Resolution of Prostaglandin Endoperoxide Synthase

Home , Cyclooxygenase, Malondialdehyde, Prostaglandin, Prostaglandin H2, Thromboxane, Thromboxane A2

Proc. Nati. Acad. Sci. USA Vol. 74, No. 9, pp. 3691-3695, September 1977 Biochemistry Resolution of prostaglandin endoperoxide synthase and thromboxane synthase of human platelets (subcellular distribution/12L-hydroperoxy-5,8,10,14-eicosatetraenoic acid/Triton X-100/DEAE-cellulose) SVEN HAMMARSTROM AND PIERRE FALARDEAU* Department of Chemistry, Karolinska Institutet, S-104 01 Stockholm, Sweden Communicated by Hugo Theorell, June 13, 1977

ABSTRACT Thromboxane synthase was localized to the icated human platelets (compare below) sedimenting between microsomes of human platelets. The enzyme was insensitive to 100,000 X g (1 hr) and 300,000 X g (4 hr), 20 mg of protein, sulfhydryl reagents and thiols but was inhibited by 12L-hydroperoxy-5,8,10,14-eicosatetraenoic acid (concentration for were incubated at 370 for 8 min in 15 ml of 50 mM Tris-HCI, 50% inhibition = 0.1 mM). Treatment of microsomes with Tri- pH 7.4/1 mM 2,2'-dipyridyl. The product eluting from silicic ton X-100 solubilized the enzymes that catalyze the conversion acid with diethyl ether/light petroleum (15:85, vol/vol) was of arachidonic acid to thromboxane B2. The solubilized material homogeneous as the methyl ester on thin-layer chromatography was resolved by DEAE-cellulose chromatography into two [RF = 0.64; solvent: diethyl ether/light petroleum (35:65, vol/ components, one converting arachidonic acid to prostaglandins vol)]. SnC12 reduction converted this product to 12L-hy- G2 and H2 and the other converting prostaglandin H2 to droxy-5,8,10,14-eicosatetraenoic acid (RF = 0.41) as judged by thromboxane B2. gas-liquid chromatography/mass spectrometry (4). Thromboxanes are a novel class of compounds derived from Washed Human Platelets and Subcellular Fractionation. prostaglandin endoperoxides (1). They are extremely potent Blood was collected in 77 mM EDTA, 7.5%, vol/vol, from do- as inducers of the platelet release reaction and aggregation (1) nors who had not taken drugs for at least 1 week. Washed and as stimulators of smooth muscle contractions (2, 3). platelets were prepared as described (12). They were suspended Thromboxane A2 is the major component of rabbit aorta con- in 50 mM Tris.HCI, pH 7.4, or in 10 mM potassium phosphate, tracting substance [RCS (2)]. It contains an unstable oxane ox- pH 7.4 (10 and 5 ml/400 ml of blood, respectively), disrupted etane structure and reacts rapidly with water (t1/2 at 370 = 32 by sonication with a Branson model S-125 sonifier (six 5-sec sec) to form a stable derivative (1). This derivative, 8-(1-hy- treatments with 1-min intervals for cooling; power setting: 4) droxy-3-oxopropyl)-9,12L-dihydroxy-5,10-heptadecadienoic or by hypotonic lysis after glycerol loading (13), fractionated acid (thromboxane B2), was originally discovered as a product by differential centrifugation (1,900 X g, 15 min; 12,000 X g, of arachidonic acid metabolism in human platelets (4). An en- 15 min; and 100,000 X g, 1 hr). Plasma membranes (14), dense zyme catalyzing the conversion of prostaglandin endoperoxides bodies, and a granules (15) were isolated as described. Platelet to thromboxanes has been described in platelet microsomes (1, disruption and subsequent manipulations were performed at 5-11). This paper deals with the solubilization of the enzymes 0-4g. in human platelets that convert arachidonic acid to throm- Characterization of Products Formed from PGG2 and boxane A2 and their resolution into prostaglandin endoperoxide PGH2. A mixture of 625 nmol of [1-14C]PGG2 or [1-14C]PGH2 synthase and thromboxane synthase components. (0.6 Ci/mol), 1 mmol of Tris-HCI, pH 7.4, and 30 mg of mi- crosomal protein (12,000 X g-100,000 X g sediment) in a total volume of 20 ml was incubated at 370 for 20 sec. The reaction MATERIALS AND METHODS was stopped by adding 100 ml of SnC12 (5 mg/ml) in dioxane [1-14C]Arachidonic acid (50-60 Ci/mol, The Radiochemical to reduce untransformed endoperoxide to PGF2 (12). Products Centre, Amersham, England) and arachidonic acid (Nu Chek were extracted at pH 3 with diethyl ether, methylated, and Prep., Inc., Elysian, MN) were used for enzyme assays and to separated by preparative thin-layer chromatography (solvent: prepare [1-14C]prostaglandin (PG) G2 and [1-14C]PGH2 (1 diethyl ether/methanol, 49:1, vol/vol). After elution from the Ci/mol) (12). Nordihydroguaiaretic acid was from Bast's Suc- plate with ethyl acetate/methanol (1:1) and conversion to cessors, Ltd., Copenhagen. Thromboxane B2 and prostaglandins methoxime (PGE2), trimethylsilyl derivatives, the following were kindly supplied by Upjohn, Kalamazoo, MI; L-cysteine, compounds were identified by gas-liquid chromatography/ 2,2'-dipyridyl, dithiothreitol, ethylenediamine tetraacetic acid mass spectrometry: 12L-hydroxy-5,8,10-heptadecatrienoic acid (EDTA), ethanethiol, N-ethylmaleimide, beef blood hemo- (HHT), thromboxane B2, PGE2, and PGF2a. globin (Type I), p-hydroxymercuribenzoic acid, 2-mercapto- Enzyme Assays. Incubations with arachidonic acid were ethanol, SnCl2, and L-tryptophan were from Sigma; DEAE- performed in one of two ways: (a) A reaction mixture (0.5 ml) cellulose (DE-52) was from Whatman; silica gel G and Tris were containing [1-14C]arachidonic acid (0.7 Ci/mol; 33 or 66 nmol), from Merck; and silicic acid was from Mallinckrodt. 12L- Tris-HCI at pH 7.4 (25 ,mol), and a platelet subcellular fraction Hydroxy-5,8,10,14-eicosatetraenoic acid (HETE) was prepared was incubated at 37° for 45 sec. Ethanol (2.5 ml) and water (2.5 as described (4). ml) were added, and the mixture was acidified to pH 3 and 12L-Hydroperoxy-5,8,10,14-eicosatetraenoic Acid extracted with diethyl ether. Thin-layer chromatography of (HPETE). Arachidonic acid (2.5 mg) and a fraction from son- Abbreviations: HETE, 12L-hydroxy-5,810,14-eicosatetraenoic acid; HPETE, 12L-hydroperoxy-5,8,10,14-eicosatetraenoic acid; HHT, The costs of publication of this article were defrayed in part by the 12L-hydroxy-5,8,10-heptadecatrienoic acid; PG, prostaglandin; TX, payment of page charges. This article must therefore be hereby marked thromboxane. "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate * Present address: Department of Nephrology, Centre Hospitalier this fact. Universitaire, Sherbrooke, PQ, Canada, J1H 5N4. 3691 Downloaded by guest on September 30, 2021 3692 Biochemistry: Hammarstrom and Falardeau Proc. Natl. Acad. Sci. USA 74 (1977) 4 I 100- I I I 80- I I I 02-- I I a 201 I I 60- % I I + I I I LALC~I40- 0 I .cc0 a- I NX, 8R 60( I -x 20- -X-

I I I I I 30 +86 nmot / 15 10 5 0 PGG2,( Distance from origin (cm) Z 20- 2 )( FIG. 1. Thin-layer radiochromatograms of products formed from E // [1-14CJPGG2 during incubations (370, 20 sec) with native (Upper) c (' or boiled (Lower) platelet microsomes. Excess PGG2 was reduced to PGF20 with SnCl2 at the end of the incubation. 12-OH-17:3, 12L- I hydroxy-5,8,10-heptadecatrienoic acid (HHT); TXB2, thromboxane m 1, B2. Solvent system: diethyl ether/methanol, 49:1, vol/vol. xi-10- +43mPt ++3 nmol PGO'2 methyl esters was performed with diethyl ether/light petroleum, 3:7 (vol/vol) as solvent. (b) A reaction mixture (0.1 ml) containing [1-'4C]arachidonic acid (55 Ci/mol; 2 nmol), he- moglobin (0.2 nmol), L-tryptophan (0.5 ,mol), Tris-HCI at pH 7.4 (10 ,umol), and microsomes, solubilized proteins, or aliquots 5 1015 30 of eluates from DEAE-cellulose chromatography was incubated Seconds for 5 min at 240. Citric acid (0.1 M in water, 1 ml) and throm- FIG. 2. Time course of PGG2 disappearance (Upper) and boxane B2 (10-gg) were added and the mixture was extracted thromboxane B2 formation (Lower). [1-14C]PGG2, 72.5 AM (0 O) with diethyl ether. After washing and methylation, the ether or 145 AM (X- - -X), was incubated with platelet microsomes at 37°. Incubations were stopped with SnCl2 in dioxane and products were extracts were analyzed by thin-layer chromatography in two separated by thin-layer chromatography as shown in Fig. 1. A second solvent systems: ethyl acetate/2,2,4-trimethylpentane/water, addition of substrate was made after 15 sec (arrows). 5:10:10, vol/vol/vol (I) and diethyl ether/methanol, 49:1, vol/vol (II). Assays with were mixture (0.6 ml) contained [1-'4C]PGG2 (0.6 Ci/moi; 43 or 86 PGG2 performed as follows: the reaction nmol), Tris.HCI at pH 7.4 (30,umol), and a platelet subcellular fraction. After 15 or 20 sec at 370, the reaction was stopped with Table 1. Effects of sulfhydryl reagents, thiols, chelators, dioxane (3 ml) containing SnCl2 (5 mg/ml). After extraction nordihydroguaiaretic acid, HPETE, and HETE on thromboxane and methylation, products were analyzed by thin-layer chro- B2 formation by platelet microsomes matography with solvent II. Microsomes, solubilized micro- Concentration, % inhibition of somes, and eluates from DEAE-cellulose columns were assayed Substance mM TXB2 formation* with PGH2 as substrate. The reaction mixture (0.1 ml) containing [1-14CJPGH2 (12 nmol; 1 Ci/mol) Tris-HCl at pH 7.4 Nordihydroguaiaretic 0.1 0 (10,mol), and enzyme was incubated at 240 for 1 min. The acid 0.3 50 reaction was terminated as described above for incubations with 0.5 80 arachidonic HPETE 0.1 50 acid (procedure b). Products were analyzed by 0.2 80 thin-layer chromatography with solvent II. HETE 0.3 15 Thin-layer chromatograms were scanned for radioactivity p-Hydroxymercuri- 1 20 with a Berthold Dunnschicht Scanner II. Thromboxane B2 benzoic acid added as carrier was detected by spraying plates with phos- N-Ethylmaleimide 1 0 phomolybdic acid and heating them briefly. 2-Mercaptoethanol 5 0 Dithiothreitol 3 0 RESULTS Ethanethiol 5 0 L-Cysteine 4 10 Characteristics of Thromboxane B2 Formation by Platelet EDTA 1 10 Microsomes. Incubations of platelet microsomes with [1- 2,2'-Dipyridyl 3 20 14C]PGG2 led to the formation of four products (Fig. 1 upper). These were identified by mass spectrometry as HHT, throm- * Assayed with PGG2 as substrate (see Materials and Methods). boxane B2, PGE2, and PGF2a,. PGF2a was mainly formed by Downloaded by guest on September 30, 2021 Biochemistry: Hammarstram and Falardeau Proc. Natl. Acad. Sci. USA 74 (1977) 3693

Front TXB2 PGE2 PGF2d 4 I11 200 Microsomnes A 100

Triton X-100 B Supernatant 1 redxio0_

0-11 9-1 M. 20a C u Fract ion I

- 100- C6iiC._ 0 0 0n m 20C 0 Fraction I ~0Ix -0 10C a: 1 'I' l

Fraction X

0 15 10 5 0 Distance from origin (cm) FIG. 4. Thin-layer radiochromatograms of products formed from [1 -14C]PGG2 during incubations (240, 1 min) with: (A) platelet microsomes, 195 gg of protein; (B) 100,000 X g supernatant of Triton X-100-treated microsomes, 35 gl; (C) fraction , 90 i1; (D) fraction II, 90 yd; (E) fraction III, 90 pl. Solvent system as in Fig. 3. Distance from origin (cm) mation. Table 1 illustrates the lack of effect of sulfhydryl reagents (p-hydroxymercuribenzoic acid and N-ethylmaleimide), FIG. 3. Thin-layer radiochromatograms of products formed from thiols (2-mercaptoethanol, dithiothreitol, ethanethiol, and L- [1-14C]arachidonic acid during incubations (240, 5 min) with: (A) cysteine), and chelators of metal ions (EDTA and 2,2'-dipyridyl) platelet microsomes, 195 gg of protein; (B) 100,000 X g supernatant of Triton X-100-treated microsomes, 35 pl; (C) fraction I, 35 1A; (D) on the conversion of PGG2 to thromboxane B2. Nordihydro- fraction II, 35 jl; (E) fraction III, 35 AI; and (F) fractions I and III, 35 guaiaretic acid (an antioxidant) and HPETE inhibited the re- + 35 1d. Solvent system: diethyl ether/methanol, 49:1, vol/vol. The action, whereas HETE was relatively ineffective. positions of reference compounds are indicated at the top of the figure Subcellular Distributions of Cyclooxygenase and (the broken line for TXB2 refers to F). Thromboxane Synthase in Platelets. Fractions obtained from disrupted platelets by differential centrifugation and subcellular the SnCl2 reduction of excess PGG2. A parellel incubation with organelles isolated by centrifugation in sucrose-containing boiled microsomes (Fig. 1 lower) gave less HHT, no throm- media were incubated with arachidonic acid and PGG2. The boxane B2, more PGE2, and more PGF2a than incubations with conversion of arachidonic acid to HHT plus thromboxane B2 native microsomes. was used as a measure of cyclooxygenase activity, whereas the Fig. 2 shows time courses of substrate disappearance (upper) conversion of PGG2 to thromboxane B2 was used to assay and thromboxane B2 formation (lower) at 370 and at two sub- thromboxane synthase. The enzyme activities measured in this strate concentrations. With 72.5 1AM PGG2, thromboxane B2 way were similarly distributed among the fractions, the highest synthesis ceased by 15 sec. This was due to substrate depletion specific activities being in the 12,000-100,000 X g sediment (upper), and readdition of 43 nmol PGG2 led to additional (Table 2). Plasma membranes, a granules, dense bodies, and synthesis. The reaction velocity was lower after the second the 100,000 X g supernatant had undetectable activities of these addition of substrate, especially with 145 ,gM PGG2. enzymes. Variations of the pH of the incubation mixture from 6.5 to Solubilization and Resolution of Cyclooxygenase and 9 did not appreciably affect the rate of thromboxane B2 for- Thromboxane Synthase. Platelet microsomes from 800 ml of Downloaded by guest on September 30, 2021 3694 Biochemistry: Hammarstrom and Falardeau Proc. Nati. Acad. Sci. USA 74 (1977)

Arachidonic acid iC-H -CO~~COH MDA < + 1O HHT C-H Cyclooxygenase V.2 02 I OH 0 0"', 0-"""-COH HO O PGG2 <|s"'_ COOH PGD2 O"lO-O

HO OH HS~~ PGE2 PGH2 'o\

Thromboxane Synthase COOH HOQHX O PGF2 d ,%%"" -COOH Thromboxane A2 OH /COOH j 2 OH. PG12 <\"->-CCOOH Thromboxane B2 HO HO O,OHH OH FIG. 5. Formation and transformations of prostaglandin endoperoxides derived from arachidonic acid. MDA, malondialdehyde.

blood were suspended in 1.8 ml of 10 mM potassium phosphate, the same transformations (panels B). None of fractions I, II, or pH 7.4, and mixed with an aqueous solution of Triton X-100 III from the DEAE-cellulose column converted arachidonic to give a detergent concentration of 0.5% (vol/vol). After 30 min acid to thromboxane B2 (Fig. 3 C-E). In contrast, PGH2 was the mixture was centrifuged at 100,000 X g for 1 hr and the transformed by fraction III to thromboxane B2 (Fig. 4E). These supernatant was applied to a 5-ml DEAE-cellulose column (1.1 results show that fraction III contained thromboxane synthase, X 4.8 cm) equilibrated in 10 mM potassium phosphate, pH free of cyclooxygenase. Arachidonic acid was recovered un- 7.4/0.1% Triton X-100. The column was eluted with the same changed after incubations with fractions II and III. Fraction buffer (30 ml) followed by 10 ml each of 20 mM and 0.2 M I, however, formed HHT from arachidonic acid, as judged by potassium phosphate, pH 7.4/0.1% Triton X-100. The eluates, thin-layer chromatography in solvent system I (not shown) and referred to as fractions, I, II, and III, respectively, were con- in addition formed PGE2 (Fig. 3C). Because HHT and PGE2 centrated to 2 ml by ultrafiltration and assayed for enzyme are formed by degradation of prostaglandin endoperoxides activities as described in Materials and Methods. All manip- these results indicated that fraction I contained the cyclooxy- ulations, excluding enzyme assays, were performed at 0-40. genase. This was substantiated by the observation that fractions Thin-layer chromatograms from incubations with [1-'4C]ara- I and III together converted arachidonic acid to thromboxane chidonic acid and [1-14C]PGH2 are shown in Figs. 3 and 4, re- B2 (Fig. 3F). spectively. Panels A show that human platelet microsomes converted either arachidonic acid or PGH2 to thromboxane B2. DISCUSSION The 100,000 X g supernatant after solubilization also catalyzed PGG2 was rapidly transformed to thromboxane B2 by platelet Table 2. Subcellular distributions of fatty acid cyclooxygenase microsomes at 370 (Fig. 2). Partial inactivation of the enzyme and thromboxane synthase in platelets occurred during the reaction, possibly because the substrate has Thromboxane a hydroperoxy group at C-15 which might react with the en- Cyclooxygenase synthase zyme. Thromboxane synthase activity in microsomes was not Platelet specific specific influenced by pH variations between 6.5 and 9, thus resembling fraction activity* activityt catalase in this respect (16). Thiols and sulfhydryl reagents did Disrupted platelets 11.6 17.2 not inhibit thromboxane B2 formation from PGG2 (Table 1), 1,900 X g sediment 5.9 14.8 suggesting that cysteine and cystine residues are not essential 12,000 X g sediment 12.0 47.6 for the catalytic activity of thromboxane synthase. In contrast, 100,000 X g sediment 20.4 84.4 HPETE inhibited this enzyme relatively effectively.. This 100,000 X g supernatant 0 0 compound, which is formed during platelet aggregation (4), Plasma membranes 0 0 might thus serve as a regulator of thromboxane synthesis in vivo. a granules 0 The hydroperoxy group of HPETE was essential for inhibition Dense bodies 0 (HETE did not inhibit) and other hydroperoxides might have a similar effect (compare the discussion above regarding PGG2). Enzyme assays are described in Materials and Methods. been localized * Sum ofnmol of HHT and thromboxane B2 formed from arachidonic Platelet cyclooxygenase has by histochemical acid per mg of protein in 45 sec at 37° (assay a). techniques to the dense tubular system (17). The results in Table t Nanomoles of thromboxane B2 formed from PGG2 per mg of protein 2 are consistent with this because platelet microsomes consist in 20 sec at 370. mainly of this subcellular structure (18). The similar distribu- Downloaded by guest on September 30, 2021 Biochemistry: Hammarstrom and Falardeau Proc. Natl. Acad. Sci. USA 74 (1977) 3695 tions of cyclooxygenase and thromboxane synthase (Table 2) This work was supported by grants from the Swedish Medical Re- suggest that both enzymes are located in the dense tubular search Council (Project 03X-217) and the Medical Research Council system of platelets. This is probably essential for efficient of Canada (fellowship to P.F.). transformation of the labile prostaglandin endoperoxides to thromboxanes. 1. Hamberg, M., Svensson, J. & Samuelsson, B. (1975) Proc. Natl. Fig. 5 shows reactions involved in the conversion of arachi- Acad. Sci. USA 72,2994-2998. donic acid to thromboxane B2. The prostaglandin endoperox- 2. Svensson, J., Hamberg, M. & Samuelsson, B. (1975) Acta Physlol. in Scand. 94, 222-228. ides PGG2 and PGH2, which are intermediates the trans- 3. Hamberg, M., Hedqvist, P., Strandberg, K., Svensson, J. & formation, can also be converted to prostaglandins (PGD2, Samuelsson, B. (1975) Life Sci. 16,451-462. PGE2, PGF2a, and PGI2) or to a C17 hydroxy fatty acid (HHT) 4. Hamberg, M. & Samuelsson, B. (1974) Proc. Natl. Acad. Scd. USA plus malondialdehyde, as shown in this figure. An enzyme ca- 71,3400-3404. talyzing the conversion of arachidonic acid to PGH2 has been 5. Ho, P. P. K., Walters, C. P. & Hermann, R. G. (1976) Biochem. purified to homogeneity from vesicular glands (19-21) and Biophys. Res. Commun. 69,218-224. named prostaglandin endoperoxide synthase or fatty acid cy- 6. Needleman, P., Moncada, S., Bunting, S., Vane, J. R., Hamberg, clooxygenase (EC 1.14.99.1). The transformation of PGH2 to M. & Samuelsson, B. (1976) Nature 261,558-560. thromboxane B2 occurs in two steps: (i) isomerization of the 7. Needleman, P., Minkes, M. & Raz, A. (1976) Science 193, endoperoxide to thromboxane A2 and (ii) nucleophilic attack 163-165. 8. Moncada, S., Needleman, P., Bunting, S. & Vane J. R. (1976) by water to form thromboxane B2 (1). Although Fig. 1 indicates Prostaglandins 12, 323-335. that the overall reaction is enzymic, it is likely that only step i 9. Kulkarni, P. S. & Eakins, K. E. (1976) Prostaglandins 12,465- is catalyzed because step ii proceeds rapidly in the absence of 469. enzyme (1). The enzyme catalyzing step i can be provisionally 10. White, H. L. & Glassman, A. T. (1976) Prostaglandins 12, called thromboxane synthase or prostaglandin endoperoxide: 811-828. thromboxane A isomerase. 11. Ho, P. P. K., Walters, C. P. & Sullivan, H. R. (1976) Prostaglan- Treatment of microsomes from human platelets with Triton dins 12, 951-970. X-100 or Tween 20 resulted in solubilization of the enzyme 12. Hamberg, M., Svensson, J., Wakabayashi, T. & Samuelsson, B. system required for the formation of thromboxane B2 from (1974) Proc. Natl. Acad. Sci. USA 71, 345-349. 13. Barber, A. J., Pepper, D. S. & Jamieson, G. A. (1971) Thromb. arachidonic acid. The solubilized material was resolved into two Diath. Haemorrh. 26,38-57. enzyme components by DEAE-cellulose chromatography. One 14. Barber, A. J. & Jamieson, G. A. (1970) J. Biol. Chem. 245, of the components, which was not adsorbed to the column, 6357-6365. catalyzed the conversion of arachidonic acid to HHT and PGE2, 15. Anderson, P., Slorach, S. A. & Uvnas, B. (1974) Acta Physiol. presumably by nonenzymatic degradation of PGH2 (Fig. 5). Scand. 90, 522-532. This fraction thus contained cyclooxygenase. It has been re- 16. Chance,.B. & Maehly, A. C. (1961) in Biochemists' Handbook, ported before that cyclooxygenase from vesicular glands does ed. Long, C. (E. & F. N. Spon Ltd., London), pp. 383-384. not adsorb to DEAE-cellulose under similar conditions (19). The 17. Gerrard, J. M., White, J. G., Rao, G. H. R. & Townsend, D. (1976) second enzyme component was eluted from DEAE-cellulose Am. J. Pathol. 83, 283-298. with 0.2 M potassium phosphate, pH 7.4. It catalyzed the con- 18.. White, J. G. & Gerrard, J. M. (1976) Am. J. Pathol. 83, 589- in 632. version of PGH2 to thromboxane B2 and, the presence of 19. Miyamoto, T., Ogino, N., Yamamoto, S. & Hayaishi, 0. (1976) fraction I, the conversion of arachidonic acid to thromboxane J. Biol. Chem. 251, 2629-2636. B2. 20. Hemler, M., Lands, W. E. M. & Smith, W. L. (1976) J. Biol. The solubilization and purification of thromboxane synthase Chem. 251, 5575-5579. should facilitate more detailed studies on the biochemistry and 21. van der Ouderaa, F. J., Buytenhek, M., Nugteren, D. H. & van biology in thromboxanes. Dorp, D. A. (1977) Biochim. Biophys. Acta 487,315-331. Downloaded by guest on September 30, 2021