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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 5 (March 1), 1998:
pp. 1590-1598
Antipeptide Monoclonal Antibodies to Defined Fibrinogen A Chain
Regions: Anti-A 487-498, a Structural Probe for Fibrinogenolysis
By
Joan H. Sobel,
Ilya Trakht,
Nicolas Pileggi, and
Hong Qi Wu
From the Department of Medicine, College of Physicians & Surgeons of
Columbia University, New York, NY.
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ABSTRACT |
The fibrinogen  C domain (A 220-610) is one of the earliest
targets attacked by plasmin following fibrinolytic system activation. Monoclonal antibodies (MoAbs) to defined sequences within the C
domain provide the opportunity to explore the structure-function relationships involved in plasmin's interaction with its A chain substrate at greater resolution and can serve as reagents with potential clinical use for detecting fibrinogenolysis in vivo. The MoAb
F-104 was raised against a multiple antigenic peptide derivative
modelled after the hydrophilic 12-residue sequence corresponding to
A 487-498 within the C domain. A sensitive solution phase
competitive enzyme-linked immunosorbent assay (ELISA) was developed for
MoAb F-104 that can be applied for the direct measurement of intact
fibrinogen (purified or plasma; ED50% 5 pmol A chain
equivalents/mL), with negligible cross-reactive interference from
peptide cleavage products released by plasmin from the COOH-terminal
end of the A chain (<3%). Immunoblotting and ELISA studies to
characterize the fate of the F-104 epitope during fibrinogenolysis in
vitro indicated a rapid loss of fibrinogen-associated immunoreactivity
that reflected the heterogeneity of plasmin cleavage sites within the
C domain; cleavage at the 493-494 arg-his bond destroyed the F-104
epitope, while cleavage at other sites released it in an altered,
inaccessible, conformation within the structure of 35- to
40-kD and 17.5- to 18-kD A chain degradation products. Application of the F-104 ELISA to monitor the course of A chain proteolysis in a small study population of patients undergoing thrombolytic therapy for myocardial infarction (n = 14) showed that
the loss of fibrinogen-associated F-104 immunoreactivity was a very
early marker (within 15 to 30 minutes) of in vivo fibrinogenolysis. Additional data obtained suggest that MoAb F-104 may have promise as a
reagent for evaluating the creation of an effective lytic state early
during therapy, information that could help determine the need for
further clinical intervention. Thus, these studies illustrate a
rational, targeted, approach towards the development of a novel
antifibrinogen MoAb whose application as a structural probe for the
region A 487-498 in vitro and in vivo can provide new insights into
the various molecular forms of fibrinogen that circulate under
physiologic conditions and in disease.
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INTRODUCTION |
THE PAST DECADE has witnessed a growing
interest in the role of fibrinogen as a risk factor in cardiovascular
disease. Elevated plasma fibrinogen levels are associated with a number
of pathologic conditions, including myocardial infarction, stroke, and
peripheral vascular disease.1-5 Circulating fibrinogen
concentrations are also increased during menopause, correlating with
the enhanced risk of heart disease in older women.6,7
Relatively little is known about the mechanism by which elevated
fibrinogen levels exert their deleterious effect and, in part, this is
due to the limited number of assays available for probing the various
structure-function relationships associated with fibrinogen's role in
maintaining hemostasis. Indeed, current methods for the measurement of
circulating fibrinogen rely, primarily, on clottability-based
functional assays.8 While the ease and speed of
clottability determinations account for their routine use in the
clinical setting, the limitations of this methodology are also well
documented.9
The COOH-terminal two thirds of the fibrinogen A chain (A
220-610), also referred to as the C domain10 represents
a rich source of structural markers for probing the various
functionalities associated with fibrinogen's role in maintaining
hemostasis. This region is involved in fibrin
polymerization,11 it serves as a substrate for Factor
XIIIa12and plasmin,13 and it binds
to endothelial cells.14 Monoclonal antibodies (MoAbs) that
recognize defined sequences within the C domain have been previously
applied to explore several of these processes at greater
resolution15-17 and to measure various forms of fibrinogen
based on the detection of novel epitopes.18,19
In the case of the two enzyme-linked immunosorbent assays (ELISAs)
reported for the specific quantitation of "intact" plasma fibrinogen based on epitopes within the C domain,18,19
the immunogens and the antigens used for antibody selection were large (A) chain derivatives. The respective epitopes involved in antibody binding were subsequently localized using smaller proteolytic fragments
originating from the COOH-terminal two thirds of the A chain.
The alternative approach, the development of antipeptide MoAbs that
target small, defined, sequences within the fibrinogen molecule, has
not yet been explored although there is ample precedent for the success
of this methodology in the development of fibrin-specific antibodies.20,21
These studies illustrate this targeted approach to MoAb development and
focus on the role of the fibrinogen C domain as a plasmin substrate.
The antibody produced, MoAb F-104, specifically recognizes an epitope
within the region A 487-498 of the dimeric fibrinogen molecule that
is not available for binding once plasmin action has occurred. In view
of this specificity, MoAb F-104 represents a unique reagent that can be
applied not only to learn more about basic aspects of fibrinogen
biochemistry, but also to monitor fibrinogenolysis in vivo and consider
how disturbances in this process might contribute to the development
and management of cardiovascular disease.
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MATERIALS AND METHODS |
Synthetic Peptides
A 487-498 multiple antigenic peptide.
The 12-residue sequence, L-D-G-F-R-H-R-H-P-D-E-A, was synthesized on a
Model 431A synthesizer using standard t-Boc chemistry and the resin,
[-N-t-Boc-lys( -N-t-Boc)]4-lys2-lys-ala-OCH2-PAM (Applied Biosystems, Foster City, CA). The resulting multiple antigenic peptide, or TAM peptide,22 was comprised of eight copies of the 12-residue sequence attached to a seven-member polylysine core. The crude peptide was used as an immunogen and as an antigen for
MoAb characterization (see below) without further purification. Amino
acid analysis indicated the following molar composition: Asp, 15.04;
Glu, 8.28; Pro, 7.23; Gly, 8.20; Ala, 9.81; Leu, 7.81; Phe, 7.56; His,
15.66; Lys, 7.35; Arg, 16.40.
A 487-498 linear peptide.
The monomeric form of the TAM peptide was synthesized as described
above except that the starting resin was
t-Boc-L-ala-OCH2-PAM (Applied BioSystems). The crude,
linear peptide was purified by reversed phase high performance liquid
chromatography (HPLC) using a Dynamax preparative C-8 column (2.1 × 25 cm; Rainin, Woburn, MA) and an increasing gradient of
acetonitrile for elution (4% to 60% in 0.1% trifluoroacetic acid
[TFA]). Analytic HPLC (Dynamax C-8; 0.46 × 25 cm) of the
purified material indicated a single, homogenous peak eluting at 31.1%
acetonitrile. Results of amino acid analysis gave the following molar
composition: Asp, 1.91; Glu, 1.05; Pro, 0.95; Ala, 0.73; Leu, 0.83;
Phe, 0.95; His, 1.89; Arg, 2.01.
Monoclonal Antibody Development
Immunization.
Balb/c mice were immunized weekly for 1 month with multiple,
intraperitoneal injections of the TAM peptide (50 µg/dose). Serum titers were determined in a direct binding screening ELISA that evaluated comparative binding to fibrinogen and its COOH-terminal A
chain plasmin derivatives, A FDPs (see below). One mouse whose antiserum exhibited preferential binding to intact fibrinogen was taken
for fusion.
Hybridoma selection.
Murine hybridoma lines were selected and grown according to standard
procedure23 except that cultures were supplemented with a
preparation of mouse spleen extract (10%) in lieu of the fetal calf or
horse serum traditionally used for in vitro growth.24
Screening assay.
Hybridoma lines secreting immunoglobulins that recognized the region,
A 487-498, in fibrinogen and/or in A FDPs were identified in a comparative direct binding ELISA on antigen-coated plates (purified fibrinogen, 0.86 µg; 5.1 pmol A chain equivalents/well; A FDPs, 0.73 µg; 24.5 pmol A chain equivalents/well), with
horseradish peroxidase-conjugated goat antimouse IgG (GAM IgG-HRP)
(Kierkegaard & Perry, Gaithersburg, MD) used to detect bound
antibody.25 Of the four cell lines selected for cloning
based on their strong binding to fibrinogen on the solid phase, one
proved stable and was taken for further study.
Antibody production and characterization.
The cell line of interest was expanded in pristane-primed
mice26 and its immunoglobulin, designated MoAb F-104 IgG,
then purified from ascites on Protein A Sepharose (Bio-Rad, Richmond, CA). The protein concentration of a standard solution of the purified IgG was determined by amino acid analysis. Isotyping was performed with
the Mouse Monoclonal Sub-Isotyping Kit (Hyclone, Logan, UT).
Purified Antigens for Epitope Characterization
Human fibrinogen.
Kabi fibrinogen (Chromogenix/Pharmacia-Hepar, Franklin, OH) was further
purified by sequential affinity and ion-exchange chromatography on
lysine-Sepharose and DEAE-Sephacel.27 The concentration of a standard solution of the purified fibrinogen preparation was determined by amino acid analysis and expressed as molar A chain equivalents, assuming a molecular weight of 340 kD and 2 moles of A
chain per mole of fibrinogen.
A FDP preparations.
COOH-terminal A chain fibrinogenolytic derivatives, A FDPs, were
released from purified fibrinogen by mild plasmin digestion and then
isolated by affinity chromatography on Concanavalin A Sepharose; the
single A FDP preparation used for the studies here contained 6,137 ± 1,640 pmol A chain equivalents/mL (± standard error of
mean [SEM]; n = 3) and a concentration of 182 µg/mL.19
A 477-517.
The cyanogen bromide (CNBr) fragment corresponding to the A chain
region 477-517, referred to here as CNBr IX (to indicate its relative
position from the A chain's NH2-terminal CNBr fragment, CNBr I), was obtained in partially purified form as
reported.28 The CNBr IX-enriched pool isolated by gel
filtration was further purified by reversed phase semipreparative HPLC
(C-18 column; 0.78 × 30 cm; Waters, Milford, MA), using
a mobile phase comprised of 0.1% TFA (Buffer A) and 60% acetonitrile
(Buffer B) and an increasing gradient of acetonitrile concentration
(30% to 55% B) for elution. The fragment eluting at 35.8%
acetonitrile was identified as CNBr IX based on its molar composition,
determined by amino acid analysis, and the reported primary structure
of the A chain.29,30
Proteolysis of A 487-498-containing peptides.
The octavalent and linear synthetic peptides (100 µg) were each
digested with trypsin (TPCK; Sigma, St Louis, MO) in 0.2 mol/L NH4HCO3, pH 8.2, at a peptide concentration of
0.1% and an enzyme:substrate ratio of 1:100. Incubation was conducted
for 11/2 hours at 37°C and proteolysis interrupted by
addition of acetic acid to 20%, final, followed by vacuum
centrifugation. The peptides and the A chain fragment, CNBr IX, were
also digested with plasmin (0.009 CU/mg substrate) (Kabi/Chromogenix,
Pharmacia-Hepar) in 0.05 mol/L Tris-0.15 mol/L NaCl, pH 7.6, (TBS) for
11/2 hours at 37°C. Plasmin activity was inhibited by
addition of Trasylol (Mobay Pharmaceuticals, New York, NY) to 250 U/mL,
final.
Animal fibrinogens for species cross-reactivity studies.
Purified bovine, murine, and rabbit fibrinogens were used as supplied
(90% to 94% clottable; Sigma). Equine fibrinogen (82% clottable;
Sigma) was further purified essentially as described for human
fibrinogen (see above) except that a final affinity chromatography step
on Protein A Sepharose was included to remove contaminating IgG from
the purified equine fibrinogen pool. The molar concentration of
standard solutions of each of the animal fibrinogens was determined by
amino acid analysis and expressed as A chain equivalents.
Plasmin Proteolysis of Fibrinogen
Purified system.
Fibrinogen, 2.05 mg/mL in TBS 1 mmol/L CaCl2, was digested
with plasmin (0.009 CU/mg fibrinogen) for increasing periods of time
(T1' to T18h) at 37°C. Aliquots were
removed at each of nine time points and digestion interrupted by the
addition of Trasylol to 250 U/mL, final. Plasmin digestion of purified fibrinogen was also performed following an initial preincubation step
with MoAb F-104; the MoAb F-102 (anti-A 563-578)19,31 was used as a control in parallel incubations to evaluate the effect of
IgG binding to a different epitope within the C domain. Antibody
binding was conducted at a twofold molar excess of IgG, relative to
A chain epitope equivalents, for 1 1/2 hours at
37°C. Plasmin and calcium were then added and samples
processed as described above. Control digests, ie, the MoAbs treated
with plasmin in the absence of fibrinogen, were assayed at each time
point for residual binding to fibrinogen-coated wells (see ELISA
screening assay, above), using horseradish peroxidase-conjugated rabbit antimouse immunoglobulins (RAM Ig-HRP; Dako, Carpenteria, CA) for
detection. MoAb F-104 IgG was unaffected by plasmin treatment during
the first 35 minutes of digestion, with 50% of its initial fibrinogen
binding capacity still retained at 1 hour. MoAb F-102 IgG binding was
fully preserved, even after 1 hour of plasmin digestion.
Plasma system.
Activation of endogenous plasminogen in a single donor normal plasma
was initiated by the addition of streptokinase (SK) (Streptase, Astra
Pharmaceutical, Westborough, MA) to a final concentration of 67 U/mL
plasma. Fibrinogenolysis was interrupted at seven time points
(T1' to T18h) following removal of aliquots
and addition of Trasylol to 350 U/mL, final.
Immunoblotting
Discontinuous sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) was conducted on Laemmli gels32 under nonreducing and/or reducing conditions. Prestained molecular
weight markers were included on each run (Amersham, Arlington Heights, IL; GIBCO-BRL, Gaithersburg, MD; 200 kD, myosin; 97 kD, phosphorylase B; 68 kD, bovine serum albumin; 43 kD, ovalbumin; 28 kD, carbonic anhydrase, 18 kD, B-lactoglobulin; 15 kD, lysozyme). Electrophoresed components were transferred to nitrocellulose (Schleicher and Schuell, Keene, NH) or polyvinylidene difluoride (PVDF) (Millipore, Bedford, MA) membranes.33,34 In some cases, transfers were stained for total protein with Amido Black. MoAbs F-104 (anti-A 487-498) and F-102 (anti-A 563-578) were used for immunoblotting at
1 to 2 µg IgG/mL and bound immunoglobulins then detected with RAM
Ig-HRP, following reaction with 4-chloro-1 naphthol
(Bio-Rad).25
Amino Acid Analysis
Samples were hydrolyzed in 6 N HCl for 24 hours under vacuum at
110°C in a Pico Tag workstation (Waters) and amino acid analysis conducted on a Beckman Model 6300 amino acid analyzer (Beckman, Palo
Alto, CA).
MoAb F-104 ELISA
This assay takes advantage of the fact that solution phase antigens
that include an expressed F-104 epitope can compete with fibrinogen on
the solid phase for MoAb F-104 binding. Details of the F-104 solution
phase competitive ELISA have been recently reported.25
Briefly, equal volumes of serially diluted competitor and MoAb F-104
IgG (13.6 ng/mL) were preincubated for 1 1/2 hours at room temperature
and then incubated for 18 hours at 4°C on fibrinogen-coated wells
(0.86 µg/100 µL). Bound MoAb F-104 was detected as 414 nm absorbance following successive incubations with RAM Ig-HRP and its
substrate, o-phenylenediamine (OPD), on the solid phase. Dose-response
curves were generated using logit transformation to extend the
effective linear portion of the curve, with logit-transformed %B (the
ratio of fibrinogen-bound MoAb F-104, in the presence and in the
absence of solution phase competitor) plotted against the molar
concentration of purified competitor, determined by amino acid
analysis. Plasma fibrinogen A chain concentrations were derived
using purified fibrinogen as the assay standard for
quantitation.19
Data Analysis
ELISA data are expressed as the mean ± SEM of at least two separate
experiments (each performed in duplicate), unless otherwise indicated.
The Student's t-test was used for statistical analysis, with a
difference at the P < .05 level considered significant.
Plasma Samples
Blood from an individual normal donor was collected into 3.8% sodium
citrate (9 parts blood: 1 part anticoagulant) and Trasylol added to 250 U/mL, final. Platelet poor plasma was harvested following centrifugation at 2,100g for 10 minutes at 4°C and then
aliquoted and stored frozen at  80°C for single use. Clottable
fibrinogen determinations for this plasma were performed at the
Columbia Presbyterian Medical Center (CPMC) Coagulation
Laboratory.19 Plasma aliquots from patients undergoing
thrombolytic therapy with either recombinant tissue plasminogen
activator (rt-PA) or streptokinase (SK) were archival and originated
from the TIMI (Phase I) Trial at CPMC. Blood collection and plasma
processing for these samples have been detailed
elsewhere.35
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RESULTS |
Species specificity of the antifibrinogen A chain MoAb,
F-104 (IgG1).
Figure 1 illustrates the SDS-PAGE and F-104
immunoblotting profiles obtained for human, equine, bovine, rabbit, and
murine fibrinogens. Specific F-104 immunoreactivity was detected in the human fibrinogen lanes only. The results obtained under reducing conditions showed that the epitope responsible for antibody binding was
localized within the A chain. The faint band of immunoreactivity at
the B chain position is consistent with the comigration of a 56-kD A
chain remnant (degraded A chain), and reflects the known structural
heterogeneity of the COOH-terminal portion of the A
chain.36 These findings show that although
MoAb F-104 was raised against a small peptide derivative corresponding
to the sequence A 487-498 in human fibrinogen, it recognizes its A chain epitope within the immunogen's large, parent protein.
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| Fig 1.
MoAb F-104 species specificity. Nonreducing and reducing
SDS-PAGE (8% and 10% gels, respectively) and F-104 immunoblotting (PVDF transfers) were conducted as described in the text. Approximately 5 µg fibrinogen were applied to each lane for Amido Black-stained transfers and 2.5 µg for F-104 epitope localization, as follows: HU
(human), EQ (equine), BO (bovine), RA (rabbit), and MU (murine). The
migration of standard molecular weight markers is indicated at the left
of each panel. The weak band of immunoreactivity observed for murine
fibrinogen (top) is nonspecific and reflects binding of the HRP
conjugate to contaminating IgG in this preparation; the same profile
was obtained when the F-104 incubation step was omitted.
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The immunoblotting findings in Fig 1 also suggest that the determinant
responsible for the generation of MoAb F-104 is not conserved within
the A chains of a number of animal fibrinogens. Epitope denaturation
was probably not a contributing factor to these negative findings, as
the four animal fibrinogens characterized here also failed to compete
with human fibrinogen for antibody binding in the MoAb F-104 solution
phase competitive ELISA (see below); (data not shown). As shown in
Table 1, available sequence data for the
region corresponding to (human) A 487-498 in nine animal species
(including equine, bovine, and murine),24,37-39 identify
several nonconserved residues, particularly in the segment A
489-492, which may account for the antihuman fibrinogen specificity of
MoAb F-104 and for the antibody's generation in a murine host.
Immunologic characterization of the MoAb F-104 epitope.
Figure 2 illustrates the results of
solution phase competitive ELISAs designed to characterize the native
structure of the epitope recognized by MoAb F-104. In these assays,
various antigens were tested for their ability to compete with
fibrinogen on the solid phase for antibody binding. As shown in Fig 2A,
the octavalent immunogen and its 12-residue, linear, counterpart were
effective solution-phase competitors, each exhibiting nearly
superimposable dose response curves in the F-104 ELISA
(ED50%B 155 ± 25.5 and 137 ± 25.9, respectively).
The 41-residue CNBr fragment, A 477-517, also competed with
fibrinogen for MoAb F-104 binding, but the slope of its dose response
curve differed from that of the peptides and approximately twice the
dose of CNBr IX was required to achieve 50% binding (239 ± 30.8).
Plasmin treatment destroyed the F-104 epitope in the TAM peptide and,
to a lesser extent, in the CNBr fragment (46% preserved), but had no
effect on the immunoreactivity of the small, linear peptide. Trypsin
treatment, however, did result in destruction of the epitope in this
derivative (data not shown). Collectively, these findings suggest that
one or both arginine residues within the sequence A 487-498 are
involved in MoAb F-104 binding (see Table 1, human sequence) and that steric factors may render these residues somewhat inaccessible within
the structure of CNBr IX. The absence of lysines in the linear peptide,
but not in the octavalent derivative whose structure includes a
polylysine core, implies that the F-104 epitope may have been
"protected" from plasmin cleavage within the smaller peptide
because of the enzyme's inability to bind to this substrate via
`pseudo' requisite lysine binding sites.
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| Fig 2.
Immunochemical characterization of the MoAb F-104
epitope. (A) Epitope expression in purified A chain derivatives. The
following antigens whose structures include A 487-498 were tested as
solution phase competitors in the MoAb F-104 ELISA: TAM peptide ( );
linear peptide ( ); CNBr IX (A 477-517) ( ); plasmin-digested
peptides (open symbols, dotted lines). The molar concentration of the
octavalent TAM peptide was normalized to the number of moles of A
487-498/mole of peptide. Immunoreactivity is expressed as logit %B
(see text). (B) Epitope expression in fibrinogen and its A chain
plasmin derivatives. Purified fibrinogen (solid symbols and line),
plasma fibrinogen from a single normal donor (solid symbols, dashed
line), and A FDPs obtained from plasmin-treated purified fibrinogen (open symbols, dashed line) were assayed for F-104 immunoreactivity. Data are presented as in (A). The molar concentration of fibrinogen in
the single donor plasma was derived from clottability measurements (3.45 mg/mL) and expressed as A chain equivalents (20,294 pmol/mL).
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As shown in Fig 2B, both purified and plasma fibrinogen were potent
solution phase competitors in the F-104 ELISA, with similar dose
response curves characterized by ED50%B levels of 6.0 ± 0.31 and 4.7 ± 0.65 pmol/mL, respectively. A FDPs
exhibited less than 3% cross-reactivity in this assay. These findings
indicate that the F-104 epitope is expressed in native, intact
fibrinogen and that plasmin cleavage either destroys it and/or
releases it within A chain derivatives whose solution phase
structures include the region A 487-498 in a buried or altered
conformation.
To extend the findings in Fig 2B, solution phase competitive ELISAs, in
parallel with immunoblotting studies, were conducted to characterize
the fate of the F-104 epitope during the course of fibrinogen
proteolysis by plasmin. Figure 3
illustrates the results obtained for both purified (Fig 3A) and plasma
(Fig 3B) systems. As shown in Fig 3A, F-104 solution phase
immunoreactivity decreased to 27.8% of its T0 level within
the first 3 minutes of plasmin digestion, with less than 10% remaining
after 20 minutes, under the experimental conditions used. As visualized
by immunoblotting, this solution phase immunoreactivity profile
paralleled both the disappearance of intact fibrinogen and the
appearance of 35- to 40-kD A FDPs, which increased in intensity as
digestion progressed. (Smaller A FDPS, 17 to 18.5 kD, were
occasionally visualized in other F-104 immunoblots of plasmin-treated
fibrinogen, but these fragments always represented a minor component of
the total F-104-immunoreactive A FDP population released over the
entire course of proteolysis). As shown in Fig 3B, similar findings
were observed for the plasma fibrinogenolytic system. The collective findings in Figs 2B and 3 confirm the specificity of the MoAb F-104
ELISA for intact fibrinogen and show that for at least one group of
A FDPs the A chain region 487-498 is present, but inaccessible for antibody binding in solution.
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| Fig 3.
F-104 epitope expression during fibrinogenolysis. (A)
Purified system. Purified fibrinogen was digested with plasmin for
increasing periods of time and residual F-104 immunoreactivity
quantified by solution phase competitive ELISA. The T0
F-104 immunoreactivity observed (16,346 ± 47 pmol A chain
equivalents/mL) was 136% of the expected value based on amino acid
analysis. Inset. SDS-PAGE was conducted on 12.5% gels under
nonreducing conditions followed by transfer to nitrocellulose and F-104
immunoblotting as described in Materials and Methods. A total of 10 µg (74.1 pmol A chain equivalents, determined immunologically) of
intact or plasmin-treated fibrinogen was applied to each lane. The
migration of standard molecular weight markers is indicated at the
extreme right for reference. (B) Plasma system. Fibrinogenolysis
was initiated in a single donor normal plasma and F-104
immunoreactivity monitored over the course of digestion by solution
phase competitive ELISA. Results are expressed as for (A). F-104
immunoreactivity in the T0 plasma (31,671 ± 2,063 pmol
A chain equivalents/mL) was 156% of the expected value based on
clottability determination. Inset. SDS-PAGE and immunoblotting were
conducted as for (A). Approximately 3.5 µg clottable plasma
fibrinogen (31.7 pmol A chain equivalents, determined
immunologically) were applied to each lane.
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Because the immunoblotting studies described in Fig 3 are blind to
COOH-terminal A chain degradation products that are released by
plasmin cleavage within the F-104 epitope per se (which leads to
epitope destruction and loss of F-104 immunoreactivity), evidence for
this second group of A FDPs was sought by using MoAb F-102 (anti-A 563-578) as an immunoblotting probe. As shown in
Fig 4 (left panel), plasmin digestion of
purified fibrinogen resulted in the release of three major fragments,
37 kD, 17.5 kD, and 9.9 kD, each of which included the region A
563-578 within their respective structures. As shown in the right panel
of Fig 4, when plasmin digestion was conducted using
fibrinogen that had been preincubated with MoAb F-104 to block
plasmin's access to the F-104 epitope (within A 487-498), only two
of the three A FDPs were detected (in contrast, there was no
change in the pattern of F-104 immunoreactive fragments released,
compared with control incubations, when MoAb F-102 was used as the
blocking antibody before plasmin cleavage; data not shown). The
observed molecular weight of the "missing" A FDP is most
consistent with that predicted for a fragment corresponding to A
494-583, ie, 9.8 kD (and/or possibly A 492-583; 10.1 kD).
The findings in Fig 4 thus provide strong evidence implicating the
arginine residue at A 493 (and/or 491), within the F-104
epitope, as a major fibrinogen A chain plasmin cleavage site.
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| Fig 4.
The F-104 epitope in fibrinogen includes a plasmin
cleavage site as visualized by immunoblotting with MoAb F-102
(anti-A 563-578). Purified fibrinogen was digested with plasmin (see
Fig 3 legend) either without (left) or with (right) an initial MoAb F-104 preincubation step. PVDF transfers from 12.5% SDS-PAGE
(nonreduced) were subjected to immunoblotting with MoAb F-102; 9.6 µg
(56 pmol A chain equivalents) of intact or plasmin-treated
fibrinogen were applied to each lane. At this load
application, COOH-terminal A chain degradation products present at
low level in the starting fibrinogen preparation are visualized in the
T0 lanes. The high molecular weight material at the top of
the lanes in the right panel reflects the F-104 IgG added during the
preincubation step (16.8 µg, 113 pmol IgG/lane).
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Application of the F-104 ELISA for the detection of fibrinogenolysis
during thrombolytic therapy.
In view of the findings in Figs 3 and 4 showing that during in vitro
fibrinogenolysis the loss of fibrinogen-associated F-104 immunoreactivity parallels COOH-terminal A chain degradation, we
next considered whether MoAb F-104 could be applied to characterize fibrinogenolysis as it occurs in vivo.
Figure 5 illustrates the F-104 ELISA
profiles obtained for 14 patients undergoing thrombolytic therapy for
myocardial infarction, where rt-PA (n = 7) and SK (n = 7) were both
used as thrombolytic agents. As shown in Fig 5 for the rt-PA-treated
group (left), five patients exhibited an early loss of F-104
immunoreactivity with less than 50% (31.4% to 49.5%) of the
T0 circulating fibrinogen remaining after 15 minutes of
therapy. Fibrinogenolysis was less efficient in the two other patients
(>60% to 100% of the T0 fibrinogen was still detected
at 15 minutes), both of whom failed to show radiographic evidence of
successful reperfusion at the end of treatment. As shown in Fig 5 for
the SK-treated group (right), less than 5% (0.5% to 4.8%) of the
T0 F-104 immunoreactivity remained after 30 minutes of
therapy in five of the seven patients examined. A chain proteolysis
appeared to proceed more slowly in the two other patients, both of whom
failed to reperfuse, with 10% to 20% of the T0 F-104
immunoreactivity persisting over the entire course of treatment.
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| Fig 5.
In vivo fibrinogenolysis during thrombolytic therapy.
Plasmas from 14 patients undergoing thrombolytic therapy with either rt-PA (left) or SK (right) were assayed for fibrinogen-associated F-104
immunoreactivity by ELISA. Eight time points, including pretreatment
and 24-hour posttreatment samples, were examined (plasmas for one
24-hour point, one 90-minute point, and three 15-minute time points
were not available). Patients who exhibited radiographic evidence of
reperfusion are identified by solid lines and symbols, while those who
did not are identified by dashed lines and open symbols. Results are
expressed as the percent of pretreatment F-104 immunoreactivity.
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F-104 immunoblotting profiles obtained for two representative patients
from each group showed that the A FDPs released during thrombolytic
therapy were the same as those shown in Fig 3 for fibrinogenolysis in
vitro (data not shown). Collectively, the findings in Fig 5 indicate
that A chain proteolysis in vivo and in vitro proceed by a common
mechanism and that plasmin cleavage at the F-104 epitope is an early
marker of fibrinolytic system activation.
| |
DISCUSSION |
The studies in this report are concerned with the development and
application of a new structural probe (MoAb F-104) for the detection of
fibrinogenolysis. The studies focus on the 12-residue sequence A
487-498 and were prompted by several observations related to the unique
structure of this localized region within the C domain. First,
hydropathy calculations40 predicted that the region A
487-498 was extremely hydrophilic and, therefore, likely to assume an
exposed conformation on the surface of the fibrinogen (A chain)
molecule (Fig 6). The residues
arg-his-arg-his within this sequence appeared to form a particularly
vulnerable site for potential interaction with other proteins, possibly
plasmin, based on its complement of positively charged amino acids
lying at the apex of the hydrophilic peak. Only five other A chain segments share this unique hydrophilicity, as well as a prominently accessible arginine (or lysine) residue. Because three of these localized regions correspond to sequences known to be involved in the
plasmin-mediated release of Fragments E and D,41 core fragments viewed as the primary hallmarks of
fibrinogenolysis,42 we considered the possibility that A
487-498, within the C domain, might also play a significant and as
yet unrecognized role in fibrinogen's proteolysis by plasmin. This
argument was strengthened by interspecies structural comparisons for
the A chain region corresponding to human A 487-498 (Table 1).
These data indicated that several amino acids in the vicinity of, and
including, arg 493 were highly conserved, thus suggesting an important
structure-function relationship for this localized region of the
fibrinogen molecule.
View larger version (16K):
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| Fig 6.
A 487-498. The 12-residue sequence targeted for
antibody development is shown as part of the Kyte Doolitle hydropathy
plot derived for the human fibrinogen A chain.
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Several important findings that support this rationale emerged from
these studies as a consequence of having successfully developed a MoAb
to the targeted sequence, A 487-498. First, arg A 493 (and/or possibly arg A 491) is an early plasmin cleavage site within the C domain. Second, the sequence A 487-498 (as represented by the F-104 epitope) is exposed on the surface of intact
fibrinogen, but this accessibility is altered once plasmin attacks its
multiple cleavage sites within C domain; cleavage at the 493-494 bond severs the structural integrity of this localized sequence, while
cleavage at other sites within the C domain releases it in a buried
conformation within the structure of moderately sized A FDPs. Third,
MoAb F-104 can be used as a structural probe for early fibrinogenolysis
because the loss of fibrinogen-associated F-104 immunoreactivity is a
hallmark of plasmin activity at the A 493-494 bond (and/or
the 491-492 bond). Finally, MoAb F-104 can detect fibrinogenolysis in
vivo, as illustrated here for the A chain proteolysis that
accompanies thrombolytic therapy. (Parenthetically, MoAb F-104
represents an unusual example where immunization with a TAM peptide
derivative produced a highly specific, antifibrinogen antibody.
Although TAM peptides, by virtue of their epitope density, offer
enhanced antigenicity relative to their monomeric counterparts, their
use as immunogens for the development of antipeptide antibodies that
cross-react with their cognate proteins in solution has met with
limited success).43
Previous studies from several different laboratories have documented
the presence of multiple cleavage sites within the COOH-terminal two
thirds of the A chain13,42,44 and implicated specific sites that are likely to function during fibrinogenolysis in vivo, based on the structural characterization of A FDPs isolated from plasmin-treated fibrinogen.45-47 The identification, in
this report, of an additional in vivo A chain plasmin cleavage site,
ie, 493/491, illustrates the value of anti-A chain peptide
antibodies when applied as structural probes for elucidating aspects of
fibrinogen's proteolysis by plasmin. It should also be possible to use
these reagents in similar immunochemical studies to learn, for example, more about the A chain heterogeneity that is a well-known feature of
normal fibrinogen populations,36 to define the sequence of plasmin attack sites within the C domain, and to elucidate how the
released products differ when cross-linked fibrin, as distinct from
fibrinogen, serves as the plasmin substrate.
Two other MoAbs directed at epitopes in the vicinity of A 476 and
A 575, within the C domain, have been previously used for the
clinical detection of fibrinogenolysis.19,48 MoAb F-104, ie, anti-A 487-498, represents a new structural probe for monitoring the initial stages of fibrinogenolysis in vivo, based on the findings obtained here for 14 patients undergoing thrombolytic therapy for
myocardial infarction. Recognizing that this small sample population
renders the findings and inferences drawn from them somewhat
speculative, one aspect of the results obtained seems worth
highlighting. Data for the rt-PA treatment group suggest that there is
a significant difference in the levels of intact fibrinogen circulating
at 15 to 30 minutes (P = .022; .038) between patients who show
clinical evidence of reperfusion at the end of treatment and those who
do not. (This same trend was not observed for the SK-treated patients
[P = .109], although one of the three who failed to reperfuse
exhibited the slowest rate of fibrinogenolysis in this group.) While
these findings will require confirmation from a larger study
population, the underlying implication is that cleavage at the F-104
epitope may be a sensitive marker of the systemic lytic state that is
considered to be a prerequisite for effective
thrombolysis.49 Currently, the identification of high risk
patients undergoing thrombolytic therapy relies on the clinical
evaluation of resolution in ST segment elevation and on biochemical
markers such as troponin T.50 MoAb F-104's potential
clinical use as a vehicle for monitoring the creation of a systemic
lytic state early during treatment (and thus providing information
about whether or not additional intervention may be warranted) would
obviously require a more rapid detection system than the one developed
here, but there is ample precedent in the literature for assay
technologies, including latex agglutination,51 which
emphasize adequate speed. In light of the recent interest in
thrombolytic therapy as a treatment modality for stroke52 and for thrombotic disease in children,53 markers such as
the F-104 epitope could provide important information for the
development of appropriate dosing regimens in these relatively new
therapeutic arenas.
| |
FOOTNOTES |
Submitted June 3, 1997;
accepted October 22, 1997.
Address reprint requests to Joan H. Sobel, PhD, Columbia University
College of Physicians & Surgeons, Department of Medicine/Black Building
1011/Box 104, 630 W 168th St, New York, NY 10032.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Andy Pound (Howard Hughes Protein Core Facility of
Columbia University) for performing the amino acid analyses and Ay-Chin
Huang (CPMC Coagulation Laboratory) for performing the clottable plasma
fibrinogen determinations required over the course of this study.
| |
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Abstract
Introduction
Abstract