Inhibitory action of soluble elastin on thromboxane B2 formation in blood platelets

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Biochimica et Blophvsica Acta, 797 (1984)348 353 Elsevier

BBA21679

INHIBITORY ACTION OF S O L U B L E ELASTIN ON T H R O M B O X A N E B2 F O R M A T I O N IN B L O O D PLATELETS KEIZO SEKIYA and HIROMICHI OKUDA

Department of Medical Biochemistry, School of Medicine, Ehime University, Shigenobu, Onsen-gun, Ehime 791-02 (Japan)

(Received October 19th, 1983)

Key words: Elastin; Platelet aggregation; Arachidonic acid," Thromboxane B 2 synthesis

Soluble elastin, prepared from insoluble elastin by treatment with oxalic acid or elastase, was found to inhibit the formation of thromboxane 1]2 both from [l-t4C]arachidonic acid added to washed platelets and from [1-14C]arachidonic acid in prelabeled platelets on stimulation with thrombin. In both systems, the formation of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) was accelerated. Oxalic acid-treated soluble elastin at 1 and 10 m g / m l inhibited the formation of thxomboxane B2 from exogenously supplied arachidonic acid 21 and 59%, respectively, and the formation of thromboxane B2 in prelabeled platelets stimulated by thrombin 44 and 94%, respectively. These concentrations of elastin increased the formation of 12-HETE from exogenously supplied arachidonic acid about 3.4- and 7.3-times, respectively. Almost all the added arachidonic acid was converted to metabolites. In prelabeled platelets, soluble elastin at 1 and 10 m g / m l increased the formation of 12-HETE stimulated by thrombin about 1.3- and 2.8-times, respectively, and inhibited the thrombin-induced total productions of thromboxane B z (12-hydroxy-5,8,10-heptadecatrienoic acid (12-HETE) and free arachidonic acid by 26 and 25%, respectively. Elastase-treated digested elastin also inhibited the formation of thromboxane B2 and stimulated the formation of 12-HETE in prelabeled platelets stimulated by thrombin. This inhibitory action of eleastin was not replaced by desmosine. The level of cAMP in platelets was not affected by soluble elastin. Soluble elastin was also found to inhibit platelet aggregation induced by thrombin. However, the inhibitory action of soluble elastin on platelet aggregation cannot be explained by inhibition of thromboxane 112 formation by the elastin.

Introduction Platelet aggregation is induced by a wide variety of stimuli. One of the mechanisms of platelet aggregation is mediated by metabolites of arachidonic acid, particularly thromboxane A 2 [1,21. The interaction between platelets and the vessel

Abbreviations: Hepes, N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid; EGTA, ethylene glycol bis(fl-aminoethyl ether)-N,N'-tetraacetic acid; 12-HETE, 12-hydroxy-5,8,10,14eicosatetraenoic acid; HHT, 12-hydroxy-5,8,10-heptadecatrienoic acid.

walls was shown to be important in the pathogenesis of atherosclerosis as well as in the formation of thrombi [3,4]. The ability of collagen, an insoluble protein in the vessel walls, to induce platelet adhesion and platelet aggregation has been studied extensively. In contrast, little is known about the effect of elastin on platelet function. Ordinas and Rafelson [5,6] reported a distinct interaction between platelets and elastin fibers, but Spaet and Baumgarther [7,8] could not detect any reaction between the two. In 1973, Kabemba [9] reported that the elastin layer of the vessel wall has an antithrombotic action but he did not examine this action. Therefore, in this work we examined the

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effect of elastin on platelet function, especially in arachidonic acid metabolism. Materials and Methods

Preparation of platelets. Whole blood obtained by venipuncture from volunteers was collected in tubes with 1/10 volume of sodium citrate (3.8%, w / v ) as anticoagulant. The blood was centrifuged at 150 x g for 15 min at room temperature and the upper layer of platelet-rich plasma was transferred to plastic tubes. Platelet-poor plasma was prepared from platelet-rich plasma by centrifugation at 1500 x g for 10 rain at room temperature. Platelet-rich plasma and platelet-poor plasma were used for aggregation tests. For studies on arachidonic acid metabolism and cAMP, platelets were prepared from platelet-rich plasma containing 3 mM EDTA by centrifugation at 1500 × g for 10 min at 4°C, washed twice with Hepes/saline buffer (25 mM Hepes, 130 mM NaC1, pH 7.4 at 37°C) containing 2 mM EGTA and resuspended in the same buffer without EGTA. Preparation of soluble elastin and digested elastin. Elastin was purchased from ICN Nutritional Biochemical, Cleveland. Soluble elastin was prepared from elastin by the method of Partridge et al. [10]. Briefly, elastin (1 g) was mixed with 0.25 M oxalic acid (10 ml) and the resulting clear yellow solution was dialysed against distilled water and lyophilized. The weight of lyophilized powder recovered was 50% of that of original elastin. The amino acid composition of the soluble elastin was consistent with that reported by Abatangelo et al. [11]. Digested elastin was prepared from elastin by the treatment of elastase (1/30, w/w, by Eisai Co., Ltd., Tokyo, Japan, 89.7 units/mg) in 0.2 M Tris-HC1 buffer (pH 8.6) at 37°C for 1 h. The final clear yellow solution was heated in boiling waterbath for 10 min, and then dialysed and lyophilized (recovery, 65%). Desmosine was purchased from Elastin Products Co. Inc. Pacific, MO. Arachidonic acid metabolism. A suspension of washed platelets (2-3 mg of platelet protein per ml) was preincubated with soluble elastin or other compounds for 5 rain at 37°C. Then [ 1 - 1 4 C ] arachidonic acid (0.05 #Ci) was added (final con-

centration, 0.9 nmol/0.2 ml per tube) and the mixture was incubated for 5 min at 37°C. The reaction was stopped by adding 0.27 ml of 0.5 N formic acid, radioactive arachidonic acid metabolites was extracted with 4 ml of ethyl acetate and the extract was evaporated under a stream of N 2 gas. The residue was dissolved in a small volume of ethyl acetate, applied quantitatively to a silica gel sheet and developed with chloroform/ methanol/acetic acid/water ( 9 0 / 8 / 1 / 0 . 8 , v/v). Radioactive spots were detected by autoradiography, cut out with scissors and counted in a liquid scintillation counter. Three major metabolites were found [12,13]. Thromboxane B2 was identified by its comigration with authentic material and 12HETE by gas chromatography-mass spectrometry [131. [1J4C]Arachidonic acid was purchased from New England Nuclear. Precoated silica gel sheets were obtained from Merck. Prelabeled platelets were prepared by incubating platelet-rich plasma (10 ml) with [114C]arachidonic acid (3 ffCi) for 1 h at 37°C. Prelabeled platelets were washed once with Hepes/saline buffer containing 2 mM EGTA and suspended in 3 ml Hepes/saline buffer. Aliquots of [1-14C]arachidonic acid-labeled platelets were incubated with soluble elastin or other compounds for 5 min and then stimulated with thrombin for 5 min at 37°C. Metabolites of radioactive arachidonic acid were analyzed as described above. Measurement of cAMP. cAMP was measured in platelet-rich plasma and the washed platelet suspension. Platelet-rich plasma or washed platelet suspension was incubated with soluble elastin for 2 min at 37°C and then trichloroacetic acid (final 6%) was added and the mixture was stirred and centrifuged at 1500 x g for 10 rain at 4°C. Trichloroacetic acid was removed from the supernatant by 3 extractions with water-saturated diethyl ether. cAMP was succinylated and assayed by the radioimmunoassay of Honma et al. [14] with a YAMASA Cyclic AMP Assay Kit (Yamasa Shoyu, Japan). Platelet aggregation. Platelet aggregation was studied by the method of Born [12]. The absorbance of platelet-rich plasma was recorded continuously in an aggregometer.

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Results A

Effect of soluble elastin on exogenously supplied arachidonic acid metabol&m. Thromboxane A2, produced from arachidonic acid by cyclooxygenase of platelet, is known to induce platelet aggregation [1]. Fig. 1 shows that soluble elastin inhibited the formation of thromboxane B2 (a stable derivative of thromboxane A2) from added arachidonic acid, but stimulated the formation of 12-HETE. At each concentration of soluble elastin tested, arachidonic acid was almost completely converted to its metabolites (data not shown). The concentration of soluble elastin inducing 50% inhibition on the formation of thromboxane B2 was about 5 m g / m l .

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Effects of soluble elastin and other compounds on arachidonic acid metabolism in prelabeled platelet. Platelets prelabeled with [1-14C]arachidonic acid produced various radioactive metabolites .of arachidonic acid, especially thromboxane B2, H H T and 12-HETE, on treatment with thrombin. Soluble elastin decreased the formations of thromboxane B2 and H H T and increased that of 12-HETE as shown in Fig. 2. Fig. 3 shows the dose-dependent stimulation of the formations of thromboxane B2 and 12-HETE by thrombin. Based on these results, platelets were stimulated with a concentration of thrombin of 1 M

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Fig. 2. Autoradiograph of thin layer chromatogram of [1-J~C] arachidonic acid metabolites in washed human platelets. Lanes 1 and 9: platelets were incubated with [1J4C]arachidonic acid and [1JaC]arachidonic acid was added after stopping the reaction. Lanes 2-8: platelets were prelabeled with [114C]arachidonic acid. Lanes 1 and 9, control metabolites and arachidonic acid as described in Ref. 14; lane 2, unstimulated platelets; lane 3-8, platelets stimulated with thrombin for 5 min after preincubation with no elastin (lane 3, buffer control) or 0.1 m g / m l (lane 4), 0.3 m g / m l (lane 5), 1 m g / m l (lane 6), 3 m g / m l (lane 7) and 10 m g / m l (lane 8) of soluble elastin. Spots: A, triglyceride; B, unidentified material(s); C. arachidonic acid; D, 12-HETE: E, HHT: F, thromboxane B2; G, origin, phospholipids. Arrow, front of solvent.

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Fig. 1. Effect of soluble elastin on metabolism of added arachidonic acid. Platelets were preincubated with soluble elastin for 5 min before the addition of [1JaC]arachidonic acid. Radioactive spots corresponding to the product of lipoxygenase, 12-HETE, ( O ) and that of cyclooxygenase, thromboxane B2, (e) were counted. Values are means __.S.E. of three experiments.

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Fig. 3. Metabolites of arachidonic acid produced by prelabeled platelets stimulated with thrombin. Prelabeled platelets were stimulated with thrombin and radioactive spots were counted. *, thromboxane B2; O, 12-HETE.

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Fig. 5. Inhibitory effect of soluble elastin on platelet aggregation induced by thrombin. Platelets were treated with 1 m g / m l of soluble elastin (lower curve) for 5 min before the addition of thrombin (0.5 unit/ml).

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Soluble Elnstin (mglml) Fig. 4. Effect of soluble elastin on arachidonic acid metabolism in prelabeled platelets stimulated with thrombin. Prelabeled platelets were preincubated with soluble elastin or buffer for 5 min before stimulation with thrombin, e, TXB2; A, 12-HETE; O. total radioactivity of thromboxane B2, HHT, 12-HHT, 12-HETE and arachidonic acid. Values are means ± S.E. of four experiments.

unit/ml, which caused submaximum stimulation. Fig. 4 shows the dose-dependence of the effect of soluble elastin on thrombin-induced formation of radioactive products in platelet prelabeled with [1-]4C]arachidonic acid. Again it inhibited the formation of thromboxane B2 and stimulated the formation of 12-HETE, but the formation of thromboxane B2 was more inhibited than in the

experiment in Fig. 1. Fig. 4 shows that soluble elastin also slightly inhibited the liberation of total radioactive materials (thromboxane B2, HHT, 12H E T E and arachidonic acid), suggesting that it might bind directly to thrombin or inhibit the release of arachidonic acid from phospholipids in platelets. In the above experiments, soluble elastin prepared by treatment with oxalic acid was used. Elastase-treated digested elastin also inhibited the formation of thromboxane B2 and stimulated the formation of 12-HETE (Table I). Table I shows that insoluble elastin also inhibited the formation of thromboxane B2 although its inhibitory effect

TABLE I EFFECTS OF I N S O L U B L E ELAST1N, ITS DERIVATIVES A N D I N D O M E T H A C I N ON A R A C H I D O N I C A C I D METABOLISM IN P R E L A B E L E D PLATELETS S T I M U L A T E D WITH T H R O M B I N Platelets were preincubated with each substance for 5 min before stimulation with thrombin. Values are m e a n s ± S . E , of four experiments. Additions

Radioactivity (% of control) Thromboxane

Thrombin (1 U / m l ) + digested elastin (1 m g / m l ) + digested elastin (10 m g / m l ) + elastin (1 m g / m l ) + elastin (10 m g / m l ) + desmosine (0.3 m g / m l ) + desmosine (1 m g / m l ) + indomethacin (10- 6 M)

100 54.0 ± 3.4 6.3 ± 0.9 73.6 __.1.4 21.0 + 1.1 96.7 ± 1.3 81.8 ± 7.6 1.2 ± 0.5

B2

12-HETE

Total

100 101.0± 3.7 206.0 ± 22.2 100.3 ± 3.0 116.3±10.4 103.2 ± 0.9 109.7 ± 4.0 310.1 ± 13.0

100 67.2±2.8 57.2±6.6 78.5±0.7 43.4±3.7 97.8±0.8 87.5±5.0 76.0±1.4

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was weaker than that of soluble or digested elastin and did not increase the formation of 12-HETE. Desmosine, a constituent of elastin, did not affect arachidonic acid metabolism in prelabeled human platelets. Indomethacin, which inhibits cyclooxygenase, reduced the formation of thromboxane B2 and increased that of 12-HETE (Table I) [12].

Effect of soluble elastin on the cAMP level in platelets. The cAMP cell level (2 min) in both platelet-rich plasma and washed platelets was not affected by soluble elastin at 1 mg/ml, although prostaglandin E (10 -5 M) increased the cAMP level by 448 and 560%, respectively. Effect of soluble elastin on platelet aggregation. Fig. 5 shows the inhibitory action of soluble elastin on platelet aggregation induced by thrombin. Soluble elastin was also found to inhibit platelet aggregation induced by collagen, epinephrine and calcium ionophore A23187 (data not shown). Discussion In the present investigation, soluble elastin was found to inhibit the formation of thromboxane BE and platelet aggregation induced by thrombin. The stimulation of 12-HETE formation by soluble elastin may be due to increase in the amoung of substrate available for the lipoxygenase reaction. This effect is analogous with that of indomethacin (a cyclooxygenase inhibitor) on platelets (Table I) [12], suggesting that soluble elastin also may inhibit cyclooxygenase activity in platelets. Peptides derived from elastin were reported to have chemotactic activities, which were attributable to desmosine [18,19]. In the present work, desmosine could not replace soluble elastin in inhibition of thromboxane B2 formation (Table I), suggesting that a polypeptide structure is necessary for inhibition of thromboxane B2 formation. Digested elastin and insoluble elastin also inhibited thromboxane B E formation (Table I), but the inhibitory effect of the latter was less than those of soluble and digested elastin. Chemicals that increase the cAMP level in platelet are reported also to inhibit platelet aggregation [18]. However, soluble elastin did not increase the cAMP level in platelets. It is known that when platelets were stimulated by thrombin or other stimuli, arachidonic acid is

released from phospholipids and metabolized via cyclooxygenase and lipoxygenase. A m o n g metabolites of arachidonic acid, thromboxane A is especially important for induction of platelet aggregation; reduction of the rate of thromboxane A 2 formation inhibits platelet aggregation induced by some stimuli. In this experiment, a higher concentration of soluble elastin was needed to inhibit the formation of thromboxane B2 than that needed to inhibit the platelet aggregation induced by thrombin (Figs. 4 and 5). This finding and others [19-21] suggest that the inhibition of platelet aggregation by soluble elastin may not be mediated by the inhibition of the thromboxane A2 formation by soluble elastin. Also it is unlikely that soluble elastin inhibits platelet aggregation by binding calcium, because its binding capacity is low [11,22,23]. Thus, the mechanism of the inhibition of platelet aggregation by soluble elastin remains to be clarified. Elastin is a main component of blood vessel walls, but contradictory results have been reported on its physiological significance in blood vessel walls in relation to platelet adhesion: there are reports that platelets can adhere to fibrous elastin [5,6] and, conversely, that they cannot adhere to elastin [7,8] and also a report that the elastin layer in the vessel walls has antithrombotic activity [9]. It is known or postulated that the interaction between platelets and the vessel walls is involved in the pathogenesis of atherosclerosis and in the formation of thrombi [3,4]. The content of elastin in the aorta is reported to decrease with progress of atherosclerosis [24,25] and with aging [25]. These reports and the present findings raise the possibility that elastin in the vessel walls may be important in inhibition of the beginning and development of atherosclerosis. Experiments are now in progress to test this possibility. References 1 Hamberg, M. and Samuelsson, B. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 2994-2998 2 Vargaftig, B.B., Chignard, M. and Benveniste, J. (1981) Biochcm. Pharmacol. 30, 263-271 3 Ross, R. and Glomset, J.A. (1976) New Engl. J. Med. 295, 420-425

353 4 Weiss, H.J. (1978) New Engl. J. Med. 298, 1344-1347, 1403-1406 5 Ordinas, A., Hornebeck, W., Robert, L. and'Caen, J.P. (1975) Path. Biol. 23, 44-48 (suppl.) 6 Rafelson, M.E., Migne, J., Santonja, R., Derouette, J.C. and Robert, L. (1980) Biochem. Pharm. 29, 943-947 7 Spaet, T.H. and Erichson, R.B. (1966) Thromb. Diath. Hemorrh. Suppl. 21, 67-86 8 Baumgarnter, H.R. (1974) Thromb. Diath. Haemorrh. Suppl. 59, 91-105 9 Kabemba, J.M., Mayer, J.E. and Hammond, G.L. (1973) Surgery 73, 438-443 10 Partridge, S.M., Davis, H.F. and Adair, G.S. (1955) Biochem. J. 61, 11-21 11 Abatangelo, G., Daga-Gordini, D., Garbin, G. and Cortivo, R. (1974) Biochim. Biophys. Acta 371,526-533 12 Hamberg, M. and Samuelsson, B. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 3400-3404 13 Sekiya, K. and Okuda, H. (1982) Biochem. Biophys. Res. Commun. 105, 1090-1095 14 Honma, M., Satoh, T., Takezawa, J. and Ui, M. (1975) Biochem. Med. 18, 257-273

15 Born, G.V.R. (1962) Nature 194, 927-929 16 Senior, R.M., Griffin, G.L. and Mecham, R.P. (1980) J. Clin. Invest. 66, 859-862 17 Hunninghake, G.W., Davidson, J.M., Rennard, S., Szapiel, S., Gadek, J.E. and Crystal, R.G. (1981) Science 212, 925-927 18 Katori, M. (1982) Trends Pharm. Sci. 3, 280-282 19 Fukami, M.H., Holmson, H. and Salganicoff, L. (1976) Biochem. Biophys. Acta 444, 633-543 20 Akkerman, J.W.N. and Holmsen, H. (1981) Blood 57, 956-966 21 Vargaftig, B.B. (1980) Trends Pharm. Sci. 1,415-416 22 Abatangelo, G. and Daga-Gordini, D. (1974) Biochim. Biophys. Acta 342, 281-289 23 Tamburro, A.M., Guantieri, V., Daga-Gordini, D. and Abatangelo, G. (1978) J. Biol. Chem. 253, 2893-2894 24 Kramsch, D.M., Franzblau, C. and Hollander, W. (1971) J. Clin. Invest. 50, 1666-1677 25 Hornebeck, W., Adnet, J.J. and Robert, L. (1978) Exp. Gerontol. 13, 293-298