RCS from human platelets: Is it only thromboxane?

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European Journal of Pharmacology, 95 (1983) 121-124 Elsevier

Short communication RCS FROM H U M A N PLATELETS: IS IT ONLY T H R O M B O X A N E ? E D W A R D F. SMITH Ill *, W E R N E R R O C K E R and KARSTEN S C H R O R **

Pharmakologisches Institut der Universiti~t KOln, Gleueler Str. 24, D- 5000 KOln 41, West Germany Received 16 August 1983, accepted 8 September 1983

E.F. S M I T H

III, W . R I S C K E R

a n d K, S C H R C ) R , R C S from human platelets: is it only thromboxane?, E u r o p e a n J.

Pharmacol. 95 (1983) 121-124. To determine if inhibition of a rabbit aorta contracting substance (RCS) corresponds to inhibition of thromboxane synthetase human washed platelets were stimulated with thrombin. RCS formation was bioassayed on rabbit aorta strips in Tyrode solution containing selective blocking agents and thromboxane ( T X ) B 2 in the same probes by specific radioimmunoassay. Dazoxiben inhibited the formation of T X B 2 but not of RCS, whereas indomethacin inhibited both RCS and TX formation. The data indicate that the rabbit aorta cannot be used alone to predict inhibition of TX formation with specific TX synthetase inhibitors. Rabbit aorta contracting substance (RCS) Radioimmunoassay

Thromboxane Bioassay

I. Introduction

In 1969, Piper and Vane described the release of a 'rabbit aorta contracting substance' (RCS) from the anaphylactic guinea-pig lung. Later work (Vargaftig and Zirinis, 1973) demonstrated RCS formation during platelet aggregation. RCS from both guinea-pig lung and human platelets consisted of one major component with a half-life of 30-40 s (Svensson et al., 1975) and the material derived from human platelets was characterized as thromboxane (TX) A 2 (Hamberg et al., 1975). These studies stimulated the search for specific inhibit0rs of the TX-synthetase in order to find substances which would prevent the formation of TXA 2, a potent arterial vasoconstrictor and platelet aggregator and, in contrast to cyclooxygenase inhibiting agents, not block the biosynthesis of prostacyclin. Because of the instability of TXA2, * Edward F. Smith Ill was a recipient of a research fellowship from the Alexander von Humboldt-Stiftung, Bonn, West Germany. Present address is: Department of Physiology, Medical University of South Carolina, Charleston, South Carolina, U.S.A. ** To whom all correspondence should be addressed. 0014-2999/83/$03.00 © 1983 Elsevier Science Publishers B.V.

Dazoxiben

Indomethacin

in early studies on TX-synthetase inhibition (Moncada et al., 1977) TXA 2 was bioassayed as RCS using strips of thoracic rabbit aorta (RbA) superfused with buffer solution and a mixture of blocking agents without the parallel measurement of TXB 2 formation e.g. by specific radioimmunoassay (RIA). The purpose of this study was to determine if changes of RCS formation by dazoxiben, a selective inhibitor of TX-synthetase (see Randall et al., 1981) paralleled the inhibition of TX biosynthesis by human platelets.

2. Materials and methods

2.1. Preparation of rabbit aorta strips The thoracic aorta was removed from adult New Zealand White rabbits killed by a blow on the head. The aortas were cut helically into strips (40 × 2 mm) and mounted isotonically (load 3-4 g) in a 10 ml organ bath containing Tyrode solution equilibrated with 95% 02 + 5% CO 2 at 37°C. This T y r o d e solution c o n t a i n e d the following

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antagonists (~mol/1): propranolol (0.8), atropine (0.1), phentolamine (0.7), methysergide (0.6), mepyramine (0.3) and indomethacin (3). Because the RbA is contracted by thrombin, the strips were treated initially with a high concentration of this agent (6 I U / m l ) in order to obtain tachyphylaxis. This procedure prevents further thrombin responses and its efficacy was demonstrated by a second addition of 0.3 I U / m l thrombin after washout. 2.2. Measurement of R C S release Washed human platelets were prepared as described previously (Schr6r et al., 1980). Aliquots of 0.4 ml platelet suspension (8 × 107 platelets) were incubated with 10 /~1 vehicle, dazoxiben or indomethacin in 0.5 ml (final volume) 50 mmol/1 Tris-buffer (pH 7.4) at 37°C for 4 min. Thrombin was added to a final concentration of 0.6 I U / m l ; the mixture was shaken gently for 15 s then added directly to the organ bath. After the peak contractile response, a 1 ml aliquot of the bathing solution was removed, placed into a plastic tube containing 60 ~mol/1 indomethacin and frozen until RIA for TXB 2, using a specific antibody generated in our laboratory (Gallenk~imper et al., 1983). 2.3. Substances and solutions Dazoxiben (Laevosan Pharmaceuticals, Linz, Austria) was dissolved in distilled water. Thrombin ( H o f f m a n n - L a Roche, Grenzach-Wyhlen, F R G ) and imidazole (Sigma, St. Louis, U.S.A.) were dissolved in 0.9% NaC1. Indomethacin (Merck, Sharp and Dohme, Mtinchen, F R G ) was dissolved in 1 m o l / l Tris buffer (pH 8.4) at 10 m g / m l and further diluted with distilled water. Other substances and the Tyrode solution were prepared as previously described (Schr6r et al., 198o). 2.4. Statistics The percent inhibition of RCS and immunoreactive TXB 2 formation was calculated from the average of a control experiment before and after testing of dazoxiben, imidazole or in-

domethacin, respectively. All Values quoted are the mean and standard error of the mean (~ _+ S.E.M.) of n determinations. Statistical significance was determined by means of Student's t-test. P values of less than 0.05 were considered to be significant.

3. Results

Addition of either human washed platelets alone or thrombin (after desensitization) to the assay system did not produce any contractile effect. However, addition of human washed platelet suspension after incubation with thrombin for 15 s was followed by rapid contraction of the RbA, indicating the formation of RCS. This response was prevented by treating the platelets with indomethacin (3 ~ m o l / l ) whereas dazoxiben pretreatment (0.3-100 ~mol/1) of the TX generating system surprisingly did not result in any attenuation of RCS formation. RIA of the bath fluid yielded 3.9 _ 0.3 n g / m l immunoreactive TXB 2 under control conditions (n = 40) and a concentration-dependent inhibition of its formation at 0.1-30 ~ m o l / l dazoxiben (table 1, fig. 1). An original tracing of an experiment in which indomethacin or increasing concentrations of dazoxiben were added to platelets stimulated by 0.6 I U / m l thrombin is shown in fig. 1. The upper panel demonstrates the contractile response in a control experiment. There was no evidence that

TABLE 1 Percent inhibition by dazoxiben of thrombin-induced (0.6 I U / m l ) formation of rabbit aorta contracting substance RCS and formation of immunoreactive TXB 2 from human washed platelets. Data are expressed as mean _+S.E.M. The numbers in parentheses indicate the number of experiments. TXB 2

RCS

Concentration of dazoxiben (ix mol/l) 0.1 0.3 1.0 3.0 10 30 100

3_+2 (7) 14_+6 (8) 7 4 + 7 (17) 93-+2 (6) 95+1 (10) 95-+ 1 (4) 97_+1 (8)

1 + 4(4) -5+

6(6)

-7-+11 (5) -7_+ 9(4)

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5O

-o f "6

< ..Q Cr ~o 25 L.)

E E

r~12

0

-

0 '-

m

/ m

0.3

CON

m

-3

DAZOXlBEN

30

m m

3

IND

CON

Fig. 1. Typical experiment showing the rabbit aorta contraction (upper panel) and immunoreactive TXB 2 (lower panel) determined from the same sample. Dazoxiben had no effect on RCS formation (upper tracings) but inhibited TXB 2 formation by more than 95% at 30/~mol/1. Indomethacin (IND) inhibited both RCS and TXB 2. The biological reactivity of the rabbit aorta strip (RbA) remained unchanged as demonstrated by an unchanged contraction under standard control conditions (CON).

dazoxiben (up to 30 ffmol/1) inhibited the RbA contraction although the cyclooxygenase inhibitor i n d o m e t h a c i n fully inhibited the response. Dazoxiben at 0.3, 3 and 30/~mol/1 inhibited TXB 2 formation by 13, 78 and 94 percent, respectively, while indomethacin inhibited by more than 99 percent at 3/~mol/1. Data similar to those described here for dazoxiben were obtained by using imidazole, a structurally different inhibitor of the thromboxane synthetase (Moncada et al., 1977). Addition of imidazole to the washed platelet suspension prior to thrombin stimulation produced a dose-dependent inhibition of TXB 2 formation which amounted to 47 _+ 5% (n = 4) of the control at 100 /~mol/1. On the other hand, even 4 mmol/1 imidazole added to the platelets did not change the tone of the RbA, i.e. biosynthesis of RCS after stimulation by arachidonic acid (Sauerland, 1983). In additional studies, it was determined that dazoxiben did not interfere with the measurement of TXB 2 or RCS. Addition of 100/~mol/1 dazoxiben directly into the organ bath neither enhanced nor decreased the contractile response to added

thromboxane. Moreover, there was no evidence that dazoxiben at 100 /~mol/l exhibited any cross-reactivity with the TXB 2 radioimmunoassay, as indicated by a less than 0.1% displacement of [3 H]TXB2 from the specific antibody.

4. Discussion

Thromboxane A 2 is an arterial vasoconstrictor and platelet aggregator. Because of its instability in other than bioassay conditions only indirect methods, e.g. chemical measurements of more stable degradation products can be used to quantify TXA 2 formation. Radioimmunoassay generally measures the accumulation of TXB 2 but not the concentration at a particular time point, and the bioassay method is subject to interference by other vasoconstrictors released from activated platelets, such as hydroperoxyfatty acids and serotonin. Indeed, it is apparent from the present experiments that there need not to be a close correlation between the actual amount of thromboxane formed and its biological activity.

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At this point it remains unclear which mediator(s) are responsible for the contraction of the RbA. One possibility is that there is a build-up of prostaglandin endoperoxides due to inhibition of a major metabolizing pathway in the platelet. Although the endoperoxides are assumed to be less potent contractors of the RbA than TXA 2 (Svensson et al., 1975), a reduction in thromboxane formation may allow sufficient endoperoxide to accumulate and to produce the observed response. However, agonist effects of endoperoxides are generally only observed when platelets are stimulated with exogenous arachidonic acid, resulting in large amounts of formed endoperoxides, and not when they are stimulated with adrenaline, collagen or thrombin (Parry, 1983). Alternatively, there may be a shunting of endoperoxides into PGD 2, PGE 2 or PGF2~ (Grimm et al., 1981, Parry, 1983). However, the weak potency of these prostaglandins in contracting RbA and the low quantities formed argue against these compounds being solely responsible. Although recent investigations by Suzuki et al. (1982) have demonstrated that pretreatment of the rabbit aorta with imidazole may potentiate the contractile response of this tissue to various agonists including prostaglandins, the concentrations necessary were much higher than those used here. Finally, tachyphylaxis for thrombin and the inclusion of receptor antagonists against dense granula-derived contracting substances, eg. serotonin and noradrenaline, suggest that these factors are not the primary cause of the RbA contraction. No data are available on a possible synergistic effect of these various substances in contracting the RbA. These studies may help to clarify the nature of the RCS produced subsequent to selective thromboxane synthetase inhibition. In summary, the data suggest that the RbA cannot be used alone to investigate the selective inhibition of the thromboxane synthetase. Similar to what has been found in some platelet aggregation studies (Bertele et al., 1981; Grimm et al., 1981), a large discrepancy may exist between the degree of inhibition of thromboxane formation and the biological effect observed after adding thrombin or arachidonic acid.

Acknowledgements The authors wish to thank Dr. H. Ferber (Laevosan Pharmaceuticals, Linz, Austria) for a generous gift of dazoxiben.

The expert technical assistance of Miss Sigrid R6ttger in performing the radioimmunoassay is also acknowledged. This study was supported in part by the Deutsche Forschungsgemeinschaft (SFB 68, A 17).

References Bertele, V., C. Cerletti, A. Shiepatti, G. Di Minno and G. De Gaetano, 1981, Inhibition of thromboxane synthetase does not necessarily prevent platelet aggregation, Lancet I. 1057. Gallenkamper, W., W. ROcker and K. SchrOr, 1983, Cardioprotective actions of pentoxifylline in an animal model of acute myocardial ischaemia, Br. J. Pharmacol. (in press). Grimm, L.J., D.R. Knapp," D. Senator and P.V. Halushka, 1981, Inhibition of platelet thromboxane synthesis by 7-(1imidazolyl) heptanoic acid: dissociation from inhibition of aggregation, Thromb. Res. 24, 307. Hamberg, M., J. Svensson and B. Samuelsson, 1975, Thromboxanes: a new group of biologically active compounds derived from prostaglandin endoperoxides. Proc. Natl. Acad. Sci. (U.S.A.) 72, 2994. Moncada, S., S. Bunting, K. Mullane, P. Thorogood, J.R. Vane, A. Raz and P. Needleman, 1977, Imidazole: a selective inhibitor of thromboxane synthetase, Prostaglandins 13, 611. Parry, M.J., 1983, Effects of thromboxane synthetase inhibition on arachidonate metabolism and platelet behaviour, Br. J. Clin. Pharmacol. 15, 23S. Piper, P.J. and J.R. Vane, 1969, Release of additional factors in anaphylaxis and its antagonism by antiinflammatory drugs. Nature 223, 29. Randall, M.J., M,J. Parry, E. Hawkeswood, P.E. Cross and R.P. Dickinson, 1981. UK-37, 248. a novel, selective thromboxane synthetase inhibitor with antiaggregatory and antithrombotic activity, Thromb. Res. 23, 145. Sauerland, S., 1983, Zur Wirkung antiinflammatorischer Substanzen auf Plattchenaggregation und TXA2-Bildung in vitro, lnauguraldissertation, KOln. SchrOr, K., S. Sauerland, A. Kuhn and R. ROsen, 1980, Different sensitivities of prostaglandin cyclooxygenases in blood platelets and coronary arteries against non-steroidal antiinflammatory drugs, Naunyn-Schmiedeb. Arch. Pharmacol. 3134 69. Suzuki, Y., Y. Ishida, R.K. Hester and S. Shibata, 1982, Imidazole-induced potentiation of the contractile response to various agonists in vascular smooth muscle, European J. Pharmacol. 83, 223. Svensson, J., M. Hamberg and B. Samuelsson, 1975, Prostaglandin endoperoxides. IX. Characterization of rabbit aorta contracting substance (RCS) from guinea pig lung and h u m a n platelets, Acta Physiol. Scand. 94, 222. Vargaftig, B.B. and P. Zirinis, 1973, Platelet aggregation induced by arachidonic acid is accompanied by release of potential inflammatory mediators distinct from PGE 2 and PGF2, ~, Nature New Biol. 244, 114.