Nonlysine-analog plasminogen modulators promote autoproteolytic
generation of plasmin(ogen) fragments with angiostatin-like activity
Shigeki Ohyama, Tomotaka Harada, Toshihiro Chikanishi, Yutaka Miura and Keiji Hasumi
Department of Applied Biological Science, Tokyo Noko University, Saiwaicho, Fuchu-shi, Tokyo, Japan
We recently discovered several nonlysine-analog conforma-
tional modulators for plasminogen. These include SMTP-6,
thioplabin B and complestatin that are low molecular mass
compounds of microbial origin. Unlike lysine-analog mod-
ulators, which increase plasminogen activation but inhibit its
binding to fibrin, the nonlysine-analog modulators enhance
both activation and fibrin binding of plasminogen. Here we
show that some nonlysine-analog modulators promote
autoproteolytic generation of plasmin(ogen) derivatives with
its catalytic domain undergoing extensive fragmentation
(PMDs), which have angiostatin-like anti-endothelial activ-
ity. The enhancement of urokinase-catalyzed plasminogen
activation by SMTP-6 was followed by rapid inactivation
of plasmin due to its degradation mainly in the catalytic
domain, yielding PMD with a molecular mass ranging from
68 to 77 kDa. PMD generation was observed when plasmin
alone was treated with SMTP-6 and was inhibited by the
plasmin inhibitor aprotinin, indicating an autoproteolytic
mechanism in PMD generation. Thioplabin B and com-
plestatin, two other nonlysine-analog modulators, were also
active in producing similar PMDs, whereas the lysine analog
6-aminohexanoic acid was inactive while it enhanced
plasminogen activation. Peptide sequencing and mass
spectrometric analyses suggested that plasmin fragmenta-
tion was due to cleavage at Lys615-Val616, Lys651-Leu652,
Lys661-Val662, Lys698-Glu699, Lys708-Val709 and several
other sites mostly in the catalytic domain. PMD was inhi-
bitory to proliferation, migration and tube formation of
endothelial cells at concentrations of 0.3–10 lgÆmL
)1
.These
results suggest a possible application of nonlysine-analog
modulators in the treatment of cancer through the
enhancement of endogenous plasmin(ogen) fragment for-
mation.
Keywords: angiostatin; plasminogen; plasmin; autoproteo-
lysis; angiogenesis.
The plasminogen/plasmin system plays crucial roles not
only in blood clot lysis but also in various physiological and
pathological events including inflammation, tissue remode-
ling, tumor metastasis and angiogenesis, where localized
proteolysis is required [1–3]. Plasminogen consists of an
N-terminal peptide, five kringle domains and a serine
protease domain [4]. Two serine proteases, tissue-type
plasminogen activator and urokinase-type plasminogen
activator (u-PA), catalyze the activation of plasminogen
by cleaving the Arg561-Val562 bond. The resulting plasmin
in turn cleaves the Lys77-Lys78 and/or Lys78-Val79 bonds
to yield mature plasmin, which consists of two polypeptide
chains that are held together by disulfide bridges. The heavy
chain (A-chain; Lys78- or Val79-Arg561) contains five krin-
gle domains and the light chain (B-chain; Val562-Asn791)
has the serine protease domain. Native plasminogen resists
activation, as it adpots a tight, spiral conformation [5] due
to intramolecular binding of Lys50 and/or Lys62 in the
N-terminal peptide to a lysine binding site (aminohexyl site)
in the fifth kringle domain (K5) [6,7]. Fibrin and cellular
receptors bind plasminogen and relax its conformation to
be highly activatable, promoting efficient localized proteo-
lysis [8–10]. Similarly, lysine analogs bind to plasminogen
kringles, resulting in conformational relaxation and
enhancement of activation to plasmin [11]. Thus, conform-
ational modulation of plasminogen is important for its
activation.
Under certain circumstances, including tumor progres-
sion and inflammation, plasmin(ogen) undergoes proteo-
lysis to form various kringle-containing A-chain fragments,
collectively called angiostatins [12–17]. It is postulated that
physiological formation of angiostatin involves partial
disulfide reduction in plasminogen and cleavages of plasmi-
nogen by plasmin itself and/or other proteinases such as
matrix metalloproteinases, metalloelastase and cathepsin D
that are derived from tumor cells or infiltrating macro-
phages [15,18–21]. Typical angiostatin consists of the first
four kringle domains (K1–4) of plasmin [12,22,23]. Angio-
statin and its relatives, including K1, K2, K3, K5, K1–3,
K1–4½ and A
61
, inhibit the proliferation of vascular
Correspondence to K. Hasumi, Department of Applied Biological
Science, Tokyo Noko University, 3-5-8 Saiwaicho, Fuchu-shi, Tokyo,
183–8509 Japan. Fax: + 81 42 3344661; Tel.: + 81 42 3675710;
E-mail: hasumi@cc.tuat.ac.jp
Abbreviations: u-PA, urokinase-type plasminogen activator; PMD,
plasmin(ogen) derivative with its catalytic domain undergoing exten-
sive fragmentation; 6-AHA, 6-aminohexanoic acid; VLK-pNA,
H-Val-Leu-Lys-p-nitroanilide; MPB, 3-(N-maleimidylpropionyl)bio-
cytin; HUVEC, human umbilical endothelial cells; FBS, fetal bovine
serum; EGM, endothelial cell growth medium; bFGF, basic fibroblast
growth factor; EGF, epidermal growth factor.
Enzymes: human plasminogen (Swiss-Prot accession No. P00747;
EC 3.4.21.7); human urokinase-type plasminogen activator
(Swiss-Prot accession No. P00749; EC 3.4.21.73).
(Received 20 October 2003, revised 30 December 2003,
accepted 7 January 2004)
Eur. J. Biochem. 271, 809–820 (2004) FEBS 2004 doi:10.1111/j.1432-1033.2004.03985.x
endothelial cells, which is a fundamental process in angio-
genesis [12–16]. By inhibiting angiogenesis, angiostatin
suppresses both in situ and metastatic tumor growths in
animal models [12,15,19,22,23].
We recently discovered novel nonlysine-analog modula-
tors of plasminogen activation [24–29]. These include the
microbial products staplabin, SMTPs and thioplabins. Such
nonlysine-analog modulators enhance both plasminogen
activation and plasminogen-fibrin binding, resulting in
augmentation of fibrinolysis [24–26]. These effects are
strikingly different from the action of lysine-analog modu-
lators, which enhance plasminogen activation but inhibit
plasminogen-fibrin binding and fibrinolysis [9]. Therefore,
conformational change induced by such nonlysine-analog
modulators may be distinct from changes induced by lysine
analogs [24,26]. We thought that conformational modula-
tion of plasminogen by nonlysine-analog modulators not
only enhances plasminogen activation, but also confers
unusual proteolytic susceptibility on plasmin(ogen). Here we
show that one such compound (SMTP-6) promotes the
autoproteolytic generation of plasmin(ogen) derivative with
its catalytic domain (B-chain) undergoing extensive frag-
mentation (designated PMD), which inhibits the prolifer-
ation, migration and tube formation of vascular endothelial
cells. Similar PMD generation is induced by two other
structurally distinct nonlysine-analog modulators but not by
a lysine analog modulator, 6-aminohexanoic acid (6-AHA).
Materials and methods
Materials
Human plasmin, bovine aprotinin and bovine insulin were
from Wako, Osaka, Japan; u-PA from JCR Pharmaceuti-
cals,Kobe,Japan;H-Val-Leu-Lys-p-nitroanilide (VLK-
pNA) from Bachem, Bubendorf, Switzerland; monoclonal
anti-(human plasminogen K1–3) IgG (2PG) from Tech-
noclone, Vienna, Austria; peroxidase-conjugated rabbit
anti-(mouse IgG + A + M) (H + L) from ZYMED
Laboratories, San Francisco, CA, USA. SMTP-6, thiopla-
bin B (antibiotic A10255B) and complestatin were isolated
from cultures of Stachybotrys microspora IFO30018, Strep-
tomyces sp. R1401 and Streptomyces sp. A1631, respectively
[25–27]. Human plasminogen was affinity-purified from
human plasma [30]. Angiostatin K1–3 was purified by
lysine-Sepharose affinity chromatography from porcine
elastase-digested plasminogen as described previously [22].
A time course of plasmin generation
u-PA-catalyzed activation of plasminogen was assayed by
VLK-pNA hydrolysis and SDS/PAGE. Plasminogen
(100 n
M
)wasincubatedat37C for up to 60 min with
50 UÆmL
)1
(6.3 n
M
) u-PA in TBS/T [50 m
M
Tris/HCl,
100 m
M
NaCl and 0.01% (w/v) Tween 80, pH 7.4]. After
incubation, the mixture received 1/9 volume of 1 m
M
VLK-
pNA in TBS/T and was further incubated at 37 Cto
measure the absorbance at 405 nm with 2 min intervals.
From the initial rate of the release of pNA, the plasmin
concentration was determined. For determination of mole-
cular species on SDS/PAGE, the above-mentioned reaction
mixture (incubated for 0–60 min) was treated with 10% (v/v)
trichloroacetic acid, and the resulting precipitates were
washed with acetone and dissolved in 11 lLofSDS-sample
buffer [2% (w/v) SDS, 62.5 m
M
Tris/HCl, pH 6.8, 10% (w/v)
sucrose and 0.02% (w/v) bromophenol blue] containing 5%
(v/v) 2-mercaptoethanol. A portion (10 lL) of the mixture
was subjected to SDS/PAGE on a 10% (w/v) gel, and the gel
was stained with Coomassie Brilliant Blue R250.
Detection of kringle 1–3
Plasmin(ogen) fragments containing K1–3 were detected
by immunoblotting. Plasminogen (200 n
M
)andu-PA
(100 UÆmL
)1
) were incubated in 60 lL of TBS/T at 37 C
for60minandthemixturewastreatedwith10%(v/v)
trichloroacetic acid to precipitate proteins, which were then
washed with acetone and dissolved in 12 lLofSDSsample
buffer. A portion (10 lL) of the mixture was subjected to
SDS/PAGE on a 7.5% (w/v) gel, and proteins were
transferred to poly(vinylidene difluoride) membrane. The
membrane was incubated in NaCl/P
i
(20 m
M
sodium
phosphate and 150 m
M
NaCl, pH 7.4) containing
0.5% (v/v) Tween 20 and 5% (w/v) BSA at 4 Covernight,
washed twice with NaCl/P
i
containing 0.5% (v/v) Tween 20
andincubatedinNaCl/P
i
containing mouse anti-(human
plasminogen K1-3) IgG (1 : 500 dilution), 0.5% (v/v)
Tween 20 and 1% (w/v) BSA at room temperature for
1 h. After washing five times with NaCl/P
i
containing 0.5%
(v/v) Tween 20, the membrane was incubated in NaCl/P
i
containing peroxidase-conjugated rabbit anti-(mouse
IgG + A + M) (H + L) antibody (1 : 2000 dilution),
0.5% (v/v) Tween 20 and 1% (w/v) BSA at room
temperature for 1 h and washed five times with NaCl/P
i
containing 0.5% (v/v) Tween 20. Finally, the membrane
was incubated in 0.1
M
sodium phosphate, pH 6.4 contain-
ing 0.2 mgÆmL
)1
diaminobenzidine and 0.06% (v/v) H
2
O
2
at room temperature for 10 min.
Detection of free thiol
Plasminogen (100 n
M
) and u-PA (50 UÆmL
)1
) were incu-
bated in the absence or presence of 200 l
M
SMTP-6 in
150 lL of TBS/T at 37 C for 60 min. The resulting mixture
was treated with 3-(N-maleimidylpropionyl)biocytin (MPB;
Molecular Probes, Eugene, OR, USA) to detect free thiol
groups in proteins as described previously [31,32] with slight
modification. Briefly, reactions were labeled with MPB
(100 l
M
) for 30 min at room temperature, followed by
quenching of the unreacted MPB with reduced glutathione
(200 l
M
) for 10 min at room temperature. Unreacted
glutathione and other free sulfhydryls in the system were
blocked with N-ethylmaleimide (400 l
M
)for10minat
room temperature. The resulting mixtures were treated with
10% (v/v) trichloroacetic acid to precipitate proteins, which
were then washed with acetone. Proteins were resolved on
nonreduced 10% (v/v) SDS/PAGE, transferred to nitrocel-
lulose membrane, and blotted with streptavidin-peroxidase
(Sigma) to detect the MPB-labeled proteins.
Identification of plasmin fragments
Both MALDI-TOF/MS and automated Edman degrada-
tion were used to identify plasmin fragments. Plasmin
810 S. Ohyama et al.(Eur. J. Biochem. 271)FEBS 2004
(100 n
M
)wasincubatedin12mLofTBS/Tat37Cfor
60 min in the presence of SMTP-6 (250 l
M
), thioplabin B
(10 l
M
)orcomplestatin(0.3l
M
), and proteins were preci-
pitated by treatment with 10% (v/v) trichloroacetic acid.
For MALDI-TOF/MS analyses, proteins (50 lg) were
dissolved in a mixture consisting of 41 lLof8
M
urea, 3 lL
of 5% (v/v) EDTA, 30 lLof1.44
M
Tris/HCl, pH 8.6, and
1lL of 14.4
M
2-mercaptoethanol. After incubation at
room temperature for 4 h under N
2
gas, the mixture was
incubated with 10 lLof1.44
M
iodoacetic acid and 1.0
M
NaOH at room temperature in the dark for 15 min. The
resulting reduced, S-carboxymethylated peptides were
applied to a silica-C
18
spin column equilibrated with 0.1%
(v/v) trifluoroacetic acid. The column was washed with
0.1% (v/v) trifluoroacetic acid and peptides were eluted with
0.1% (v/v) trifluoroacetic acid and 50% (v/v) CH
3
CN. The
eluate was treated with ZipTip C18 (Millipore) and peptides
were recovered in 4 lL of 0.1% (v/v) trifluoroacetic acid
and 50% (v/v) CH
3
CN. A portion (0.5 lL) was analyzed
by MALDI-TOF/MS on a Voyager DE-STR (Applied
Biosystems) employing a linear mode positive ionization.
Sinapinic acid was used as a matrix and bovine insulin as
an internal standard for calibration. Results obtained were
compared with a panel of theoretical peptide masses
calculated for trypsin-cleaved, S-carboxymethylated plas-
min fragments.
For peptide sequencing, proteins (50 lg) were dissolved
in 20 lL of SDS sample buffer containing 5% (v/v)
2-mercaptoethanol. A portion (10 lL) of the mixture
was subjected to SDS/PAGE on a 20% (w/v) gel (Tris/
Tricine buffer system) and transferred to poly(vinylidene
difluoride) membrane. The membrane was stained with
Coomassie Brilliant Blue R250 and a section of mem-
brane was subjected to N-terminal amino acid sequence
analysis on a model 476A protein sequencer (Applied
Biosystems).
Preparation and purification of PMDs for biological
assays
Plasminogen (100 n
M
) and u-PA (50 UÆmL
)1
)wereincu-
batedwithSMTP-6(250l
M
), thioplabin (20 l
M
)or
complestatin (0.3 l
M
) in TBS/T at 37 C for 60 min. After
adding phenylmethanesulfonyl fluoride to a final concen-
tration of 1 m
M
, the mixture was dialyzed against water at
4C and lyophilized. The resulting sample was used directly
for assays after filter-sterilization. The endotoxin levels in
the PMD-S preparation, as determined using a Kinetic-
QCL kit (BioWhittaker; Walkersville, MD, USA), were in a
range of 0.082–0.21 ngÆmg
)1
protein.
For HPLC purification, the lyophilized sample was
dissolved in 0.1% (v/v) trifluoroacetic acid and subjected
to preparative HPLC on a lBONDASPHERE 5 lC8–
300 A
˚(19 ·150 mm; Waters), which was developed with a
linear gradient of 2-propanol in water containing 0.1% (v/v)
trifluoroacetic acid at a rate of 5 mLÆmin
)1
. The concen-
tration of 2-propanol was 0% for the initial 20 min and
increased to 100% at a rate of 0.8%Æmin
)1
thereafter. The
elution was monitored by UV absorption at 210 nm, and a
peak at a retention time of 60–62.5 min was pooled and
lyophilized. The lyophilized sample was dissolved in NaCl/
P
i
and filter-sterilized before assay.
Cell culture
Human umbilical vein endothelial cells (HUVEC; obtained
from Clonetics, San Diego, CA, USA) were grown in
endothelial cell growth medium (EGM; Clonetics) contain-
ing 2% (v/v) fetal bovine serum. HT1080 cells and rat
primary fibroblasts were maintained in DMEM containing
10% (v/v) fetal bovine serum, and Lewis lung carcinoma
cells and CHO-K1 cells were grown in RPMI 1640
supplemented with 10% (v/v) fetal bovine serum.
Cell proliferation assay
The proliferation of endothelial cell was assayed as
described [13,15] with slight modification. HUVEC were
dispersed in trypsin/EDTA solution (Clonetics) and resus-
pended with EGM defective in human epidermal growth
factor (EGF). Cells (2500 cells in 100 lL) were seeded into
96-well plates and incubated at 37 C overnight. The
medium was replaced by 100 lL of fresh medium contain-
ing PMD or angiostatin. After incubation for 30 min,
100 lL of EGM containing 20 ngÆmL
)1
EGF was added,
and cells were incubated for 3 days. After discarding
the medium, fresh medium (100 lL) containing 0.5 m
M
2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-
2H-tetrazolium (WST-1 reagent; Dojindo Laboratories,
Kumamoto, Japan), a water-soluble formazan-forming
reagent, was added at 37 C for 4 h. Absorbance at
405 nm was measured with a reference at 655 nm as an
index of cell proliferation.
The growth of nonendothelial cells was assayed by a
similar method employed for the assay for HUVEC using
respective growth medium shown above.
Endothelial cell migration assay
A modified Boyden chamber method was used [33].
Polycarbonate membrane with 8 lm pores was coated with
15 lg of fibronectin and placed over a 24-well culture plate
containing 500 lL of EGM supplemented with 0.1% (v/v)
BSA and 10 ngÆmL
)1
bFGF.After30min,HUVECthat
had been starved overnight in serum- and growth factor-free
EGM containing 0.1% (v/v) BSA were seeded into the
upper chamber (1.1 ·10
5
cells in 500 lL of EGM contain-
ing 20 lgÆmL
)1
of PMD or angiostatin) and incubated for
5 h. The upper chamber was removed and nonmigrated
cells were scraped off with cotton swabs. The migrated cells
were fixed with 70% (v/v) ethanol for 30 min, stained with
Giemsa for 30 min and counted under a light microscope
at ·100 magnification.
Endothelial cell tube formation assay
Approximately 4.0 ·10
4
cells in 100 lL of MCDB 131
medium were seeded into a 24-well culture plate containing
200 lL of collagen gel. After incubation at 37 Cfor2h,
medium was discarded, and cells were overlaid with 200 lL
of collagen gel. After gel formation for 30 min, each well
received 200 lL of MCDB 131 medium. Both medium and
collagen gel contained 10 lgÆmL
)1
of PMD or angiostatin.
After 24 h of incubation, cultures were examined for tube
formation under a light microscope at ·40 magnification.
FEBS 2004 Nonlysine-analogs promote plasmin autoproteolysis (Eur. J. Biochem. 271) 811
The length of tubes in 8–9 fields were measured and
expressed as percent of control.
Results
Nonlysine-analog modulators induce autoproteolytic
plasmin(ogen) fragmentation
SMTP-6 (Fig. 1A), one of the nonlysine-analog plasmino-
gen modulators, enhances the initial rate of u-PA-catalyzed
plasminogen activation [25]. We found that the SMTP-6
enhancement of plasminogen activation was transient and
that a rapid decrease in plasmin activity followed this
enhancement (Fig. 2A). On the other hand, u-PA-catalyzed
plasminogen activation in control incubation proceeded
constantly for up to 60 min. The lysine analog 6-AHA
markedly enhanced plasmin generation throughout the
incubation period. The time-dependent increase in plasmin
activity in the control and 6-AHA incubations accompanied
an increase in the intensity of protein bands both at
68/70 kDa doublet and 27.2 kDa in reduced SDS/PAGE
analysis, which corresponded to A- and B-chains of
plasmin, respectively (Fig. 2B,C). In incubation in the
presence of SMTP-6, A-chain band appeared to be smeared
while its intensity increased time-dependently, whereas the
intensity of B-chain band increased transiently and
decreased after 10 min of incubation (Fig. 2D). Thiopla-
bin B and complestatin, two other structurally distinct
nonlysine-analog modulators (Fig. 1B,C), also caused rapid
increases in plasmin generation, which was halted after
20–30 min of incubation (Fig. 2A). The level of plasmin
B-chain appeared to be consistent with plasmin activity,
while A-chain production increased time-dependently in
these incubations (Fig. 2E,F). In the absence of u-PA, the
three nonlysine-analogs induced neither plasminogen acti-
vation nor its fragmentation (data not shown). These results
suggested that these nonlysine-analog modulators enhanced
not only plasminogen activation (cleavage at Arg561-
Val562 bond by u-PA) but also its fragmentation mainly
in the B-chain, which harbors the catalytic domain. This
may account for the transient increase in plasmin activity.
The apparent molecular masses of the plasmin(ogen)
derivative PMD produced in the presence of the nonlysine-
analog modulators were estimated to range from 68 to
77 kDa on SDS/PAGE under nonreducing conditions
(Fig. 3A). In immunoblot analysis, the PMD reacted with
anti-(K1-3) IgG (Fig. 3B). The molecular size of plasmino-
genwasestimatedtoberathersmallerinnonreducedSDS/
PAGE: 84–92 kDa (Fig. 3A) vs. 100–110 kDa in reduced
SDS/PAGE (Fig. 2). Other investigators have reported
similar observations with A-chain fragments. For example,
the apparent sizes observed in nonreduced and reduced
SDS/PAGE, respectively, are reported to be 45 kDa and
66 kDa for K1–4 + 69 residues of K5[32], and 50 kDa
and 61 kDa for K1-4 + 7 residues of K5[16]. Thus, the
molecular mass of PMDs at 68–77 kDa in nonreduced
SDS/PAGE was higher than that of such A-chain fragments
by 18–32 kDa. Therefore, the data in Figs 2 and 3 suggested
that PMDs consist of not only A-chain but also fragment(s)
of B-chain that are connected by disulfide bridges.
To test whether the nonlysine-analog-mediated plas-
min(ogen) degradation was due to cleavage by u-PA or
plasmin, we incubated plasminogen and u-PA in the
presence of aprotinin at a concentration of 1000 kallikrein
inhibitor units per mL, which inhibits plasmin but not
u-PA. As shown in Fig. 3C, the nonlysine-analog-mediated
Fig. 1. Structures of SMTP-6 (A), thioplabin B (B) and complestatin (C).
812 S. Ohyama et al.(Eur. J. Biochem. 271)FEBS 2004
production of PMDs was completely inhibited by aprotinin.
Furthermore, the incubation of plasmin alone in the
presence of SMTP-6 produced extensive B-chain degrada-
tion, whereas the A-chain appeared to remain intact
(Fig. 3D). These results demonstrated an involvement of
an autoproteolytic mechanism in the nonlysine-analog-
mediated generation of PMDs.
Structural characterization of PMD
To determine the cleavage sites in PMD, we employed both
N-terminal amino acid sequencing and MALDI-TOF/MS
analyses. Plasmin was incubated with the nonlysine-analog
modulators to allow autoproteolytic formation of PMDs,
which were then reduced and fractionated by SDS/PAGE
on a 20% (w/v) gel for sequence analysis by automated
Edman degradation. The amino acid sequences of bands
numbered in Fig. 3E were reasonably assigned as shown in
Table 1. We could not determine sequences of fragment
bands at 17–22 kDa because of the appearance of multiple
amino acid peaks in the early part of several cycles in their
analyses. The assignments demonstrated that, although one
fragment at 14.9 kDa (Fig. 3E, band 1) was derived from
the A-chain (starting with Val79), all the remaining
fragments detected were from B-chain. MALDI-TOF/
MS analyses were performed after the reduction and
S-carboxymethylation of plasmin derivatives. Because
plasmin cleaves lysyl and arginyl bonds, results obtained
were compared with a panel of theoretical peptide masses
calculated for S-carboxymethylated plasmin cleaved at lysyl
and arginyl bonds. Seven to 13 peptide fragments were
identified within a difference of 1.2 mass units in compar-
ison with the theoretical peptide masses calculated for each
fragments (Table 2). All but one fragment species identified
were assigned to be from the B-chain. Taking the data
obtained by the two methods together, autoproteolytic
cleavage in plasmin incubated in the presence of the three
nonlysine-analog modulators was assigned to occur at least
at the positions shown in Table 3.
Figure 3F shows a time course for B-chain fragmentation
in the presence of SMTP-6. The results demonstrated that
peptide bands at 9.9 and 8.1 kDa appeared after 5–10 min,
and their intensity remained constant after 20–60 min. This
observation suggested that the two peptides at 9.9 kDa
(with N-terminal Glu699) and 8.1 kDa (with N-terminal
Val709) (Table 1) have been early intermediates in a
sequence of fragmentation events. Therefore, it is likely
that Lys698-Glu699 and/or Lys708-Val709 are most sus-
ceptible cleavage sites in the B-chain. The cleavage at such
sites may enable subsequent cleavage at many other sites.
It has been reported that autoproteolytic generation of
the A-chain fragment K1–4 + 69 residues of K5is
Fig. 2. Nonlysine-analog modulators enhance both initial plasminogen activation and subsequent fragmentation of plasmin(ogen). (A) Plasminogen
(100 n
M
) was incubated with u-PA (50 UÆmL
)1
)intheabsence(d) or presence of 20 m
M
6-AHA (s), 250 l
M
SMTP-6 (n), 30 l
M
thioplabin B (h)
or 0.3 l
M
complestatin (e). After incubation for the indicated time, plasmin activity was measured by adding VLK-pNA. Each value represents the
mean ± SD from triplicate determinations. (B–F), SDS/PAGE analyses of plasmin(ogen) in the first incubation in (A). Aliquot of the incubation
(2 lg protein) in the absence (B) or presence of 6-AHA (C), SMTP-6 (D), thioplabin B (E) or complestatin (F) was reduced and subjected to SDS/
PAGE on a 10% gel. The positions of molecular mass markers (left) as well as of plasminogen (Glu-Plg) and A- and B-chains of plasmin (right) are
shown.
FEBS 2004 Nonlysine-analogs promote plasmin autoproteolysis (Eur. J. Biochem. 271) 813