MODULATION OF HUMAN PLATELET ADENVLATE CVCLASE BY PROSTACVCLIN (PGX) R. R. Gorman, S. Bunting,l and 0. V. Miller Department of Experimental Biology The Upjohn Company Kalamazoo, MI 49001

SUMMARY: Prostacyclin (PGX) (5Z)-9-deoxy-6,9a-epoxy-A5-PGF1, has been found to be a potent stimulator of CAMP accumulation in human platelet rich plasma (PRP), and a direct stimulator of platelet microsome adenylate cyclase. Prostacyclin is, on a molar basis, at least 10 times more potent a stimulator of CAMP accumulation in platelets than PGE,. The prostacyclin stimulation of platelet CAMP accumulation can be antagonized by the prostaglandin endoperoxide PGH,, and a PGH,-induced platelet aggregation is antagonized by prostacyclin. A model of platelet homeostasis is proposed that suggests platelet aggregation is controlled by a balance between the adenylate cyclase stimulating activity of prostacyclin, and the CAMP lowering activity of PGH,. INTRODUCTION: Kloeze was the first to report that PGEl inhibits human platelet aggregation (1). This early report was followed by a number of papers that linked the PGEl inhibition of platelet aggregation with a stimulation of platelet CAMP levels (2,3,4). From these data a hypothesis was advanced that suggested that agents that decrease platelet CAMP enhance aggregation, while agents that increase CAMP inhibit platelet aggregation (5). Recent studies have shown that the prostaglandin endoperoxides PGG2 and PGH2 induce human platelet aggregation (6), and inhibit PGEl-stimulated CAMP accumulation in platelets (7). Thromboxane A2 (TXA2) which is derived from PGH, also induces platelet aggregation (8) and inhibits PGE,-stimulated CAMP accumulation (9). Whether or not PGH2 must be converted to TXA, in order to induce aggregation is controversial (lo), but TXA, is certainly, on a molar basis, more potent than PGH*. The predominant precursor fatty acid for prostaglandin biosynthesis in platelets is eicosatetraenoic acid (arachidonic) (11). Because of the paucity of the precursor fatty acid for the synthesis of PGEl (eicosatrienoic), it has been difficult to assign a function to PGE in platelet homeostasis. Moncada et al (12) and Gryglewski et al 113) have reported 'Present address:

Wellcome Research Laboratories, Langley Court, Beckenham, Kent BR3 3BS, England

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PROSTAGLANDINS an activity which they called PGX that inhibits human platelet aggregation, and relaxes some vascular smooth muscle. PGX can be derived from either arachidonic acid or PGH2 (14). It is biosynthesized in vascular walls, but not in platelets. Bunting et al (14) have shown that platelets can serve as the source of PGH2 for the synthesis of PGX in the arterial wall. The structure of PGX has recently been determined by Johnson et al (15), and found to be (5Z)-9-deoxy-6,9a-epoxy-A5-PGF1,or simply prostacyclin (Figure 1). In this report we will show that prostacyclin is a more potent stimulator of CAMP accumulation in platelets than any previously described prostaglandin, and that this stimulation is antagonized by PGH2.


Fig. 1.

,a (Prastacyclin)

Structure of Prostacyclin

MATERIALS AND METHODS: Experiments were performed with human platelet rich plasma (PRP) prepared by withdrawing blood directly into 3.8% (v/v8) trisodiumI citrate, followed by centrifugation at 200 x g for 10 min at room temperature. The complete radioimmunoassay kit for CAMP was purchased from Collaborative Research. [sH] CAMP was purchased from International Chemical and Nuclear (I.C.N.), and was subsequently purified by thin layer chromatography (16). Platelet CAMP levels were measured by incubating 1.0 ml of PRP with the appropriate prostaglandin and/or metabolite of arachidonic acid metabolism at 37°C. The reactions were then terminated by the addition of 5% TCA and immediately frozen in liquid nitrogen. L3HlCAMP was added to determine recoveries. After thawing, the platelets


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were disrupted in a Brinkman Polytron for 30 seconds and were shaken at 4°C for 30 min. The protein precipitate was removed by centrifuging in a Sorvall RC-3 centrifuge at 5000 x g for 30 min. The resulting supernatants were extracted 3 times with a lo-fold excess of water saturated ether, and the residual ether of the aqueous extract was evaporated under nitrogen in a 50" water bath. The radioimmunoassay for CAMP was done according to Steiner et al (17), with the incorporation of the acetylation modification of Harper and Brooker (18). All samples were tested at two dilutions. Previous work from our laboratory has shown that all of the imnunodetectable CAMP could be destroyed by beef heart phosphodiesterase, and that our results were the same with or without column separation and purification prior to radioimmunoassay (7). Human platelet microsmes were prepared according to Needleman et al (19), and adenylate cyclase was assayed according to Rodbell (20) with modifications as reported by Pohl et al (21), from the rate of formation of cyclic AMP from [32~]~~~. The complete reaction mixture contained final concentrations of 1.0 mM Tris/[32P]ATP (I.C.N., 40-70cpm/pmol), 5 mM MgC12, 25 mM Tris-HCl (pH 7.4), 0.1% bovine serum albumin, and an ATP-regeneration system consisting of 20 mM creatine phosphate and creatine kinase (1 mg/ml) (Sigma, 50 units/mg). All incubations contained 10 mM theophylline. Reactions were initiated with 40-80 pg of membrane protein. Incubations were done at 30°C for 10 min in a total volume of 0.05 ml, and the reaction stopped by the addition of 0.10 ml of a solution of 34 mM sodium dodecyl sulfate, 40 mM ATP, 0.15 PCi of cyclic [3H]AMP, plus sufficient cyclic AMP to give a final concentration of 12.5 mM. After boiling for 3.5 min, the reactants were diluted with 0.4 ml of water, and the cyclic AMP was purified by sequential passage through Oowex AG 5OW-4X (Bio-Rad) and BaSO, columns (22). The eluate from the BaSO, column was collected directly into counting vials, suspended jn 15 ml of scintillation fluid, and counted in a Packard model 3375 liquid scintillation spectrometer. The identity of the 32P-labelled product of the adenylate cyclase assay was verified by paper chromatography to be cyclic AMP (23). Recovery of cyclic AMP from the column chromatography was 40-50%. Aggregation studies were done on the Payton Aggregation Module according to Nishizawa et al (24) and reported as percent transmission, with the aggregation induced by 2.8 PM PGH, taken as 100% transmission. PGH2 was prepared biosynthetically according to Gorman et al (25), and the prostacyclin by total organic synthesis according to Lincoln and Johnson (26). RESULTS: The aggregometer trace in Fig. 2A shows a PGH,-induced aggregation of human PRP. Coincubation of PGH2 with 2.2 nM prostacyclin results in

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a marked inhibition of the PGH2-induced aggregation - (Fig. ZB). A similar degree of inhibition is found with either 22 nM PGDs or PGEl (Fig. 2C, 2D). These data indicate that on a molar basis, prostacyclin is approximately 10 times more potent against a PGHs-induced aggregation than either PGEl or PGDs.


PGH2 + 2.2 nM Prostacyclin

IJ Fig. 2.

Inhibition of PGH2-induced platelet aggregation by prostacyclin. Human PRP was incubated with either (a) 2.8 uM PGH2; (b) 2.8 PM PGHs + 2.2 nM prostacyclin; (c,d) 2.8 PM PGH2 + 22 nM PGEl or PGD2. Data presented as percent transmission with the aggregaion induced by 2.8 vM PGH2 taken as 100%.

The time course of CAMP accumulation in PRP in response to 0.28 I.IM prostacyclin or PGEl is shown in Fig. 3. The maximum stimulation of CAMP levels due to PGEl occurs after 30 set, while the maximum prostacyclin response occurs at 60 sec. Both the rate of synthesis, and the total amount of CAMP generated is greater in the presence of prostacyclin than with PGE1. Prostacyclin also maintains the elevated levels of CAMP for a longer period than PGEl (Fig. 3).


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In ', 3 1 m;

400 -


;1 2 6


s iz

PGEl _*___-___---___-----_-_-_* 10 30


I 120

I 300 Seconds

Fig. 3.

Time course of platelet CAMP changes in response to PGE, and prostacyclin. One ml of PRP (0.48 x 10s platelets/ml) was preincubated for 2 min at 37" with 0.2 mM isobutylmethylxanthine. At that point either 0.28 uM PGE, or prostacyclin was added and allowed to incubate for the appropriate length of time. Reactions were terminated by the addition of 0.8 ml of 5% TCA and rapid freezing in liquid nitrogen. Data presented as net increase over basal CAMP levels (32 pmoles/lOs platelets).

This apparently does not involve an inhibition of the CAMP phosphodiesterase by prostacyclin, since our incubation contained 2 x 10m4M isobutylmethylxanthine. The dose response curves for the prostacyclin and PGEl-stimulation of CAMP accumulation are shown in Figure 4. Prostacyclin, at a concentration of 0.028 MM, is about 10 times more potent a stimulator of CAMP accumulation than PGE1. At higher concentrations, 0.28 and 2.8 PM, prostacyclin is also more potent than PGE1. The exact differences are difficult to calculate since the dose response curves are not parallel. These measurements were done at 1 min, where the prostacyclin response is maximal, but the PGEl response has declined from

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the maximum. This may account for some, but certainly not all of the non-parallelism in the dose response.


IIM Prostaglandin

Fig. 4.

Dose response curve of CAMP accumulation due to PGEl and prostacyclin. One ml of PRP (0.42 x log/ml) was preincubated for 2 min at 37", and then either 0.028, 0.28 or 2.8 UM PGEl or prostacyclin was added and allowed to incubate for an additional one minute. Reactions were terminated by the addition of 0.8 ml of 5% TCA and rapid freezing in liquid nitrogen.

The accumulation of CAMP cyclin or PGEl is antagonized PGH;!antagonism, on a percent concentration of prostacyclin

in PRP in response to either prostaby the endoperoxide PGH, (Fig. 5). The basis, becomes more pronounced as the or PGE, is reduced.

The prostacyclin directly stimulates adenylate cyclase in platelet microsomal preparations (Fig. 6). As with the experiments in PRP, the dose response curves indicate that prostacyclin is more effective than PGEl in stimulating adenylate cyclase in these membrane preparations. However, the differences between prostacyclin and PGE, are not as pronounced as those observed in PRP. This may be due to receptor damage during membrane isolation, differences in lipophilicity, or to other unknown factors.


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1000 -

n + 2.9/A

900 -


800 Q =

700 -

2 m Z $

600 -

e ;

500 400-




2.8 pM

Fig. 5.



2.8 pM

0.28 PGEl

Inhibition of prostacyclin and PGEl-stimulated CAMP accumulation by PGH,. One ml of PRP was incubated for 1 min at 37" with either prostacyclin or PGEl at the indicated concentrations. Half of the tubes contained 2.8 VM PGHz. Incubations were terminated with 0.8 ml of 5% TCA and rapid freezing in liquid nitrogen. Data are presented as mean f S.E.M. of tri licate sam les. The basal level of CAMP (31 pmoles/lO! plateletsP has been subtracted from all values.

DISCUSSION: Since our original observation that PGHz was an inhibitor of hormone-stimulated adenylate cyclase in fat cell ghosts (27), we have been interested in the endoperoxide-thromboxane family as regulators of cyclic nucleotide metabolism. Upon the realization that most of the pharmacological actions of PGH, could be thought of as anti-CAMP (28) we expanded our studies with PGH, to platelets where CAMP was strongly implicated in platelet homeostasis (2,3,4). We found both PGH, and TXA, were potent inhibitors of PGE,-stimulated

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700 -



II ”

I 0.0028 pM

Fig. 6.

I 0.028 Prostaglandin

I 0.28

I 2.0

Dose response stimulation of platelet microsome adenylate cyclase by PGEl and prostacyclin. Seventy-two micrograms of microsomal protein was incubated for 10 min at 30" with either 0.0028, 0.028, 0.28, or 2.8 PM PGEl or prostacyclin. Data presented as the mean value of triplicate determinations.

CAMP accumulation in platelets (7,9). However, we had difficulty identifying a prostaglandin that increased CAMP in platelets, and is produced in sufficient quantities to counterbalance the potent endoperoxide-thromboxane system. The discovery of PGX by Moncada et al (12) and Gryglewski et al (13) and their reports that PGX inhibited human platelet aggregation suggested to us that PGX was probably the potent stimulator of adenylate cyclase that we had been seeking.


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The most appealing property of PGX was that it was derived from arachidonic acid through the endoperoxij de PGH,. This meant that for the first time two products could bl e derived from a common precursor fatty acid, one which induced platelet aggregation and inhibited CAMP accumulation ITXA,). and an other (PGX), that inhibited platelet aggregation, presum'ablyby increasing platelet CAMP. The structural elucidation (15) and to'tal synthesis (26) of PGX (prostacyclin) permitted us to study d irectly the effects of prostacyclin on platelet CAMP metabolism. The prostacyclin has proved to be the most potent stimulator of CAMP accumulation that we have ever used. In both PRP and platelet microsomal preparations, prostacyclin is, on a molar basis, at least 10 times more potent a stimulator of CAMP accumulation than PGE,. Not only is prostacyclin a more potent stimulator of CAMP accumulation in PRP than PGE,, but the increase in CAMP due to prostacyclin persists for a longer period of time. Of particular importance is the finding that PGH, can antagonize the prostacyclinstimulation of CAMP accumulation in PRP. Moncada et al (12)have proposed that a balance between the thromboxane generating system in platelets and the prostacyclin generating system in vascular endothelium regulates platelet aggregation. We support their proposal, and as a result of the data presented in this report have constructed the postulated mechanism of platelet homeostasis illustrated in Figure 7. Platelets when stimulated to aggregate, produce PGH2. This endoperoxide can either be converted to TXA,, which initiates vasoconstriction, and a platelet aggregation that is associated with an inhibition of CAMP accumulation, or escape the platelet and be converted to prostacyclin by the vessel wall. Prostacyclin can then dilate the vessel, and stimulate platelet adenylate cyclase which will retard the aggregation and platelet thrombus formation that has been induced by the endoperoxide-TXA2 system. In the model, we suggest that prostacyclin can also be produced from arachidonic acid in vessel walls. However, the amounts of prostacyclin synthesized, and relative importance of this vascular source is not known (14). We propose that the balance between the CAMP lowering activity of the PGH2-thromboxane system in the platelet, and the CAMP stimulating properties of prostacyclin, controls human platelet aggregation. Prostacyclin has been found in other cell types besides vascular tissue (13) and the reciprocal regulation of CAMP levels by the endoperoxide-thromboxane system and prostacyclin may be of upmost importance in understanding cellular regulation in these tissues as well.

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Fig. 7.


Model of human platelet homeostasis.


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Modulation of human platelet adenylate cyclase by prostacyclin (PGX).

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