|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 7 (April 1), 2000:
pp. 2297-2303
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
The  EC domain of human fibrinogen-420 is a stable
and early plasmin cleavage product
Dianne Applegate,
Lara Stoike Steben,
Kathe M. Hertzberg, and
Gerd Grieninger
From the Lindsley F. Kimball Research Institute of the New York
Blood Center, New York, NY.
 |
Abstract |
Human fibrinogen-420, ( E  )2, was
isolated from plasma and evaluated for its ability to form clots and
for its susceptibility to proteolysis. Clotting parameters, including
cross-linking of subunit chains, of this subclass and of the more
abundant fibrinogen-340 ()2, were found to be
similar, suggesting little impact of the unique EC
domains of fibrinogen-420 on coagulation. Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of
plasmic digestion patterns revealed production from fibrinogen-420
of the conventional fibrinogen degradation products, X, Y, D, and E, to
be comparable to that from fibrinogen-340 in all respects except the
presence of at least 2 additional cleavage products that were shown by
Western blot analysis to contain the EC domain. One was
a stable fragment (ECX) comigrating with a 34-kd yeast
recombinant EC domain, and the other was an apparent precursor. Their release occurred early, before that of fragments D and
E. Two bands of the same mobility and antibody reactivity were found in
Western blots of plasma collected from patients with myocardial
infarction shortly after the initiation of thrombolytic therapy.
(Blood. 2000;95:2297-2303)
© 2000 by The American Society of Hematology.
| |
Introduction |
Fibrinogen, the precursor of the fibrin clot, is
composed of paired sets of 3 types of chains:  , , and . These
chains share amino terminal homologies, but the long, random coil at
the carboxyl terminus of the conventional chain (C) is distinct
from the related globular domains at the ends of the conventional and chains (C and C, respectively). However, in a subclass of fibrinogen molecules, accounting in humans for 1% to 3% of the total,1 a globular C-terminal domain (EC),
comparable to C and C, further elongates the chain to form an
extended chain isoform (E).2,3 The
EC, C, and C domains share approximately 40%
amino acid identity,2 and the folds of their -carbon
backbones are largely superimposable, as recently shown by x-ray
crystallography.4-7
The human E and conventional chains are
identical in sequence through the length of the C region to Val610,
but in E this valine is followed by Arg611 and the 236 residues encoded by the gene's 6th exon
(Asp612-Gln847)2 that form the globular domain. This
EC domain is the only region of any normal chain to
be glycosylated in human fibrinogen.3,8 From sequence analyses, it is now known that the E isoform occurs
throughout the vertebrate kingdom, and that the EC
domain is the single largest conserved portion of the
chain.9-11
Both conventional fibrinogen and E-containing fibrinogen
are symmetrical and heavily disulfide-bonded
hexamers.3,8,12,13 We have introduced nomenclature based on
size for these 2 human fibrinogen subclasses: fibrinogen-340 for the
conventional 340-kd ()2 form and fibrinogen-420
for (E)2 with its predicted mass of
420 kd.
The general spatial arrangement of the fibrinogen subunits has been
well studied, and the molecule's primary functioning in clot formation
and lysis has been characterized in detail.12,13 Briefly,
the fibrinogen chain amino termini are clustered in a central E-domain
from which 2 coiled coil regions emerge, each ending in a distal
carboxyl terminal D-domain that contains the C and C globular
domains and an interior region of the chain. A clot begins to form
when thrombin cleaves fibrinopeptides A and B from the amino termini of
fibrinogen's and chains, thereby generating a fibrin monomer.
Each newly exposed -chain N-terminus spontaneously interacts with a
complementary polymerization pocket located in the carboxyl-terminal
domain of a chain from 2 other fibrin molecules, noncovalently
associating the fibrin monomers in a half-staggered, double array of
protofibrils. Thus organized, the polymers become covalently stabilized
by the thrombin-activated transglutaminase, factor XIIIa, which
catalyzes establishment of  -amino-(-glutamyl) lysine cross-links
between chain carboxy termini and, to a lesser extent, between chains. Both formation and cross-linking of fibrin polymers are
promoted by calcium ions, the binding of which also limits
susceptibility of fibrin(ogen) to proteases such as plasmin. Under
attack by plasmin and other proteases, fibrin clots are ultimately
depolymerized, restoring plasma fluidity.
A distinctive role for fibrinogen-420 has yet to be elucidated.
Properties of the EC domains have been explored in
mutation studies of recombinant fibrinogen-420 assembly in COS
cells8 and in biochemical and structural analyses with a
recombinant human EC domain (rEC)
expressed in the yeast Pichia pastoris.6,14 Like
C and C,4,7 the EC domain has a
calcium-binding site.6,14 However, in lieu of the
negatively charged pockets of C and C that allow fibrin monomer
polymerization,7,15 EC has a cleft with
neutral residues at its center.6
Physical separation of fibrinogen-420 from fibrinogen-340 molecules in
plasma has presented a formidable challenge because of their
disproportionate representation and their similarity of structure but
for the EC domains of fibrinogen-420. In the current
article, we describe a procedure for the isolation of highly purified
fractions of fibrinogen-420 and of fibrinogen-340 from human plasma. We
verify fibrinogen-420's native structure as a symmetrical molecule. We
show that both species clot in similar fashion and that their
degradation by plasmin yields comparable patterns of proteolysis, with
1 notable exception: the conserved EC domain is released
from fibrin(ogen)-420 as a stable cleavage product
(ECX).
| |
Materials and methods |
Materials
Human umbilical cord plasma was donated through the Placental Blood
Program of the New York Blood Center under the direction of Pablo
Rubinstein. Blood collection and plasma processing for these samples
have been described elsewhere.1,16 Plasma samples from
patients undergoing thrombolytic therapy with recombinant tissue
plasminogen activator or streptokinase were generously provided by Joan
Sobel (Columbia Presbyterian Medical Center, NY). The samples were
archival, originating from The TIMI Study Group's Phase 1 Trial.17
Human plasmin and -thrombin were generous gifts of Michael Mosesson
(Sinai Samaritan Medical Center, Milwaukee, WI). Recombinant factor
XIII was graciously provided by Paul Bishop (ZymoGenetics, Seattle, WA).
Rabbit anti-fibrinogen was purchased from DAKO Corporation
(Carpenteria, CA). Rabbit anti-EC #9395, also known as
anti-VI, was generated against a recombinant human EC
domain expressed in Escherichia coli; it has been described
previously.2,3 Rabbit anti-(615-625), a gift from Russ
Doolittle (University of California at San Diego, La Jolla, CA), was
generated against the synthetic peptide TSPLGKPSLSP. This sequence
corresponds to the carboxyl-terminal residues of the (1-625)
chain18,19 before it is processed to the predominant plasma
form (1-610).20 On Western blots, the antibody reacted
strongly with fibrinogen in spent medium from HepG2 culture, obtained
as described previously 3; these cells are known to secrete
a significant proportion of (1-625)-containing
fibrinogen.21
Mouse monoclonal anti-EC #29-1 was also generated
against a recombinant human EC expressed in E
coli and is specific for an epitope at the domain's
C-terminus.14 Monoclonal anti-(603-610) antibody F-48,
kindly provided by Gary Matsueda (Bristol-Myers Squibb, Princeton,
NJ), was generated against the synthetic octapeptide, GHAKSRPV,
representing the common chain carboxyl terminus; it is specific for
processed but nondegraded chains in plasma
fibrinogen.22 Monoclonal anti- chain antibody, 1D4,
supplied by Bohdan Kudryk (New York Blood Center, New York, NY), has
been described previously.23,24
Column chromatography
Human fibrinogen (fraction I-2) was prepared from umbilical cord
plasma according to Mosesson and Sherry25 and
dialyzed against 0.005 mol/L Tris phosphate, pH 8.6. (In all Tris
phosphate buffers, the molarity refers to
phosphate26.) The material (30 ml at a
concentration of 4 mg/ml) was applied to a Mono Q HR 10/10 anion
exchange column (Pharmacia, Piscataway, NJ) that previously had been
equilibrated with the same buffer. Bound protein was eluted using a
stepwise gradient starting from 0.005 mol/L Tris phosphate, pH 8.6, to
a final 0.5 mol/L Tris phosphate, pH 4.2. Eluted protein was collected
in 2.5-mL fractions. For storage and further analysis, pooled fractions
were either dialyzed against 125 mmol/L NaCl, 25 mmol/L
HEPES (pH 7.4) or concentrated and exchanged to the buffer using a YM10
ultrafiltration membrane within an Amicon stirred cell (Amicon,
Beverly, MA).
SDS-PAGE and Western blot analysis
Samples were prepared for electrophoresis in Laemmli sample buffer
in the absence or presence of 0.1 mol/L dithiothreitol27 and were separated on SDS-PAGE using a Mini-Protean II Electrophoresis Cell (Bio-Rad, Hercules, CA). Protein was stained with Gel Code Blue
Stain Reagent (Pierce, Rockford, IL). Electrophoretic transfer onto
0.2-µm nitrocellulose membranes was performed with a Mini Trans-Blot
Cell (Bio-Rad). Membranes were incubated with either primary mouse
monoclonal or rabbit polyclonal antibodies followed by horseradish
peroxidase-labeled secondary antibody, either goat antimouse IgG (Pierce) or goat antirabbit IgG (Pierce), as appropriate. To visualize enzyme activity, signals were developed by enhanced chemiluminescence (SuperSignal Chemiluminescent Substrate, Pierce) and filmed.
Fibrinogen clottability
Clottability of the purified fibrinogen fractions was determined as
previously described28 using thrombin (1 U/mL) and 125 mmol/L NaCl, 25 mmol/L HEPES, (pH 7.4), and 5 mmol/L CaCl2.
Polymerization turbidity curves
Polymerization of fibrinogen species was evaluated by measuring
turbidity changes with time at 350 nm using a Lambda 2 spectrophotometer (Perkin Elmer, Norwalk, CT) equipped with a Peltier
temperature-regulated cuvette holder. Measurements were made at
25°C in 100-µL quartz cuvettes. Data were collected with a
sampling interval of 0.2 seconds and analyzed using UVWINLAB software.
Factor XIIIa-catalyzed cross-linking
Cross-linking reactions were carried out in 125 mmol/L NaCl, 25 mmol/L HEPES (pH 7.4), and 5 mmol/L CaCl2 at room
temperature. Reactions were initiated by the addition of thrombin (0.5 U/mL) to a mixture containing fibrinogen (0.36 mg/mL), either
fibrinogen-420 or fibrinogen-340, and recombinant human factor XIII (10 µg/mL). Reactions were stopped at specified times by the addition of
Laemmli sample buffer and boiling.
Digestion with plasmin
Proteolysis by plasmin was conducted with substrates (0.45 mg/mL) in
a buffer containing 125 mmol/L NaCl, 25 mmol/L HEPES, pH 7.4, and 5 mmol/L CaCl2 at 37°C. Proteolysis was initiated by the
addition of plasmin to a final concentration of 0.03 U/mL. At specified
times, aliquots were removed and the reaction was stopped by mixing
with Laemmli sample buffer and boiling.
| |
Results |
Purification of E-fibrinogen
The paucity of positively charged amino acids in the EC
domains of fibrinogen-4202,6 implies that the molecule will be more negatively charged than the more abundant fibrinogen-340. This
difference was the rationale for attempting separation of the 2 species
by anion exchange chromatography. As shown by the elution profile in
Figure 1, separation of human fibrinogen
(fraction I-2) into 2 separate, unequal peaks was accomplished using a
Mono Q column with a stepwise gradient of Tris phosphate. A steep step from the starting buffer, 0.005 mol/L Tris phosphate, pH 8.6, to 0.2 mol/L Tris phosphate, pH 6, was immediately followed by elution of most
of the protein in a single major peak (peak A). Foothills of peak A
eluted during maintenance of the step for 12-column volumes. A distinct
second peak (peak B) eluted approximately midway during the subsequent
12-column volume linear gradient, ending at 0.5 mol/L Tris phosphate,
pH 4.2.
View larger version (15K):
[in this window]
[in a new window]
| Fig 1.
Separation of
fibrinogen species by Mono Q
anion exchange chromatography. Human fibrinogen
(fraction I-2) was purified from umbilical cord plasma and then
subjected to column chromatography as described in "Materials and
methods." The elution profile is plotted with absorbance at 280 nm
as a solid line (scale on the left), and the stepwise gradient in Tris
phosphate is indicated by the broken line (scale on the right). The
major and minor peaks are labeled A and B, respectively.
|
|
Characterization of fibrinogen species in peaks A and B
The first 3 fractions of peak A were pooled and compared, by
SDS-PAGE and Western blot analysis, with a concentrated pool of the
central 3 fractions of peak B (Figure 2).
The 2 bands visible in peak A (Figure 2A) corresponded directly to the
2 most abundant species in the original fibrinogen I-2 fraction intact
and partially degraded forms of the conventional chain-containing
fibrinogen; the heterogeneity reflects the well-known susceptibility of
the chains to carboxyl terminal proteolysis.29 After
its disulfide chains were reduced (Figure 2B, left panel, lane 2), peak
A resolved into the intact conventional , , and bands and
into minor bands corresponding to the partially degraded chains.
Comigration of I-2 fibrinogen and peak A bands throughout these
analyses suggests that column separation did not affect the initial
ratio of intact to partially degraded molecules.
View larger version (1938K):
[in this window]
[in a new window]
| Fig 2.
Characterization of
fibrinogen species in peaks A
and B. (A) Unreduced samples and (B) reduced samples.
Fibrinogen (Fib I-2) represents the material added, and peak A and peak
B represent the material eluted from the anion exchange column of
Figure 1. Western blot analysis was performed with either polyclonal
anti-EC #9395 or monoclonal anti-(603-610). Samples
in the upper panel were electrophoresed on 4-15% SDS-PAGE gels;
proteins in the lower panels were separated on homogeneous 12%
SDS-PAGE gels. Positions of various hexamers (A) and individual chains
(B) are indicated. d and dE refer to
degraded and E, respectively. All designated gene-derived species were recognized by 1D4, an antibody specific for
an epitope in the center of the C region (not shown).
|
|
In the late-eluting peak B, anti-EC identified 2 bands
(Figure 2A, lane 4) that corresponded directly to the 2 bands detected by protein staining (lane 3). The proportionality between these 2 bands
of E-fibrinogen, approximately 3:1 (upper:lower), was roughly comparable to that of the 2 bands in peak A (lane 2), suggesting that they might also represent intact and partially proteolyzed forms.
On reduction of the disulfide bonds in peak B, it became clear that the
E-fibrinogens collectively contained not only
E, , and chains but also a minor band migrating
at approximately 70 kd (Figure 2B, left panel, lane 3), just
above the 68-kd chain of peak A (lane 2). Presumably derived from a
larger species, the band was tentatively designated a degradation
product (dE) of the intact approximately 110-kd
E chain. It closely corresponded to the expected size of
the E sequence remaining after cleavage of the
EC domain
This chain assignment was confirmed by Western blot analysis with
discriminating antibodies: anti-EC #9395, which
recognizes an epitope(s) unique to the extended C-terminus of
E chains, and anti-(603-610), which is specific for
the last residues of intact chains (Figure 2B, right panel).
Anti-EC recognized peak B's 110-kd band but not its
70-kd band (lane 2), consistent with the latter being an
E chain without its carboxy terminal domain.
Anti-(603-610) recognized only peak A's intact chain (lane 3)
and not any of the bands in peak B (lane 4). Although intact
E includes the same sequence as the terminal residues 603-610 of conventional , it presumably escapes recognition by this
antibody because the peptide bond between Val610 and Arg611 of
EC eliminates the free carboxyl epitope. By the same
logic, the immunoreactive differences between the 68-kd chain and
the 70-kd band of peak B suggest that the latter is indeed an
E chain cleaved at a site downstream from Val610. In
this context, it should be noted that in peak B fibrinogen-420, no
significant contribution of (1-625), the conventional chain's
non-processed form,18,19 was detected by Western analysis
with anti-(615-625) (data not shown). Had it been
present, the (1-625) chain would have comigrated with the 70-kd band
designated dE and, like it, escaped recognition by
anti-(603-610).
It is noteworthy that, even after heavily overloading the gels, no
E-containing material was detectable in peak A by
Western blot analysis with anti-EC, suggesting an
essentially complete separation of the 2 subclasses. Although a
portion of the or E chains in each peak is degraded,
resulting in the minor bands labeled
d()2 and
EdE()2 in
Figure 2A, for simplicity we hereafter refer to the pooled subfractions
by the nomenclature for the intact speciesfibrinogen-340 for peak A
and fibrinogen-420 for peak B.
Thrombin-catalyzed fibrin polymerization
When incubated with human thrombin, both fibrinogen-420 and
fibrinogen-340 were found to be more than 90% clottable, forming clots
that were sufficiently solid that they remained in place in inverted
cuvettes. Parameters of thrombin-induced clot formation were compared
by monitoring turbidity as a function of time. As seen in Figure
3, the turbidity curves obtained for
fibrinogen-420 and fibrinogen-340 are typical for clot formation: an
initial delay, followed by a rapid rise in turbidity that culminates in a plateau. The lag period represents the time required for fibril formation, and the maximum slope reflects the rate of fibril assembly during the phase of lateral associations and
branching,30,31 whereas the plateau value attained by
each species is related to the average fiber thickness in the
clot.32 By all 3 measures, the curves for fibrinogen-420
and fibrinogen-340 were similar, suggesting fundamentally comparable
roles in the clotting process, though a functional impact of the
observed differences cannot be excluded.
View larger version (12K):
[in this window]
[in a new window]
| Fig 3.
Polymerization with
fibrinogen-420 and fibrinogen-340.
Polymerization was initiated by the addition of thrombin (0.1 U/mL) at
time 0 to substrate at 0.1 mg/mL: either fibrinogen-340 (A) or
fibrinogen-420 (B). Polymer formation was measured as change in
turbidity at 350 nm with time as described in "Materials and
methods." Data in each panel are averages obtained from 4 separate trials. Bars indicate the standard deviation of the mean.
For fibrinogen- 340 and fibrinogen-420, respectively, the lag periods
were 8.0 ± 4.0 and 9.8 ± 4.6 minutes, and the maximum
slopes were 7.3 ± 2.7 and
5.2 ± 3.7 × 10 5
seconds1.
|
|
Factor XIIIa-catalyzed cross-linking
The kinetics of factor XIIIa cross-linking of
fibrinogen-420 and fibrinogen-340 is compared in Figure
4. Cross-linking of the chains in both
preparations was essentially complete within 30 minutes, as evidenced
both by the disappearance of the band corresponding to the chain
and the concomitant appearance of -dimer. Cross-linking of the chain in fibrinogen-340 (evident from its gradual disappearance and the
emergence of higher molecular weight species) occurred at a rate
lagging that of the chain, as expected.33 A similarly
delayed cross-linking occurred for the E and
dE chains of fibrinogen-420. This observation
contrasts with findings in lamprey fibrinogen where cross-linking of
the E homologue (') was considerably more
efficient than for the chain.34 The disparity may be
the result of differences in the C regions of the lamprey fibrinogen
chain and the E chain homologue, which are
atypically derived from separate genes.10,11
View larger version (63K):
[in this window]
[in a new window]
| Fig 4.
Time course of
factor XIIIa-catalyzed cross-linking.
Cross-linking reactions with either fibrinogen-420 (upper panel) or
fibrinogen-340 (lower panel) were carried out as described in
"Materials and methods." Lane 1 contains substrate alone. Lanes 2 to 6 contain substrate with either nonactivated factor XIII (lane 2) or
thrombin-activated factor XIIIa (lanes 3-6), incubated for 2 minutes
(lane 3), 5 minutes (lane 4), 30 minutes (lane 5), or 60 minutes (lane
6). Proteins were separated on homogenous 12% SDS-PAGE gels under
reducing conditions and stained. Positions of individual and
cross-linked fibrinogen chains are indicated. dE
refers to degraded E, E-xlinks refers to
cross-linked E chains, and -xlinks refers to
cross-linked chains.
|
|
Plasmic digestion of fibrinogen-420
Comparison of plasmic digestion of fibrinogen-420 and fibrinogen-340
by SDS-PAGE revealed similar kinetics for production of the
conventional fibrinogen degradation products: fragments X, Y, D, and E
(Figure 5, left and middle panels).
However, accumulation of at least 2 additional products was observed in
the plasmic digest of fibrinogen-420: 1 band (ECX)
comigrating with rEC, the 34-kd yeast recombinant
EC domain,14 and another of slower mobility,
which appears to be its immediate precursor (Figure 5, middle panel).
Both products were detected in immunoblots using antibodies specific
for the EC domain (Figure 5, right panel). In addition,
some short-lived pre-ECX species of slower mobility were
observed. Cleavage of fibrinogen-420 to yield the
pre-ECX species was a particularly early event in the
digestion, occurring well before the appearance of significant
quantities of fragments D and E. Quantitation, using rEC
as a standard, suggested that the final product, ECX,
accumulated in molar proportion to the amount of EC
present in the intact fibrinogen-420 (Figure 5, middle panel),
indicating a degree of stability comparable to that of the core
fragments D and E. Stabilization of the domain against further
digestion by plasmin requires the presence of calcium (data not
shown), an observation first noted for the recombinant domain.14
View larger version (33K):
[in this window]
[in a new window]
| Fig 5.
Plasmin digestion of
fibrinogen-420 and fibrinogen-340. The first 5 lanes in each panel contain purified fibrinogen (3.4 µg/lane), either
fibrinogen-420 or fibrinogen-340; the sixth contains 0.5 µg
recombinant human EC (rEC), which
migrates at 34 kd.14 Proteins were separated on 4% to 15%
SDS-PAGE gels under nonreducing conditions. Left and middle panels:
Gelcode blue stain. Right panel: Western blot analysis of
fibrinogen-420 using monoclonal anti-EC #29-1. Positions
of fibrinogen (F) and fragments X, Y, D, and E are indicated, as are
those of the E-containing cleavage products
ECX and its precursors (pre-ECX); the
larger precursors can only be seen in overexposures.
|
|
Immunologic identification of ECX in vivo
The observed in vitro stability of ECX in the
presence of plasmin prompted us to examine whether the
fragment could also be detected in such lytic states in vivo. The
generation of fibrin(ogen) degradation products is found in clinical
states associated with activation of the fibrinolytic system. In
particular, relatively high concentrations of fibrinolytic products
have been detected in the plasma of patients with myocardial infarction
during thrombolytic therapy with tissue plasminogen activator or
streptokinase, as a result of the lytic state created during treatment.
Figure 6 shows a representative Western
blot analysis, with an anti-EC antibody, of plasma
obtained from patients with myocardial infarction 30 minutes after
treatment with either streptokinase or tissue plasminogen activator.
Indeed, 2 bands are detected that comigrate with ECX and
its immediate precursor from plasmic digests of purified fibrinogen-420
in vitro.
View larger version (24K):
[in this window]
[in a new window]
| Fig 6.
Presence of
E-containing plasmin
cleavage products in vivo. In vitro: a
30-minute time point from plasmin digestion of purified fibrinogen-420
(see Figure 5). In vivo: plasma samples were collected from patients
with myocardial infarction 30 minutes into treatment with either
streptokinase (SK) or tissue plasminogen activator (tPA). Proteins were
separated on 10% SDS-PAGE gels under nonreducing conditions, Western
blotted, and detected using monoclonal anti-EC #29-1.
Positions of the split products, ECX and its precursor
pre-ECX, are indicated.
|
|
| |
Discussion |
This study is the first reported purification of
fibrinogen-420 from human plasma, enabling a structural and
functional characterization of this E-containing
fibrinogen subclass that constitutes a minor percentage of the
circulating fibrinogen. With well-separated subfractions of
fibrinogen-420 and the more abundant -fibrinogen, fibrinogen-340, we
have demonstrated the overall similar behavior of these fibrinogen
subclasses in clot formation and proteolytic susceptibility and have
shown that plasmin attack rapidly releases the EC domain
of fibrinogen-420 as an entity, ECX, resistant to
further degradation in vitro. Furthermore, the ECX
fragment is also detectable in the plasma of patients undergoing
thrombolytic therapy. On the basis of these findings, we propose that
an important function(s) is discharged by the EC domain
independent of its parent fibrinogen molecule.
The protocol described in this article binds fraction I-2 of human
fibrinogen to a Mono Q anion exchange column, eluting it in a stepwise
fashion to effect a clean separation of E-fibrinogen (fibrinogen-420) from -fibrinogen (fibrinogen-340). Our SDS-PAGE analysis shows that, after purification, fibrinogen-420 is as intact as
fibrinogen-340, with roughly 20% of the molecules degraded (see Figure
2). The degradation, whether caused by plasmin or other
proteases,35 occurs before the column chromatography step, either in vivo or during generation of fibrinogen fraction I-2.
By differential antibody reactivity, we have shown that conventional
chains are not incorporated into E-fibrinogen from human plasma; the band in reduced E-fibrinogen that
migrates spuriously near the position of conventional chains is a
distinct E-chain derivative that has lost a significant
portion of its C-terminal domain (see Figure 2). Thus, the native
structure of E-fibrinogen is indeed symmetrical
(E)2 rather than mixed, E()2, and reflects a
nonstochastic assembly process as noted in an earlier
study.8
The closely related structures of fibrinogen-420 and fibrinogen-340
originally led us to investigate whether the EC domains of fibrinogen-420 might alter the fibrinogen molecule's primary behavior in clotting and fibrinolysis. The analyses of polymerization and cross-linking presented here (Figures 3, 4) show that the presence
of the EC domains on a fibrinogen molecule does not grossly affect these functions. The findings support previous studies
showing that rEC, the recombinant EC
expressed in yeast, lacks a polymerization pocket and does not
participate in cross-linking.14 The findings are also
consistent with electron micrographic images of clots derived from
either fibrinogen-340 or fibrinogen-420 (unpublished observations).
Despite similarities to C and C, the EC domain
appears to be specialized for a different and as yet unknown function,
based on several considerations. (1) While still attached to the
fibrinogen core via its "C" tether, EC
undoubtedly enjoys more degrees of spatial freedom than C or C
and, consequently, greater availability of its binding sites to other
macromolecules. (2) This location also appears designed to ensure more
rapid release of the EC domain, given the extreme
susceptibility of the C region to proteolysis.36 (3)
Proteolytic release of monomeric ECX (Figure 5) provides definitive evidence that the EC domains of
fibrinogen-420 have no disulfide attachments, either to each other or
to the core of the molecule, a finding consistent with the results of
mutational analysis of recombinant fibrinogen-4208 and
trypsin digests of '-fibrinogen, the counterpart to
E-fibrinogen in lamprey.34 (4) During
fibrin(ogen)olysis, the EC domains are released as
monomers, unlike the C and C domains, which remain anchored
together in the proteolytic fragment D. (5) Finally, the binding clefts
of the C and C domains contain charged/polar amino acid pairs
that engage the polymerization "knobs" during fibrin
assembly,5,7,15 whereas the corresponding cleft in the
EC domain has neutral residues at its center, suggesting a different purpose.6
The EC domain is derived from exon VI, the largest
conserved segment of the entire fibrinogen gene.11 In
light of no discernible effect of the domain on coagulation, we suspect
that preservation of the E chains among higher
vertebrates reflects the ability of E-fibrinogen to
deliver the ECX fragment to a location critical to its
mission. In the recent literature, a growing number of comparable
proteolytic products exhibit potent effects unrelated to the primary
function of their parent molecules, which often serve to localize
fragment release to sites of tissue repair, wound healing, and
angiogenesis.37 Recent experiments with recombinant forms
expressed in E coli38 and yeast (unpublished observations) suggest that the domain is capable of
supporting integrin-mediated cell adhesion. Current investigation is
focused on further exploring the role of ECX in this context.
| |
Acknowledgments |
We thank Joan Sobel of Columbia Presbyterian Medical Center for
providing access to archival plasma samples and Bohdan Kudryk and
Alessandra Bini for many helpful discussions. We are particularly grateful to Ludy Dobrilla for her cooperation in supplying cord plasma.
We also thank Peter J. Baker for his capable technical assistance.
| |
Footnotes |
Submitted July 15, 1999; accepted December 8, 1999.
Supported in part by grants from the National Institutes of
Health (HL 51050), the American Heart Association, and the Hugoton Foundation.
Reprints: Gerd Grieninger, New York Blood Center, 310 East 67th
Street, New York, NY 10021; e-mail: ggrien{at}nybc.org.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
References |
1.
Grieninger G, Lu X, Cao Y, et al.
Fib420, the novel fibrinogen subclass: newborn levels are higher than adult.
Blood.
1997;90:2609[Abstract/Free Full Text].
2.
Fu Y, Weissbach L, Plant PW, et al.
Carboxy-terminal-extended variant of the human fibrinogen subunit: a novel exon conferring marked homology to and subunits.
Biochemistry.
1992;31:11,968[Medline]
[Order article via Infotrieve].
3.
Fu Y, Grieninger G.
Fib420: a normal human variant of fibrinogen with two extended chains.
Proc Natl Acad Sci U S A.
1994;91:2625[Abstract/Free Full Text].
4.
Yee VC, Pratt KP, Cote HC, et al.
Crystal structure of a 30 kDa C-terminal fragment from the chain of human fibrinogen.
Structure.
1997;5:125[Medline]
[Order article via Infotrieve].
5.
Spraggon G, Everse SJ, Doolittle RF.
Crystal structures of fragment D from human fibrinogen and its crosslinked counterpart from fibrin.
Nature.
1997;389:455[Medline]
[Order article via Infotrieve].
6.
Spraggon G, Applegate D, Everse SJ, et al.
Crystal structure of a recombinant EC domain from human fibrinogen-420.
Proc Natl Acad Sci U S A.
1998;95:9099[Abstract/Free Full Text].
7.
Everse SJ, Spraggon G, Veerapandian L, Riley M, Doolittle RF.
Crystal structure of fragment double-D from human fibrin with two different bound ligands.
Biochemistry.
1998;37:8637[Medline]
[Order article via Infotrieve].
8.
Fu Y, Zhang J-Z, Redman CM, Grieninger G.
Formation of the human fibrinogen subclass Fib420: disulfide bonds and glycosylation in its unique (E chain) domains.
Blood.
1998;92:3302[Abstract/Free Full Text].
9.
Weissbach L, Grieninger G.
Bipartite mRNA for chicken -fibrinogen potentially encodes an amino acid sequence homologous to and -fibrinogens.
Proc Natl Acad Sci U S A.
1990;87:5198[Abstract/Free Full Text].
10.
Pan Y, Doolittle RF.
cDNA sequence of a second fibrinogen chain in lamprey: an archetypal version alignable with full-length and chains.
Proc Natl Acad Sci U S A.
1992;89:2066[Abstract/Free Full Text].
11.
Fu Y, Cao Y, Hertzberg KM, Grieninger G.
Fibrinogen genes: conservation of bipartite transcripts and carboxy-terminal-extended subunits in vertebrates.
Genomics.
1995;30:71[Medline]
[Order article via Infotrieve].
12.
Doolittle RF.
Fibrinogen and fibrin.
Ann Rev Biochem.
1984;53:195[Medline]
[Order article via Infotrieve].
13.
Blomback B.
Fibrinogen and fibrinproteins with complex roles in hemostasis and thrombosis.
Thromb Res.
1996;83:1[Medline]
[Order article via Infotrieve].
14.
Applegate D, Haraga L, Hertzberg KM, et al.
The EC domains of human fibrinogen420 contain calcium binding sites but lack polymerization pockets.
Blood.
1998;92:3669[Abstract/Free Full Text].
15.
Pratt KP, Cote HCF, Chung DW, Stenkamp RE, Davie EW.
The primary fibrin polymerization pocket: three-dimensional structure of a 30-kDa C-terminal gamma chain fragment complexed with the peptide Gly-Pro-Arg-Pro.
Proc Natl Acad Sci U S A.
1997;94:7176[Abstract/Free Full Text].
16.
Rubinstein P, Dobrila L, Rosenfield RE, et al.
Processing and cryopreservation of placental/umbilical cord blood for unrelated bone marrow reconstitution.
Proc Natl Acad Sci U S A.
1995;92:10,119[Abstract/Free Full Text].
17.
The TIMI Study Group.
The thrombolysis in myocardial infarction (TIMI) trial: phase I findings.
N Engl J Med.
1985;312:932[Medline]
[Order article via Infotrieve].
18.
Rixon MW, Chan WY, Davie EW, Chung DW.
Characterization of a complementary deoxyribonucleic acid coding for the alpha chain of human fibrinogen.
Biochemistry.
1983;22:3237[Medline]
[Order article via Infotrieve].
19.
Kant JA, Lord ST, Crabtree GR.
Partial mRNA sequences for human A alpha, B beta, and gamma fibrinogen chains: evolutionary and functional implications.
Proc Natl Acad Sci U S A.
1983;80:3953[Abstract/Free Full Text].
20.
Doolittle RF, Watt KW, Cottrell BA, Strong DD, Riley M.
The amino acid sequence of the alpha-chain of human fibrinogen.
Nature.
1979;280:464[Medline]
[Order article via Infotrieve].
21.
Farrell DH, Huang S, Davie EW.
Processing of the carboxyl 15-amino acid extension in the alpha-chain of fibrinogen.
J Biol Chem.
1993;268:10,351[Abstract/Free Full Text].
22.
Rudchenko S, Trakht I, Sobel JH.
Comparative structural and functional features of the human fibrinogen alpha C domain and the isolated alpha C fragment: characterization using monoclonal antibodies to defined COOH-terminal A alpha chain regions.
J Biol Chem.
1996;271:2523[Abstract/Free Full Text].
23.
Kudryk BJ, Grossman ZD, McAfee JG, Rosebrough SF.
Monoclonal antibodies as probes for fibrin(ogen) proteolysis. In:
Chatal JF, ed.
Monoclonal Antibodies in Immunoscintigraphy. Boca Raton, FL: CRC Press; 1989:365.
24.
Procyk R, Medved L, Engelke KJ, Kudryk B, Blomback B.
Nonclottable fibrin obtained from partially reduced fibrinogen: characterization and tissue plasminogen activator stimulation.
Biochemistry.
1992;31:2273[Medline]
[Order article via Infotrieve].
25.
Mosesson MW, Sherry S.
The preparation and properties of human fibrinogen of relatively high solubility.
Biochemistry.
1966;5:2829[Medline]
[Order article via Infotrieve].
26.
Mosesson MW, Finlayson JS, Umfleet RA.
Human fibrinogen heterogeneities, 3: identification of chain variants.
J Biol Chem.
1972;247:5223[Abstract/Free Full Text].
27.
Laemmli UK.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature.
1970;227:680[Medline]
[Order article via Infotrieve].
28.
Birken S, Wilner GD, Canfield RE.
Studies of the structure of canine fibrinogen.
Thromb Res.
1975;7:599[Medline]
[Order article via Infotrieve].
29.
Mosesson MW.
Fibrinogen heterogeneity.
Ann N Y Acad Sci.
1983;408:97[Medline]
[Order article via Infotrieve].
30.
Hantgan RR, Hermans J.
Assembly of fibrin.
J Biol Chem.
1979;254:11,272[Abstract/Free Full Text].
31.
Weisel JW, Nagaswami C.
Computer modeling of fibrin polymerization kinetics correlated with electron microscope and turbidity observations: clot structure and assembly are kinetically controlled.
Biophys J.
1992;63:111[Medline]
[Order article via Infotrieve].
32.
Carr MEJ, Hermans J.
Size and density of fibrin fibers from turbidity.
Macromolecules.
1978;11:46[Medline]
[Order article via Infotrieve].
33.
McKee PA, Mattock P, Hill RL.
Subunit structure of human fibrinogen, soluble fibrin, and cross-linked insoluble fibrin.
Proc Natl Acad Sci U S A.
1970;66:738[Abstract/Free Full Text].
34.
Shipwash E, Pan Y, Doolittle RF.
The minor form ' chain from lamprey fibrinogen is rapidly crosslinked during clotting.
Proc Natl Acad Sci U S A.
1995;92:968[Abstract/Free Full Text].
35.
Nakashima A, Sasaki S, Miyazaki K, Miyata T, Iwanaga S.
Human fibrinogen heterogeneity: the COOH-terminal residues of defective A alpha chains of fibrinogen II.
Blood Coagul Fibrinolysis.
1992;3:361[Medline]
[Order article via Infotrieve].
36.
Liu CY, Sobel JH, Weitz JI, Kaplan KL, Nossel HL.
Immunologic identification of the cleavage products from the A alpha- and B beta-chains in the early stages of plasmin digestion of fibrinogen.
Thromb Haemost.
1986;56:100[Medline]
[Order article via Infotrieve].
37.
Hanahan D, Folkman J.
Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis.
Cell.
1996;86:353[Medline]
[Order article via Infotrieve].
38.
Yokoyama K, Zhang XP, Medved L, Takada Y.
Specific binding of integrin alpha v beta 3 to the fibrinogen gamma and alpha E chain C-terminal domains.
Biochemistry.
1999;38:5872[Medline]
[Order article via Infotrieve].
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
V. K. Lishko, V. P. Yakubenko, K. M. Hertzberg, G. Grieninger, and T. P. Ugarova
The alternatively spliced {alpha}EC domain of human fibrinogen-420 is a novel ligand for leukocyte integrins {alpha}M{beta}2 and {alpha}X{beta}2
Blood,
October 15, 2001;
98(8):
2448 - 2455.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Top
Abstract
Introduction