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Blood, Vol. 95 No. 5 (March 1), 2000:
pp. 1721-1728
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Iatron Laboratories Inc., Katori-Gun, Chiba, Japan; Division of
Hemostasis and Thrombosis Research, Institute of Hematology, Jichi
Medical School, Minamikawachi, Tochigi, Japan; Intensive Care Unit,
Shiga University of Medical Science, Ohtsu, Shiga, Japan.
When granulocytes are stimulated under certain clinical conditions,
elastase is released therefrom and digests fibrin(ogen) independently
of the plasmin system, which may also be mobilized simultaneously.
Thus, discrimination of these 2 systems becomes urgent for the
diagnosis and treatment of the underlying diseases. Using as immunogen
a 97-kd granulocyte-elastase digest of human fibrinogen, we raised an
antibody IF-123 that specifically recognizes elastase digests of human
fibrin(ogen). The 97-kd elastase fragment resembles plasmic fragment
D1, and the epitope of this antibody is located on the
A
Granulocyte-derived elastase (GE) is localized in
azurophilic and specific granules of granulocytes and is released
extracellularly in response to various stimuli such as endotoxin and
cytokines.1 Released GE may degrade the components of the
extracellular matrix, such as elastin and a variety of proteoglycans,
and also plasma proteins including fibrin(ogen).2,3 GE has
thus been implicated in the pathogenesis of a wide variety of diseases.
Granulocyte-derived elastase is regulated predominantly by
Chemicals and reagents
Preparation of GE digests of human fibrinogen corresponding
to plasmic fragment D1
Preparation of GE digests of cross-linked fibrin The human fibrinogen fraction was brought to 10 mg/mL in TBS containing 5.0 mmol/L CaCl2 (TBS-CaCl2). The fibrinogen fraction (10 mL) enriched with 1.0 µg/mL factor XIII was clotted with 0.6 NIH-u/mL human thrombin (Sigma, St. Louis, MO). The fibrin clots formed were squeezed with filter paper, washed extensively with 0.15 mol/L NaCl, lyophilized, and ground with a glass rod. A 50-mg portion of the ground fibrin was suspended in 8.0 mL TBS-CaCl2 containing 1.0 mmol/L benzamidine HCl and 1.0 mmol/L t-AMCHA, and digested with 37 µg of GE for 3 hours at 37°C. The enzyme/substrate ratio (wt/wt) was 1:1333. The reaction was terminated with 1.0 mmol/L DFP, and the digests were centrifuged for 20 minutes at 10,000g. The supernatant was applied to a Sephacryl S-300 HR column (5.0 × 90 cm), and the first peak fractions were collected and pooled. The GE digests in this pooled fraction were found to correspond to the phase-3 plasmic digests of cross-linked fibrin (plasmic XDP)10,16 as examined by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE).17 This fraction was named GE-XDP.Preparation of plasmic degradation products Plasmic degradation products of human fibrinogen were prepared essentially as described.18 The degradation products were found to contain fragments X, Y, D1A, D1, and E3. When necessary, fragment D119 and phase-3 digests of cross-linked fibrin16 were prepared as described. Absorbance coefficients (A1%, 1 cm at 280 nm) for calculating the protein concentration were 15.1 for fibrinogen,20 20.8 for fragment D1,20 18.1 for plasmic XDP, which was assessed on the basis of 20.0 and 12.0 for plasmic fragments DD and E,20 respectively. The same values were used for the corresponding GE digests.Preparation of monoclonal antibodies specific to the GE digests of fibrinogen We inoculated Balb/c mice with 50 µg of GE-D in complete Freund's adjuvant followed by 50 µg of GE-D without the adjuvant as immunogen essentially by a hybridoma technique of Köhler and Milstein21 with minor modifications as described elsewhere.22 Selection of clones secreting monoclonal antibodies (mAbs) specific to the GE digests of fibrinogen, but not to the parent molecule fibrinogen or it plasmic digests was carried out by a direct-binding enzyme-linked immunosorbent assay (ELISA) using GE-D, plasmic fragment D1, and fibrinogen as antigens.Binding of mAbs to antigens determined by a direct-binding ELISA Binding between the antigens and mAbs was studied by a direct-binding ELISA as described previously.22 Briefly, wells of polystyrene microtiter plates (Immulon-II, Dynatech, Chantilly, VA) were coated overnight at 4°C with 50 µL of respective antigens at 5 µg/mL in 50 mmol/L Tris-HCl, pH 8.5. The antigen-coated wells were washed with 0.15 mol/L NaCl containing 0.05% (w/v) Tween-20 (NaCl-Tween) and incubated with 50 µL of the culture supernatant for 1 hour at 25°C. After decantation of the reaction mixture, the wells were washed with NaCl-Tween. Horseradish peroxidase (HRPO)-conjugated antimouse IgG rabbit antibody (DAKO, Glostrup, Denmark) diluted 200-fold with 50 mmol/L Tris-HCl, pH 8.0, containing 0.15 mol/L NaCl and 0.05% (w/v) Tween-20 was added to each well as the second antibody. The bound antibodies were determined using 50 mmol/L Tris-HCl, pH 7.5, containing 0.5 mmol/L 4-aminoantipyrine, 10 mmol/L phenol, and 0.005% hydrogen peroxide as substrate, and the color produced was read at 492 nm on an MPR-A4i Microplate reader (Tosoh, Tokyo, Japan).Determination of the dissociation constant of IF-123 Binding between antigens and IF-123 was studied by a solid-phase ELISA using microtiter plates coated with either GE-D or GE-XDP as described previously.22 The amounts of bound antibodies were calculated from calibration curves constructed with known amounts of the bound respective antibodies. The dissociation constant was calculated as described previously.23Immunoblotting Reactivity of IF-123 to various antigens was analyzed by immunoblotting essentially as described elsewhere.14,24Separation of the  -,  -
and  -chain remnants with a linear gradient from 30% to 60% acetonitrile in 5 minutes. The pyridylethylated -remnant
(Pe-/GE-D) was further purified by rechromatography on the same
column, and the collected fraction was dried using a Speed Vac (Savant
Instruments, Farmingdale, NY). The dried Pe-/GE-D (510 µg) was
dissolved in 235 µL of 50 mmol/L Tris-HCl, pH 9.0, containing 3 mol/L
urea, and digested with lysyl endopeptidase (Wako Chemical, Osaka,
Japan) at an enzyme/substrate molar ratio of 1:50 for 18 hours at
37°C. The digests were analyzed by reverse-phase HPLC using a
Cosmosil 5C18P column (4.6 × 150 mm, Nacalai Tesque, Kyoto,
Japan). A 0.1% trifluoroacetic acid/water (solvent A) and 0.1%
trifluoroacetic acid/acetonitrile (solvent B) gradient system was used,
and a linear gradient from 0% to 40% solvent B in 100 minutes was
used. The flow rate was 0.5 mL/min, and the column effluent was
monitored by absorbance at 215 nm (A215). Individual peak fractions
were dried with a Speed Vac, dissolved in 0.2 mL of 10 mmol/L
bicarbonate buffer, pH 8.6, and coated to wells of polystyrene
microtiter plates (Immulon-II) after 500-fold dilution. The
peptide-coated wells were washed 3 times with NaCl-Tween and incubated
with IF-123 at 5 µg/mL in TBS-Tween for 1 hour at 25°C. The bound
antibodies were detected by antimouse IgG conjugated with HRPO and
visualized by incubation with 4-aminoantipyrine-phenol-hydrogen
peroxide as substrate.
Inhibition by synthetic peptides of the binding of IF-123 to
immobilized affinity-purified GE-D or a synthetic 13-residue
peptide corresponding to A (192-204) residue segment.
Preparation of protease(s) released from activated granulocytes To release the enzymes stored in granules, the granulocytes (5.7 × 107 cells) isolated from 30 mL of heparinized venous blood obtained from healthy volunteers described elsewhere,25 were preincubated for 3 minutes at 37°C with 5 µg/mL of cytochalasin B (Sigma) in the presence of 1.3 mmol/L CaCl2 and 1.0 mmol/L MgCl2, and activated with 500 nmol/L N-formyl-Met-Leu-Phe (fMLP, Sigma) for 5 minutes at 37°C. After centrifugation, the supernatant was collected and passed through a cellulose acetate membrane filter (0.2 µm pore, Advantec, Tokyo, Japan). The concentrations of GE and cathepsin G in the supernatant were measured by spectrophotometric assays using MeO-Suc-Ala-Ala-Pro-Val-NA and Suc-Ala-Ala-Pro-Phe-NA (Sigma) as substrates, respectively.26 Concentrations of GE and cathepsin G were calculated from calibration curves constructed with known amounts of the commercially available purified GE and cathepsin G (purified from human purulent sputum; elastase, myeloperoxidase, lysozyme free; Elastin Products).Characterization of GE digests by immunoprecipitation with IF-123-conjugated Sepharose 4B Purified IgG fractions of IF-123 and JIF-23,27 an mAb that specifically recognizes the amino-terminal disulfide-linked conformation of the plasmic fragment D species (D1, D2, and D3), were individually coupled to CNBr-activated Sepharose 4B (Pharmacia Biotech) according to the manufacturer's instruction, and the IgG-conjugated Sepharose 4B was suspended in an equal volume of 50 mmol/L Tris-HCl, pH 8.0, containing 0.15 mol/L NaCl and 0.05% sodium azide. One hundred microliters of either purified GE digests (10 µg of GE digests of fibrinogen and GE-XDP) or patient-derived plasma samples were incubated with 200 µL of the gel suspension and 1 mL of 20 mmol/L Tris-HCl, pH 7.6, containing 0.5 mol/L NaCl and 0.05%(w/v) Tween-20 (suspension buffer) for 16 hours at 4°C, and the mixture was centrifuged at 10 000g for 3 minutes at 25°C. The gels precipitated were suspended and washed 5 times with the same buffer, each time by rotating the tubes for 20 minutes at 25°C, and then centrifuged at 10 000g for 3 minutes at 25°C. The precipitate was resuspended in 1.0 mL of 20 mmol/L Tris-HCl, pH 7.6, containing 0.15 mol/L NaCl and 2% SDS to solubilize the proteins bound to the antibody-coupled gels. Proteins in 0.2 mL of the supernatant after centrifugation at 10 000g for 5 minutes at 25°C were isolated by acetone precipitation and subjected to SDS-PAGE using 3.5% to 9.0% gradient gels under nonreducing conditions as described elsewhere.28 The proteins were visualized by silver staining.Measurement of GE digests of fibrinogen and fibrin in clinical samples by a sandwich ELISA Wells of polystyrene microtiter plates were coated overnight at 4°C with 20 µg/mL F(ab')2 fragment of IF-123 in 50 mmol/L Tris-HCl, pH 8.5, and 50 fold- diluted plasma samples in 50 mmol/L Tris-HCl, pH 8.0, containing 0.05%(w/v) Tween-20 and 1 mol/L urea were allowed to react with the immobilized F(ab')2 fragment of IF-123 for 1 hour at 25°C. After decantation of the reaction mixture, the wells were washed with NaCl-Tween, and HRPO-conjugated antihuman fibrinogen rabbit antibody (DAKO) was added to each well as the second antibody. The amounts of bound antibodies were determined by reading A492 on an MPR-A4i Microplate reader using 4-aminoantipyrine-phenol-hydrogen peroxide as substrate. Proteins specifically bound to IF-123 were determined on a calibration curve constructed with pooled normal plasma spiked with known amounts of GE-XDP.Statistical analysis To compare the levels of GE digests in plasma between patients and the control, Welch's t test was used. A P value of less than 0.05 was considered significant.
Characterization of an mAb specific to GE digests of human fibrinogen and cross-linked fibrin Among the mAbs thus prepared, there was an antibody that specifically reacted with GE-D but not with fibrinogen or its plasmic D1, when analyzed by a direct-binding ELISA. The reactivity of this antibody IF-123 was independent of calcium ions, although the fibrinogen D domain contains a high-affinity calcium binding site29,30 (data not shown). This antibody was classified into IgG1 with -type light chains. When analyzed by
immunoprecipitation followed by SDS-PAGE, GE-D and GE-XDP were adsorbed
to IF-123 (Figure 1A, GE-D and GE-XDP in
lanes 5 and 6, respectively). Fibrinogen, its plasmic digests, or
plasmic XDP were not adsorbed (Figure 1B, lanes 4-6). The dissociation
constants of IF-123 with GE-D and GE-XDP were
1.20 × 10 9 mol/L and
1.23 × 109 mol/L, respectively.
Epitope mapping for IF-123 By immunoblotting run under reducing conditions, neither fibrinogen nor plasmic D1 was stained (Figure 2B, lanes 1 and 2), whereas 2-remnant
species of GE-D were stained with this antibody (Figure 2B, lane 3).
The epitope was thus localized to an approximately 12-kd
A-chain-derived segment specifically cleaved by GE. The 12-kd
fragment purified (Figure
3A,
Pe-/GE-D) and their lysyl endopeptidase digests were fractionated by
reverse-phase HPLC on a Cosmosil 5C18P column (Figure 3B). Separated
peak fractions were coated onto immunoplates and their reactivities
with IF-123 were examined. Among them, a fraction denoted by peak #19
(Figure 3B) reacted with IF-123. By sequence analysis, we assigned this peptide to the A (192-204) residues,
Asp-Leu-Leu-Phe-Ser-Arg-Asp-Arg-Gln-His-Leu-Pro-Leu. Because lysyl
endopeptidase hydrolyzes specifically the carboxyl side bond of lysyl
residues,31 it is very likely that peptide #19 lacking the
carboxyl-terminal Lys residue constitutes the carboxyl-terminal segment
of the -remnant of GE-D. To confirm that the epitope resides in
peptide #19, we conducted inhibition assays by using ELISAs, where the
affinity-purified GE-D and a synthetic peptide with the same sequence
as peptide #19, s-peptide 19, were individually immobilized, and
binding of IF-123 was tested in the presence of various synthetic
analogs of peptide #19. Two synthetic peptides, s-peptide 19 and a
peptide analog, Ser 196-Leu 204, lacking the first 4 amino acids of
s-peptide 19 were able to inhibit the binding of IF-123 to the
affinity-purified GE-D (Figure 4A) as well as to
s-peptide 19 (Figure 4B) in the same manner. When the first 3 Ser-Arg-Asp residues of the (Ser 196-Leu 204) peptide had been removed,
binding with IF-123 decreased nearly two magnitudes (Figure 4A and B).
On the other hand, deletion of Leu 204 from or addition of Ile 205 or
Ile 205-Lys 206 to the (Ser 196-Leu 204) peptide resulted in complete
loss of binding with IF-123 (Figure 4A and B). The A Lys 206 has
been reported to be a potential plasmic cleavage P1 site.32
The results together suggested that the A (Ser 196-Leu 204) residue
segment functioned as the epitope for IF-123, and that its
carboxyl-side residues were critical for full expression of the
epitope, as schematically shown in Figure 5.
Expression of the epitope by proteases released from activated granulocytes When granulocytes (107 cells/mL) had been stimulated with 500 nmol/L fMLP, they were estimated to release GE and cathepsin G at the concentration of 610 nmol/L and 56 nmol/L, respectively. These proteases were tested for digestion of fibrinogen with or without prior treatment with various protease inhibitors. When the supernatant of activated granulocytes had been treated with a mixture of Z-Gly-Leu-Phe-CH2Cl, N-tosyl-L-lysine
chloromethylketone (TLCK), and N-tosyl-L-phenylalanine
chloromethylketone (TPCK), inhibitors of cathepsin G, trypsin-type
enzymes, and chymotrypsin-type enzymes, respectively, profiles of
fibrinogen degradation and appearance of the reactivity with IF-123
(Figure 6B) were nearly identical with those for the
nontreated supernatant (Figure 6A) as examined by SDS-PAGE and a
sandwich ELISA. When the supernatant had been treated with a
GE-specific inhibitor, MeO-Suc-Ala-Ala-Pro-Val-CH2Cl, fibrinogen was degraded gradually, but none of the degradation products
reacted with IF-123 (Figure 6C).
Effects on the epitope expression of plasmic digestion of GE-XDP and GE digestion of plasmic XDP To see whether plasmic digestion of GE-XDP and GE digestion of plasmic XDP affect the structure required for the epitope expression, we digested GE-XDP with plasmin and plasmic XDP with GE, both at 1:1000 of the enzyme/substrate ratio (wt/wt), and examined the reactivity of the digests to IF-123 by the immunoprecipitation method. Although GE-XDP was converted by plasmin to the phase-4 digests (Figure 7A, lanes 1-4), the epitope for IF-123 was retained in the digests containing the fragment D components (Figure 7A, lanes 5-8). Digestion of plasmic XDP with GE also yielded the phase-4 digests (Figure 7B, lanes 1-4), and the new epitope was expressed at later stages, where plasmic DD and DY had been further degraded to smaller fragments DD and DY (Figure 7B, lanes 7 and 8, indicated by arrowheads).
Measurement of GE digests in plasma by a sandwich ELISA By a sandwich ELISA, we were able to measure GE-D and GE-XDP spiked in plasma up to 80 µg/mL without any interference by fibrinogen (Figure 8, open and closed circles, respectively). When plasma samples, 282 in total, derived from patients with a variety of diseases were subjected to measurement of plasmic fragment D species and GE-digests by ELISAs using JIF-23 and IF-123, respectively, there was a moderate correlation, r = 0.652, between these 2 fibrin(ogen) digests (Figure 9). However, they were found to be independent entities, as shown by 3 representative examples: (a) GE digests were markedly elevated but plasmic digests were low; (b) both GE and plasmic digests were elevated; and (c) GE digests were low but plasmic digests were elevated. By immunoprecipitation analysis, multiple protein fractions were abundantly precipitated with IF-123 from examples a and b (Figure 10A, lanes 2 and 3), but only a little from example c (Figure 10A, lane 4). On the contrary, only small amounts of proteins were precipitated with JIF-23 from example a (Figure 10B, lane 2), whereas considerable amounts of multiple proteins were precipitated from examples b and c (Figure 10B, lanes 3 and 4).
Granulocytes are known to release intrinsic proteolytic enzymes
including elastase and cathepsin G in a variety of pathologic conditions.1-3 In fact, they may degrade the
tissue-constituent proteins such as elastin and a variety of
proteoglycans, and also plasma proteins including fibrinogen and
fibrin.4,7-13 Although a variety of proteases may be
released, cathepsin G, the other major granulocyte-derived protease, or
trypsin-type and chymotrypsin-type proteases if any, were not able to
affect the epitope expression in the GE digests of fibrinogen and
fibrin (see Figure 6). Degradation of fibrinogen and fibrin by GE was
distinct from degradation of those by plasmin,7-13 and
discrimination of their fibrin(ogen) degradation products have been
attempted by SDS-PAGE10 and immunochemical techniques.9,13 In this study, we raised an mAb IF-123 that recognized the The authors are indebted to Chizuko Nakamikawa for skillful assistance
and to Michiko Takano for clerical work for construction of this manuscript.
Submitted May 3, 1999; accepted November 8, 1999.
Supported in part by Grants-in-Aid for Scientific Research 08407034 and
11470250, by the International Scientific Research Program, and Joint
Research Grant 09044329 and 11694308 from the Ministry of Education,
Science and Culture of the Government of Japan.
Reprints: Michio Matsuda, Division of Hemostasis and Thrombosis
Research, Institute of Hematology, Jichi Medical School, Yakushiji
3311-1, Minamikawachi, Kawachi-Gun, Tochigi-ken 329-0498, Japan;
e-mail: thmichi{at}jichi.ac.jp.
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.
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Top
Abstract
Introduction
(196-204) residue segment. This segment appears to be masked in
fibrin(ogen) but exposed when the A
), A
), A
), A
), A
) and A
).
