Fibrinogen Secretion Research Articles

Abnormalities in gγ region of fibrinogen molecule and influences in assembly, secretion and function of intact fibrinogen

Abnormalities in gγ region of fibrinogen molecule and influences in assembly, secretion and function of intact fibrinogen

Molecular mechanisms accounting for fibrinogen deficiency: from large deletions to intracellular retention of misfolded proteins.

Fibrinogen, the soluble precursor of fibrin, which is the main protein constituent of the blood clot, is synthesized in hepatocytes in the form of a hexamer composed of two sets of three polypeptides (Aalpha, Bbeta, and gamma). Each polypeptide is encoded by a distinct gene, FGA, FGB and FGG, all three clustered in a region of 50 kb on 4q32. Congenital afibrinogenemia is characterized by the complete absence of fibrinogen. The first causative mutation for this disease was identified in Geneva in a non-consanguineous Swiss family in 1999: the four patients were homozygous for a large deletion in the fibrinogen cluster, which eliminated almost the entire FGA genomic sequence. Mutations in the fibrinogen genes may lead to deficiency of fibrinogen by several mechanisms: acting at the DNA level, at the RNA level by affecting mRNA splicing or stability, or at the protein level by affecting protein synthesis, assembly or secretion. Recent reviews have provided helpful updates for the rapidly growing number of causative mutations identified in patients with fibrinogen deficiencies, either afibrinogenemia or hypofibrinogenemia. The aim of this review is to highlight specifically the subset of mutations that allow fibrinogen chain synthesis and hexamer assembly but impair secretion. Indeed, functional studies of these mutations have shed light on the specific sequences and structures in the fibrinogen molecule involved in the quality control of fibrinogen secretion.

Molecular characterization of the first missense mutation in the fibrinogen Aalpha-chain gene identified in a compound heterozygous afibrinogenemic patient

Congenital afibrinogenemia is a rare coagulopathy characterized by extremely low levels of functional and immunoreactive fibrinogen in plasma, associated with a hemorrhagic phenotype of variable severity. It is transmitted as an autosomal recessive trait and is invariantly associated with mutations affecting 1 of the 3 fibrinogen genes ( FGA, FGB, and FGG, coding for Aα, Bβ, and γ chain, respectively). Most genetic defects causing afibrinogenemia are truncating mutations, whereas only few missense mutations (6) have been identified so far, all located in FGB. In this study, the mutational screening of an afibrinogenemic Italian male identified the first missense mutation (Met51Arg) in FGA leading to afibrinogenemia. The patient was a compound heterozygote for a previously described frameshift mutation (1215delT) in the same gene. Met51Arg involves a residue located at the very beginning of the coiled-coil domain, in a region demonstrated to play a pivotal role in hexamer formation. In-vitro expression experiments showed that Met51Arg strongly reduces secretion of hexameric fibrinogen, whereas traces of not completely assembled trimeric intermediate were found in conditioned media. Western blot analysis on the proband's plasma confirmed the presence in vivo of the trimeric fibrinogen, supporting the hypothesis that Met51Arg prevents the final step of fibrinogen assembly.

Analysis of fibrinogen variants at γ387Ile shows that the side chain of γ387 and the tertiary structure of the γC-terminal tail are important not only for assembly and secretion of fibrinogen but also for lateral aggregation of protofibrils and XIIIa-catalyzed γ-γ dimer formation

AbstractTo examine the role of fibrinogen γ-chain residue 387Ile in the assembly and secretion of this multichain protein, we synthesized a series of variants with substitution at γ387 by Arg, Leu, Met, Ala, or Asp. Only the variant γ387Asp showed impaired synthesis in the cells and very low secretion into the medium. In addition, we performed thrombin-catalyzed fibrin polymerization and factor (F) XIIIa-catalyzed cross-linking of the γ-chain for 4 variants. The degree of lateral aggregation of protofibrils into fibrin fibers was slightly reduced for γ387Arg and Ala, and moderately reduced for γ387Leu and Met. Although the FXIIIa-catalyzed cross-linking for all of the variants was slower than that for γ387Ile, that of γ387Arg was much more markedly impaired than that of the others. In summary, our studies demonstrated that the specific residue at γ387 or the conformation of γ388-411 residues, but not the length of the γC tail, is critical for fibrinogen assembly and subsequent secretion. Moreover, this residue or the conformation is also important for not only the lateral aggregation of fibrin polymers but also the FXIIIa-catalyzed cross-linking of the γ-chain. Interestingly, our results clearly indicate that the conformations critical for these 2 functions are different from each other.

Quality control of fibrinogen secretion in the molecular pathogenesis of congenital afibrinogenemia

Congenital afibrinogenemia is a rare bleeding disorder characterized by the absence in circulation of fibrinogen, a hexamer composed of two sets of three polypeptides (Aalpha, Bbeta and gamma). Each polypeptide is encoded by a distinct gene, FGA, FGB and FGG, all three clustered in a region of 50 kb on 4q31. A subset of afibrinogenemia mutations has been shown to specifically impair fibrinogen secretion, but the underlying molecular mechanisms remained to be elucidated. Here, we show that truncation of the seven most C-terminal residues (R455-Q461) of the Bbeta chain specifically inhibits fibrinogen secretion. Expression of additional mutants and structural modelling suggests that neither the last six residues nor R455 is crucial per se for secretion, but prevent protein misfolding by protecting hydrophobic residues in the betaC core. Immunofluorescence and immuno-electron microscopy studies indicate that secretion-impaired mutants are retained in a pre-Golgi compartment. In addition, expression of Bbeta, gamma and angiopoietin-2 chimeric molecules demonstrated that the betaC domain prevents the secretion of single chains and complexes, whereas the gammaC domain allows their secretion. Our data provide new insight into the mechanisms accounting for the quality control of fibrinogen secretion and confirm that mutant fibrinogen retention is one of the pathological mechanisms responsible for congenital afibrinogenemia.

The Role of Amicar in Decreasing Perioperative Blood Loss in Idiopathic Scoliosis

Four separate studies on the role of Amicar in decreasing perioperative blood loss in idiopathic scoliosis. To assess the efficacy and possible mechanisms of action for Amicar. Preliminary prospective, randomized double-blind and analysis of same-day anterior spinal fusion (ASF), fibrinogen, and posterior spinal fusion (PSF) studies have demonstrated Amicar to be effective in idiopathic scoliosis surgery. Increased fibrinogen secretion is a possible explanation. Amicar is administered at 100 mg/kg over 15 minutes not to exceed 5 g at anesthesia induction. Maintenance is 10 mg/kg per hour until wound closure. Preliminary study: Amicar (N = 28) was effective compared with a control group (N = 31). Perioperative blood loss and transfusion following PSF were 1,604 +/- 517 mL and 1.1 +/- 1.0 U in the Amicar group compared with 2,312 +/- 994 mL and 2.1 +/- 1.1 U in the control group (P < 0.003). Prospective, randomized double-blind study confirmed this efficacy, although primarily in postoperative suction drainage: 1,391 +/- 212 mL and 1.1 +/- 1.0 U compared with 1,716 +/- 513 mL and 2.1 +/- 1.3 U (P < 0.002). A fibrinogen study (N = 21) demonstrated steady and excessive increase following PSF: before surgery it was 266 +/- 63 mg/dL and on the fifth postoperative day 699 +/- 94 mg/dL. In same-day anterior and posterior spinal surgery, Amicar was again effective, but primarily in decreasing chest tube drainage and during PSF. Group 1 (N = 15, no Amicar) 3,807 +/- 105 mL and 3.1 +/- 1.5 U; Group 2 (N = 27, Amicar for PSF only) 2,080 +/- 659 mL and 1.9 +/- 0.9 U; and Group 3 (N = 16, both ASF and PSF) 2,183 +/- 851 mL and 1.0 +/- 0.8 U. Amicar appears highly effective in decreasing perioperative blood loss. This results in less autologous blood donation, blood transfusion, costs, and complications. Its mechanism of action is uncertain but may be related to increased fibrinogen secretion.

The Severe Acute Respiratory Syndrome Coronavirus 3a Protein Up-Regulates Expression of Fibrinogen in Lung Epithelial Cells

Here we analyzed the gene expression profile of cells that stably express the severe acute respiratory syndrome coronavirus (SARS-CoV) 3a protein to determine its effects on host functions. A lung epithelial cell-line, A549, was chosen for this study because the lung is the primary organ infected by SARS-CoV and fatalities resulted mainly from pulmonary complications. Our results showed that the expression of 3a up-regulates the mRNA levels of all three subunits, Aalpha, Bbeta, and gamma, of fibrinogen. Consequently, the intracellular levels as well as the secretion of fibrinogen were increased. We also observed increased fibrinogen levels in SARS-CoV-infected Vero E6 cells.

Liver histology of an afibrinogenemic patient with the Bβ‐L353R mutation showing no evidence of hepatic endoplasmic reticulum storage disease (ERSD); comparative study in COS‐1 cells of the intracellular processing of the Bβ‐L353R fibrinogen vs. the ERSD‐associated γ‐G284R mutant

Type I fibrinogen deficiencies (hypofibrinogenemia and afibrinogenemia) are rare congenital disorders characterized by low or unmeasurable plasma fibrinogen antigen levels. Their genetic bases are represented by mutations within the three fibrinogen genes. Among the 11 reported missense mutations, a few have been characterized by expression studies and found to have an impaired fibrinogen assembly and/or secretion. Histopathological analyses were previously reported in two hypofibrinogenemic cases with discernible hepatic disease, revealing that both underlying mutations (gamma-Gly284Arg and gamma-Arg375Trp) were associated with hepatic fibrinogen endoplasmic reticulum storage disease (ERSD). The objective of this study was to investigate the liver histology in an afibrinogenemic patient, homozygous for the Bbeta-Leu353Arg mutation, and to study the intracellular processing of the mutant protein. Liver histology was evaluated by light microscopy, electron microscopy and immunocytochemistry. Intracellular processing of mutant fibrinogen was analyzed by pulse-chase labeling and immunoprecipitation experiments. Messenger RNA levels were determined by real-time reverse transcription-polymerase chain reaction (RT-PCR). The histopathological characterization of the liver showed no signs of fibrinogen accumulation, a difference from the previously reported findings in two hypofibrinogenemic kindreds with ERSD. To evaluate whether the Bbeta-Leu353Arg mutation and the ERSD-associated gamma-Gly284Arg mutation affected intracellular fibrinogen trafficking differently, both mutant proteins were expressed in COS-1 cells. Bbeta-Leu353Arg led to a more severe secretion defect, but no differences that could explain phenotype-genotype correlation were found in the intracellular processing. Endoglycosidase-H analysis demonstrated a secretion block before translocation to the Golgi medial stacks. Real-time RT-PCR studies showed normal levels of the Bbeta mRNA in the patient's liver. The results confirm that Bbeta-Leu353Arg is associated with impaired fibrinogen secretion, but not with hepatic ERSD.

In vitro expression demonstrates impaired secretion of the γAsn319, Asp320 deletion variant fibrinogen

The hypodysfibrinogenemia Otsu is caused by the two-residue deletion, gammaAsn319 and gammaAsp320. Analysis of plasma or purified fibrinogen from the heterozygous propositus revealed that the amount of variant gamma-chain was lower than that of normal gamma-chain. In order to examine the basis for this difference, we transfected Chinese hamster ovary cells and established stable cell lines that expressed both chains, gammaDelta/gammaN, only the normal chain, gammaN, and only the variant chain, gammaDelta. We measured fibrinogen concentration of confluent cultures by ELISA. We found the ratios of the concentrations in the media to the concentrations in the cell lysates of gammaDelta, gammaDelta/gammaN, and gammaN-cells were 0.42, 0.60, and 1.00, respectively. We measured the concentrations of the gammaDelta and gammaN chains by densitometric analysis of samples following separation by SDS-PAGE and found the fraction of gammaDelta-chains in cell lysates was always greater than the fraction in the respective culture media. We examined the kinetics of fibrinogen synthesis, assembly and secretion in pulse-chase experiments, and found that the gammaDelta-chain was assembled into intact fibrinogen at a rate similar to assembly of the gammaN-chain into normal fibrinogen, but was secreted into the medium at a slightly slower rate than normal fibrinogen. Considered together, these experiments indicate secretion of the variant fibrinogen was slightly impaired. These results suggest that the reduced level of gammaDelta319,320 fibrinogen in the plasma of the Otsu patient arises from modestly impaired secretion of this variant fibrinogen.

Secretion of recombinant human fibrinogen by the murine mammary gland

Transgenic animals secreting individual chains and assembled fibrinogen were produced to evaluate the capacity of the mammary gland for maximizing assembly, glycosylation and secretion of recombinant human fibrinogen (rhfib). Transgenes were constructed from the 4.1 kbp murine Whey Acidic Protein promoter (mWAP) and the three cDNAs coding for the Aalpha, Bbeta and gamma fibrinogen chains. Transgenic mice secreted fully assembled fibrinogen into milk at concentrations between 10 and 200 microg/ml, with total secretion of subunits approaching 700 microg/ml in milk. Partially purified fibrinogen was shown to form a visible and stable clot after treatment with human thrombin and factor XIII. The level of assembled fibrinogen was proportional to the lowest amount of subunit produced where both the Bbeta and gamma chains were rate limiting. Both the Bbeta and gamma chains were glycosylated when co-expressed and the degree of saccharide maturation was dependent on expression level, with processing preferred for gamma chains over Bbeta chains. Also, the subunit complexes gamma2, Aalphagamma2 and the individual subunits Aalpha, Bbeta and gamma were found as secretion products. When the Bbeta was secreted individually, the glycosylation profile of the molecule was of a mature complex saccharide indicating recognition of the molecule by the glycosylation pathway without association with other fibrinogen chains. To date secretion of Bbeta chain has been not observed in any cell type, suggesting that the secretion pathway in mammary epithelia is less restrictive than that occurring in hepatocytes and other cells previously used to study fibrinogen assembly.

Oxysterols suppress constitutive fibrinogen expression

Elevated levels of both fibrinogen and cholesterol are risk factors in coronary artery disease. Previously we reported a metabolic link between fibrinogen and lipid metabolism in that HepG2 cells that were programmed by transfection of Bbeta-fibrinogen cDNA to overexpress fibrinogen exhibited increased synthesis of cholesterol and increased secretion of apolipoprotein B. In this study we demonstrate that oxysterols, which participate in maintaining cholesterol homeostasis, also down regulate fibrinogen expression. Treatment of HepG2 cells with 25-hydroxycholesterol lowered fibrinogen Aalpha, Bbeta and gamma mRNA levels and inhibited fibrinogen synthesis and secretion but had no effect on alpha1 -antitrypsin which, like fibrinogen, is an acute-phase protein. The inhibition of fibrinogen synthesis by oxysterols was maintained in interleukin-6 treated cells. Other oxysterols, that inhibit cholesterol synthesis by a feedback mechanism, also diminished fibrinogen expression in HepG2, rat H-4-II-E hepatoma cells and in primary human hepatocytes. Overexpression of SREBP-1 and SREBP-2 by transfection of HepG2 cells, or treatment with a synthetic LXRalpha agonist, which affect cholesterol metabolism, did not affect fibrinogen expression. We conclude that fibrinogen and cholesterol may share a novel common regulatory pathway.

Long-term stable cultures of rat hepatocytes: an in vitro model to study acute and chronic hepatic inflammation.

Engineered tissues provide an opportunity to investigate important physiological processes difficult to study in whole perfused organs and animal models. For example, a hepatocyte culture model consisting of rat hepatocytes cultured in a collagen sandwich configuration, which exhibits stable differentiated liver-specific functions, may be useful to investigate liver pathophysiology. To investigate systemic inflammation-related hepatic failure, we chronically exposed hepatocytes to the inflammatory mediators interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) for up to 4 weeks. IL-6 (2.5 ng/mL) transiently suppressed albumin (-90%) and chronically increased fibrinogen (+6-fold) production. IL-6 inhibited urea synthesis at 2.5 ng/mL and stimulated it at 0.025 ng/mL. IL-1beta (10 ng/mL) inhibited albumin (-90%), urea (-40 to 50%), and IL-6-stimulated fibrinogen (-90%) secretion. The inhibitory effect of IL-1beta on urea secretion was dose-dependent. Furthermore, IL-1beta transiently stimulated nitric oxide (NO) synthesis; however, NO did not mediate the effect of IL-1beta on albumin and fibrinogen production, and played a minor role in IL-1beta-mediated urea synthesis suppression. In conclusion, IL-1beta and IL-6 exert, via a direct effect on hepatocytes, long-term inhibitory effects on hepatic functions that are potentially important for the survival of the host, which may contribute to hepatic dysfunction in prolonged inflammatory states.

Hyperfibrinogenemia in rats treated with meloxicam.

Fibrinogen secretion is mediated by prostaglandin biosynthesis and is considered a risk factor for cardiovascular disease. Since meloxicam produces inhibition of prostaglandin biosynthesis it may help to normalize hyperfibrinogenemia. We investigated the pharmacological effect of meloxicam on fibrinogen levels and the possible regression of histopathological lesions of thoracic aorta. Rats were subjected to multiple injuries (MI) in the form of laparotomies (Lx) during a 30 day period (1/week). Meloxicam 0.065 mg/kg/day (per rat) was administered orally immediately after the third Lx in multiple injury animals during a ten day period. Blood was obtained 72 hours after the last injury in all groups. Fibrinogen was measured by spectrophotometry and the values were expressed in mg/dL. A statistically significant increment of fibrinogen was observed when comparing uninjured animals (control) (208.7+/-6.0) with the multiple injury group (336.6+/-7.5) (P<0.001). Fibrinogen decreased to the control value in the meloxicam group (198+/-8.7). Histopathological lesions were similar in the MI and meloxicam groups, showing endothelial denudation and intima enlargement from the thoracic aorta in 96% of the slices studied. The decrease in fibrinogen in the meloxicam group would be due to cyclooxygenase-2 (Cox-2) selective inhibition, even though the histopathological lesions did not regress.

Congenital afibrinogenemia: mutations leading to premature termination codons in fibrinogen Aα-chain gene are not associated with the decay of the mutant mRNAs

AbstractCongenital afibrinogenemia is a rare coagulation disorder with autosomal recessive inheritance, characterized by the complete absence or extremely reduced levels of fibrinogen in patients' plasma and platelets. Eight afibrinogenemic probands, with very low plasma levels of immunoreactive fibrinogen were studied. Sequencing of the fibrinogen gene cluster of each proband disclosed 4 novel point mutations (1914C>G, 1193G>T, 1215delT, and 3075C>T) and 1 already reported (3192C>T). All mutations, localized within the first 4 exons of the Aα-chain gene, were null mutations predicted to produce severely truncated Aα-chains because of the presence of premature termination codons. Since premature termination codons are frequently known to affect the metabolism of the corresponding messenger RNAs (mRNAs), the degree of stability of each mutant mRNA was investigated. Cotransfection experiments with plasmids expressing the wild type and each of the mutant Aα-chains, followed by RNA extraction and semiquantitative reverse-transcriptase–polymerase chain reaction analysis, demonstrated that all the identified null mutations escaped nonsense-mediated mRNA decay. Moreover, ex vivo analysis at the protein level demonstrated that the presence of each mutation was sufficient to abolish fibrinogen secretion.

Differential Degradation of the Three Fibrinogen Chains by Proteasomes: Involvement of Sec61p and Cytosolic Hsp70

HepG2 cells, which synthesize and secrete fibrinogen, accumulate surplus Aα and γ chains. The nonsecreted fibrinogen chains are degraded both by proteasomes and lysosomes, with unassembled chains primarily degraded by proteasomes and an Aα–γ complex by lysosomes. To further determine the mechanisms by which unassembled fibrinogen chains are degraded, and to explain the pools of Aα and γ chains that occur in HepG2 cells, the association of fibrinogen chains with Sec61β, a component of the translocon, and with a cytosol chaperone, Hsp70, was studied in both HepG2 cells and COS cells expressing single fibrinogen chains. Retrotranslocation from the lumen of the endoplasmic reticulum was shown by treatment with MG132, a proteasome inhibitor. MG132 caused glycosylated Bβ to accumulate on Sec61β in COS cells expressing Bβ and acted similarly with all three fibrinogen chains in HepG2 cells. In HepG2 cells, Bβ was associated with Sec61β ahead of Aα and γ chains, suggesting that pools of Aα and γ chains may be caused by unequal rates of retrotranslocation. In COS cells, retrotranslocation into the cytoplasm was demonstrated by the ATP-sensitive association of ubiquitinylated Aα, Bβ, and γ chains bound to Hsp70. More Aα and γ than Bβ accumulated on Hsp70 of HepG2 cells, consistent with more rapid degradation of Bβ. Overexpression of Hsp70 in HepG2 cells resulted in decreased secretion, but not synthesis, of fibrinogen. Decreased secretion may be due to enhanced degradation of unassembled fibrinogen chains, indicating that proteolysis by proteasomes might regulate both the intracellular pools of fibrinogen chains and fibrinogen secretion.

Molecular mechanisms of hypo- and afibrinogenemia.

Point mutations responsible for hypo- and afibrinogenemia are yielding new insights into amino acid side chains involved in the molecular processing, assembly, secretion, and domain stability of fibrinogen. Reverse phase chromatography, isoelectric focussing, electrospray mass spectrometry, and tryptic peptide mass mapping have shown that chains with heterozygous mutations of gamma 284 Gly-->Arg, B beta 316 Asp-->Tyr and gamma 371 Thr-->Ile are absent from plasma fibrinogen. The nonexpression of these mutations appears to result from perturbation of the five-stranded beta sheet of the D domain. We propose that this is due to retention of the variant in the endoplasmic reticulum and that in turn this leads to hypofibrinogenemia. Other mutations effect intracellular proteolysis and chain assembly. For example the mutation, A alpha 20 Val-->Asp, makes the protein a substrate for furin, which removes the first 19 residues of the A alpha chain as the mature molecule transits the trans golgi complex. Transient expression of gamma 153 Cys-->Arg chains together with A alpha and B beta chains suggests this mutation might perturb chain assembly, and the incorporation of mutations of B beta 353 Leu-->Arg or B beta 400 Gly-->Asp into intracellular fibrinogen precludes its subsequent export from host cells expressing fibrinogen genes. The graded severity of the hypo- and afibrinogenemias associated with homozygous A alpha chain truncations suggest the absolute minimal requirement for molecular assembly is the formation of the C terminal disulfide ring of the coiled coil.

Homozygous truncation of the fibrinogen Aα chain within the coiled coil causes congenital afibrinogenemia

AbstractThe molecular basis of a novel congenital afibrinogenemia has been determined. The proposita, the only affected member in a consanguineous Norwegian family, suffers from a moderate to severe bleeding disorder due to the total absence of any detectable fibrinogen. Dot blots of solubilized platelets revealed a small amount of γ chain but no Aα or Bβ chains, whereas no chains were detected in plasma dot blots. DNA sequencing of the Aα chain gene revealed a homozygous C→T transversion 557 nucleotides from the transcription initiation site. This nucleotide change predicts the nonsense mutation Aα 149 Arg (CGA)→stop (TGA). Early truncation of the Aα chain appears to result in defective assembly or secretion of fibrinogen, probably due to the removal of the C-terminal disulfide ring residues that are critically required for the formation of a stable 3-chained half molecule.

Homozygous truncation of the fibrinogen Aα chain within the coiled coil causes congenital afibrinogenemia

The molecular basis of a novel congenital afibrinogenemia has been determined. The proposita, the only affected member in a consanguineous Norwegian family, suffers from a moderate to severe bleeding disorder due to the total absence of any detectable fibrinogen. Dot blots of solubilized platelets revealed a small amount of γ chain but no A or Bβ chains, whereas no chains were detected in plasma dot blots. DNA sequencing of the A chain gene revealed a homozygous C→T transversion 557 nucleotides from the transcription initiation site. This nucleotide change predicts the nonsense mutation A 149 Arg (CGA)→stop (TGA). Early truncation of the A chain appears to result in defective assembly or secretion of fibrinogen, probably due to the removal of the C-terminal disulfide ring residues that are critically required for the formation of a stable 3-chained half molecule.

Missense mutations in the human β fibrinogen gene cause congenital afibrinogenemia by impairing fibrinogen secretion

Congenital afibrinogenemia is a rare autosomal recessive disorder characterized by bleeding that varies from mild to severe and by complete absence or extremely low levels of plasma and platelet fibrinogen. Although several mutations in the fibrinogen genes associated with dysfibrinogenemia and hypofibrinogenemia have been described, the genetic defects of congenital afibrinogenemia are largely unknown, except for a recently reported 11-kb deletion of the fibrinogen Aalpha-chain gene. Nevertheless, mutation mechanisms other than the deletion of a fibrinogen gene are likely to exist because patients with afibrinogenemia showing no gross alteration within the fibrinogen cluster have been reported. We tested this hypothesis by studying the affected members of two families, one Italian and one Iranian, who had no evidence of large deletions in the fibrinogen genes. Sequencing of the fibrinogen genes in the 2 probands detected 2 different homozygous missense mutations in exons 7 and 8 of the Bbeta-chain gene, leading to amino acid substitutions Leu353Arg and Gly400Asp, respectively. Transient transfection experiments with plasmids expressing wild-type and mutant fibrinogens demonstrated that the presence of either mutation was sufficient to abolish fibrinogen secretion. These findings demonstrated that missense mutations in the Bbeta fibrinogen gene could cause congenital afibrinogenemia by impairing fibrinogen secretion. (Blood. 2000;95:1336-1341)

Hypofibrinogenemia Associated With a Heterozygous Missense Mutation γ153Cys to Arg (Matsumoto IV): In Vitro Expression Demonstrates Defective Secretion of the Variant Fibrinogen

We genetically analyzed a case of hypofibrinogenemia that showed no bleeding or thrombotic tendency. Direct sequencing of a polymerase chain reaction-amplified γ-chain gene segment showed a novel nucleotide substitution. This heterozygous mutation encodes both Cys (TGT) and Arg (CGT) at residue 153. To examine the basis for the fibrinogen deficiency, we prepared expression vectors containing mutant γ-chain DNAs encoding γ153R and γ153A for in vitro expression in Chinese hamster ovary (CHO) cells. Enzyme-linked immunosorbent assay and immunoblot analysis of the culture media and cell lysates showed that CHO cells transfected with γ153R or γ153A synthesized the variant γ-chain, but did not secrete variant fibrinogen into the culture medium. Metabolic pulse-chase experiments showed that fibrinogen assembly was impaired when either variant γ-chain was expressed. In cells expressing normal fibrinogen, assem- bly intermediates and intact fibrinogen were seen in cell lysates prepared after short (3 minutes) or long (1 hour) incubation with 35S-methionine. Neither intermediates nor intact fibrinogen was seen with the variant γ-chains. These data suggest that γ-chains have an important early role in fibrinogen assembly. Thus, our results support the model for fibrinogen assembly proposed by Huang et al (J Biol Chem 268:8919, 1993), in which the first step in assembly is the formation of γ or βγ dimers, or both. This model implies that γCys153 has a critical role in the formation of these early assembly intermediates. We concluded that the γ153Cys→Arg substitution does not allow fibrinogen assembly and secretion, and this is manifest in vivo as a fibrinogen deficiency. We designated this variant as fibrinogen Matsumoto IV.