Recombinant γY278H Fibrinogen Showed Normal Secretion from CHO Cells, but a Corresponding Heterozygous Patient Showed Hypofibrinogenemia.

We identified a novel heterozygous hypofibrinogenemia, γY278H (Hiroshima). To demonstrate the cause of reduced plasma fibrinogen levels (functional level: 1.12 g/L and antigenic level: 1.16 g/L), we established γY278H fibrinogen-producing Chinese hamster ovary (CHO) cells. An enzyme-linked immunosorbent assay demonstrated that synthesis of γY278H fibrinogen inside CHO cells and secretion into the culture media were not reduced. Then, we established an additional five variant fibrinogen-producing CHO cell lines (γL276P, γT277P, γT277R, γA279D, and γY280C) and conducted further investigations. We have already established 33 γ-module variant fibrinogen-producing CHO cell lines, including 6 cell lines in this study, but only the γY278H and γT277R cell lines showed disagreement, namely, recombinant fibrinogen production was not reduced but the patients' plasma fibrinogen level was reduced. Finally, we performed fibrinogen degradation assays and demonstrated that the γY278H and γT277R fibrinogens were easily cleaved by plasmin whereas their polymerization in the presence of Ca2+ and "D:D" interaction was normal. In conclusion, our investigation suggested that patient γY278H showed hypofibrinogenemia because γY278H fibrinogen was secreted normally from the patient's hepatocytes but then underwent accelerated degradation by plasmin in the circulation.

Mutations Causing Mild or No Structural Damage in Interfaces of Multimerization of the Fibrinogen γ-Module More Likely Confer Negative Dominant Behaviors.

Different pathogenic variants in the same protein or even within the same domain of a protein may differ in their patterns of disease inheritance, with some of the variants behaving as negative dominant and others as autosomal recessive mutations. Here is presented a structural analysis and comparison of the molecular characteristics of the sites in fibrinogen γ-module, a fibrinogen component critical in multimerization processes, targeted by pathogenic variants (HGMD database) and by variants found in the healthy population (gnomAD database). The main result of this study is the identification of the molecular pathogenic mechanisms defining which pattern of disease inheritance is selected by mutations at the crossroad of autosomal recessive and negative dominant modalities. The observations in this analysis also warn about the possibility that several variants reported in the non-pathogenic gnomAD database might indeed be a hidden source of diseases with autosomal recessive inheritance or requiring a combination with other disease-causing mutations. Disease presentation might remain mostly unrevealed simply because the very low variant frequency rarely results in biallelic pathogenic mutations or the coupling with mutations in other genes contributing to the same disease. The results here presented provide hints for a deeper search of pathogenic mechanisms and modalities of disease inheritance for protein mutants participating in multimerization phenomena.

Fibrinogen Columbus II: A novel c.1075G>T mutation in the FGG gene causing hypodysfibrinogenemia and thrombosis in an adolescent male.

Hypodysfibrinogenemia, the least frequently reported congenital fibrinogen disorder is characterized by low circulating levels of a dysfunctional protein, and is associated with phenotypic features of both hypo- and dysfibrinogenemia. Herein, we report an adolescent male with unprovoked venous thromboembolism and hypodysfibrinogenemia. Patient had recurrent, progressive thrombosis despite therapeutic anticoagulation with both low molecular weight heparin and warfarin. He had clinical and radiological improvement after transition to a direct thrombin inhibitor. Sequencing of the FGG gene identified a novel heterozygous mutation, c.1075G>T. Structural visualization of the identified variant was pursued and suggested that the mutation likely destabilizes the Ca2+ -binding site of fibrinogen resulting in pathogenicity.

Role of electrostatic interactions in binding of thrombin to the fibrinogen γ' chain.

Thrombin binds to the highly anionic fibrinogen γ' chain through anion-binding exosite II. This binding profoundly alters thrombin's ability to cleave substrates, including fibrinogen, factor VIII, and PAR1. However, it is unknown whether this interaction is due mainly to general electrostatic complementarity between the γ' chain and exosite II or if there are critical charged γ' chain residues involved. We therefore systematically determined the contribution of negatively charged amino acids in the γ' chain, both individually and collectively, to thrombin binding affinity. Surface plasmon resonance binding experiments were performed using immobilized γ' chain peptides with charged-to-uncharged amino acid substitutions, i.e., Asp to Asn, Glu to Gln, and pTyr to Tyr. Individually, the substitution of uncharged for charged amino acids resulted in only minor changes in binding affinity, with a maximal change in K(d) from 0.440 to 0.705 µM for the Asp419Asn substitution. However, substitution of all three charged amino acids in a conserved ß-turn that is predicted to contact thrombin, pTyr418Tyr, Asp419Asn, and pTyr422Tyr, resulted in the loss of measurable binding, as did substitution of all the flanking charged amino acids. In addition, the binding of the γ' chain to thrombin was weakened in a dose-dependent manner with increasing NaCl concentration, resulting in a net loss of three or four ion pairs between thrombin and the γ' chain. Therefore, although each of the individual charges in the γ' chain contributes only incrementally to the overall binding affinity, the ensemble of the combined charges plays a profound role in the thrombin-γ' chain interactions.

Histidine-rich glycoprotein binds fibrin(ogen) with high affinity and competes with thrombin for binding to the gamma'-chain.

Histidine-rich glycoprotein (HRG) is an abundant protein that binds fibrinogen and other plasma proteins in a Zn(2+)-dependent fashion but whose function is unclear. HRG has antimicrobial activity, and its incorporation into fibrin clots facilitates bacterial entrapment and killing and promotes inflammation. Although these findings suggest that HRG contributes to innate immunity and inflammation, little is known about the HRG-fibrin(ogen) interaction. By immunoassay, HRG-fibrinogen complexes were detected in Zn(2+)-supplemented human plasma, a finding consistent with a high affinity interaction. Surface plasmon resonance determinations support this concept and show that in the presence of Zn(2+), HRG binds the predominant γ(A)/γ(A)-fibrinogen and the γ-chain elongated isoform, γ(A)/γ'-fibrinogen, with K(d) values of 9 nm. Likewise, (125)I-labeled HRG binds γ(A)/γ(A)- or γ(A)/γ'-fibrin clots with similar K(d) values when Zn(2+) is present. There are multiple HRG binding sites on fibrin(ogen) because HRG binds immobilized fibrinogen fragment D or E and γ'-peptide, an analog of the COOH terminus of the γ'-chain that mediates the high affinity interaction of thrombin with γ(A)/γ'-fibrin. Thrombin competes with HRG for γ'-peptide binding and displaces (125)I-HRG from γ(A)/γ'-fibrin clots and vice versa. Taken together, these data suggest that (a) HRG circulates in complex with fibrinogen and that the complex persists upon fibrin formation, and (b) by competing with thrombin for γ(A)/γ'-fibrin binding, HRG may modulate coagulation. Therefore, the HRG-fibrin interaction may provide a novel link between coagulation, innate immunity, and inflammation.

The pleiotropic role of the fibrinogen gamma' chain in hemostasis.

A fraction of fibrinogen contains a differently spliced gamma chain called gamma', which presents itself mainly as heterodimer with the common gammaA chain as gammaA/gamma' fibrinogen. The gamma' chain differs from the gammaA chain in its C-terminus and has important functional implications for fibrinogen. The presence of the gamma' chain modulates thrombin and FXIII activity, influences clot architecture, and eliminates a platelet-binding site. Associations of gammaA/gamma' fibrinogen levels with arterial and venous thrombosis have been reported, indicating that the functional effects of gammaA/gamma' fibrinogen may contribute to the pathology of thrombosis. This review summarizes the key biologic aspects of this interesting variant of fibrinogen and discusses inconsistencies in current reports.

Thermal shift assay to probe melting of thrombin, fibrinogen, fibrin monomer, and fibrin: Gly-Pro-Arg-Pro induces a fibrin monomer-like state in fibrinogen.

BACKGROUND: Thrombin activates fibrinogen and binds the fibrin E-domain (Kd ~ 2.8 µM) and the splice variant γ'-domain (Kd ~ 0.1 µM). We investigated if the loading of D-Phe-Pro-Arg-chloromethylketone inhibited thrombin (PPACK-thrombin) onto fibrin could enhance fibrin stability. METHODS: A 384-well plate thermal shift assay (TSA) with SYPRO-orange provided melting temperatures (Tm) of thrombin, PPACK-thrombin, fibrinogen, fibrin monomer, and fibrin. RESULTS: Large increases in Tm indicated that calcium led to protein stabilization (0 vs. 2 mM Ca2+) for fibrinogen (54.0 vs. 62.3 °C) and fibrin (62.3 vs. 72.2 °C). Additionally, active site inhibition with PPACK dramatically increased the Tm of thrombin (58.3 vs. 78.3 °C). Treatment of fibrinogen with fibrin polymerization inhibitor GPRP increased fibrinogen stability by ΔTm = 9.3 °C, similar to the ΔTm when fibrinogen was converted to fibrin monomer (ΔTm = 8.8 °C) or to fibrin (ΔTm = 10.4 °C). Addition of PPACK-thrombin at high 5:1 M ratio to fibrin(ogen) had little effect on fibrin(ogen) Tm values, indicating that thrombin binding does not detectably stabilize fibrin via a putative bivalent E-domain to γ'-domain interaction. CONCLUSIONS: TSA was a sensitive assay of protein stability and detected: (1) the effects of calcium-stabilization, (2) thrombin active site labeling, (3) fibrinogen conversion to fibrin, and (4) GPRP induced changes in fibrinogen stability being essentially equivalent to that of fibrin monomer or polymerized fibrin. SIGNIFICANCE: The low volume, high throughput assay has potential for use in understanding interactions with rare or mutant fibrin(ogen) variants.

[Assembly and secretion of mutant fibrinogens with variant gamma-chain C terminal region (gamma313-gamma345)].

BACKGROUND: It has been reported that the structure of the fibrinogen gamma-chain C terminal (D) region (140-411 residues) has important functions in fibrinogen assembly and/or secretion. Variant fibrinogens, gamma313S>N, gamma336M>I, gamma341A>D, and gamma345N>D have been reported as hypofibrinogenemias or dysfibrinogenemias. To study the assembly and secretion of the variant fibrinogens containing aberrant D regions, we established CHO cells producing these four fibrinogens. METHODS: A fibrinogen gamma-chain expression vector was altered and transfected into CHO cells that expressed normal human fibrinogen Aalpha and Bbeta-chains. Cell lysates and culture media of the selected cell lines were subjected to ELISA and immunoblot analysis. RESULTS: The CHO cells synthesized mutant gamma-chains and assembled these into fibrinogen, although these variant fibrinogens were barely secreted into the culture media. In the cell lysates, however, concentrations of these variant fibrinogens were higher than the normal levels. CONCLUSIONS: The present study indicated that the tertiary structure of the fibrinogen gamma-chain C terminal region between 313 and 345 is necessary for fibrinogen secretion. These findings suggest that reduced levels of fibrinogen secretion lead to the hypofibrinogen in the patients and secreted fibrinogens might show dysfibrinogenemia.

Heterozygous variant fibrinogen γA289V (Kanazawa III) was confirmed as hypodysfibrinogenemia by plasma and recombinant fibrinogens.

INTRODUCTION: Congenital fibrinogen disorders are classified as afibrinogenemia, hypofibrinogenemia, dysfibrinogenemia, and hypodysfibrinogenemia. However, difficulties are associated with discriminating between dysfibrinogenemia, hypofibrinogenemia, and hypodysfibrinogenemia using routine analyses. We previously reported a heterozygous variant fibrinogen (γA289V; Kanazawa III) as hypodysfibrinogenemia; however, the same variant had previously been described as hypofibrinogenemia. To clarify the production of γA289V fibrinogen, we expressed recombinant γA289V (r-γA289V) fibrinogen and compared it with wild-type (WT) and adjacent recombinant variant fibrinogens. METHODS: Target mutations were introduced into a fibrinogen γ-chain expression vector by site-directed mutagenesis, and the vector was then transfected into Chinese hamster ovary cells to produce recombinant fibrinogen. Fibrinogen was purified from the plasma of the proposita, and culture media and fibrinogen functions were analyzed using fibrin polymerization, plasmin protection, and FXIIIa-catalyzed fibrinogen cross-linking. RESULTS: The fibrinogen concentration ratio of the culture media to cell lysates was markedly lower for r-γA289V fibrinogen than for WT. Because the secretion of recombinant γF290L (r-γF290L) fibrinogen was similar to WT, we compared r-γF290L fibrinogen functions with WT. The fibrin polymerization of Kanazawa III plasma (K-III) fibrinogen was significantly weaker than normal plasma fibrinogen. Moreover, K-III fibrinogen showed a markedly reduced "D:D" interaction. However, all functions of r-γF290L fibrinogen were similar to WT. An in silico analysis confirmed the above results. CONCLUSION: The present results demonstrated that γA289 is crucial for the γ-module structure, and the γA289V substitution markedly reduced fibrinogen secretion. Moreover, K-III fibrinogen showed markedly reduced fibrin polymerization and "D:D" interactions. γA289V fibrinogen was confirmed as hypodysfibrinogenemia.

Demonstration of heterodimeric fibrinogen molecules partially conjugated with albumin in a novel dysfibrinogen: fibrinogen Mannheim V.

A 30-year-old female experienced three miscarriages in early pregnancy. Extensive laboratory screening showed a low plasma fibrinogen level of approximately l g/l detected by PT-derived fibrinogen assay. The fibrinogen level in the immunological assay was 3 g/l. The functional Clauss assay yielded an intermediate result of 1.78 g/l. During her fourth and fifth pregnancy, the patient received fibrinogen concentrates (Haemocomplettan, CLS Behring, Marburg, Germany), starting with 4 grams of human fibrinogen, followed by 2 grams every second day until the 15(th) week of pregnancy. The further course of these pregnancies was uneventful. SDS-PAGE and immunoblotting showed doublet bands in the positions of the high-molecular weight (HMW)- and low-molecular-weight (LMW)-fibrinogen, a single LMW' fibrinogen band, plus additional bands with higher molecular weight than HMW-fibrinogen, which were also reactive with anti-human serum albumin (HSA) antiserum. These bands correspond to variant fibrinogen conjugated with albumin. Reduced SDS-PAGE and immunoblotting using polyclonal anti-fibrinopeptide A antiserum disclosed one additional Aalpha-chain band with lower molecular weight. Amplification and sequencing of exon 5 of the alpha gene indicated heterozygosity for a novel single nucleotide deletion at codon Aalpha494 (C1537delA). His494 is replaced by Pro and this is followed by 23 (LMKLPSSTLPQLEKHSQ VSSHLC) new amino acids before premature truncation after Cys517, yielding a free C-terminal cysteine, which may link with albumin. This new fibrinogen mutation, leads to a balanced array of homo- and heterodimeric fibrinogen molecules, some of which are conjugated to albumin.

Deletion of five residues from the coiled coil of fibrinogen (Bbeta Asn167_Glu171del) associated with bleeding and hypodysfibrinogenemia.

Routine pre-surgical coagulation investigations led to the detection of a novel type of hypodysfibrinogenemia whose functional defect appears to result from an alteration in the spacing between the functional domains of the fibrinogen molecule. The detection, by reverse phase HPLC, of a minor isoform of Bbeta chain with a 554 Da decrease in mass led to the identification of a deletion of five amino acids (NVVNE) from the center of the coiled coil. The variant chain contributed only 10% of the total Bbeta material and the mutation (BbetaAsn167_Glu171del) was associated with both increased clotting times and low functional and physical fibrinogen concentrations in 3 family members. There was a significant history of pregnancy-associated bleeding and miscarriage within the first trimester. Mechanistically the 15-nucleotide deletion appears to arise from replication advancement during DNA synthesis caused by a flanking pentanucleotide repeat of AATGA.

[Functional analysis of heterozygous plasma dysfibrinogens derived from two families of gammaArg275Cys and three families of gammaArg275His, and haplotype analysis for these families].

We have identified five heterozygous dysfibrinogenemias, two families with variant fibrinogen gammaArg275Cys (CGC > TGC; Matsumoto III and Sendai) and three families with gammaArg275His (CGC > CAC; Otsu II, Iida, and Shizuoka), from PCR-amplified DNA fragments and direct sequence analysis. gammaArg275 is the most important residue in fibrinogen for the so-called "D-D interface" in protofibril elongation. We compared the functions of plasma fibrinogen purified from affected family members with gammaArg275Cys and gammaArg275His. Both fibrinogens showed markedly impaired thrombin-catalyzed fibrin polymerization in comparison with normal controls. The degree of impairment of gammaArg275Cys fibrinogen was greater than that of gammaArg275His. These results were consistent with the fibrinogen concentration ratio (thrombin time method/immunological method). That is, the ratio of gammaArg275Cys was significantly lower than that of gammaArg275His. Moreover, scanning electron microscopy indicated significantly thicker fibers in fibrin clots made from gammaArg275Cys than in those of normal controls or gammaArg275His, and abnormal bundles with tapered ends. Factor XIIIa-catalyzed cross-linking of the fibrinogen gamma-chain (in the absence of thrombin) showed a similar delay for gammaArg275Cys and gammaArg275His. We report markedly impaired fibrin polymerization of gammaArg275Cys compared to gammaArg275His, and speculate that the difference is due to the disulfide-linked Cys in gammaArg275Cys, as we have already demonstrated for plasma and recombinant mutant fibrinogens. These results also indicate that an amino acid substitution of gammaArg275 disrupts D:D interactions in fibrin fiber formation. Furthermore haplotype analysis for three families with gammaArg275His suggested that founder of Iida family might be different from that of Otsu II or Shizuoka family.

Polymerization-defective fibrinogen variant gammaD364A binds knob "A" peptide mimic.

Fibrin polymerization is supported in part by interactions called "A:a". Crystallographic studies revealed gamma364Asp is part of hole "a" that interacts with knob "A" peptide mimic, GPRP. Biochemical studies have shown gamma364Asp is critical to polymerization, as polymerization of variants gammaD364A, gammaD364H, and gammaD364V is exceptionally impaired. To understand the molecular basis for the aberrant function, we solved the crystal structure of fragment D from gammaD364A. Surprisingly, the structure (rfD-gammaD364A+GP) showed near normal "A:a" interactions with GPRP bound to hole "a" and no change in the overall structure of gammaD364A. Of note, inspection of the structure showed negative electrostatic potential inside hole "a" was diminished by this substitution. We examined GPRP binding to the gamma364Asp variants in solution by plasmin protection assay. We found no protection of either gammaD364H or gammaD364V but partial protection of gammaD364A, indicating the peptide does not bind to either gammaD364H or gammaD364V and binds more weakly than normal to gammaD364A. We also examined protection by calcium and found all variants were indistinguishable from normal, suggesting the global structures of the variants are not markedly different from normal. Our data imply that gamma364Asp per se is not required for knob "A" binding to hole "a"; rather, this residue's negative charge has a critical role in the electrostatic interactions that facilitate the important first step in fibrin polymerization.

Impact of γ'γ' fibrinogen interaction with red blood cells on fibrin clots.

AIM: γ' fibrinogen has been associated with thrombosis. Here the interactions between γ'γ' or γAγA fibrinogen and red blood cells (RBCs), and their role on fibrin clot properties were studied. MATERIALS & METHODS: Atomic Force microscopy (AFM)-based force spectroscopy, rheological, electron and confocal microscopy, and computational approaches were conducted for both fibrinogen variants. RESULTS & CONCLUSION: AFM shows that the recombinant human (rh)γ'γ' fibrinogen increases the binding force and the frequency of the binding to RBCs compared with rhγAγA, promoting cell aggregation. Structural changes in rhγ'γ' fibrin clots, displaying a nonuniform fibrin network were shown by microscopy approaches. The presence of RBCs decreases the fibrinolysis rate and increases viscosity of rhγ'γ' fibrin clots. The full length of the γ' chain structure, revealed by computational analysis, occupies a much wider surface and is more flexible, allowing an increase of the binding between γ' fibers, and eventually with RBCs.

The relationship between the fibrinogen D domain self-association/cross-linking site (gammaXL) and the fibrinogen Dusart abnormality (Aalpha R554C-albumin): clues to thrombophilia in the "Dusart syndrome".

Cross-linking of fibrinogen at its COOH-terminal gamma chain cross-linking site occurs in the presence of factor XIIIa due to self-association at a constitutive D domain site ("gammaXL"). We investigated the contribution of COOH-terminal regions of fibrinogen Aalpha chains to the gammaXL site by comparing the gamma chain cross-linking rate of intact fibrinogen (fraction I-2) with that of plasma fraction I-9, plasmic fraction I-9D, and plasmic fragment D1, which lack COOH-terminal Aalpha chain regions comprising approximately 100, approximately 390, and 413 residues, respectively. The cross-linking rates were I-2 > I-9 > 1-9D = D1, and indicated that the terminal 100 or more Aalpha chain residues enhance gammaXL site association. Fibrinogen Dusart, whose structural abnormality is in the COOH-terminal "alphaC" region of its Aalpha chain (Aalpha R554C-albumin), is associated with thrombophilia ("Dusart Syndrome"), and is characterized functionally by defective fibrin polymerization and clot structure, and reduced plasminogen binding and tPA-induced fibrinolysis. In the presence of XIIIa, the Dusart fibrinogen gamma chain cross-linking rate was about twice that of normal, but was normalized in proteolytic fibrinogen derivatives lacking the Aalpha chain abnormality, as was reduced plasminogen binding. Electron microscopy showed that albumin-bound Dusart fibrinogen "alphaC" regions were located in the vicinity of D domains, rather than at their expected tethered location near the fibrinogen E domain. In addition, there was considerable fibrinogen aggregation that was attributable to increased intermolecular COOH-terminal Aalpha chain associations promoted by untethered Dusart fibrinogen aC domains. We conclude that enhanced Dusart fibrinogen self-assembly is mediated through its abnormal alphaC domains, leads to increased gammaXL self-association and gamma chain cross-linking potential, and contributes to the thrombophilia that characterizes the "Dusart Syndrome."

The role of fibrinogen D domain intermolecular association sites in the polymerization of fibrin and fibrinogen Tokyo II (gamma 275 Arg-->Cys).

Intermolecular end-to-middle domain pairing between a thrombin-exposed 'A' polymerization site in the central 'E' domain of fibrin, and a constitutive complementary 'a' site in each outer 'D' domain ('D:E'), is necessary but not alone sufficient for normal fibrin assembly, as judged from previous studies of a congenital dysfibrinogen, Tokyo II (gamma 275 arg-->cys), which showed defective fibrin clot assembly and a normal D:E interaction (Matsuda, M., M. Baba, K. Morimoto, and C. Nakamikawa, 1983. J. Clin. Invest. 72:1034-1041). In addition to the 'a' polymerization site, two other constitutive intermolecular association sites on fibrinogen D domains have been defined: between gamma chain regions containing the carboxy-terminal factor XIIIa crosslinking site ('gamma XL:gamma XL'); and between sites located at the outer ends of each molecule ('D:D') (Mosesson, M. W., K. R. Siebenlist, J. F. Hainfeld, and J. S. Wall, manuscript submitted for publication). We evaluated the function of these sites in Tokyo II fibrinogen, and confirmed that there was a normal fibrin D:E interaction, as determined from a normal fibrin crosslinking rate in the presence of factor XIIIa. We also found a normal gamma XL: gamma XL interaction, as assessed by a normal fibrinogen crosslinking rate. Judging from electron microscopic images, factor XIIIa-crosslinked Tokyo II fibrinogen failed to form elongated double-stranded fibrils like normal fibrinogen. Instead, it formed aggregated disordered collections of molecules, with occasional short fibrillar segments. In addition, Tokyo II fibrin formed an abnormal, extensively branched clot network containing many tapered terminating fibers. These findings indicate that the Tokyo II fibrinogen defect results in a functionally abnormal D:D self-association site, and that a normal D:D site interaction is required, in addition to D:E, for normal fibrin or fibrinogen assembly.

Molecular basis for fibrinogen Dusart (A alpha 554 Arg-->Cys) and its association with abnormal fibrin polymerization and thrombophilia.

The molecular defect in the abnormal fibrinogen Dusart (Paris V) that is associated with thrombophilia was determined by sequence analysis of genomic DNA that had been amplified using the polymerase chain reaction. The propositus was heterozygous for a single base change (C-->T) in the A alpha-chain gene, resulting in the amino acid substitution A alpha 554 Arg-->Cys. Restriction analysis of the amplified DNA derived from the family members showed that his father and his two sons were also heterozygous. Electron microscopic studies on fibrin formed from purified fibrinogen Dusart demonstrated fibers that were much thinner than in normal fibrin. In contrast to the previously observed defective binding of plasminogen, the binding of thrombospondin to immobilized fibrinogen Dusart was similar to that of normal fibrinogen. Immunoblot analysis of plasma fibrinogen demonstrated that a substantial part of the fibrinogen Dusart molecules were disulfide-linked to albumin. The plasma of the affected family members also contained fibrinogen-albumin complexes. Furthermore, small amounts of high molecular weight complexes containing fibrinogen were detected in all the heterozygous individuals. These data indicate that the molecular abnormality in fibrinogen Dusart (A alpha 554 Arg-->Cys) results in defective lateral association of the fibrin fibers and disulfide-linked complex formation with albumin, and is associated with a family history of recurrent thrombosis in the affected individuals.

Aberrant hepatic processing causes removal of activation peptide and primary polymerisation site from fibrinogen Canterbury (A alpha 20 Val --> Asp).

A novel mechanism of molecular disease was uncovered in a patient with prolonged thrombin time and a mild bleeding tendency. DNA sequencing of the fibrinogen A alpha chain indicated heterozygosity for a mutation of 20 Val --> Asp. The molar ratio of fibrinopeptide A to B released by thrombin was substantially reduced at 0.64 suggesting either impaired cleavage or that the majority of the variant alpha chains lacked the A peptide. The latter novel proposal arises from the observation that the mutation changes the normal 16R G P R V20 sequence to R G P R D creating a potential furin cleavage site at Arg 19. Synthetic peptides incorporating both sequences were tested as substrates for both thrombin and furin. There was no substantial difference in the thrombin catalyzed cleavage. However, the variant peptide, but not the normal, was rapidly cleaved at Arg 19 by furin. Predictably intracellular cleavage of the Aalpha-chain at Arg 19 would remove fibrinopeptide A together with the G P R polymerisation site. This was confirmed by sequence analysis of fibrinogen Aalpha chains after isolation by SDS-PAGE. The expected normal sequence was detected together with a new sequence (D V E R H Q S A-) commencing at residue 20. Truncation was further verified by nonreducing SDS-PAGE of the NH2-terminal disulfide knot which indicated the presence of aberrant homo- and heterodimers.

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.