kindred and was not found in 10 controls. This region of the gamma chain is involved in fibrin polymerization, supporting this as the polymerization defect causing the mutation. Thus, this abnormal fibrinogen is characterized by 2 unique features: (1) abnormal polymerization probably due to a major defect in lateral aggregation and (2) hypercatabolism of the mutant protein. The location, nature, and unusual characteristics of this mutation may add to our understanding of fibrinogen protein interactions necessary for normal catabolism and fibrin formation.     

The impaired polymerization of fibrinogen Longmont (Bbeta166Arg-->Cys) is not improved by removal of disulfide-linked dimers from a mixture of dimers and cysteine-linked monomers

This study identified a new substitution in the Bbeta chain of an abnormal fibrinogen, denoted Longmont, where the residue Arg166 was changed to Cys. The variant was discovered in a young woman with an episode of severe hemorrhage at childbirth and a subsequent mild bleeding disorder. The neo-Cys residues were always found to be disulfide-bridged to either an isolated Cys amino acid or to the corresponding Cys residue of another abnormal fibrinogen molecule, forming dimers. Removing the dimeric molecules using gel filtration did not correct the fibrin polymerization defect. Fibrinogen Longmont had normal fibrinopeptide A and B release and a functional polymerization site "a." Thus, the sites "A" and "a" can interact to form protofibrils, as evidenced by dynamic light-scattering measurements. These protofibrils, however, were unable to associate in the normal manner of lateral aggregation, leading to abnormal clot formation, as shown by an impaired increase in turbidity. Therefore, it is concluded that the substitution of Arg166-->Cys-Cys alters fibrinogen Longmont polymerization by disrupting interactions that are critical for normal lateral association of protofibrils. (Blood. 2001;98:661-666)     

Hypofibrinogenemia in an individual with 2 coding (gamma82 A-->G and Bbeta235 P-->L) and 2 noncoding mutations

We investigated the molecular basis of hypofibrinogenemia in a man with a normal thrombin clotting time. Protein analysis indicated equal plasma expression of 2 different Bbeta alleles, and DNA sequencing confirmed heterozygosity for a new Bbeta235 P-->L mutation. Protein analysis also revealed a novel gamma(D) chain, present at a ratio of 1:2 relative to the gamma(A) chain. Mass spectrometry indicated a 14 d decrease in the gamma(D)-chain mass, and DNA sequencing showed this was caused by a novel gamma82 A-->G substitution. DNA sequencing established heterozygosity for 2 further mutations: T-->C in intron 4 of the Aalpha gene and A-->C in the 3' noncoding region of the Bbeta gene. Studies on the man's daughter, together with plasma expression levels, discounted both the Aalpha and Bbeta mutations as the cause of the low fibrinogen, suggesting that the gamma82 mutation caused the hypofibrinogenemia. This was supported by analysis of 31 normal controls in whom the Bbeta mutations were found at polymorphic levels, with an allelic frequency of 5% for the Bbeta235 mutation and 42% for the Bbeta 3' untranslated mutation. The gamma82 mutation was, however, unique to the propositus. Residue gamma82 is located in the triple helix that separates the E and D domains, and aberrant packing of the helices may explain the decreased fibrinogen concentration. (Blood. 2000;95:1709-1713)     

Fibrinogen Matsumoto II: γ308Asn → Lys (AAT → AAG) mutation associated with bleeding tendency

Fibrinogen Matsumoto II is a hereditary dysfibrinogenaemia identified in a woman with Basedow's disease and a bleeding tendency. Coagulation tests of the patient's plasma revealed a prolonged thrombin time and a decreased fibrinogen level determined by functional method. Release of fibrinopeptide A and B was normal, whereas fibrin monomer polymerization was delayed. Fibrinogen γ-chain gene of the propositus was heterozygous for a missense mutation that resulted in Asn → Lys substitution at codon 308. Though the same amino acid substitution was also attributed to fibrinogen Kyoto I and Bicetre II, fibrinogen Matsumoto II showed different clinical manifestations from them.