Proc. Nat. Acad. Sci. USA Vol. 71, No. 2, pp. 345-349, February 1974
Isolation and Structure of Two Prostaglandin Endoperoxides That Cause Platelet Aggregation (15-hydroperoxy endoperoxide/15-hydroxy endoperoxide/platelet aggregation/ contraction of rabbit aorta) MATS HAMBERG, JAN SVENSSON, TOSHIO WAKABAYASHI, AND BENGT SAMUELSSON Department of Chemistry, Karolinska Institutet, S 104 01, Stockholm, Sweden Communicated by Hugo Theorell, September 19, 1978
ABSTRACT Incubation for a short time of arachidonic (kindly provided by Dr. W. Stoffel, Cologne, Germany, see acid with the microsomal fraction of a homogenate of the ref. 2) and Na14CN followed by hydrolysis of the nitrile. The vesicular gland of sheep in the presence of 1 mM p-mer- curibenzoate followed by extraction and silicic acid chemical and radiochemical purity was in excess of 98%, chromatography yielded two prostaglandin endoper- as judged by thin-layer radiochromatography. Part of the oxides. The structures of these compounds, i.e., 15-hy- labeled acid was diluted with unlabeled material to make a droperoxy-9a,11a-peroxidoprosta-5,13-dienoic acid (pros- preparation with specific radioactivity of 0.77 Ci/mol, which taglandin G2) and 15-hydroxy-9a,lla-peroxidoprosta- 5,13-dienoic acid (prostaglandin H2), were assigned mainly was used for incubations with vesicular gland microsomes. by a number of chemical transformations into previously Thin-Layer Chromatography (TLC) was carried out with known prostaglandins. The new prostaglandins were 50- 200 times (prostaglandin G2) and 100-450 times (prosta- plates coated with chloroform-methanol-washed Silica gel G glandin H2) more active than prostaglandin E2 on the super- and the following solvent systems (when not otherwise fused aorta strip. The half-life of the prostaglandin endo- indicated): ethyl acetate-2,2,4-trimethylpentane (55:45, peroxides in aqueous medium (about 5 min) was signifi- v/v) (TLC of methyl esters) and ethyl acetate-2,2,4-tri- cantly longer than that of "rabbit aorta-contracting sub- of free stance" released from guinea pig lung, indicating that methylpentane-acetic acid (50:50:0.5, v/v/v) (TLC none of the prostaglandin endoperoxides is identical with acids). A Berthold Dunnschichtscanner II was used for this factor. Addition of 10-300 ng/ml of the endoperoxides determination of radioactivity on TLC plates. to suspensions of washed human platelets resulted in rapid aggregation. Furthermore, platelet aggregation in- Enzyme Preparation and Incubations. Rinsed microsomal duced by thrombin was accompanied by release of ma- pellets obtained from 8 g of sheep vesicular gland (1) were terial reducible by stannous chloride into prostaglandin suspended and homogenized in 20 ml of 92 mM potassium F2a, thus indicating the involvement of endogenous prostaglandin endoperoxides in platelet aggregation. phosphate buffer pH 7.4 containing 7.2 mg (1 mM) of sodium p-hydroxymercuribenzoate (Sigma Chemical Co.). The mix- Direct evidence for the formation of an endoperoxide during ture was kept at 370 for 2 min and subsequently incubated prostaglandin (PG) biosynthesis was recently obtained (1). for 30 sec with 1.2 mg of [1-14C]arachidonic acid in 0.1 ml of In these experiments a compound, reducible to PGF2, ethanol. The mixture was rapidly poured into a separatory could be detected in incubations of arachidonic acid for a funnel containing 100 ml of 25 mM HCl and extracted twice short time with the microsomal fraction of a homogenate of with diethyl ether. The combined ether phases were washed the vesicular gland of sheep. Preincubation with p-mercuri- twice with water and dried over 75 g of anhydrous MgSO4. benzoate and other SH blocking agents reduced the rate of The residue obtained after evaporation of the solvent was formation of PGE2 as well as the rate of disappearance of immediately dissolved in about 3 ml of diethyl ether-light the intermediate. This compound was also isolated by solvent petroleum (2:8, v/v) and subjected to silicic acid chroma- extraction and thin-layer chromatography and was found to tography. be a 15-hydroxy prostaglandin endoperoxide (1). The present paper is concerned with isolation and determi- Platelet Aggregation. Blood of healthy donors who had not nation of the structure of an additional prostaglandin endo- taken aspirin for 1 week was collected with 7.5% (v/v) of peroxide having a hydroperoxy group at C-15, as well as the 77 mM sodium EDTA and centrifuged at 200 X g for 15 min. effect of the 15-hydroperoxy and 15-hydroxy endoperoxides The platelet-rich plasma thus obtained (280,000-400,000 on platelet aggregation and rabbit aorta strip. platelets per ul) was centrifuged at 650 X g for 15 min. The platelet pellet from 12 ml of platelet-rich plasma was sus- MATERIALS AND METHODS pended in 5 ml of 0.15 M NaCl-0.15 M Tris*HC1 buffer pH [1_-4C]Arachidonic acid, specific radioactivity about 11 Ci/ 7.4-0.077 M sodium EDTA (90:8:2, v/v/v) and subse- mol, was prepared from 1-chloro-4,7,10,13-nonadecatetraene quently recentrifuged. The pellet of washed platelets was suspended in 4 ml of Krebs-Henseleit medium (3) not con- Abbreviations: PGEI, prostaglandin El; PGE2, prostaglandin taining calcium. Platelet aggregation was monitored with a E2; PGF2, prostaglandin F2,; PGG2, prostaglandin G2; PGH2, Born aggregometer (4). To 1 ml of platelet suspensions at prostaglandin H2; TLC, thin-layer chromatography; GLC, gas- 37° were added prostaglandin endoperoxides (10-1000 ng in liquid chromatography; Me3Si, trimethylsilyl; MO, 0-methyl- 1-5 ,ul of acetone) or 5 units of thrombin (Topostasin®, Hoff- oxime. man-La Roche Co.) in 33 IAl of 0.9% NaCl. 345 Downloaded by guest on September 23, 2021 346 Biochemistry: Hamberg et al. Proc. Nat. Acad. Sci. USA 71 (1974) E 3600- HO Q. PGO2 71sX11 R FPGH2 ;2400O =0.64 RF=.46 KI R2 iRF 1 u HO OH 1200- 12 Sn C12 or PGF2v Sn C12 or P EU b 03 03P '. I . 5 0 Dist n .f I c I 1)Pb(OAc)4 HQ Distance from originkcm) Distance from origin (cm) 0-tS~ 2) 03 p 1>S~ 0-I. (I2^
1 'R 1 R E 1200- PGG2 OOH HO 0 PGH2 OH
._ Buffer 15-Keto-PGF2d Buffer U 0 n Sn ,gR .M 400- AR1 Cl2 0 HO H(R2O M. RR2 U 1 1 a: FIbfl HO QOH 2 4 6 8 10 12 14 Hd° OH Fraction number 15-Hydroperoxy-PGE? PGE2 FIG. 1. Lower panel: Silicic acid chromatography of labeled FIG. 2. Reactions performed on PGG2 and PGH2. RI = material obtained after incubation of 1.2 rg of [1-'4C] arachidonic CH2-CH=ICH-(CH2)3-COOH; R2 = (CH2)4-CH3; + = acid with the microsomal fraction from 8 g of vesicular gland in phenyl. the presence of 1 mM p-nercuribenzoate. The column (1 g of silicic acid) was eluted with diethyl ether-light petroleum (2:8, v/v; fractions 1-4), diethyl ether-light petroleum (4:6, v/v; 7.4; ref. 1) at room temperature for 2 min. The material iso- fractions 5-10), diethyl ether-light petroleum (6:4, v/v; frac- lated by extraction with diethyl ether was treated with tions 11-13), and ethyl acetate (fraction 14). Fraction volume, diazomethane and analyzed by TLC. A major peak of radio- 10 ml. Upper panel: TLC of labeled material present in fractions activity (about 90% of the radioactivity applied) with RF = 6-8 of silicic acid chromatography (16% of incubated radio- 0.05 was observed (reference, methyl ester of PGFa, RF = activity, left) and material present in fractions 11-12 (11% of 0.05). Part of this material was rechromatographed in a more- incubated radioactivity, right). Solvent system: ethyl acetate- = 2,2,4-trimethylpentane-acetic acid (50:50:0.5, v/v/v). polar solvent system (water-saturated ethyl acetate; RF 0.35; reference, methyl ester of PGF2a, RF = 0.35). Another part was converted into the Me3Si derivative and analyzed by gas-liquid chromatography (GLC)-mass spectrometry Rabbit Aorta Strip. Spirally cut thoracal aortas, 3.5-4 cm (LKB 9000 instrument; column, 1% SE 30 on Gaschrom Q). from male rabbits weighing about 2000 g were suspended and A single peak with a retention time equivalent to C-24.1 superfused (5, 6) with 2 ml/min of Krebs-Henseleit bicar- appeared (reference, Me3Si derivative of the methyl ester of bonate medium (3) gassed with 96.5% 02-3.5% CO2. The iso- PGF2,, C-24.1; ref. 1). The mass spectrum was identical with metric contractions were recorded by a Grass model FT.03C that of the Me3Si derivative of the methyl ester of PGF2,. force-displacement transducer connected with a Grass model Treatment of PGG2 with SnCl2 in nonaqueous medium 79C polygraph. Prostaglandin endoperoxides (100-2000 ng in (methanol-diethyl ether-light petroleum) gave a mixture of 1-20 til of acetone) were dissolved in 1-9 ml of Krebs medium comparable amounts of PGF2, and 12-hydroxy-5,8,10- and either added to the strip immediately or incubated at 370 heptadecatrienoic acid. The latter component was identified for different times before addition to the strip (about 400 ,ul by analysis of the methyl ester-Me3Si derivative by GLC- added by drops during 15 see). mass spectrometry. The retention time corresponded to RESULTS C-19.3, and the mass spectrum showed ions of high intensity at m/e 366 (M), 335 (M-31; loss of OCHO), 295 IM-71; loss Fig. 1 shows a silicic acid chromatography of the material iso- 225 with the of * (CH2)4CH3], 276 (M-90; loss of Me3SiOH), [M-141; lated after incubation of [1-14C]arachidonic acid loss of * CH2-CH=CH-(CH2)3-COOCH3], and 173 microsomal preparation. The fractions corresponding to the [Me3Si-O+=CH-(CH2)4CH3]. major peaks of radioactivity were pooled and taken to dryness. 1 of and ali- Treatment of PGG2 (50 jig) with mg triphenylphos- The residue was dissolved in 0.5 ml of dry acetone, in ml of ether room temperature for 60 two phine 0.1 diethyl at quots were subjected to TLC (Fig. 1). The compounds of as to be stable in dry diethyl ether and ace- min gave a product containing 80-90% PGF2,,, judged isolated appeared TLC and identification as described above GLC-mass tone at -20°. However, the presence of a sensitive peroxide by by group in both compounds was indicated by the rapid degrada- spectrometry. tion induced by addition of water or ethanol. The less-polar Dehydration by Lead Tetraacetate. The methyl ester of compound (yield from [1-14C]arachidonic acid, 15-20%; PGG2 (about 250 MAg) was dissolved in 0.2 ml of dry benzene RF = 0.64, Fig. 1) will be referred to as prostaglandin G2 and treated with 2 mg of KHCO3 and 6 mg of Pb(OAc)4. (PGG2) and the more-polar compound (yield, 10-15%; After 5 min at room temperature and 5 min at +4°, 7 mg of RF = 0.46, Fig. 1) as prostaglandin H2 (PGH2). triphenylphosphine was added and the mixture was left at room temperature for 30 min. TLC showed the presence of a Structure of prostaglandin G2 (Fig. 2) minor (RF = 0.05; methyl ester of PGF2a, RF = 0.05) and Reduction by SnCl2 and Triphenylphosphine. PGG2 (50 Ag) a major component (RF = 0.13; methyl 9a,lla-dihydroxy- was treated with buffered SnCI2 (25 mg of SnC12 in 5 ml of 15-ketoprosta-5,13-dienoate, RF = 0.13). The 0-methyl- ethanol and 1 ml of 92 mM potassium phosphate buffer pH oxime (MO)-Me3Si derivative of the latter component (C- Downloaded by guest on September 23, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Two Prostaglandin Endoperoxides 347
time (min) Time (min) i 1 2. 6 _0t . ;. 4 .-
E25 ;__ H
FIG. 3. Light transmission recordings in studies of platelet aggregation by prostaglandin endoperoxides. (Left) Platelet suspensions (1 ml, 37°) treated with 5 units of thrombin (A), 55 ng of PGG2 (B), 55 ng of PGH2 (C), and 11 ng of PGG2 (D). (Right) Platelet sus- pensions (1 ml, 370) treated with 18 ng of PGG2 (E,F) and 55 ng of PGH2 (G,H). Suspensions F and H were preincubated for 1 min with 50 Ag of aspirin.
24.2) gave a mass spectrum identical to that of the MO- medium (methanol-diethyl ether-light petroleum), compara- Me3Si derivative of a sample of methyl 9a,11a-dihydroxy-15- ble amounts of PGF2. and 12-hydroxy-5,8,1Q-heptadecatri- ketoprosta-5,13-dienoate (7). enoic acid were formed, i.e., the products formed by the same treatment of PGG2 (see above). Treatment of PGH2 (50 Mg) Isomerization in Aqueous Medium: Formation of 15-Hydro- with 1 mg of triphenylphosphine in 0.1 ml of diethyl ether peroxy-PGE2. PGG2 (150 Mug) in 0.5 ml of acetone was added at room temperature for 60 min gave a product containing to 10 ml of potassium phosphate buffer pH 7.4 and kept at about 90% PGF2,,, as judged by TLC as well as GLC-mass room temperature for 4 hr. The product isolated by extraction spectrometric analysis of the Me3Si derivative. with diethyl ether was treated with diazomethane and sub- jected to TLC. A major peak (60-70% of the applied radio- Isomerization in Aqueous Medium. PGH2 (150 ,ug) in 0.5 ml activity, RF = 0.30) and several minor peaks of less polar of acetone was treated with 10 ml of potassium phosphate derivatives appeared. The major compound was assigned the buffer pH 7.4 at room temperature for 4 hr. The esterified structure 1 1-hydroxy-15-hydroperoxy-9-ketoprosta-5,13-di- product was analyzed by TLC. A major peak (about 70% of enoic acid (methyl ester) on the basis of the following experi- the radioactivity applied, RF = 0.10) was observed (reference, ments. Reduction with 10 mg of SnCl2 in 2 ml of methanol methyl ester of PGEi, RF = 0.10). The major compound was gave a single component that chromatographed with the conclusively identified as the methyl ester of PGE2 by GLC- methyl ester of PGE2 (RF = 0.10). This material was con- mass spectrometric analysis of the MO-Me3Si derivative (1). clusively identified as the methyl ester of PGE2 by analysis of the MO-Me3Si derivative by GLC-mass spectrometry. Isomerization by Silica Gel. PGH2 (50 Mig) was treated with In another experiment, the isomerization product was diazomethane and applied to a TLC plate. The plate was left treated with sodium borodeuteride (98% deuterium, Merck, at room temperature for 2 hr and subsequently developed Sharp, and Dohme, Quebec, Canada) and subsequently with the organic layer of ethyl acetate-2,2,4-trimethyl- subjected to TLC (solvent: water-saturated ethyl acetate). pentane-water (75:75: 100, v/v/v) as solvent. The major part Two peaks of radioactivity in proportions 1:1 appeared of the radioactivity applied (about 70%) appeared in two (RF = 0.35 and 0.23, chromatographing with the methyl peaks with RF = 0.07 and 0.17 (ratio, 1:5, reference: methyl esters of PGF2, and PGF2,6, respectively). The mass spectra ester of PGE2, RF = 0.07). Analysis of the MO-Me3Si deriva- recorded on the Me3Si derivatives showed a molecular ion at tives by GLC-mass spectrometry established the identity of m/e 585 (unlabeled derivatives, m/e 584), i.e., 1 atom of the two compounds with the methyl esters of PGE2 and 11- deuterium was incorporated in the borodeuteride reduction. dehydro-PGF2,, respectively. The ion at m/e 218 (Me3SiO+=C2H-CH=CH-OSiMe3) supported the location of this deuterium at C-9. Platelet aggregation by PGG2 and PGH2 Structure of prostaglandin H2 (Fig. 2) PGG2 and PGH2 induced aggregation of human blood plate- The identity of PGH2 with the previously isolated 15-hydioxy lets (Fig. 3). The extent as well as the reversibility of the aggregation was dependent upon the concentration of the endoperoxide (1) was suggested by TLC [solvent system: organic layer of ethyl acetate-2,2,4-trimethylpentane-water endoperoxides, i.e., small amounts (about 10 ng/ml) gave a (75:75:100, v/v/v)]. The methyl esters of both compounds small and reversible aggregation whereas higher concentra- tions (50-300 ng/ml) gave a pronounced aggregation that was had an RF = 0.49 (1). Proof of the identity of the two com- reversible only to a small extent. Linear relationships between pounds was furnished by the following experiments. log concentration and maximum aggregation were obtained Reduction by SnCI2 and Triphenylphosphine. PGH2 (50 lgg) for both compounds in the concentration range 10-300 ng/ml. was treated with buffered SnCl2, as described above for PGG2. The relative potency of PGG2 and PGH2 was about 3:1. Analysis by TLC of the esterified product showed a major Preincubation for 1 min with 50 ug/inl of aspirin gave no peak (about 90% of the applied radioactivity) with RF = significant change in the maximum aggregation induced by 0.05 (methyl ester of PGF2a, RF = 0.05). The identity of the PGG2 or PGH2. However, a somewhat greater initial slope reduction product with the methyl ester of PGF2
* Hamberg, AL, to be published. t Svensson, J., Hamberg, A. & Samuelsson, B., to be published. Downloaded by guest on September 23, 2021 Proc. A/at. Acad. Sci. USA 71 (1974) Two Prostaglandin Endoperoxides 349
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