Tài liệu Báo cáo khoa học: Tissue factor pathway inhibitor is highly susceptible to chymase-mediated proteolysis pptx

Tissue factor pathway inhibitor is highly susceptible to chymase-mediated proteolysis Tsutomu Hamuro1, Hiroshi Kido2, Yujiro Asada3, Kinta Hatakeyama3, Yuushi Okumura2, Youichi Kunori4, Takashi Kamimura4, Sadaaki Iwanaga1 and Shintaro Kamei1 Therapeutic Protein Products Research Department, The Chemo-Sero-Therapeutic Research Institute, Kaketsuken, Japan Division of Enzyme Chemistry, Institute for Enzyme Research, University of Tokushima, Japan Department of Pathology, Faculty of Medicine, University of Miyazaki, Japan Institute for Biomedical Research, Teijin Pharma Limited, Japan Keywords chymase; inflammation; protease inhibitor; serine proteinase; tissue factor pathway inhibitor (TFPI) Correspondence T Hamuro, Therapeutic Protein Products Research Department, The Chemo-SeroTherapeutic 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 protease inhibitor that primarily inhibits the extrinsic pathway of blood coagulation 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 : 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 individual Kunitz domains Neutrophil-derived proteinase 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 chymase, 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 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-terminal 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 tertiary complex with factor Xa, whereas the second Kunitz domain inhibits factor Xa TFPI also inhibits a variety of serine proteases, such as trypsin, a-chymotrypsin, plasmin, and cathepsin G, demonstrating that this inhibitor has a relatively broad spectrum of inhibition [2] The third Kunitz domain as well as the C-terminal 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, fibroblasts, monocytes, and cardiomyocytes in response to Abbreviations pNA, p-nitroanilide; TFPI, tissue factor pathway inhibitor; TFPI-C, TFPI with truncated C-terminal basic-amino-acid region FEBS Journal 274 (2007) 3065–3077 ª 2007 The Authors Journal compilation ª 2007 FEBS 3065 Chymase-mediated proteolysis of TFPI T Hamuro et al various stimuli produced in inflammatory states [6–9] It is well known that neutrophils and mast cells are important effector cells at sites of vascular perturbation These cells release secretory granules that contain a variety of biologically active substances In particular, neutrophil-derived proteolytic enzymes participate 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 precisely mimic physiological conditions Furthermore, little 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 inflammation, 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 substrate molar ratio, resulting in its inactivation In addition, we identified the cleavage sites in TFPI, and determined the apparent kinetic constant for its proteolysis by chymase Neutrophil-derived proteinase 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 proteinases, inhibition assays were performed using appropriate 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 neutrophil-derived proteinase As shown in Fig 1C,D, TFPI inhibited the amidolytic activities of elastase (IC50 ¼ 1.4 lm) and cathepsin G (IC50 ¼ 0.13 lm), 3066 which agrees with a previous report [12] In contrast, TFPI produced only weak inhibition of the amidolytic activity of proteinase (Fig 1E), even though this protein 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 polysaccharides 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 inhibition of tryptase by TFPI using TFPI and a TFPI derivative In the presence of a relatively low concentration of heparin (0.5 lgỈmL)1), TFPI strongly inhibited tryptase activity throughout a 60 incubation (Fig 2A), whereas TFPI-C, which lacked the C-terminal 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 (IC50 ¼ 1.7 lm) In the presence of excess heparin (500 lgỈmL)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 cellderived and neutrophil-derived proteinases, each proteinase was incubated with TFPI at a molar ratio of : 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 and 2) with molecular masses of 20–30 kDa observed on SDS ⁄ PAGE Subsequently, the level of the approximately 15-kDa band increased, and TFPI (43 kDa) as well as bands and disappeared entirely after incubation 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, completely blocked the chymase-mediated proteolysis of FEBS Journal 274 (2007) 3065–3077 ª 2007 The Authors Journal compilation ª 2007 FEBS B 120 100 80 60 40 20 120 100 0.1 TFPI (µM) 60 40 20 10 100 80 60 40 20 100 80 60 40 20 0.1 TFPI (µM) 10 E 120 0 0.1 TFPI (µM) 10 0.1 TFPI (µM) 10 120 Proteinase activity (% of control) Cathepsin G activity (% of control) 80 120 0 D C Elastase activity (% of control) Chymase-mediated proteolysis of TFPI Tryptase activity (% of control) A Chymase activity (% of control) T Hamuro et al 100 80 60 40 20 0 0.1 TFPI (µM) 10 Fig 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 nM) was assayed using Suc-Ala-Ala-Pro-Phe-pNA (5 mM) (B) Tryptase (14 nM) was assayed using H-D-Ile-Pro-Arg-pNA (0.15 mM) (C) Neutrophil elastase (40 nM) was assayed using MeOSuc-Ala-Ala-Pro-Val-pNA (0.6 mM) (D) Cathepsin G (167 nM) was assayed using Suc-Ala-Ala-Pro-Phe-pNA (0.6 mM) (E) Neutrophil-derived proteinase (100 nM) was assayed using MeO-Suc-Ala-Ala-Pro-Val-pNA (5 mM) Data are presented as the mean ± SD from three independent experiments B 120 100 100 80 60 40 20 Tryptase activity (% of control) Fig Inhibition of tryptase by TFPI derivatives and antithrombin TFPI (A), TFPI-C (B), and antithrombin (C) were incubated with tryptase in the presence of 0.5 lgỈmL)1 (d) or 500 lgỈmL)1 heparin (s) for 60 A chromogenic substrate was then added, and the amidolytic activity of tryptase was measured Data are presented as the mean ± SD from three independent experiments 80 60 40 20 0 C 120 Tryptase activity (% of control) Tryptase activity (% of control) A TFPI (µM) 10 Antithrombin (µM) 10 TFPI (µM) 10 120 100 80 60 40 20 FEBS Journal 274 (2007) 3065–3077 ª 2007 The Authors Journal compilation ª 2007 FEBS 3067 Chymase-mediated proteolysis of TFPI A T Hamuro et al Mr 15 30 60 120 180 1200 B Mr 15 30 60 120 180 C kDa kDa kDa 97 66 45 97 66 45 97 66 45 30 Band 30 30 20.1 Band 20.1 20.1 14.4 14.4 Mr 15 30 60 120 180 14.4 D Mr 15 30 60 120 180 E Mr 15 30 60 120 180 F kDa kDa kDa 97 66 97 66 97 66 45 45 45 30 30 30 20.1 20.1 20.1 14.4 14.4 Mr 15 30 60 120 180 14.4 Fig Cleavage of TFPI by mast cell-derived and neutrophil-derived serine proteinases Chymase (A), tryptase (B), a-chymotrypsin (C), neutrophil elastase (D), cathepsin G (E), or proteinase (F) at a concentration of nM was incubated with TFPI (3.5 lM) 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 Brilliant Blue Intermediate degradation products were designated as bands and (A) Mr, Low molecular mass marker TFPI (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 materials In addition to these results, we found that proteinase 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 3068 reaction products after a 20-h incubation demonstrated that proteinase 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 FEBS Journal 274 (2007) 3065–3077 ª 2007 The Authors Journal compilation ª 2007 FEBS Chymase-mediated proteolysis of TFPI A B Mr Pe ak Pe I I I ak Pe I V ak Pe V ak VI T Hamuro et al kDa 97 66 45 Peak V Peak VI Peak IV 40 Peak I Peak II 20 0 10 20 30 40 50 60 30 CH3 CN (%) Absorbance at 214nm Peak III 20.1 14.4 70 Elution time (min) Fig 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 containing 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 this 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 fragments that resulted in these six peaks were analyzed to determine their amino-acid compositions, N-terminal sequences, and mass spectra The results of these analyses are summarized in Table Peaks III and IV corresponded 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 corresponded 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 fragment 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 residues On the basis of these observations, we believe that the recombinant TFPI carried an O-linked carbohydrate 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 Table N-Terminal amino-acid sequences and mass spectra of degradation products The individual fragments designated in Figs and 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) Band 1a DSEEDEE – – – a GTQLNAV Tyr159-Gly160 – – Peak I EEIFVKN Tyr268-Glu269 1010.61 1010.17 Peak II EFHGPSW Phe181-Glu182 7778.76 7778.72 Peak III EFHGPSW Phe181-Glu182 10031.02 Band Deduced structure 10032.50 b Peak IV QQEKPDF Leu89-Gln90 9705–12656 – Peak V DSEEDEE – 11334.61 – Peak VI DSEEDEE – 10386.94 10386.63 a Intermediate degradation products; b multiple signals were observed FEBS Journal 274 (2007) 3065–3077 ª 2007 The Authors Journal compilation ª 2007 FEBS 3069 Chymase-mediated proteolysis of TFPI T Hamuro et al 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 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 37 °C with nm chymase As shown in Fig 5A, TFPI antigen promptly disappeared; the two fragments generated by cleavage at Tyr159-Gly160 were not detected with this ELISA system The anticoagulant and anti-(factor Xa) activities also decreased in a time-dependent manner; after 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, suggesting 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 anticoagulant activity of TFPI 3070 TFPI antigen (% of control) A 100 80 60 40 20 0 30 60 90 120 30 60 90 120 30 60 90 Incubation time (min) 120 Anticoagulant activity (% of control) B 100 80 60 40 20 C Anti-factor Xa activity (% of control) incubation 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 processed these sites in TFPI, we next separated the intermediate fragments (Fig 3A, bands and 2) using RP-HPLC after incubation with the enzyme for h N-Terminal sequencing analyses identified bands and 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 : : : 1, respectively These data revealed that chymase first cleaved TFPI at Tyr159-Gly160 to generate two molecules (bands and 2), which was followed by cleavage at Phe181-Glu182 and Leu89-Gln90 to generate the fragments corresponding to peaks IV, V, and VI Finally, chymase-mediated cleavage at Tyr268-Glu269 generated 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 100 80 60 40 20 Fig Effects of chymase on the anticoagulant and anti-(factor Xa) activities of TFPI TFPI (70 nM) was incubated with chymase (7 nM) at 37 °C After various incubation times, the reaction was terminated 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 Kinetic analyses of the degradation of TFPI by inflammatory proteinases To quantify the abilities of chymase, elastase, and proteinase to proteolytically cleave TFPI, we performed kinetic analyses using ELISAs With this method, kinetic constants were calculated as apparent values, FEBS Journal 274 (2007) 3065–3077 ª 2007 The Authors Journal compilation ª 2007 FEBS T Hamuro et al Chymase-mediated proteolysis of TFPI chymase occurred in the presence of every polysaccharide tested Unfractionated heparin and low-molecularweight 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 cellderived heparin and cell-surface heparan sulfate not prevent the proteolysis of TFPI by chymase Table Apparent kinetic constants for the proteolytic cleavage of TFPI by chymase, neutrophil elastase, and proteinase The velocity of TFPI degradation was measured using an ELISA as described in Experimental procedures Kinetic constants were calculated from a Lineweaver–Burk plot Values are expressed as the mean ± SD from three independent experiments Enzyme Km Substrate (lM) Chymase TFPI Elastase TFPI Proteinase TFPI kcat (min)1) kcat ⁄ Km (lM)1Ỉmin)1) 5.01 ± 0.84 23.16 ± 1.98 4.62 2.00 ± 0.06 10.20 ± 0.71 5.10 17.47 ± 4.46 8.10 ± 1.11 0.46 Discussion because the efficiency of enzymatic proteolysis was estimated on the basis of the amount of remaining TFPI antigen As shown in Table 2, the apparent catalytic efficiency (kcat ⁄ Km) of chymase was almost equivalent to that of elastase On the other hand, although the enzymatic properties of proteinase are similar to those of elastase, this proteinase showed lower activity toward TFPI 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 proteinases released from mast cells Effects of sulfated polysaccharides kDa 97 Mr TF PI Bu on ffe ly UF r H A LM W He H pa Ch ran su o De ndr lfa rm oit te Hy at in al an sul u f De ron sulf ate xt i a c a te ci n su d lfa te 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 polysaccharides affected the chymase-mediated degradation of TFPI As shown in Fig 6A, cleavage of TFPI by B 100 45 30 20.1 TFPI antigen (% of control) 66 80 60 40 20 14.4 60 120 180 Incubation time (min) Fig Effects of various polysaccharides on chymase-mediated proteolysis of TFPI (A) TFPI (3.5 lM) was incubated with chymase (7 nM) for 60 at 37 °C in the presence of each polysaccharide (100 lgỈmL)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 nM) was incubated with chymase (7 nM) in the presence or absence of heparin (100 lgỈmL)1) for up to 180 After various incubation times, the reaction was terminated 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 FEBS Journal 274 (2007) 3065–3077 ª 2007 The Authors Journal compilation ª 2007 FEBS 3071 Chymase-mediated proteolysis of TFPI T Hamuro et al We 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 indispensable for tryptase activity [15,16] Owing to the loss of heparin, tetrameric tryptase rapidly and irreversibly dissociates into inactive monomers [35] For example, polybrene and protamine convert tetrameric tryptase into monomers, resulting in the loss of tryptase 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 proposed 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, suggesting that the domain responsible for the inactivation 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 thrombin, 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 abolished the anticoagulant activity of TFPI The previously 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-macroglobulin [50], profilin [51], albumin [52], and connective tissue-activating peptide III [53] The cleaving sites of these natural substrates are summarized in Table Using a combinatorial peptide screening method, Raymond et al [52] demonstrated that chymase preferen3072 IIa, Pm MMPs T14 Xa, Pm MMPs L89-Q90 Y159-G160 F181-E182 Pm IIa Y268-E269 Fig 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 determined 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 glycosylation 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), plasmin (Pm), factor Xa (Xa), neutrophil elastase, proteinase 3, and chymase The thin arrows indicate the cleavage sites for each proteolytic enzyme MMP, Matrix metalloproteinase 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-Thr87Thr88-Leu89-Gln90 appears to be a ‘hot region’, because it contains cleavage sites for thrombin, plasmin, factor Xa, and elastase in addition to chymase Table Sites of hydrolysis of natural substrates of human chymase P1 site P4–P1_P1¢ Substrate Reference Tyr NEAY_V RRPY_I VVPY_G VGFY_E RETY_G VDNY_G KIAY_E IHPF_H FSPF_R KMLF_V TKPF_M RVGF_Y PSLF_E QFVL_T LKSL_S KTTL_Q PSVW_A Interleukin 1b Neurotensin Endothelin-1 a2-Macroglobulin Albumin TFPI TFPI Angiotensin I Bradykinin C1 inhibitor Kit ligand a2-Macroglobulin TFPI Procollagenase Procollagen 1a TFPI Profilin [44] [45] [48] [50] [52] This This [41] [42] [43] [47] [50] This [46] [49] This [51] Phe Leu Trp work work work work FEBS Journal 274 (2007) 3065–3077 ª 2007 The Authors Journal compilation ª 2007 FEBS T Hamuro et al (Fig 7) Presumably, this region has a distinct conformation and is exposed on the surface of the molecule, making it highly susceptible to attack by these proteinases 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-terminal portion of TFPI and instead contains a glycosylphosphatidylinositol 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 identical 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 interaction 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 heparin [55,56] Heparin, 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 angiotensin I [56,57], suggesting that the regulation of chymase 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 phosphatidylserine in vitro, we found that phosphatidylserine 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, unpublished data) It was previously reported that human intestinal mast cells produce and release tumor necrosis factor-a in response to Gram-negative bacteria such as Escherichia coli [59] Furthermore, tumor necrosis factor-a induces the expression of tissue factor on vascular endothelial cells [60] Clot formation resulting from tissue factor induction and chymase- Chymase-mediated proteolysis of TFPI mediated proteolysis of TFPI might be a protective function of intestinal mast cells against bacterial invasion 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-pNHCl where pNA is p-nitroanilide) and S-2222 [Bz-Ile-Glu(GlucOMe)-Gly-Arg-pNHCl] were obtained from Chromogenix AB (Stockholm, Sweden) Succinyl-Ala-Ala-Pro-Phe-pNA, methoxysuccinyl-Ala-AlaPro-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) HemolianceÒ human control plasma was obtained from Instrumentation Laboratory (Lexington, MA, USA) Dad thromboplastinỈC plus was purchased from Dade International Inc (Miami, FL, USA), and heparan 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 cathepsin G were obtained from Calbiochem (La Jolla, CA, USA) Human neutrophil proteinase 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 activator (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 hamster ovary (CHO) cells and purified from the culture medium as described previously [4] TFPI-C, which lacked the C-terminal basic region and ended at Lys249, was separated from FEBS Journal 274 (2007) 3065–3077 ª 2007 The Authors Journal compilation ª 2007 FEBS 3073 Chymase-mediated proteolysis of TFPI T Hamuro et al full-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 All inhibition experiments were performed in Tris ⁄ NaCl buffer (50 mm Tris ⁄ HCl containing 150 mm NaCl, pH 7.5) at 37 °C in 96-well microtiter plates TFPI was incubated with each proteinase and synthetic substrate, and the velocities of the initial reactions were measured with a THERMOmax microplate spectrometer (Molecular Devices, Sunnyvale, CA, USA) as the linear increase in A405 over The amount of residual active proteinase was determined by comparing the result to a standard curve constructed using known amounts of the proteinase Chymase (7 nm) was assayed using Suc-Ala-Ala-Pro-Phe-pNA (5 mm) Tryptase (14 nm) was assayed using H-d-Ile-ProArg-pNA (0.15 mm) in the presence of 0.5 lgỈmL)1 heparin Neutrophil-derived proteinase (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-ProVal-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 Tryptase (2 nm) in Tris ⁄ NaCl containing a low or high concentration of heparin was incubated with various concentrations of TFPI, TFPI-C, or antithrombin for 60 at 37 °C S-2288 was then added to a concentration of mm, and the initial rate of hydrolysis was measured Proteolysis of TFPI by proteinases Reactions containing nm chymase, tryptase, a-chymotrypsin, elastase, cathepsin G, or proteinase and 3.5 lm TFPI (molar ratio of : 500) were incubated for the designated times (15, 30, 60, 120, and 180 min) at 37 °C Experiments using chymase, a-chymotrypsin, elastase, cathepsin G, and proteinase were carried out in Tris ⁄ NaCl Reactions using tryptase were carried out in Tris ⁄ NaCl containing 500 lgỈmL)1 unfractionated heparin At the indicated time points, samples were taken from the reaction mixture and subjected to SDS ⁄ PAGE under reducing conditions Protein bands were visualized by staining with Coomassie Brilliant 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 3074 a solution of 0.1% trifluoroacetic acid for 10 TFPI fragments were eluted with a linear gradient of this solution containing 24–37% acetonitrile at a flow rate of 1.0 mLỈmin)1 Each peak fraction was pooled, lyophilized, 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 analyzed using an AccQTagTM system (Waters, Milford, MA, USA) according to the manufacturer’s protocol Automated Edman degradation was carried out using an Applied Biosystems 492 protein sequencer and standard methods MS analysis was performed using matrix-assisted laser desorption ⁄ ionization time-of-flight MS and a Voyager-DE 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 horseradish peroxidase-conjugated K270 (110 ngỈmL)1) were premixed and incubated in a K9-coated microtiter well for h at °C in a reaction volume of 200 lL Then, the plate was washed five times with Tris ⁄ NaCl ⁄ 0.05% Tween 20 buffer, and mixed with 200 lL 3,3¢,5,5¢-tetramethylbenzidine solution (2 mm EDTA containing 350 lgặmL)1 3,3Â,5,5Â-tetramethylbenzidine and 0.015% H2O2) After a 30-min incubation, development was terminated by the addition of 100 lL 0.5 m H2SO4, and the absorbance at 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 TFPI (70 nm) was incubated with chymase (7 nm) at 37 °C in Tris ⁄ NaCl After various incubation times, the reaction was terminated with 100 lm chymostatin, and a 15-lL aliquot of the sample was added to 135 lL human control plasma and mixed for at 37 °C Then, 150 lL thromboplastin [diluted : 40 in 50 mm Tris ⁄ HCl (pH 7.0) FEBS Journal 274 (2007) 3065–3077 ª 2007 The Authors Journal compilation ª 2007 FEBS T Hamuro et al containing 100 mm NaCl, 30 mm CaCl2, and 0.2% BSA] was added to the sample ⁄ plasma mixture, and the clotting time was measured using a Fibrintimer coagulometer (Dade Behring Ltd, Tokyo, Japan) The concentration of the diluted thromboplastin was adjusted to yield clotting times of about 30 s in the absence of TFPI The clotting time was related to the anticoagulant activity (expressed as the percentage of the control sample) using a reference curve constructed with known amounts of TFPI Measurement of the inhibitory activity of TFPI toward factor Xa The inhibition of factor Xa by TFPI was measured using S-2222 as a substrate First 20 lL mm substrate and 75 lL sample were mixed with 75 lL Tris ⁄ NaCl ⁄ 0.2% BSA and preincubated for at 37 °C The reaction was initiated by the addition of 20 lL 70 nm factor Xa, and the change in A405 was monitored by means of a THERMOmax microplate spectrometer The anti-(factor Xa) activity of TFPI (expressed as the percentage of the control sample) was calculated using a reference curve constructed with known amounts of TFPI Chymase-mediated proteolysis of TFPI Determination of Km and kcat values Chymase (7 nm) was mixed with various concentrations of TFPI (0.31, 0.68, 1.25, 2.5, 5, and 10 lm) in Tris ⁄ NaCl 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Kunitz-type-protease -inhibitor domains from tissue- factor- pathway inhibitor Eur J Biochem 235, 310–316 Wesselschmidt R, Likert K, Girard T, Wun TC & Broze GJ Jr (1992) Tissue factor pathway inhibitor: the... necrosis factor- a induces the expression of tissue factor on vascular endothelial cells [60] Clot formation resulting from tissue factor induction and chymase- Chymase-mediated proteolysis of... leukocyte elastase on tissue factor pathway inhibitor Blood 79, 1712–1719 Petersen LC, Bjorn SE & Nordfang O (1992) Effect of leukocyte proteinases on tissue factor pathway inhibitor Thromb Haemost