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thrombin clotting
Biochimica et Biophysica Acta, 1164 (1993) 215-218 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4838/93/$06.00
215
BBAPRO 34520
Inhibitory effects of enzymatic hydrolysates of collagen and collagen-related synthetic peptides on fibrinogen/thrombin clotting Susumu Maruyama a, Isao N o n a k a b and H i d e o l d T a n a k a a a Department of Biornolecular Engineering, National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Higashi, Tsukuba, lbaraki (Japan) and b Laboratory for Bio-Resource Sciences, Research and Development Center, Nippon Meat Packers, Inc., Midorigahara, Tsukuba, Ibaraki (Japan) (Received 13 February 1993)
Key words: Collagen; Collagenase; Collagen-related synthetic peptide; Fibrinogen; Thrombin; Synthetic peptide
Inhibitory effects of some enzymatic hydrolysates of collagen and collagen-related synthetic peptides on fibrinogen/thrombin clotting were investigated. The hydrolysate of porcine skin collagen with thermolysin or bacterial collagenase inhibited fibrinogen/thrombin clotting, but did not inhibit the activity of thrombin. Although the activity was not pronounced, the hydrolysate of collagen with such proteinases as trypsin and pepsin also inhibited the dotting. Gly-Pro-Arg, which is a known inhibitor of fibrinogen/thrombin clotting, was isolated from the bacterial collagenase hydrolysate of porcine collagen by HPLC. Collagen-related synthetic peptides such as Gly-Pro-Arg-Gly, Gly-Pro-Arg-Gly-Pro, Gly-Pro-Arg-Gly-Pro-Ala, Gly-Pro-Arg-GlyPro-Pro, and Gly-Pro-Arg-Pro-Pro also inhibited the clotting, but did not inhibit the activity of thrombin. The inhibitory activity of Gly-Pro-Arg-Gly-Pro-Pro and Gly-Pro-Arg-Pro-Pro was more marked than that of Gly-Pro-Arg. However, Gly-Pro-Lys, Gly-Ala-Arg, Gly-Pro-Hyp, Ala-Gly-Pro-Arg and Gly-Pro-Ala-Gly-Pro-Arg had no inhibitory effect on the clotting.
Introduction
Vertebrate fibrinogen is transformed into fibrin by the thrombin-catalyzed release of small peptides (fibrinopeptides A and B) from the N-termini of the a and B-chains. After release of these peptides, clotting of the fibrin occurs spontaneously to form a non-covalently-bonded gel. Laudano and Doolittle [1] reported that the N-terminal tripeptide of fibrin a-chain, GlyPro-Arg, inhibits fibrin polymerization. Further, they synthesized more potent inhibitors of fibrin polymerization such as Gly-Pro-Arg-Pro and Gly-Pro-ArgSar (Sar stands for sarcosine) [2]. Later, Kawasaki et al. [3,4] synthesized various amide analogues of Gly-ProArg, and found that the inhibitory activity of Gly-ProArg-Pro-Pro-NH 2 was 4.6-times that of Gly-Pro-ArgPro. Meanwhile, the interaction between platelets and collagen has been widely studied, and collagen-derived octapeptide, Lys-Pro-Gly-Glu-Pro-Gly-Pro-Lys, has
Correspondence to: S. Maruyama, Department of Biomolecular Engineering, National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, 1-1 Higashi, Tsukuba, Ibaraki 305, Japan.
been reported to inhibit the aggregation of platelets [5,61. We have been searching for bioactive peptides such as angiotensin-I-converting enzyme inhibitors [7,8] and brain prolyl endopeptidase inhibitors [9] in the enzymatic hydrolysates of various food proteins. In this process, we noticed that interstitial collagen has the sequence Gly-Pro-Arg. Collagens such as type I, II and III have triple-chain helical structures. Each chain has an approx. 1000 amino-acid-residues long repetitive sequence Gly-Xaa-Yaa, where Xaa is often Pro, and al-chain of rat or calf skin collagen has eight units of the sequence Gly-Pro-Arg [10]. Thus, we examined inhibitory effects of enzymatic hydrolysate of collagen and collagen-related synthetic peptides on fibrinogen/ thrombin clotting. Materials and Methods
Materials. Thrombin (from bovine plasma), fibrinogen (from bovine plasma), collagen (from bovine achilles tendon), collagenase (from Clostridium histolyticum), thermolysin, Gly-Pro-Arg-Pro, Gly-Pro-Hyp, and tosyl-Gly-Pro-Arg-p-nitroanilide were purchased from Sigma (St. Louis, MO, USA). Pepsin and trypsin
216 were purchased from Boehringer-Mannheim Biochemica (Mannheim, Germany). t-Butyloxycarbonyl amino acids, resins and other reagents for solid-phase peptide synthesis were purchased from Applied Biosystems (Foster City, CA, USA). Synthesis ofpeptides. The peptides used in this study were synthesized by the solid-phase method with a 430A Peptide Synthesizer (Applied Biosystems). Hydrogen fluoride was used to remove side-chain-protecting groups and cleave peptides from their solid supports. The synthesized peptides were purified by HPLC on a reverse-phase LiChrosorb RP-Select B column (250 × 10 mm, E. Merck) with a gradient of CH3CN (3.5 to 67%) in 0.1% trifluoroacetic acid (TFA). The purity and molecular formula of each synthetic peptide was checked by fast atom bombardment mass spectrometry and amino-acid analysis.
Hydrolysis of porcine skin collagen and isolation of Gly-Pro-Arg from the hydrolysate. Pure porcine skin collagen was purified by repetition of isoelectric precipitation after pepsin treatment of the delipidated porcine skin. Porcine skin collagen (3 g) was suspended in 120 ml of the buffer (50 mM Tris-HCl, 100 mM CaC12 (pH 7.0)) and hydrolyzed by the addition of 3.6-104 units of collagenase (from Clostridium histolyticum). After 18 h of incubation at 37°C under reciprocal shaking, the solution was boiled at 100°C for 15 min and cooled. The polypeptides were then removed by ultrafiltration with a YM-10 membrane (Amicon). The filtrate was then applied onto a reverse osmosis membrane R75A (Millipore), and the peptides, molecular weights of which were less than 400, were collected. The peptide solution was then desalted with a reverse osmosis membrane R15A (Millipore) and applied onto a CM MemSep 1010 cartridge (Millipore) for HPLC, which was eluted with a linear gradient of NaC1 from 0 to 0.5 M. The peak corresponding to the retention time of synthetic Gly-Pro-Arg was then collected, and the peptide was further purified on a reverse-phase LiChrosorb RP-Select B column (250 x 4 mm), which was eluted with a gradient of CH3CN (7 to 63%) in 0.1% TFA at a flow rate of 1 ml/min, while monitoring absorbance at 210 nm. The peak corresponding to the retention time of synthetic Gly-Pro-Arg was collected. The amino-acid composition of the collected peptide was analyzed with a Hitachi 835 aminoacid analyzer after hydrolysis with 4 M methanesulfonic acid at ll0°C for 24 h.
Hydrolysis of porcine skin collagen with various proteinases. Porcine skin collagen (2 g) was suspended in 150 ml of the buffer (50 mM Tris-HCl, 10 mM CaC12 (pH 7.2)) and hydrolyzed with 52 mg of thermolysin for 19 h at 37°C under reciprocal shaking. The solution was then boiled for 15 min, and polypeptides were removed with a YM-10 membrane. The filtrate was then applied onto an ultrafiltration membrane PCAC
(Millipore), and the peptides, molecular weights of which were less than 1000, were collected. The peptide solution was then desalted with a reverse osmosis membrane R15A. Hydrolysis with trypsin or pepsin was performed after thermal denaturation of the porcine collagen at 100°C for 10 min. 2 g of the denatured collagen were hydrolyzed with 400 mg of trypsin in 150 ml of the buffer (50 mM Tris-HC1, 10 mM CaC12 (pH 8.5)) or 220 mg of pepsin in 200 ml of diluted HC1 (pH 2.5) at 37°C for 22 h. Each hydrolysate was boiled, ultrafiltrated and desalted by the abovementioned method. Fibrinogen / thrombin clotting assays. These assays were carried out at 37°C by adding a solution of bovine thrombin to bovine fibrinogen solutions containing synthetic peptides or collagen hydrolysate additives and determining the clotting time. The final solution contained 0.83 mg/ml fibrinogen, 0.17 units/ml thrombin, 5 mM CaCI z, 130 mM NaC1 and 13 mM Tris-HCl buffer (pH 7.4). The IC50 value was determined by the method of Kawasaki et al. [4]. From the standard curve of thrombin amount against clotting time, the clotting time of each specimen was converted to the thrombin amount and then the % inhibition was calculated. Concentration of the synthetic peptide was then plotted against % inhibition and the IC50 value, concentration of each synthetic peptide exhibiting 50% inhibition, was estimated.
Assay of inhibitory activity of peptides on thrombin. The activity of various synthetic peptides on thrombin was assayed using the synthetic substrate tosyl-Gly-ProArg-p-nitroanilide. 85/zl of peptide solution was mixed with 100/zl of 200 mM Tris-HC1 buffer (pH 8.4) and 50 /zl of substrate solution (1 mM in distilled water). Reaction was started by adding 12.5 /1.1 of thrombin solution (1 U / m l in 20 mM Tris-HCl buffer (pH 7.4) containing 15 mM CaC12 and 260 mM NaCI). After 10 min of incubation at 37°C, reaction was stopped by adding 50/~1 of 60% acetic acid. 20/zl of the solution was then applied onto a column of/z Bondasphere C 8 (Waters) for HPLC, which was eluted with 53% CH3CN containing 0.1% TFA at a flow rate of 1 ml/min, while monitoring the absorbance at 410 nm. Thus, the peak area of liberated p-nitroaniline was measured. Results and Discussion
Fig. 1 shows the inhibition of fibrinogen/thrombin clotting by some enzymatic hydrolysates of porcine skin or bovine tendon collagen. In this figure, the concentration of the peptides in each enzymatic hydrolysate was estimated from the concentration of glycine in the 6 M HCI hydrolysate of each sample. Bacterial collagenase (from Clostridium histolyticum) hydrolysate of porcine skin collagen, from which the peptides larger than M r 400 were removed with a reverse osmosis
217 800
/
600
400
o
20(1
O /f/ 2b
o
/s
Concentration of Glycine (raM) Fig. 1. Inhibitory effects of some enzymatic hydrolysates of collagen on fibrinogen/thrombin clotting. The abscissa represents the concentration of glycine in the 6 M HC1 hydrolysate of each enzymatic hydrolysate. ( I ) , collagenase hydrolysate of BT (M r <400); (D), collagenase hydrolysate of BT (M r < 1000); (o), collagenase hydrolysate of PS (M r < 400); ( - ) , thermolysin hydrolysate of PS (M r < 1000); (/x ), trypsin hydrolysate of PS ( M r < 1000); ( • ), pepsin hydrolysate of PS ( M r < 1000); BT, bovine tendon collagen; PS, porcine skin collagen.
membrane, inhibited fibrinogen/thrombin clotting. Collagenase hydrolysate of bovine tendon collagen, from which the peptides larger than M r 400 or M r 1000 were removed with reverse osmosis membranes, also inhibited the clotting, and the inhibitory activity was more marked than that of porcine skin collagen. Since these inhibitory activities were considered to originate from the activity of Gly-Pro-Arg contained in the hydrolysate (inhibitory activity of synthetic Gly-ProArg is shown in Table I), we tried to isolate the peptide from the collagenase hydrolysate of porcine skin colla-
gen. Fig. 2 shows the purification of Gly-Pro-Arg from the sample, which was fractionated from the collagenase hydrolysate with a CM MemSep cartridge, by HPLC on a reverse-phase column. A peak corresponding to the retention time of synthetic Gly-Pro-Arg was collected. The amino-acid composition of this sample hydrolyzed with 4 M methanesulfonic acid was: Gly (1.0), Pro (0.7), Arg (0.8). These values closely resembled the theoretical values. Thus, the inhibitory activity of the bacterial collagenase hydrolysate of collagen may originate from Gly-Pro-Arg. The activity of the hydrolysate of porcine collagen with such proteinases as trypsin, pepsin and thermolysin, from which the peptides larger than M r 1000 were removed by ultrafiltration, was also investigated (Fig. 1). Thermolysin hydrolysate inhibited fibrinogen/ thrombin clotting at an extent similar to that of collagenase hydrolysate, however, pepsin or trypsin hydrolysate inhibited the clotting only slightly. Thermolysin hydrolyzes the peptide bonds at the amino sites of hydrophobic amino acids with bulky side chains. However, it has been reported that this enzyme is not able to hydrolyze the peptide bond if a proline residue is present at the P~ site [11]. Thus, some peptide other than Gly-Pro-Arg may inhibit the clotting. We then examined the inhibitory activity of some collagen-related synthetic peptides on fibrinogen/ thrombin clotting. As has been reported by Laudano and Doolittle, Gly-Pro-Arg and Gly-Pro-Arg-Pro strongly inhibited fibrinogen/thrombin clotting (Table
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TABLE I
t~ e~
Inhibitory effects of collagen-related synthetic peptides on fibrinogen / thrombin clotting Peptide
IC50 (/~M)
Gly-Pro-Arg a Gly-Pro-Arg-Pro a Gly-Pro-Arg-Gly Gly-Pro-Arg-Gly-Pro Gly-Pro-Arg-Gly-Pro-Ala Gly-Pro-Arg-Gly-Pro-Pro Gly-Pro-Arg-Pro-Pro Gly_Pro_Arg.Pro.Pro.NH 2 b
250 110 510 380 260 230 50 40
a Ref. 2. b Ref. 4.
tO el
,¢
""
0
4 8 ~~ ~ J
10
12°
T'm~e (ram) Fig. 2. Isolation of Gly-Pro-Arg from bacterial collagenase hydrolysate of porcine skin collagen. After fractionation with CM MemSep cartridge, the Gly-Pro-Arg containing fraction was chromatographed on a reverse-phase LiChrosorb RP-Select B HPLC column. The peak corresponding to the retention time of synthetic Gly-Pro-Arg was collected.
218 I). Gly-Pro-Arg containing peptides such as Gly-ProArg-Gly, Gly-Pro-Arg-Gly-Pro, Gly-Pro-Arg-Gly-ProAla, and GlyoPro-Arg-Gly-Pro-Pro inhibited the clotting (Table I), and the inhibitory activity of Gly-ProArg-Gly-Pro-Pro was somewhat more marked than that of Gly-Pro-Arg. We then synthesized Gly-Pro-Arg-ProPro, and the inhibitory activity of this peptide was most potent. This agrees with the report that the activity of the amide compound of this peptide, Gly-Pro-Arg-ProPro-NH2, is 4.6-times more potent than that of GlyPro-Arg-Pro [4]. Thus, extension of the peptide chain of Gly-Pro-Arg toward its C-terminus gave some active compounds. On the other hand, other collagen-related synthetic peptides such as Gly-Pro-Lys, Gly-Ala-Arg, Gly-ProHyp, and Gly-Pro-Hyp-Gly-Pro-Ala had no inhibitory activity on the clotting at 1 m M concentration (Table II). With regard to Gly-Pro-Lys, Gly-Pro-Lys-Pro has been reported not to bind to human fibrinogen [2]. Also, Ala-Gly-Pro-Arg and Gly-Pro-Ala-Gly-Pro-Arg had no inhibitory activity. Thus, extension of the peptide chain of Gly-Pro-Arg toward its N-terminus completely eliminated the inhibitory activity. TABLE II Effects of other synthetic peptides on fibrinogen / thrombin clotting
Peptide
Concentration Clottingtime (mM) (s) Control 80 Gly-Pro-Arg 0.8 800 (IC 5o= 250 ~M) Gly-Pro-Lys 1.0 95 Gly-Ala-Arg 1.0 84 Gly-Pro-Hyp 1.0 91 Gly-Pro-Hyp-Gly-Pro-Ala 1.0 83 Ala-Gly-Pro-Arg 1.0 88 Gly-Pro-Ala-Gly-Pro-Arg 5.0 83
Inhibitory activity of various synthetic peptides on thrombin was assayed using tosyl-Gly-Pro-Arg-p-nitroanilide as substrate. Neither these synthetic peptides, nor enzymatic hydrolysates of collagen at a concentration of 1 mM, or at a concentration which delays the clotting time for 120 s, inhibited the activity of thrombin. Thus, these synthetic peptides and collagen hydrolysates appeared to inhibit specifically the polymerization of fibrin monomers. Thus, the enzymatic hydrolysate of collagen may be usable as a source of bioactive peptides. In addition, some degradation fragments of collagen in the blood vessel, including collagen-derived octapeptide Lys-ProGly-Glu-Pro-Gly-Pro-Lys [5,6], may play some role in the regulation of coagulation of the blood in vivo.
References 1 Laudano, A.P. and Doolittle, R.F. (1978) Proc. Natl. Acad. Sci. USA 75, 3085-3089. 2 Laudano, A.P. and Doolittle, R.F. (1980) Biochemistry 19, 10131019. 3 Hirase, K., Tsuji, T., Miyano, M., Kawasaki, K. and Iwamoto, M. (1989) in Peptide Chemistry 1988 (Ueki, M., ed.), pp. 31-34, Protein Research Foundation, Osaka, Japan. 4 Kawasaki, K., Hirase, K., Tsuji, T., Miyano, M. and Iwamoto, M. (1989) Thromb. Res. 56, 757-762. 5 Bevers, E.M., Karniguian, A., Legrand, Y.J. and Zwaal, R.F.A. (1985) Thromb. Res. 37, 365-370. 6 Leger, D., Karniguian, A., Soria, J., Soria, C. and Legrand, Y. (1985) Haemostasis 15, 293-299. 7 Maruyama, S. and Suzuki, H. (1982) Agric. Biol. Chem. 46, 1393-1394. 8 Maruyama, S., Miyoshi, S., Kaneko, T. and Tanaka, H. (1989) Agric. Biol. Chem. 53, 1077-1081. 9 Maruyama, S., Miyoshi, S., Osa, T. and Tanaka, H. (1992) J. Ferment. Bioeng. 74, 145-148. 10 Hulmes, D.J.S., Miller, A., Parry, D.A.D., Piez, K.A. and Woodhead-Galloway, J. (1973) J. Mol. Biol. 79, 137-148. 11 Matsubara, H., Singer, A. and Sasaki, R.M. (1969) Biochem. Biophys. Res. Commun. 34, 719-724.
215
BBAPRO 34520
Inhibitory effects of enzymatic hydrolysates of collagen and collagen-related synthetic peptides on fibrinogen/thrombin clotting Susumu Maruyama a, Isao N o n a k a b and H i d e o l d T a n a k a a a Department of Biornolecular Engineering, National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Higashi, Tsukuba, lbaraki (Japan) and b Laboratory for Bio-Resource Sciences, Research and Development Center, Nippon Meat Packers, Inc., Midorigahara, Tsukuba, Ibaraki (Japan) (Received 13 February 1993)
Key words: Collagen; Collagenase; Collagen-related synthetic peptide; Fibrinogen; Thrombin; Synthetic peptide
Inhibitory effects of some enzymatic hydrolysates of collagen and collagen-related synthetic peptides on fibrinogen/thrombin clotting were investigated. The hydrolysate of porcine skin collagen with thermolysin or bacterial collagenase inhibited fibrinogen/thrombin clotting, but did not inhibit the activity of thrombin. Although the activity was not pronounced, the hydrolysate of collagen with such proteinases as trypsin and pepsin also inhibited the dotting. Gly-Pro-Arg, which is a known inhibitor of fibrinogen/thrombin clotting, was isolated from the bacterial collagenase hydrolysate of porcine collagen by HPLC. Collagen-related synthetic peptides such as Gly-Pro-Arg-Gly, Gly-Pro-Arg-Gly-Pro, Gly-Pro-Arg-Gly-Pro-Ala, Gly-Pro-Arg-GlyPro-Pro, and Gly-Pro-Arg-Pro-Pro also inhibited the clotting, but did not inhibit the activity of thrombin. The inhibitory activity of Gly-Pro-Arg-Gly-Pro-Pro and Gly-Pro-Arg-Pro-Pro was more marked than that of Gly-Pro-Arg. However, Gly-Pro-Lys, Gly-Ala-Arg, Gly-Pro-Hyp, Ala-Gly-Pro-Arg and Gly-Pro-Ala-Gly-Pro-Arg had no inhibitory effect on the clotting.
Introduction
Vertebrate fibrinogen is transformed into fibrin by the thrombin-catalyzed release of small peptides (fibrinopeptides A and B) from the N-termini of the a and B-chains. After release of these peptides, clotting of the fibrin occurs spontaneously to form a non-covalently-bonded gel. Laudano and Doolittle [1] reported that the N-terminal tripeptide of fibrin a-chain, GlyPro-Arg, inhibits fibrin polymerization. Further, they synthesized more potent inhibitors of fibrin polymerization such as Gly-Pro-Arg-Pro and Gly-Pro-ArgSar (Sar stands for sarcosine) [2]. Later, Kawasaki et al. [3,4] synthesized various amide analogues of Gly-ProArg, and found that the inhibitory activity of Gly-ProArg-Pro-Pro-NH 2 was 4.6-times that of Gly-Pro-ArgPro. Meanwhile, the interaction between platelets and collagen has been widely studied, and collagen-derived octapeptide, Lys-Pro-Gly-Glu-Pro-Gly-Pro-Lys, has
Correspondence to: S. Maruyama, Department of Biomolecular Engineering, National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, 1-1 Higashi, Tsukuba, Ibaraki 305, Japan.
been reported to inhibit the aggregation of platelets [5,61. We have been searching for bioactive peptides such as angiotensin-I-converting enzyme inhibitors [7,8] and brain prolyl endopeptidase inhibitors [9] in the enzymatic hydrolysates of various food proteins. In this process, we noticed that interstitial collagen has the sequence Gly-Pro-Arg. Collagens such as type I, II and III have triple-chain helical structures. Each chain has an approx. 1000 amino-acid-residues long repetitive sequence Gly-Xaa-Yaa, where Xaa is often Pro, and al-chain of rat or calf skin collagen has eight units of the sequence Gly-Pro-Arg [10]. Thus, we examined inhibitory effects of enzymatic hydrolysate of collagen and collagen-related synthetic peptides on fibrinogen/ thrombin clotting. Materials and Methods
Materials. Thrombin (from bovine plasma), fibrinogen (from bovine plasma), collagen (from bovine achilles tendon), collagenase (from Clostridium histolyticum), thermolysin, Gly-Pro-Arg-Pro, Gly-Pro-Hyp, and tosyl-Gly-Pro-Arg-p-nitroanilide were purchased from Sigma (St. Louis, MO, USA). Pepsin and trypsin
216 were purchased from Boehringer-Mannheim Biochemica (Mannheim, Germany). t-Butyloxycarbonyl amino acids, resins and other reagents for solid-phase peptide synthesis were purchased from Applied Biosystems (Foster City, CA, USA). Synthesis ofpeptides. The peptides used in this study were synthesized by the solid-phase method with a 430A Peptide Synthesizer (Applied Biosystems). Hydrogen fluoride was used to remove side-chain-protecting groups and cleave peptides from their solid supports. The synthesized peptides were purified by HPLC on a reverse-phase LiChrosorb RP-Select B column (250 × 10 mm, E. Merck) with a gradient of CH3CN (3.5 to 67%) in 0.1% trifluoroacetic acid (TFA). The purity and molecular formula of each synthetic peptide was checked by fast atom bombardment mass spectrometry and amino-acid analysis.
Hydrolysis of porcine skin collagen and isolation of Gly-Pro-Arg from the hydrolysate. Pure porcine skin collagen was purified by repetition of isoelectric precipitation after pepsin treatment of the delipidated porcine skin. Porcine skin collagen (3 g) was suspended in 120 ml of the buffer (50 mM Tris-HCl, 100 mM CaC12 (pH 7.0)) and hydrolyzed by the addition of 3.6-104 units of collagenase (from Clostridium histolyticum). After 18 h of incubation at 37°C under reciprocal shaking, the solution was boiled at 100°C for 15 min and cooled. The polypeptides were then removed by ultrafiltration with a YM-10 membrane (Amicon). The filtrate was then applied onto a reverse osmosis membrane R75A (Millipore), and the peptides, molecular weights of which were less than 400, were collected. The peptide solution was then desalted with a reverse osmosis membrane R15A (Millipore) and applied onto a CM MemSep 1010 cartridge (Millipore) for HPLC, which was eluted with a linear gradient of NaC1 from 0 to 0.5 M. The peak corresponding to the retention time of synthetic Gly-Pro-Arg was then collected, and the peptide was further purified on a reverse-phase LiChrosorb RP-Select B column (250 x 4 mm), which was eluted with a gradient of CH3CN (7 to 63%) in 0.1% TFA at a flow rate of 1 ml/min, while monitoring absorbance at 210 nm. The peak corresponding to the retention time of synthetic Gly-Pro-Arg was collected. The amino-acid composition of the collected peptide was analyzed with a Hitachi 835 aminoacid analyzer after hydrolysis with 4 M methanesulfonic acid at ll0°C for 24 h.
Hydrolysis of porcine skin collagen with various proteinases. Porcine skin collagen (2 g) was suspended in 150 ml of the buffer (50 mM Tris-HCl, 10 mM CaC12 (pH 7.2)) and hydrolyzed with 52 mg of thermolysin for 19 h at 37°C under reciprocal shaking. The solution was then boiled for 15 min, and polypeptides were removed with a YM-10 membrane. The filtrate was then applied onto an ultrafiltration membrane PCAC
(Millipore), and the peptides, molecular weights of which were less than 1000, were collected. The peptide solution was then desalted with a reverse osmosis membrane R15A. Hydrolysis with trypsin or pepsin was performed after thermal denaturation of the porcine collagen at 100°C for 10 min. 2 g of the denatured collagen were hydrolyzed with 400 mg of trypsin in 150 ml of the buffer (50 mM Tris-HC1, 10 mM CaC12 (pH 8.5)) or 220 mg of pepsin in 200 ml of diluted HC1 (pH 2.5) at 37°C for 22 h. Each hydrolysate was boiled, ultrafiltrated and desalted by the abovementioned method. Fibrinogen / thrombin clotting assays. These assays were carried out at 37°C by adding a solution of bovine thrombin to bovine fibrinogen solutions containing synthetic peptides or collagen hydrolysate additives and determining the clotting time. The final solution contained 0.83 mg/ml fibrinogen, 0.17 units/ml thrombin, 5 mM CaCI z, 130 mM NaC1 and 13 mM Tris-HCl buffer (pH 7.4). The IC50 value was determined by the method of Kawasaki et al. [4]. From the standard curve of thrombin amount against clotting time, the clotting time of each specimen was converted to the thrombin amount and then the % inhibition was calculated. Concentration of the synthetic peptide was then plotted against % inhibition and the IC50 value, concentration of each synthetic peptide exhibiting 50% inhibition, was estimated.
Assay of inhibitory activity of peptides on thrombin. The activity of various synthetic peptides on thrombin was assayed using the synthetic substrate tosyl-Gly-ProArg-p-nitroanilide. 85/zl of peptide solution was mixed with 100/zl of 200 mM Tris-HC1 buffer (pH 8.4) and 50 /zl of substrate solution (1 mM in distilled water). Reaction was started by adding 12.5 /1.1 of thrombin solution (1 U / m l in 20 mM Tris-HCl buffer (pH 7.4) containing 15 mM CaC12 and 260 mM NaCI). After 10 min of incubation at 37°C, reaction was stopped by adding 50/~1 of 60% acetic acid. 20/zl of the solution was then applied onto a column of/z Bondasphere C 8 (Waters) for HPLC, which was eluted with 53% CH3CN containing 0.1% TFA at a flow rate of 1 ml/min, while monitoring the absorbance at 410 nm. Thus, the peak area of liberated p-nitroaniline was measured. Results and Discussion
Fig. 1 shows the inhibition of fibrinogen/thrombin clotting by some enzymatic hydrolysates of porcine skin or bovine tendon collagen. In this figure, the concentration of the peptides in each enzymatic hydrolysate was estimated from the concentration of glycine in the 6 M HCI hydrolysate of each sample. Bacterial collagenase (from Clostridium histolyticum) hydrolysate of porcine skin collagen, from which the peptides larger than M r 400 were removed with a reverse osmosis
217 800
/
600
400
o
20(1
O /f/ 2b
o
/s
Concentration of Glycine (raM) Fig. 1. Inhibitory effects of some enzymatic hydrolysates of collagen on fibrinogen/thrombin clotting. The abscissa represents the concentration of glycine in the 6 M HC1 hydrolysate of each enzymatic hydrolysate. ( I ) , collagenase hydrolysate of BT (M r <400); (D), collagenase hydrolysate of BT (M r < 1000); (o), collagenase hydrolysate of PS (M r < 400); ( - ) , thermolysin hydrolysate of PS (M r < 1000); (/x ), trypsin hydrolysate of PS ( M r < 1000); ( • ), pepsin hydrolysate of PS ( M r < 1000); BT, bovine tendon collagen; PS, porcine skin collagen.
membrane, inhibited fibrinogen/thrombin clotting. Collagenase hydrolysate of bovine tendon collagen, from which the peptides larger than M r 400 or M r 1000 were removed with reverse osmosis membranes, also inhibited the clotting, and the inhibitory activity was more marked than that of porcine skin collagen. Since these inhibitory activities were considered to originate from the activity of Gly-Pro-Arg contained in the hydrolysate (inhibitory activity of synthetic Gly-ProArg is shown in Table I), we tried to isolate the peptide from the collagenase hydrolysate of porcine skin colla-
gen. Fig. 2 shows the purification of Gly-Pro-Arg from the sample, which was fractionated from the collagenase hydrolysate with a CM MemSep cartridge, by HPLC on a reverse-phase column. A peak corresponding to the retention time of synthetic Gly-Pro-Arg was collected. The amino-acid composition of this sample hydrolyzed with 4 M methanesulfonic acid was: Gly (1.0), Pro (0.7), Arg (0.8). These values closely resembled the theoretical values. Thus, the inhibitory activity of the bacterial collagenase hydrolysate of collagen may originate from Gly-Pro-Arg. The activity of the hydrolysate of porcine collagen with such proteinases as trypsin, pepsin and thermolysin, from which the peptides larger than M r 1000 were removed by ultrafiltration, was also investigated (Fig. 1). Thermolysin hydrolysate inhibited fibrinogen/ thrombin clotting at an extent similar to that of collagenase hydrolysate, however, pepsin or trypsin hydrolysate inhibited the clotting only slightly. Thermolysin hydrolyzes the peptide bonds at the amino sites of hydrophobic amino acids with bulky side chains. However, it has been reported that this enzyme is not able to hydrolyze the peptide bond if a proline residue is present at the P~ site [11]. Thus, some peptide other than Gly-Pro-Arg may inhibit the clotting. We then examined the inhibitory activity of some collagen-related synthetic peptides on fibrinogen/ thrombin clotting. As has been reported by Laudano and Doolittle, Gly-Pro-Arg and Gly-Pro-Arg-Pro strongly inhibited fibrinogen/thrombin clotting (Table
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TABLE I
t~ e~
Inhibitory effects of collagen-related synthetic peptides on fibrinogen / thrombin clotting Peptide
IC50 (/~M)
Gly-Pro-Arg a Gly-Pro-Arg-Pro a Gly-Pro-Arg-Gly Gly-Pro-Arg-Gly-Pro Gly-Pro-Arg-Gly-Pro-Ala Gly-Pro-Arg-Gly-Pro-Pro Gly-Pro-Arg-Pro-Pro Gly_Pro_Arg.Pro.Pro.NH 2 b
250 110 510 380 260 230 50 40
a Ref. 2. b Ref. 4.
tO el
,¢
""
0
4 8 ~~ ~ J
10
12°
T'm~e (ram) Fig. 2. Isolation of Gly-Pro-Arg from bacterial collagenase hydrolysate of porcine skin collagen. After fractionation with CM MemSep cartridge, the Gly-Pro-Arg containing fraction was chromatographed on a reverse-phase LiChrosorb RP-Select B HPLC column. The peak corresponding to the retention time of synthetic Gly-Pro-Arg was collected.
218 I). Gly-Pro-Arg containing peptides such as Gly-ProArg-Gly, Gly-Pro-Arg-Gly-Pro, Gly-Pro-Arg-Gly-ProAla, and GlyoPro-Arg-Gly-Pro-Pro inhibited the clotting (Table I), and the inhibitory activity of Gly-ProArg-Gly-Pro-Pro was somewhat more marked than that of Gly-Pro-Arg. We then synthesized Gly-Pro-Arg-ProPro, and the inhibitory activity of this peptide was most potent. This agrees with the report that the activity of the amide compound of this peptide, Gly-Pro-Arg-ProPro-NH2, is 4.6-times more potent than that of GlyPro-Arg-Pro [4]. Thus, extension of the peptide chain of Gly-Pro-Arg toward its C-terminus gave some active compounds. On the other hand, other collagen-related synthetic peptides such as Gly-Pro-Lys, Gly-Ala-Arg, Gly-ProHyp, and Gly-Pro-Hyp-Gly-Pro-Ala had no inhibitory activity on the clotting at 1 m M concentration (Table II). With regard to Gly-Pro-Lys, Gly-Pro-Lys-Pro has been reported not to bind to human fibrinogen [2]. Also, Ala-Gly-Pro-Arg and Gly-Pro-Ala-Gly-Pro-Arg had no inhibitory activity. Thus, extension of the peptide chain of Gly-Pro-Arg toward its N-terminus completely eliminated the inhibitory activity. TABLE II Effects of other synthetic peptides on fibrinogen / thrombin clotting
Peptide
Concentration Clottingtime (mM) (s) Control 80 Gly-Pro-Arg 0.8 800 (IC 5o= 250 ~M) Gly-Pro-Lys 1.0 95 Gly-Ala-Arg 1.0 84 Gly-Pro-Hyp 1.0 91 Gly-Pro-Hyp-Gly-Pro-Ala 1.0 83 Ala-Gly-Pro-Arg 1.0 88 Gly-Pro-Ala-Gly-Pro-Arg 5.0 83
Inhibitory activity of various synthetic peptides on thrombin was assayed using tosyl-Gly-Pro-Arg-p-nitroanilide as substrate. Neither these synthetic peptides, nor enzymatic hydrolysates of collagen at a concentration of 1 mM, or at a concentration which delays the clotting time for 120 s, inhibited the activity of thrombin. Thus, these synthetic peptides and collagen hydrolysates appeared to inhibit specifically the polymerization of fibrin monomers. Thus, the enzymatic hydrolysate of collagen may be usable as a source of bioactive peptides. In addition, some degradation fragments of collagen in the blood vessel, including collagen-derived octapeptide Lys-ProGly-Glu-Pro-Gly-Pro-Lys [5,6], may play some role in the regulation of coagulation of the blood in vivo.
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