(Received for publication, March 23, 1995; and in revised form, September 1, 1995)
From the
A dysfibrinogenemia was attributable to a single amino acid
substitution from glycine to cysteine at residue 15 of the B chain
in a fibrinogen molecule designated as fibrinogen Fukuoka II. The
fibrinogen Fukuoka II showed prolonged thrombin and reptilase times and
impaired fibrinopeptide B release by thrombin, resulting in abolition
of fibrin monomer repolymerization under physiological conditions.
Repolymerization of the des-(B
1-42)-fibrin monomers,
however, was not distinguished from the normal pattern of des-(B
1-42)-fibrin monomers, suggesting that no other abnormality
existed in fibrinogen Fukuoka II. Although an additional cysteine was
substituted at residue 15 of the B
chain, fibrinogen Fukuoka II
had no free sulfhydryl group within the molecule. Instead, fibrinogen
Fukuoka II formed a disulfide bond with cysteine, albumin, another
mutated B
chain within the same molecule, or intermolecular
dimeric fibrinogen Fukuoka II. The mutation in fibrinogen Fukuoka II
was the same as that in fibrinogen Ise published previously (Yoshida,
N., Wada, H., Morita, K., Hirata, H., Matsuda, M., Yamazumi, K.,
Asakura, S., and Shirakawa, S.(1991) Blood 77,
1958-1963). Fibrinogen Ise, however, has been described as having
prolonged thrombin time but normal reptilase time. Reasons for the
discrepancy were not clear. Analysis of the B
1-42 fragment
showed that fibrinogen was heterogeneous at position 31 of the B
chain with respect to proline or hydroxyproline.
Fibrinogen molecules contain two copies each of three different
polypeptide chains that are designated as A, B
, and
.
During clotting, the A
chain is cleaved by thrombin at position 16
to produce peptide A (fibrinopeptide A, FPA) (
)and the
chain. Similarly, the B
chain is cleaved at position 14 to produce
peptide B (fibrinopeptide B, FPB) and the
chain. These cleavages
and the resulting dissociation of the small peptides FPA and FPB from
the molecule convert fibrinogen to fibrin(1, 2) . The
six peptide chains in fibrinogen are held together by 29 disulfide
bonds(1) , and interchain disulfide bonds have important roles
in the assembly and secretion of fibrinogen molecules during its
biosynthesis(3, 4) . After thrombin cleavage of
fibrinogen, many disulfide cross-linked, six-chain fibrin molecules
assemble into large polymers. Immediately after assembly these
noncovalent intermolecular complexes or polymers (fibrin clots) can be
dissociated into the individual fibrin molecules in acetic acid, which
are designated as fibrin monomers. Congenital abnormalities of
fibrinogen molecules have been reported as
dysfibrinogenemia(5, 6) . The amino-terminal region of
the B
chain plays a pivotal role in the lateral associations of
fibrin monomers(7, 8, 9) . Synthetic peptides
of the B
15-42 region have inhibitory effects on
polymerization of fibrin monomers(10) .
Fibrinogen
Christchurch II(11) , fibrinogen Seattle
I(12, 13) , and fibrinogen IJmuiden (14) have
a mutation from Arg to Cys at residue 14 of the B chain.
Dysfibrinogenemia with prolonged thrombin and reptilase times has been
attributed to the impaired release of FPB from abnormal fibrinogen
molecules by thrombin due to this Arg to Cys substitution. Fibrinogen
with a deletion of the B
9-72 region, fibrinogen New York I,
also shows the prolonged thrombin and reptilase
times(15, 16) . However, fibrinogen Ise, an abnormal
fibrinogen with a substitution of Gly to Cys at residue 15 of the
B
chain shows a prolonged thrombin time but a normal reptilase
time, although the release of FPB is strongly impaired(17) .
Thus, participation of the FPB release with polymerization of fibrin
monomers in detail are still controversial.
In the present
communication, we report an abnormal fibrinogen designated as
fibrinogen Fukuoka II with a substitution of Gly to Cys at residue 15
of the B chain, which is the same mutation as described for
fibrinogen Ise. As shown for fibrinogen Ise(17) , the B
chain of fibrinogen Fukuoka II was resistant to a low concentration of
thrombin and clotting time with thrombin was prolonged. Unlike
fibrinogen Ise, however, fibrinogen Fukuoka II showed a prolonged
reptilase time and formed a disulfide complex with cysteine or albumin,
an intramolecular disulfide bond formation, or an interchain disulfide
bond with another fibrinogen Fukuoka II molecule at the mutated Cys
residue.
Figure 1:
HPLC analysis of FPA and FPB.
Fibrinogen was incubated with a low concentration of thrombin (0.1
units/ml) at 37 °C for 1 h (a), with a high concentration
of thrombin (50 units/ml) at 37 °C for 6 h followed by incubating
at 4 °C for another 12 h (b), and with reptilase (2
µg/ml) at 37 °C for 1 h (c). The released FPA and FPB
were analyzed by HPLC on a reversed-phase column of Cosmosil 5C18-P
(4.6 250 mm) with a linear gradient of 5-20%
CH
CN (dotted line) containing 0.025 M ammonium acetate (pH 6.0), at a flow rate of 0.5
ml/min(25) . A, FPA that consists of A
1-16; AP, phosphorylated FPA (A
1-16) at the
3rd Ser of A
; AY, FPA that consists of A
2-16; B, FPB that consists of B
1-14; BR, FPB
that consists of B
1-13 (des-Arg-B); N, normal
fibrinogen; F, fibrinogen Fukuoka
II.
Figure 2:
Fibrin
monomer repolymerization. Three kinds of fibrin monomers (a and b; c and d; e and f) were repolymerized in Tris buffer of various ionic
strength: 1, 0.05 M Tris-HCl, pH 7.4 (ionic strength,
µ = 0.04); 2, 0.05 M Tris-HCl in 0.03 M NaCl, pH 7.4 (µ = 0.07); 3, 0.05 M Tris-HCl in 0.06 M NaCl, pH 7.4 (µ = 0.1); 4, 0.05 M Tris-HCl in 0.1 M NaCl, pH 7.4
(µ = 0.14). a and b, repolymerizaton of
fibrin monomers pretreated with thrombin. c and d,
repolymerization of fibrin monomers pretreated with reptilase. e and f, repolymerization of des-(B 1-42)-fibrin
monomers pretreated with reptilase. a, c, and e, fibrin monomers or des-(B
1-42)-fibrin monomers
obtained from normal fibrinogen. b, d, and f, fibrin monomers or des-(B
1-42)-fibrin monomers
obtained from fibrinogen Fukuoka II.
C. atrox proteinase III cleaves the B chain of
fibrinogen at the carboxyl side of Arg-42(9) . Des-(B
1-42)-fibrinogens prepared from either normal fibrinogen or
fibrinogen Fukuoka II by pretreating with C. atrox proteinase
III were digested with reptilase to form fibrin clots. Repolymerization
of normal des-(B
1-42)-fibrin monomers did not occur at
physiological ionic strength but did occur at lower ionic strengths,
although the ultimate turbidity was lower than when uncleaved fibrin
monomers were used (Fig. 2, c and e).
Repolymerization of Fukuoka II des-(B
1-42)-fibrin monomers
was identical to normal des-(B
1-42)-fibrin monomers (Fig. 2, e and f), suggesting that the
susceptible fibrinogen Fukuoka II structural abnormalities are located
within the first 42 residues of the B
chain.
Figure 3: Electron micrographs of negatively stained repolymerized fibrin monomers obtained from normal fibrinogen (a) and from fibrinogen Fukuoka II (b). Fibrin monomers prepared by reptilase (2 µg/ml) were repolymerized for 15 min in TBS (0.05 M Tris-HCl in 0.1 M NaCl, pH 7.4, µ = 0.14) and negatively stained with uranyl acetate. The bars represent 200 nm.
Figure 4:
DNA sequence analysis of individual clones
containing the amplified B chain gene exon II fragment derived
from genomic DNA of the propositus. The arrow indicates the
position of the mutation. Heterozygosity with wild type and mutant type
alleles is shown.
Figure 5:
SDS-PAGE and immunoblot analysis of
fibrinogen and des-(B 1-42)-fibrinogen. Normal fibrinogen (lanes 1), fibrinogen of the propositus (lanes 2),
fibrinogen of his father (lanes 3), and des-(B
1-42)-fibrinogen of his father (lanes 4) (1 µg
protein/lane) were analyzed with SDS-PAGE (5% acrylamide) under
nonreducing conditions. a, Coomassie Blue staining. b, immunoblot analysis with anti-human fibrinogen IgG. c, immunoblot analysis with anti-human albumin
IgG.
The albumin-fibrinogen complex was selectively removed from an aliquot of purified fibrinogen Fukuoka II using an immobilized anti-human albumin antibody column. At physiological ionic strength, repolymerization of the fibrinogen Fukuoka II was not improved after removing the albumin-fibrinogen complex (data not shown).
Figure 6:
SDS-PAGE and immunoblot analysis of B
1-42 fragments. Normal fibrinogen or fibrinogen Fukuoka II (60
µg of protein each) was incubated with or without C. atrox proteinase III (1 µg/ml) at 37 °C for 2 h. The reaction
was stopped by heating at 56 °C for 10 min and centrifuged at
10,000
g for 5 min. B
1-42 fragments in the
supernatant were analyzed by SDS-PAGE (7-20% acrylamide) under
nonreducing (a and c) and reducing conditions (b and d). a and b, Coomassie Blue
staining. c and d, immunoblot analysis with
anti-human albumin IgG. Lanes 1, normal fibrinogen without C. atrox proteinase III treatment; lanes 2, B
1-42 from normal fibrinogen; lanes 3, B
1-42
from fibrinogen Fukuoka II; lanes 4, fibrinogen Fukuoka II
without C. atrox proteinase III
treatment.
Further analysis of the B 1-42 fragments using either
reducing or nonreducing conditions was performed by reversed-phase HPLC
(TSK gel ODS-120T, 4.6
250 mm) using a linear gradient of
20-30% CH
CN containing 0.1% trifluoroacetic acid.
Using nonreducing conditions, the normal and mutated B
1-42
fragments were separated into two peaks (B1 and B2) and seven peaks
(B3-B9), respectively (Fig. 7a, i and ii). Using reducing conditions, the B
1-42 fragment
of fibrinogen Fukuoka II was separated into four peaks (B10-B13) (Fig. 7a, iii), whereas the elution pattern of
normal B
1-42 fragments were not changed by reduction. The
separated peaks were analyzed on SDS-PAGE. B
1-42 fragments
in peaks B1 through B6 appeared as a single band on SDS-PAGE at 4,500
daltons under either nonreducing or reducing conditions (Fig. 7, b and c). In contrast, peaks B7, B8, and B9 showed
bands at 9,000 daltons under nonreducing conditions that shifted to
4,500 daltons under reducing conditions (Fig. 7, b and c).
Figure 7:
HPLC elution profiles of B 1-42
fragments (a) and SDS-PAGE analysis of the HPLC fractions (b and c). a, B
1-42 from normal
fibrinogen (i), B
1-42 from fibrinogen Fukuoka II (ii), and reduced S-pyridylethylated B
1-42 from fibrinogen Fukuoka II (iii) were analyzed on a
TSK gel ODS-120T reversed-phase column (4.6
250 mm) with a
linear gradient of 20-30% CH
CN (dotted line)
containing 0.01% trifluoroacetic acid. B1-B9 were analyzed by
SDS-PAGE (7-20% acrylamide) under nonreducing (b) and
reducing conditions (c).
Peaks B1-B9 were sequenced on a gas phase
sequenator after treating with pyroglutamate aminopeptidase (Table 2). These results showed that the sequence of B
1-42 in peak B2 was the normal sequence(1) , but the
B
1-42 in peak B1 had hydroxyproline instead of proline at
residue 31 in the B
chain as previously observed by
Henschen(39) . B
1-42 in peaks B4 and B6 had the
same normal sequence as those in peaks B1 and B2, respectively. B
1-42 in peaks B3 and B5 had the Gly to Cys mutation at residue 15
as deduced from the genomic DNA analysis. These results are consistent
with the genomic DNA analysis, which shows that the propositus and his
father are heterozygous (Fig. 4).
The B 1-42
fragments from peaks B10 and B12 confirmed that they were the mutated
fragments, and those from peaks B11 and B13 had the normal sequence.
Judging from the peak heights of B10-B13 in Fig. 7a (iii), we concluded that fibrinogen Fukuoka II was
expressed at almost the same level as normal fibrinogen in these
heterozygosis patients.
Cysteine was shown to disulfide cross-link
to fibrinogen Fukuoka II in an equilimolar ratio. The results of amino
acid composition and of sequence analysis of all B 1-42
fragments were consistent with the exception of peaks B3 and B5. Amino
acid composition of those two fragments indicated that an additional
Cys residue was associated with B
1-42 in peaks B3 and B5 (Table 3). The additional Cys was lost after reducing those
samples with dithiothreitol (see B3` and B5` in Table 3). Thus, a
cysteine-fibrinogen Fukuoka II complex was formed through a disulfide
bond at the mutated Cys of residue 15.
Figure 8: Electron micrographs of rotary shadowed molecules of normal fibrinogen (a) and fibrinogen Fukuoka II (b). Extra globular domains and dimers of the tridomainal structures are indicated by the arrows and the arrowhead, respectively. The bars indicate 200 nm.
A congenital heterozygous abnormal fibrinogen designated as
fibrinogen Fukuoka II is characterized by prolonged thrombin and
reptilase times (Table 1), retarded release of FPB, normal
release of FPA (Fig. 1), and disrupted repolymerization of
fibrin monomers ( Fig. 2and Fig. 3). Genomic DNA analysis
on exon II of B chain indicated that fibrinogen Fukuoka II had a
single base substitution in the codon normally coding for Gly at
residue 15 of the B
chain. This substitution changed the codon
from GGT (for Gly) to TGT (for Cys) (Fig. 4), and amino acid
sequencing of B
1-42 peaks confirmed the mutation (Table 2). Normal repolymerization of des-(B
1-42)-fibrin monomers of fibrinogen Fukuoka II was recovered (Fig. 2, e and f), indicating that the
abnormal repolymerization behavior of intact fibrinogen Fukuoka II is
due to the mutation at residue 15 of the B
chain.
Titration of sulfhydryl groups showed that fibrinogen Fukuoka II contained no detectable free sulfhydryl groups, indicating that the mutated Cys residue forms a disulfide bond with a sulfhydryl group from other compounds. SDS-PAGE and immunoblot analysis showed that this mutated Cys residue could form a disulfide bond with a sulfhydryl group in albumin as shown in other abnormal fibrinogens(14, 40) . In addition, dimeric fibrinogen Fukuoka II was also formed via a intermolecular disulfide bond (Fig. 5). Rotary shadowing of the affected fibrinogen molecules indicated that 5 and 2% of fibrinogen Fukuoka II fraction formed albumin-fibrinogen complexes and intermolecular dimeric fibrinogen complexes, respectively. This means that approximately 10 and 4% of the mutated fibrinogen forms albumin-fibrinogen and dimeric fibrinogen complexes, because the examined patients were heterozygous for this mutation.
Analysis by HPLC of the B 1-42 peptides from
fibrinogen Fukuoka II produced seven peaks (B3-B9), whereas
normal fibrinogen produced only two peaks (B1 and B2). Peak B1
contained hydroxyproline instead of proline at normal residue 31 of the
B
chain, and peak B2 contained proline at this position (Table 2). When multiple individuals were examined, the ratio of
peak B1 to peak B2 ranged from 0.2 to 0.7. B
1-42 in peaks
B7 and B9 were homodimers of the mutated B
1-42 fragment
that contained hydroxyproline and proline at position 31, respectively (Table 2). Peak B8 was a heterodimer of B
1-42 that
contained hydroxyproline or proline. These data show that normal
fibrinogen molecules have a random distribution of proline or
hydroxyproline at position 31 of the B
chain. The functional
significance of this heterogeneity is not currently understood.
Peaks B3, B5, B7, B8, and B9 contained the Gly to Cys mutation at
position 15 of the B chain (Table 2). B
1-42 in
peaks B3 and B5 were disulfide cross-linked to cysteine (Table 3). Peaks B7-B9 were B
1-42 dimers
cross-linked by disulfide bonds through the mutated Cys (Fig. 7, b and c) and indicated that two fibrinogen Fukuoka II
molecules were cross-linked to form a 700,000-dalton dimer. A rough
estimation from Fig. 7a (ii) shows that the
amount of fibrinogen in peaks B3 and B5 was almost equal to that in
peaks B7-B9, indicating that the amount of cysteine-fibrinogen
complex is similar to the sum of intermolecular cross-linked dimeric
fibrinogen molecules and intramolecular cross-linked fibrinogen
molecules. Under reducing conditions, all peaks of B3, B5, and
B7-B9 were reduced into two peaks B10 and B12 (Fig. 7a, iii). The peak heights of B10 and
B12 were almost equal to the heights of peaks B11 and B13, indicating
that normal fibrinogen and fibrinogen Fukuoka II are equally expressed
in the heterozygous patients. Taken together with statistic analysis on
rotary shadowing electron micrograph, we estimated that the proportions
of the cysteine-fibrinogen Fukuoka II complex, the intramolecular
cross-linked fibrinogen Fukuoka II, the albumin-fibrinogen Fukuoka II
complex, and the intermolecular cross-linked dimeric fibrinogen were
approximately 45, 40, 10, and 5%, respectively. In fibrinogen Fukuoka
II, almost half of the mutated molecules complexed with cysteine.
The same mutation designated as fibrinogen Fukuoka II has been
reported as fibrinogen Ise(17) . However, fibrinogen Ise showed
prolonged thrombin time with normal reptilase time and defective
release of FPB by thrombin but normal repolymerization of fibrin
monomers pretreated with reptilase. Contrary to fibrinogen Ise, no
repolymerization occurred from des-(A)-fibrin monomer Fukuoka II at
physiological ionic strength ( Fig. 2and Fig. 3). Despite
the same mutation from Gly to Cys at residue 15 of the B chain,
analytical data on the fibrinogen Ise and Fukuoka II mutants were not
consistent with each other. The most important differences are the
effects of the mutated B
chain on repolymerization, i.e. repolymerization of fibrinogen Fukuoka II was disrupted as long as
the B
chain associated with the original fibrinogen molecule. The
bulky amino-terminal region of the B
chain sterically disturbed
the repolymerization of fibrinogen Fukuoka II but did not affect the
structure of the A
chain, because the release of FPA by thrombin
or reptilase was not depressed (Fig. 1c) as in the case
of fibrinogen Ise. Steric hindrance of fibrin monomer repolymerization
by the amino-terminal region of mutated B
chain has been reported.
Abnormal fibrinogens with a substitution of Arg to Cys at position 14
of the B
chain (11, 13, 14) show
prolonged reptilase time and impaired repolymerization of
des-(A)-fibrin monomers as described here for fibrinogen Fukuoka II,
but these properties were not observed for fibrinogen Ise(17) .
Another difference between fibrinogen Ise and fibrinogen Fukuoka II is
that the mutated Cys in fibrinogen has been reported in the free
sulfhydryl form(17) , whereas our results show that all mutated
Cys residues are disulfide-bonded to other molecules. The basis for the
discrepancies between fibrinogen Ise and fibrinogen Fukuoka II
described above is not clear but may involve additional mutations in
the fibrinogen Ise molecule that have not yet been determined.