Human TFPI contains 276 amino acids that comprise an acidic N-terminal domain followed by three tandem Kunitz-type trypsin inhibitor domains and a C-ter-minal basic amino-acid cluster re
Trang 1to chymase-mediated proteolysis
Tsutomu Hamuro1, Hiroshi Kido2, Yujiro Asada3, Kinta Hatakeyama3, Yuushi Okumura2,
Youichi Kunori4, Takashi Kamimura4, Sadaaki Iwanaga1and Shintaro Kamei1
1 Therapeutic Protein Products Research Department, The Chemo-Sero-Therapeutic Research Institute, Kaketsuken, Japan
2 Division of Enzyme Chemistry, Institute for Enzyme Research, University of Tokushima, Japan
3 Department of Pathology, Faculty of Medicine, University of Miyazaki, Japan
4 Institute for Biomedical Research, Teijin Pharma Limited, Japan
Tissue factor pathway inhibitor (TFPI) is the main
inhibitor of tissue factor-induced blood coagulation
Human TFPI contains 276 amino acids that comprise
an acidic N-terminal domain followed by three tandem
Kunitz-type trypsin inhibitor domains and a
C-ter-minal basic amino-acid cluster region [1] The first
Kunitz domain is necessary for the inhibition of the
factor VIIa–tissue factor complex, which forms a
ter-tiary complex with factor Xa, whereas the second
Kunitz domain inhibits factor Xa TFPI also inhibits a
variety of serine proteases, such as trypsin, a-chymo-trypsin, plasmin, and cathepsin G, demonstrating that this inhibitor has a relatively broad spectrum of inhibi-tion [2] The third Kunitz domain as well as the C-ter-minal basic region is important for binding to heparin [3–5]; however, whether this Kunitz domain possesses
an inhibitory activity is still unknown TFPI is mainly synthesized and secreted from endothelial cells [6] In addition, it is expressed in smooth muscle cells, fibro-blasts, monocytes, and cardiomyocytes in response to
Keywords
chymase; inflammation; protease inhibitor;
serine proteinase; tissue factor pathway
inhibitor (TFPI)
Correspondence
T Hamuro, Therapeutic Protein Products
Research Department, The
Chemo-Sero-Therapeutic Research Institute, Kaketsuken,
1-6-1 Okubo, Kumamoto, 860-8568, Japan
Fax: +81 96 3449234
Tel: +81 96 3442189
E-mail: hamuro@kaketsuken.or.jp
(Received 20 December 2006, revised 12
March 2007, accepted 17 April 2007)
doi:10.1111/j.1742-4658.2007.05833.x
Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type prote-ase inhibitor that primarily inhibits the extrinsic pathway of blood coagula-tion It is synthesized by various cells and its expression level increases in inflammatory environments Mast cells and neutrophils accumulate at sites
of inflammation and vascular disease where they release proteinases as well
as chemical mediators of these conditions In this study, the interactions between TFPI and serine proteinases secreted from human mast cells and neutrophils were examined TFPI inactivated human lung tryptase, and its inhibitory activity was stronger than that of antithrombin In contrast, mast cell chymase rapidly cleaved TFPI even at an enzyme to substrate molar ratio of 1 : 500, resulting in markedly decreased TFPI anticoagulant and anti-(factor Xa) activities N-Terminal amino-acid sequencing and MS analyses of the proteolytic fragments revealed that chymase preferentially cleaved TFPI at Tyr159-Gly160, Phe181-Glu182, Leu89-Gln90, and Tyr268-Glu269, in that order, resulting in the separation of the three indi-vidual Kunitz domains Neutrophil-derived proteinase 3 also cleaved TFPI, but the reaction was much slower than the chymase reaction In contrast, a-chymotrypsin, which shows similar substrate specificities to those of chy-mase, resulted in a markedly lower level of TFPI degradation These data indicate that TFPI is a novel and highly susceptible substrate of chymase
We propose that chymase-mediated proteolysis of TFPI may induce a thrombosis-prone state at inflammatory sites
Abbreviations
pNA, p-nitroanilide; TFPI, tissue factor pathway inhibitor; TFPI-C, TFPI with truncated C-terminal basic-amino-acid region.
Trang 2various stimuli produced in inflammatory states [6–9].
It is well known that neutrophils and mast cells are
important effector cells at sites of vascular
perturba-tion These cells release secretory granules that contain
a variety of biologically active substances In
parti-cular, neutrophil-derived proteolytic enzymes
partici-pate in the destruction of inflamed regions through the
degradation and inactivation of matrix proteins and
various protease inhibitors, including antithrombin, C1
inhibitor, heparin cofactor II, and a2-antiplasmin [10]
Previous reports have described the interactions of
TFPI with inflammatory cell-derived proteinases, such
as neutrophil elastase [11,12], cathepsin G [12], and
matrix metalloproteinases [13,14] The experimental
conditions used in these studies, however, did not
pre-cisely mimic physiological conditions Furthermore,
lit-tle is known about the interactions between TFPI and
serine proteinases derived from the secretory granules
of inflammatory cells
To investigate the functional role of TFPI in
inflam-mation, we examined the interactions between TFPI
and several serine proteinases derived from mast cells
and neutrophils Here, we demonstrate that TFPI
inhibited human lung tryptase In contrast, the activity
of chymase was not inhibited by TFPI, and chymase
rapidly cleaved TFPI even at a low enzyme to
sub-strate molar ratio, resulting in its inactivation In
addi-tion, we identified the cleavage sites in TFPI, and
determined the apparent kinetic constant for its
pro-teolysis by chymase Neutrophil-derived proteinase 3
also cleaved TFPI, but the reaction rate was much
slower than that of chymase These data identify TFPI
as a novel, highly susceptible substrate of chymase
Thus, chymase-mediated degradation and inactivation
of TFPI may induce a thrombosis-prone state at
inflammatory sites
Results
Inhibitory properties of TFPI on proteases
To investigate the inhibitory properties of TFPI against
human mast cell-derived and neutrophil-derived
pro-teinases, inhibition assays were performed using
appro-priate synthetic substrates For the mast cell-derived
proteinases, TFPI inhibited the activity of tryptase with
a 50% inhibitory concentration (IC50) of 10 lm,
whereas it did not inhibit the activity of chymase
(Fig 1A,B) Next, we tested the effects of TFPI on the
amidolytic activities of elastase, cathepsin G, and
neu-trophil-derived proteinase 3 As shown in Fig 1C,D,
TFPI inhibited the amidolytic activities of elastase
(IC50¼ 1.4 lm) and cathepsin G (IC50¼ 0.13 lm),
which agrees with a previous report [12] In contrast, TFPI produced only weak inhibition of the amidolytic activity of proteinase 3 (Fig 1E), even though this pro-tein is structurally similar to elastase and cathepsin G
Inhibitory properties of TFPI derivatives
on tryptase
It is well known that the conversion of tryptase into
an active tetrameric form requires sulfated polysaccha-rides such as heparin [15,16] On the other hand, TFPI strongly binds to heparin via its C-terminal basic amino-acid cluster region and the third Kunitz domain [3–5] Therefore, we tested the specificity of the inhibi-tion of tryptase by TFPI using TFPI and a TFPI derivative In the presence of a relatively low concen-tration of heparin (0.5 lgÆmL)1), TFPI strongly inhi-bited tryptase activity throughout a 60 min incubation (Fig 2A), whereas TFPI-C, which lacked the C-ter-minal basic region and ended at Lys249, showed no inhibitory activity (Fig 2B) In addition, antithrombin inhibited tryptase activity with an IC50 of 6.5 lm (Fig 2C); however, its inhibitory activity was weaker than that of TFPI (IC50¼ 1.7 lm) In the presence of excess heparin (500 lgÆmL)1), the activity of tryptase was not affected by TFPI, TFPI-C, or antithrombin (Fig 2A–C) These results strongly suggest that TFPI converted tryptase into an inactive monomer by removing the ‘essential heparin’, which was necessary for the tetramerization of tryptase, whereas an excess
of free heparin prevented TFPI from accessing the
‘essential heparin’ These results were consistent with a previous report that used heparin antagonists [17]
Proteolysis of TFPI by chymase and other proteinases
To examine whether TFPI is degraded by mast cell-derived and neutrophil-cell-derived proteinases, each protei-nase was incubated with TFPI at a molar ratio of
1 : 500 Surprisingly, chymase rapidly cleaved TFPI, even at a low enzyme to substrate ratio As shown in Fig 3A, TFPI was cleaved by chymase within 15 min,
as evidenced by the two smeared bands (bands 1 and 2) with molecular masses of 20–30 kDa observed on SDS⁄ PAGE Subsequently, the level of the approxi-mately 15-kDa band increased, and TFPI (43 kDa) as well as bands 1 and 2 disappeared entirely after incu-bation for 20 h These results indicate that TFPI was completely converted into fragments with molecular masses of 15 kDa by chymase Chymostatin, an inhibitor of chymotrypsin-type serine proteinases, com-pletely blocked the chymase-mediated proteolysis of
Trang 3Chymase activity (% of control)
A
TFPI (µ M )
B
Tryptase activity (% of contro
TFPI (µ M )
TFPI (µ M ) TFPI (µ M )
TFPI (µ M )
D
E
Elastase activity (% of contro
10 1
0.1 0
10 1
0.1 0
0 20 40 60 80 100 120
100 120
10 1
0.1 0
C
10 1
0.1
0
20
40
60
80
100
120
0
20
40
60
80
100
120
0 20 40 60 80
0 20 40 60 80 100 120
Fig 1 Inhibition of mast cell-derived and neutrophil-derived proteinases by TFPI Each proteinase was incubated with various concentrations
of TFPI at 37 C, and the residual proteinase activity was measured (A) Chymase (7 n M ) was assayed using Suc-Ala-Ala-Pro-Phe-pNA (5 m M ) (B) Tryptase (14 n M ) was assayed using H- D -Ile-Pro-Arg-pNA (0.15 m M ) (C) Neutrophil elastase (40 n M ) was assayed using MeO-Suc-Ala-Ala-Pro-Val-pNA (0.6 m M ) (D) Cathepsin G (167 n M ) was assayed using Suc-Ala-Ala-Pro-Phe-pNA (0.6 m M ) (E) Neutrophil-derived proteinase 3 (100 n M ) was assayed using MeO-Suc-Ala-Ala-Pro-Val-pNA (5 m M ) Data are presented as the mean ± SD from three independ-ent experimindepend-ents.
Antithrombin (µ M )
C
TFPI (µ M ) TFPI (µ M )
10 1
0 0 20 40 60 80 100 120
10 1
0 0 20 40 60 80 100 120
10 1
0 0 20 40 60 80 100 120
Tryptase activity (% of control)
Fig 2 Inhibition of tryptase by TFPI
deriva-tives and antithrombin TFPI (A), TFPI-C (B),
and antithrombin (C) were incubated with
tryptase in the presence of 0.5 lgÆmL)1(d)
or 500 lgÆmL)1heparin (s) for 60 min A
chromogenic substrate was then added, and
the amidolytic activity of tryptase was
measured Data are presented as the
mean ± SD from three independent
experi-ments.
Trang 4TFPI (data not shown) In addition, neither human
mast cell tryptase (Fig 3B) nor a-chymotrypsin
(Fig 3C) degraded TFPI In the same analysis, elastase
cleaved TFPI, resulting in the appearance of three new
bands on SDS⁄ PAGE which were estimated to be
38 kDa, 12 kDa, and 10 kDa (Fig 3D) This result
agrees with a previous report from Higuchi et al [11]
Petersen et al [12] previously reported that TFPI was
degraded by cathepsin G; we, however, did not detect
any degradation products after incubating TFPI with
cathepsin G (Fig 3E) at a similar enzyme to inhibitor
molar ratio (1 : 20, data not shown) This discrepancy
may be due to a difference in the experimental
materi-als In addition to these results, we found that
protein-ase 3 produced a similar digestion pattern on SDS⁄
PAGE to that of elastase, although the digestion was
less efficient (Fig 3F) N-Terminal sequencing of the
reaction products after a 20-h incubation demonstrated that proteinase 3 primarily cleaved TFPI between Thr87 and Thr88, which was the cleavage site targeted
by elastase In addition, trace amounts of peptide sequences starting with Leu90, Asp157, or Asp194 were found Of the proteinases examined in this study, chymase most rapidly and specifically processed TFPI
In the light of this finding, we further characterized the chymase-mediated proteolysis of TFPI
Characterization of the chymase-mediated proteolysis of TFPI
To characterize the proteolysis of TFPI by chymase,
we digested TFPI with chymase for 20 h and separated the resulting peptides using RP-HPLC As shown in Fig 4A, six major peptide peaks were separated using
97
66
45
30
20.1
14.4
kDa
Band 1
Band 2
Mr 0 15 30 60 120 180 1200min
97 66 45 30 20.1 14.4
kDa
Mr 0 15 30 60 120 180min
97 66 45 30 20.1
14.4
kDa
Mr 0 15 30 60 120 180
min
C
97 66 45 30 20.1 14.4
min
97
66
45
30
20.1
14.4
min
97 66 45 30 20.1
14.4
min
Fig 3 Cleavage of TFPI by mast cell-derived and neutrophil-derived serine proteinases Chymase (A), tryptase (B), a-chymotrypsin (C), neu-trophil elastase (D), cathepsin G (E), or proteinase 3 (F) at a concentration of 7 n M was incubated with TFPI (3.5 l M ) for the indicated times
at 37 C Proteins were separated on 15–25% polyacrylamide gels under reducing conditions and the gels were stained with Coomassie Bril-liant Blue Intermediate degradation products were designated as bands 1 and 2 (A) Mr, Low molecular mass marker.
Trang 5this type of chromatography Four of them, designated
peaks III, IV, V, and VI, all migrated at 15 kDa
during SDS⁄ PAGE (Fig 4B) The broad peak IV was
also visualized as a smeared band on the gel, even
though only a single N-terminal residue was detected
for this fragment Because recombinant TFPI contains
a variety of carbohydrate chains, this fragment may
have included N-linked carbohydrate chains [18] To
identify the chymase cleavage sites in TFPI, the
frag-ments that resulted in these six peaks were analyzed to
determine their amino-acid compositions, N-terminal
sequences, and mass spectra The results of these
ana-lyses are summarized in Table 1 Peaks III and IV
cor-responded to the third and second Kunitz domains of
TFPI, respectively Multiple MS signals were observed
for the peak IV fragment, supporting the idea that this
fragment was glycosylated Peaks V and VI both
cor-responded to the first Kunitz domain of TFPI; only
peak VI, however, was consistent with the calculated
MS value of this domain We assumed that the peak V fragment had an O-linked carbohydrate on Thr14 for two reasons: (1) N-terminal sequencing of this frag-ment did not produce a signal corresponding to Thr14, although such a signal was observed for the peak VI fragment; (2) the difference between the MS values for these two fragments (948 Da) perfectly matched the mass of a ubiquitous O-linked carbohydrate chain, Gal1–3GalNAc, with two N-acetylneuraminic acid resi-dues On the basis of these observations, we believe that the recombinant TFPI carried an O-linked carbo-hydrate chain on Thr14 Moreover, the peak heights and the areas under the peaks suggested that about half of the TFPI carried the O-linked carbohydrate chain on Thr14 (Fig 4A) Peak II corresponded to the third Kunitz domain without the C-terminal basic region (Table 1), and presumably resulted from a trace amount of contaminating TFPI-C [4] In addition, peak patterns obtained using RP-HPLC after 48 h of
1 2 3
20 40
Elution time (min)
Peak I Peak II
Peak III
Peak IV Peak VI Peak V
66 45
30 20.1
14.4
kDa
Mr Peak III
B A
Peak IVPeak VPeak VI 97
Fig 4 Separation of TFPI degradation products produced by treatment with chymase (A) TFPI digested with chymase for 20 h was applied
to a Vydac C8 column that had been equilibrated with 0.1% trifluoroacetic acid The products were eluted with 0.1% trifluoroacetic acid con-taining a linear concentration gradient of acetonitrile from 0% to 50% (B) SDS ⁄ PAGE of the peak III, IV, V, and VI fractions after RP-HPLC.
Mr, Low molecular mass marker.
Table 1 N-Terminal amino-acid sequences and mass spectra of degradation products The individual fragments designated in Figs 2 and 3 were separated by RP-HPLC, and the amino-acid sequences and mass spectra were determined.
Fragment Sequence Cleavage site Observed mass (m ⁄ z) Calculated mass (m ⁄ z) Deduced structure
a Intermediate degradation products; b multiple signals were observed.
Trang 6incubation were essentially identical with those
obtained with a 20-h incubation (data not shown),
implying that chymase acted on specific cleavage sites
in TFPI To clarify the order in which chymase
pro-cessed these sites in TFPI, we next separated the
inter-mediate fragments (Fig 3A, bands 1 and 2) using
RP-HPLC after incubation with the enzyme for 6 h
N-Terminal sequencing analyses identified bands 1 and
2 as the first and second Kunitz domains and the third
Kunitz domain, respectively (Table 1) The sample
obtained after a 3-h incubation was also subjected to
N-terminal sequencing In addition to the original
N-terminal sequence of native TFPI, four unique
sequences starting with Gln90, Gly160, Glu182, and
Glu269 were observed These sequences were present
at an approximate molar ratio of 2 : 5 : 4 : 1,
respect-ively These data revealed that chymase first cleaved
TFPI at Tyr159-Gly160 to generate two molecules
(bands 1 and 2), which was followed by cleavage at
Phe181-Glu182 and Leu89-Gln90 to generate the
frag-ments corresponding to peaks IV, V, and VI Finally,
chymase-mediated cleavage at Tyr268-Glu269
gener-ated the peaks I and III Taken together, these data
indicate that chymase selectively cleaved TFPI into five
fragments that were not disulfide linked, three of
which contained individual Kunitz domains
Anticoagulant activity of the TFPI degradation
products produced by chymase treatment
The effects of the chymase-mediated degradation on
TFPI function were evaluated by testing the residual
anticoagulant activities of the fragments and also by
determining the amount of residual TFPI antigen
using an ELISA Figure 5 shows the time course of
the decrease in anticoagulant and anti-(factor Xa)
activities determined under conditions in which 70 nm
TFPI was incubated at 37C with 7 nm chymase As
shown in Fig 5A, TFPI antigen promptly
disap-peared; the two fragments generated by cleavage at
Tyr159-Gly160 were not detected with this ELISA
sys-tem The anticoagulant and anti-(factor Xa) activities
also decreased in a time-dependent manner; after 5 min,
however, these activities decreased at relatively slow
rates (Fig 5B,C) In particular, 20% of the
anti-(factor Xa) activity was observed after 120 min,
sug-gesting that the digested TFPI was still able to inhibit
the protease Petersen et al [2] reported that a single
Kunitz domain can act as a protease inhibitor, although
its activity was weaker than that of the full-length
protein These results indicate that chymase rapidly
reduces, but does not completely eliminate, the
anti-coagulant activity of TFPI
Kinetic analyses of the degradation of TFPI
by inflammatory proteinases
To quantify the abilities of chymase, elastase, and pro-teinase 3 to proteolytically cleave TFPI, we performed kinetic analyses using ELISAs With this method, kinetic constants were calculated as apparent values,
120 90
60 30
0 0 20 40 60 80 100
120 90
60 30
0 0 20 40 60 80 100
120 90
60 30
0 0 20 40 60 80 100
Incubation time (min)
A
B
C
Fig 5 Effects of chymase on the anticoagulant and anti-(factor Xa) activities of TFPI TFPI (70 n M ) was incubated with chymase (7 n M )
at 37 C After various incubation times, the reaction was termin-ated with chymostatin, and aliquots of the sample were subjected
to ELISA, a dilute tissue factor clotting assay, and anti-(factor Xa) assay (see Experimental procedures) (A) Residual TFPI antigen (B) Residual anticoagulant activity of TFPI (C) Residual anti-(factor Xa) activity of TFPI Data are presented as the mean ± SD from three independent experiments.
Trang 7because the efficiency of enzymatic proteolysis was
estimated on the basis of the amount of remaining
TFPI antigen As shown in Table 2, the apparent
cata-lytic efficiency (kcat⁄ Km) of chymase was almost
equiv-alent to that of elastase On the other hand, although
the enzymatic properties of proteinase 3 are similar to
those of elastase, this proteinase showed lower activity
toward TFPI
Effects of sulfated polysaccharides
TFPI is thought to localize on various cell surfaces,
especially the surfaces of endothelial cells, by binding to
sulfated proteoglycans such as heparan sulfate [19,20]
In addition, sulfated polysaccharides bind to TFPI and
enhance its anticoagulant activity in vitro [21,22]
Therefore, we investigated whether sulfated
polysaccha-rides affected the chymase-mediated degradation of
TFPI As shown in Fig 6A, cleavage of TFPI by
chymase occurred in the presence of every polysaccha-ride tested Unfractionated heparin and low-molecular-weight heparin slightly delayed the chymase-mediated proteolysis of TFPI over the first 60 min; after 180 min, however, the levels of residual TFPI antigen in these samples were the same as observed in the control sample (Fig 6B) These results suggest that mast cell-derived heparin and cell-surface heparan sulfate do not prevent the proteolysis of TFPI by chymase
Discussion Mast cells, which reside mainly in connective tissue matrices, lung, heart, and epithelial surfaces, are effector cells that participate in innate and acquired immunity [23–26] In pathological conditions, such as inflammation, fibrosis, and malignancy, mast cells as well as neutrophils and macrophages accumulate at the affected sites Recent studies indicate that mast cells also accumulate at sites of atrial appendages [27], deep venous thrombosis [28], periprostate vein thrombosis [29], and atherosclerotic plaques [30–33] These findings imply that mast cells are involved in thrombosis and fibrinolysis In fact, mast cells express tissue-type plasminogen activator and urokinase-type plasminogen activator receptor [34] Little, however,
is known about the functional roles of proteinases released from mast cell granules during thrombosis and other pathological states In this report, we focused on the reactivity of TFPI with serine protein-ases released from mast cells
Table 2 Apparent kinetic constants for the proteolytic cleavage of
TFPI by chymase, neutrophil elastase, and proteinase 3 The
velo-city of TFPI degradation was measured using an ELISA as
des-cribed in Experimental procedures Kinetic constants were
calculated from a Lineweaver–Burk plot Values are expressed as
the mean ± SD from three independent experiments.
Enzyme Substrate
Km (l M )
kcat (min)1)
kcat⁄ K m
(l M )1Æmin)1)
Chymase TFPI 5.01 ± 0.84 23.16 ± 1.98 4.62
Elastase TFPI 2.00 ± 0.06 10.20 ± 0.71 5.10
Proteinase 3 TFPI 17.47 ± 4.46 8.10 ± 1.11 0.46
e
Dermatan sulfat e
Hyaluronic a cid
Dextran sulfat e
TFPI only kDa
A
97 66 45
30
20.1
14.4
B
Incubation time (min)
180 120
60 0
0 20 40 60 80
100
Mr
Fig 6 Effects of various polysaccharides on chymase-mediated proteolysis of TFPI (A) TFPI (3.5 l M ) was incubated with chymase (7 n M ) for 60 min at 37 C in the presence of each polysaccharide (100 lgÆmL)1) Proteins were separated on 15–25% polyacrylamide gels under reducing conditions, and the gels were stained with Coomassie Brilliant Blue The arrow indicates TFPI (B) TFPI (70 n M ) was incubated with chymase (7 n M ) in the presence or absence of heparin (100 lgÆmL)1) for up to 180 min After various incubation times, the reaction was ter-minated with chymostatin, and the level of TFPI that remained was measured using an ELISA (d) Control incubation; (j) incubation with low molecular weight heparin (LMWH); (h) incubation with unfractionated heparin (UFH) Mr, Low molecular mass marker.
Trang 8We first found that TFPI inactivates the amidolytic
activity of tryptase, presumably by removing heparin;
heparin or acidic polysaccharides allow tryptase to
form a stabilized noncovalent tetramer and are
indis-pensable for tryptase activity [15,16] Owing to the
loss of heparin, tetrameric tryptase rapidly and
irre-versibly dissociates into inactive monomers [35] For
example, polybrene and protamine convert tetrameric
tryptase into monomers, resulting in the loss of
tryp-tase activity [17] The conformational structure of
b-tryptase suggests that the active site of each tryptase
monomer is largely inaccessible to macromolecular
inhibitors [16], which probably explains why tryptase
is resistant to endogenous proteinase inhibitors, such
as a1-proteinase inhibitor and antithrombin [36,37]
Therefore, this inactivating process has been
pro-posed to be a control system that regulates tryptase
activity in vivo In this study, TFPI, but not TFPI-C,
inactivated the amidolytic activity of tryptase,
sug-gesting that the domain responsible for the
inactiva-tion was the C-terminal basic-amino-acid cluster
region A synthetic peptide representing Lys254 to
Met276, however, was rapidly degraded by tryptase
(data not shown) This synthetic peptide probably
did not mimic the native structure of the C-terminal
region of TFPI, because TFPI was not cleaved by
tryptase in this study (Fig 3B) The mechanism by
which TFPI inactivates tryptase requires further
investigation
Secondly, we found that chymase efficiently cleaved
TFPI, even at a very low enzyme to substrate molar
ratio (1 : 500) As shown in Fig 7, TFPI is known to
be degraded by several proteinases, including
throm-bin, plasmin, factor Xa, matrix metalloproteinases,
and neutrophil elastase [11–14,38–40] Those results,
however, were obtained from reactions performed at
high enzyme to substrate molar ratios, or after long
incubations The present study revealed that chymase
selectively cleaves TFPI at four peptide bonds
(Tyr159-Gly160, Phe181-Glu182, Leu89-Gln90, and
Tyr268-Glu269 in that order), which separated the
three individual Kunitz inhibitor domains and
abol-ished the anticoagulant activity of TFPI The
previ-ously reported natural substrates of human chymase
include angiotensin I [41], bradykinin [42], C1-inhibitor
[43], interleukin-1b [44], neurotensin [45], interstitial
procollagenase (proMMP-1) [46], kit ligand [47], big
endothelins [48], type-I procollagen [49],
a2-macroglo-bulin [50], profilin [51], albumin [52], and connective
tissue-activating peptide III [53] The cleaving sites of
these natural substrates are summarized in Table 3
Using a combinatorial peptide screening method,
Ray-mond et al [52] demonstrated that chymase
preferen-tially acts at sites with Tyr or Phe as the P1 residue, which is supported by the presence of these residues at the P1 positions in the natural substrates (Table 3) In agreement with those results, we found that three of the four chymase cleavage sites in TFPI have either Tyr or Phe as the P1 residue At the fourth site, chymase cleaved TFPI between Leu89 and Gln90 Interestingly, the region containing Lys86-Thr87-Thr88-Leu89-Gln90 appears to be a ‘hot region’, because it contains cleavage sites for thrombin, plas-min, factor Xa, and elastase in addition to chymase
MMPs
IIa, Pm
MMPs
Xa, Pm
IIa Pm
L 89 -Q 90
Y 159 -G 160
F 181 -E 182 Y 268 -E 269
T 14
Fig 7 Schematic structure of TFPI and cleavage sites by chymase The cleavage sites in TFPI are summarized in this figure Data obtained in this study including the four chymase cleavage sites are shown below the TFPI structure, whereas previously deter-mined data are shown above the TFPI structure The thick arrows indicate the locations of the sites cleaved by chymase, which include the amino acids and residue numbers The open circles and branches indicate O-linked glycosylation sites and N-linked glycosy-lation sites, respectively Our findings suggest that the threonine residue at amino-acid position 14 carried an O-linked carbohydrate
in half of the TFPI molecules used here The solid bar indicates a
‘hot region’, which contains cleavage sites for thrombin (IIa), plas-min (Pm), factor Xa (Xa), neutrophil elastase, proteinase 3, and chymase The thin arrows indicate the cleavage sites for each pro-teolytic enzyme MMP, Matrix metalloproteinase.
Table 3 Sites of hydrolysis of natural substrates of human chy-mase.
Trang 9(Fig 7) Presumably, this region has a distinct
confor-mation and is exposed on the surface of the molecule,
making it highly susceptible to attack by these
protein-ases It was also reported that the P2 and P3 subsite
preferences of chymase were Thr⁄ Pro and Thr ⁄ Glu ⁄
Ser, respectively [52] This could explain why TFPI
was cleaved by chymase at Leu89-Gln90, because both
of the P2 and P3 subsites are Thr
TFPI-b is an alternatively spliced form of TFPI
that lacks the third Kunitz domain and the
C-ter-minal portion of TFPI and instead contains a
glyco-sylphosphatidylinositol anchor [54] It is thought that
TFPI-b binds and localizes to the cell surface via its
glycosylphosphatidylinositol anchor domain Because
the N-terminal 181 amino acids of TFPI-b are
iden-tical with those of TFPI, TFPI-b has at least two
chymase cleavage sites, and chymase might be able
to release TFPI-b from the cell surface The
interac-tion between chymase and TFPI-b requires further
elucidation
In addition, we investigated the effects of sulfated
polysaccharides on the interaction between TFPI and
chymase, because both of these proteins bind to
hep-arin [55,56] Hephep-arin, which is produced and secreted
by mast cells, did not inhibit the cleavage of TFPI by
chymase Moreover, heparan sulfate did not influence
the proteolysis of TFPI It was reported that heparin
has no affect on the amidolytic activity of chymase
for a chromogenic substrate, whereas it inhibited the
chymase-mediated proteolysis of casein and
angio-tensin I [56,57], suggesting that the regulation of
chy-mase activity by heparin is dependent on the
substrate Therefore, cell-surface TFPI, which is bound
to proteoglycans, could be cleaved by chymase
Although Valentin & Schousboe [58] reported that
TFPI interacts with acidic phospholipids such as
phos-phatidylserine in vitro, we found that
phosphatidyl-serine did not affect the cleavage of TFPI (data not
shown)
The human gastrointestinal tract contains numerous
mast cells, which are located primarily in the lamina
propria mucosa We confirmed that a large number
of chymase-positive mast cells are located around
microvessels in the lamina propria mucosa, and TFPI
was detected on the intraluminal surface of these
microvessels (K Hatakeyama and Y Asada,
unpub-lished data) It was previously reported that human
intestinal mast cells produce and release tumor
necro-sis factor-a in response to Gram-negative bacteria
such as Escherichia coli [59] Furthermore, tumor
nec-rosis factor-a induces the expression of tissue factor
on vascular endothelial cells [60] Clot formation
resulting from tissue factor induction and
chymase-mediated proteolysis of TFPI might be a protective function of intestinal mast cells against bacterial inva-sion into the bloodstream
In conclusion, the present study suggests that the presence of mast cells and the associated release of chymase may accentuate local thrombosis due to the local inactivation of TFPI at inflammatory sites
Experimental procedures
Materials
S-2288 (H-d-Ile-Pro-Arg-pNAÆHCl where pNA is p-nitroan-ilide) and S-2222 [Bz-Ile-Glu(GlucOMe)-Gly-Arg-pNAÆHCl] were obtained from Chromogenix AB (Stockholm, Sweden) Succinyl-Ala-Ala-Pro-Phe-pNA, methoxysuccinyl-Ala-Ala-Pro-Val-pNA, unfractionated heparin, and low molecular weight heparin (average molecular mass 3000 Da) were purchased from Sigma-Aldrich (St Louis, MO, USA) Chymostatin was purchased from Peptide Institute, Inc (Osaka, Japan) Hemoliance human control plasma was obtained from Instrumentation Laboratory (Lexington,
MA, USA) Dade thromboplastinÆC plus was purchased from Dade International Inc (Miami, FL, USA), and hepa-ran sulfate, chondroitin sulfate A, dermatan sulfate, and hyaluronic acid were obtained from Seikagaku kogyo Co (Tokyo, Japan) Sodium dextran sulfate was purchased from ICN Biochemicals, Inc (Aurora, OH, USA) All other chemicals were of analytical grade or of the highest quality commercially available
Proteins
Human lung tryptase, human neutrophil elastase, and cath-epsin G were obtained from Calbiochem (La Jolla, CA, USA) Human neutrophil proteinase 3 was purchased from Athens Research and Technology (Athens, GA, USA) Bovine a-chymotrypsin was obtained from Worthington Biochemical Corp (Lakewood, NJ, USA) Recombinant human chymase was expressed in Trichoplusia ni insect cells using a baculovirus expression system and purified from the culture medium as described previously [61] Activated human factor X (factor Xa) was prepared by incubating purified factor X with Russell’s viper venom factor X activa-tor (Haematologic Technologies, Essex Junction, VT, USA) and then separating factor Xa by gel filtration on a column
of Sephacryl S-200 (Amersham Biosciences, Piscataway,
NJ, USA) as described in a previous paper [62] Human antithrombin was purified from human plasma using a procedure based on heparin affinity chromatography [63] Recombinant human TFPI was expressed in Chinese ham-ster ovary (CHO) cells and purified from the culture medium
as described previously [4] TFPI-C, which lacked the C-ter-minal basic region and ended at Lys249, was separated from
Trang 10full-length TFPI [4] TFPI expressed in CHO cells had
N-linked carbohydrate chains at Asn117 and Asn167, and
O-linked carbohydrate chains at Ser174 and Thr175 [18]
Inhibition assay of TFPI
with each proteinase and synthetic substrate, and the
velocities of the initial reactions were measured with a
THERMOmax microplate spectrometer (Molecular Devices,
1 min The amount of residual active proteinase was
deter-mined by comparing the result to a standard curve
con-structed using known amounts of the proteinase Chymase
(5 mm) Tryptase (14 nm) was assayed using
hep-arin Neutrophil-derived proteinase 3 (100 nm) was assayed
using MeO-Suc-Ala-Ala-Pro-Val-pNA (5 mm) Neutrophil
elastase (40 nm) was assayed using
MeO-Suc-Ala-Ala-Pro-Val-pNA (0.6 mm) Cathepsin G (167 nm) was assayed
using Suc-Ala-Ala-Pro-Phe-pNA (0.6 mm)
Inhibition of human lung tryptase by TFPI
derivatives
con-centration of heparin was incubated with various
concen-trations of TFPI, TFPI-C, or antithrombin for 60 min at
and the initial rate of hydrolysis was measured
Proteolysis of TFPI by proteinases
Reactions containing 7 nm chymase, tryptase,
a-chymotryp-sin, elastase, cathepsin G, or proteinase 3 and 3.5 lm TFPI
(molar ratio of 1 : 500) were incubated for the designated
using chymase, a-chymotrypsin, elastase, cathepsin G, and
points, samples were taken from the reaction mixture and
Pro-tein bands were visualized by staining with Coomassie
Bril-liant Blue R-250
RP-HPLC
RP-HPLC was carried out on a Vydac 208TP54 C8-300
column (Cypress International Ltd, Tokyo, Japan) After
the sample was injected, the column was washed with
a solution of 0.1% trifluoroacetic acid for 10 min TFPI fragments were eluted with a linear gradient of this solution containing 24–37% acetonitrile at a flow rate of
and dissolved in water for further analyses
Amino-acid composition analysis, N-terminal sequencing, and MS analysis
The amino-acid compositions of the fragments were
USA) according to the manufacturer’s protocol Automa-ted Edman degradation was carried out using an Applied Biosystems 492 protein sequencer and standard methods
MS analysis was performed using matrix-assisted laser
STR workstation (Applied Biosystems, Foster City, CA, USA)
Measurement of the level of TFPI antigen using
an ELISA
TFPI antigen was detected with a sandwich ELISA method using two different monoclonal antibodies against TFPI One monoclonal antibody (designated K9), which was immobilized on the microtiter plate, recognized the third Kunitz domain of TFPI [64], whereas the other monoclonal antibody (designated K270), which was conjugated with horseradish peroxidase, recognized the region between the first and second Kunitz domains of TFPI [62] Only TFPI antigen consisting of all three Kunitz domains was detected with this ELISA In this procedure, each sample and
pre-mixed and incubated in a K9-coated microtiter well for 2 h
buffer, and mixed with 200 lL
30-min incubation, development was terminated by the
405 nm was measured using a THERMOmax microplate spectrometer The concentration of TFPI was calculated from a standard curve prepared with known amounts of TFPI
Dilute tissue factor clotting assay
was terminated with 100 lm chymostatin, and a 15-lL ali-quot of the sample was added to 135 lL human control