ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 279, No. 1, May 15,~~. 146-150,199O
Inhibition of Platelet Aggregation by S-(1,2-Dicarboxyethyl)glutathione, Intrinsic Tripeptide in Liver, Heart, and Lens Seiji Tsuboi,* Masayo Ohnaka,* Shinji Ohmori,*” Takahiro Kazumi Ogata,t Toshifumi Itano,$ and Osamu Hatase$
Sakaue,?
*Faculty of Pharmaceutical Sciences, Okayama University, Tsushima-Naka-l-l-l, Okayama 700, Japan; tSenju Pharmaceutical Co. Ltd., Ohshika-Sakuragaoka l-1, Itami 664, Japan; and SDepartment of Physiology, Kagawa Medical School, 1750-l Ikenobe, Miki-cho, Kagawa 761-07, Japan
Received November
27,1989
S-( 1,2-Dicarboxyethyl)glutathione (DCE-GS) found in animal tissues or baker’s yeast showed strong inhibitory effects on blood coagulation and platelet aggregation. The inhibitory effect of blood coagulation was almost the same as those of EDTA, oxalate, and citrate. DCE-GS did not show chelating activity. As for ADPor thrombin-induced platelet aggregations, DCE-GS exerted a potent effect on the secondary aggregation, while it was less active in the primary aggregation. DCE-GS gave a distinct lag period in the time course of the secondary aggregation induced by collagen and inhibited most strongly the aggregation induced by arachidonic acid compared with those elicited by ADP, thrombin, and collagen. The peptide, however, did not inhibit the platelet aggregation induced by 12-O-tetradecanoylphorbol13-acetate. Although both DCE-GS and EDTA inhibited the platelet aggregation which was triggered by ADP, their inhibitory manners were entirely different. D 1990 Academic Press, Inc.
In 1963 S-(1,2-dicarboxyethyl)glutathione (DCEGS)’ was isolated from calf lens, and its chemical structure was determined by comparison to the synthetic compound (Fig. 1) (1). In a previous paper we reported the assay system of this peptide by HPLC after reaction ’ To whom correspondence should be addressed. 2 Abbreviations used: DCE-GS, S-(1,2dicarboxyethyl)glutathione; TPA, 12-0-tetradecanoylphorbol-13.acetate; DCE-Cys, S-(1,2-dicarboxyethyl).L-cysteine; CP-GS, S-(2.carboxypropyl)glutathione; GDCE-Cys, y-glutamyl-S-(1,2-dicarboxyethyl)-L-cysteine; DCE-CG, S(1,2-dicarboxyethyl)-L-Cys-Gly; OP, ophthalmic acid; NOR, norophthalmic acid, PRP, platelet rich plasma; PPP, platelet poor plasma; TBS, Tris-buffered saline. 146
with 2,4-dinitrofluorobenzene (2). By this method we studied DCE-GS distribution in various tissues of rats and subcellular fractions of rat liver. DCE-GS was found in especially high concentrations in lens and in the cytosolic fraction of rat liver (3). We also reported that DCEGS was synthesized enzymatically using GSH and L-malate and we detected the highest activity of DCE-GS synthesizing enzyme in the cytosolic fractions of rat livers. The enzyme was purified from the rat liver cytosolic fraction (4). However, the physiological significance and biochemical role of this peptide are still unknown. We assumed at first that DCE-GS may act as a chelater of Ca2+, since the peptide has four carboxy groups and one amino group. In order to demonstrate the chelating activity, we examined the inhibition of blood coagulation using DCE-GS. As is expected, it was found that DCEGS acts as an anticoagulant and its activity was not less potent than those of EDTA, oxalate, and citrate. However, contrary to our expectations, it turned out that the peptide provides no chelating activity for Ca2+ ion after the chemical investigation. Consequently, it was thought that the inhibition of the blood coagulation by DCE-GS might be caused by inhibition of platelet aggregation. In this communication, we will show the inhibitory effect of DCE-GS in the platelet aggregation. MATERIALS
AND
METHODS
Chemicals. ADP, 12.O-tetradecanoylphorbol-13.acetate (TPA), arachidonic acid, thrombin from human plasma (Sigma Co., St. Louis), and collagen (Chrono-Log Co., Havertown, PA) were used as plateletaggregating agents. S-(1,2-Dicarboxyethyl)-L-cysteine (DCE-Cys) and S-(2-carboxypropyl)glutathione (CP-GS) were prepared in our laboratory (2, 5). Glutathione, reduced form, was kindly supplied by Yamanouchi Pharmaceutical Co., Ltd. (Tokyo, Japan). DCE-GS, y-glutamyl-S-(1,2-dicarboxyethyl)-L-cysteine (GDCE-Cys), S-(1,2dicarboxyethyl)-L-Cys-Gly (DCE-CG), S-(l&diethyloxycarbonyl0003.9861/90 $3.00 Copyright 8 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
INHIBITION
OF PLATELET
HOOC-CH-CH2-CH2-CONH-FH-CONH-CH2-COOH AH,
CH I 2 S-CH-COOH iH2-COOH
FIG.
1.
Structure
of S-(1,2-dicarboxyethyI)glutathione
ethyl)-glutathione, ophthalmic acid (OP, y-Glu-n-aminobut-Gly), norophthalmic acid (NOR, y-Glu-Ala-Gly), y-Glu-GluOH, y-Glu-AspNH,-Gly, r-Glu-Thr-Gly, y-Glu-Asp-Gly, y-Glu-Ser-Gly, Asp-Gly, Ser-Gly, and Ala-Gly were supplied by Senju Pharmaceutical Co., Ltd. (Osaka, Japan). EDTA disodium salt and quin 2 were purchased from Dojin Laboratories (Kumamoto, Japan), trizma base was from Sigma Co. and other reagents were from Wako Pure Chemical Industries (Osaka, Japan). Blood coagulation time Determination of blood coagulation time. was determined using blood of rabbit by the Lee-White method (6). Uetrrmination ofplatelet uggre.gation. Platelet rich plasma (PRP) and platelet poor plasma (PPP) were prepared from healthy male donors in the usual fashion. Platelet, aggregation assay was performed in Aggrecorder II (Kyoto Daiichi Chemical Co., Ltd., Kyoto) at 37°C by the usual method which will be briefly described below (7). The assay mixture consisted of PRP (2-3 X lo”), 1 Fmol CaCl,, 8 pmol Tris-HCl (pH 7.4), 60 bmol NaCl (TBS), and 0.05 to 0.5 pmol of sample to be tested in a total volume of 0.4 ml. The mixture was added to a siliconlined glass cuvette (6 X 41.7 mm) preincubated at 37°C. CaCl, or the sample to be tested was dissolved in TBS and the solution was neutralized with solid NaHCO,. A small magnetic stirring bar was rotated in the glass cuvette at about 1000 rpm. After incubation for 2 min, an aggregation trigger such as ADP was added to the cuvette and the aggregation was measured by the Aggrecorder II. The concentrations of ADP, collagen, thrombin, arachidonic acid, and TPA added as the trigger for aggregation were 3.13 to 12.5 pM, 1.% to 3.75 pg/ml, 0.2 to 0.625 U/ml, 0.615 to 1.23 mM, and 23.4 pM in TBS, respectively. The aggregation rate was measured by following the change in turbidity at 650 nm. The turbidity of PRP was set at 0% and that of PPP at 100% on the Aggrecorder. In order to calculate the percentage inhibition of aggregation, the turbidity of PRP in the presence of coagulant and inhibitor was taken as 100% and that with coagulant but without inhibitor as 0°C. The Determination of the binding constant for DC&-GS to Ca”. apparent constant for DCE-GS to Ca2+ was determined using quin 2 (8). The assay mixture consisted of 7.5 pmol CaCl,, 60 Fmol Tris-HCl (pH 7.4). 450 pmol NaCI, and 0.375 to 7.5 pmol of DCE-GS or 7.5 pmol of EDTA to be tested in a total volume of 2 ml and finally 1 ml of 60 PM quin 2 was added to the assay mixture. Fluorescence at 339 nm excitation and 492 nm emission were measured with Shimadzu Fluorescence Spectromonitor RF-500 LC. As a separate experiment the apparent binding constant of DCE-GS (250 and 500 fiM) to Ca” was determined by the method of Harafuji and Ogawa (9). Raman spectra were measured for investigating the binding of DCE-GS (0.1 M) to Ca’+. Effect of Cc? on inhibition of platekt aggregation induced by The platelet aggregation induced by ADP was assayed in the ADP. same manner as described above. In order to examine the effects of extracellular concentrations of Ca2+ on inhibition of platelet aggregation induced hy ADP, two kinds of TBS solutions containing Ca’+ were used, of which the final concentrations of CaCl, were 2.5 and 6.85
mM.
RESULTS
Effect of DCE-GS as an anticoagulant of blood. I shows the effects of DCE-GS as an anticoagulant,
Table com-
AGGREGATION
147
pared with other compounds. When DCE-GS was added to the rabbit blood in a final concentration of 2 mM, blood remained uncoagulated for over 10 min. The inhibition of blood coagulation by DCE-GS was not less potent than those of EDTA, oxalate, and citrate, which were used usually as anticoagulants. CP-GS, which is a natural product and an analog of DCE-GS, and DCE-Cys, which is a constituent of DCE-GS, had no anticoagulant effect. GSH also has no anticoagulant activity. Effect of DCE-GS on platelet aggregation induced by ADP, collagen, thrombin, arachidonic acid, and TPA. Figure 2a illustrates the inhibitory effects of DCE-GS on the ADP-induced platelet aggregation. When ADP was added to PRP, the platelet aggregation was immediately initiated and reached the maximum level after 1 min. It continued for 5 min at the shortest. If platelets were preincubated for 2 min by 0.125 to 1.25 mM DCE-GS, the initial velocity of the primary aggregation was slightly inhibited with 1.25 mM DCE-GS, and the primary aggregation percentage was inhibited dose-dependently. Similarly, the inhibitory effect on the secondary aggregation and disaggregation was observed by increasing the concentration of DCE-GS. We observed the more intensive inhibitory effect of DCE-GS on thrombin-induced platelet aggregation (Fig. 2b). Figures 2c and 2d illustrate the inhibitory effects of DCE-GS on collagen- and arachidonic acid-induced platelet aggregation. It has been reported that collagen and arachidonic acid were aggregating agents which induced only the secondary aggregation without the primary one (10). The evidence of the inhibitory effects of DCE-GS on the secondary aggregation in Figs. 2c and 2d fortified their results. When collagen was added to PRP, platelet aggregation took place after 1.5 to 2 min. DCE-GS inhibited this aggregation dose-dependently as shown in Fig. 2c. As illustrated in Fig. 2d when arachidonic acid was added to PRP, platelet aggregation was brought about after approximately 1.5 min and reached the maximum level after 3 min. This aggregation was completely inhibited with 0.125 mM of DCE-GS. As can be seen in Figs. 2a and 2b, the primary aggregation immediately occurred after addition of ADP and thrombin and continued for approximately 1 min. On the other hand, in Figs. 2c and 2d the secondary aggregation, which has a lag period of approximately 1 min, reached the plateau after 3 min. At a concentration of 1.25 mM, DCE-GS inhibited the primary aggregation induced by ADP and thrombin up to 50% and the secondary one was inhibited up to 90% (Figs. 2a and 2b). This evidence shows that the inhibition of DCE-GS was more effective on the secondary aggregation than the primary one. It is very interesting that GSH, CP-GS, GDCE-Cys, DCECG, S-(1,2-diethyloxycarbonylethyl)glutathione, DCECys, OP, NOR, y-Glu-GluOH, y-Glu-AspNHz-Gly, y-
148
TSUBOI
ET AL.
TABLE
I
Effect of Various Samples on Blood Coagulation Sample concentration Sample
50
DCE-GS GSH CP-GS DCE-Cys EDTA Oxalate Citrate
>lO
30
20
10
I
5 7 >lO
5 6.5 7
>lO
>lO
Time
(mM)
5
3
2
1
>lO 5 5 4 >10 >lO 6
>lO 4.5 5 4.5 >lO >lO 4.5
>lO 4.5 4.5 4 7 I 5
7 4.5 4.5 4.5 5 5 4.5
Note. Blood coagulation time (min) was determined using rabbit blood for 10 min. If blood coagulation reported as >lO. Each value is the mean of three to five experiments.
Glu-Thr-Gly, r-Glu-Asp-Gly, y-Glu-Ser-Gly, Asp-Gly, Ser-Gly, and Ala-Gly showed no inhibitory effect. From these results, an essential chemical structure required for the aggregation is two free carboxyl groups of succinic residues in the peptide. It is worth noting that a methylene group of succinyl residue, attached to S was in the racemic form (2). We examined the inhibitory effect of DCE-GS on the platelet aggregation induced by TPA, which triggered the platelet aggregation through different mechanisms from those of ADP and collagen. Figure 2e illustrates the effect of DCE-GS on TPA-induced platelet aggregation.
z
0
TPA induced the platelet aggregation very slowly and its aggregation reached the maximum level after 15 min. The lag time of aggregation in TPA was longer than that of ADP and collagen. DCE-GS showed very slight inhibitory effects on TPA-induced platelet aggregation and did not show any dose-dependency. From these data, we concluded that DCE-GS did not inhibit TPA-induced platelet aggregation. Figure 3 illustrates the dose-response curves for the inhibitory effect of DCE-GS on the secondary aggregation induced by several aggregation agents. DCE-GS had the strong inhibitory effect on the platelet aggregations
;; IOOU
; 1001. s
did not occur until 10 min, it was
12
3
4
5
0(bl
'0 1.25 mM 0.625 Ml
E :
; 'E
012
345
o 'd)
0.125 mM ,093s mfl
;
bs
0.125
n-tl
.a 50 .
OMl ;I
0.0625 :
loo0
12
3 Time
(min)
4
5
CI'
mM
OmM
100 I 012
345 Time
(mln)
0
5
10 Time
15
20
25
(min)
FIG. 2. Effects of adding increasing amounts of DCE-GS on (a) ADP, (b) thrombin, (c) collagen, (d) arachidonic acid, and (e) TPA-induced aggregation of PRP. PRP and 2.5 mM CaCl, in TBS were preincubation at 37°C in glass cuvettes for 2 min with varying amounts (0 to 1.25 mM) of DCE-GS. At the end of 2 min incubation, ADP (6.25 FM), thrombin (0.2 U/ml), collagen (3.75 ag/ml), arachidonic acid (0.625 mM), or TPA (23.4 pM) was added to each incubation mixture in order to initiate the aggregation, which was autographically recorded for 5 min except for the TPA experiment which was recorded for 25 min. Control refers to platelet aggregation induced by each aggregating agent in the absence of DCE-GS.
INHIBITION
OF PLATELET
AGGREGATION
iLli .\:-I
0,Ol
1
0.1 S-(1,2-Dicorboxyethyl)slutothione ( mfl I
FIG. 3. Dose-response curve for inhibitory eflect of DCE-GS on platelet aggregation induced by ADP (o), thrombin (A), collagen (a), and arachidonic acid (0). PRP and 2.5 mM CaC12 in TBS were incubated at 37°C for 2 min with DCE-GS (0 to 1.25 mM). ADP (3.13 to 12.5 PM), thrombin (0.2 to 0.625 U/ml), collagen (1.25 to 3.75 pg/ml), or arachidonic acid (0.615 to 1.23 mM) was added and the aggregation was observed for 5 min. Data revealed the inhibition percentage of the platelet aggregation by DCE-GS. Each value is the mean * SD ofthree experiments.
induced by arachidonic acid, thrombin, ADP, and collagen, in that order. Determination of the binding constant for DCE-GS to Ca2+. As shown in Fig. 4, when DCE-GS (0.125 to 2.5 mM) was added to the quin 2 test solution with Ca”, no changes in fluorescence intensity were observed. However, the fluorescence intensity was 18 when EDTA (2.5 mM) was added to the test solution containing 2.5 mM
L
0
0
1
2
S-(1,2-Olcartxxyethyl
3
)glutathlone
(IIM)
FIG. 4. Effect of DCE-GS concentration on fluorescence intensity of quin 2%Ca complex. Varying amounts (0 to 2.5 mM) of DCE-GS or 2.5 mM of EDTA was added to 2.5 mM CaCl, and then 20 pM of quin 2 was added to the mixture. For the comparison, Ca’+ (2.5 mM) and EDTA (2.5 mM) were added to quin 2 (0) and as a blank only quin 2 was measured (0). (I, T) Half of the standard deviation.
149
” I”
FIG. 5. Effects of extracellular Ca2+ on inhibition of ADP-induced platelet aggregation by EDTA (a) and DCE-GS (b). The ADP-induced platelet aggregation was performed in the same manner as described in Fig. 1. DCE-GS or EDTA was prepared to 2.5 mM in TBS and CaCl, was 2.5 mM and 6.25 mM. Control refers to ADP-induced platelet aggregation in the absence of DCE-GS and EDTA, respectively. I; 2.5 mM CaCl,, II; 2.5 mM CaCl, plus 2.5 mM EDTA, III; 6.25 mM CaCl, plus 2.5 mM EDTA, IV; 2.5 mM CaCl,, V; 6.25 mM CaCI, plus 2.5 mM DCE-GS.
Ca”’ for comparison with DCE-GS and 17 for quin 2 in buffer as a blank. This evidence indicates DCE-GS does not show chelating activity for Ca’+. In separate experiments, by the method of Harafuji and Ogawa (9), DCEGS (200 and 500 pM) did not bind Ca”’ (data not shown). The raman spectrum of DCE-GS (0.1 M) was the same as that in the presence of Ca2+ (0.1 M), also showing Ca” did not bind DCE-GS (data not shown). Effects of extracellular Ca2+ on inhibition of ADP-induced platelet aggregation by EDTA and DCE-GS. The inhibitory effect of DCE-GS on the platelet aggregation was compared with that of EDTA. Figures 5a and 5b illustrate the inhibitory patterns caused by EDTA and DCE-GS on platelet aggregation induced by ADP. The addition of both CaCl, (2.5 mM) and ADP to PRP immediately triggered the platelet aggregation and the primary aggregation occurred 1 min after and the secondary aggregation took place about 2 min later (Fig. 5a, I). When 2.5 mM EDTA (which is able to chelate 2.5 mM CaCl,) was added, ADP-induced platelet aggregation was perfectly inhibited (Fig. 5a, II). But, when 6.25 mM CaClz (which overcomes 2.5 mM EDTA) was added, platelet aggregation occurred again (Fig. 5a, III). However, in the case of DCE-GS, the simultaneous addition of DCE-GS (2.5 mM) and CaCla (6.25 mM) triggered the primary aggregation, which reached a maximum level about 1 min later and was afterward followed by desaggregation and an inhibition of secondary aggregation (Fig. 5b, V). These results indicate the inhibition of platelet aggregation by DCE-GS is not due to the chelation of Ca*+, unlike that by EDTA. In general, peptides undergo neither decarboxylation nor deamination reactions, but are subject to hydrolysis by some enzymes (11). The hydrolysis products of DCE-
150
TSUBOI ET AL.
GS would include GDCE-Cys, DCE-CG, and DCE-Cys. However, our experiments have shown that these peptides and the amino acid have no antiaggregation activity.
DCE-GS may be developed as an anticoagulant of blood or an inhibitor of platelet aggregation in the future. Since it is a natural substance and is present in lens and liver tissue, it could be a valuable potential new therapeutic agent.
DISCUSSION
When platelet aggregation is triggered by addition of agents such as ADP, collagen, and thrombin, arachidonic acid is released from membrane phospholipid by activation of phospholipase A, (12,13). The arachidonic acid released is converted to thromboxane A, via prostaglandin G, and Hz by cyclooxygenase and thromboxane synthetase, and this causes platelet aggregation and the subsequent release reaction of ADP and serotonin (14). Previously it was reported that aspirin and indomethatin prevented platelet aggregation by inhibiting the activity of cyclooxygenase (15-17), while dibucaine (18) and mepacrine are also effective in preventing the aggregation (17) by inhibiting the phospholipase AZ activity. In general, platelets aggregate reversibly without the release reaction of ADP and serotonin (the primary aggregation) and irreversibly with the release reaction and the prostaglandin synthesis (the secondary aggregation). In this study, it was shown that DCE-GS inhibited the secondary aggregation but not the primary reaction (Fig. 2) and the compound provides a strong inhibitory effect on the platelet aggregation induced by arachidonic acid (Fig. 3). It was reported that TPA may be intercalated into membranes and directly activate protein kinase C without mobilization of Ca2+ in sufficient quantities (1921). But, DCE-GS did not inhibit TPA-induced platelet aggregation. This result seems to indicate that the inhibitory effect of platelet aggregation by DCE-GS is not related with protein kinase C. It was known that EDTA inhibited the platelet aggregation due to chelation of Ca2+ (22). As illustrated in Figs. 4 and 5, it became clear that the inhibitory mechanism of DCE-GS on platelet aggregation is not due to the chelation of Ca2+. Recently it was demonstrated by us that DCE-GS inhibited serotonin release during the aggregation process induced by collagen, which will be published later (23). It may be concluded from these results that DCE-GS inhibits a point of a pathway where arachidonic acid is metabolized to thromboxane A,, which causes the mobilization of the Ca2+ required for the platelet aggregation and release reaction. The more precise mechanism of the antiaggregation activity by DCE-GS is under study. In order to test the acute toxicity of DCE-GS, mice were injected intravenously with DCE-GS in a dose of 1600 mg/kg, which was dissolved in 5 ml of saline and neutralized with 1 N NaOH. No toxicity was observed.
ACKNOWLEDGMENTS This work was supported in part by a Grant-in-Aid for Scientific Research (No. 63771958) from the Ministry of Education, Science and Culture, dapan. We thank Dr. F. Nakayama for supplying the Aggrecorder II and Dr. J. Odoh for the raman spectra. We are grateful to Mr. Y. Kondo and Mr. Y. Kawakami for supplying healthy blood.
REFERENCES 1 Calam, D. H., and Waley, S. G. (1963) Biochem. J. 86,226-231. 2. Tsuboi, S., Uda, N., Hirota, K., and Ohmori, S. (1984) J. Clin. Chem. Clin. B&hem. 22,28&290. 3 Tsuboi, S., Kobayashi, M., and Ohmori, S. (1984) JPNJ. Pharm. 38(8), 422 [Abstract] 4. Tsuboi, S., and Ohmori, S. (1988) Seikugaku 60(8), 645. t5. Tsuboi, S., Kishimoto, S., and Ohmori S. (1989) J. Agric. Food Chem. 37(3), 611-615. p. 193, Ishiyaku press, Japan. 6. Hino, S. (1977) Rinshohkensakouza 7. Born, G. V. R. (1962) Nature (London) 194,927-929. 8. Tsien, R. Y., Pozzan, T., and Rink, T. d. (1982) J. Cell Biol. 94, 325-334. 9. Harafuji, H., and Ogawa, Y. (1980) J. Biochem. (Tokyo) 87,130& 1312. 10. Thomas, M. C., Richard, J. H. W., Andrew, H. K., and John, N. F. (1988) Thromb. Res. 50, 719-731. 11. Larsson, A., Orrenius, S., Holmgren, A., and Mannervik, B. (1983) Functions of Glutathione; Biochemical, Physiological, Toxicological, and Clinical Aspects, p. 23, Raven Press, New York. 12. Bills, T. K., Smith, J. B., and Silver, M. J. (1977) J. Clin. Inuest. 60,1&6. 13. Flower, R. J., and Blackwell, G. J. (1976) Biochem. Pharmacol. 25,2855291. 14. Kannagi, R. (1987) Seikuguku 59,1291-1307. 15. O’Brien, J. R. (1968) Lancet 1,779-783. 16. Weiss, H. J., Aledort, L. M., and Kochwa, S. (1968) J. Clin. Inuest. 47,2169-2180. 17. Blackwell, G. J., Duncombe, W. G., Flower, R. J., Parsons, M. F., and Vane, J. R. (1977) Brit. J. Phurmucol. 59,353-366. 18. Kunze, H., Nahas, N., Traynor, J. R., and Wurl, M. (1976) Biothem. Biophys. Actu. 441,933102. 19. Naka, M., Nishikawa, M., Adelstein, R. S., and Hidaka, H. (1983) Nature (London) 306,490-492. 20. Castsgna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, U., and Nishizuka, Y. (1982) J. Biol. Chem. 257,7847-7851. 21. Yamanishi, J., Takai, Y., Kaibuchi, K., Sano, K., Castagna, M., and Nishizuka, Y. (1983) Biochem. Biophys. Res. Commun. 112, 778-786. 22. Cazenaev,