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INTRODUCTION |
Subtilisin NAT (1) (formerly designated BSP, or
nattokinase), a serine proteinase from Bacillus subtilis,
has been reported to have potent fibrinolytic activity (1, 2). The
enzyme is composed of 275 amino acids with a molecular mass of 27.7 kDa in its mature form (1). DNA sequence analysis showed that subtilisin NAT was 99.5 and 99.3% homologous to subtilisins E and
Amylosacchariticus, respectively (3). It is also homologous to other
members of the subtilisin family (BPN' 86% and Carlsberg 72%),
and sequences are conserved especially around the three amino acids
(serine 221, histidine 64, and aspartic acid 32) essential for the
catalytic center of serine proteinases.
The mechanism for this enzyme to potentiate fibrinolysis is not fully
understood. Subtilisin NAT is reported not to possess plasminogen
activator activity but appears to directly digest fibrin by limited
proteolysis (4). However, this direct cleavage of fibrin does not seem
to account for all of the enhancement of the fibrinolytic activity that
has been observed without affecting the fibrinolytic cascade. To
explore other possible mechanisms, we have looked for interactions
between subtilisin NAT and the physiological inhibitors of
fibrinolysis, plasminogen activator inhibitor type 1 (PAI-1)1 (5) and
2-antiplasmin (
2-AP) (6). These
inhibitors are both members of the serine protease inhibitor
superfamily (SERPINs). The SERPINs are proteolytically cleaved and
inactivated by a variety of proteases including members of the
subtilisin family (7).
PAI-1 is the primary inhibitor of tissue-type plasminogen activator
(tPA) and regulates fibrinolytic activity in the vasculature at the
initial step of the fibrinolytic cascade (5). Evidence for the
significance of PAI-1 in the regulation of fibrinolysis has been
documented by both epidemiologic studies and experimental animal models
including transgenic (8) and gene knockout animals (9). High PAI-1
activity is directly related to impaired fibrinolysis (10), and low
activity is associated with bleeding disorders (11, 12). The
fibrinolytic activity determined by the balance between tPA and PAI-1
can be altered by changing the gene expression of either molecule under
a variety of physiological or pathological conditions (13). Their
balance could also be altered as a consequence of the interaction of
PAI-1 with serine proteases other than plasminogen activators in plasma
(14). Like other SERPINs, PAI-1 inhibits plasminogen activator activity
by forming a stoichiometric complex through its reactive site in the
C-terminal region, the so-called strained loop (15, 16). The strained
loop of SERPINs is exposed to the outside of the molecule and is very
susceptible to proteolytic digestion as shown for both PAI-1 (17) and
1-antitrypsin (18) using neutrophil elastase and for
other members using snake venom and bacterial metalloproteases
(19).
In the present study we examine the possible interaction between
subtilisin NAT and PAI-1 or
2-AP to clarify the
mechanism for subtilisin NAT to enhance fibrinolytic activity. We found that subtilisin NAT cleaved PAI-1 at the P1-P1' peptide bond by limited
proteolysis, resulting in the effective enhancement of tPA-induced clot
lysis of PAI-1-enriched fibrin.
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EXPERIMENTAL PROCEDURES |
Purification of Subtilisin NAT--
Subtilisin NAT was purified
from a saline extract of B. subtilis (natto) as previously
reported (1) by techniques of ammonium precipitation and column
chromatography using butyl-Toyopearl (Tosoh Co., Tokyo, Japan),
CM-Toyopearl (Tosoh Co.), and Sephadex G-50 (Amersham Pharmacia
Biotech). After the purification to homogeneity, the protein
concentration was determined by the BCA protein assay reagent kit from Pierce.
Purification of Active Recombinant Prokaryotic PAI-1
(rpPAI-1)--
The cultivation of bacteria expressing PAI-1 and the
purification of active non-glycosylated rpPAI-1 have been previously described (20, 21). Purified active rpPAI-1 was stored at
80 °C
before use. The concentration of active rpPAI-1 was determined by
titration with two-chain uPA (20) of which the active site concentration was prevoiusly determined by titration with
4-methyl-umbelliferyl-p-guanidinobenzoate (22).
Other Proteins and Materials--
Glu-plasminogen was prepared
from freshly frozen plasma by lysine-Sepharose affinity
chromatography (23). Human fibrinogen was purchased from Enzyme
Research Laboratories, Inc. (South Bend, IN), and trace amounts of
contaminated plasminogen and plasmin were removed by passage
through lysine-Sepharose. After treatment with 1 mM (final
concentration) phenylmethylsulfonyl fluoride (purchased from Sigma),
the material was dialyzed exhaustively against 50 mM Hepes
(analytical grade and purchased from Sigma) buffer containing 100 mM NaCl.
2-AP was purchased from Biopool (Umeå, Sweden). Human thrombin was purchased from Welfide Corp. (Osaka, Japan). Single-chain tPA was kindly provided by Dai-ichi Pharmaceutical Co. (Tokyo, Japan). The chromogenic substrate S-2444 (L-pyroglutamyl-glycyl-L-arginine-p-nitroanilide)
and human plasmin were purchased from Chromogenix AB (Mölndal, Sweden).
Analysis of the Proteolytic Action of Subtilisin NAT on Active
and Latent rpPAI-1--
rpPAI-1 (1.7 µM) was incubated
with subtilisin NAT (0.5 nM) for various intervals (0, 10, 30, and 60 min) at 37 °C, and the reaction was stopped by the
addition of the sample buffer for SDS-polyacrylamide gel
electrophoresis (PAGE). The samples were then subjected to 10%
SDS-PAGE. The latent form of rpPAI-1 was obtained by the incubation of
active rpPAI-1 at 37 °C for 48 h (24), and its specific
activity was confirmed to be undetectable after the treatment. The
latent form of rpPAI-1 was also treated by subtilisin NAT as mentioned
above, and the proteolytic action of subtilisin NAT was analyzed by
SDS-PAGE.
Measurement of PAI-1 Activity after Treatment with Subtilisin
NAT--
The specific activity of rpPAI-1 toward plasminogen
activators after interaction with subtilisin NAT was estimated by a
two-step assay (25). rpPAI-1 (10 nM) was incubated with
increasing concentrations of subtilisin NAT for 1 h at 37 °C in
50 mM Tris-HCl buffer, pH 7.4, containing 100 mM NaCl and 0.1% bovine serum albumin. After the addition
of two-chain urokinase (final concentration, 100 IU/ml, ~10
nM), the mixture was incubated for another 30 min at 37 °C. S-2444 (final concentration, 1.0 mM) was then
added, and its hydrolysis was continuously monitored by absorbance at
405 nm. The PAI-1 activity was inversely proportional to the amount of
uPA preserved in the incubation mixture. The amount of S-2444 hydrolyzed by subtilisin NAT was estimated using the above assay conducted in the absence of uPA, and this value was subtracted although it was almost negligible. The rate of the hydrolysis of S-2444
in the presence of rpPAI-1 and absence of subtilisin NAT was considered
to represent the rate at full activity of rpPAI-1 (0% inhibition). The
rate of the hydrolysis in the absence of rpPAI-1 was considered to
represent fully inactivated rpPAI-1 (100% inhibition).
SDS-PAGE--
SDS-PAGE was performed according to Laemmli (26),
and protein bands were stained by Coomassie Brilliant Blue. A broad
range molecular weight standard was purchased from Bio-Rad.
Measurement of Clot Lysis--
96-well microtiter plates were
used for the clot lysis assay. 2 µM fibrinogen, 1 µM Glu-plasminogen, 0.2 nM tPA, 4 nM rpPAI-1, and various concentrations of subtilisin NAT
(0, 0.016, 0.032, 0.063, 0.13, 0.25, 0.5, and 1 nM) were
added to individual wells, and the clot formation was initiated by the
addition of 0.2 unit/ml human thrombin. Absorbance at 405 nm in each
well was measured every 15 min until the time of complete clot lysis
employing an automatic microtiter plate reader (Plate Analyzer ETY-300,
TOYO, Tokyo, Japan). To analyze the possible effect of subtilisin NAT on direct fibrin dissolution, the clot lysis time was determined as
mentioned above in the absence of tPA. Another set of similar experiments without rpPAI-1 was also conducted employing a much lower
(
) concentration of tPA (2 pM) to analyze the
effect of subtilisin NAT on fibrin clot lysis in the absence of PAI-1.
All of the assays were conducted at 37 °C.
Analysis of the Proteolytic Action of Subtilisin NAT on Other
Proteins Involved in Fibrinolysis--
2-AP (2.0 µM) was incubated with subtilisin NAT (0.5 nM) for various intervals (0, 10, 30, and 60 min) at
37 °C, and the reaction was stopped by the addition of the sample
buffer for SDS-PAGE. The samples were then subjected to 10% SDS-PAGE.
Fibrinogen (2.0 µM) was also treated with subtilisin NAT
(0.5 nM) in a similar way, and its cleavage was analyzed by
10% SDS-PAGE after reduction.
Determination of Molecular Mass--
The molecular mass of the
intact and the cleaved forms of rpPAI-1 was determined using
matrix-assisted laser desorption/ionization (MALDI) in combination with
a time-of-flight (TOF) mass analyzer (AXIMA-CFR, Shimadzu Solutions for
Science/Kratos Analytical, Kyoto, Japan) in the Life Science
Department, Analytical Instruments Division, of Shimadzu Co. The
instrument was externally calibrated using apomyoglobin (horse) and its
dimer form. Sinapinic acid in 0.1% trifluoroacetic acid and
acetonitrile (70:30) was used as matrix. A 0.7-µl protein sample
(~1.2 pmol of rpPAI-1) and 0.7 µl of matrix solution were mixed on
the sample plate and were subjected to analysis.
N-terminal Sequence Analysis--
Automatic amino acid sequence
analyses were performed by using an Applied Biosystems protein
sequencer (model 476A, Applied Biosystems, Foster City, CA) according
to the established principles. For N-terminal sequence analysis,
rpPAI-1 (1.8 µM) was incubated with 0.5 nM
subtilisin NAT in a final volume of 100 µl for 24 h at 37 °C.
The reaction was stopped, and the sample (~180 pmol of rpPAI-1) was
lyophilized and loaded onto a polyvinylidene difluoride membrane
(ProSpin, Applied Biosystems) after reconstitution and desalting. Data
were analyzed by the Applied Biosystems model 610A data analysis program.
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RESULTS |
Cleavage of rpPAI-1 by Subtilisin NAT--
Subtilisin NAT was
purified to homogeneity with a molecular mass of ~28 kDa (Fig.
1). Peptide sequence analysis after
trypsin digestion revealed that the protein was subtilisin NAT. The
specific activity for hydrolysis of S2251 (0.5 mM) was 20.7 mmol/min/mg. In a fibrin plate assay, which was calibrated using human
plasmin, the specific activity was 55.8 casein units/mg.

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Fig. 1.
Polyacrylamide gel electrophoresis of
purified subtilisin NAT. Purified subtilisin NAT was subjected to
10% SDS-PAGE under either nonreduced (lane 1) or reduced
(lane 2) conditions.
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After incubation with a catalytic amount of subtilisin NAT (~0.5
nM), active rpPAI-1 (1.7 µM) was cleaved into
smaller fragments in a time-dependent manner (Fig.
2A). The molecular mass of the main fragment was ~39 kDa, which is similar to the size of the cleaved form of PAI-1 generated by plasminogen activators through cleavage of its reactive site (21, 27). Similar cleavage was observed
even in the presence of vitronectin, a protein cofactor known to
maintain PAI-1 activity (data not shown).

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Fig. 2.
Cleavage of rpPAI-1 by subtilisin NAT.
rpPAI-1 either in the active form (A) or in the latent form
(B) was incubated with catalytic amounts of subtilisin NAT
for different time periods (lanes 1-4 indicate 0, 10, 30, and 60 min, respectively) and was subjected to 10% SDS-PAGE without
reduction.
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We also analyzed the interaction between the latent form of rpPAI-1 and
subtilisin NAT to see a possible difference in its susceptibility to
subtilisin NAT because the latent form of PAI-1 adopts a unique
conformation in which its reactive site is buried inside the molecule
(28). The latent form of rpPAI-1 appeared to be more resistant to
subtilisin NAT digestion with only a small fraction being cleaved into
the expected fragments (Fig. 2B). Thus, complete insertion
of the reactive site loop into the molecule seems to make it resistant
to cleavage by subtilisin NAT.
Inactivation of rpPAI-1 Activity by Subtilisin NAT--
Active
rpPAI-1 was incubated with different concentrations (0.02-1.0
nM) of subtilisin NAT for 1 h, and the residual PAI-1 activity was estimated by its specific activity toward uPA. rpPAI-1 lost its specific activity after incubation with subtilisin NAT in a
dose-dependent manner (Fig.
3). Half-maximal inhibition was obtained
at ~0.1 nM subtilisin NAT by a double-reciprocal plot of
subtilisin NAT concentration and its inhibitory effect. This inactivation of PAI-1 activity by subtilisin NAT suggests that rpPAI-1
was proteolytically cleaved at a site close to its reactive center.

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Fig. 3.
Dose-dependent inactivation of
rpPAI-1 by subtilisin NAT. Active rpPAI-1 (10 nM) was
incubated for 1 h with increasing concentrations of subtilisin NAT
(abscissa), and its residual activity toward uPA
(ordinate) was determined. Inset, a
double-reciprocal plot of subtilisin NAT concentration and its
inhibitory effect.
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Analysis of the Subtilisin NAT Cleavage Site in rpPAI-1--
The
subtilisin NAT cleavage site in rpPAI-1
was analyzed by automated Edman degradation and MALDI-TOF/MS (Fig. 4)
using an unfractionated subtilisin NAT digest of rpPAI-1 (Fig. 5).
Automated sequence analysis of the subtilisin NAT digest of rpPAI-1
gave two residues of an approximately 1:0.5 ratio for the first seven steps. One sequence was consistent
with the N-terminal sequence of PAI-1 (N-Val-His-His-Pro-Pro-Ser-Tyr;
average, 58.8 pmol), and the other was a new sequence consisting of
N-Met-Ala-Pro-Glu-Glu-Ile-Ile (average, 26.8 pmol). The latter sequence
originates from the C-terminal portion of the intact PAI-1 molecule and
corresponds to amino acid residues 347-353. The subtilisin NAT
cleavage site in PAI-1 appears to be between the residues
Arg346-Met347, which forms its reactive site
(P1-P1').

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Fig. 4.
MALDI-TOF/MS spectra of subtilisin NAT digest
of active rpPAI-1. Active rpPAI-1 was incubated with catalytic
amounts of subtilisin NAT for 24 h at 37 °C (see Fig. 5) and
was subjected to MALDI-TOF/MS spectrum analysis.
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Fig. 5.
SDS-PAGE of subtilisin NAT digest of active
rpPAI-1 used for peptide sequence analysis and MALDI-TOF/MS spectrum
analysis.
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MALDI-TOF/MS analysis gave two high molecular mass peaks of 42679.2 and
38875.1 Da and a low molecular mass peak of 3835.6 Da (Fig. 4). These
peaks correspond to intact rpPAI-1 (calculated mass, 42769.3 Da,
90.1
Da), the N-terminal fragment of cleaved rpPAI-1 (1) (calculated
mass, 38983.8 Da,
108.7 Da), and the C-terminal fragment of cleaved
rpPAI-1 (347) (calculated mass, 3803.6 Da, +32.0 Da),
respectively. This was consistent with the results obtained by peptide
sequence analysis of the rpPAI-1 digest and confirmed that the cleavage
site was the P1-P1' site. The difference between the measured molecular
mass and the theoretical mass (10 to ~100 Da) was within the accuracy
range for this size of protein (>30,000 Da), although a mass accuracy
of ±0.05% is normally obtained for peptides with this instrument.
Minor peaks of 21393.41 and 19496.71 Da correspond to doubly
charged species of 42679.2 and 38875.1 Da, respectively. Another minor
peak of 7674.48 Da corresponds to a dimer of the 3835.6-Da peptide and is probably formed as an artifact of the MS analysis.
Proteolytic Action of Subtilisin NAT on Other Proteins Involved in
Fibrinolysis--
2-AP, another important inhibitor of
fibrinolysis and member of the SERPIN family, did not appear to
interact with subtilisin NAT (no complex formation or cleavage
products) (Fig. 6A). The A
chain of fibrinogen was cleaved, although the B
and
chains were
resistant to subtilisin NAT under the conditions employed (Fig.
6B).

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Fig. 6.
Proteolytic action of subtilisin NAT on
2-AP and fibrinogen.
2-AP and fibrinogen were treated with catalytic amounts
of subtilisin NAT for different time periods (lanes 1-4
indicate 0, 10, 30, and 60 min, respectively), and the reaction
mixtures were then subjected to 10% SDS-PAGE either without reduction
(A, 2-AP) or with reduction (B,
fibrinogen).
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Effect of Subtilisin NAT on tPA-induced Fibrinolysis--
The
fibrin clot lysis time induced by 0.2 nM tPA was 0.40 ± 0.02 h and was prolonged by the addition of 4 nM
active rpPAI-1 to 29.1 ± 2.4 h. Subtilisin NAT dose
dependently shortened clot lysis time, reaching 7.6 ± 0.6 h
at 1 nM subtilisin NAT (Fig. 7A). To evaluate the direct
influence of subtilisin NAT on fibrin dissolution, similar clot lysis
assay was conducted in the complete absence of tPA. The dissolution of
the fibrin clot, however, was not observed over a period of 40 h
even in the presence of the highest concentration of subtilisin NAT (1 nM) (data not shown). We then analyzed the effect of
subtilisin NAT on rpPAI-1-depleted fibrin clots, employing a lower
concentration of tPA (
, 2 pM) to make the clot
lysis time long enough (5.8 ± 0.1 h). The clot lysis was
also shortened by subtilisin NAT in a dose-dependent manner
(3.0 ± 0.1 h at 1 nM subtilisin NAT), albeit to
a lesser extent (Fig. 7B). This enhancement of clot lysis
time in the absence of rpPAI-1 is most likely caused by direct
digestion of fibrin as previously reported (1, 29). The clot lysis
times both in the presence and absence of rpPAI-1 were expressed as a
percentage of the time obtained without subtilisin NAT in each assay
and were compared (Fig. 7C). The extent of the enhancement
was more dramatic in the presence of rpPAI-1, suggesting that the
inactivation of PAI-1 by subtilisin NAT plays a crucial role in the
effective lysis of PAI-1-enriched fibrin clots.

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Fig. 7.
Potentiation of tPA-induced fibrin clot lysis
time by subtilisin NAT. The profibrinolytic activity of subtilisin
NAT was evaluated either in rpPAI-1-enriched fibrin clots
(A) or in fibrin clots without rpPAI-1 (B). The
concentrations of tPA employed were 0.2 nM in
rpPAI-1-enriched fibrin clot lysis and 2 pM in
rpPAI-1-depleted fibrin clot lysis. The clot lysis time obtained at
each subtilisin NAT concentration, shown in A and
B, was expressed as a percentage over the corresponding
lysis time in the absence of subtilisin NAT (C).
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DISCUSSION |
The subtilisin-like serine protease (subtilase) superfamily is one
of two large serine protease superfamilies (the other being the
(chymo)trypsin-like serine protease family) (30). This subtilase superfamily contains large numbers of proteases of extremely widespread occurrence in plants, bacteria, yeast, and eukaryotes. It contains a
large group of proprotein convertases, which have attracted a great
deal of attention recently (31-33).
Both chymotrypsin and subtilisin have well conserved arrangements of
catalytic His, Asp, and Ser residues, but they are located in radically
different protein scaffolds,
/
for chymotrypsin and
/
for
subtilisin (30). Subtilisin NAT is a member of the subtilisin family,
whose members have rather broad substrate specificity due to larger and
more hydrophobic S1 and S4 binding sites (34). Its potential to enhance
fibrinolytic activity was first reported by Sumi et al. (2).
The analyses of its nucleotide sequence (3) and amino acid sequence (1)
confirmed that this enzyme belonged to the subtilisin family, showing
strong homology with both subtilisin E (from B. subtilis)
(35) and subtilisin J (from Bacillus
stearothermophilus) (36).
Although subtilisin NAT was shown to enhance fibrinolysis, its precise
mechanism is not known. It does not activate plasminogen but is
reported to directly digest fibrin especially in its cross-linked form
(29). In the present study we found a second mechanism by which
subtilisin NAT enhances fibrinolysis through cleavage and inactivation
of PAI-1. Because PAI-1 is the primary inhibitor of fibrinolysis and
regulates total fibrinolytic activity by its relative ratio with tPA,
its inactivation is directly related to the enhancement of
fibrinolysis. Cleavage of PAI-1 at its reactive center loop has been
demonstrated for activated coagulation factors (37) and neutrophil
elastase (17), both members of the (chymo)trypsin serine protease
family. The interaction of SERPINs with other members of the subtilisin
family has also been reported. Subtilisins BPN' (38) and
Carlsberg (7), which are both homologous to subtilisin NAT, cleave and
inactivate
1-antitrypsin. The latter was shown to
cleave
1-antitrypsin at two distinct sites within the
reactive site loop including its P2 site (7). Although it does not
possess inhibitory activity, ovalbumin, another SERPIN, was also shown
to be cleaved by subtilisin Carlsberg (39). This cleavage was not
observed when conformationally modified ovalbumin (S-ovalbumin),
in which the reactive center loop is fully inserted into
-sheet A,
was treated with subtilisin Carlsberg. This is consistent with data in
our present study that showed active rpPAI-1 was more susceptible to
subtilisin NAT digestion than the latent form whose conformation is
known to be similar to that of S-ovalbumin (28).
The reactive center loop of active SERPINs is exposed above the plane
of the molecule and is essential for forming a stoichiometric complex
with their target serine proteases, providing a "bait" residue (P1
residue) in the strained loop that acts as a pseudo-substrate for the
target protease (16). An interesting feature of PAI-1 is that the
active molecule spontaneously converts into a latent molecule (40), in
which the strained loop is fully inserted into the molecule as a
central strand (28) generating an apparently smaller Stokes' radius
(40). The decreased susceptibility of latent rpPAI-1 to subtilisin NAT
digestion, therefore, appears to be due to its compact conformation
with the reactive site loop inserted into the molecule.
In addition to the plasminogen activators, there are a number of serine
proteases that can reduce PAI-1 activity through complex formation or
proteolytic cleavage. As a result of these interactions the
fibrinolytic balance is shifted toward increased tPA activity (14, 17,
37, 41). This is based on the fact that tPA is a unique enzyme
possessing intrinsic specific activity in its single-chain form (42)
unlike other members of the (chymo) trypsin serine protease family.
The amount of active enzyme activity in this case is simply determined
by its relative concentration over its specific inhibitor. Several
activated coagulation factors, including factor XIa (37), calcium-bound
factor Xa and thrombin (41), and neutrophil elastase (17), have the
ability to interact with PAI-1. The inactivation of PAI-1 by these
activated coagulation factors is considered to be one of the mechanisms
responsible for the well known phenomenon of coagulation-associated
enhancement of fibrinolysis (14). In the present study subtilisin NAT
was also shown to be able to enhance tPA-induced clot lysis of
rpPAI-1-enriched fibrin by inactivating PAI-1.
Subtilisin NAT also shortened tPA-induced fibrin clot lysis time in the
absence of rpPAI-1, although to a lesser extent. The effect was likely
caused by direct digestion of fibrin by subtilisin NAT as previously
demonstrated both in vitro (29) and in vivo (4).
This is consistent with the fact that subtilisin NAT shows similar
activity both in plasminogen-rich and plasminogen-poor fibrin plates.
We also demonstrated subtilisin NAT-induced partial digestion of the
A
chain of the fibrinogen molecule. Although we did not analyze the
cleavage site in fibrinogen, lysine is a candidate for the P1 residue
of the cleavage site, since P1-lysine is a characteristic of
preferable substrate for subtilisin NAT (1, 2). Because similar partial
digestion of fibrin by plasmin is known to enhance fibrinolysis by
providing a C-terminal lysine in the A
chain that binds to both
plasminogen and tPA (43), this may be another mechanism for subtilisin
NAT to enhance fibrinolysis.
The fact that subtilisin NAT, like plasmin, is able to directly digest
fibrin and the finding that subtilisin NAT preferably hydrolyzes a
synthetic substrate for plasmin
(H-D-Val-Leu-Lys-p-nitroanilide) (1)
prompted us to analyze a possible interaction between subtilisin NAT
and
2-AP. Subtilisin NAT, however, neither formed a high molecular weight complex with
2-AP nor cleaved it.
Therefore, inactivation of
2-AP does not seem to be
involved in the enhancement of fibrinolysis by subtilisin NAT. This, in
turn, suggests that the cleavage of PAI-1 by subtilisin NAT is a rather
specific phenomenon.
In the present study we have shown that subtilisin NAT inactivates
PAI-1 by limited proteolysis of its reactive site. This mechanism seems
to allow this profibrinolytic enzyme to initiate effective lysis of
PAI-1-enriched fibrin clots.