Involvement of disulfide-sulfhydryl interaction in anti-platelet actions of KF4939

THROMPOSIS RESEARCH 38; 61-69, 1985 0049-3848/85 $3.00 + .OO Printed in the USA. Copyright (cl 1985 Pergamon Press Ltd. 411 rights reserved.

INVOLVEMENT OF DISULFIDE-SULFHYDRYL INTERACTION IN ANTI-PLATELET ACTIONS OF KF4939

Koji Yamada, Kazuhiro Kubo+, Katsuichi Shuto and Nobuhiro Nakamizo

Pharmaceuticals Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Nagaizumi-cho, Sunto-gun, Shizuoka-ken, 411 Japan. + To whom a reprint should be requested.

(Received 23.7.1984; Accepted in revised form 17.12.1984 by Editor M. Matsuda)

(Received in final form by Executive Editorial Office 23.1.1985)

ABSTRACT We studied on the role of an intramolecular disulfide bond of KF4939 in its anti-platelet actions by using rabbit platelets. The inhibitions of aggregation of platelet-rich plasma(PRP) and washed platelets, and of malondialdehyde production in thrombin-stimulated platelets by KF4939 were counteracted by pretreatment with sulfhydryl compounds, glutathione,' 1-cysteine and dithiothreitol. A reduced compound of KF4939 (Red-KF4939) showed nearly equal anti- aggregating activities to those of KF4939 in PRP, it, however, showed low activities to inhibit platelet aggregation and thrombin -stimulated malondialdehyde production in washed platelet suspensions. In addition, the anti-aggregating action of Red-KF4939 was counteracted by pretreatment with sulfhydryl compounds, simi- la,rly to that of KF4939. Furthermore, when platelets were treated with KF4939, a significant decrease of protein-bound sulfhydryl groups was observed. We may conclude from these results that the intramolecular disulfide bond plays an essential role in anti- platelet actions of KF4939 and the interaction of the disulfide bond with protein-bound sulfhydryl groups may be involved in the mechanism of anti-platelet actions of KF4939.

INTRODUCTION

KF4939(2,2'dithiobis(N-2-hydroxypropylbenzamide)) is a new orally active inhibitor of platelet aggregation (1). This agent inhibits platelet aggregations induced by a wide variety of aggregating agents(l). Up to now, we have clarified that KF4939 inhibits phospholipase activation, but it does not inhibit cyclooxygenase and thromboxane synthetase (2), and this

Key words: blood platelets, aggregation, phospholipase, disulfide compounds sulfhydryl reagents.

61

62 KF4939 AS SULFHYDRYL INHIBITOR Vo1.38, No.1

agent has thromboxane A2 antagonistic action (1,3). The molecular mechanism of these actions of KF4939 is still unclear.

Because KF4939 is a disulfide compound, it is presumable that this agent interacts with sulfhydryl groups of platelets. Sulfhydryl inhibitors such as N-ethylmaleimide(NEM) and p-chloromarcuribenzene (PCMB) inhibit aggregation, secretion and arachidonic acid mobilization from membrane phospholipids in intact platelets (4-8). These evidences indicate that sulfhydryl groups of platelets play roles in these platelet reactions. It is possible to presume that disulfide compounds including KF4939 exert their anti-platelet actions through interaction with sulfhydryl groups of platelets. There are a few reports on anti-platelet actions of disulfide compounds. One of the most popular disulfide compounds, dithiobis-p-nitrobenzoic acid (DTNB), reacts with sulfhydryl groups of platelets (8), it, however, does not inhibit aggregation , secretion, and arachidonic acid mobilization from membrane phospholipids, probably due to lack of membrane permeability in this agent. Anti- aggregating action of Bay i 7351, a disulfide compound had been reported (9). The report, however, did not deal with interaction of this agent with sulfhydryl groups. Thus, it is still obscure if disulfide-sulfhydryl interaction may be involved in anti-platelet actions of sulfhydryl compounds.

This study was undertaken to examine the role of the intramolecular disulfide bond of KF4939 in its anti-platelet actions and the effect of this agent on sulfhydryl groups of platelets. As a result, it was showed that the intramolecular disulfide bond may play an essential role and KF4939 interacts with protein-bound sulfhydryl groups of platelets.

MATERIALS AND METHODS

Drugs and Chemicals KF4939 and its reduced compound (Red-KF4939) were obtained from Permachem Asia Ltd., Tokyo, and were used as an aqueous solution in 0.1 % Tween 80. Other chemicals used were as follows; ADP 2Na (Boeringer Mannheim), arachidonic acid, sodium salt (Sigma), collagen (Hormone Chemie), human thrombin (Green Cross), glutathione and 1-cysteine (Kyowa Hakko), dl-dithiothreitol (Sigma), sodium thiobarbiturate (Nakarai Chemicals), 5,5'-dithiobis-p-nitrobenzoic acid and tetraethoxypropane (Wako Pure Chemicals), N-ethylmaleimide (Eastman Kodak CO.,).

Preparation of Platelets Platelet-rich plasma (PRP) and a washed platelet suspension of rabbits were prepared as described in the previous reports (1, 2). The washed platelets were finally suspended in calcium-free Krebs-Hen- seleit solution at a concentration of 5 x 10' cells/ml.

Platelet Aggregation In vitro aggregometry was performed as described before (1). Drugs were added to PRP or a washed platelet suspension 3 min before addition of aggregating agent. Sulfhydryl compounds were added 30 set before addition of drugs.

Malondialdehyde Production The method of MacMillan et al_ (10) was slightly modified and employed. A washed platelet suspension in a volume of 0.25 ml was preincubated with drugs (10 ul) for 3 min at 37°C and then 10 ul of thrombin (final concn. 1 U/ml) was added and incubated for an additional 3 min at 37'C. The reaction mixture in a volume of 0.25 ml was taken and transfered into 0.25 ml of 10 % TCA to terminate the reaction. The precipitate was removed by centrifugation at 14000 xg for 2 min by using Kubota microcentrifuge @M-15000). The supernatent in a volume of 0.4 ml was mixed with an equal volume of 0.53 % thiobarbituric acid. The samples were heated

Vo1.38, No.1 KF4939 AS SULFHYnRYL INHIRITOR

for 30 min at 7O"C, allowed to cool at room temperature of distilled water. Malondialdehyde (MDA) was measured

63

and mixed with 2 ml by recording fluore-

scence in a Hitachi spectrophotofluorimeter (MPF-2A) with extinction and emission wave lengths of 520 nm and 553 nm, respectively. A hydrolysis product of tetraethoxypropane was used as MDA standard.

Effect on Sulfhydryl Groups of Platelets The quantification of platelet sulfhydryl groups was carried out by the method of Matsuda et al (11) and Beutler et al (12) with slight modifications. A washed platelet suspension in a volume of 2.5 ml was preincubated for 2 min at 37°C. Then, KF4939 (0.25 ml) was added. The mixture was incubated for an additional 10 min. Immedi- ately after the incubation, the mixture was centrifuged at 3000 rpm for 10 min to give a platelet pellet. The pellet was suspended in 1 ml of distilled water. The suspension was mixed with precipitating solution consisting of 1.67 % metaphospholic acid, 0.02 % EDTA 2 Na, and 30 % NaCl, allowed to stand for about 2 hr at room temperature, and centrifuged at 3000 rpm for 10 min. The precipitate was used for the assay of protein-bound sulfhydryl groups, after the denatured protein sediment was neutralized by addition of 0.1 ml of 0.3M Na,HPO, and solubilized by adding 1 ml of 1 % sodium dodecyl sulfate. Then, 0.1 ml of O.OlN DTNB was added to the mixture. After incubation for 30 min at room temperature, an optical density was measured at 412 nm. The content of sulfhydryl groups was calculated on the basis of a molar extinction coefficient of 1.16 x lG4.

RESULTS

Comparison of Anti-Platelet Activities between KF4939 and Red-KF4939 Anti- platelet activities of KF4939 were compared with those of Red-KF4939. The data are summalized in TABLE 1. Since the molecular weight of Red-KF4939 is equal to half the molecular weight of KF4939, IC50 (the concentration of drug required to inhibit by 50 % the induced responses) is expressed on weight basis (ug/ml). KF4939 at concentrations above 3 pM inhibited platelet aggregation induced by collagen, arachidonic acid and ADP in PRP in a concentration-dependent manner. Red-KF4939 also inhibited these aggregations in PRP. The IC50 values for Red-KF4939 were nearly equal or about 2 times higher than IC50 values for KF4939. In washed platelet suspensions, KF4939 inhibited arachidonic acid-induced aggregation and thrombin-stimulated MDA production at concentrations above 1 uM and 3 uM, respectively. On the other hand, a threshold concentration of Red-KF4939 to inhibit these platelet reactions was 60 uM. So ICSO values for Red-KF4939 were approximately 15 times higher than those of KF4939.

Counteraction by Sulfhydryl Compounds of Anti-Platelet Actions of KF4939 It is known that an inhibitory effect of sulfhydryl inhibitors such as NEM and PCMB on platelet aggregation is counteracted by a sulfhydryl compound, l-cysteine (CySH) (5). Another sulfhydryl compound, dithiothreitol (DTT) is known as a disulfide reducing agent. Effects of these sulfhydryl compounds on anti-platelet actions of KF4939 were examined. As shown in FIG. 1, when PRP was pretreated with 0.3 mM DTT and with 1 mM glutathione (GSH), the inhibitory effect of KF4939 on collagen-, arachidonic acid- and ADP-induced aggregation of PRP was almost completely abolished. This effect of KF4939 was significantly diminished by 1 mM CySH.

The inhibitory effect of Red-KF4939 on collagen- and ADP-induced aggregation of PRP was also abolished by pretreatment with 1 mM GSH and with 0.3 mM DTT, similarly to the effect of KF4939 (FIG. 2). NEM at 0.3 mM markedly


TABLE 1

Anti-Platelet Activities of KF4939 and its Reduced Compound (Red-KF4939) in Rabbit Platelets

IC50 ug/ml (ratio of concn.)

Items KF4939 Red-KF4939

Platelet-Rich Plasma Aggregation:

Collagen (10 ug/ml) 3.02 (1) 3.36 (1.11) Arachidonic acid (50 ,g/ml> 2.27 (1) 4.51 (1.89) ADP (10 uM) 13.0 (1) 24.6 (1.89)

Washed Platelets Aggregation: Arachidonic acid (0.5 uM) 1.59 (1) 22.5 (14.2)

Malondialdehyde Production: Thrombin (1 U/ml> 2.65 (1) 46.2 (17.4)

-- COntrOl DlT03mM GSH tySH

FIG. 1

Effects of Sulfhydryl Compounds on the Anti-Aggregating Action of KF4939 in Rabbit Platelet-Rich Plasma. Sulf- hydryl compounds were added at 30 set before treatment with KF4939. Concent- rations of collagen, arachidonic acid and ADP were 10 vg/ml, 50 ug/ml and 10 uM, respectively. Each column represents the mean + S.E.M. of 3-4 expe- - riments.

inhibited platelet aggregation induced by collagen and ADP. The inhibition was counteracted by pretreatment with sulfhydryl compounds(FIG. 3.). The inhibitory effects of prostaglandin El (0.1-0.3 uM>, papaverine (0.1 mM) and aspirin (0.1 mM) on aggregation of PRP were unaltered by pretreatment with these sulfhydryl compounds (data not shown).

Vol.38, No.1 KF4939 AS SIJLFHYDRYL INHIBITOR

,OO (A) Collagen (6) ADP

t loot

60

0 Vehicle + saline, m Red-KF4939 120~~M tsallne, B Red-KF4939 ~~OJJM+DTT 0.3 mM &I ” +CySH 1mM. ?? .I GSH 1mM.

FIG. 2

Effects of Sulfhydryl Compounds on the Anti-Aggregating Action of Red-KF4939 in Rabbit Platelet-Rich Plasma. Experimental procedures and concentrations of aggregating agents were as same as in FIG. 1. Each column represents the mean + S.E.M. of 3-4 experiments.

100 (A) Collagen

c 80 0

z

-

0 Vehicle +sallne , ??NEM 0.3 mM + saline,

??NEM 0.3mM+DTT 0.3mM. ONEM 0.3mM+GSH 1mM

QNEM 0.3mM.CySH lmM,

FIG. 3

Effects of Sulfhydryl Compounds on the Anti-Aggregating Action of N-Ethylmaleimide (NEM) in Rabbit Platelet-Rich Plasma. Experimental procedures and concentrations of aggregating agents were as same as in FIG. 1. Each column represents the mean + S.E.M. of 3 experiments.

-


The inhibition by KF4939 of arachidonic acid-induced aggregation of washed platelets was also counteracted by these sulfhydryl compounds (FIG. 4).

Inhibition by KF4939 of thrombin-stimulated MDA production is probably due to inhibition of mobilization of arachidonic acid from membrane phospholipids (2). The inhibitory effect of KF4939 on the MDA production was also markedly diminished by pretreatment with 1 mM GSH and with 1 mM CySH (TABLE 2).

OTT + KF4939 GSH + KF4939

saline . vehicle

Loo FIG. 4

Counteraction by Sulfhydryl Compounds of Inhibitory Effect of KF4939 on Arachidonic acid-Induced Aggregation of Washed Platelets.

TABLE 2

Counteraction by Sulfhydryl Compounds of Inhibitory Effect of KF4939 on Thrombin-Stimulated Malondialdehyde Production in Washed Rabbit Platelets.

Drug

Vehicle

KF4939

Malondialdehyde Production

Concn. nmoles/lOq cells/3 min

pM saline GSH 1 mM CySH 1 mM

1.16 2 0.04 1.75 2 0.09 1.68 2 0.11

10 0.46 + 0.05 1.76 + 0.06 1.58 + 0.05 (59.9 -c 5.5) (-1.1 -, 4.0) ( 8.8 7 5.1) - -

KF4939 30 0.26 + 0.04 1.25 + 0.07 1.32 + 0.07 (77.7 + 2.6) (27.4 7 6.1) - (19.3 T-7.6)

Figures in parentheses represent per cent inhibition. All values represent the mean + S.E.M. of 5-6 experiments.

Vo1.38, No.1 KF4939 AS SULFHYDRYL INHIBITOR 57

Effect on Sulfhydryl Groups of Platelets Effect of KF4939 on the content of protein-bound sulfhydryl groups of platelets was examined by using washed platelets. As shown in TABLE 3, treatment of a platelet suspension with 30 uM KF4939 slightly but significantly decreased the contents of protein- bound sulfhydryl groups. In separate experiment, we observed that 0.3 mM NEM decreased by approximately 50 % the content of protein-bound sulfhydryl groups (data not shown). Non-protein sulfhydryl groups of platelets, for example, GSH are also probable as reactant with KF4939, but because Red- KF4939 , which may be formed by interaction of KF4939 with sulfhydryl groups, reacts with DTNB and disturbs correct measurement of the non-protein sulfhydryl groups, effect of KF4939 on the content of non-protein sulfhydryl groups was unable to be examined.

TABLE 3

Effect of KF4939 on the Content of Protein-bound Sulfhydryl Groups of Rabbit Platelets

Drug Concn.

PM

Sulfhydryl group content

nmoles/lOg cells (per cent change)

Vehicle 109.8 + 9.2 (100) - KF4939 10 113.7 t 9.9 (103.4 + 1.0) - KF4939 30 95.3 + 10.1* ( 86.5 + 3.6) -

* Significantly different from the vehicle-treated group (p 0.05). Statistical analysis was performed by paired t-test. The mumber of experiments was five.

DISCUSSION

KF4939 which is a disulfide compound chemically reacts with sulfhydryl compounds such as GSH and CySH to form a mixed disulfide, and to convert to Red-KF4939. This agent is readily reduced by DTT, a disulfide reducing agent. (Ishii, personal communication)

To clarify the role of the intramolecular disulfide bond of KF4939 in its anti-platelet actions, anti-platelet activities of Red-KF4939 were compared with those of KF4939. In PRP, anti-aggregating activities of Red- KF4939 were nearly equal to the activities of KF4939. However, in washed platelet suspensions which contain no plasma constituents, the activities of Red-KF4939 to inhibit aggregation and thrombin-stimulated MDA production were only one-fifteenth or less the activities of KF4939. Furthermore, the anti- aggregating action of Red-KF4939 observed in PRP was counteracted by pretreatment with sulfhydryl compounds, similarly to that of KF4939. We presumed from these evidences that Red-KF4939 has no or, if any, weak anti-platelet action, and it may be converted to an unknown disulfide compound which may be an active metabolite in PRP. Thus, the intramolecular disulfide bond of KF4939 seems to play an essential role in anti-platelet actions of this agent. Consequently, when the intramolecular disulfide bond of KF4939 is

68 KF4939 AS SULFHYDRYL INHIBITOR Yo1.38, Vo. 1

reduced, anti-platelet activity of this agent should be decreased. A sulfhydryl compound, CySH, is known to counteract the inhibitory effect

of sulfhydryl inhibitors on platelet aggregation induced by various aggregating agents (5). We confirmed the counteracting effect of sulfhydryl compounds, GSH, CySH and DTT on anti-aggregating action of NEM. The anti-aggregating effect of KF4939 both in PRP and in washed platelet suspensions was counteracted by addition of these sulfhydryl compounds before treatment with KF4939. No alteration was observed in anti-aggregating actions of prostaglandin El , papaverine and aspirin after pretreatment with these sulfhydryl compounds. These evidences suggest that anti-aggregating action of KF4939 may be mediated by interaction with sulfhydryl groups of platelets, in a similar manner to the action of NEM. In addition, the inhibitory action of KF4939 on thrombin-stimulated MDA production was also counteracted by pretreatment of platelets with sulfhydryl compounds. Because the inhibitory action of KF4939 on the MDA production is due to inhibition of phospholipase activation (2)) interaction of this agent with sulfhydryl groups may be involved in the inhibition of phospholipase activation. In fact, when platelets were treated with KF4939, a significant decrease in the content of protein-bound sulfhydryl groups was observed. This gives a strong proof for the involvement of the disulfide-sulfhydryl interaction in the anti-platelet actions of KF4939. Because the pretreatment of KF4939 with sulfhydryl compounds in the presence of platelets resulted in a complex number of reactions, it is difficult to explain the mechanism of counteraction by sulfhydryl compounds of anti-platelet actions of KF4939. However, the reductive cleavage of the intramolecular disulfide bond of KF4939 is able to be thought as a possible cause for the counteraction.

There are several reports on location of the functionally important sulfhydryl groups involved in platelet reactions. It is known that NEM penetrate cell membranes and DTNB does not (13). MacIntyre et al (7) proposed that intracellular sulfhydryl groups play an important role in platelet aggregation, secretion and shape-change from the evidence that these platelet reactions are inhibited by NEM but are not inhibited by DTNB. Silk et al (8) discussed that essential sulfhydryl groups for arachidonic acid releasing activity may be located on the inner surface of the platelet membranes, from the evidence that DTNB inhibition of isolated platelet membranes is much greater than in membranes from DTNB-treated intact platelets, on the other hand, no such difference in inhibition is observed with NEM. KF4939 exerts the inhibition of aggregation and of arachidonic acid mobilization from membrane phospholipids in intact platelets. In addition, a result obtained in a preliminary experiment showed thatQw]-labeled KF4939 was taken up into platelets. From these circumstantial evidences, we suppose that KF4939 may penetrate into the membranes and reacts with the functionally important sulfhydryl groups.

KF4939 reduced only 15 % protein-bound sulfhydryl groups at a concentra- tionwhich caused above 80 % inhibition in platelet aggregation and thrombin- stimulated MDA production. This agrees with the assumption that only certain sulfhydryl groups play an essential role in arachidonic acid releasing activity (8) and suggest that KF4939 may selectively react with these essential sulfhydryl groups.

Since the concentration of KF4939 required to inhibit platelet reactions was lower than that of NEM which is the most potent inhibitor of platelet aggregation among well known sulfhydryl inhibitors (7), KF4939 is probably the most potent sulfhydryl inhibitor in platelets. Many other sulfhydryl- dependent biological actions are known. However, this agent even at several times higher dose than the effective dose to inhibit platelet aggregation caused no serious toxic sign after repeated administration for 30 days to

Vo1.38, No.1 KF4939 AS SIJLFHYDRYL INHIBITOR

rats, although we have no evidence on effect of KF4939 on the sulfhydryl- dependent actions besides in platelets.

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REFERENCES

YAMADA, K., SHUTO, K. and NAKAMIZO, N. Inhibition of platelet aggregation by a new agent, Z,Z'-dithiobis(N-2-hydroxypropylbenzamide)(KF4939) Thromb. Res. ___- 2, 197-206, 1983.

YAMADA, K., SHUTO, K., KUBO, K. and NAKAMIZO, N. Evidence that KF4939 a new anti-platelet agent, inhibits phospholipase actifation in rabbit platelets: Differential aspects from cyclic-AMP increasing agents and calmodulin antagonist. Arch. intern. Pharmacodyn. Ther. 268, 141-154, 1984. YAMADA, K., KUBO., K., SHUTO, K. and NAKAMIZO, N. Inhibition of thromboxane A -induced vasocontraction by KF4939, a new anti-platelet agent, in rabbit mesenteric and dog coronary arteries. Japn. J. Pharmacol. 36, 283-290, 1984.

ROBISON, C.W.Jr., MASON, R.G. and WAGNER, R.H. Effect of sulfhydryl inhibitors on platelet aggrutinability. Proc. Sot. Exptl. Biol. Med. 113, 857-861, 1963.

HARRISON, M.J.G., EMMONS, P.R. and MICHELL, J.R.A. The effect of sulfhydryl and enzyme inhibitors on platelet aggregation in vitro. Thromb. Disth. Haemorrh. 16 122-133, 1966. _' ALEDORT, L.M., TROUP, S.B. and WEED, R.I. Inhibition of sulfhydryl- dependent platelet functions by penetrating and non-penetrating analogues of parachloromercuribenzene. Blood, 31, 471-479, 1968. -- MACINTYRE, D.E., GRAINGE, C.A., DRUMMOND, A.H. and GORDEN, J.L. Effect of thio reagents on platelet transport process and responses to stimuli. Biochem. Pharmacol. 26 319-323, 1977. _' SILK, S.T., WONG, K.T.H. and MARCUS, A.J. Arachidonic acid releasing activity in platelet membranes: Effects of sulfhydryl-modifying reagents. Biochem. 20, 391-397, 1981. - BUSSE, W.D., SEUTER, F., HOERLEIN, U., BOESHAGEN, H., HOFFMEISTER, F., and PHILIPP, E. Inhibition of thromboxane synthesis and other platelet functions by Bay i 7351, a new antithrombotic compound. Thrombs. Haemost. 42, 213, 1979. - MACMILLAN, R.M., MACINTYRE, D.E. and GORDEN, J.L. Simple, sensitive fluorimetric assay for malondialdehyde production by blood platelets. Thromb. Res. 11, 425-428, 1977. --- MATSUDA, S., IKEDA, Y., AOKI, M., TOYAMA, K., WATANABE, K. and ANDO, Y. Role of reduced glutathione on platelet functions. Thrombs. Haemost. 42 1324-1331, 1979. _'

BEUTLER, E., DURON, 0. and KELLY, B.M. Improved method for determination of blood glutathione. J. Lab. Clin. Med. 61, 882-888, 1963. - SMITH, R.P.P. and ELLMAN, G.L. A study of the dependence of the human erythrocyte glucose transport system on membrane sulfhydryl groups. J. Membrane Biol. 12, 177-188, 1973. -

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Involvement of disulfide-sulfhydryl interaction in anti-platelet actions of KF4939