Interaction between prostaglandin el and nitric oxide (NO)

THROMBOSIS RESEARCH 62; 299-304,199l 0049-3848/91 $3.00 + .OO Printed in the USA. Copyright (c) 1991 Pergamon Press pk. All rights reserved.

INTERACTION

BETWEEN PROSTAGLANDIN

El AND NITRIC OXIDE

(NO)

R. Ratzenschlager*, K. Weiss*, Waltraud Rogatti**, Monika Stelzeneder*, and H. Sinzinger* *Wilhelm Auerswald Atherosclerosis Research Group (ASF) Vienna, Atherosclerosis Research Group of the Austrian Academy of Sciences (ATK), Vienna, Austria and **Schwarz Pharma, Monheim, Germany (Received

22.51990;

accepted

in revised form 31 .1.1991

by Editor H.A. Vinazzer)

ABSTRACT

A synergistic antiplatelet effect between prostaglandin 12 (PGI2), a CAMP-stimulator, and nitric oxide (NO), a cGMPstimulator, has been described. Data on a PGEl-NO interaction, however, are lacking so far. We therefore examined the question in healthy volunteers, whether a similar synergism exists between PGEl and NO on human platelets in vitro. Each of the substances alone caused a platelet ADP-induced inhibition of dose-dependent aggregation, PGEl (lng/mlPRP) reduced platelet aggregation by 27.8+14.6%, NO (0,3%) by 26,7+25,5%. The combination of these compounds caused an additive effect resulting in a reduction of 40,6+23,58. The findings indicate that PGEl(like PG12) and NO have a synergistic antiplatelet action. The concomitant treatment with both compounds offers an interesting concept for clinical therapy. INTRODUCTION

It has been demonstrated that the release of PGI2 and the endothelial derived relaxing factor (EDRF) from endothelial cells is coupled (1). Both compounds exert a potent antiaggregatory capacity(2), PGI2 acting via an elevation of CAMP (3), EDRF via an increase in cGMP (4). In vitro aggregation studies demonstrated that PGI2 and NO (one of the active compounds of EDRF) (5) are acting synergistically (6). words: Nitric oxide (NO), Prostaglandin atherosclerosis, platelet aggregation

Key

El

(PGEl),

Author's address: R. Katzenschlager, M.D. Wilhelm Auerswald Atherosclerosis Research Group (ASF) Vienna Nadlergasse 1 A-1090 Vienna, Austria 299

300

INTERACTION BETWEEN PGEl AND NO

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Although the interaction between PGI2 and NO has been extensively studied (7), there is no information available concerning the interaction of PGEl and NO. We approached this question using platelet of healthy volunteers and ADP as aggregation-inducing stimulus. MATERIALS AND METHODS

Platelets from 8 healthy donors (5 males, 3 females; aged 2240a;mean age 29a+4) were examined. They had no risk factors and were drug-abstaining for at least two weeks prior to the examination. Blood drawn from an antecubital vein was anticoagulated (1:9) using 3,8% sodium citrate (Heilmittelwerke, Vienna, Austria). After sedimentation for 10 minutes at 22OC platelet rich plasma (PRP) was prepared by a 10 minutes centrifugation at 150xg. After the careful removal of PRP, a further 15 minutes centrifugation at 3000xg (22O C) to obtain platelet poor plasma (PPP) was performed. PRP was adjusted with PPP to a final platelet count of 250 x 103//.&l. The platelet aggregation was measured in an aggregometer (H.Upchurch , Leicester, UK), according to the method of Born (8). The results were depicted on a flatbed recorder (Pharmacia, Fine Chemicals, Netherlands). PGEl-addition: 100 1.r1 PG El (Schwarz-Pharma,Monheim, Germany) stock solution (lOmg/ml) were diluted with Tris-buffer (25 mM, pH 7,4) to a final concentration of lng PG El/loo@ buffer. 100 ).bl PG El were added 1 minute before induction of aggregation. NO-addition: A glass vial was filled with 10 ml Tris-buffer (pH=7,4). The Tris-buffer was bubbled for 15 min with Argon gas and then with NO-gas (10 minutes). The gas-bulb was sealed with a rubber stopper. lml was removed with a syringe and injected into another gas bulb which was filled with 9 ml Tris-buffer. This buffer has been also bubbled for 15 min. with Argon gas. The final concentration of NO was 0,3%. 10~1 of this solution were added to PRP 30 seconds before induction of aggregation. Aggregation-induction: 600 ~1 of PRP were filled into a silicon coated glass vial. A little teflon-coated magnetic stirrer (length 5mm, diameter 0.5mm) stirred constantly at 800 rpm. 100~1 Trisbuffer (pH=7,4) or 100 ~1 PGEl were added to the aggregation-vial. Thirty seconds later 10 1.11 Tris-buffer or 10~1 NO and finally after further 30 seconds 100 1.11ADP (10 PM) were added into the aggregometer-vial at a constant temperature of 37O. Quantification of aggregation: Quantitative estimation was performed by evaluating: 1. the type of the aggregation curve (mono-phasic or bi- phasic, and reversible or irreversible, respectively) 2. maximum amplitude: maximum amplitude of light transmission (measured 4 minutes after aggregation induction) 3. the angle a: slope of the aggregation curve after the addition of the aggregation inducing agent. Statistical analysis: Results are presented as mean values ? standard deviation; calculation for significance was carried out by

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INTERACTION BETWEEN PGEl AND NO

301

means of paired Student's t-test, using a statistical analysis program (SPSS/PC, version 2.0). RESULTS Types of the aggregation curves: The ADP-induced platelet aggregation in the volunteers examined was monophasic and irreversible. The aggregation curves after the addition of NO and PGEl, however, were monophasic and reversible (~~0.05; Tab.1, Fig.1). TABLE 1 Types of aggregation curves after addition of NO and PGEl i

r

m

b

control

8

0

8

0

NO

4

4

3

5

PGEl

3

5

5

3

8 0 0 8 NO+PGEl ____________________~~~~~~~~~~~~~~~~~~ r...... reversible, i......irreversible, b ....biphasic.

FIG.1

m.. ..monophasic,

Typical curves from a male volunteer (27a, 178cm/71kg) a)control b) NO c) PGEl d)NO and PGEl

Both 100 ~1 PGE (lng/lOOmlPRP) and 10~1 NO (0.3%) comparably inhibited the amplitude of ADP-induced platelet aggregation while the combination of them caused a further significant decrease (Fig.2).

302

INTERACTION BETWEEN PGEl AND NO

80

amplitude

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(%)

00 :::::.:: ::::.:::: :::::::: ::::::::: :::::::: ......... ::::::::: ........ .:::::::: ::::::::: * * ................. ........ ......... :::::::: ......... x::::::: ........ .:::::::: ::::::::: .:::::::: ::::::::: ................. :::::::: ........ ....... ::::::::: :::::::::::::::::: .................. ........ ................................... .................. ........ :::::::: ::::::::: ................. . ................. .................. :::::::::::::::::: :::::::: ::::::::: :::::::: ::::::::: .................. ................. ................................... ................................... ................. :::::::. ......... .................. ................................... ::::::::::iiGlii ..................................................... .................. ................................... ................... ................................... ................................... ......... .................................... ~~~~~~~~;~~~~~~~~~ .......................................... .................. ........ , .................................. ., .......................... .................

40

20

0

control

FIG.2

NO

POE1

**

NO+PGEl

Mean values of the maximum amplitude of ADP induced platelet aggregation. xfSD;n=8(*=p<0.05, **=p
A similar behaviour was found for the slope of the aggregation curves, however, the differences were -lesspronounced (Fig.3).

1001

80

degrees

_

(alpha)

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* T **

:::::::g::::::

:::::::::::::::::.

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40 -

20

0’

::::::::::::::::: :::::::::::::::::. :::::::::::::::::: ......................... .................................... ::::::::::::::::: ::::::::::::: ::::, :::::::::::::::::: yxpzi* . :::::::::::::::::: ::::::::::::::::: :::::::::::::::::: :::::::::::::::::: .................. .................................... .................. :::::::::::::::::: .:::::::::::::::::: .................. iilliiitlixti ................... ........................................................................ ~.:::::: :::::::::: .................. ::::::::::::::::: ..................................................... A:::::::::::::::: .::::::::::::::::: ::::::::::::::::: :::::::::::::::::: ::::::::::::::::: :::::::::::::::::: x:::::::::::::: :::::::::::::::::: ::::::::::::::::: ::::::::::::::::: ..................................................... E.. ............... .::::::::::::::::: :::::::::::::::::: :::::::::::::::::: :::::::::::::::::. :::::::::::::::::: ::::::::::::::::: zz:::::::::::::::: ::::::::::::::::: :::::::::::::::::. .................. ................. .::::::::::::::i::. :::::::::::::::::, z::::::::::::::::: ::::::::::::::::: :::::::::::::::::: ::::::::.:::::::: ................................................. :::::::: ::::::::

control

FIG.3

NO

PGEl

::::::::: :::::::: ....... :::::::::::::::::: :::::::::::::::::: :::::::::::::::::: :::::::::::::::::: .................. :::::::::::::::::: :::::::::::::::::: :::::::::::::::::: :::::::::::::::::: :::::::::::::::::: .................. :::::::::::::::::: .................. .................. :::::::::::::::::: ::::::::::::::::::

_I

NO+PGEl

Mean values of the slope a of the aggregation curve XfSD;n=8(*=p
Whittle et al. described the inhibition of platelet aggregation with the CAMP stimulator PGEl (9). Furchgott and Zawadski discovered in 1980 the paradox that acetylcholine (ACh), a potent hypotensive agent in vivo, did not relax isolated vascular strips in vitro. These authors demonstrated ACh-induced vascular relaxation to be dependent on the presence of endothelium. Their results provided evidence for the release of a labile humoral factor which causes relaxation. This substance was called endothelium-derived relaxing

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INTERACTION BETWEEN PGEl AND NO

factor (EDRF) and was shown to be distinct from the vasodilator PGI2 and PGEl (10). Palmer et al. described in 1987 that NO is the active compound of EDRF and the pharmacology of NO and EDRF is indistinguishable (5). In their experiments both EDRF and NO caused a relaxation of vascular strips, inhibited platelet aggregation (3), induced the disaggregation of aggregated platelet (6), and inhibited platelet adhesion (ll), acting via cGMP (4). Furthermore, the biological half-life as inhibitors of platelet aggregation was similar (6). NO is synthesized by the vascular endothelium from the terminal guanido nitrogen atom (12) of the amino acid 1-arginine (13) and acting via an elevation of cGMP (5). Recent results from Minor et al. suggest a possible difference between NO and EDRF. This group found that atherosclerosis does not reduce NO-release while EDRF-release is suppressed (14). These findings are suggestive that NO is only one of the active compounds of EDRF. A synergistic effect between PGI2 and EDRF in vitro has been described by Radomski et al.in 1987 (7). This effect and the fact that PGEl and PG12 similarly inhibited the platelet aggregation via CAMP prompted us to examine the potential additive effect between PGEl and NO. This study clearly showed this synergism. The types of the aggregation curves inhibited by both NO and PGEl were all monophasic and reversible, NO and PGEl significantly reduced the maximum amplitude by 40,6 +23,0%, and suppressed the slope a with 63.00°f15,010indicating a strong inhibition. Recent results showed that Isosorbide-dinitrate, releasing NO in vivo, and PGEl have a synergistic effect in reducing platelet deposition and increasing platelet survival in patients with peripheral vascular disease (15). The synergistic effect between NO and PGEl in vitro on human platelet and in vivo provides an interesting concept for clinical therapy. REFERENCES

1. DENUCCI, G., GRYGLEWSKI, R.J., WARNER, T. and VANE, J.R.Receptormediated release of endothelium-derived relaxing factor and prostacyclin from bovine aortic endothelial cell is coupled. Proc.Natl.Acad.Scic.USA 857: 2334-2338, 1988. 2. MONCADA, S., GRYGLEWSKI, R.J., BUNTING, S. and VANE, J.R. An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation. Nature 263:663-665, 1976. 3. AZUMA, H., ISHIKAWA, M. and SEKIZAKI. Endothelium-dependent inhibition of platelet aggregation. Brit.J.Pharmacol.88:411-415, 1986. 4. RADOMSKI, M.W., PALMER, R.M.J. and MONCADA, S. The role of nitric oxide and cGMP in platelet adhesion to vascular endothelium. Biochem.Bionhvs.Res.Commun. 148:1482-1489, 1987.

5.PALMER, R.M.J., FERRIGE, A.G. and MONCADA, S. Nitric oxide release accounts for biological activity of endothelium-derived relaxing factor. Nature 327:524-526, 1987.

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6. RADOMSKI, M.Wa, PALMER, R.M.J. and MONCADA, S. The nntiaggregatory properties of vascular endothelium: Interactions between prostacyclin and nitric oxide. Brit.J.Pharmacol.92:639-646, 1987. 7. RADOMSKI, M.W., PALMER, R.M.J. and MONCADA, S. Comparative pharmacology of endothelium derived relaxing factor, nitric oxide and prostacyclin in platelet. Brit.J.Pharmacol.92:181-187, 1987.

8. BORN, G.V.R. Aggregation of blood platelet by diphosphate and its reversal. Nature 194:927-929, 1962.

adenosine

9. WHITTLE, B.J.R., MONCADA, S. and VANE, J.R. Comparison of the effects of prostacyclin (PGI2), prostaglandin El and D2 on platelet aggregation in different species. -:373-388, 1978. lO.FURCHGOTT R.F.and ZAWADSKI J.V. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 299:373-376, 1980. ll.RADOMSKI, M.W., PALMER, R.M.J. and MONCADA, S. Endogenous nitric oxide inhibits human platelet adhesion to vascular endothelium. Lancet II:10 57-1068, 1988. 12.BUSSE, R. Stimulation of soluble guanylate cyclase activity by EDRF: a general principle of its vasodilator and antiaggregatoryproperties. Thromb. Res. su~ol VII:l, 1987. 13.PALMER, R.M.J., ASHTON, S.D. and MONCADA, S. Vascular endothelial cells synthesize nitric oxide from 1-arginine. Nature 333:664-666, 1988. D. MYERS,R.,BATES,J.and HARRISON, 14.MINOR, R. L. Jr., Atherosclerosis impairs release of EDRF, but not NO from the vascular endothelium. Circulation sun~l II: 482, 1989. 15.SINZINGER, H., FITSCHA, P., O'GRADY, J., RAUSCHA, F., ROGATTI,WALTRAUD and VANE, J.R. Synergistic effect of prostaglandin Eland isosorbide dinitrate in peripheral vascular disease. Lancet ::627-628, 1990.