Deletion of five residues from the coiled coil of fibrinogen (B{beta} Asn167_Glu171del) associated with bleeding and hypodysfibrinogenemia

Routine pre-surgical coagulation investigations led to the detectionof a novel type of hypodysfibrinogenemia whose functional defectappears to result from an alteration in the spacing betweenthe functional domains of the fibrinogen molecule. The detection,by reverse phase HPLC, of a minor isoform of Bβ chain witha 554 Da decrease in mass led to the identification of a deletionof five amino acids (NVVNE) from the center of the coiled coil.The variant chain contributed only 10% of the total Bβmaterial and the mutation (BβAsn167_Glu171del) was associatedwith both increased clotting times and low functional and physicalfibrinogen concentrations in 3 family members. There was a significanthistory of pregnancy-associated bleeding and miscarriage withinthe first trimester. Mechanistically the 15-nucleotide deletionappears to arise from replication advancement during DNA synthesiscaused by a flanking pentanucleotide repeat of AATGA.

Key words: fibrinogen, amino acid deletion, hypodysfibrinogenemia, protein expression, replication slippage.

Introduction

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Introduction

The fibrinogen molecule is a symmetrical dimer with each halfcomposed of three polypeptide chains (A ${alpha}$ , Bβ and ${gamma}$ ) linkedby a network of disulfide bonds to form the circulating 340kDa protein.^1,2 The molecule has a linear trinodal DE-D structurewith a central E domain joined to outer D domains by a coiledcoil of all three chains. Upon thrombin activation, the centralE domain of one fibrin monomer binds to the outer D domainsof two adjacent molecules forming a protofibril of half staggeredsubunits.

As well as being the focal point of the coagulation cascade,fibrinogen participates in other important processes such aswound healing and the maintenance of pregnancy. Individualswith very low fibrinogen levels encounter umbilical cord bleeding,spontaneous abortions, central nervous system bleeding and softtissue hemorrhage.³ Low levels of fibrinogen are associatedwith recurrent abortions, usually in the 6–8^th week ofpregnancy. For example, six consecutive early abortions wereassociated with fibrinogen Keokuk (A ${alpha}$ truncation at residue 328),and the woman with fibrinogen Otago (A ${alpha}$ truncation at residue271) suffered four first trimester miscarriages.^4,5 As in these2 cases, the presence of some dysfunctional fibrinogen moleculescan further exacerbate bleeding⁵ and contribute to thromboticcomplications,⁴ or lead to additional pathologies such as renalamyloidosis.⁶ Mutations causing dysfibrinogenemia are usuallyclustered to functionally important regions such as the thrombincleavage site in the E domain or the GPR-binding site in the ${gamma}$ D domain. However remote mutations that cause structural deformationscould alter function by disrupting the precise domain alignmentsneeded for effective intermolecular interaction. Here we identifya novel deletion in the fibrinogen β gene that is associatedwith miscarriage and post-partum hemorrhage. The deletion offive amino acids from the center of the coiled coil resultsin impaired fibrin polymerization and enhanced clot lysis.

Design and Methods

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Design and Methods

Protein purification and analysis
Fibrinogen was purified by ammonium sulphate precipitation⁷and individual chains were isolated on a (25x0.46 cm) C-4 columnusing a 0.05% TFA/acetonitrile gradient system.⁸

Electrospray ionization mass spectrometry was performed as previouslyoutlined.⁹ Peak crests (20 µL) from reverse phase purifiedchains were directly injected into the ion source at a flowrate of 10 µL/min. The probe was charged at + 3500 V andthe source maintained at 60°C. The mass range 800–1600m/z was scanned every 2s with a cone voltage ramp of 30–60V and the raw data was transformed using Max-Ent software.⁹Individual fibrinogen chains were digested with trypsin andm/z mapped as previously described.⁸

DNA analysis
The entire coding and flanking intronic sequences of all threefibrinogen genes were amplified^10,11 and sequencing was carriedout on an ABI 3130xl genetic analyzer using BigDye Terminatorv. 3.1 cycle sequencing (Applied Biosystems, Foster City, CA,USA). Data was analyzed using SeqScape v. 2.5 software.

Functional studies
Thrombin catalyzed polymerization kinetics were determined intriplicate as previously described.¹² Fibrinolysis curves weregenerated by overlaying the resulting clots with 10 nM tissueplasminogen activator and 0.05 mg/mL Glu-plasminogen.¹²

Also, purified fibrinogen (100 µg) was incubated with1 mU of porcine plasmin and the digestion profile analyzed on7.5% reducing SDS PAGE. Bands corresponding to the Y and D1fragments were quantified by densitometry.

Results and Discussion

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Results and Discussion

The proband, a 20-year-old male, had a coagulation screen priorto wisdom teeth extraction. This showed normal liver function,platelet levels and bleeding time (6.5 min). However, APTT andINR were slightly raised at 41s and 1.3 respectively. Functionalfibrinogen (Clauss) was low at 0.6 mg/mL (normal 1.5–4.0)and further testing showed prolonged thrombin and reptilasetimes of 24 sec each (normal 10–18 and 16–23 respectively).He reported good health with no bleeding or clotting problems,apart from a period of recurrent epistaxis which ceased followingcauterization. He had a number of surgical interventions, includingtonsillectomy, appendectomy and an eye operation without bleedingcomplications. He was given cryoprecipitate prior to the toothextraction, which proceeded uneventfully. His parents are originallyfrom the South of England and there is no history of bleedingproblems in his siblings or his father.

His mother however, has had bleeding problems with all her pregnancies,both during pregnancy and following delivery, but never requiredblood transfusions. All the children were born in the UK andshe does not recall any investigation into a bleeding problem.She has had two miscarriages, both in the first trimester. Inaddition she has had a history of menorrhagia and has alwaysbled for a prolonged time from venipuncture sites. Surgerieshowever (including tonsillectomy and teeth extractions) werenot associated with excess bleeding. The 17-year old sisterhas had a similar history of menorrhagia and the maternal grandmother(who was unavailable for investigation) has a history of miscarriageand problems with post-partum hemorrhage, which necessitatedblood transfusion on at least one occasion. There was no familyhistory of thrombosis and current family data is shown in Table 1.

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Table 1. Family fibrinogen levels and mutation status.

Purified fibrinogen from all 5 family members that were availableshowed a normal pattern of A ${alpha}$ , Bβ and ${gamma}$ chains on reducingSDS PAGE, while non-reducing gels gave the expected patternof 340 and 305 kDa bands. Reverse phase assays indicated normalfibrinopeptide release.

Reverse phase separation of reduced fibrinogen chains from thepropositus, however, consistently showed an additional minorpeak eluting just ahead of the normal Bβ^A chain (Figure 1).The slight reduction in area of the Bβ^A peak (Bβ^Ato ${gamma}$ ratio 1.03:1) compared to the control (Bβ^A to g ratio1.12:1) suggested the new peak (Bβ^X) might be an aberrantform of Bβ chain expressed at a lower than normal level.This was supported by electrospray mass analysis of the peaks.The monosialylated isoform of the ${gamma}$ chain appeared normal, witha mass of 48,358 Da compared to 48,359 Da for control ${gamma}$ chains.However, while the Bβ^A peak from the proband (54,182 Da)had a comparable mass to the control (54,198 Da), the earlyeluting Bβ^X peak was only 53,629 Da. This mass decreaseof 553 Da was confirmed in a second HPLC separation, where adecrease of 554 Da was recorded. Tryptic peptide maps comparingthe Bβ^X and Bβ^A peaks were uninformative, but didconfirm that the new peak was derived from Bβ chain material.

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Figure 1. Reverse phase HPLC profile (215 nm) of purified fibrinogen from control (lower trace) and from individual with fibrinogen Epsom (upper trace). In addition to the normal Bβ chain, a minor more hydrophilic chain, Bβ^X, was detected in carriers of the Epsom mutation.

Subsequent DNA sequencing of coding regions and the intron/exonboundaries of the fibrinogen β gene (FGB) indicated heterozygosityfor a novel 15 nucleotide deletion (NM_005141.2:c.498_512del)in exon four (Figure 2A). The deletion segregated with low fibrinogenin 3 family members and was absent from the 2 normal individuals(Table 1).

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Figure 2. (A) DNA and protein sequence of relevant region of the fibrinogen Bβ chain showing the normal (upper) and new sequence in fibrinogen Epsom (lower). The deletion is flanked by an AATGA repeat (underlined), which could facilitate replication slippage and deletion of the intervening 15 nucleotides. (B) Molecular model showing the coiled coil and outer D domain, with the unresolved E domain on left. Black shading indicates the location of the NVVNE deletion that removes 1.5 turns of helix resulting in instability and altered fibrin polymerization. Created from PDB co-ordinate file 1M1J (13) using spdb viewer v.3.7 and POV-Ray v.3.6 software.

No other mutations were detected in the coding regions of eitherFGA or FGG. The 15 nucleotide deletion (Chr4(NCBI36):g.155708202_155708216del)results in the loss of the five amino acids, NVVNE, that spanpositions 137–141 of the mature Bβ chain (Figure 2).The predicted mass decrease of 555 Da associated with this mutationwas in excellent agreement with the measured decrease and thisnew variant has been named fibrinogen Epsom. The prolonged clottingtimes (43–47s) of all 3 carriers, together with the discrepancybetween their functional and physical fibrinogen levels (Table 1),suggested the deletion might impact on function. This was supportedby studies on purified fibrinogen, which showed abnormalitiesin all kinetic parameters (data not shown). The patient curvesdisplayed a lag time to protofibril formation of 132s (control69s), a Vmax of 0.0105 OD/s (control 0.0132 OD/s) and a finalOD of 0.2 (control 0.35). After the clot had formed fibrinolysiswas examined by overlaying the clot with plasminogen/tPA andmeasuring the decrease in optical density. Comparison of patientclot lysis to 2 normal controls revealed an enhanced lysis rate1.3 times faster. Similarly, plasmin catalyzed lysis of nativefibrinogen was also found to have a significant 1.25 fold increasein susceptibility to proteolysis.

Mechanistically the Epsom mutation most likely arises from replicationslippage during DNA synthesis, where the priming strand partlydetaches from the template strand, loops out and reanneals toa common sequence later in the template.¹⁴ In this case thedeletion is flanked by direct AATGA repeats which would facilitatethe reannealing process (Figure 2A).

At a protein level the five residue deletion would be expectedto shorten the Bβ coil of the coiled coil by ~1.5 turnsof helix, or 7.5 Å. This would tend to tension and deformthe super coil shortening the distance between the E and D domains.On coagulation this would perturb the close packing of fibrinmonomers and be reflected in aberrant polymerization and a phenotypeof dysfibrinogenemia. The coagulation data (Table 1) confirmthis and indicate both a functional and quantitative defect.While the low plasma expression of the variant chain (β^X/β^A+β^X; 0.1:1.0) was confirmed by reverse phase HPLC it remainsunresolved as to whether the hypofibrinogenemia results fromaccelerated intracellular degradation of misfolded protein orincreased plasma turnover, or a combination of both. The deletiondoes occur close to the plasmin sensitive site in the coiledcoil and we have shown that molecules containing variant chainshave an increased susceptibility to plasmin cleavage. This maybe reflective of a general increase in susceptibility to proteolyticactivity and increased circulatory turnover. As 75% of newlyassembled molecules would contain at least one variant Bβchain, a destabilizing event that decreases the expression levelof Bβ^X to 10% of the total would have a significant impacton plasma fibrinogen concentration and result in an overallphenotype of hypodysfibrinogenemia.

Low fibrinogen is a recognised cause of spontaneous abortionwithin the first trimester and in knock-out mice fibrinogenhas been shown to facilitate placental-maternal attachment duringembryonic development.¹⁵ Also, during pregnancy fibrinogen levelsbecome elevated and there is a shift to less highly sialylatedoligosaccharide side chains; both of these adaptations protectagainst bleeding, because the lower sialic acid content decreasesthe clotting time.¹⁶ If the Epsom deletion favored increasedsialylation then its potential effect on bleeding might be moreapparent during pregnancy. As one of the oligosaccharide sidechains is attached to the coiled coil ( ${gamma}$ 52 Asn) and one to theβD domain it is possible the mutation could have an increasedeffect on bleeding during pregnancy. The bleeding diathesisassociated with fibrinogen Epsom can be explained by the cumulativeeffects of decreased fibrinogen concentration and altered function.These together with the increased rate of fibrinolysis wouldall be expected to contribute to a bleeding tendency. However,they are probably not the sole cause of bleeding; mutation orgenetic variation in other (unidentified) coagulation geneswill undoubtedly contribute to the overall phenotype.

Footnotes

Authorship and Disclosures

SOB performed the protein structural analysis, wrote the draftmanuscript and approved the final version. RLD performed theprotein functional analysis, prepared the figures, and helpedwrite the manuscript. RL performed the DNA sequencing and assistedin other experiments. AR identified the initial hematologicproblem, obtained the family histories and blood samples, andhelped write the manuscript.

The authors reported no potential conflicts of interest.

Received for publication October 1, 2008. Revision received November 9, 2008. Accepted for publication November 27, 2008.

References

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