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Blood, Vol. 91 No. 9 (May 1), 1998:
pp. 3282-3288
Factor XIIIa Cross-Linking of the Marburg Fibrin:
Formation of
 m· n-Heteromultimers and
the -Chain-Linked Albumin· Complex, and Disturbed
Protofibril Assembly Resulting in Acquisition of Plasmin Resistance
Relevant to Thrombophila
By
Teruko Sugo,
Chizuko Nakamikawa,
Mikihiro Takebe,
Isao Kohno,
Rudorf Egbring, and
Michio Matsuda
From the Division of Hemostasis and Thrombosis Research, Institute of
Hematology, Jichi Medical School, Tochigi, Japan; and the Medical
University Clinic Marburg/Lahn, Marburg, Germany.
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ABSTRACT |
The truncated A -chain of fibrinogen Marburg is partly linked with
albumin by a disulfide bond. Based on the recovery of the first six
amino acid residues assigned to the subunit polypeptides of fibrinogen
(the A-and  -chains) and albumin, 0.33 mol of albumin was
estimated to be linked to 1 mol of the Marburg fibrinogen. When the
Marburg fibrinogen was clotted with thrombin-factor
XIIIa-Ca2+, various mn
heteromultimers were produced, and part of the albumin was cross-linked
to the -chain. Acid-solubilized Marburg fibrin monomer failed to
form large aggregates that could be detected by monitoring turbidity at
A350, but it was able to enhance tissue-type plasminogen-activator-catalyzed plasmin generation, though not as
avidly as the normal control, indicating that the double-stranded protofibrils had, to some extent, been constructed. This idea seems to
be supported by normal factor XIIIa-catalyzed cross-linking of the
fibrin -chains. However, the cross-linked Marburg fibrin, being
apparently fragile and translucent, was highly resistant against
plasmin, and its subunit components were considerably retained for 48 hours as noted by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis. Although the exact mechanisms are still unclear, the
albumin-incorporated factor XIIIa-cross-linked Marburg fibrin seems to
have undergone a critical structural alteration(s) to acquire
resistance against plasmin. This aquisition of plasmin resistance may
be contributed to the postoperative pelvic vein thrombosis and
recurrent pulmonary embolisms in the patient after caesarian section
for her first delivery at the age of 20 years.
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INTRODUCTION |
CONGENITALLY ABNORMAL fibrinogens
with a cysteine (Cys) substitution have been shown to be linked with
either albumin as reported for fibrinogens Nijmegen (B Arg44 to
Cys),1 Ijmuiden (B Arg14 to Cys),1
Dusart2 and Chapel Hill III (A Arg554 to
Cys),3 and Fukuoka (B Gly15 to Cys),4 or a
single Cys molecule as was described in fibrinogen Osaka II ( Arg275
to Cys).5 Otherwise, the aberrant polypeptide subunits may
be disulfide linked with their counterpart in the same molecule as
shown in fibrinogens Metz6 and Kawaguchi.7 Any
abnormal fibrinogens with a Cys substitution seem to be loaded with a
severe structural alteration due to disulfide-linked additives. In
particular, the fibrinogen-associated albumin could affect the fibrin
polymerization by steric hindrance, and thus interpretation for the
structure-function relationship in such abnormal molecules is most
difficult.
Fibrinogen Marburg is a homozygous dysfibrinogen lacking amino acid
residues A 461-610 due to premature appearance of a stop codon TAA
for AAA coding for A lysine (Lys) 461 of human fibrinogen. Because of
this truncation, Cys at position 442 in the Marburg fibrinogen
A-chain loses its disulfide partner, A Cys 472, and part of the
Marburg A-chain forms a disulfide bridge with albumin.8 This dysfibrinogen was found in a 20-year-old woman suffering from
recurrent thromboembolic diseases in addition to a severe uterine
bleeding after delivery of her first child by caesarian section.9 In this study, we attempted to estimate the
amount of albumin disulfide linked to the Marburg fibrinogen and
observed the behavior of the albumin molecule on formation of factor
XIIIa-cross-linked fibrin. We also studied fibrin facilitation of
plasmin formation catalyzed by tissue-type plasminogen activator (t-PA)
and degradation of cross-linked fibrin by plasmin in relation to the
postoperative recurrent thromboembolic diseases observed in the
patient.
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MATERIALS AND METHODS |
Purification of fibrinogen.
Fibrinogen was purified from the patient's and normal plasma by
immunoaffinity chromatography using a monoclonal antibody, IF-1, which
recognizes the calcium-dependent conformation of the D domain of human
fibrinogen, essentially as described previously.10 By
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting under nonreducing conditions, the purified Marburg
fibrinogen was confirmed to consist of at least five species, native
and partly degraded fibrinogen species free of albumin, and those
linked with one or two molecules of albumin (profiles not shown). As
anticipated from the data presented by Koopman et al8 on
this abnormal fibrinogen, the A-chain was found to migrate close to
the -chain on SDS-PAGE under reducing conditions, and a faint
protein band noted at the position for the normal A-chain was
confirmed to be albumin by immunoblotting (profiles not shown).
Aggregation studies of preformed, acid-solubilized fibrin monomer
and enhancement of t-PA-catalyzed activation of plasminogen by the
polymerizing fibrin monomer.
Fibrinogen Marburg (0.5 mg/mL) was clotted with human -thrombin (5 NIH U/mL) prepared from prothrombin as described
elsewhere11 at 37°C for 30 minutes and allowed to form
gels for 40 minutes at 37°C and for successive 18 hours at 4°C.
The Marburg fibrin clots appeared to be translucent and fragile as
compared with turbid and solid normal fibrin clots. The fibrin clots
were washed three times with tris-buffered saline (TBS)
and solubilized with 20 mmol/L acetic acid and subjected to aggregation
studies essentially as described elsewhere.5 Enhancement of
t-PA-catalyzed plasminogen activation by polymerizing fibrin monomer
was measured in 200 µL of the reaction mixture composed of
acid-solubilized fibrin monomer (0.2 µmol/L), two-chain t-PA (4 U/mL;
Sumitomo Pharmaceutical Co,Osaka, Japan), plasminogen (1.0 µmol/L),
and a chromogenic substrate, S-2251
(H-D-valine-leucine-lysine-p-nitroanilide; 0.3 mmol/L).
Plasmin generation was monitored by measurement of A405 at 2-minute
intervals.
Factor XIIIa-catalyzed cross-linking of fibrin.
The normal and Marburg fibrinogen (0.5 mg/mL) were clotted at 25°C
with -thrombin (2.5 NIH U/mL) and factor XIII (1.25 U/mL, prepared
from pooled plasma essentially described elsewhere,12 and
the activity of factor XIIIa was expressed as amine-incorporating units
as described by Lorand et al13) in the presence or absence of 2-plasmin inhibitor (2-PI, 10 µg/mL;
Calbiochem-Novabiochem, La Jolla, CA) in 32 µL of TBS containing 5 mmol/L CaCl2. At timed intervals, the reaction was stopped
by addition of ethylenediaminetetraacetate-Na2 (EDTA, 2 mmol/L), and the clots were dissolved in the reducing SDS-PAGE solution
(1.5 mol/L Tris-HCl, pH 8.8 containing 3% SDS, 8 mmol/L
dithiothreitol, 2 mmol/L EDTA, and 8 mol/L urea). One microgram of
proteins per lane was loaded to SDS-PAGE gels. For immunoblot analyses,
antibodies against the A (148-160) residue segment (a kind gift from
Dr Willem Nieuwenhuizen, Gaubius Laboratory, Leiden, The Netherlands)
and against the (89-273) residue segment, JIF25,5 were
used to specify the subunit polypeptides. Incorporation of
2-PI into cross-linked fibrin clots was monitored by
autoradiography by using 37 ng of 125I-2-PI
(5 × 109 cpm/mg) in the cross-linking reaction
mixture. For this experiment, 2-PI was labeled with
125I by the Iodobeads method using Na125I
essentially according to the manufacturer's instruction (Pierce, Rockford, IL). After extensive washing with Tris-HCl, pH 7.4 containing 0.5 mol/L NaCl and 0.1 % Tween 20, the radiolabeled clots were subjected to SDS-PAGE followed by autoradiography.
Plasmic degradation of factor XIIIa-cross-linked fibrin.
Normal and the Marburg fibrinogen (0.5 mg/mL) were clotted with 5 NIH
U/mL -thrombin in the presence of 2 U/mL factor XIII, 4 U/mL t-PA,
10 nmol/L plasminogen, and 2 mmol/L CaCl2 at 37°C. At
timed intervals, the fibrin clots were dissolved in the reducing SDS-PAGE solution, and 2 µg of proteins per lane was subjected to
SDS-PAGE.
Characterization of factor XIIIa-cross-linked fibrin formed in the
presence of 2-PI.
To characterize the Marburg fibrin in relation to clinical
thromboembolic diseases, we attempted to analyze the subunit
compositions of cross-linked Marburg fibrin formed in the presence of
2-PI. In this experiment, 3 µg of 2-PI
was added to 90 µg of fibrinogen, 0.75 NIH U of -thrombin, and 0.1 U of factor XIII in 300 µL of TBS containing 5 mmol/L
CaCl2, and the mixture was allowed to clot for 30 minutes
at 37°C. After treatment with 2 mmol/L EDTA, the clots were
precipitated by centrifugation for 30 minutes at 12,000 rpm and
solubilized with 150 µL of the reducing SDS-PAGE solution. To get
precise band separation in SDS-PAGE gels and efficient recovery of the
resolved peptides therefrom, as much as 90 µg of proteins was divided
in two parts and loaded separately onto 7.5% to 12.5% polyacrylamide
gradient gels (2-mm thick and 15-cm long separation gels). The resolved
polypeptides were blotted onto Problot membrane (PE-Applied Biosystems,
Foster City, CA) according to the manufacturer's instructions. After
protein staining with Coomassie Brilliant Blue (CBB), amino acid
sequence analysis of polypeptide bands was conducted with a protein
sequencer, model 476 A (PE; Applied Biosystems). The recovery of
individual polypeptides was estimated from the recovery of
phenylthiohydantoin (PTH) amino acids in the first five
cycles.
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RESULTS |
Amount of albumin disulfide linked to the Marburg fibrinogen.
Based on the recoveries of the first six amino acid residues assigned
to the subunit polypeptides of fibrinogen and albumin, we estimated the
amount of albumin linked to fibrinogen in duplicate runs
(Table 1). At each cycle, recoveries of
only representative amino acid residues for individual polypeptides
were used for estimation as indicated by bold letters. The amount of
albumin per mole of fibrinogen was calculated to be 0.34 mol for the
first run and 0.32 mol for the second run from the ratio of albumin versus 2-chains (2) representing a dimeric molecule of
fibrinogen. Thus, approximately 0.33 mol of albumin was found to be
linked to the Marburg fibrinogen by a disulfide bond. Because no free sulfhydryl (SH) groups in the Marburg fibrinogen were
present, the remainder of unpaired A Cys442 was expected to be
linked with other substances such as a Cys molecule as reported for
fibrinogen Osaka II.5
Aggregation of acid-solubilized fibrin monomer and facilitation by
the polymerizing fibrin monomer of t-PA-catalyzed plasminogen
activation.
Although the turbidity of the acid solubilized fibrin monomer failed to
increase when monitored by A350 (Fig 1),
t-PA-catalyzed plasminogen activation was considerably enhanced in the
presence of the polymerizing Marburg fibrin monomer at pH 7.4, though
not as avidly as in the control (Fig 2).
The result indicated that the Marburg fibrin monomer molecules were
able to form double-stranded protofibrils to a certain extent and that
failure to form fibrin gels could largely be attributed to impairment
of lateral association of the double-stranded protofibrils, normally
mediated by untethered carboxy-terminal regions of the -chains (C
domains).14
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| Fig 1.
Polymerization of acid-solubilized fibrin monomer was
measured by monitoring A350 nm. The normal and Marburg acid-solubilized des-AB fibrin monomers were prepared as described in Materials and
Methods. The reaction was started by dilution of 18.4 µL of fibrin
monomer (20 µg) with 500 µL of 25 mmol/L imidazole-buffered saline,
pH 7.4, and aggregation was monitored by A350.
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| Fig 2.
Facilitation of t-PA-catalyzed activation of plasminogen
by polymerizing fibrin monomer. Enhancement of t-PA-catalyzed
plasminogen activation by fibrin monomer was measured in 180 µL of
the reaction mixture composed of acid-solubilized fibrin monomer (0.2 mmol/L), plasminogen (1.0 µmol/L), t-PA (4 U/mL), and S-2251 (0.3 µmol/L) as described in Materials and Methods. Plasmin generation was measured by monitoring A405 nm at 2-minute intervals.
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Factor XIIIa-catalyzed cross-linking of the fibrin - and
-chains.
Despite severely altered fibrin monomer aggregation, factor
XIIIa-catalyzed cross-linking of the Marburg fibrin -chains took place in a normal fashion (Fig 3). The
result indicated that the initial double-stranded oligomers had been
constructed by the A polymerization site in the E domain and its
complementary a site in the D domain.15-17 Besides bands
for the -dimer, at least two higher-molecular-weight bands
containing the -chain were present as indicated by numbers 1 and 2 in the patient's sample. There were also bands denoted by N1 and N2 in
the normal sample, apparently corresponding to the two bands for the
Marburg fibrin, suggesting that they are heteromultimers composed of
the - and -chains. Indeed, this was confirmed by immunoblotting
with an antibody recognizing the fibrin -chain (148-160) segment.
Furthermore, formation of high-molecular-weight -polymers was almost
missing in the patient's cross-linked fibrin, indicating that
intramolecular -chain cross-linking was severely disturbed by the
lack of putative amine donor lysine residues at positions 508 and 556 or 562.18
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| Fig 3.
Factor XIIIa-catalyzed cross-linking of the fibrin
-chain analyzed by immunoblotting. Fibrinogen was clotted with
thrombin and factor XIII in the presence of CaCl2. At timed
intervals, the clots were subjected to SDS-PAGE followed by
immunoblotting using an anti--chain antibody. Besides the
-dimer, high-molecular-weight polypeptides 1 and 2 were formed in
the Marburg fibrin, and the corresponding peptides, N1 and N2, were
formed in the normal fibrin. The molecular mass and the positions of
the marker proteins are indicated in the left margin.
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Plasmic degradation of factor XIIIa-cross-linked fibrin.
The Marburg fibrinogen was hardly clotted by an ordinary amount of
thrombin used for the clotting assay, but addition of large amounts of
thrombin together with factor XIII and Ca2+ resulted in
formation of solid but fragile gels. Although the fibrin gels thus
formed appeared to be fragile and translucent, they were found to be
highly resistant against plasmin. In the degradation experiments of
t-PA and plasminogen-enriched cross-linked fibrin, the Marburg fibrin
clots remained as solid gels for more than 72 hours at 37°C. In
fact, their subunit polypeptides were found to be largely preserved
even at 48 hours as compared with those of normal control, which had
been digested into much smaller segments at 36 hours of incubation as
observed by SDS-PAGE (Fig 4).
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| Fig 4.
Fibrin degradation by t-PA-catalyzed plasmin digestion.
Fibrinogen was clotted with thrombin and factor XIII in the presence of
plasminogen (1.0 nmol/L), t-PA (4 U/mL), and CaCl2. At 6 hours, 24 hours, 36 hours, and 48 hours, the reaction mixture was
subjected to SDS-PAGE under reducing conditions.
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Factor XIIIa-catalyzed cross-linking of 2-PI to the
Marburg fibrin.
2-PI is known to be cross-linked by factor XIIIa to the
fibrin -chain,19 where Gln2 of 2-PI
serves as amine acceptor and A Lys303 of fibrin as amine
donor.20 When the cross-linking profile of
2-PI to the Marburg fibrin was studied by
autoradiography, the radiolabel was distributed to a
115-kD band corresponding to a complex of the Marburg
-chain and 2-PI (band 2) and to higher-molecular-weight proteins (band 4, Mr > 205 × 103) representing the polymerized Marburg -chains
(Fig 5). The extremely high-molecular-weight protein complex containing 2-PI in
the normal sample (band 5) was, however, missing in the patient's sample. These results were in agreement with the observation as reported by Sobel et al21 on an in vitro plasma clotting
system using the patient's plasma.
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| Fig 5.
Factor XIIIa-catalyzed cross-linking of
2-PI to the Marburg fibrin analyzed by autoradiography.
Fibrinogen was clotted with thrombin and factor XIII in the presence of
125I-2-PI and CaCl2 at 37°C
for 30 minutes. After extensive washing, the radiolabeled clots were
subjected to SDS-PAGE followed by autoradiography. Band 1 represents
2-PI and bands 2 and 3 represent a 115-kD (Marburg
-chain-2-PI) and a 135-kD (normal
-chain-2-PI) complex, respectively. Bands 4 and 5 represent high-molecular-weight complexes.
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Behavior of the -chain-linked albumin on factor XIIIa
cross-linking of the Marburg fibrin.
When the Marburg fibrinogen was clotted with thrombin, factor XIIIa,
and Ca2+ and analyzed by immunoblotting using an
antialbumin antibody, albumin was found to be distributed to a 115-kD
protein complex (Fig 6, band 2 in lane A)
as well as a 66-kD protein (Fig 6, band 1 in lane A), suggesting that
some of the -chain-linked albumin molecules had been cross-linked
to an about 48-kD polypeptide subunit, ie, either the -chain or the
truncated -chain, by serving as substrate for factor XIIIa. The
factor XIIIa-catalyzed cross-linking was also observed regardless of
the presence or absence of 2-PI (band 2 in lane B),
indicating that the fibrinogen-linked albumin did not share the
cross-linking site of A Lys303 with 2-PI and that the
albumin was most likely cross-linked to the -chain but not to the
-chain.
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| Fig 6.
Behavior of fibrinogen-associated albumin in the
cross-linked Marburg fibrin as analyzed by immunoblotting. Fibrinogen
Marburg was clotted with thrombin and factor XIII in the presence (A) or absence (B) of 2-PI and then subjected to
immunoblotting using an anti-human albumin antibody. Band 1 represents
noncross-linked albumin, and band 2 represents the cross-linked albumin
with a fibrin-derived subunit.
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To further characterize the factor XIIIa-catalyzed cross-linking of
the fibrinogen-linked albumin in the presence of 2-PI, we conducted amino acid analysis of the cross-linked polypeptide complexes resolved by SDS-PAGE under reducing conditions. To obtain enough amounts of polypeptides for sequence analysis of the 115-kD band, an unusually large amount, as much as 90 µg, of the Marburg fibrinogen was used for this study (for details, see Materials and
Methods). In SDS-PAGE gels, at least 10 major polypeptide bands were
noted (Fig 7, bands 1 to 10 in lane P). In
a 115-kD polypeptide band (band 7), we were able to assign albumin and 2-PI in addition to the - and -chains of fibrin at
an approximate molar ratio of 5:3:1:1, based on PTH-amino acids
recovered in the first five cycles (Table
2). In other polypeptide bands we could assign the monomeric fibrin
subunits, 2-PI and albumin (bands 1 to 4), the -dimer
(band 6), or heteromultimers consisting of the - and -chains
(bands 8 to 10). Together with approximate molecular sizes of these
polypeptide bands, we assigned most probable heteromultimers for the
polypeptide complexes (Table 3).
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| Fig 7.
Polypeptide compositions of proteins derived from
2-PI-incorporated cross-linked fibrin. Fibrinogen (90 µg) was clotted with thrombin and factor XIII for 30 minutes in the
presence of CaCl2 and 2-PI. The clots were
solubilized and subjected to PAGE in two lanes under reducing
conditions followed by blotting onto PVDF-membranes as described in
Materials and Methods. The protein bands were stained with CBB. Major
bands 1-9 in the patient's sample were separetely subjected to
amino-terminal sequence analysis.
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DISCUSSION |
Fibrinogen Marburg is a unique dysfibrinogen, in that (1) the
carboxy-terminal 150 residues are missing due to premature appearance of a stop codon TAA for AAA coding for A Lys461;
(2) because of truncation of the A-chain, A Cys442 loses its
disulfide bond partner A Cys472, and as a consequence some A
Cys442 residues are linked with albumin by a disulfide bond. Indeed,
the amount of disulfide-linked albumin to the Marburg fibrinogen was
calculated to be 0.33 mol per mol of fibrinogen. In other words, one in
every three Marburg fibrinogen molecules, or one in every six Marburg A-chains is linked with albumin near its carboxyl-terminus; and (3)
the abnormality is associated with concomitant severe bleeding and
thromboembolic diseases.8,9
In normal fibrinogen, the two carboxy-terminal A-chain segments
(C domains) interact with each other and associate with the central
E domain. On thrombin-cleavage of fibrinopeptides A and B, the C
domains are loosened from the E domain and then untethered, thereby
becoming available for association with other C domains in promoting
lateral association of fibrin protofibrils.12 In the
Marburg fibrinogen, cleavage of fibrinopeptides A and B proceeded in a
normal fashion (profiles not shown), and subsequent fibrin monomer
assembly to form double-stranded protofibrils may also have progressed
nearly normally, as evidenced by normal factor XIIIa-catalyzed
cross-linking of the fibrin -chain and substantially enhanced
t-PA-catalyzed plasminogen activation in the presence of polymerizing
Marburg fibrin monomer (Fig 2). The failure for the Marburg fibrin
monomer to increase the turbidity as monitored by A350 (Fig 1) could be
accounted for by impaired lateral association of fibrin protofibrils.
Based on our observation, we presume that the Marburg fibrin monomer
molecules are able to bind with one another via the set of A-a
polymerization sites and to form double-stranded fibrin protofibrils.
These protofibrils are, however, unable to associate laterally in a
normal fashion because of the truncated -chain lacking the
interaction sites assigned to the C domain15 and the
presence of the albumin molecule between the strands. In addition,
factor XIIIa-catalyzed intermolecular ligation of the C domains may
not occur because the putative amine donor Lys residues at positions
508 and 556 or 562 are all missing in the Marburg -chain. This
interpretation may well agree with the delayed -polymer formation
and lack of the extremely high-molecular-weight -polymer complex in
the patient's fibrin in SDS-PAGE gels (data not shown). When the
Marburg fibrinogen was clotted with thrombin in the presence of factor
XIII, 2-PI, and Ca2+, a variety of
heteromultimers composed of - and -chains, such as ,
2, 4,
24, and mm (m = 4) were identified
(Fig 7 and Table 3). Moreover, some of the -chain-linked albumin
molecules were found to be cross-linked to an about 48-kD polypeptide,
most probably -chain, forming a 115-kD polypeptide complex (Fig 6),
which was noticeable only when factor XIII was present in the reaction
mixture. This complex was not present in the normal sample either.
Interestingly, formation of the 115-kD complex was not inhibited by
2-PI (Fig 6A), indicating that the albumin did not share
the amine donor A Lys303 with Gln2 of 2-PI. If the
albumin is ligated to the -chain at another sites, a potential
(2-PI) trimer can be formed, but this sort of
trimolecular complex was not identified in this experiment.
We attempted to calculate the amount of cross-linked albumin on the
basis of the recovery of amino acids assigned to albumin in the 115-kD
polypeptide (Fig 7 and Table 2). Assuming that the -chain-ligated
albumin molecules had been transferred at 100% to the membrane and
that they had been totally recovered in the sequence analysis, the
ligated albumin was estimated to be only 0.2% of the total
A-chain-linked albumin. However, an extraordinarily large amount
(as much as 90 µg) of the Marburg fibrin had been applied to SDS-PAGE
gels in this experiment; therefore, the efficiency of protein transfer
to the membrane may have been extremely low. Indeed, a large amount of
proteins was retained in the SDS-PAGE gels even after electroblotting.
Furthermore, the recovery of amino acids in our sequence study is
estimated to be 30% to 40%. If the transfer efficiency was around
30%, the amount of albumin ligated to the -chain could be expected
to be about 2% of the total albumin molecules.
On formation of factor XIIIa-cross-linked double-stranded
protofibrils, the albumin-linked C domain may be aligned closely to
the carboxy-terminal segment of the -chain of an adjacent fibrin
molecule in another strand of the protofibril, and a factor XIIIa-mediated cross-link is introduced between the albumin and either
Gln398 (or 399) or Lys406 of the -chain. Consequently, extraordinary
-chain-albumin--chain bridges are formed in the Marburg fibrin.
At this stage of the investigation, we have no evidence for the amine
donor-acceptor relationship in this cross-linking. In addition to
cross-linking of the albumin to the -chain, the -chain-linked
albumin not involved in the cross-linking with the -chain must have
been integrated into the cross-linked Marburg fibrin clots an affected
their tertiary structure. Taking this sort of disoriented cross-linking
into consideration, the Marburg cross-linked fibrin must have a
distorted tertiary structure, manifesting unusual properties and
behaviors. In fact, the cross-linked Marburg fibrin clots appeared to
be fragile and less turbid, but they were extremely resistant against
plasmin, remaining as solid gels for more than 72 hours and
considerably retaining the subunit polypeptides even at 48 hours of
incubation at 37°C (Fig 4). In view of the fact that fibrin clots
containing thin fibers are digested more slowly by plasmin than intact
normal fibrin clots,22 the Marburg fibrin may also be
composed of thin fibers.
Recently, cross-linked and noncross-linked fibrin gels, derived from
the two types of congenitally abnormal fibrinogens, were characterized
by electron microscopic analyses.23-25 The Caracas II
fibrin, which has a mutation of A Ser434 to Asn linked with an extra
oligosaccharide,26 was shown to have thinner fibers in
diameter and large pore or open areas bounded by local fiber networks.23 The presence of such irregular large pores
allows fluids to get through the fibrin gels without any disturbance as
evidenced by high permeation rates.23 This finding seems to
account for the absence of thrombosis in the proband.27 On the other hand, the Dusart fibrin with an A Arg554 to Gly
substitution partly linked with albumin2 was shown to have
a less ordered structure than normal fibrin, but with many branch
points resulting in greatly diminished pore size and high
stiffness.24,25 In this fibrin, the -chain-linked
albumin may have distorted the fibrin structure and may have also
contributed to the resistance against plasmin noted in the Dusart
fibrin28 and in the Chapel Hill III fibrin29
with the same type of mutation.3 The presence of albumin
near the carboxy-terminal part of the fibrin -chain, defective
lateral association of protofibrils, and extremely high resistance of
fibrin against plasmin relevant to thromboembolic diseases are shared
by the Marburg fibrin.
At this stage, our study is still incomplete, but pieces of information
provided in this study may partly account for the unique Marburg fibrin
clot structure and behaviors relevant to concomitant bleeding and
thromboembolic diseases observed in this patient.
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FOOTNOTES |
Submitted September 18, 1997;
accepted December 10, 1997.
Supported in part by Grants-in-Aid for Scientific Research 06404043, 07680696, and 08407034; and for International Scientific Research
Program, Joint Research Grants 06044196 and 09044329 from the Ministry
of Education, Science and Culture of the Government of Japan.
Address reprint requests to Teruko Sugo, PhD, Division of
Hemostasis and Thrombosis Research, Institute of Hematology, Jichi Medical School, Yakushiji 3311-1, Minamikawachi, Tochigi, 329-0498, Japan.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
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ACKNOWLEDGMENT |
We thank Michiko Takano for her expert secretarial assistance.
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REFERENCES |
1.
Koopman J,
Haverkate F,
Grimbergen J,
Engesser L,
Navakova I,
Kerst AFJA,
Load ST:
Abnormal fibrinogens IJmuiden (BArg14 Cys) and Nijmegen (BArg44Cys) form disulfide-linked fibrinogen albumin complexes.
Proc Natl Acad Sci USA
89:3478,
1992[Abstract/Free Full Text]
2.
Koopman J,
Haverkate F,
Grimbergen J,
Load ST,
Mosesson MW,
DiOrio JP,
Siebenlist KS,
Legrand C,
Soria J,
Soria C,
Caen JP:
Molecular basis for fibrinogen Dusart (A554 ArgCys) and its association with abnormal fibrin polymerization and thrombophila.
J Clin Invest
91:1637,
1993
3.
Wada Y,
Load ST:
A correlation between thrombotic disease and a specific fibrinogen abnormality (A 554 ArgCys) in two unrelated kindred, Dusart and Chapel Hill III.
Blood
84:3709,
1994[Abstract/Free Full Text]
4.
Kamura T,
Tsuda H,
Yae Y,
Hattori S,
Ohga S,
Shibata Y,
Kawabata S,
Hamasaki N:
An abnormal fibrinogen Fukuoka II (Gly-B15Cys). Characterized by fibrin lateral association and mixed disulfide formation.
J Biol Chem
270:29392,
1995[Abstract/Free Full Text]
5. Terukina S, Matsuda M, Hirata H, Takeda Y, Miyata T, Takano T,
Shimonishi Y: Substitution of Arg-275 by Cys in an abnormal
fibrinogen, " Fibrinogen Osaka II." J Biol Chem 263:13579, 1988
6.
Mosesson MW,
DiOrio JP,
Shainoff JR,
Siebenlist KR,
Amrani DL,
Homandberg GA,
Soria J,
Soria C,
Samama M:
Studies on the ultrastructure of fibrin lacking fibrinopeptide B (b-fibrin).
Blood
69:1073,
1987[Abstract/Free Full Text]
7.
Miyata T,
Terukina S,
Matsuda M,
Kasamatsu M,
Takeda Y,
Murakami T,
Iwanaga S:
An amino acid substitution of A arginine-16 to cysteine which forms an extra interchain disulfide bridge between the two A-chains.
J Biochem
102:93,
1987[Abstract/Free Full Text]
8.
Koopman JK,
Haverkate F,
Grimbergen J,
Egbring R,
Lord ST:
Fibrinogen Marburg: A homozygous case of dysfibrinogenemia, lacking amino acids A 461-610 (Lys 461 AAA stop TAA).
Blood
80:1972,
1992[Abstract/Free Full Text]
9.
Fuchs G,
Egbring R,
Havemann K:
Fibrinogen Marburg, a new genetic variant of fibrinogen.
Blut
34:107,
1977[Medline]
[Order article via Infotrieve]
10.
Takebe M,
Soe G,
Kohno I,
Sugo T,
Matsuda M:
Calcium ion-dependent monoclonal antibody against human fibrinogen: Preparation, characterization, and application to fibrinogen purification.
Thromb Haemost
73:662,
1995[Medline]
[Order article via Infotrieve]
11.
Tanabe S,
Sugo T,
Matsuda M:
Synthesis of protein C in human umbilical vein endothelial cells.
J Biochem
109:924,
1991[Abstract/Free Full Text]
12.
Lorand L,
Credo RB,
Janus TJ:
Factor XIII (Fibrin-stabilizing factor).
Methods Enzymol
80:333,
1981
13.
Lorand L,
Urayama T,
Kiewiet JWC,
Nossel HL:
Diagnostic and Genetic studies on fibrin-stabilizing factor with a new assay based on amine incorporation.
J Clin Invest
48:1054,
1969
14.
Veklich YI,
Gorkun OV,
Medved' LV,
Nieuwenhuizen W,
Weisel JW:
Carboxy-terminal portion of the chains of fibrinogen and fibrin. Localization by electron microscopy and the effects of isolated C fragments on polymerization.
J Biol Chem
268:13577,
1993[Abstract/Free Full Text]
15.
Kudryk BJ,
Collen D,
Woods KR,
Blombäck B:
Evidence for localization of polymerization sites in fibrinogen.
J Biol Chem
249:3322,
1974[Abstract/Free Full Text]
16.
Olexa S,
Budzynsky AZ:
Evidence for four different polymerization sites involved in human fibrinogen.
Proc Natl Acad Sci USA
77:1374,
1980[Abstract/Free Full Text]
17.
Laudano AP,
Doolittle RF:
Studies on synthetic peptides that bind to fibrinogen and prevent fibrin polymerization. Structural requirements, number of binding sites, and species differences.
Biochemistry
19:1013,
1980[Medline]
[Order article via Infotrieve]
18.
Corcoran DH,
Ferguson EW,
Fretto LJ,
McKee PA:
Localization of a cross-link donor site in the -chain of human fibrinogen.
Thromb Res
19:883,
1980[Medline]
[Order article via Infotrieve]
19.
Sakata Y,
AokiN:
Cross-linking of 2-plasmin inhibitor to fibrin by fibrin-stabilizing factor.
J Clin Invest
65:290,
1980
20.
Kimura S,
Aoki N:
Cross-linking site in fibrinogen for 2 plasmin inhibitor.
J Biol Chem
261:15591,
1986[Abstract/Free Full Text]
21.
Sobel JH,
Trakht I,
Wu HQ,
Rudchenko S,
Egbring R:
-chain cross-linking in fibrin(ogen) Marburg.
Blood
86:989,
1995[Abstract/Free Full Text]
22.
Gabriel DA,
Muga K,
Boothroyd EM:
The effect of fibrin structure on fibrinolysis.
J Biol Chem
267:24259,
1992[Abstract/Free Full Text]
23.
Mosesson MW,
Siebenlist KR,
Hainfeld JF,
Wall JS,
Soria J,
Soria C,
Caen JP:
The relationship between the fibrinogen D domain self-association/cross-linking site (XL) and the fibrinogen Dusart abnormality (AR554C-albumin).
J Clin Invest
97:2342,
1996[Medline]
[Order article via Infotrieve]
24.
Woodhead JL,
Nagaswami C,
Matsuda M,
Arocha-Piñango CL,
Weisel JW:
The ultrastructure of fibrinogen Caracas II molecule, fibers, and clots.
J Biol Chem
271:4946,
1996[Abstract/Free Full Text]
25.
Collet JP,
Woodhead JL,
Soria J,
Soria C,
Mirshahi M,
Caen JP,
Weisel JW:
Fibrinogen Dusart: Electron microscopy of molecules, fibers and clots, and viscoelastic properties of clots.
Biophys J
70:500,
1996[Medline]
[Order article via Infotrieve]
26.
Maekawa H,
Yamazumi K,
Muramatsu S,
Kaneko M,
Hirata H,
Takahashi N,
de Bosch NB,
Carvajal Z,
Ojeda A,
Arocha-Piñango,
Matsuda M:
An A Ser-434 to N-glycosylated Asn substitution in a dysfibrinogen, fibrinogen Caracas II, characterized by impaired fibrin gel formation.
J Biol Chem
266:11575,
1991[Abstract/Free Full Text]
27.
Arocha-Piñango CL,
de Bosh NB,
Torres A,
Caires-Tronchoni D,
Marci R,
Vidal H,
Rizzo GM:
Dysfibrinogenemias in Latin America. A cooperative study group.
Rev Iberoamer Thromb Hemostasia
3:110,
1990
28.
Soria C,
Soria J,
Caen P:
A new type of congenital dysfibrinogenemea with defective fibrin lysis Dusart syndrome: Possible relation to thrombosis.
Br J Haematol
53:575,
1983[Medline]
[Order article via Infotrieve]
29.
Carrell N,
Gabriel DA,
Blatt PM,
Carr ME,
McDonagh J:
Hereditary dysfibrinogenemea in a patient with thrombotic diseases.
Blood
62:439,
1983[Abstract/Free Full Text]
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Abstract
Introduction
Abstract