Regulatory role of cyclic adenosine 3′,5′-monophosphate on the plattelet cyclooxygenase and platelet function

44

Biochimica et Biophysica Acta, 582 (1979) 44--58

© Elsevier/North-Holland Biomedical Press

BBA 28759 R E G U L A T O R Y ROLE OF CYCLIC ADENOSINE 3',5'-MONOPHOSPHATE ON THE P L A T E L E T CYCLOOXYGENASE AND PLATELET FUNCTION

FRANCIS A. FITZPATRICK and ROBERT R. GORMAN Pharmaceutical Research and Development, The Upjohn Company, Kalamazoo, MI 49001 (U.S.A.)

(Received May 17th, 1978) (Revised manuscript received August 4th, 1978) Key words: Cyclic AMP; Prostaglandin; oxygenase

Thromboxane A2: Platelet aggregation; Cyclo-

Summary T h r o m b o x a n e A2 plays an important role in arachidonie acid- and prostaglandin H2-induced platelet aggregation. Agents that stimulate platelet adenylate cyelase (prostaglandin 12, prostaglandin I1, and prostaglandin E l ) a n d dibutyryl cyclic AMP inhibit both thromboxane A2 formation and arachidonateinduced aggregation in platelet-rieh plasma. Despite complete suppression of aggregation with agents that elevate cyclic AMP, considerable thromboxane A2 is still formed. Prostaglandin H2-induced aggregations which bypass the cyelooxygenase regulatory step are also inhibited by agents that elevate cyclic AMP without any measurable effect on thromboxane A2 production. These data demonstrate that cyclic AMP can inhibit platelet aggregation by a mechanism independent of its ability to suppress the cyclooxygenase enzyme. Parallel experiments with washed platelet preparations suggest that they may be an inadequate model for studying the relationship between the platelet cyclooxygenase and platelet function. Introduction Prostaglandin endoperoxides and thromboxane A2 are involved in arachidonic acid-induced platelet aggregation [1--8]. Agents which increase cyclic adenosine 3':5'-monophosphate (cyclic AMP) inhibit such aggregations. Investigators have shown that cyclic AMP affects platelet function by regulating the platelet eyclooxygenase, the platelet phospholipase, and the platelet release reaction, either independently or in combination [9--16]. The fatty acid cyelooxygenase, however, appears to be a key regulatory enzyme. Malmsten et al. Abbreviations: gelatin.

HHT, 12I,-hydroxy-8,10-heptadeeadienoie

acid; PBSG, phosphate-buffered

saline w i t h

45 [13] first demonstrated this, by showing that prostaglandin E1 through an increase in cyclic AMP could oppose the influence of arachidonic acid on platelet aggregation by inhibiting the cyclooxygenase. A major physiological role for prostaglandin E~ has been difficult to accept, however, because the levels required to exert an anti-aggregatory effect are not likely to be sustained in vivo, and these levels are inconsistent with the amounts of 8,11,14-eicosatrieonic acid ordinarily available for conversion into prostaglandin E1 [17]. Some data have emerged which suggest that prostagiandin E~ could play a role under basal conditions [18]. Nevertheless, prostaglandin I2, a recently discovered [19,20] cyclooxygenase-derived product, has now assumed the physiological role previously conceded to prostaglandin E~. Evidence to support this proposed role has appeared [21,22]. We have investigated three agents which elevate cyclic AMP, and dibutyryl cyclic AMP, to determine their effects on the platelet cyclooxygenase in detail. Prostaglandin Is was examined because of its proposed role as the physiologically important anti-aggregatory prostaglandin. Prostaglandins I~ and E1 were examined to clarify any parallel role for the 'I' series prostaglandins in regulating platelet function, and dibutyryl cyclic AMP was examined because of contradictory reports regarding its effect on the platelet cyclooxygenase [13--15]. All experiments were performed in both platelet-rich plasma and washed platelet suspensions, and each platelet preparation was aggregated with arachidonic acid and prostaglandin H2 to distinguish, unambiguously, effects on the cyclooxygenase from effects on the thromboxane synthetase. Materials Arachidonic acid, 99%, (NuCheck Prep., Elysian, Minn.); prostagiandins E2, Is, and I1, thromboxane B2 (Experimental Chemistry Laboratories, The Upjohn Co.); [3H]prostaglandin E2, 160 Ci/mmol (NEN, Boston, Mass.); and [3H]thromboxane B2, prepared biosynthetically [23] were examined for purity by thin-layer chromatography prior to use. Prostaglandin H2 was prepared biosynthetically according to Gorman et al. [24]. Dibutyryl cyclic AMP (Sigma, St. Louis, Mo.) was used as received. 0.1 M phosphate buffer (0.1 M Buffer I), pH 7.4, containing 0.9% NaC1/0.1% gelatin/0.05% NAN3, was used for all radioimmunoassay incubations. A specific, high affinity antiserum for the radioimmunoassay of thromboxane B2 was developed experimentally [23]. Methods

Washed platelets. Washed platelets were prepared from fresh human plasma according to Miller et al. [25]. Platelets were resuspended to concentrations of 1.10~--1 • 109 platelets/ml in Ca2+-free Krebs-Henseleit buffer and held at 25°C d.uring use. Platelet-rich plasma. Platelet-rich plasma was prepared from fresh human whole blood collected over citrate (1 part 3.8% citrate/9 parts blood) and centrifuged at 200 × g for 10 min at 25°C. All blood donors were fasted for 8 h and they were free from medication at least 5 days prior to collection.

46

Aggregation procedure. Four c o m p o u n d s were tested to correlate their ability to inhibit the cyclooxygenase and platelet aggregation. Prostaglandins El, I2 and I1 [26] are p o t e n t stimulators of platelet adenylate cyclase [21,22,27]. Dibutyryl cyclic AMP mimics the actions of cyclic AMP. In the control aggregations, either arachidonic acid or prostaglandin H2 was added as a hexane or acetone solution, respectively, to a siliconized aggregometer cuvette and the organic solvent was evaporated under nitrogen. 2 ml of platelet-rich plasma or washed platelet suspension, incubated at 37°C for 2 min were added; the mixture was stirred at 1100 rev./min at 37°C and the ensuing aggregation was monitored with a Payton single channel aggregometer. The c o m p o u n d s to be tested (prostaglandins El, I~, I2 or dibutyryl cyclic AMP) were incubated with platelet-rich plasma (2.0 ml) or washed platelet suspensions (2.0 ml) at 37°C for 2 min. The platelets were then transferred quantitatively to a second cuvette containing the same amount of arachidonic acid or prostaglandin H2 as the control aggregation. The platelets were stirred at 1100 rev./min at 37°C and the aggregation patterns were monitored with a Payton aggregometer. All c o m p o u n d s were studied at three concentrations to distinguish dose-dependent effects from non-specific effects. Samples (0.1 ml) were withdrawn from the cuvette during aggregation for the analysis of t h r o m b o x a n e B2 and prostaglandin E2. During the aggregation of platelet-rich plasma by arachidonic acid (400--500 ~g arachidonic acid/ml platelet-rich plasma), 0.1 ml was withdrawn at 1-min intervals for 10 min. The sample was quenched in 0.9 ml 0.1 M Buffer I containing 0.2 mM flurbiprofen, a p o t e n t cyclooxygenase inhibitor, and this 1 : 10-diluted sample was immediately frozen in liquid nitrogen. During the aggregation of washed platelet suspensions by arachidonic acid (0.5 ~g arachidonic acid/ml suspension), 0.1 ml was withdrawn as above and quenched in 2.4 ml 0.1 M Buffer I containing 0.2 mM flurbiprofen. This 1 : 25-diluted sample was immediately frozen in liquid nitrogen. During the aggregation of platelet-rich plasma by prostaglandin H2 (2 t~g protaglandin H2/ml platelet-rich plasma) 0.1 ml was withdrawn at 10-s intervals for 60 s and quenched in 0.01 ml 2 M citric acid. Samples were immediately frozen in liquid nitrogen. After thawing for 30 rain, the sample was neutralized with 1 M NaOH (approx. 40 •l), and diluted with 0.9 ml 0.1 M Buffer I. All four c o m p o u n d s were tested in both platelet-rich plasma and washed platelet suspensions; and each platelet preparation was aggregated with both arachidonic acid and prostaglandin H,. By these comprehensive, direct comparisons, we hoped to detect any differences which were peculiar to the systems examined. Data presented are from individual experiments, b u t are representative of at least three independent studies. Parallel determinations of both thromboxane B2 and prostaglandin E 2 discriminate effects on the cyclooxygenase from effects on the thromboxane synthetase. If elevations in cyclic AMP affected the thromboxane synthetase selectivity, excess prostaglandin H2 would be diverted into the prostaglandin pathway analogously to other selective thromboxane synthetase inhibitors [28--32]. Measurements of thromboxane B2 concentrations reflect the initial throm-

47 boxane A2 concentration since it has recently been demonstrated that thromboxane A2 is transformed exclusively into thromboxane B2 in an aqueous system and does n o t convert into HHT and malondialdehyde [33]. One cannot exclude the possibility that t h r o m b o x a n e A2 behaves differently in the presence of lipids, proteins or acids. In this case thromboxane A2 might form other products or it m a y bind covalently to proteins in the plasma. Thromboxane B2 radioimmunoassay. T h r o m b o x a n e B2 in these diluted samples was measured by radioimmunoassay. Samples (0.1 ml) or standard solutions (0.01--6.4 ng t h r o m b o x a n e B2/0.1 ml PBSG) and [3H]thromboxane B2 (15 000 dpm/0.1 ml PBSG, 40% counting efficiency) were added to an antiserum (0.1 ml) at a dilution (1 : 1500) which bound 50% of the [3H]t h r o m b o x a n e B2 in the absence of competition by unlabeled compound. The final assay volume was 0.3 ml. Tubes were vortexed, incubated at 25°C for 1 h, and then at 4°C for 16--24 h. Antibody-bound and free thromboxane B2were separated by incubation with 1.0 ml of a dextran-coated charcoal suspension (2.5 mg Norit A, 0.25 mg Dextran T70, 1.0 ml PBSG) at 0°C for 12 min. After centrifugation at 3000 × g for 15 rain, the antibody-bound [3H]thromboxane B2 in the supernatant was measured by scintillation counting. The concentration was determined by comparison of the percent bound/bound0 (%B/Bo) in the u n k n o w n tubes with the standard tubes. In some cases, additional dilutions were needed to produce a thromboxane B2 content falling within the linear portion (85% B/Bo) of the standard curve. Since our initial report on this m e t h o d [23] we have determined that the antiserum cross reacts minimally with l l - d e h y d r o t h r o m b o x a n e B2 (0.8%), l l , 1 5 - d i k e t o t h r o m b o x a n e B2 (0.6%), and 13,14-dihydro-15-keto-thromboxane B~ (0.05%). Cross reaction with 6-keto-prostaglandin Fl~ and prostaglandin I~, and is less than 0.01%. Non-specific displacement of [3H]thromboxane B2 by cyclic AMP or dibutyryl cyclic AMP was less than 0.0001%. The sensitivity (i.e. the amount of thromboxane B2 required to decrease B/Bo from 100% to 85%) of this radioimmunoassay is 50 pg per assay tube. Prostaglandin E2 radioimmunoassay. Prostaglandin E2 in the diluted samples was determined by radioimmunoassay. An antiserum was used that recognized the prostaglandin E structure specifically. This antiserum cross reacted significantly (90%), only with prostaglandin El and to less than 1% with all other prostaglandins and thromboxanes tested including the A, B, D, E, and F-classes of the 1 and 2 series and their main circulating metabolites. Nonspecific displacement of antibody-bound prostaglandin E2 by cyclic AMP or dibutyryl cyclic AMP was less than 0.0001%. Samples (0.1 ml) or standard solution (0.001--1.0 ng prostaglandin E~/0.1 ml PBSG) and [3H]prostaglandin E2 (2 • 104 dpm/0.1 ml PBSG, 40% counting efficiency) were added to an antiserum (0.1 ml) at a dilution (1 : 500) which b o u n d 50% of the [3H]prostaglandin E2 in the absence of competition by unlabeled compound. The final assay volume was 0.3 ml. Tubes were vortexed, incubated at 25°C for 1 h, and then at 4°C for 16--24 h. Antibody-bound and free prostaglandin E2 were separated by incubation with 1.0 ml of dextrancoated charcoal suspension as in the t h r o m b o x a n e B2 radioimmunoassay. In most cases, additional serial dilutions of 1 : 10 were needed to produce a prostaglandin E~ content falling within the linear portion (85% B/Bo--20% B/Bo) of

48 the standard curve. The sensitivity of this radioimmunoassay was 6 pg per tube. Cyclic AMP radioimmuoassay. Platelet cyclic AMP levels were measured by radioimmunoassay according to Steiner et al. [34], with the acetylation modification of Harper and Brooker [35]. All samples were tested at two dilutions, and all of the immunodetectable cyclic AMP could be destroyed by beef heart phosphodiesterase. Results

Aggregation of platelet-rich plasma with arachidonate Fig. 1 shows the effect of arachidonate concentration on the aggregation response, t h r o m b o x a n e B~, and prostaglandin E2 production. The initial rate, the maximal extent, and the time of onset of both measurable thromboxane B2 and prostaglandin E2 concentrations and platelet aggregation were nearly coincident. At a subaggregatory concentration of arachidonate (100 ~g/ml), both t h r o m b o x a n e B2 and prostaglandin E2 levels were below the limit of the radioimmunoassays. The results of this control experiment consistently showed that

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F i g . 1. T h e e f f e c t o f a r a c h i d o n i c a c i d c o n c e n t r a t i o n o n t h e a g g r e g a t i o n o f p l a t e l e t - r i c h p l a s m a . P l a t e l e t rich plasma (2.0 ml) was incubated at 37°C for 2 rain; then transferred quantitatively to a second aggregometer cuvette containing an amount of arachidonic acid which would yield the concentrations indicated below. The aggregation profile was monitored w h i l e s a m p l e s (0.1 m l ) w e r e w i t h d r a w n f o r assay. o ...... ©, t r a c e A, 5 0 0 ttg a r a c h i d o n i c a e i d / m l ; e - e, t r a c e B, 4 0 0 p g a r a c h i d o n i c a c i d / m l ; • m t r a c e C, 3 0 0 p~g a r a c h i d o n i c a c i d / m l ; A-A t r a c e D, 2 0 0 tLg a x a c h i d o n i c a c i d / m l ; ~ 100 ~g a r a c h i d o n i c a c i d / m l = n o a g g r e g a t i o n a n d n o d e t e c t a b l e t h r o m b o x a n e B 2 o r p r o s t a g l a n d i n E 2.

49 the appearance of thromboxane A2 (as measured by thromboxane B2) coincided with the initiation of aggregation. Incubation of platelet-rich plasma for 2 min with dibutyryl cyclic AMP suppressed arachidonate-induced aggregation in a concentration dependent manner (Fig. 2). Dibutyryl cyclic AMP delayed the onset of measurable thromboxane B2 concentrations, lowered the initial formation rate, and suppressed maximal thromboxane A2 levels in a concentration-dependent manner. The effects of prostaglandin E1 (Fig. 3) were quantitatively different from the effects of dibutyryl cyclic AMP, but they were again consistent with an influence of intracellular cyclic AMP on the platelet cyclo0xygenase. Prostaglandin E1 suppressed thromboxane B2 synthesis more than dibutyryl cyclic AMP did. Even at the higher concentrations of prostaglandin E1 there was some minor thromboxane B2 synthesis although aggregation was entirely suppressed. Some of the quantitative differences between the effect of prostaglandin E1 and dibutyryl cyclic AMP could be due to a direct effect of prostaglandin E1 on the thromboxane synthetase [36]. Crossreaction between prostaglandins E~ and E2 in our radioimmunoassay prevented accurate determination of prostaglandin E2. Fig. 4 shows the effect of prostaglandin I2, a potent stimulator of adenylate cyclase [21,22]. Aggregation was suppressed by extremely low amounts (less

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MINUTES F i g . 2. T h e e f f e c t o f d i b u t y r y l c y c l i c A M P o n t h e a g g r e g a t i o n o f p l a t e l e t - r i c h p l a s m a w i t h a r a c h i d o n i c a c i d (500 pg/ml). Platelet-rich plasma (2.0 ml) containing the concentrations of dibutyryl cyclic AMP indicated bel'ow was incubated at 37°C for 2 min; then transferred quantitatively to a second aggTegometer cuvette containing 1 mg axachidonic acid. The aggregation profile was monitored while samples were withdrawn for assay for thromboxane B 2 . ©. . . . . . ©, t r a c e A , c o n t r o l ; a e, trace B, 4 - 10 -4 M dibutyryl cyclic AMP; E --, t r a c e C , 6 • 1 0 - 4 M d i b u t y r y l c y c l i c A M P ; • •, trace D, 1 • 10 -3 M dibutyryl cyclic AMP.

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F i g . 3. T h e e f f e c t o f p r o s t a g l a n d i n E 1 on the aggregation of platelet-rich plasma with arachidonic acid ( 7 5 0 /~g/ml). T h e c o n d i t i o n s w e r e e q u i v a l e n t t o t h o s e i n F i g . 2 e x c e p t t h e p l a t e l e t s w e r e i n c u b a t e d w i t h p r o s t a g l a n d i n E 1 at t h e l e v e l s i n d i c a t e d b e l o w , a n d 1 . 5 m g a r a c h i d o n i c a c i d w a s u s e d . ~ . . . . . o, t r a c e A , control; e-• t r a c e B , 1 0 n g p r o s t a g l a n d i n E 1 / m l ; m-m t r a c e C, 1 0 0 n g p r o s t a g l a n d i n E 1 / m h A - - ~ trace D, 1 pg prostaglandin E 1/ml.

than 0.05 t~g/ml) in a concentration-dependent manner. The onset of measurable thromboxane B: concentrations was delayed relative to the control, and the initial thromboxane B2 formation rate and the maximal levels declined. In trace C, after reaching a plateau, thromboxane B2 and prostaglandin E2 concentrations rose sharply at the formation rate of the control after aggregation began and thromboxane B: levels attained the maximal level of the control. The highest concentration of prostaglandin I: (500 ng/ml) suppressed thromboxane B2 formation to approximately the same level as 50 ng/ml of prostaglandin I:, but there was no aggregation and no change in the rate or level of thromboxane Bz after the plateau was reached. The inhibition of platelet aggregation in the presence of substantial thromboxane B2 levels again indicated that cyclic AMP has an additional influence on platelet function beside its effect on the cyclooxygenase. Prostaglandin I1, a chemically stable analog of prostaglandin I2 [26] and also a stimulator of adenylate cyclase-suppressed aggregation, delayed the onset of measurable thromboxane B2 concentrations, depressed the initial thromboxane B~ formation rate and lowered the maximal thromboxane B2 levels in a concentration-dependent manner (Fig. 5). In trace C, there was again an increase in thromboxane B2 concentration near a shape change in the aggregation similar to a result obtained with prostaglandin I2. Trace D shows that high levels of thromboxane B2 were formed, although there was no aggre-

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F i g . 4. T h e e f f e c t of p r o s t a g l a n d i n 12 o n t h e a g g r e g a t i o n o f p l a t e l e t - r i e h p l a s m a w i t h a r a c h i d o n i c a c i d ( 5 0 0 # g / m l ) . T h e c o n d i t i o n s w e r e e q u i v a l e n t to those in Fig. 2 e x c e p t p r o t s g l a n d i n 12 w a s u s e d at t h e concentrations indicated below. An additional concentration (5.0 ng/ml) was tested. The results were s i m i l a r t o t r a c e B. T h e y w e r e n o t i n c l u d e d in t h i s f i g u r e f o r t h e s a k e of c l a r i t y , o . . . . . o, t r a c e A, c o n t r o l . • • , t r a c e B, 0 . 5 n g p r o s t a g l a n d i n I 2 / m l ; E =, t r a c e C, 50 n g p r o s t a g l a n d i n 12/ml; • •, t r a c e D, 5 0 0 n g p r o s t a g l a n d i n 12/ml.

gation, which supports a dual or multiple role for cyclic AMP [10,14]. If the data in Figs. 3, 4, and 5 are compared to the adenylate cyclase data in Table I, it seems that the ability of either prostaglandin Is, I1, or E1 to inhibit t h r o m b o x a n e B2 formation from arachidonate is related to their ability to increase cyclic AMP in platelet-rich plasma. Prostaglandin I2 is the most potent stimulator of adenylate cyclase, and inhibits t h r o m b o x a n e B2 at lower'concentrations than either prostaglandin E1 or I1, while prostaglandin E1 is more potent than prostaglandin I1 and it is more efficient in inhibiting thromboxane B2 formation (Table I). Note that aggregation is inhibited by cyclic AMP even in situations where considerable t h r o m b o x a n e As is produced.

Aggregation of platelet-rich plasma with prostaglandin H2 We have previously shown [37] that platelet-rich plasma transforms exogenous prostaglandin H2 into t h r o m b o x a n e A2 immediately prior to irreversible platel.et aggregation and that the t h r o m b o x a n e A2 binds covalently to plasma proteins. The transformation is substrate concentration dependent. The concentration versus time profile of measurable t h r o m b o x a n e B2 from prostaglandin H2-induced aggregations of platelet-rich plasma is different from that obtained during arachidonic acid-induced aggregations. The declining portion

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F i g . 5. T h e e f f e c t o f P r o s t a g l a n d i n I 1 o n t h e a g g r e g a t i o n o f p l a t e l e t - r i c h p l a s m a w i t h a r a c h i d o n i c a c i d ( 5 0 0 ~zg/ml). T h e c o n d i t i o n s w e r e e q u i v a l e n t t o t h o s e i n F i g . 2 e x c e p t p r o s t a g l a n d i n I 1 w a s u s e d a t t h e concentrations indicated below, o ...... ~>, t r a c e A , c o n t r o l ; • $, trace B, 50 ng prostaglandin I 1/ ml; • • , t r a c e C, 5 0 0 p r o s t a g l a n d i n I 1 / m l ; • •, trace D, 2500 ng prostaglandin I 1/ml.

TABLE

I

STIMULATION AND E 1 1 ml of glandin. freezing content

OF

HUMAN

PLATELET

ADENYLATE

CYCLASE

BY PROSTAGLANDINS

I2, I1,

platelet-rich plasma was incubated for 30 s at 37°C with the appropriate concentration of prostaIncubations were terminated by the addition of 0.8 ml 5% trichloroacetic acid, followed by rapid i n l i q u i d n i t r o g e n . D a t a p r e s e n t e d as m e a n +- S . E . o f t r i p l i c a t e d e t e r m i n a t i o n s . The cyclic AMP o f t e n d i f f e r e n t u n s t i m u l a t e d c o n t r o l s a m p l e s w a s 2 4 -+ 6 p m o l c y c l i c A M P / 1 0 9 p l a t c l e t s .

Additions (#g]ml)

pmol cyclic AMP/109

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12

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El

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platelets

53 of the curve represents t h r o m b o x a n e A2 present in the platelet-rich plasma in its active form, which is lost by irreversible covalent binding [37,38]. The flat portion of the curve represents samples of platelet-rich plasma taken after all the t h r o m b o x a n e A2 had hydrolyzed to t h r o m b o x a n e B2 within the plateletrich plasma or had b o u n d to plasma proteins. The identification of this material as t h r o m b o x a n e A2 was evident from the effect of selective thromboxane synthetase inhibitors on the curves [37]. Neither dibutyryl cyclic AMP, nor prostaglandins E1 and I2 suppressed the burst of t h r o m b o x a n e A2 synthesis, even though aggregation was completely suppressed (Figs. 6B--6D) relative to the control (Fig. 6A). The t h r o m b o x a n e B2 measured was produced from exogenously added prostaglandin H2 since the addition of a p o t e n t cyclooxygenase inhibitor, flurbiprofen, had no significant effect on the t h r o m b o x a n e A2 formation from prostaglandin H2.

Aggregation o f washed platelets with arachidonate The experiments described above in platelet-rich plasma were repeated using washed platelets to establish equivalency or differences between the two systems. Incubation of washed platelets for 2 min with dibutyryl cyclic AMP suppressed arachidonate-induced aggregation as well as t h r o m b o x a n e B2 formation (Fig. 7) in qualitative agreement with Malmsten et al. [13] b u t in disagreement with Minkes et al. [14], Gerrard et al. [15] and Lapetina et al. [16]. We differed from Malmsten et al. [13] on the quantitative effects of dibutyryl cyclic AMP. We found only 10% inhibition of dibutyryl cyclic AMP at 8 mM dibutyryl cyclic AMP while Malmsten et al. found 100% inhibition at 0.5--1.25 mM dibutyryl cyclic AMP. Incubations with either prostaglandin El (Fig. 8), I1, or

A : CONTROL

B = BT 2 CYCLIC AMP

C = PROSTAGLANDIN I 2

D : PROSTAGLANDIN E1

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F i g . 6. T h e t h r o m b o x a n e B 2 concentration profile during the aggregation of platelet-rich plasma with prostaglandin H 2 (2 pg/ml). Platelet-rich plasma (2.0 ml) was incubated at 37°C for 2 rain; then transf e r r e d q u a n t i t a t i v e l y t o a s e c o n d a g g r e g o m e t e r c u v e t t e c o n t a i n i n g 4 p g p r o s t a g l a n d i n H 2. T h e a g g r e g a t i o n p r o f i l e w a s m o n i t o r e d w h i l e 100-/~1 s a m p l e s w e r e w i t h d r a w n f o r t h e a s s a y o f t h r o m b o x a n e B 2. I n p a n e l s B, C, D t h e p l a t e l e t s w e r e i n c u b a t e d f o r 2 r a i n w i t h e i t h e r 1 m M d i b u t y r y l c y c l i c A M P o r 1 /~M p r o s t a g l a n d i n 12 o r E j , r e s p e c t i v e l y .

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F i g . 7. T h e e f f e c t o f d i b u t y r y l c y c l i c A M P o n t h e a g g r e g a t i o n o f w a s h e d p l a t e l e t s w i t h a x a c h i d o n i c a c i d (0.5 pg/ml). A washed platelet suspension (2.0 ml, approx. 109 platelets/ml) containing the concentration of dibutyryl cyclic AMP indicated below was incubated at 37°C for 2 min; then transferred quant i t a t i v e l y t o a s e c o n d a g g r e g o m e t e r c u v e t t e c o n t a i n i n g 1 . 0 ~zg a r a c h i d o n i c a c i d , T h e a g g r e g a t i o n p r o f i l e was monitored while samples were withdrawn for assay of thromboxane B 2. o . . . . . . o, t r a c e A , c o n t r o l ; • =, t r a c e B, 4 • 1 0 - 4 M d i b u t y r y l c y c l i c A M P ; m [:]. t r a c e C, 8 • 1 0 - 4 M d i b u t y r y l c y c l i c A M P ; ~A trace D, 2 • 10 -3 M dibutyryl cyclic AMP.

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F i g . 8. T h e e f f e c t o f p r o s t a g l a n d i n E 1 o n t h e a g g r e g a t i o n o f w a s h e d p l a t e l e t s w i t h a r a c h i d o n i c a c i d . T h e conditions were equivalent to those in Fig. 7 except the Platelets were incubated with prostaglandin E l at the concentrations indicated below, o ...... ©, t r a c e A , c o n t r o l ; • - $ , t r a c e B, 1 0 0 n g p r o s t a glandin E1/ml; m • , t r a c e C, 1 # g p r o s t a g l a n d i n E 1 / m l . T h e a g g r e g a t i o n p r o f i l e s a n d t h e t h r o m boxane B 2 levels were virtually identical when washed platelets were incubated with prostaglandins I 1 a n d 12 b e f o r e c h a l l e n g e w i t h a r a c h i d o n i c a c i d . T h e a g g r e g a t i o n P r o f i l e s a n d t h e t h r o m b o x a n e B 2 profiles were virtually identical to those in this figure when washed platelets were incubated with dibutyryl cyclic A M P , o r p r o s t a g l a n d i n s E l , I i , a n d 12 b e f o r e c h a l l e n g e w i t h p r o s t a g l a n d i n H 2.

55 I2 suppressed aggregation of washed platelets in a concentration-dependent manner w i t h o u t an effect on measurable t h r o m b o x a n e B2 levels.

Washed platelets aggregated with prost.aglandin H2 In washed platelets, aggregated with prostaglandin H2, neither dibutyryl cyclic AMP nor prostaglandins El, I1, and I2 affected t h r o m b o x a n e Bz production, however they all suppressed prostaglandin H2-induced aggregation in a concentration-dependent manner. The aggregation profiles and the thromboxane Bz profiles, in every case, were virtually identical to those shown in Fig. 8 when washed platelet suspensions were incubated with dibutyryl cyclic AMP, prostaglandin E~, In, or I2 before challenge with prostaglanding H2 (1 t~g/ml). Again, the thromboxane synthetase was n o t affected, b u t cyclic AMP retarded platelet aggregation by an independent mechanism. Discussion

Measurement of the t h r o m b o x a n e B2 levels in platelet-rich plasma during arachidonate-induced aggregations established a coincidence between the appearance of measurable t h r o m b o x a n e Bz and the onset of platelet aggregation. Incubation of platelet-rich plasma with either dibutyryl cyclic AMP or agents that stimulate platelet adenylate cyclase produced a concentration-dependent inhibition of t h r o m b o x a n e B2 synthesis. Prostaglandin E2 control levels were less than 10 ng/ml, so significant inhibition in the prostaglandin pathway of the arachidonic acid cascade was difficult to observe; however, there was never any increase in prostaglandin E2 levels corresponding to decreased t h r o m b o x a n e B2 levels. These results show that increased intracellular cyclic AMP inhibited the cyclooxygenase, in agreement with the work of Malmsten et al. [13]. If the cyclic AMP inhibition were selectively localized at the t h r o m b o x a n e synthetase, prostaglandin H2 would necessarily have been diverted into the prostaglandin pathway, and prostaglandin E2 concentrations would have increased as has been shown for other selective thromboxane synthetase inhibitors [28--32]. Incubation of platelet-rich plasma with dibutyryl cyclic AMP or agents which stimulate adenylate cyclase inhibited the prostaglandin H2-induced aggregations, b u t the t h r o m b o x a n e A2 produced during these aggregations was unaltered, indicating again that cyclic AMP inhibits the cyclooxygenase and not the t h r o m b o x a n e synthetase, and further, suggesting an additional anti-aggregatory role for cyclic AMP unrelated to thromboxane A: synthetase or cyclooxygenase inhibition. Claesson and Malmsten [10] have recently shown that the effect of prostaglandin endoperoxide G2 on cyclic AMP levels in platelet-rich plasma is principally mediated by adenosine diphosphate. The unusual pattern of measurable thromboxane B2 concentration versus time curves (Figs. 6A--6D) from prostaglandin H2-induced aggregations of platelet-rich plasma has been discussed in a recent publication [37]. At any given time, the sample of platelet-rich plasma contains t h r o m b o x a n e A2 in its active state, and also t h r o m b o x a n e B2 resulting from the direct hydrolysis of thromboxane A2 within the platelet-rich plasma. The actual t h r o m b o x a n e A2 concentration in the platelet-rich plasma declines at two rates: (1) The rate which

56 t h r o m b o x a n e A2 binds irreversibly (covalently) to proteins [37,38]. (2) The rate which t h r o m b o x a n e A2 hydrolyzes to thromboxane B2 within plateletrich plasma. As long as some t h r o m b o x a n e A2 is in the platelet-rich plasma in its labile form, the measurable t h r o m b o x a n e B2 concentration (amount of t h r o m b o x a n e B2 present after quenching in citric acid) will show a decline (the composite rate at which thromboxane A2 hydroyzes or binds). Once all of the t h r o m b o x a n e A2 has reverted to t h r o m b o x a n e B2 within the platelet-rich plasma the measurable t h r o m b o x a n e B2 level will reach a steady state. The appearance of the curve is intrinsic to processes in which a labile material removes itself by two competing mechanisms, one of which is irreversible. During arachidonate-induced aggregations of washed platelet preparations, dibutyryl cyclic AMP, prostaglandins El, I1, and I2 all inhibited aggregation in a concentration-dependent manner, b u t only dibutyryl cyclic AMP suppressed t h r o m b o x a n e B2 formation. Dibutyryl cyclic AMP may have been singularly effective because its more lipophilic character permits a complete saturation of cyclooxygenase throughout the cell membrane. Alternatively, dibutyryl cyclic AMP may limit the availability of arachidonic acid to platelet enzymes, making the inhibition of thromboxane B2 in washed platelets more apparent than real. Support for this interpretation is that in washed platelets, b u t not platelet-rich plasma, dibutyryl cyclic AMP also reduces the synthesis of 12L-hydroxy-5,8,10,14-eicosatetraenocic acid, a non-cyclooxygenase-derived metabolite of arachidonic acid (unpublished experiment). The data obtained with dibutyryl cyclic AMP were n o t due to butyrate or adenosine 5'-monophosphate. Butyrate at 1 mM does not significantly influence either aggregation or thromboxane B2 synthesis. Adenosine 5'-monophosphate does show marginal inhibition of platelet aggregation at 1 mM, b u t does n o t influence thromboxane B2 synthesis (unpublished experiments). Many of the differences between Malmsten et al. [13] and Minkes et al. [ 14] and Gerrard et al. [ 15] and this report on the production of thromboxane B2 in the presence of dibutyryl cyclic AMP may be the result of technical problems associated with the washed platelet system. When prostaglandin H2 was used as the aggregating agent in washed platelets, neither dibutyryl cyclic AMP nor agents that stimulated adenylate cyclase affected t h r o m b o x a n e B2 synthesis. These results are consistent with the results using prostaglandin H2 in platelet-rich plasma and demonstrate that cyclic AMP does n o t selectively inhibit the thromboxane synthetase. However, it is imperative to note that agents that elevated cyclic AMP did block platelet aggregation even in the presence of high concentrations of thromboxane. In summary, in both platelet-rich plasma and washed platelet controls thromboxane B2 appeared at the same time aggregation occurred. The platelet cyclooxygenase is inhibited by dibutyryl cyclic AMP and agents that stimulate adenylate cyclase in platelet-rich plasma b u t only dibutyryl cyclic AMP influences thromboxane B2 synthesis from arachidonic acid in washed platelets, possibly through indirect effects. Neither dibutyryl cyclic AMP nor agents that stimulate adenylate cyclase selectivity inhibited the thromboxane synthetase in either platelet-rich plasma or washed platelets. In both platelet preparations, and regardless of substrate, there were

57

instances where substantial thromboxane B2 was formed, but cyclic AMP still depressed aggregation, suggesting other roles for cyclic AMP besides cyclooxygenase regulation [10,14]. It is interesting to note that cyclic AMP has recently been reported to stimulate calcium uptake by platelet membrane vesicles [39]. It may be that an elevation of platelet cyclic AMP reduces the free intracellular Ca2+ concentration which is necessary for the platelet release reaction and subsequent aggregation. We are presently trying various modifications of washed platelet preparation, and the readdition of plasma components in an attempt to delineate what fractor(s) are responsible for the difference in cyclooxygenase regulation observed in platelet-rich plasma and washed platelets, and what protein fraction covalently binds prostaglandin H2 and thromboxane A2 during the prostaglandin H2-induced aggregation of platelet-rich plasma. The interaction in the platelet between cyclic AMP, calcium, the platelet release reaction, phospholipase A2, fatty acid cyclooxygenase and thromboxanes or prostaglandins is very complicated. The results presented must be regarded cautiously since it is not possible in such complex systems to ignore the possible influences of the other elements in the system. References 1 Willis, A., V a n e , F., K u h n , D. a n d P e t r i n , M. ( 1 9 7 4 ) P r o s t a g l a n d i n s 8 , 4 5 3 - - 5 0 7 2 H a m b e r g , M., S v e n s s o n , J., W a k a b a y a s h i , T. a n d S a m u e l s s o n , B. ( 1 9 7 4 ) P r o c . N a t l . A c a d . Sci. U.S. 71, 345--349 3 S m i t h , J., I n g e r m a n , G., K o c s i s , J. a n d Silver. M. ( 1 9 7 4 ) J. Clin. Invest. 5 3 , 1 4 6 8 - - 1 4 7 2 4 H a m b e r g , M., S v e n s s o n , J. a n d S a m u e l s s o n , B. ( 1 9 7 5 ) P r o c . N a t l . A c a d . Sci. U.S. 7 2 , 2 9 9 4 - - 2 9 9 8 5 N e e d l e m a n , P., M i n k e s , M. a n d R a z , A. ( 1 9 7 6 ) S c i e n c e 1 9 3 , 1 6 3 - - 1 6 5 6 H a m b e r g , M. a n d S a m u e l s s o n , B. ( 1 9 7 4 ) P r o c . N a t l . A c a d . Sci. U.S. 7 1 , 3 4 0 0 - - 3 4 0 4 7 M a l m s t e n , C., H a m b e r g , M., S v e n s s o n , J. a n d S a m u e l s s o n , B. ( 1 9 7 5 ) P r o c . N a t l . A c a d . Sci. U.S. 72, 1446--1450 8 V a r g a f t i g , B. a n d Zirinis, P. ( 1 9 7 3 ) N a t . N e w Biol. 2 4 4 , 1 1 1 1 - - 1 1 1 6 9 Mills, D. a n d S m i t h , J. ( 1 9 7 1 ) B i o c h e m . J. 1 2 1 , 1 8 5 - - 1 9 6 1 0 C l a e s s o n , H.-E. a n d M a l m s t e n , C. ( 1 9 7 7 ) E u r . J. B i o c h e m . 7 6 , 2 7 7 - - 2 8 4 11 V a r g a f t i g , B. a n d C h i g n a r d , M. ( 1 9 7 5 ) A g e n t s A c t i o n s 5, 1 3 7 - - 1 4 4 1 2 Miller, O. a n d G o r m a n , R . ( 1 9 7 6 ) J. C y c l i c N u c l . Res. 2, 7 9 - - 8 7 13 M a l m s t e n , C., G r a n s t r o m , E. a n d S a m u e l s s o n , B. ( 1 9 7 6 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 6 8 , 569--576 14 M i n k e s , M., S t a n f o r d , N., Chi, M., R o t h , G., R a z , A., N e e d l e m a n , P. a n d Majerus, P. ( 1 9 7 7 ) J. Clin. Invest. 5 9 , 4 4 9 - - 4 5 4 1 5 G e r r a x d , J., Pellet, J., K r i c k , T. a n d W h i t e , J. ( 1 9 7 7 ) P r o s t a g l a n d i n 14, 3 9 - - 5 0 16 L a p e t i n a , E., S c h m i t g e s , C., C h a n d r a b o s e , K. a n d C u a t r e c a s a s , P. ( 1 9 7 7 ) B i o c h e m . B i o p h y s . Res. Commun. 76,828--835 17 H i l d i t c h , T.P. ( 1 9 5 6 ) T h e C h e m i c a l C o n s t i t u t i o n of N a t u r a l F a c t s , p. 6 6 4 , 3 r d e d n . , C h a p m a n a n d Hall, L o n d o n 1 8 L a g a r d e , M., G h a r i b , A. a n d D e c h a v a n n e , M. ( 1 9 7 7 ) B i o c h i m i e 5 9 , 9 3 5 - - 9 3 7 19 M o n e a d a , S., G r y g l e w s k i , R., B u n t i n g , S. a n d V a n e , J. ( 1 9 7 6 ) N a t u r e 2 6 3 , 6 6 3 - - 6 6 5 2 0 J o h n s o n , R . , M o r t o n , D., K i n n e r , J., G o r m a n , R . , M c G u i r e , J., S u n , F., W h i t t a k e r , N., B u n t i n g , S., S a l m o n , J., M o n c a d a , J. a n d V a n e , J. ( 1 9 7 6 ) P r o s t a g l a n d i n s 1 2 , 9 1 5 - - 9 2 8 21 G o r m a n , R., B u n t i n g , S. a n d Miller, O. ( 1 9 7 7 ) P r o s t a g l a n d i n s 13, 3 7 7 - - 3 8 9 2 2 T a t e s o n , J., M o n c a d a , S. a n d V a n e , J. ( 1 9 7 7 ) P r o s t a g l a n d i n s 1 3 , 3 8 9 - - 3 9 9 2 3 F i t z p a t r i c k , F., G o r m a n , R . , M c G u i r e , J., Kelly, R . , W y n a l d a , M. a n d S u n , F. I 1 9 7 7 ) A n a l . B i o c h e m . 82, 1--7 2 4 G o r m a n , R . , S u n , F., Miller, O. a n d J o h n s o n , R. ( 1 9 7 7 ) P r o s t a g l a n d i n s 13, 1 0 4 3 - - 1 0 5 3 2 5 Miller, J., K a t z , A. a n d F e i n s t e i n , M. ( 1 9 7 5 ) T h r o m b . D i a t h . H a e m o r r h . 3 3 , 2 8 6 - - 3 0 9 26 J o h n s o n , R . , L i n c o l n , F., T h o m p s o n , J., N i d y , E., M i z s a k , S. a n d A x e n , J. A m . C h e m . Soc. 9 9 , 4182--4184 27 K l o e z e , J. ( 1 9 6 7 ) N o b e l S y m p o s i u m 2, P r o s t a g l a n d i n s , S t o c k h o l m , A l m q u i s t a n d Wickell, p. 2 4 1

58 28 Needleman, P., Raz, A., Ferrcndelli, J. and Minkes, M. (1977) Proc. Natl. Acad. Sci. U.S. 74, 1716--1720 29 Gorman, R., Bundy, G., Peterson, D., Sun, F., Miller, O. and Fitzpatrick, F. (1977) Proc. Natl. Acad. Sci. U.S. 74, 4007--4011 30 Moncada, S., Bunting, S., Mullane, K., Thorogood, P. and Vane, J. (1977) Prostaglandins 13, 611--618 31 Nijkamp, F., Moncada, S., White, H. and Vane, J. (1977) Eur. J. Pharmacol. 44, 179--186 32 Diczfalusy, U. and HammarstrSm, S. (1977) FEBS Lett. 82° 107--110 33 Diezfalusy, U., Falardeau, P. and HammarstrDm, S. (1977) FEBS Lett. 84, 271--274 34 Steiner, A., Parker, C. and Kipnis, D. (1972) J. Biol. Chem. 217, 1106--1113 35 Harper, J. and Brooker, G. (1975) J. Cyclic Nucl. Res. 1, 207--218 36 Sinha, A. and Colman, R. (1978) Science 200, 202--203 37 Fitzpatrick, F. and Gorman, R. (1977) Prostaglandins 14, 881--889 38 Eling, T., Wilson, A., Chaudhari, A. and Anderson, M. (1977) Life Sci. 21, 245--252 39 Kb:zer-Glanzmann, R., Jakabova, M., George, J. and Luscher, E. (1977) Biochim. Biophys. Acta 466, 429--440