(Received for publication, September 6, 1995; and in revised form, November 14, 1995)
From the
The C domain of fibrinogen (A
-(220-610)) plays a
central role in maintaining hemostasis by serving as a substrate for
factor XIII
and plasmin. Monoclonal antibodies that
recognize eight distinct epitopes within the COOH-terminal two-thirds
of the A
chain were employed as structural probes to: 1) isolate
the human
C domain, 2) compare the topography of the eight
epitopes within the
C domain of intact fibrinogen and in purified
C fragments, and 3) explore the degree to which the
C
domain's role as a factor XIII
substrate in intact
fibrinogen is preserved within the structure of isolated
C
fragments. Five antibodies were raised against small, synthetic peptide
immunogens (A
-(220-230), A
-(425-442),
A
-(487-498), and A
-(603-610)), and three were
generated against larger cyanogen bromide (A)
chain derivatives
with each epitope subsequently localized to discrete A
chain
sequences (A
-(259-276), A
-(529-539), and
A
-(563-578)). Human
C preparations were isolated from
mild plasmin digests of fibrinogen by successive chromatography on
concanavalin A-Sepharose, anti-A
-(425-442)-Sepharose, and
Superdex-75 fast protein liquid chromatography. Immunochemical
characterization indicated that the NH
-terminal residue of
C fragments was either A
-220 or A
-231 and that, although
the extreme COOH-terminal region, A
-(603-610), was absent,
all molecules were intact at least through A
-(563-578).
Solution phase competitive assays indicated that the release of the
C domain from intact fibrinogen was associated with several
conformational changes, e.g. in the vicinity of
A
-(220-230), A
-(259-276), A
-(487-498),
and A
-(529-539), but that the relative accessibility of
other localized structures remained unchanged, e.g. A
-(425-442) and A
-(563-578). Immunoblotting
analysis of
C cross-linking in vitro revealed that
isolated
C fragments could serve as a substrate for factor
XIII
. Immunoblotting studies of the A
chain
proteolysis that occurs during thrombolytic therapy indicated that
C fragments, similar in size and epitope content to those isolated
from purified fibrinogen, were released in vivo early during
fibrinolytic system activation. The collective findings provide new
information about the fine structure of the fibrinogen
C domain
and its functional implications and also draw attention to the as yet
unexplored role of
C fragments in the pathophysiology of
thrombosis and hemostasis.
The past decade has witnessed a growing interest in the
structure of the fibrinogen C domain, i.e. the
COOH-terminal two-thirds of the A
chain that extends from the
coiled-coil portion of each half of the dimeric fibrinogen molecule.
Elucidation of this region's structural features is of
considerable interest given its multifunctional role in maintaining
hemostasis. Factor XIII
cross-linking sites included within
the
C domain are responsible for the covalent bonds created
between fibrin
chains and between
chains and
PI (
)that lead to the formation of a highly
cross-linked
polymer
network(1, 2, 3, 4, 5, 6) .
The resulting ``stabilized'' fibrin is characterized by an
enhanced resistance to lysis by plasmin, the hallmark of a
physiologically effective thrombus(7, 8) . A number of
plasmin-susceptible cleavage sites have been identified within the
C domain of fibrinogen(9) . Presumably, these same sites
are responsible for the initial cleavages that eventually lead to
disruption of the fibrin
polymer network, a process considered to
be a prerequisite for efficient particulate clot lysis(10) .
The fibrinogen A
chain contains two RGD sites, and one of these,
located within the
C domain, has recently been implicated in
fibrinogen's binding to the endothelial cell vitronectin receptor (11) ; this suggests an additional role for the
C domain
as a mediator of fibrinogen-vessel wall interaction.
Historically,
the COOH-terminal two-thirds of the A chain (referred to here as
C) has been viewed as a ``free-floating'' appendage with
little organized structure, based on primary structure considerations
as well as observations from early biochemical and immunoelectron
microscopy studies(12, 13, 14) . This concept
has been challenged in recent years in light of newer electron
microscopy and microcalorimetry findings which suggest that the
C
portions of fibrinogen are, in fact, highly organized and interact to
form a fourth globular domain that is centrally positioned over the E
domain of the intact dimeric molecule(15, 16) .
Additional evidence indicates that these intramolecular
C contacts
are disrupted during fibrin formation, thus facilitating the
intermolecular associations that must be in place before
chain
cross-linking can occur(17) .
Apart from these structural
studies which have provided important information about the general
architecture of the C domain as it exists in intact fibrin(ogen),
a second area of related interest concerns the biochemistry of the
isolated
C region, i.e. the large fragment representing
nearly the entire COOH-terminal two-thirds of the A
chain, that is
released during the initial stages of plasmin cleavage.
Structure-function studies of human
C fragments have been hampered
to date because of the difficulties in obtaining homogeneous
preparations due to the A
chain's intrinsic COOH-terminal
heterogeneity and its extreme lability during in vitro handling(18) . However, electron microscopy studies of
isolated
C fragments from bovine fibrinogen (which are less
susceptible to proteolytic degradation than their human counterpart)
demonstrate that the structure of these derivatives can support the
formation of linear arrays of
C polymers and can also promote the
interaction of
C with intact fibrin molecules to inhibit their
polymerization(17, 19) . These findings are consistent
with earlier biochemical and immunologic observations suggesting the
existence of a polymerization site within the COOH-terminal portion of
the A
chain (20, 21, 22) .
The fact
that isolated C fragments retain their parent protein's
capacity for polymerization raises the possibility that other
functionalities unique to the fibrin(ogen)
C domain may be
preserved as well. To date, little information is available regarding
the extent to which conformational features of isolated
C
fragments support the various functions predicted for these derivatives
based on their primary structure alone, e.g. factor XIII
cross-linking, plasmin susceptibility, and endothelial cell
interaction. This is of particular significance when one considers that
C fragments are expected to be released as natural by-products of
fibrinolytic system activation and, therefore, their relative
prothrombotic or anticoagulant activity in this milieu may have
important clinical ramifications.
The studies in this report focus
on the isolation and structural characterization of the human C
domain and consider the extent to which the functional capacity for
chain cross-linking is preserved within the native conformation
of the isolated fragment. The studies feature an immunochemical
approach, employing a unique panel of monoclonal antibodies to defined
COOH-terminal A
chain regions as structural probes for localized
sequences within the
C domain of intact fibrinogen and within
isolated
C. The results obtained provide preliminary information
about the comparative topography of selected regions within the native
structure of the two
C forms and demonstrate that conformational
features unique to the isolated fragment are compatible with Factor
XIII
cross-linking. The collective findings, which include
observations to indicate that
C fragments are released in vivo during fibrinolytic system activation, provide new insights into
the structure-function relationships of the fibrinogen
C domain
and suggest an as yet unexplored role for the isolated fragment in the
pathophysiology of hemostasis.
Figure 4:
Relative epitope expression in the intact
fibrinogen C domain and in isolated
C determined by ELISA.
Purified
C (solid symbols) and fibrinogen (open
symbols) were employed as competitors in solution phase
competitive ELISAs developed for mAbs F-102
(anti-A
-(563-578); Panel A), F-105
(anti-A
-(425-442); Panel B), F-103
(anti-A
-(259-276); Panel C), and F-106
(anti-A
-(220-230); Panel D), as detailed under
``Materials and Methods.'' The assay standards for the F-105
and F-106 ELISAs were the appropriate synthetic peptide; 50% bound
= 0.90 ± 0.21 nM and 0.24 ± 0.06 nM, respectively
(mean, ±S.D.; n = 3). Fibrinogen was the assay
standard for the F-102 and F-103 ELISAs (see Table 2for
additional details). Immunoreactivity is plotted as the
logit-transformed percent bound versus dose, where dose is
expressed as the molar concentration of competitor in the solution
phase determined from amino acid analysis of the purified standard
preparation. The molar concentration of fibrinogen, expressed as A
chain equivalents, was calculated assuming a molecular mass of 340 kDa
and 2 mol of A
chain/mol of
fibrinogen.
Figure 6:
Immunovisualization of C release in
vivo during thrombolytic therapy. Plasmas (5 µl) from two patients
undergoing thrombolytic therapy with rt-PA and streptokinase were
applied to the lanes of 12.5% gels, and electrophoresis was conducted
under nonreducing conditions. Duplicate nitrocellulose transfers were
incubated with anti-A
-(563-578) (mAb F-102) and
anti-A
-(259-276) (mAb F-103), employing expanded cloned
culture supernatants diluted 1:20 and 1:100, respectively.
Immunoreactive components were visualized as described in the legend to Fig. 3. The arrows identify the migration of intact and
partially degraded fibrinogen. The migration of A
FDPs (right) and standard molecular mass markers (left)
are indicated for reference (see Fig. 2legend for
identification of marker proteins).
Figure 3:
Immunologic characterization of purified
C by immunoblotting with anti-A
chain mAbs. Ten pmol of
purified
C were applied to each of seven lanes of a 12.5% gel, and
electrophoresis was conducted under nonreducing conditions.
Nitrocellulose transfers of each lane were incubated with purified IgGs
from the antibody panel and immunoreactive components subsequently
visualized using RAM-Ig-HRP as described under ``Materials and
Methods.'' The migration of standard molecular markers is
indicated at the left for reference (ovalbumin, 43 kDa;
carbonic anhydrase, 29 kDa;
-lactoglobulin, 18.8 kDa; lysozyme,
16.5 kDa; bovine trypsin inhibitor, 6.4 kDa; insulin
and
chains, 3.0 kDa).
Figure 2:
FPLC purification of isolated C
fragment(s). 1.6 mg of partially purified
C obtained following
successive adsorption of plasmin-treated fibrinogen on ConA-Sepharose
and anti-A
-(425-442)-Sepharose were chromatographed on
Superdex-75 as described (see ``Materials and Methods''). The
280-nm absorbance profile of the column effluent is shown; AUFS, absorbance full scale. The elution positions of standard
molecular mass markers are indicated at the top for reference
(
-globulin, 158 kDa; ovalbumin, 44 kDa; myoglobin, 17 kDa; and
vitamin B-12, 1.35 kDa). SDS-PAGE/Western blotting: approximately 1% of
the material in fractions 17-22 was subjected to SDS-PAGE on
12.5% gels under nonreducing conditions. Nitrocellulose transfers were
stained for total protein with Amido Black. The migration of standard
molecular mass markers is indicated at the right for reference
(phosphorylase b, 92.5 kDa; bovine serum albumin, 69 kDa;
ovalbumin, 46 kDa; carbonic anhydrase, 30 kDa; trypsin inhibitor, 21.5
kDa; and lysozyme, 14.3 kDa). Note that the high molecular mass
components eluting in fraction 17 (as well as 15 and 16) were barely
detectable by Amido Black staining indicating that they comprised a
negligible proportion of the total material recovered. Moreover, there
was no COOH-terminal A
chain immunoreactivity associated with this
material based on ELISA and immunoblotting.
Figure 1:
mAbs to defined regions within the
human fibrinogen C domain. The A
chain is illustrated
schematically from residues 1-610. The
C domain is shown
extending from the NH
-terminal third of the molecule which
is included within the E and D domains of fibrinogen. The various
A
chain functional domains, including RGD sites, plasmin cleavage
sites (P), acceptor Gln residues (Q), the
PI lysine cross-linking site and the lysine-rich
region associated with
chain donor cross-linking activity (K), are indicated for reference. mAbs raised against large
CNBr (A)
chain derivatives, with their respective epitopes
subsequently localized within small defined sequences, are shown in A. mAbs raised against predefined peptide conjugates are shown
in B. The inclusive A
chain residues that contain the
respective epitopes recognized by each of the eight antibodies in the
panel are: 1) A
-(220-230) (mAbs F-106, 106f); 2)
A
-(259-276) (mAb F-103); 3) A
-(425-442)
(mAb F-105); 4) A
-(487-498) (mAb F-104);
5) A
-(529-539) (mAb 5A2); 6) A
-(563-578)
(mAb F-102); and 7) A
-(603-610) (mAb F-48)
(see also Table 2).
Fig. 2(left) illustrates the 280-nm absorbance
profile obtained when A FDPs in the anti-A
-(425-442)
flow-through fraction were separated by size exclusion chromatography
on Superdex-75 FPLC. Analysis of the column effluent with the
anti-A
chain antibody panel indicated the emergence of (at least)
five discrete peaks of COOH-terminal A
chain immunoreactivities,
reflecting a heterogeneous population of A
FDPs ranging in size
from approximately <1.3 to
40 kDa (data not shown). SDS-PAGE (Fig. 2, right) identified two closely migrating
components in fractions 18 and 19 with the approximate mass of 40 kDa
expected for
C.
Fig. 3shows the immunoblotting profile
typically obtained for C preparations isolated and purified as
described above. Characterization using the antibody panel confirmed
the material's purity, based on the predominant 40-kDa component
observed in each lane. These data, obtained from a denaturing system,
also provided qualitative structural evidence to indicate that although
a significant proportion of
C fragments included the NH
terminus of the
C region, i.e. A
-(220-230) (Fig. 3, lane 1), none
contained the extreme COOH-terminal epitope within
A
-(603-610) (Fig. 3, lane 7). (Small A
FDPs, representing
10- and
3-kDa cleavage products released
from the A
chain COOH terminus, were identified in the FPLC column
effluent as discrete peaks of anti-A
-(603-610)
immunoreactivity; data not shown). Amino-terminal sequencing revealed
that two major components were included in our
C preparations and
that these were present at approximately equimolar level. Comparison of
the phenylthiohydantoin-derivatives recovered at each cycle with the
known primary structure of the A
chain indicated that the NH
terminus of these fragments originated from plasmin cleavage at
residues A
-(219-220) and A
-(230-231),
respectively (data not shown). Based on the structural information
obtained from the immunoblotting and NH
-terminal sequencing
findings, the molar composition derived from amino acid analysis of the
C preparation (data not shown) was most consistent with both
fragments extending at least through residue A
-583.
Figure 5:
Factor XIII-mediated
C
cross-linking visualized by immunoblotting. Purified fibrinogen (left; lanes 1) and
C fragment(s) (right; lanes 1) were subjected to in vitro cross-linking in the presence (lanes 3) or absence (lanes 2) of a synthetic peptide probe for factor-XIII
donor lysine residues, as described under ``Materials and
Methods.'' Approximately 2.5 µg of fibrin(ogen) and 5.2 µg
of
C from the respective incubation mixtures were applied to 9%
gels, and electrophoresis was conducted under reducing conditions.
Duplicate nitrocellulose transfers were treated with
anti-A
-(259-276) (mAb F-103; first three lanes in
each panel) and anti-
PI-(1-12) peptide (mAb AP-102; last three lanes in each panel), employing expanded
cloned culture supernatants diluted 1:100 and 1:300, respectively.
Immunoreactive components were visualized using GAM-IgG-HRP, as
described under ``Materials and Methods.'' The faint bands of
immunoreactivity observed in the first two lanes of each AP-102 panel
are due to nonspecific binding by the HRP-IgG conjugate, as described
previously(26) . The migration of standard molecular mass
markers is indicated at the extreme left for reference (myosin, 215
kDa; phosphorylase b, 105 kDa; bovine serum albumin, 70 kDa;
ovalbumin, 43 kDa; carbonic anhydrase, 28
kDa).
The studies described in this report illustrate the
application of a unique panel of characterized monoclonal antibodies to
address questions related to the biochemistry of the fibrinogen C
domain. The antibodies in the panel recognize eight distinct epitopes
that span the COOH-terminal 391 residues of the A
chain (Fig. 1). These include three epitopes within the
``loosely'' organized portion of the
C domain
(A
-(220-390)) and five within the portion characterized as
``compact'' (A
-(390-610)), based on biophysical
observations and inferences drawn from the region's primary
structure (12, 16) . To date, characterization of the
topography of the
C domain and its relationship to other domains
within the trinodular fibrinogen molecule, have provided a general view
of this region's structural features(15, 17) .
As demonstrated here, the availability of monoclonal antibodies to
defined A
chain regions provides the opportunity to refine this
view by characterizing localized conformations within the
C
domain.
Several groups have previously reported the use of
anti-A chain antibodies as conformational probes of native
fibrinogen and its structural modulation during plasmin
digestion(22, 37) . In both studies, antisera were
raised against native fibrinogen or isolated A
chains, with
antibody selection methods contributing to the recovery, primarily, of
immunoglobulin populations that recognized two classes of
immunodominant epitopes. One of these was localized to the mid-section
of the A
chain, within A
-(239-429), and the other to
the COOH-terminal end of the A
chain, within
A
-(518-584). The three antibodies in our panel,
anti-A
-(259-276) (mAb F-103), anti-A
-(529-539)
(mAb 5A2), and anti-A
-(563-578) (mAb F-102), which were each
raised against large A
chain derivatives, more than likely reflect
this same phenomenon. Their isolation highlights the fact that
repertoires of anti-COOH-terminal A
chain antibodies will be
restricted to immunodominant epitopes unless rational approaches to
immunogen design and antibody selection are considered. In view of
this, we have focused on the use of small peptide immunogens, modeled
after defined COOH-terminal A
chain sequences, to produce
structural probes other than those represented in the traditional
schemes employed for the development of anti-fibrinogen monoclonal
antibodies. Peptides were chosen based on hydropathy criteria (for
example, A
-(487-498) is predicted to be one of the most
hydrophilic A
chain regions) or functional considerations (the
reported early plasmin cleavage sites at A
-(219-220) and
A
-(424-425) (9) would be expected to produce at
least some antibodies to neo-epitopes). This approach, together with a
dual primary screening assay employing both peptide and intact
fibrinogen as solid phase antigens, led to the isolation of two
anti-fibrinogen antibodies (mAbs F-106f and F-104) and two anti-peptide
antibodies (mAbs F-106 and F-105), each characterized by unique
specificities (Fig. 1). (A similar strategy resulted in recovery
of mAb F-48, anti-A
-(603-610)). (
)
In
developing in vitro methods for the isolation of C
fragment(s) from purified fibrinogen, we were aware of the
heterogeneity of plasmin cleavage sites within the COOH-terminal
two-thirds of the A
chain (38) and anticipated that
C
would represent one of a series of overlapping A
chain
derivatives, each isolated in reduced yield. In fact, while
characterization with our antibody panel indicated the efficient
recovery of COOH-terminal A
chain immunoreactivity, i.e. A
FDPs, during the initial purification steps (Table 1),
C typically accounted for only 5% of the total
A
FDP population based on amino acid analysis of the purified
material isolated following the FPLC step (Fig. 2). This finding
highlights the difficulties associated with the preparation of human
C fragments in vitro and explains, in part, the choice of
bovine fibrinogen as a starting substrate for most of the previously
reported structural studies of
C. However, the substitution of
bovine for human fibrinogen does not consider the potential for
conformational differences imposed by nonconserved sequences within the
A
chains of various animal species. As two examples, 1) the region
corresponding to human A
-(529-539) is missing in bovine
fibrinogen as part of a 26-residue deletion and 2) hydrophobic residues
appear in the bovine A
chain at the position corresponding to the
early plasmin cleavage site, A
-(424-425). Structural
differences such as these may partially explain why bovine
C
fragments form long linear polymers at neutral pH (17) while
human
C fragments do not appear to aggregate under similar
conditions (Fig. 2).
Application of the antibody panel to
characterize localized conformations within the fibrinogen C
domain and the isolated
C fragment was conducted in an aqueous
environment that presumably preserved the native form of each molecule.
(In this regard, the potential for structural distortion introduced
during electron microscopy sample preparation should not be
overlooked.) Immunologic analysis was predicated on the fact that
epitopes that are oriented in an exposed conformation on the surface of
antigen molecules will be readily accessible for antibody binding and,
therefore, will require less antigen to effect a given response than
epitopes contained within hydrophobic regions or regions that are
sterically masked. Since the goal of this immunologic characterization
was to detect whether or not conformational differences existed within
respective regions of fibrinogen and
C, all the information
required to address this question was provided by the comparative dose
response curves obtained for the two antigens in each assay (Fig. 4, Table 2). (Issues related to antibody affinity or
the degree to which either antigen achieved maximal epitope expression,
were, therefore, not pertinent here.) Results of this immunologic
analysis indicated that the release of
C from intact fibrinogen
was accompanied by conformational changes in the isolated fragment, as
reflected in the altered accessibility of several of the epitopes
examined. Although speculative, one structural implication drawn from
the findings is that the newly exposed and loosely organized
NH
-terminal connector portion of
C may fold back and
wrap around the more globular portion of the molecule, thereby
obscuring a number of localized COOH-terminal regions that were
surface-oriented on the
C domain of intact fibrinogen.
The
conformational profiles obtained for several different C
preparations using these immunologic methods were similar, except for a
significant difference in expression of the localized region,
A
-(425-442). This difference appeared to be associated with
the F-105 immunoaffinity step since
C preparations obtained by
direct FPLC following ConA chromatography exhibited enhanced expression
of this localized region compared to fragments isolated without prior
immunoadsorption. Immunoblotting analysis confirmed that a group of
contaminating 17-25 kDa F-105-immunoreactive A
FDPs
originating from cleavage at the A
-(424-425) bond were
responsible (data not shown). Curiously, although the
anti-A
-(425-442) immunoaffinity step was subsequently found
to remove a major proportion of these fragments from the total A
FDP population (thereby permitting a more effective separation of
C in the final gel-filtration step), 47% failed to bind to the
immunosorbent despite the relatively high molar excess (5:1) of
antibody employed (Table 1). The reason for this apparent
heterogeneity in F-105 epitope expression on the solid phase matrix and
in solution remains unclear although a contributing factor may be the
fact that the region A
-(425-442) is in the vicinity of the
A
chain's single intrachain disulfide (involving the cys
residues at A
-442 and A
-472; see Fig. 1). The reported
lability of this disulfide (39) suggests that perturbations in
its structure may affect adjacent local conformations.
The
structural modulations revealed for C coincident with its release
from intact fibrinogen did not interfere with the fragment's
capacity to serve as a factor XIII
substrate based on the
findings obtained employing a synthetic peptide probe for plasma
transglutaminase donor lysine residues(26) . Up to 8 mol of
peptide became incorporated into purified
C fragments based on the
molecular weight differences observed for monomeric
C and its
peptide-decorated products (Fig. 5). This represents
cross-linking at 35% of the potentially available donor lysines within
the region, A
-(220-610). Moreover,
C fragments could
undergo cross-linking by factor XIII
in the absence of the
peptide probe to produce a significant, albeit minor, proportion of
dimeric
C forms. The degree to which natural
C cross-linking
can be extended in vitro to involve a greater proportion of
C monomers and/or result in the formation of polymeric species
remains to be explored. In addition, the role of intact fibrin(ogen) as
a partner for
C cross-linking requires evaluation since the
structural features intrinsic to the intact molecule could serve to
potentiate what may be only a partial capacity for cross-linking on the
part of isolated
C fragments alone.
The cross-linking of C
fragments, particularly in a fibrin(ogen)-containing milieu may have
important potential implications with respect to the thrombotic and
hemorrhagic complications often associated with current thrombolytic
therapies. Immunologic characterization of the A
chain proteolysis
that reflects the lytic state created during treatment clearly
demonstrates, for the first time, that
C fragments are naturally
occurring products of fibrinolytic system activation (Fig. 6).
Since these A
chain derivatives circulate together with varying
concentrations of intact fibrinogen over the course of therapy, their
capacity to serve as a cross-linking partner may provide a functional
reserve in a plasma milieu where the conventional substrate is facing
continual depletion.
The findings described here, together with the
unique panel of anti-A chain monoclonal antibodies developed,
offer new approaches for investigating other structure-function
relationships associated with the fibrinogen
C domain in basic
research as well as clinical applications.