Proc. Natl. Acad. Sci. USAVol. 77, No. 3, pp. 1706-1710, March 1980Physiological Sciences

Dissociation of vasoconstrictor and platelet aggregatory activities ofthromboxane by carbocyclic thromboxane A2, a stable analog ofthromboxane A2

(coronary arteries/thromboxane synthetase/platelet aggregation/sudden death)

ALLAN M. LEFER*, EDWARD F. SMITH III*, HARUO ARAKI*, J. BRYAN SMITHt, DAVID AHARONYt,DAVID A. CLAREMON§, RONALD L. MAGOLDA§, AND K. C. NICOLAOU§*Department of Physiology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107; tCardeza Foundation and Departmentof Pharmacology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107; and §Department of Chemistry, University of Pennsylvania,Philadelphia, Pennsylvania 19104

Communicated by C. Ladd Prosser, December 17, 1979

ABSTRACT Carbocyclic thromboxane A2 [2#(Z),3a-(1E,3R*i3 3-hydrox ctenylfbicyclo[3.1.1]hept-2-yl-5hepte-noic acidi, a stable analog of thromboxane A2, has been testedfor its physiologic properties. Carbocyclic thromboxane A2 isa potent coronary vasoconstrictor, stimulating coronary vascularsmooth muscle at concentrations as low as 29 pM. At 1-5 ,Mit is also an inhibitor of arachidonic-acid- and endoperoxide-induced aggregation of platelets. At 200 nM it stimulated therelease of lysosomal hydrolases from large granule fractions ofliver homogenate. It inhibited thromboxane synthesis inplatelets, although it did not inhibit synthesis of prostacyclinin ram seminal vesicles. Thus, carbocyclic thromboxane A2, amolecule closely related to thromboxane A2, separates coronaryvasoconstrictor from platelet-aggregating activity. The con-strictor activity predominates in vivo; carbocyclic thromboxaneA2 induces coronary vasoconstriction leading to myocardialischemia and sudden death in rabbits in the absence of pul-monary or coronary thrombosis.

Thromboxane A2 (TA2) is an unstable substance (half-life t30sec at pH 7.4 and 370C) produced by blood platelets withpowerful vasoconstricting and thrombotic properties in vitro(1). Although TA2 has not been isolated or chemically synthe-sized, its structure has been proposed (Fig. 1) by Samuelsson andhis collaborators (1) on the basis of its biosynthetic origin andchemical properties. Analogs of TA2 containing the naturallyoccurring pinane nucleus (e.g., PTA2) (2) have been synthe-sized; they produced primarily antagonistic effects towardscoronary artery constriction and platelet aggregation and se-lective inhibition of thromboxane synthetase (2). In this report,we describe the synthesis of enantiomerically pure (+)-carbo-cyclic TA2 (CTA2) [2f(Z),3a(1E,3R *)-3-(3-hydroxy(1-oc-tenyl)-bicyclo[3. 1.1]hept-2-yl-5-heptenoic acid] (Fig. 1), theparent carbocyclic analog of TA2 (3), and present some of itsin vitro and in vivo biological properties.CTA2 is unusual in its activity profile because it is apparently

the most potent coronary vasoconstrictor of the known pros-tanoids and yet possesses no intrinsic ability to aggregateplatelets. In fact, CTA2 appears to be a potent inhibitor ofarachidonic-acid-induced platelet aggregation. Nevertheless,CTA2 induces sudden cardiac death in rabbits by producingsevere myocardial ischemia without coronary or pulmonarythrombosis.

OH OH

TA2 CTA2

FIG. 1. Chemical structures of TA2 and CTA2.

MATERIALS AND METHODSSynthesis of CTA2. The synthesis of CTA2 has recently been

reported (3). For the study of the biological properties of thisimportant stable analog of TA2, we synthesized CTA2 in itsoptically active form by using pure (+)-trans-iodo-1-octen-3-oltert-butyldimethylsilyl ether, [a ]30 = +10.20 (c = 2.59 g/100ml, methanol) (4), for the attachment of the lower side chainas described (3). Thus, the mixed cuprate derived from thisiodide reacted with bicyclo[3.1. ljhept-2-ene-2-carboxaldehydeand the aldehyde so obtained homologated and condensed withthe standard upper prostaglandin side chain to complete theCTA2 skeleton. The final product, CTA2, was chromato-graphically separated from its diastereoisomer [RF = 0.25; silica;ethylacetate/petroleum ether, 7.5:92.5 (vol/vol)] and exhibitedoptical rotation [ a]30 of + 32.60 (c = 0.95 g/100 ml, methanol).Its diastereoisomer (RF = 0.28) had an optical rotation [aj]30 of- 23.90 (c = 1.29 g/100 ml, methanol). Both (+)-CTA2 and itsdiastereoisomer are stable compounds in solution or neat at250C for prolonged periods of time.

In Vitro Biological Methods. CTA2 (up to 10Al of a 2.5 mMsolution in ethanol) was tested for its effect on cat coronaryarteries continuously perfused with 10 ml of Krebs-Henseleitbuffer as described (5). CTA2, from a stock solution in 100%ethanol, was diluted in Krebs-Henseleit solution and tested infinal concentrations of 10 pM-30 nM. In addition, other pros-tanoids, including prostaglandin H2 (PGH2), 9,1 1-azo-PGH2,9,1 1-methanoepoxy-PGH2 (U46619), 9,1 1-epoxymethano-PGH2 (U44069), and thromboxane B2 (TB2), were studied forcoronary vasoactivity. CTA2 was also tested for coronaryvasoactivity in the presence of 9,11-methanoepoxy-PGH2 orPTA2.

Abbreviations: PG, prostaglandin; TA2, thromboxane A2; CTA2, car-

bocyclic thromboxane A2; PTA2, pinane thromboxane A2; TB2,thromboxane B2.

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Proc. Natl. Acad. Sci. USA 77 (1980) 1707

The effect of CTA2 on the release of a cathepsin D (a lyso-somal hydrolase) was determined in large granule fractions ofcat liver as described (6). The greater the release of cathepsinD, the greater the degree of lysosomal disruption.

Platelet aggregation was studied in an aggregometer(Chronolog, Philadelphia, PA) with 0.5 ml of citrated humanplatelet-rich plasma at 370C (7). One minute after addition ofPTA2 (up to 2 Al of a 25 mM solution in ethanol), aggregationwas initiated by addition of sodium arachidonate (0.3-0.5 mM),ADP (2 ,uM), collagen (1 Mg/ml), 9,11-azo-PGH2 (0.1-0.6 AM),9,11-methanoepoxy-PGH2 (U46619) (0.3-0.6 AtM), or 9,11-epoxymethano-PGH2 (U44069) (1-3 AuM). Equal volumes ofethanol produced no effect. CTA2 was also tested for its effectson the inhibition of ADP-induced aggregation by 2 nM pros-tacyclin or 20 nM PGD2.To study the effects of CTA2 on thromboxane synthesis, we

washed rabbit platelets free of plasma, resuspended them inbuffered 0.9% NaCl solution (8), and incubated them at 370Cfor 5 min with CTA2 at 1-100 MM. [1-'4C]Arachidonic acid (0.5MCi, specific activity 50 Ci/mol, New England Nuclear; 1 Ci= 3.7 X 1010 becquerels) was added and incubation was con-

tinued for 15 min; then lipids were extracted by the method ofFolch et al. (9) and subjected to thin-layer chromatography(solvent system C of ref. 10). TB2 concentrations were deter-mined by specific radioimmunoassay as described (11).The effects of CTA2 on prostacyclin synthetase were deter-

mined with sheep vesicular glands as the enzyme source and[1-'4C]arachidonic acid as the substrate essentially as described(12).

Intact Animal Study. New Zealand white rabbits (2.0-3.5kg) were anesthetized with sodium pentobarbital (25 mg/kg)and placed in the supine position. Thegight femoral and ex-

ternal jugular veins and the left common carotid artery were

cannulated with polyethylene catheters. Standard limb needleelectrodes were placed subcutaneously for recording the elec-trocardiogram. The trachea was cannulated with a siliconizedglass tube connected to a pressure transducer for the recordingof airway pressure. Mean arterial blood pressure, central venous

pressure, airway pressure, and lead III of the electrocardiogramwere continuously recorded on a Grass model 7 oscillographicrecorder. Sodium arachidonate (90% pure, Sigma) at 2.0 mg/kgor CTA2 at 125 ,g/kg was injected into the femoral venous

catheter, and the rabbit was observed until death. Two-milliliterblood samples were drawn into 25 mM EDTA just prior to in-jection of drug and just prior to death for radioimmunoassayof TB2 according to established procedures (11). At death,biopsies of heart and lung were taken for histological sec-

tions.

A 10

T(a) (b),; min _j (c) (d)

40 mm Hg mmIx0.02 nM 0.2 nM 2nM 2OnM

B(a) (b) (c) (d)

0.2MM 0.6 pM 2pM 20,4M

FIG. 2. Typical responses of isolated cat coronary arteries to (A)CTA2 and (B) TB2 at low (a), moderate (b and c), and high (d) con-centrations of these vasoconstrictor agents.

60-(A

1 50 -9,1 1-Methanoepoxy-PGH2C

10 _ S vvCTA

(a PGH B

co20 0

CA

10 9 8 7 6 5Concentration, -log M

FIG. 3. Concentration-response relationships of various endo-peroxide and thromboxane analogs in isolated cat coronary arteries.Each point represents a mean of five to seven values + SEM.

RESULTS

CTA2 produced dose-dependent constriction (i.e., increasedperfusion pressure at constant flow) in isolated perfused catcoronary arteries. Fig. 2 illustrates typical responses of coronaryarteries to threshold, moderate, and high concentrations ofCTA2 and TB2. Responses to these agents peaked at 5-8 minand then gradually declined. Fig. 3 summarizes the full con-centration-response relationships for all the endoperoxide-likeand thromboxane analogs studied. CTA2 was the most potentsubstance of those studied followed by azo-PGH2 > 9,11-methanoepoxy-PGH2 > 9,11-epoxymethano-PGH2 >> PGH2>> TB2. Table 1 summarizes the relative potencies of theseagents on cat coronary arteries. CTA2 was 10,000 times morepotent than TB2 with regard to the concentration required toproduce an increase in coronary perfusion pressure of 20 mmHg (EC20). CTA2 was also 4 times more potent than 9,11-azo-PGH2 and 5-6 times more potent than the other two PGH2analogs (9,11-methanoepoxy-PGH2 and 9,11-epoxymethano-PGH2).

In coronary arteries, CTA2 constricted at all concentrationsbut did not antagonize the coronary constrictor effects of9,11-methanoepoxy-PGH2 at concentrations of 3 nM (Fig. 4A).However, PTA2, a thromboxane antagonist, almost completelyantagonized the coronary constrictor effects of CTA2 (Fig. 4B).Thus, CTA2 behaved as a pure thromboxane agonist on thecoronary vasculature without any antagonism of the actions ofendoperoxide-like analogs.CTA2 also induced the release of lysosomal enzymes from

large granule fractions of liver incubates, indicating a lytic ef-fect of CTA2 on lysosomal membranes. In two experiments withlarge granule fractions, 200 nM CTA2 increased the release of/3-glucuronidase by an average of 41% and cathepsin D by 32%.These effects occurred at a lower concentration than thoseproduced by other lysosomal labilizing agents.

Table 1. Relative potencies of endoperoxide and thromboxaneanalogs on cat coronary arteries

RelativeSubstance EC20, M* potencyt

CTA2 1.2 X 10-9 10,0009,11-Azo-PGH2 4.8 X 10-9 2,5009,11-Methanoepoxy-PGH2 6.0 X 10-9 2,0009,11-Epoxymethano-PGH2 7.6 X 10-9 1,500PGH2 3.0 X o-7 40TB2 1.2 X l0-5 1

* EC20, effective concentration resulting in an increase in coronaryperfusion pressure of 20 mm Hg.

t TB2 potency was set at 1.

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FIG. 4. Typical responses of cat coronary arteriesinability of CTA2 to modify the constrictor effect of 9,epoxy-PGH2 (A) and the ability of PTA2 to markedlconstrictor effect of CTA2 (B). W, washout of drug.

With regard to platelet aggregation, CTA2 didas a thromboxane agonist. It failed to induce platetion in vitro up to concentrations of 100 MiM. Morewas a very potent inhibitor of platelet aggregatioprostanoids, including arachidonic acid, 9,119,11-methanoepoxy-PGH2, and 9,11-epoxymetiFig. 5 illustrates typical findings in response tonacid-induced (Fig. 5A) and 9,11-methanoepoxy-PG(Fig. 5B) platelet aggregation. In both cases, 4-_completely prevented platelet aggregation. Howconcentrations (1-4 ttM) were only partially pro

arachidonic acid-induced aggregation, lower coI

of CTA2 (1-2 AM) delayed the onset of plateletwithout actually inhibiting the magnitude of thesponse, indicating an effect on the early phaseaggregation. CTA2 (5 MM) totally inhibited the se

of ADP-induced aggregation. Moreover, 100 nMmented subaggregatory concentrations of epineinduced aggregation.

In addition to inhibiting platelet aggregation, Ceffective inhibitor of thromboxane synthesis. In therange (1-100MM), CTA2 inhibited generation of Tplatelets in a dose-dependent manner. Fig. 6 sumeffect of CTA2 on thromboxane synthetase inhiinhibition of thromboxane synthesis was selective Ethe same concentration range, CTA2 had no effeccyclin synthesis by ram seminal vesicles as measure(

formation of 6-keto-PGFia. Without CTA2, the sen

produqed 66.2% 6-keto-PGFi,.At concentrations o:,MM CTA2, the yields were 62A9% and 60.6%, respectmodest changes were not significantly different frcobtained without CTA2.The profile of CTA2 appears to be unusual

thromboxane and endoperoxide analogs thus fa

Ct I °~0

100

Arachidonic acid 9,11 -Methanoepoxy-PH 2

I_ -4 AM 4CTA2

\ \ \' 2MM CTA2iI

\ \ 1 M CTA2\\0M M CTA \

1 min

A B

FIG. 5. Typical records of arachidonic acid-indu9,11-methanoepoxy-PGH2-induced (B) aggregatiorplatelets. Responses labeled 0 MM CTA2 were with aggralone. Successive responses were with 1-5 AM CTA2.

;showing the,li-methano-[y inhibit the

Lnot behavelet aggrega-,over, CTA2in to various-azo-PGH2,iano-PGH2.

1 OOr

c

0

Cur._

E0

I-em

W-0

C

0

._

._DE

50-

-IO

10CTA2, AMm

50 100

FIG. 6. Effect of CTA2 (1-100 uM) on the percent inhibition ofTB2 formation by washed rabbit platelets.

arachidonic CTA2 is a potent coronary vasoconstrictor that does not an-

,H2-induced tagonize the constrictor effects of endoperoxide analogs. It thus

ItM CTA2 behaves as a potent thromboxane agonist on the vasculature.rever, lower However, CTA2 acts as though it is a thromboxane antagonist

itective. For on platelet aggregation. It does not induce platelet aggregationicentrations itself, and it antagonizes the aggregatory action of arachidonicaggregation acid and endoperoxide analogs. This is consistent with the lackoverall re- of aggregation in vivo by CTA2 when it produced suddenof platelet death. Moreover, it selectively inhibits the formation of TB2.,cond phase It was therefore of considerable interest to assess the overall[CTA2 aug- effect of CTA2 in intact animals to determine whether its cor-

phrine and onary vasoconstrictor effect or its inhibition of platelet activity

predominated. CTA2 was compared to arachidonic acid with,TA2 was an regard to the latter's ability to induce a thrombotic relatedmicromolar sudden death in rabbits. Arachidonic acid was selected since'B2 by rabbit its mechanism of producing sudden death is well known. Fig.kmarizes the 7 illustrates a typical response of an anesthetized rabbit to 125bition. This Mg of CTA2 per kg given intravenously. Within 1-2 min, ar-

xecause over terial blood pressure started to decline, central venous pressure

t on prosta- rapidly increased, and respiration rate increased markedly butd bypercent the depth of respiration became very shallow. Additionally, the

vesicles electrocardiogram exhibited a prominent S-T segment elevation

Iland 100 followed by an enlarged broadened T-wave indicative of

tiyely. These myocardial ischemia. By 3 min, severe hypotension occurred,)0n the value respiration became ineffective, and the S-T segment elevation

and T-wave became even more prominent. At 9 min, deathamong the ensued. Radioimmunoassay of blood samples from rabbits in-ir reported. jected with 125 Mg of CTA2 per kg revealed no increase in cir-

culating TB2 concentrations, values averaging 2.2 4 0.75.0 MM CTA, pmol/ml before CTA2 injection and 2.3 I 0.7 pmol/ml just-2.0 MM CTA2 prior to death. On autopsy, no thrombosis or platelet aggregates-1.5 MCTA2 could be detected in either the coronary or pulmonary bed, and-1.5

CTA2, this was verified by histological sections of the heart and lungs.1.0 M CTA Fig. 8 gives typical examples of sections from heart and lungs

CTA2 from a rabbit killed by CTA2 compared with one killed byarachidonic acid. In addition to the absence of thrombosis,

-0.0 MM CTA2 vessels from the CTA2-injected rabbits exhibited constrictedcoronary vessels. In contrast, there were prominent thrombosed

iced (A) and vessels in the lung and an absence of constricted coronary arterya of human vessels in arachidonic-acid-injected rabbits. In addition toegatory agent myocardial ischemia, apnea and perhaps cerebral ischemia

could have contributed to the sudden death.

A B

100&|--1 0 min--w

mm Hg

t tlw IrCTA (3 nM)

9, 1 -Methanoepoxy-PGH P 2(i1 M) 2 PTA2 (5 nM)

CTA2 (3 nM) 0_

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Proc. Natl. Acad. Sci. USA 77 (1980) 1709

min 0 3 6 9

c,,~~~~"Vwl100"0oE 0J t

CTA2 f

,-i min--

Respiration

ECG

FIG. 7. Typical oscillographic records of the response to 125 ,gof CTA2 per kg given intravenously at arrow to an anesthetized rabbit.MABP, mean arterial blood pressure; CVP, central venous pressure;respiration record is of intratrachial pressure; ECG is lead III of theelectrocardiogram. Death ensued 9 min after CTA2 injection.

Table 2 summarizes the effects of arachidonic acid and CTA2on sudden death in rabbits. Arachidonic acid produced a muchmore rapid death than CTA2, and the dose required to consis-tently produce death with CTA2 was less than one-tenth thatof arachidonic acid. CTA2 produced evidence of severe myo-

CTA2 AA

_ ~~~~w_4 tj 0 .:

H 4'^ -y sS t >w<;< za Ee . ,

ip

.~~~~~~~~~~~~~~~~~i

.~~~~~~~~~o

1 00 anm

FIG. 8. Representative photomicrographs of a small artery fromheart (H) and lung (L) from a rabbit killed by CTA2 or arachidonicacid (AA). (X400.) The pulmonary vessel from the rabbit injected witharachidonic acid is totally thrombosed, and a few erythrocytes are seenin the coronary vessel from the same rabbit. However, essentially noblood cells or thrombosis is present in the vessels from the rabbit givenCTA2-

Table 2. Prostanoid-induced sudden deathArachidonic

Variable* acid CTA2

Time to death, sec 103 + 5 643 ± 99Dose producing sudden death 2 mg/kg 125 ,gg/kgPulmonary thrombosis Prominent AbsentMyocardial ischemia Absent ProminentCoronary vasculature Dilated Constricted

* Values are means ± SEM for four rabbits in each group.

cardial ischemia without pulmonary thrombosis, whereasarachidonic acid produced widespread pulmonary thrombosiswithout significant myocardial ischemia, as previously shownby Silver et al. (13). Arachidonic acid induced a large increasein circulating TB2 concentrations from 1.5 ± 0.5 pmol/mlinitially to 9.6 + 1.7 pmol/ml just prior to death in four rabbits(P < 0.001), whereas CTA2 did not elevate TB2 values. Thus,the pathophysiology of CTA2-induced sudden death is quitedifferent from that of arachidonic acid-induced sudden death.In two rabbits, PTA2 (1 Amol/kg per hr) starting 30 min priorto CTA2 prevented death.

DISCUSSIONCTA2 is a compound that has unique properties among pros-tanoids thus far known. It is different from TA2, the parentcompound of the naturally occurring thromboxanes (1, 14), inthat CTA2 appears not to induce platelet aggregation as TA2is thought to do. We know of no unequivocal experiments thatclearly attribute induction of platelet aggregation to TA2. Inthis regard, Needleman et al. (15) have suggested that theprimary activity of TA2 is vasoconstriction and that any po-tential effects on platelets may be secondary to its actions onvascular tone. Whether this concept is valid in intact animalsremains to be proven. However, if this concept proves to becorrect, CTA2 may be more of an agonist of TA2 than a partialagonist and partial antagonist.

Both CTA2 and PTA2 selectively inhibit the generation ofTA2 by platelets in a specific manner. The effect is a relativelypotent one, complete inhibition occurring at 100,M for bothCTA2 and PTA2 (2). Moreover, both CTA2 and PTA2 fail toinhibit prostacyclin synthetase at 100 1AM, indicating a specificenzyme inhibition. In the present study, we observed CTA2inhibition of thromboxane synthesis in washed rabbit plateletsas well as in intact rabbits subjected to CTA2-induced suddendeath.Whereas PTA2 is a likely candidate for use as an an-

tithrombotic and antishock agent, CTA2 is not because it alsoproduces myocardial ischemia and killed four of five rabbitsinto which it was injected. In contrast, PTA2 moderates theextent of ischemic damage to the myocardium after acutemyocardial ischemia in cats (16) and improves survival intraumatic shock in rats (17). In both cases, PTA2 acted as a ly-sosomal stabilizing agent in addition to inhibiting and antago-nizing TA2. Although, we do not know what CTA2 does in theischemic myocardium, it could contribute to the cell damageby virtue of its lysosomal labilizing action.The severe vasoconstrictor effect of CTA2 could produce

coronary vasospasm, now recognized as a significant cause ofischemia, particularly under conditions where coronary ob-struction is not evident (18). In fact, in patients demonstratingsevere angina without obstructive coronary artery disease, highconcentrations of TB2 have been found in coronary venousblood (unpublished data). Clearly, CTA2-induced vasocon-striction and lysosomal labilization would be deleterious incirculatory shock, acute myocardial ischemia, or other ischemic

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disorders in which the reduced flow and enhanced release ofhydrolytic enzymes into the circulation could act in concert todepress myocardial performance and impair cardiovascularreserve capacity.CTA2 is an interesting thromboxane analog that may have

important use as a biochemical and physiological tool inthromboxane research. It has very potent vascular effects (i.e.,threshold concentration in cat coronary artery is 29 pM). It maybe useful in separating vascular actions from effects of plateletaggregation and thrombus formation in pathophysiologicprocesses. Thus, CTA2 may help clarify the cardiovascularactions of thromboxanes. Furthermore, the sudden cardiacdeath m6del may be a useful technique in assessing the role ofthromboxanes in disease states as well as a model in which totest pharmacologic substances as possible protective agents insudden cardiac death.The specific mechanism of action of the thromboxane re-

ceptor antagonism of CTA2 is not clear at the present time.However, CTA2 appears to be a better thromboxane receptorantagonist than a thromboxane synthetase inhibitor. CTA2 maymodulate the transport of calcium ions across cellular mem-branes, effectively mobilizing intracellular calcium, or it mayaffect the ratio of cyclic AMP to cyclic GMP (19). Further in-vestigations will be necessary to clarify these and other sub-cellular actions of CTA2. It is also not clear why CTA2 is dif-ferent from TA2. Perhaps the carbon substitutions for the oxanering oxygens in the currently proposed structure of TA2 mayaccount for this difference.

We thank Mary Ann Spath and Judith Komlash of Thomas JeffersonUniversity for their expert technical assistance. This work was finan-cially supported in part by Contract HV-E2931 from the NationalHeart, Lung and Blood Institute and by Thomas Jefferson Universityand the University of Pennsylvania.

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2. Nicolaou, K. C., Magolda, R. L., Smith, J. B., Aharony, D., Smith,E. F., III & Lefer, A. M. (1979) Proc. Natl. Acad. Sci. USA 76,2566-2570.

3. Nicolaou, K. C., Magolda, R. L. & Claremon, D. A. (1979) J. Am.Chem. Soc., in press.

4. Kluge, A. F., Untch, D. G. & Fried, J. H. (1972) J. Am. Chem.Soc. 94,7827-7832.

5. Ogletree, M. L., Smith, J. B. & Lefer, A. M. (1978) Am. J. Physiol.253, H400-H406.

6. Lefer, A. M., Ogletree, M. L., Smith, J. B., Silver, M. J., Nicolaou,K. C., Barnette, W. E. & Gasic, G. P. (1978) Science 200, 52-54.

7. Smith, J. B., Ingerman, C., Kocsis, J. J. & Silver, M. J. (1973) J.Clin. Invest. 52,965-969.

8. Bills, T. K., Smith, J. B. & Silver, M. J. (1976) Biochim. Biophys.Acta 424, 303-314.

9. Folch, J., Lees, M. & Sloane-Stanley, G. H. (1957) J. Biol. Chem.226,497-509.

10. Nugteren, D. H. & Hazelhof, E. (1973) Biochim. Biophys. Acta326,448-461.

11. Ferraris, V., Smith, J. B. & Silver, J. B. (1977) Thromb. Hae-mostas. 38, 20.

12. Cottee, F., Flower, R. J., Moncada, S., Salmon, J. A. & Vane, J.R. (1978) Prostaglandins 14, 413-423.

13. Silver, M. J., Hoch, W., Kocsis, J. J., Ingerman, C. M. & Smith,J. B. (1974) Science 183, 1085-1087.

1.4. Samuelsson, B. (1976) in Advances in Prostaglandin andThromboxane Research, eds. Samuelsson, B. & Paoletti, R.(Raven, New York), pp. 1-6.

15. Needleman, P., Minkes, M. & Raz, A. (1976) Science 193,163-165.

16. Schror, K., Smith, E. F., III, Bickerton, M., Smith, J. B., Nicolaou,K. C., Magolda, R. & Lefer, A. M. (1980) Am. J. Physiol.7, H87-H92.

17. Lefer, A. M., Araki, H., Smith, J. B., Nicolaou, K. C. & Magolda,R. C. (1979) Prostaglandins Med. 3, 139-146.

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