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Fibrinogen Bern I: Substitution y 337 Asn + Lys Is Responsible
for Defective Fibrin Monomer Polymerization
By Colette Steinmann, Peter Reber, Myriam Jungo, Bernhard LBmmle, Gabriela Heinemann,
Bendicht Wermuth, and Miha Furlan
An inherited fibrinogen variant, fibrinogen Bern I, was isolated from plasma of an asymptomatic woman. Routine
coagulation studies showed prolonged thrombin and reptilase clotting times. Fibrinogen concentration was diminished when determined by a functional assay, but was normal by the heat precipitation method. The release of
fibrinopeptides A and B was not delayed. Two-dimensional
gel electrophoresis of mercaptolyzedfragments D of fibrinogen, obtained by digestion with plasmin, showed an abnormal electrophoretic mobility in the y-chain remnants of
fragments D, and D, from fibrinogen Bern 1, whereas conversion of D, to D, by plasmin resulted in the loss of the
abnormal charge, suggesting that the structural abnormality in this variant is located in the region y 303 through
356. The molecular defect in fibrinogen Bern I was identified by sequence analysis of genomic DNA amplified by
polymerase chain reaction and cloned in M13mpl9. ‘The
triplet AAC coding for asparagine at position y 337 was
found to be substituted by AAA coding for lysine. W e conclude that the substitution y 3 3 7 Asn -+ Lys in fibrinogen
Bern I is responsible for defective polymerization of fibrin
monomers and for impaired protection by calcium against
plasmic degradation.
0 1993 by The American Society of Hematology.
C
site. Recently, the binding site for Gly-Pro-Arg was located
to the y-chain residues 337 through 379 by photoaffinity
labeling.’ Functionally abnormal fibrinogens with structural defects in the y-chain include variants with amino acid
substitutions between positions y 275’”,” and y 375.”
In the present study, we identified the defect in fibrinogen
Bern I, a dysfunctional variant characterized by delayed
clotting in the absence of calcium ion^.'^,'^ Residue y 337
Asn was found to be replaced by Lys, which seems to be
responsible for delayed polymerization of fibrin monomers
derived from this variant.
ONGENITAL dysfibrinogenemia defines an inherited
functional defect caused by structural alteration in the
fibrinogen molecule. Most dysfibrinogenemias have thus
far been detected by virtue of a prolonged thrombin clotting
time and, therefore, mainly represent variants with defective fibrinogen to fibrin conversion. More than 240 abnormal fibrinogen variants have been described.’ In a large proportion of abnormal fibrinogens, the defective release of
fibrinopeptides A and/or B is caused by an amino acid substitution interfering with the thrombin-induced cleavage of
the peptide bonds ACI 16-17 or BP 14-15,
Furthermore, in several fibrinogen variants, the functional
defect is associated with an abnormal or absent amino terminal polymerization site. Polymerization of fibrin is initiated
by exposure of amino terminal binding sites, Gly-Pro-Arg
(a-chain) and Gly-His-Arg (P-chain) after proteolytic cleavage of fibrinopeptides A and B, re~pectively.~,~
Fibrin monomers interact with complementary carboxy terminal domains of neighboring fibrin(ogen) m01ecules.~~’The
carboxy terminal region of the y-chain plays an important
role in fibrin monomer polymerization. The synthetic peptide Gly-Pro-Arg-Pro, an analogue of the amino terminus of
the fibrin a-chain, was found to bind to fragment D, and to
inhibit polymerization of fibrin monomers.* However, peptides with the sequence Gly-Pro-Arg did not bind to the
plasmin-generated fragment D, lacking the carboxy terminal 109 residues of the y-chain. Thus, the carboxy terminal
third of the y-chain is supposed to bear the polymerization
From the Central Hematology Laboratory and the Central Clinical Chemistry Laboratory, Inselspital, University of Bern, Switzerland.
Submitted March 24, 1993; accepted May 20, 1993.
Supported by Swiss National Science Foundation Grant No. 3228785.90.
Address reprint requests to Miha Furlan. PhD, Central Hematology Laboratory, Inselspital, CH-3010 Bern, Switzerland.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1993 by The American Society of Hematology.
0006-4971/93/8207-0110$3.00/0
2104
MATERIALS AND METHODS
Materials. Taq DNA polymerase, 123-bp ladder-size standard
and JM 10 1 Escherichia coli cells for transformation were obtained
from Bethesda Research Laboratories (Gaithersburg, MD). Sequenase Version 2.0 kit was from US Biochemicals (Cleveland,
OH). Deoxyadenosine 5’[a35S]thiotriphosphatewas from Amersham (Amersham, UK). M 13mp19 cloning vector, Sma I restriction endonuclease, dATP, dCTP, dGTP, dTTP, proteinase K,
RNase A, ethidium bromide, isopropylthio-P-D-galactoside, 5bromo-4-chloro-3-indolyl-~-D-galactoside,
T4 DNA polymerase,
and T4 DNA ligase were purchased from Boehringer (Mannheim,
Germany). Agarose Type I1 DNA grade was from Sigma (St. Louis,
MO). QIAGEN-tip 5 mini columns were from DIAGEN
(Dusseldorf, Germany) and Sephadex G-25 DNA grade (NapTM25) columns from Pharmacia (Uppsala, Sweden). All other chemicals were of analytical grade.
Purification ofjbrinogen. Fibrinogen was purified from normal and proposita’s plasma by affinity chromatography on fibrin
monomer Sepharose CL-2B.I’ No attempt was made to separate
normal and abnormal fibrinogen molecules isolated from the heterozygous carrier of defective fibrinogen.
Determination of coagulation parameters in plasma. Activated
partial thromboplastin time (APTT), prothrombin time (Quick),
thrombin time (TT), and reptilase time (RT) were determined by
conventional methods. Concentration of fibrinogen was measured
by the functional assay of Clauss16 and by the heat precipitation
method of Schu1z.l’
Fibrinopeptide release. High performance liquid chromatography (HPLC)analysis of fibrinopeptides was performed according to
Kehl et all*with minor modifications. Fibrinogen (final concentration, 1 mg/mL) in 0.15 mol/L ammonium acetate buffer (pH 7.4),
containing 0.1 mmol/L CaCl,, was incubated for various time intervals at 37°C with 0.1 NIH U/mL bovine thrombin (HoffmannL a Roche, Basel, Switzerland). The reaction was stopped by addiBlood, Vol 82, No 7 (October 1). 1993: pp 2104-2108
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ABNORMAL FIBRINOGEN VARIANT BERN I
tion of concentrated formic acid (l/lO, vol/vol). Fibrin was precipitated by neutralizing the solution with 25% NH,. After centrifugation, the supernatant was lyophilized. The fibrinopeptides were
dissolved in 0.025 mol/L ammonium acetate, pH 6.0, and analyzed
by HPLC. Chromatographywas performed on a Beckman (Fullerton, CA) Ultrasphere-ODS RP-18/5 pm (0.46 X 25 cm) column.
Buffers used were the following: solvent A, 0.025 mol/L ammonium acetate, pH 6.0; solvent B, 50% acetonitrile (Fluka, Buchs,
Switzerland) in 0.05 mol/L ammonium acetate, pH 6.0; gradient,
18%to 28% B in 20 minutes at a flow rate of 1.5 mL/min.
Two-dimensional gel electrophoresisof plasmin-digestedfibrinogenfragments D,,D,,and D,. Normal fibrinogen and fibrinogen
Bern I were digested by plasmin (Kabi, Stockholm, Sweden) in the
presence of 10 mmol/L CaCl,. The resulting fragment D, was isolated by affinity chromatography on ly~ine-agarosel~
and dialyzed
against 0.05 mol/L triethanolamine, 0.1 mol/L NaCl, 10 mmol/L
EGTA, pH 7.4. Fragment D, (2 mg/mL) was incubated for 3 hours
at 37°C with 0.3 U/mL plasminogen (Kabi) and 6 U/mL streptokinase (Kabi). The resulting fragmentsD,, D,, and D, were reduced
with 2% dithiothreitol (Fluka) and separated by isoelectric focusing
and sodium dodecyl sulfate (SDS) polyacrylamidegel electrophoresis according to Reber et aLzo
DNA isolation from whole blood. Twenty milliliters fresh
EDTA (final concentration 4 mmol/L) blood of the proposita's
mother were centrifuged for 20 minutes at 1,800g at room temperature. Plasma was aspirated to approximately 5 mm above the buffy
coat and discarded. Ice-cold lysis buffet' (0.32 mol/L sucrose, 10
mmol/L Tris-HCI, 5 mmol/L MgCl,, I % Triton X-100, pH 7.5)
was added to the cell pellet to a final volume of 50 mL. The suspension was mixed gently by inverting, set on ice for 15 minutes, and
then centrifuged for 15 minutes at 1,800g and 4°C. The supernatant was discarded, and the pellet resuspended in fresh lysis buffer
to a final volume of 50 mL. The suspension was centrifuged immediately for 10 minutes at 1,SOOg (4"C), and the resulting nuclear
pellet was resuspended in 3 mL of a solution containing 10 mmol/L
Tris-HCI, 1 mmol/L EDTA, 0.5% SDS, pH 8.0, 500 pg/mL proteinase K and 50 pg/mL RNase. Digestion was performed for 3
hours at 50°C. The DNA was then extracted with phenol-chloroform according to Gustafsonet aIz2and precipitated with 2 volumes
of ice-cold ethanol after addition of0.2 vol of 10 mol/L ammonium
acetate. High molecular weight DNA was washed twice with 70%
ethanol, lyophilized, and dissolved in 1.5 mL 10 mmol/L Tris-HC1,
1 mmol/L EDTA, pH 8.0. The absorbance was measured at 260
nm, and the calculated yield of DNA was approximately 360 pg
from 20 mL fresh blood.
Amplification by polymerase chain reaction (PCR). A 3 12-bp
fragment of the fibrinogen y-chain gene23containing the DNA
sequence of exon VI11 coding for amino acids 259 through 350
was amplified with the following 22-base-long primer pair:
sense primer, 5'-TCAGCATGTGATGGTTGTATTT-3'; antisense
primer, 5'-CTTGGTAATAAACTCCATTGAG-3'.These oligonucleotide primers had been synthesizedby L. Edenharter (Institute of
Microbiology, Bern, Switzerland) using an Applied Biosvstem
38 1A DNA synthesizer by the P-cyanoethyl phosphoramidine
method. Amplification was performed in a 100 pL reeztion volume
containing 1 pg genomic DNA, 4 U Taq DNA polymerase, 200
pmol/L each of d-adenosine triphosphate (dATP), d-cytidine triphosphate (dCTP), d-thymidine triphosphate (dTTP), d-guanosine
triphosphate (dGTP), 0.1 pmol/L, and 0.5 pmol/L of sense and
antisense primer, respectively, in 10 mmol/L Tris-HC1, at 25"C, 50
mmol/L KCI, 1.5 mmol/L MgCl,, and 0.01%(wt/vol) gelatin, pH
8.4. Sixty cycles of amplification were performed by using a DNA
thermal cycler (Perkin-Elmer-Cetus, Nonvalk, CT). Incubation
times for each cycle were 1 minute at 94"C, I minute at 45"C, and 2
2105
minutes at 72°C. Similar procedure was used for amplification of a
290-bp fragment of the y-chain gene, containing exon IX and coding for amino acids 35 I through 407, using the following pair of
2 I-mer primers, 5'-GCACTTCGTAATAGACAGCTC-3'and 5'CATATTCTGTTTCCGCAGGGT-3' (Microsynth, Windisch,
Switzerland). Incubation times were 1 minute at 9 4 T , 1 minute at
47"C, and 2 minutes at 72°C for a total of 60 cycles. The size ofthe
PCR products was examined using a 123-bpladder-size standard in
a 1.4%(wt/vol) agarose gel stained with 0.66 pg/mL ethidium bromide in 0.09 mol/L Tris-borate, 2.6 mmol/L EDTA, pH 8.3.
Molecular cloning and sequencing of normal and variant exon
VIII offibrinogen Bern I. Amplification of double-stranded exon
VI11 fragment was performed under similar conditions as described
above with the following modifications: 0.42 pmol/L each of sense
and antisense primer, 2 U Taq DNA polymerase, and 50 cycles.
The PCR product was purified on a QIAGEN-tip 5 mini column
according to manufacturer's instructions and cloned into Sma I
polycloning site of M 1 3 m ~ 1 9Individual
.~~
clones were sequenced
using the 17-mer sequencing primer (-40) and [c~-~'S]
dATP by
using the Sequenase Version 2.0 kit. Dideoxynucleotide termination reaction products were run on a 8% polyacrylamide sequencing gel in a Sequi-Gen Nucleic Acid SequencingCell from Bio Rad
(Richmond, CA). The gel was dried on a Slab Gel Dryer SE 1160
(Hoefer ScientificInstruments, San Francisco, CA), and autoradiography was performed using 3M Trimax medical imaging film (3M,
Ferrani, Italy).
RESULTS
Case report. Dysfibrinogen Bern I was discovered in
1978 in a 24-year-old woman without a history of bleeding
or thrombotic tendency. Upon admission to the hospital,
her TT and RT were found to be prolonged (Table 1). Fibrinogen concentration determined by the functional assay
was markedly decreased, whereas the heat precipitation
method indicated normal fibrinogen values. Family
members were studied, and their laboratory parameters
were consistent with the diagnosis of hereditary dysfibrinogenemia. The proposita, her sisters, and her brother have
inherited the functionally abnormal fibrinogen from their
mother, whereas the father was not affected. Neither the
mother nor any of her children had bleeding symptoms or
thrombotic episodes.
Fibrinopeptide release. The kinetics of fibrinopeptide
release by thrombin from the normal and the variant fibrinogen are shown in Fig 1. The amounts of fibrinopeptides A
and B cleaved from normal fibrinogen within 1 hour were
taken as 100%.Our results indicate that delayed clotting of
fibrinogen Bern I cannot be ascribed to an impaired fibrinopeptide cleavage by thrombin, because fibrinopeptide A
cleavage from fibrinogen-Bern I appeared to proceed even
faster than that from normal fibrinogen, and fibrinopeptide
B release was normal. These results suggest that the structural defect is not situated in the amino terminal domain of
fibrinogen near the thrombin cleavage sites.
Two-dimensional gel electrophoresis offragments D,, D2,
and D3. Two-dimensional gel electrophoresis of mercaptolyzed plasmic degradation products D,, D,, and D, is
shown in Fig 2. An abnormal y-chain remnant, denoted as
y*D2was observed in fragment D, of fibrinogen Bern I, but
not in fragment D,. This difference suggests an excess positive charge in the y-chain remnant of fragment D,. Plasmin-
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STEINMANN ET AL
2106
Table 1. Results of Laboratory Tests on Plasma Samples From the Proposita and the Members of Her Family
R.Z.
1954
T.Z.
1960
H.-P.Z.
1956
H.F.-Z.
1952
H.Z.
1922
M.Z.
1925
Proposita
Sister
Brother
Sister
Father
Mother
Normal
Range
85
48
23.5
22
66
54
20.3
19
85
48
23.5
23
90
53
22
26
>70
40-60
12-15
13-14
70
Quick (%)
APTT (s)
TT 1s)
RT (s)
Fibrinogen (g/L)
Functional assay
Heat precipitation
45
21.8
32.1
0.25
1.80
0.32
1.60
0.40
1.50
0.32
1.80
>loo
48
13.2
ND
3.80
4.00
0.25
2.10
1.50-3.00
1.50-3.00
Abbreviation: ND, not determined
induced cleavage at position y 302-303 during conversion
of D2 to D, apparently results in the loss of the abnormal
peptide.
AmplSfication and DNA sequence analysis. Two DNA
fragments of 3 12- and 290-bp, comprising exons VI11 and
IX and coding for amino acids 259 through 350 and 351
through 407 of the y-chain, respectively, were amplified by
asymmetric PCR. The DNA sequence of the amplified exon
IX was indistinguishable from the normal DNA sequence.
The sequence analysis of exon VI11 showed the presence of
two bands at the same migration distance with about equal
radioactivity (data not shown), suggesting that the patient is
heterozygous for the mutation AAC + AAA, corresponding to amino acid substitution y 337 Asn + Lys. To improve quality of the sequence analysis of exon VIII, cloning
experiments were performed. Five of eighty-eight clones
contained the insert. The sequence of two of them, both
representing antisense strands from either normal or abnormal y-chain gene fragments, is shown in Fig 3. A guanine in
the normal amplified exon VI11 was found to be substituted
by thymine. Thus, the single base substitution altered the
codon in the sense strand for y 337 Asn (AAC) to Lys
(AAA).
loo
r
60
40
20
I
I
0
0
10
5
15
Time (min)
Fig 1. Fibrinopeptide release by thrombin from fibrinogen Bern I
and from normal fibrinogen. (0)Fibrinopeptide A, normal fibrinogen; ( 0 )fibrinopeptide A, fibrinogen Bern I; (A) fibrinopeptide B.
normal fibrinogen; and (A)fibrinopeptide B, fibrinogen Bern I.
DISCUSSION
An abnormal fibrinogen variant, denoted as fibrinogen
Bern I, was detected in a clinically asymptomatic woman.
Thrombin and reptilase clotting times were prolonged. Fibrinogen concentration was decreased when measured by
the functional assay but was normal by the heat precipitation method (Table 1). Family study confirmed the hereditary nature of this dysfibrinogenemia. Fibrinogen Bern I
was isolated from the proposita’s plasma and displayed normal fibrinopeptide A and B release (Fig 1). Fibrinopeptide
A was cleaved even faster from fibrinogen Bern I than from
normal fibrinogen. As shown previou~ly,’~
the rate of fibrin
monomer polymerization was decreased at low concentration ofcalcium ions, but it was fully normalized only at high
(5 mmol/L) calcium concentration, suggesting that calcium
binding site might be functionally inadequate in fibrinogen
Bern I. A complete correction of fibrin polymerization by
high concentration of calcium has also been observed in
fibrinogen Osaka VI2,a variant with an amino acid substitution y 375 Arg + Gly, lacking 2 of the 3 high-affinity calcium binding sites. It is obvious that the functional defect in
fibrinogen Bern I cannot be explained solely by deficient
calcium binding, because in the presence of EDTA, the defective clotting of fibrinogen Bern I even became more
prominent. l 4
Our previous experiments showed impaired protection
by calcium of the carboxy terminal portion of the y-chain of
fibrinogen Bern I against proteolytic degradation by plasmin.I4 Similarly, in fibrinogen variants Haifa (y 275 Arg +
His),2’Osaka V (y 375 Arg + Gly),” Kyoto I (y 308 Asn +
LYS)?~
and Vlissingen (deletion y 3 19 Asn and 320 Asp),27
the carboxy terminus of the y-chain was degraded by plasmin despite the presence of Ca2+ ions. Thus, our former
observations with fibrinogen Bern I hinted at a structural
defect in the carboxyterminal part of the y-chain, affecting
calcium binding.
The carboxy terminal portion of the y-chain not only
contains one high-affinity binding site for Ca2+ions, tentatively located between residues y 3 11 through 336,28but
also appears to be involved in polymerization of fibrin
monomers. Synthetic peptides, beginning with the amino
terminal sequence (Gly-Pro-Arg) of the fibrin a-chain were
shown to bind strongly to plasmin-generated fragment D,,
whereas no binding occurred to fragment D,, which lacks
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2107
ABNORMAL FIBRINOGEN VARIANT BERN I
61
tu
I
a
nl
.
Fig 2. Two-dimensional electrophoresis of reduced fibrinogen fragments D,, D2, and D,. The abnormal 7-chain remnant in fibrinogen Bern I is denoted with an asterisk.
1
....................
....................
5’ 3’
To identify the structural defect in fibrinogen Bern 1.
PCR was used for amplification and sequence analysis of
the exons VI11 and IX coding for amino acid residues 259
through 350 and 35 1 through 407 of the y-chain,” respectively. The DNA sequence of the amplified exon IX was
indistinguishable from the normal DNA sequence. Direct
sequence analysis of exon VI11 showed the presence of two
bands at the same migration distance, corresponding to
Lys (AAC + AAA).
amino acid substitution y 337 Asn
The purified double-stranded PCR-derived fragment was
cloned and subjected to sequence analysis. Normal and mutated alleles of fibrinogen Bern I were obtained, showing a
G + T transition in the noncoding strand (Fig 3). Thus,
codon AAC in the sense strand of the exon VI11 of the ychain gene was substituted by AAA, leading to a replacement of Asn by Lys at position y 337 in fibrinogen Bern I.
In conclusion, PCR was used to determine the structural
defect in the abnormal fibrinogen variant Bern I. A new
point mutation was found in the y-chain of fibrinogen Bern
I, leading to the substitution of the amino acid y 337 Asn by
Lys. This mutation results in defective expression of the
polymerization site and in impaired protection by calcium
ions against plasmic digestion of the carboxy terminus of
the y-chain.
GC
REFERENCES
the carboxy terminal 109 residues ofthe y-chain.’ The binding site for Gly-Pro-Arg has recently been more precisely
ascribed to y-chain residues 337 through 379.9 Using twodimensional electrophoresis in polyacrylamide gel of the reduced fragments D,, D,, and D, derived from fibrinogen
Bern I by plasmic degradation. a y-chain remnant of fragment D, with an increased isoelectric point was noted (Fig
2 ) . whereas the abnormal charge disappeared during conversion of fragment D, to D, suggesting that the molecular
defect is situhed within the amino acid sequence y 303
through 356, which corresponds to the peptide released
from fragment D, by plasmin.
BERN I
NORMAL
G
A
T
C
G
A
-
T
C
._
\A
Asn A T
IC G
AT
TA
Fig 3. DNA sequence analysis of individual clones containing
the amplified exon Vlll of the 7-chain gene coding for the normal
and mutated alleles of fibrinogen Bem I. A cytosine was substituted by adenine, changing the triplet AAC to AAA in the coding
strand. The brackets indicate the change of y 337 Asn to Lys.
-
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From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1993 82: 2104-2108
Fibrinogen Bern I: substitution gamma 337 Asn-->Lys is responsible
for defective fibrin monomer polymerization
C Steinmann, P Reber, M Jungo, B Lammle, G Heinemann, B Wermuth and M Furlan
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