From the Centre for Thrombosis and Vascular Research, School of Pathology, University of New South Wales, Sydney, New South Wales 2052, Australia
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ABSTRACT |
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Plasmin is processed in the conditioned medium of
HT1080 fibrosarcoma cells producing fragments with the domain
structures of the angiogenesis inhibitor, angiostatin, and
microplasmin. Angiostatin consists of kringle domains 1-4 and part of
kringle 5, while microplasmin consists of the remainder of kringle 5 and the serine proteinase domain. Our findings indicate that formation of angiostatin/microplasmin involves reduction of plasmin by a plasmin
reductase followed by proteolysis of the reduced enzyme. We present
evidence that the Cys461-Cys540 and
Cys511-Cys535 disulfide bonds in kringle 5 of
plasmin were reduced by plasmin reductase. Plasmin reductase activity
was secreted by HT1080 and Chinese hamster ovary cells and the human
mammary carcinoma cell lines MCF-7, MDA231, and BT20 but not by the
monocyte/macrophage cell line THP-1. Neither primary foreskin
fibroblasts, blood monocyte/macrophages, nor macrovascular or
microvascular endothelial cells secreted detectable plasmin reductase.
In contrast, cultured bovine and rat vascular smooth muscle cells
secreted small but reproducible levels of plasmin reductase. Reduction
of the kringle 5 disulfide bonds triggered cleavage at either
Arg529-Lys530 or two other positions C-terminal
of Cys461 in kringle 5 by a serine proteinase. Plasmin
autoproteolysis could account for the cleavage, although another
proteinase was mostly responsible in HT1080 conditioned medium. Three
serine proteinases with apparent Mr of 70, 50, and 39 were purified from HT1080 conditioned medium, one or more of
which could contribute to proteolysis of reduced plasmin.
The formation of new blood vessels from preexisting vessels is an
important factor in a broad spectrum of diseases (1). New blood vessel
growth by the process of angiogenesis is balanced by several protein
activators and inhibitors. One such inhibitor is angiostatin, which
accumulated in the murine circulation in the presence of a growing
Lewis lung tumor and disappeared when the tumor was removed (2). The
angiostatin produced by the primary tumor was found to inhibit the
neovascularization and growth of its remote metastases. Angiostatin has
been shown to inhibit the growth of a number of murine and human
primary carcinomas in mice (3-5). The mechanism of action of
angiostatin is not known but may relate to the induction of endothelial
cell apoptosis (6).
Angiostatin is an internal fragment of plasminogen consisting of
approximately the first four kringle
domains.1 Both
metalloproteinase and serine proteinase activity have been implicated
in the formation of angiostatin. Angiostatin fragments are generated
from plasminogen by metalloelastase (7),
MMP-72 (8), MMP-9 (8), and
MMP-3 (9). Dong et al. (7) proposed that angiostatin is
produced by metalloelastase secreted by tumor-infiltrating macrophages.
Serine proteinase activity was required for the generation of
angiostatin from plasminogen or plasmin by cultured human prostate carcinoma cells (10), and generation of angiostatin from plasmin by
Chinese hamster ovary (CHO) or HT1080 human fibrosarcoma cells (11).
Production of angiostatin by CHO or HT1080 cells involves reduction of
one or more disulfide bonds in plasmin followed by proteolysis of the
reduced enzyme by a serine proteinase (11). The plasmin disulfide
bond(s) are reduced by a secreted reductase, which we have called
plasmin reductase. Plasmin reductase requires a small cofactor for
activity, and physiologically relevant concentrations of reduced
glutathione or cysteine fulfill this role. Angiostatin can also be
generated from plasmin with the reductants, thioredoxin (11), protein
disulfide isomerase (11), or high concentrations of small thiols
(12).
In this study, we present evidence that the
Cys461-Cys540 and
Cys511-Cys535 disulfide bonds in kringle 5 of
plasmin were reduced by plasmin reductase. Plasmin reductase activity
was secreted by the transformed cell lines, HT1080, CHO, MCF-7, MDA231,
and BT20, but not by the monocyte/macrophage cell line, THP-1. Cultured
bovine and rat vascular smooth muscle cells secreted small but
reproducible levels of plasmin reductase, but neither primary foreskin
fibroblasts, blood monocyte/macrophages, nor macrovascular or
microvascular endothelial cells secreted detectable plasmin reductase.
Reduction of the kringle 5 disulfide bonds triggered cleavage at either Arg529-Lys530 or two other positions C-terminal
of Cys461 in kringle 5 by a serine proteinase. Three serine
proteinases were purified from HT1080-conditioned medium (HT1080cm),
one or more of which could account for proteolysis of reduced plasmin.
Chemicals and
Proteins--
3-(N-maleimidylpropionyl)biocytin (MPB) was
purchased from Molecular Probes (Eugene, OR), while soybean trypsin
inhibitor (SBTI) and SBTI-agarose were from Sigma-Aldrich (Sydney,
Australia). Plasminogen was purified from fresh frozen human plasma and
separated into its two carbohydrate variants according to published
procedures (13). Glu1-plasminogen was used in the
experiments described herein. Urokinase plasminogen activator was a
gift from Serono Australia. Plasmin was generated by incubating
plasminogen (20 µM) with urokinase plasminogen activator
(20 nM) for 30 min in 20 mM Hepes, 0.14 M NaCl, pH 7.4 buffer at 37 °C. Val-Phe-Lys-chloromethyl
ketone was from Calbiochem. Plasmin (20 µM) was
inactivated by incubation with Val-Phe-Lys-chloromethyl ketone (40 µM) for 60 min at room temperature. The treated plasmin
contained <0.01% active plasmin measured using hydrolysis of
H-D-Val-Leu-Lys-p-nitroanilide (S2251; Kabi,
Mölndal, Sweden). Plasminogen fragments were generated by limited
proteolysis with porcine elastase and purified by a combination of
lysine-Sepharose affinity and Sephacryl S-100 gel filtration
chromatography (Amersham Pharmacia Biotech, Uppsala, Sweden) as
described previously (14). Miniplasmin was generated by incubating
miniplasminogen (2 µM) with urokinase plasminogen activator (20 nM) for 2 h in 20 mM Hepes,
0.14 M NaCl, pH 7.4 buffer at 37 °C. Thioredoxin-derived
angiostatin fragments were prepared as described previously (11).
Protein concentrations were determined using the Bio-Rad protein assay
kit and plasminogen as the standard. All proteins were aliquoted and
stored at Cell Culture--
Human foreskin fibroblasts (15), blood
monocyte/macrophages (16), human umbilical vein (17), human dermal
microvascular (18) and bovine aortic endothelial (19) cells, and rat
vascular smooth muscle cells (20) were harvested and cultured as
indicated. Bovine vascular smooth muscle cells were purchased from Cell
Applications (San Diego, CA). HT1080, CHO-K1, MCF-7, BT20, MDA231, and
THP-1 cells were purchased from American Type Cell Culture (Rockville, MD). All media components were from Life Technologies, Inc. Conditioned medium was collected by incubating cells at ~80% confluence with Hanks' balanced salt solution containing 25 mM Hepes at pH
7.4 for 6 h. The ratio of number of cells to volume of conditioned medium was 1-3 × 106 cells/ml. All conditioned medium
was passed through a 0.22-µm filter prior to storage at
Angiostatin Generation--
Conditioned medium (1 ml) was
incubated with plasmin (10 µg) for 2 h at 37 °C. Angiostatin
fragments were labeled with MPB (100 µM) for 30 min at
room temperature, followed by quenching of the unreacted MPB with GSH
(200 µM) for 10 min at room temperature. Unreacted GSH
and other free sulfhydryls in the system were blocked with
iodoacetamide (400 µM) for 10 min at room temperature.
The plasmin kringle products were collected on 50 µl of packed
lysine-Sepharose beads by incubation on a rotating wheel for 1 h
at room temperature; washed three times with 20 mM Hepes,
0.14 M NaCl, pH 7.4 buffer; and eluted with 50 mM Purification of Angiostatin from HT1080cm--
Angiostatin was
generated from 1 mg of plasmin in 100 ml of HT1080cm as described
above. The conditioned medium was applied to a 1 × 20-cm column
of lysine-Sepharose (Amersham Pharmacia Biotech,), and the matrix was
washed with 20 mM Hepes, 0.14 M NaCl, pH 7.4 buffer. The bound proteins were eluted with a linear gradient of
Electrophoresis and Western Blotting--
Samples were separated
on 10 or 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under
nonreducing or reducing conditions (21), transferred to PVDF membrane,
and developed and visualized using chemiluminescence according to the
manufacturer's instructions (DuPont). Both rabbit anti-human
plasminogen polyclonal antibodies and swine anti-rabbit IgG horseradish
peroxidase-conjugated antibodies (Dako, Carpinteria, CA) were used at
1:2000 dilution. Streptavidin-horseradish peroxidase (Amersham
Australia, Sydney, Australia) was used at a 1:2000 dilution.
ELISA for MPB-labeled Angiostatin--
NeutraLite avidin
(Molecular Probes, Eugene, OR) (100 µl of 5 µg/ml in 15 mM Na2CO3, 35 mM
NaHCO3, 0.02% azide, pH 9.6) were adsorbed to Nunc
PolySorp 96-well plates (Nunc, Roskilde, Denmark) overnight at 4 °C
in a humid environment. Wells were washed once with 20 mM
Hepes, 0.14 M NaCl, pH 7.4 buffer containing 0.05% Tween
20 (Hepes/Tween). Nonspecific binding sites were blocked by adding 200 µl of 2% bovine serum albumin in 15 mM
Na2CO3, 35 mM NaHCO3,
0.02% azide, pH 9.6 buffer and incubating for 90 min at 37 °C, and
then wells were washed two times with Hepes/Tween. MPB-labeled
angiostatin fragments were diluted in Hepes/Tween, and 100-µl
aliquots were added to avidin-coated wells and incubated for 30 min at
room temperature with orbital shaking. Wells were washed three times
with Hepes/Tween and 100 µl of 5 µg/ml murine anti-K1-3 monoclonal
antibody (American Diagnostica, Greenwich, CT) added and incubated for
30 min at room temperature with orbital shaking. Wells were washed
three times with Hepes/Tween, and rabbit anti-mouse IgG horseradish
peroxidase-conjugated antibody was added at a 1:500 dilution in 100 µl of Hepes/Tween and incubated for 30 min at room temperature with
orbital shaking. Wells were washed three times with Hepes/Tween, and
the color was developed with 100 µl of 0.003%
H2O2, 1 mg/ml
2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) in 50 mM citrate, pH 4.5 buffer for 20 min at room temperature with orbital shaking. Absorbances were read at 405 nm using a Molecular
Devices Thermomax Kinetic Microplate Reader (Molecular Devices Corp.,
Palo Alto, CA). Results were corrected for control wells not incubated
with MPB-labeled angiostatin.
Gelatin Zymography--
Gelatin zymography was a modification of
the technique originally described by Heussen and Dowdle (22). Bovine
type B gelatin (Sigma-Aldrich) was incorporated into a 10%
SDS-polyacrylamide gel at a final concentration of 1 mg/ml. Following
electrophoresis, gels were washed twice with 20 mM Hepes,
0.14 M NaCl, pH 7.4 buffer containing 2.5% Triton X-100
for 30 min to remove the SDS. Gels were then incubated in 20 mM Hepes, 0.14 M NaCl, pH 7.4 buffer overnight
at 37 °C and stained with Coomassie Brilliant Blue. On some
occasions, gels were incubated with 20 mM Hepes, 0.14 M NaCl, pH 7.4 buffer containing 10 mM EDTA to
inactivate metalloproteinases in the HT1080cm.
Plasmin Amidolytic Activity--
Plasmin (10 µg) was incubated
with HT1080cm (1 ml) at 37 °C. At discrete time intervals, aliquots
(20 µl) of the reactions were diluted 10-fold into 20 mM
Hepes, 0.14 M NaCl, 1 mg/ml PEG 6000, pH 7.4 buffer
containing 200 µM
H-D-Val-Leu-Lys-p-nitroanilide. The initial
rates of release of p-nitroaniline from the chromogenic substrate were measured as described previously (23).
Purification of Serine Proteinases from
HT1080cm--
Conditioned medium (200 ml) from HT1080 cells was
concentrated 20-fold over a 10-kDa cut-off membrane (Amicon, Beverly,
MA) and loaded onto a 10-ml SBTI-agarose column (Sigma-Aldrich)
equilibrated with 20 mM Hepes, 0.14 M NaCl, pH
7.4 buffer. The SBTI-agarose was washed with two column volumes of
Hepes buffer containing 1 M NaCl and re-equilibrated with
Hepes buffer containing 0.14 M NaCl. The serine proteinases
were eluted from the column with 10 mM MES, 10 mM Hepes, pH 5.2 buffer containing 0.8 M
benzamidine. The eluate was extensively dialyzed against 20 mM Hepes, 0.14 M NaCl, pH 7.4 buffer and stored
at Quantitation of GSH in HT1080cm--
HT1080 cells at ~80%
confluence were washed twice with PBS and incubated with Hanks'
balanced salt solution containing 25 mM Hepes at pH 7.4 for
6 h (0.7 × 106 cells/ml of medium). Conditioned
medium was passed through a 0.22-µm filter prior to storage at
Angiostatin Fragments Produced in HT1080cm--
Stathakis et
al. (11) observed that three angiostatin fragments were made in
CHO or HT1080cm with apparent Mr of 45, 41, and
38. These fragments were generated in HT1080cm, labeled with MPB, and
purified by lysine-Sepharose affinity and gel filtration chromatography. Accordingly, three fragments were purified with apparent Mr of 45, 41, and 38 on nonreducing
SDS-PAGE (Fig. 1A). The
apparent Mr of the three fragments on reducing
SDS-PAGE were 66, 60, and 57 (not shown). All three fragments were
labeled with MPB, indicating that all fragments contained free thiols.
Three angiostatin fragments with similar Mr were
also generated by incubation of plasmin with reduced thioredoxin as
previously reported (Fig. 1B) (11).
ELISA for MPB-labeled Angiostatin--
To estimate secretion of
plasmin reductase by cultured cells, an ELISA assay for MPB-labeled
angiostatin was developed. This assay measured angiostatin generated by
plasmin reductase and, therefore, was a relative measure of plasmin
reductase activity. Briefly, plasmin was incubated with conditioned
medium, and the angiostatin fragments were labeled with the
biotin-linked maleimide, MPB. The MPB-labeled angiostatin fragments
were adsorbed to avidin-coated microtiter plate wells, and the bound
angiostatin was detected using a murine kringle 1-3 monoclonal
antibody and a secondary peroxidase-conjugated antibody.
To test the specificity of the ELISA, plasminogen, plasmin, or the
plasminogen fragment kringles 1-3 (K1-3), K1-4, K4, or K5-serine
proteinase (10 µg/ml) were incubated in either Hepes-buffered saline
or HT1080cm for 2 h at 37 °C, and the angiostatin fragments were labeled with MPB and quantitated by ELISA. The plasminogen fragments were prepared by limited proteolytic digestion of plasminogen with porcine elastase and purified by a combination of lysine-Sepharose affinity and gel filtration chromatography (14). MPB-labeled angiostatin fragments were only produced in HT1080cm from plasmin or
plasmin derived endogenously from plasminogen (11) (Fig. 2A). As anticipated, no
MPB-labeled angiostatin fragments derived from the plasminogen
fragments in HT1080cm. Also, no MPB-labeled angiostatin fragments were
produced from incubation of plasminogen, plasmin, or any of the
plasminogen fragments in Hepes-buffered saline for 2 h at
37 °C. This result served as a negative control for plasmin
reductase. The response of the ELISA was linear up to a plasmin kringle
fragment concentration of ~200 ng/ml (Fig. 2B).
It is important to note that the ELISA assay was not an absolute
measure of angiostatin formation. It is possible that one or more of
the free thiols on angiostatin were refractive or inefficiently labeled
by MPB due to steric factors or that two thiols on a proportion of the
angiostatin molecules oxidized to form an intra- or interchain disulfide bond, which was not labeled with MPB. These considerations would have resulted in underestimation of the angiostatin generated. Nevertheless, the ELISA was a relative measure of angiostatin formation
or plasmin reductase activity in serum-free conditioned medium. For
example, generation of MPB-labeled angiostatin was a linear function of
the concentration of HT1080cm in the reaction or of plasmin reductase
concentration (r = 0.99) (Fig. 2C).
Plasmin Reductase Activity Secreted by Selected Primary and
Transformed Cells--
Conditioned medium from selected primary and
transformed cells was collected by incubating cells at ~80%
confluence with Hanks' balanced salt solution containing 25 mM Hepes, pH 7.4, buffer for 6 h. The ratio of number
of cells to volume of conditioned medium was between 1 and 3 × 106 cells/ml. Plasmin (10 µg/ml) was incubated with the
conditioned medium, and MPB-labeled angiostatin fragments were
quantitated by ELISA (see Fig. 2). ELISA results were corrected for
background angiostatin formation in unconditioned medium, which was negligible.
Neither human foreskin fibroblasts, bovine aortic endothelial cells,
human umbilical vein endothelial cells, nor human dermal microvascular
endothelial cells secreted plasmin reductase activity (Fig.
3). However, cultured rat or bovine
vascular smooth muscle cells secreted plasmin reductase and converted
plasmin to angiostatin, although the activity was 6% of the reductase
activity secreted by HT1080 cells. CHO and MCF-7 cells secreted
approximately 60%, BT20 cells 27%, and MDA231 cells 20% of the
plasmin reductase activity secreted by HT1080 cells. THP-1 cells did
not secrete detectable levels of plasmin reductase.
Cofactor Requirements of Plasmin Reductase--
The
angiostatin-generating activity secreted by HT1080 cells was the
highest of the cells examined and was used to further investigate the
cofactor requirements of plasmin reductase. We previously reported that
plasmin reductase secreted by HT1080 cells requires two components for
activity, a protein component that can be heat-inactivated and a low
Mr cofactor that can be GSH (11). The low
Mr thiol compounds in HT1080cm were derivatized with the fluorescent compound 7-benzo-2-oxa-1,3-diazole-4-sulfonic acid
and resolved by reverse-phase high performance liquid chromatography (24). The only low Mr thiol detectable in
HT1080cm was GSH (not shown). The concentrations of GSH and GSSG were
determined as described by Vandeputte et al. (25). The
concentration of GSH in the HT1080cm was 1.1 ± 0.12 µM. This corresponded to secretion of 0.27 ± 0.03 nmol of GSH/106 cells/h. No GSSG was detected in
HT1080cm.
To examine the GSH requirements of plasmin reductase, we measured the
plasmin reductase activity of either Hepes-buffered saline or dialyzed
HT1080cm supplemented with 0, 1, 5, or 10 µM GSH (Fig.
4). Dialysis of HT1080cm using a
12-14-kDa cut-off membrane reduced the angiostatin-generating activity
to 12% of control. Supplementation of the dialyzed HT1080cm with 1 µM GSH doubled the angiostatin-generating activity, while
10 µM GSH restored the angiostatin-generating activity to
that of undialyzed HT1080cm. This is in accordance with our previous
findings (11). GSH at a concentration of 1 µM, the
concentration in HT1080cm, had no angiostatin-generating activity on
its own, while 10 µM GSH had 6% of the
angiostatin-generating activity of undialyzed HT1080cm (Fig. 4).
The Kringle 5-Serine Proteinase Fragment of Plasminogen Is a
Substrate for Plasmin Reductase--
The plasminogen fragment K1-3,
K1-4, K4, or K5-serine proteinase was incubated with HT1080cm for
2 h and then labeled with MPB. The fragment was resolved on 15%
SDS-PAGE, transferred to PVDF membrane, and blotted with
streptavidin-peroxidase to detect the MPB label. Of the plasminogen
fragments, only K5-serine proteinase incorporated MPB (Fig.
5).
This observation suggested that the target disulfide bonds in plasmin
for plasmin reductase resided in K5. Incubation of plasmin in pH 11 buffer causes reduction and isomerization of K5 disulfide bonds and
results in formation of microplasmin (26, 27). Microplasmin has a
Lys530 N terminus that is within K5. We compared the
plasmin fragments generated by plasmin reductase with those generated
by alkaline pH.
Comparison of Microplasmin Fragments Generated in either HT1080cm
or in pH 11 Buffer--
Plasmin was incubated in either 0.1 M glycine, pH 11 buffer or HT1080cm for 12 h at
37 °C. Samples were resolved and detected on gelatin zymography.
Three major catalytically active plasmin fragments with apparent
Mr of 40, 30, and 29 were generated in either pH
11 buffer or HT1080cm (Fig.
6A). The
Mr 29 fragment corresponded to the
Mr 29 microplasmin fragment described by Wu et al. (26, 27).
Microplasmin hydrolyzes the tripeptidyl p-nitroanilide
substrate, H-D-Val-Leu-Lys-p-nitroanilide with
1.4-fold higher efficiency than plasmin. This is a consequence of a
1.4-fold increase in the catalytic constant (26). Plasmin was incubated
in HT1080cm, and the initial rate of hydrolysis of
H-D-Val-Leu-Lys-p-nitroanilide was measured at
discrete time intervals (Fig. 6B). Plasmin activity is
reported as the fraction of control plasmin activity. The efficiency of
hydrolysis of the chromogenic substrate increased with time of
incubation and peaked at ~2-fold enhanced efficiency at ~8 h. The
initial rate of hydrolysis returned to control levels after 24 h
of incubation.
Serine Proteinase(s) Other than Plasmin Were Mostly Responsible for
Proteolysis of Reduced Plasmin in HT1080cm--
Angiostatin is
generated from reduced plasmin by a serine proteinase in CHO or
HT1080cm (11). Serine proteinase activity is also required for
generation of angiostatin from plasminogen or plasmin by cultured human
prostate carcinoma cells (10). Plasmin autoproteolysis can account for
angiostatin formation in the presence of protein reductants (11) or
small thiols (12). To examine whether autoproteolysis is the operative
mechanism in HT1080cm, we compared formation of angiostatin from either active plasmin or plasmin inactivated with Val-Phe-Lys-chloromethyl ketone (VFK-plasmin).
Plasmin or VFK-plasmin was incubated with HT1080cm for 2 h, and
the angiostatin fragments were labeled with MPB. The MPB-labeled fragments were either resolved on SDS-PAGE and blotted with
streptavidin-peroxidase (Fig.
7A) or quantitated by ELISA
(Fig. 7B). Similar levels of angiostatin were generated from
either active plasmin or VFK-plasmin, which indicated that
autoproteolysis was not necessary for angiostatin formation.
Interestingly, MPB labeled both intact VFK-plasmin and angiostatin
fragments derived from the inactivated plasmin (Fig. 7A).
This observation implied that reduction of plasmin preceded
proteolysis. Formation of angiostatin from either active plasmin or
VFK-plasmin was inhibited by the serine proteinase inhibitor, SBTI
(Fig. 7B).
To examine the contribution of autoproteolysis of reduced plasmin to
angiostatin formation, the HT1080cm was passed over either a Sepharose
4B or SBTI-Sepharose 4B column, and the angiostatin-generating activity
of the eluate was measured (Fig. 7C). Sepharose 4B was used
to control for nonspecific protein adsorption to the agarose matrix.
Depletion of serine proteinases in HT1080cm reduced the angiostatin-generating activity to 34% of control.
SBTI-inhibitable serine proteinases in HT1080cm were purified on a
SBTI-agarose column, and the bound enzymes were eluted with
benzamidine. Serine proteinases from the equivalent of 3 ml of HT1080cm
were resolved and detected using gelatin zymography (Fig.
8). Proteinases with apparent
Mr of 70, 50, and 39 were evident. These enzymes
did not correspond to plasmin or catalytically active plasmin
fragments.
The formation of angiostatin from plasmin in the conditioned
medium of transformed cells is a two-step process (11). First, one or
more disulfide bonds in plasmin are reduced by a protein disulfide bond
reductase, which we have called plasmin reductase, and a reductase
cofactor, which can be a small thiol such as GSH. Second, reduced
plasmin is cleaved by a serine proteinase producing angiostatin. Three
angiostatin fragments are produced with apparent Mr of 45, 41, and 38 on nonreducing SDS-PAGE,
which have the same Lys78 N terminus but different C termini.
Plasmin reductase activity was secreted by the human fibrosarcoma cell
line HT1080; the human mammary carcinoma cell lines, MCF-7, MDA231, and
BT20; and CHO cells. In contrast, the monocyte/macrophage cell line
THP-1 did not secrete significant levels of plasmin reductase before or
after stimulation with phorbol ester (not shown). Neither primary
foreskin fibroblasts, blood monocyte/macrophages, nor macrovascular or
microvascular endothelial cells secreted detectable plasmin reductase.
In contrast, cultured bovine and rat vascular smooth muscle cells
secreted small but reproducible levels of plasmin reductase. In general
terms, cellular transformation appeared to be associated with secretion
of plasmin reductase and angiostatin formation. This result suggested
that angiostatin formation is driven by tumor cells in vivo.
Production of angiostatin by vascular smooth muscle cells is an
interesting observation and suggested that angiostatin may function in
the atherosclerotic vessel wall. The metalloproteinase inhibitor EDTA
did not have any effect on angiostatin production by the cell lines
used in this study.
Gately et al. (12) have shown that a sufficient
concentration of small thiols alone can generate angiostatin from
plasmin. The concentration of GSH in the HT1080cm was 1.1 ± 0.12 µM, and it was the only small thiol detected in the
medium. This corresponded to secretion of 0.27 ± 0.03 nmol of
GSH/106 cells/h, which is comparable with the level of
secretion of GSH by other cultured cells (28, 29). To examine the
contribution of the GSH to the plasmin reductase activity of HT1080cm,
the medium was dialyzed, and the angiostatin-generating activity was determined. Dialysis of HT1080cm reduced the angiostatin-generating activity to 12% of control. Supplementation of the dialyzed HT1080cm with 1 µM GSH doubled the angiostatin-generating
activity, while 10 µM GSH restored the
angiostatin-generating activity to that of undialyzed HT1080cm. In
contrast, 1 µM GSH, the concentration in HT1080cm, had no
angiostatin-generating activity, while 10 µM GSH had 6%
of the angiostatin generating activity of HT1080cm. These results
support our previous findings (11) and the proposal that plasmin
reductase requires a small thiol cofactor such as GSH to provide the
hydrogens and electrons to reduce the plasmin disulfide bonds. We
suggest that high concentrations (100 µM) of small thiols
have enough reducing power to reduce plasmin (12); however, these
concentrations are not achievable in cell culture. Considered together,
these observations implied that plasmin reduction in HT1080cm was
catalyzed by plasmin reductase using GSH as a cofactor and not by GSH directly.
One or more disulfide bonds in the K5-serine proteinase fragment of
plasminogen was reduced in HT1080cm. In contrast, neither K4, K1-3,
nor K1-4 were substrates for plasmin reductase. This finding suggested
that the target disulfide bond(s) for plasmin reductase were in K5.
Plasmin undergoes autoproteolysis in alkaline pH, producing a
catalytically active microplasmin fragment with a Lys530 N
terminus (26, 27). Microplasmin consists of the last 10 amino acids of
K5, the remaining 21 amino acids of the A chain, and the serine
proteinase domain. Wu et al. (26, 27) noticed that both the
Cys461-Cys540 and
Cys511-Cys535 disulfide bonds in K5 must have
been reduced to release microplasmin from K1-4. They proposed that the
increased Three major angiostatin fragments (Fig. 1A) and three major
serine proteinase fragments (Fig. 6A) were produced in
HT1080cm. Based on the close similarity in plasmin fragments produced
at alkaline pH and in HT1080cm, we suggest that at least the
Cys461-Cys540 and
Cys511-Cys535 disulfide bonds in K5 were
reduced by plasmin reductase. For instance, reduction of only the K5
Cys482-Cys523 and
Cys511-Cys535 disulfide bonds would not have
resulted in release of angiostatin from plasmin. Similarly, angiostatin
would have remained covalently linked to the remaining kringles if the
Cys461-Cys540 and
Cys482-Cys523 disulfide bonds were reduced,
but not the disulfide bond at Cys511-Cys535.
Reduction of the Cys461-Cys540 and
Cys511-Cys535 disulfide bonds is consistent
with all of the experimental data, although we cannot exclude reduction
of the Cys482-Cys523 disulfide bond in K5 or
other disulfide bonds in K1-4.
The largest angiostatin fragment (Mr 45) and the
smallest catalytically active fragment (Mr 29;
microplasmin) probably resulted from cleavage at the
Arg529-Lys530 peptide bond. We hypothesize that
cleavage can also occur at either the
Arg473-Ala474 or
Arg503-Ala504 peptide bond. Cleavage at these
sites is favored by serine proteinases with plasmin-like specificity
and would produce fragments of the size observed experimentally. Also,
all three angiostatin fragments contained one or more cysteine residues
(Fig. 1A). For the smallest angiostatin fragment
(Mr 38) to contain a free thiol, proteolysis must have occurred C-terminal of Cys461. A model of K5 and
the proposed target disulfide bonds is shown in Fig.
9.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C until use.
80 °C.
-aminocaproic acid in the Hepes buffer.
-ACA to 12 mM in the Hepes buffer. The angiostatin fragments were separated from a small amount of aggregated protein by
gel filtration on a 2 × 50-cm Sephacryl S-100 (Amersham Pharmacia Biotech,) column in the Hepes buffer.
80 °C until use.
80 °C. The low Mr thiol compounds in the
conditioned medium were derivatized with the fluorescent compound
7-benzo-2-oxa-1,3-diazole-4-sulfonic acid and resolved by reverse-phase
high performance liquid chromatography as described previously (24).
GSH and GSSG levels were determined as described by Vandeputte et
al. (25).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Angiostatin generated in HT1080cm or by
thioredoxin. A, plasmin (10 µg/ml) was incubated in
HT1080cm for 24 h at 37 °C, and the angiostatin fragments were
labeled with MPB. Angiostatin fragments were purified from HT1080cm by
lysine-Sepharose affinity and Sephacryl S-200 gel filtration
chromatography, and 2 µg was resolved on nonreducing 10% SDS-PAGE
and stained with Coomassie Blue (lane 1) or
transferred to PVDF membrane and blotted with streptavidin peroxidase
to detect the MPB label (lane 2). The positions
of Mr markers are shown at the left.
B, thioredoxin-derived angiostatin fragments (2 µg)
resolved on nonreducing 10% SDS-PAGE and stained with Coomassie Blue
(11). The positions of Mr markers are shown at
the left.
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Fig. 2.
ELISA for MPB-labeled angiostatin.
A, plasminogen, plasmin, K1-3, K1-4, K4, or K5-serine
proteinase (10 µg/ml) was incubated in either 20 mM
Hepes, 0.14 M NaCl, pH 7.4 buffer (open
bars) or HT1080cm (solid bars) for
2 h at 37 °C, and the angiostatin fragments were labeled with
MPB. Plasmin fragments were collected on lysine-Sepharose and eluted
with 20 mM -aminocaproic acid. The MPB-labeled
angiostatin fragments were immobilized on avidin-coated wells and
detected using a K1-3 monoclonal antibody and a peroxidase-conjugated
anti-mouse IgG secondary antibody. B, plasmin (10 µg/ml)
was incubated in HT1080cm, and the angiostatin fragments were labeled
with MPB and collected on lysine-Sepharose as described for
A. The concentration of plasmin kringle fragments was
determined by protein assay. The figure shows the
concentration dependence of plasmin kringle fragments in the ELISA.
C, plasmin (10 µg/ml) was incubated in 20 mM
Hepes, 0.14 M NaCl, pH 7.4 buffer and increasing HT1080cm
such that the fraction of HT1080cm varied from 0 to 100% of the
incubation volume. The reaction was incubated for 2 h at 37 °C,
and the angiostatin fragments were labeled with MPB and detected by
ELISA as described for A. The solid
line represents the linear regression fit of the data
(r = 0.99). The bars represent the mean and
range of duplicate experiments.
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Fig. 3.
Plasmin reductase activity secreted by
selected primary and transformed cells. Conditioned medium from
selected primary and transformed cells was collected by incubating
cells at ~80% confluence with Hanks' balanced salt solution
containing 25 mM Hepes at pH 7.4 for 6 h. The ratio of
number of cells to volume of conditioned medium was 1-3 × 106 cells/ml. Plasmin (10 µg/ml) was incubated with
conditioned medium for 2 h, and angiostatin formation was
quantitated by ELISA (see Fig. 2). The bars represent the
mean and range of duplicate experiments.
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Fig. 4.
Cofactor requirements of plasmin
reductase. HT1080cm was dialyzed for 16 h against 20 mM Hepes, 0.14 M NaCl, pH 7.4, buffer using a
12-14-kDa cut-off membrane. Plasmin (10 µg/ml) was incubated in the
dialyzed conditioned medium (dCM) or Hepes buffer for 2 h in the absence or presence of 0, 1, 5, or 10 µM GSH,
and angiostatin formation was quantitated by ELISA (see Fig. 2). The
bars and error bars represent the mean and S.E.,
respectively, of triplicate experiments.
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Fig. 5.
The kringle 5-serine proteinase fragment of
plasminogen is a substrate for plasmin reductase. A,
fragments of plasminogen consisting of K4, K1-3, K1-4, and K5-serine
proteinase were prepared by limited proteolytic digestion of
plasminogen with porcine elastase and purified by a combination of
lysine-Sepharose affinity and gel filtration chromatography (14). The
plasminogen fragments (2µg) were resolved on 15% SDS-PAGE under
nonreducing conditions and stained with Coomassie Brilliant Blue.
B, the plasminogen fragments (10 µg/ml) were incubated
with HT1080cm for 2 h and then labeled with MPB. The fragments (0.2 µg) were resolved on 15% SDS-PAGE, transferred to PVDF membrane, and
blotted with streptavidin-peroxidase to detect the MPB label. Of the
plasminogen fragments, only K5-serine proteinase incorporated MPB. The
positions of Mr markers are shown at left.
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Fig. 6.
Comparison of microplasmin fragments
generated in either HT1080cm or pH 11 buffer. A,
plasmin (10 µg/ml) was incubated in either 0.1 M glycine,
pH 11 buffer (lane 3) or HT1080cm
(lane 4) for 12 h at 37 °C. Samples
corresponding to 0.2 µg of plasmin or fragments were resolved and
detected on gelatin zymography. Matrix metalloproteinases in the
HT1080cm (MMP-9 and MMP-2) were inactivated with EDTA. Intact plasmin
(lane 1) and miniplasmin (lane
2) are shown for comparison. Fragments of apparent
Mr of 40, 30, and 29 were observed. Note the
close similarity in catalytically active plasmin fragments generated in
either pH 11 buffer (lane 3) or HT1080cm
(lane 4). Intact plasmin (lane
1) and miniplasmin (Val443-Asn791)
(lane 2) are shown for comparison. The positions
of Mr markers are shown at the left.
B, plasmin (10 µg) was incubated with HT1080cm (1 ml) at
37 °C. At discrete time intervals, aliquots (20 µl) were taken and
assayed for hydrolysis of
H-D-Val-Leu-Lys-p-nitroanilide. Control activity
was plasmin incubated with 20 mM Hepes, 0.14 M
NaCl, pH 7.4 buffer at 37 °C. Plasmin was also incubated with 100 µM GSH in the Hepes buffer. Plasmin activity is reported
as the fraction of control plasmin activity. The data
points and error bars represent the
mean and S.E., respectively, of three separate experiments.
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Fig. 7.
A serine proteinase other than plasmin was
mostly responsible for proteolysis of reduced plasmin in HT1080cm.
A, generation of angiostatin from plasmin versus
plasmin inactivated with VFK-CH2Cl. Plasmin or VFK-plasmin
(10 µg/ml) was incubated with HT1080cm for 2 h and then labeled
with MPB. The fragments were resolved on 15% SDS-PAGE, transferred to
PVDF membrane, and blotted with streptavidin-peroxidase to detect
MPB-labeled angiostatin. Note that MPB labeled both intact VFK-plasmin
and angiostatin fragments derived from the inactivated plasmin. The
positions of Mr markers are shown at the
left. B, plasmin or VFK-plasmin (10 µg/ml) was
incubated in HT1080cm in the absence or presence of SBTI (25 µg/ml)
for 2 h, and angiostatin formation was quantitated by ELISA (see
Fig. 2). The bars represent the mean and range of duplicate
experiments. C, HT1080cm (10 ml) was passed over either a
Sepharose 4B or SBTI-Sepharose 4B column (2 ml). Plasmin (10 µg/ml)
was incubated with the column eluate for 2 h, and angiostatin
formation was quantitated by ELISA (see Fig. 2).
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Fig. 8.
Profile of SBTI-binding serine proteinases in
HT1080cm. HT1080cm was passed over a SBTI-agarose column, and the
bound proteinases were eluted with benzamidine. Serine proteinases from
the equivalent of 3 ml of HT1080cm were resolved and detected using
gelatin zymography. Proteinases with apparent Mr
of 70, 50, and 39 were evident. For comparison, plasmin was incubated
with HT1080cm for 12 h, and then 1 µg was resolved on gelatin
zymography. Matrix metalloproteinases in the HT1080cm (MMP-9 and MMP-2)
were inactivated with EDTA. Plasmin and catalytically active plasmin
fragments were evident at apparent Mr of 85 and
40, respectively. The positions of Mr markers
are shown at the left.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
OH ion concentration at pH 11 was responsible
for reducing the two disulfide bonds. We observed that the proteinase
fragments produced from plasmin in pH 11 buffer were of identical
Mr to the proteinase fragments generated from
plasmin in HT1080cm. Fragments with apparent Mr
of 40, 30, and 29 were generated. The Mr 29 fragment is the same size as microplasmin. We suggest that the
mechanism of plasmin proteolysis at pH 11 is the same as the mechanism
of proteolysis in HT1080cm. In other words, plasmin reductase achieves at neutral pH what is achieved by
OH ion at pH 11.
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Fig. 9.
Model for generation of angiostatin by
disulfide bond reduction and proteolysis in kringle 5 of plasmin.
We suggest that both the Cys461-Cys540 and
Cys511-Cys535 disulfide bonds in plasmin
kringle 5 are reduced by plasmin reductase (shaded
circles). The reduced kringle 5 is then subject to
proteolysis at either the Arg529-Lys530 peptide
bond (solid black line) or two other
unidentified peptide bonds. Microplasmin is formed at pH 11 by
autoproteolysis at the Arg529-Lys530 peptide
bond (26, 27).
Gately et al. (10) observed that serine proteinase activity was required for generation of angiostatin from plasminogen or plasmin by cultured human prostate carcinoma cells, and we reported that serine proteinase activity was necessary for angiostatin generation by cultured CHO and HT1080 cells (11). Incubation of purified plasmin with the reductant thioredoxin (11), protein-disulfide isomerase (11), or high concentrations of small thiols (12) results in formation of angiostatin. This indicates that plasmin autoproteolysis can account for angiostatin formation. However, angiostatin was generated almost equally well from either active or inactive plasmin in HT1080cm, which implied that a proteinase(s) secreted by HT1080 cells was mostly responsible for plasmin proteolysis. The enzyme(s) was a serine proteinase, since it was inactivated by SBTI. Depletion of serine proteinases in HT1080cm by SBTI-agarose reduced the angiostatin-generating activity to 34% of control. This result implied that the rate of angiostatin formation is slowed when autoproteolysis of reduced plasmin is the operative mechanism. There were three major serine proteinases in HT1080cm with apparent Mr of 70, 50, and 39 that cleaved gelatin. One or more of these enzymes may contribute to generation of angiostatin from reduced plasmin. Therefore, although plasmin autoproteolysis can account for angiostatin formation, HT1080 cells did not rely entirely on this mechanism and secreted other serine proteinase(s) that cleaved reduced plasmin.
We have proposed that reduction of plasmin precedes proteolysis (11). This hypothesis was supported by the experiment that examined generation of angiostatin from inactivated plasmin. MPB labeled both intact VFK-plasmin and angiostatin fragments derived from the inactivated plasmin. This finding demonstrated reduction of disulfide bond(s) in intact plasmin, which supports our contention that plasmin reduction precedes proteolysis.
In summary, angiostatin formation involves disulfide bond reduction and
proteolysis in K5 of plasmin. The motif in angiostatin responsible for
its antiangiogenic properties is not known. Interestingly, the K5
domain of plasminogen has been shown to be a potent inhibitor of
endothelial cell proliferation (30). Also, plasminogen interacts with
cultured human umbilical vein endothelial cells through K5 (31). It is
possible that the active motif in angiostatin resides in K5 and that
the reduction and proteolytic events in this kringle expose the motif.
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ACKNOWLEDGEMENTS |
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We thank Dr. P. Little for the gift of rat vascular smooth muscle cells and Dr. N. Dudman and Dr. C. Grant for assistance with the GSH determinations.
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FOOTNOTES |
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* This work was supported by the National Health and Medical Research Council of Australia, the National Heart Foundation of Australia, and the New South Wales Cancer Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Centre for Thrombosis
and Vascular Research, School of Pathology, University of New South
Wales, Sydney, NSW 2052, Australia. Tel.: 61-2-9385-1004; Fax:
61-2-9385-1389; E-mail: p.hogg{at}unsw.edu.au.
1 Angiostatin has been loosely defined in the literature as an internal fragment of plasminogen consisting of kringles 1-4 or smaller fragments thereof. We define angiostatin as a protein consisting of kringles 1-4 and part of kringle 5 of plasmin.
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ABBREVIATIONS |
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The abbreviations used are: MMP, matrix metalloproteinase; CHO, Chinese hamster ovary K-1; HT1080cm, HT1080 conditioned medium; MPB, 3-(N-maleimidylpropionyl)biocytin; PVDF, polyvinylidene difluoride; PAGE, polyacrylamide gel electrophoresis; SBTI, soybean trypsin inhibitor; ELISA, enzyme-linked immunosorbent assay; MES, 4-morpholineethanesulfonic acid.
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