Selective antagonism of platelet-activating factor (PAF)-induced aggregation and secretion in washed rabbit platelets by CV-3988, l-652731, triazolam and alprazolam

THROMBOSIS RESEARCH47; 249-257,1987 0049-3848/87 $3.00t .OO Printed in the Copyright

(c) 1987 Pergamon Journals

Ltd.

USA. All rights reserved.

SELECTIVE ANTAGONISM OF PLATELET-ACTIVATING FACTOR (PAFJ-INDUCED AGGREGATION AND SECRETION IN WASHED RABBIT PLATELETS BY CV-3988, L-652731, TRIAZOLAM AND ALPRAZOLAM

Charles

P.

Coxl

and

Kevin

L. Wood

Veterans Administration Medical Center and University of Oklahoma Health Sciences Center, Oklahoma City, OK 73109, U.S.A. (Received 20.2.1987 by Editor A.P. Fletcher; Accepted 1.6.1987 by Editors-in-Chief B. Blombtick and A.L. Copley due to the death of Editor Anthony P. Fletcher)

ABSTRACT Platelet-activating factor (l-O-alkyl-2-acetyl-_s.nglycero-3-phosphorylcholine or AGEPC) is a potent phospholipid mediator elaborated by a variety of mammalian cells. (X-3988 (a unique structural analog of AGEPC), L-652731 (a lignan derivative of a natural product) and two triazolobenzodiazepines (triazolam and alprazolam) were evaluated for their ability to selectively antagonize a gregation and secretion responses in washed, [ 9 Hlserotonin-labeled rabbit platelets stimulated with graded doses of AGEPC. When 0.2 nM AGEPC was used as the stimulus, the concentration of antagonist needed for 50% inhibition (IC50) of secretion was obtained at 0.05 uM, 0.15 uM, 0.6 uM and 2.5 uM, for L-652732, CV-3988, triazolam and alprazolam, respectively. The corresponding IC50 values for aggregation were obtained at 0.2 uM, 0.1 uM, 1.5 uM and 6.5 uM, respectively. The inhibitory effects could be overcome by increasing the amount of AGEPC used to stimulate the platelets. Of the four compounds tested, L-652731 was the most potent antagonist of AGEPC-induced activation of washed rabbit platelets. -.

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1 Current Address: Searle R&D, Div. of G.D. Searle & Co., 4901 Searle Parkway, Skokie, IL 60077 Key Words:

AGEPC, platelet activation, AGEPC antagonists 249

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_INTRODUCTION Platelet-activating factor (l-O-alkyl-2-acetyl-Sn-glycero-3phosphorylcholine or AGEPC) has been shown to possess a variety of biological activities, both in viva and in vitro* These include dose-related activation of platelets and neutrophils, systemic hypotension, bronchoconstriction, glycogenolysis, cardiac anaphylaxis and acute lung injury (l-4). Recently, several selective antagonists of AGEPC have been identified, including structural analogs of AGEPC as well as unrelated compounds, from both natural and synthetic sources (5-7). This study was designed to systematically compare the potency of four selective antagonists of AGEPC-induced aggregation and secretion of [3Hlserotonin from labeled, washed rabbit platelets. The compounds used were: CV-3988, a structural analog of AGEPC (8-10); L-652731, a derivative of a natural product (U-13); and triazolam and alprazolam, two triazolobenzodiazepines, (14,151.

: All chemicals and solvents used were reagent grade or better, purchased from Sigma Chemical Co. (St. Louis, MO). Buffers for washing platelets were prepared fresh daily in double-glass-distilled water, as previously described (16). AGEPC was purchased from Bachem, Inc. (Torrance, CA) and dissolved in phosphate-buffered saline (PBS) containing 2.5 mg/ml bovine serum albumin (BSA). . m The structures of AGEPC and the four antagonists used in these studies are shown in Figure 1. (X-3988 was generously provided by Dr. M. Nishikawa (Takeda Chemical Industries, Ltd., Osaka, Japan) and dilutions in PBS were prepared according to his instructions (17). L-652731 was the gift of Dr. T. Y. Shen (Merck, Sharp and Dohme Research Laboratories, Rahway, NJ). A 1 mM stock solution of L-652731 was prepared in dimethyl sulfoxide (DMSO) and lo-fold serial dilutions were made in PBS just before use. Stock solutions (1 mM) of alprazolam and triazolam (The Upjohn Co., Kalamazoo, MI) were prepared in ethanol and diluted in PBS prior to use.

Platelet Aaareaation and SecretiQD : Washed, [3Hlserotoninlabeled rabbit platelets were prepared as previously described (161, and maintained in an atmosphere of 5% CO2 at 37O C until used in the bioassay. Aliquots of platelets (2.5 x 108/ml) were incubated with either an antagonist of AGEPC or the appropriate vehicle for 60 set prior to the addition of AGEPC (0.2 nM to 0.2 uM). Aggregation was continuously monitored on a strip-chart recorder and recorded as the height of the tracing at 60 set after the addition of AGEPC. Secretion of [3Hlserotonin was measured in a sample of the platelet suspension removed at 60 set after the addition of AGEPC (16). The percent inhibition of aggregation and secretion was calculated by comparing antagonisttreated platelets with the appropriate vehicle-treated control platelets. Each combination of antagonist and AGEPC was repeated 12-15 times, using several different platelet preparations. IC50

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values were determined by inspection of the dose-response curves. Platelet-Activating

0 II CH3COwC

Factor

CH O Cl&18 12 1 H 0

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II

CH20

-‘;

-

0-CHZCH2rj(CH3)3

0

I-O-hex.decyl/octadecyt-Z-acetylE-glycero-3-phosphorytchollne

L-652.731

cv-3988

I: ~H20CNH(CH2),7CH3 CH30

CH30

rac-3-(N-n-octadecyl~er~~yl0xy)Gethoxypropyl-Z-thiazolioethylphosphate

(dl)-2,5-bis(3,4.5-trimethoxyphenyl)tetrehydrofuran

Alprazolam

Triazolam

Cl

Cl

C6”5

8-Chloro-6-(2-chlorophenyl)-l-methyl-4H[1,2.4ltrlazolo[4,3-a][ benzodiazepine: &chloro-6IO-chlorophenylk1-methyl-4k-striaz.Tlo 14.3~a][l,4]benzodlazepine

Fig. 1

1.41

B-Chloro-1-methyl-6-phenyl-4H[1,2,4]trlazolol4,3-a] [1,4J benrodlazepine; 8-chloro-l-methyl6-phenyl-4H-s-triazolo[4,3-a) 1 ,4] benzodiazepine

[

Structures of platelet-activating factor (AGEPC) and four selective antagonists. DISCUS=

Although there is considerable interest in AGEPC antagonists, there is very little information available in the literature comparing the inhibitory effects of different AGEPC antagonists on both aggregation and secretion responses in washed platelet preparations. In our hands, each of the AGEPC antagonists tested inhibited both aggregation and secretion in washed rabbit platelets in a dose-related manner. The dose-response curve for CV-3908 versus 0.2 nM AGEPC is shown in Figure 2, where the solid line represents secretion data and the dashed line represents aggregation data. The IC5U values for aggregation and secretion

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are summarized

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in Table 1.

Fig. 2

CV-3988 (0.01 to 0.5 uM) inhibits platelet aggregation (O-----O) and secretion (M 1 induced by AGEPC (0.2 nM).

TABLE1 Relative Potency of AGEPC Antagonists Compound

Aggregation

L-652731

0.20 UM

0.05 uM

Cv-3 988

0.10 uM

0.25 uM

Triazolam

1.50 uM

0.60 uM

Alprazolam

6.50 uM

2.50 uM

(IC50)

Secretion

(1C50)

In their paper describing the first selective antagonist of PAF, Terashita _& ti (8) reported that CV-3988 inhibited aggregation of rabbit platelet-rich plasma (PRP) stimulated with 0.3 nM AGEPC (IC50, 7.8 uM). Our present results with CV-3988 are similar to those of Tokumura e_t al (181, who found that (X-3988 inhibited both aggregation and secretion in [3Hlserotonin-labeled washed rabbit platelets stimulated with 0.1 nM AGEPC (IC50, 0.2 uM and 0.6 uM, respectively). Our slightly higher IC50 values may reflect the fact that we used twice as much AGEPC to stimulate the platelets. Tokumura and co-workers also found that at doses above 1 uM, CV-3988 was a non-specific antagonist, blocking both

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collagen and thrombin-induced platelet activation. We have previously reported that concentrations of CV-3988 in excess of 0.5 uM non-specifically antagonized platelet activation induced has also by collagen and calcium ionophore A23187 (17). CV-3988 been reported to inhibit AGEPC-induced aggregation of washed human platelets with an IC50 of 1 uM (19). L-652731, related to the naturally-occurring AGEPC antagonist kadsurenone (111, has been reported to inhibit AGEPC-induced serotonin secretion from platelets at a concentration of 0.1 uM (19). Our dose-response data for L-652731 are shown in Figure 3 and Table 1.

L-652,731

Fig. 3

@M)

L-652731 (0.005 to 0.5 uM) inhibits both aggregation (0-----0) and secretion (-1 in washed rabbit platelets stimulated with AGEPC (0.2 nM1.

Tokumura n.tti (18) demonstrated that kadsurenone, a less potent AGEPC antagonist than L-652731, inhibited aggregation of washed human platelets (IC50, 0.8 uM) induced by 7.5 nM AGEPC. Voelkel and co-workers (20) reported that 10 uM L-652731 inhibited serotonin secretion from rabbit platelets stimulated with 2 nM AGEPC by 95%. Although we used lo-fold smaller doses of both AGEPC and L-652731 in the present study, our results (Fig. 3) parallel those of Voelkel & al (20). Kornecki nf al (14) made the intriguing observation that the triazolobenzodiazepines (triazolam and alprazolam), commonly used to treat panic disorder, selectively antagonized AGEPC-induced activation of human platelets ~JJ vitro, over the range of 1 to 40 uM. Our results using rabbit platelets, shown in Figures 4 and 5, with IC50 values summarized in Table 1, demonstrated similar inhibition of both the aggregation and secretion responses.

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I

I

0.1

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Fig. 4

Triazolam (0.1 to 5 uM) prevents AGEPCinduced aggregation (O-----O) and secretion CW 1 in washed rabbit platelets.

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0-t

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6’ I

0.1

0.5 ALPRAZOUM

Fig. 5

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1

1

5

I

10

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Alprazolam (0.5 to 10 uM) inhibits rabbit platelet aggregation (O-----O) and secretion CM 1 induced by AGEPC (0.2 nM1.

Except for (X-3988, the AGEPC antagonists were generally more potent inhibitors of platelet secretion than platelet aggregation. The inhibition of either response, however, could be overcome by increasing the amount of AGEPC used to stimulate

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the platelets, implying that these compounds are competitive antagonists of AGEPC. The effect of increased AGEPC levels on inhibition by L-652731 is depicted in Figure 6.

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2xlo-‘oM

1

I

2x10-%

2x10-&4

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AGEPC (PM)

Fig. 6

Increasing the concentration of AGEPC from 0.2 nM to 0.2 uM overcomes the inhibition of aggregation (O-----O) and secretion (M 1 in rabbit platelets pre-treated with L-652731 (1 uM).

In summary, four antagonists of AGEPC (CV-3988, L-562731, triazolam and alprazolam) were shown to inhibit AGEPC-induced aggregation and secretion responses in washed, [3Hlserotoninlabeled rabbit platelets in a dose-related manner. Inhibition of platelet activation induced by 0.2 nM AGEPC required a SOO-fold to 50,000-fold excess of antagonist to achieve the IC50 values, depending on the antagonist. This inhibitory effect could be overcome by increasing the amount of AGEPC used to stimulate the washed platelets. Under these experimental conditions, L-652731 was the best of the AGEPC antagonists evaluated.

1.

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2.

PINCKARD, R. N., L. M. McMANUS, D. J. HANAHAN and M. HALONEN: Immunopathology of acetyl glyceryl ether phosphorylcholine (AGEPC). In: moav of tb

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8.

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17.

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19.

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20.

VOELKEL, N. F., S. W. CHANG, K. D. PFEFFER, S. G. WORTHEN, I. F. McMURTRY and P. M. HENSON: PAF antagonists: Different effects on platelets, neutrophils, guinea pig ileum and PAF-induced vasodilation in isolated rat lung. -:359-372, 1986.