305

Biochimica et Biophysics 0 Elsevier/North-Holland

Acta,

488 (1977)

Biomedical

305-311

Press

BBA 57034

ENDOPEROXIDES

AND THROMBOXANES

STRUCTURAL DETERMINANTS VASOCONSTRICTION

AMIRAM

RAZ

*, MARK

S. MINKES

Departments of Pharmacology St. Louis, MO. 63110 (U.S.A.) (Received

FOR PLATELET

and PHILIP

and Surgery,

AGGREGATION

AND

NEEDLEMAN

Washington,

University

School

of Medicine,

April 6th, 1977)

Summary Structural requirements of prostaglandin endoperoxides for conversion to thromboxanes and for inducing platelet aggregation were compared utilizing five endoperoxides: prostaglandin H2 which aggregates platelets and forms a H3 which does not potent vasoconstrictor thromboxane A *; prostaglandin aggregate but does form thromboxane A3; prostaglandin H1 which neither induces aggregation nor forms vasoactive thromboxane A,; and two endoperoxides, one from fatty acid C!,,:, which induces aggregation but does not form a vasoactive thromboxane, and the other from fatty acid C19:3 with actions similar to prostaglandin H1. Utilizing these endoperoxides, receptor sites for aggregation and for thromboxane vasoconstriction have been distinguished by their structural requirements as well as their biological consequences. The aggregating receptor site requires an endoperoxide molecule with a C6 or C7 alkyl a-chain containing a double bond two carbons from the ring. An * Present address: Department of Biochemistry, Tel Aviv Univeaity,

Ramat-Aviv, Tel-Aviv, Israel.

Abbreviations: Sa,lla~pidioxy-l5(S)~ydroperoxy-5-cis,l3-transprosta&ndin Gz (PGG2). prostadienoic acid; prostaglandin HI (PGHj ). 9~,llcu-epidioxy-15(S)-hydroxy-l3-trans-prostaenoic acid; prostaglandin Hz (PGH2). 9a,lla-epidioxy-l5(S)-hydroxy-5-cis,l3-trans-prostadienoic acid: prostaglandin 90i,lla-epidioxy-l5(S)-hydroxy-5-cis.l3-trans,l7-cis-prostatrienoic H3 (PGH3L acid: Prior-PGH2 derived from 4,7,10,13-nonadecatetraenoic acid), Sa,lln-epidioxy-15(S)-hydroxy5-cis.13-Hans-2-nor-prostadienoic acid; 2-nor-PGH1 (PGHl derived from 7.10.12~nonadecatrienoic acid). 9a,lla-epidioxy-15(S)-hydroxy-13-tmns-2-nor-prostenoic acid. Thromboxane A2 (TxA2) presumed structure (ref. 9) is

The structure of thromboxanes ~1 is similar but without the B-&-double thromboxane A3 contains an additional c&double bond at Cl 7.

bond while that of

306

additional recognition factor involves the w-chain since aggregation does not occur if this chain contains additions unsaturation at Cl7 (pros~gland~n H3), In contrast, the thromboxane synthetase enzyme can utilize an endoperoxide substrate molecule only if it contains a C, alkyl Q-chain with a double bond two carbons from the ring. Additional unsaturation at n-3 (prostaglandin H,) does not affect thromboxane formation. The results indicate that certain unique structural features of prostaglandin endoperoxides and not their conversion to thromboxanes are responsible for their pro-aggregatory activity. Thus thromboxane formation does not appear to be an essential process in endoperoxide-induced platelet aggregation. Finally the potent vasoconstrictor property of some thromboxanes can be dissociated from their capacity to cause platelet ag~egationIntroduction Arachidonic acid and the endoperoxide intermediates prostaglandins G2 and Hz induce platelet aggregation [l---4]. Furthermore, endogenous synthesis of these endoperoxides has been demonstrated during platelet aggregation [5,6]. Prostaglandins H2 and Gz are converted by a platelet microsomal enzyme (thromboxane synthetase) into a highly unstable potent rabbit aorta constrictor substance now identified as thromboxane A2 (TxAz) [7--g]. This compound also caused platelet aggregation [7] and was suggested to be the substance responsible for platelet aggregation induced with ~achidoni~ acid or the endoperoxides. We recently observed f4f that pros~gla~din HZ-induced aggregation of human platelet-rich plasma is associated with little if any prustaglandin thromboxane A2 formation. Furthermore, we demonstrated [ 41 that prostaglandin H3 (endoperoxide from 5,8,11,14,17-eicosapentaenoic acid) was converted by platelet thromboxane synthetase into a potent rabbit aorta contracting substance thromboxanz A3, but that neither prostaglandin H3 nor thromboxane A3 caused platelet aggregation. Although thromboxane A2 possesses both vasoconstriction and pro~ag~egatory properties, the data with prostaglandin H3 suggested that these two properties are dissociated and implicated the endoperoxide as the active substance responsible for platelet aggregation. We report here further evidence for this supposition and also describe the differential structural features of the endoperoxides for induction of plate. let aggregation and for conversion to vaso~onstri~tor thromboxanes. Materials and Methods Human platelet-rich plasma was prepared as described previously [ 41. Washed human platelet suspensions were prepared by a modification of a previously described method 151. The various endoperoxides were added to an aggregometer cuvette, followed by addition of plasma or washed platelet suspension. After the reaction, the contents of the aggregometer cuvette were injected over rabbit aorta strips (superfused with Krebs-Hen~le~t media at 10 ml/min) to measure the generation of rabbit aorta-contracting substance activity. The potent ao~a-contrasting activity generated from arachidonic acid or endo-

peroxides by washed platelet suspension has been identified as thromboxane A2 [7]. Prostaglandin endoperoxides H, and Hz were prepared from 8,11,14eicosatrienoic acid (C&3, n-6) and arachidonic acid (C&:4, n-6) respectively, and were purified as recently described [4,10]. Prostaglandin H3 (from 5,811, 14,17eicosapentaenoic acid; Czoz5, n-3) and the prostaglandin H endoperoxides from the fatty acids 4,7,10,13nonadecatetraenoic acid (C,,,,, n-6) and 7,10,13nonadecatrienoic acid (C,,,, , n-6) were prepared and purified similarly using a tracer amount of [1-‘4C]C20:3 acid (1 pg C2,,:,/mg fatty acid). The Cl9 endoperoxides were designated Z-nor-endoperoxides. Lacking a radioactive precursor or sufficient material for chemical analysis, the 2-nor-prostaglandin Hz prepared from the C1,:, acid, and the 2-nor-prostaglandin Hi prepared from C,9:, acid were characterized by their ability to contract the isolated rabbit aorta strip, and by the half-time for spontaneous decay in aqueous media (measuring vasocontrictor potency). The 2-nor-endoperoxides had a 5 min half-life (in 0.05 M potassium phosphate buffer, pH 7.8, 37°C) similar to that of prostaglandin H 1, prostaglandin H, and prostaglandin H3 [4]. The 2norendoperoxides were further characterized by their thin-layer chromatographic mobility in light petroleum/diethyl ether (20 : 80, v/v, developed at -20°C) which was identical to prostaglandin Hi, and by their decomposition in aqueous media to 2norprostaglandin E 1 or 2-nor-prostaglandin Ez, respectively (purified by thin-layer chromatography, chloroform/methanol/acetic acid/water (90 : 8 : 1 : 0.8, v/v), mobility identical to prostaglandins E, and E,). The 2-nor-prostaglandin E compounds thus isolated possessed biological activity in the superfused rat stomach bioassay, the activity being abolished by conversion to the corresponding 2-nor-prostaglandin B compounds by alkaline treatment (0.5 M methanolic KOH, 6O”C, 30 min). Results

and Discussion

2-Nor-prostaglandin Hz was found to be a potent agent for platelet aggregation in both platelet-rich plasma (Fig. 1) and washed platelets (Fig. 2). Low doses of endoperoxides added directly to plasma produced reversible aggregation; higher doses produced complete and irreversible aggregation (Fig. 1). Reversible aggregation induced by low concentrations of prostaglandin H2 could be augmented to produce full aggregation by preincubation of the endoperoxide with platelet microsomes. The augmented aggregation so produced was associated with generation of rabbit aorta-contracting substance (during the preincubation) which was previously demonstrated to be thromboxane AZ [7,9,4]. In contrast, similar experiments with 2-nor-prostaglandin Hz indicated no augmentation of either aggregation or rabbit aorta contraction (Fig. 1). Furthermore, no generation of rabbit aorta-contracting substance was observed during incubation of 2-nor-prostaglandin Hz or 2-nor-prostaglandin Hi with platelet microsomes (data not shown). These results strongly suggest that the two prostaglandin H endoperoxides from the Cl9 fatty acids are not converted into thromboxanes and/or some other very potent labile vasoconstrictor substance. Due to the limited amounts of Cl9 fatty acids we could not chemically (e.g. by thin-layer chromatography) demonstrate the lack of formation of the 2-nor-thromboxanes. It is therefore possible that 2-nor-thromboxane AZ and/or

308

Fig. 1. Effects of prostaglandin endoperoxides and tbramboxanes on aggregation of platelet-rich plasma (top panel) and the simultaneous generation of rabbit aorta contractile activity (bottom panel). Aggregation (3’7’C. stirring) was carried out with 0.4 ml of platelet-rich plasma using a Payton aggregometer. The arrow indicates the addition of agonist to plasma. Aggregation induced by arachidonic acid was performed by adding the required amount of sodium arachidonate solution (5 ma/ml, pH 8.5) to 0.4 ml of plasma in the aggregometer cuvette. Aggregation induced by the endoperoxides was measured by evaporating an ahquot of the endoperoxide solution (2%-50 &g/ml acetone) in the cuvettr foflowed immediately by addition of 0.4 mi of pfasma. Thromboxanes were generated by preincubation (in the euvette) of the endoperoxide (in 40 j&i of 0.05 M phosphate buffer, pH 5.8) with 10 pi of aspirin-treated piatetet microsomes at O°C for 2 min followed by addition of 0.4 mI pfasma 141. When testing for rabbit aorta-eontraetinn activity the contents of the aggregometer cuvette were removed 2 min after initiation of the reaction and injected over a rabbit thoracic aorta strip. The sma.U rabbit aorta-contracting activity produced by addition of the endoperoxides to plasma is due to the direct constrictor activity of the endoperoxides. The following abbreviations were employed: 2-nor-PGHZ, prostaglandin Hz obtained from Cr9:4 acid: 2-nor-PGHr. prostagtandin Hf obtained from Cr9:~ acid: APM, aspirin-treated platelet mierosomes prepared as described previously [4] ; AA. sodium araehidonate.

Fig. 2. Dose-dependent aggregation of washed platelet suspension by arachidonic acid and prostaghandin endoperoxides (3) and the concomitant generation at rabbit aorta contraetitc activity (A). The rabbit aorta contraction data is corrected for the direct contrattile response produced by the endoperoaides tested in the absence of platekts. Abbreviations are indicated in the legend for Fig. 1. Washed platelets were prepared as prwiousi~ described 151. Indomethacin (INDO). 10 &ml, was preincubated with the 0.4 mI washed human platelets for 2 mm at 37’C.

309

2-nor-thromboxane A, are formed, but are devoid of the potent stimulatory effect on the specific contractile receptor in the rabbit aorta. The unique differences between the various endoperoxides in their proaggregatory properties and their ability to produce rabbit aorta-contracting substance (vasoconstrictor thromboxanes) are illustrated in experiments with washed platelets (Fig. 2). 2-Nor-prostaglandin Hz was similar to prostaglandin H2 (although somewhat less potent) in inducing a dose-dependent platelet H2 did not proaggregation, but unlike prostaglandin Ha, 2-nor-prostaglandin duce potent constriction of the aorta. The aggregation by either endoperoxide was not blocked by indomethacin (Fig. 2) and therefore appears not to be due to stimulation of endogenous synthesis of prostaglandin H2 or thromboxane AZ. Prostaglandin H3 exhibited effects opposite to 2-nor-prostaglandin Hz; it was converted to a vasoconstrictor thromboxane yet did not induce aggregation. 2-Nor-prostaglandin H1 was similar to prostaglandin Hi in failing to induce aggregation or be converted to an aorta vasoconstrictor. 2-Nor-prostaglandin H, is a useful tool for studies on the mechanism of endiperoxides-induced aggregation since it enables the study of the pro-aggregatory effects of endoperoxides per se without possible effects due to conversion of the endoperoxides to thromboxanes. The data presented here for 2-nor-prostaglandin Hz and for prostaglandin H3 demonstrates the dissociation of two unique properties of the prostaglandin endoperoxides, namely their ability to induce platelet aggregation and their conversion to a vasoactive thromboxane. Although we had onlv a limited number of endoperoxides available for testing, these results demonstrate the remarkable and differential specificity for recognition of the endoperoxide molecule by the platelet aggregatory receptor and by the platelet microsomal thromboxane synthetase enzyme. The aggregatory site requires an a-chain which contains a double bond two carbons from the ring (Fig. 3A). The receptor does not have a restricted requirement for the number of methylene groups in the (Ychain since endoperoxides from both arachidonic acid (R, is (CH2)3-COOH) and the C,,:, fatty acid (R, is (CH*)*-COOH) are active in inducing aggregation. An additional recognition factor involves the Rz alkyl side chain since aggregation does not occur with endoperoxides having additional unsaturation in the w-chain (e.g. prostaglandin H3). In contrast, the platelet thromboxane synthetase enzyme requires a prostaglandin endoperoxide substrate molecule which contains a C7 alkyl a-chain with a double bond two carbons from the ring (Fig. 3B). The w-chain could contain four methylene groups as in prostaglandin H2 (Rz is (CH2)&H3) or have an additional unsaturation as in prostaglandin H3 (Rz is CH2-CH=CH-CH2-CH3). Among the several endoperoxides we compared, prostaglandin H2 is structurally unique since it contains the determinants for both inducing aggregation and for conversion to a vasoconstrictor thromboxane. Hence with prostaglandin HZ, these two properties are concurrently being expressed when studying its effect on platelets. Additional compounds need evaluation for an expanded consideration of the chemical requirements necessary for receptor interaction. Such structure vs. activity studies could be fruitful in predicting agonists and possible antagonists of either the aggregatory or the vasoconstrictor properties of the endoperoxides. The results presented here indicate that conversion of prostaglandin endo-

310

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Fig. 3. Differential structural requirements of endoperoxides for inducing platelet aggregation (A) and for conversion to thromboxanes (B). The table indicates the chain length and unsaturation of the two alkyl chains. Hatched boxes in the table indicate essential structural features necessary for formation of vasoconstrictor thromboxanes. Dark boxes indicate structural requirements necessary for induction of platefet aggregation. The symbol A denotes double bond.

peroxides to thromboxanes is not an essential biochemical step in the induction of platelet aggregation by endoperoxides or arachidonic acid. Furthermore, the oata with prostaglandin H3 indicate that the potent vasoconstrictor property of thromboxanes is dissociated from the ability to induce platelet aggregation.

We thank Drs. D.A. Van Dorp and D.H. Nugteren, Unilever Research Laboratories, Vlaardingen, The Netherlands, for generously supplying the C19 fatty acids. We also thank A. Wyche and SE. Denny for valuable technical assistance. This work was supported by HL-14397, SCOR-HL-17646, HEW Surgical Training Grant GM-00371 and an American Heart Grant-in-aid 75-883. References Vargaftig, B.B. and Zirinis, P. (1973) Nat. New Biol. 224,114-116 Silver, M.J., Smith, J.B., Ingerman. C. and Kocsis, J.J. (1973) Prostaglandins 4, 863-875 MaImsten. C., Hamberg, M., Svensson, J. and Samuelsson, B. (1975) Pro&. Natl. Acad. Sci. U.S. 72, 1446-1450 Needleman, P., Minkes. M. and Raz, A. (1976) Science 193,163--165 Hamberg, M., Svensson. J., Wakabayashi, T. and Samuefsson, B. (1974) Proc. Natl. Acad. Sci. U.S. 71.345-349

311

6 Smith. J.B.. Ingerman, C.. Kocsis, J.J. and Silver, M.J. (1974) J. Clin. Invest. 53, 1468-1472 7 Hamberg, M.. Svensson, J. and Samuelsson, B. (1975) Proc. Natl. Acad. Sci. U.S. 72, 2994-2998 8 Bunting. 8.. Moncada, S.. Needleman. P. and Vane, J.R. (1976) 9 Needleman. 261.558-560

Br. J. Pharmacol, 56, 344

P., Moncada. S., Bunting, S., Vane, J.R., Hamberg, M. and Samuelsson. 8. (1976)

10 Raz, A., Schwartzman, M. and Kenig-Wakshai, R. (1976)

Eur. J. Biochem. 70.89-97

Nature