- Email: [email protected]
prostaglandin H2 receptor agonists
THROMBOSIS RESEARCH 64; 667-676,199l 0049-3848/91 $3.00 + .OO Printed in the USA. Copyright (G) 1991 Pergamon Press plc. All rights reserved.
BY
ELEVATION THHOMBOXANE
YOSHIHIDE The Japanese 4-l-31, Hiroo,
(Received
22.7.1991;
OF PLATELET CYCLIC AMP LEVEL Az/PROSTAGLANDIN H? RECEPTOR AGONISTS
ISHIKAWA and ATSUKO OGAWA Red Cross Central Blood Center Shibuya-ku, Tokyo 150, Japan
accepted
in revised form 1 .10.1991
by Editor A. Takada)
ABSTRACT Arachidonic acid (AA)or thromboxane Az/prostagIandin H, (TXA,/ PGH,) analog (STA? and U-46619)-induced aggregations yielded a bell-shaped dose-resnonse curve. The inhibitorv mechanism by high concentrations of the agonists was examined. STAz elevated CAMP level of platelet in a dose-dependent manner. And the aggregation was affected by metabolic inhibitors of CAMP. AA also rised CAMP level, and the rise was suppressed by indomethacin. These results indicate that the reduction of aggregation by high dose of the agonists is through CAMP elevation. The CAMP elevation was not suppressed by ruling out phospholipase C effects by chelation of cytoplasmic Ca”’ inhibition of protein kinase C (PKC). These results and suggest that the CAMP elevation is not due to activation of phospholipase C-linked TXAZ/PGH2 receptor. 13-APA, an antagonist of TXA2/PGH2 receptor, suppressed the CAMP elevation, although ONO-3708, another antagonist, had no effect. As to be expected effect of From this result, inhibitory 13-APA on high STA, level-induced aggregation was weaker than that of ONO-3708. The antagonists did not inhibit PGE,- or PGD2-induced CAMP elevation. suggest that platelet has adenylate These findings cyclase-linked TXA,/PGH, receptor.
INTRODUCTION Thromboxane AZ (TXA2) is a potent stimulator of platelet, and its binding to TXA,/PGH, receptor results in the formation of diacylglycerol and inositol 1,4,5-trisphosphate (IPB) through the activation of GTPbinding protein and phospholipase C (PLC)(l). It is known that dose-response curve for arachidonic acid (AA)-induced aggregation is biphasic (Z-4). The first phase is aggregatioEf deTp;;ding on TXA, synin spite of increase thesis (5). However, 2 synthesis with increasing AA dose, the aggregation yields a bell-shaped dose-response curve. There are several reports about the inhibitory mechanism by high concentrations of AA. For example, inhibitory effect of IipoxyKey words: Platelet aggregation, TXA,/PGH, receptor, STAz, Adenylate cyclase, CAMP
667
668
ELEVATION OF CAMP LEVEL BY STA,
genase products (6). perturbation elevation of cyclic AMP (CAMP) the cause of the inhibition.
Vol. 64, No. 6
of platelet membrane by AA (4) level by AA (3,7) are considered
and as
Best et al. (8) have demonstrated that synthetic analogs of prostaglandin endoperoxides, which caused platelet to aggregate, produced a small elevation of CAMP content in platelet. Murray et al. (9) have reported that U-46619 enhances iloprostor forskolin-induced Furthermore, cAMP elevation. we have already reported that aggregations induced by the TXAz/PGH, receptor agonists, STA, and U-46619, yield bell-shaped dose-response curves (10) as same as AA-induced aggregation. From these findings, we hypothesized that the inhibitory effect of high-dose of AA and TXA2/PGHz receptor agonists is due to elevation of CAMP level. To test this hypothesis, in this paper we have examined the change of CAMP level by these agonists and the mechanisms are discussed.
MATERIALS
AND
METHODS
Materials STAz ((+)-9,11-epithia-ll,lZ-methano-thromboxane A,) and ONO-3708 ((9, ll)(ll, 12)-dideoxa-9, a, 11a-dimethylmethano-11,12-methano-13,14dihydro-13-aza-14-oxo-15-cyclopenty1-16,17,18,19,20-pentanor-15-epithromboxane AZ) were gifts from Ono Pharmaceutical Co. (Osaka, Japan). 13-APA ((+)13-Azaprostanoic acid), U-46619 (15(S)-hydroxy-11,9-(epoxymethano)prosta-5%.13E-dicnoic acid), and Prostagrandin El and DZ (PGE,, PGDz) were obtained from Funakoshi (Osaka, Japan). DDA (2’,5’-dideoxyadenosine) was from Pharmacia ( Uppsala, Sweden) and IBMX (isobutylmethylxanthine) was from Sigma (St. Louis, USA). Staurosporine was from Kyowa Medex Co. Ltd. (Tokyo, Japan). BAPTA-AM (1,2-bis(o-aminophenoxy) -othane-N, N, N’, N’-tetraacetic acid) was from Dojindo Laboratories (Kumamoto, Japan). STA2, 13-APA, IBMX and staurosporine were dissolved in ethanol. DDA and ONO-3708 were dissolved in distilled water. BAI’TA-AM was dissolved in dimethyl sulfoxide. Preparation of platelets I’laLcletn (3.5 x lo5 cells/ u 1) in HEPES-buffered saline (135 mM NaCl, 5 mM -KCl, 10 mM HEPES, pH 7.4) ‘was prepared from 1 daystored platelet concentrate (PC) which was prepared by The Japanese lied Cross Central Blood Center. Treatment with inhibitors For studies of the effect of cytoplasmic Ca2’, platelet suspension was prepared from PC incubated with 100 ,uM of BAPTA-AM for 30 min at room temperature. For inhibition of CAMP metabolism, platelets were incubated with DDA or IBMX for 2 min at 37 “C. For the- urpose of inhibition-of protein kinase C, 500 nM of staurosporine was ad Z ed 2 min Antagonists of TXA,/PGH, receptor were before additi%n of agonist. added 1 min before stimulation of platelets. Measurement of platelet aggregation J STA,-, U-46619- or AA-induced anrrregation was _ measured in an aggregometer (NKK Hematracer 1, Nil; The aggregation ratio was determined at 2 min Isioscience Co., Japan). after the addition of agonists. Measurement of CAMP Platelets were incubated with TXA2/PGH2 receptor agonists, PGEl of 1 mM OF CaCh. The or PGD, for 1 min at 37°C in the presence adding trichloroacetic acid (TCA; final reaction was terminated by After removal of TCA with ether, CAMP concentration 6% (w/v)).
Vol. 64, No. 6
concentration kit (DuPont,
ELEVATION OF CAMP LEVEL BY STA,
of aqueous layer Wilmington, DE, USA).
was
determined
using
669
cAMP
[‘““I]
RIA-
RESULTS Relationship between cAMP elevation and aggregation by STA, As already reported (lo), STAz-induced aggregation exhibited a maximum value with about 50 nM of STA, (Fig.1, open circle). IBMX, an inhibited STA,-induced aggregation. inhibitor of phosphodiesterase, And DDA, an antagonist of adenylate cyclase (AC), enhanced the aggregation. Aggregation of DDA-treated platelet was hardly suppressed by a high concentration of STA,. As shown in Fig.2 in the presence of IBMX, cAMP level was apparently elevated by STA, with a concentration lower than that induces the maximum aggregation (see Fig.l), and the level was increased in a dose-dependent manner by STA,. Even in the absence of IBMX, CAMP level was elevated by a high concentration of STA,. Similar results were obtained with U-46619. U-46619-induced aggre-’ In the gation exhibited a maximum value with about 1 WM of U-46619. is a high concentration of presence of IBMX, 30~. of U-46619, which 11-46619, increased CAMP level by 2.42 + 0.56 pmol/lO” platelets (mean f SD, four experiments, pcO.01). The changes of aggregation shown in Fig.1 are thought to Ibe For confirmation of t-his idea, the due to the changes of CAMP level. relationship between cAMP level and aggregation by STA, was invcstiAs shown in Fig.3, CAMP level was increased by IBMX and degated.
loo-
s
80-
: z 605 bl cn 40a
STAZ (nM)
STAZ (nM)
Fig.1 (Left) Effects of CAMP metabolic inhibitors on STAz-induced aggregation. Platelets were incubated with DDA (200 MM; ?? ), IBMX (20 &MM;Cl) or without inhibitor ( 0) for 2 min before addition of STA,. Each point represents the mean value ? SD from three experiments. Fig.2 (Right) CAMP level of platelets stimulated by STA,. Platelet suspension was incubated with STAz for 1 min at 37 “C in the presence (11) or absence (0) of IBMX (20 rc~ M). Each point represents the mean value + SD from three experiments.
ELEVATION
670
loo-
a
OF CAMP LEVEL
-10
g 80-
loo-
s
BY STA,
Vol. 64, No. 6
b
-8 $
20 Oot,
HVIX WV
I 50
I 100
I 150
4% a 2o02 0 .I
DDA($4
Fig.3 Effects of cAMP metabolic inhibitors on CAMP elevation and After 2 min incubation with inhibitors, aggreaggregation by STAz. gation (open) and CAMP elevation (closed) induced by 100 nM (a) or 1000 nM of STAz (b) were measured. CAMP level of platelets treated by DDA (b) were determined in the presence of 20 ,uM of IBMX. The results are representative of four separate experiments.
”
-+ --
-
-+
-
-+
+
-
+
creased reverse
+
-+
Fig.4 Effects of inhibitors on STA,-induced CAMP elevation. Platelet suspension was prepared from platelet concentrate incubated with or without 100 hM of BAPTA-AM for 30 min at room temperature. Staurosporine (500 nM; Stauro.) was added 2 min before addition of STAz (0.1 y M). CAMP levels were determined in the presence of IBMX (20 KM). Mean + SD was calculated from four separate experiments.
STAz
Stauro. SAPTA-AM
by DDA in dose-dependent change of CAMP level.
manners.
And aggregation
showed
the
To determine whether STAz elevates CAMP level through PLC we studied the effects of second messengers released by activation, Protein kinase C activated by diacylglycerol was inhibited by PLC. by and rise of cytoplasmic Ca”’ by IP3 was diminished staurosporine, The concentrations of staurosporine and BAPTA inhibiBAPTA (Fig.4). of 47kDa (11) protein and elevation ted completely phospholylation of cytoplasmic Ca”, respectively, by 30 mu/ml of thrombin (data And chelation Staurosporine enhanced CAMP elevation. not shown). affect on STAz-induced CAMP BAPTA did not of cytoplasmic Ca” by elevation. The treatment by both BAPTA and staurosporine showed the If CAMP same results as the treatment with staurosporine alone. ought to elevation by STA, is through PLC activation, the elevation
Vol. 64, No. 6
loo-
ELEVATION OF CAMP LEVEL BY STA,
671
a
20-
A :“I _.;;
ol
J
10
100
1000
STAZ (nM)
,
,
,
10
100
1000
STAz (nM)
Fig.5 Effects of TXAJPGH, receptor antagonists on STAz-induced aggrePlatelets were incubated with 7.28 nM of ONO-3708 (a: ?? ), gation. 2.67 PM of 13-APA (b:O) or without antagonist (a,b:O) for 1 min at 37°C Each point represents the mean value ? SD of before addtion of STA,. three experiments. be suppressed plasmic Ca”.
by the inhibition But the elevation
PKC and the chelation of cytoof was enhanced by these treatment.
Effects
of TXA2/PGHz receptor antagonists ONO-3708 and 13-APA inhibited STAz-induced aggregation in doseFig.5 shows effects of the antagonists of dose dependent manners. which reduce the maximum aggregation (50 nM STAJ Lo abouL one-half. 13-APA concentration which was needed Lo inhibit STAz-induced aggregation was roughly two orders of magnitude higher than the concenLThis results seemed to be consistent with the ration of ONO-3708. demonstrated that the inhibreport by Kattelman et al. (12). They have itory potency of ONO-3708 for U-46619 binding was about two orders of ONO-3708 inhibited the aggremagnitude over the potency of 13-APA. gation by the high concentrations of STAz as well as that by the low But the inhibition by 13-APA was weaker in concentrations of STA,. For study of the difference of the high concentration range of STAZ. The antagonists CAMP levels were determined. inhibitory effects, The antagonists, however, alone had no effect on CAMP level (Fig.6). As PKC inhibits AC acLivity(l3), enhanced STAZ-induced CAMP elevation. PKC was inhibited by staurosporine to determine a net AC activity After treatment with induced by STAz and the antagonists. elevation of CAMP level was suppressed in a dosestaurosporine, dependent manner by 13-APA (Fig.G-b), although ONO-3708 had no effect (Fig.G-a). cAMP
elevation by PGE, and PGD, Activation of PGIJPGEIand PGD?-receptor results in CAMP eleIf STA, cross-reacts with these PG -receptors and if STA,vation. induced CAMP elevation is decreased by 13-APA antagonizing the PG receptors, the antagonist ought to decrease PGs-induced CAMP eleva.The 1’Gs were used with the concentrations which inhibit comLion. These concentrations pletely full aggregation induced by thrombin. of the PGs elevated CAMP level as the same extent as 0.1 ,uM of STA, (Fig.6) as shown in Table 1. Both ONO-3708 and 13-APA had no effect on CAMP elevation by the PGs.
ELEVATION OF CAMP LEVEL BY STA,
672
Vol. 64, No. 6
8-
6-
0
0.01
0
0.1
ONO-3708
1 13-APA
(ptvl)
10 (j&l)
Fig.6 c4MP levels of platelets treated by TXA2/PGH2 antagonists. After treatment with 20 ,uM of IBMX, platelets were incubated with (IQ) or without ( R) staurosporine (500 nM), and then stimulated by STA, (0.1 b M) for 1 min at 37 ‘C in the presence of antagonists. Open bar shows cAMP level of platelets incubated wil.h antagonists alone (a: ONO-3708, b:13-APA) for 2 min at 37 “C. Mean + SD was calculated from StatisLical analysis (conLrol@ in Fig.b) four separate experiments. versus expose to 13-APA) was by paired t test: ‘p
Effects of PGs-lnduced on
TXA,/PGH2
Antagonist ONO-3708
( FM) 0 0 . 1 1;::
13-APA
TABLE 1 receptor
antagonists
cAMP(pmol/lO”platelets) PGE, PGD, 11.4k1.7 8.9k2.1 10.7k3.5 9.2kl.l 10.6k2.4 9.8kO.9 11.623.0 8.2+1 .5
0 1
12.5k2.2 9.6k1.1 12.Ok3.3 9.7kl.l 10.9fl.O 8.9k1.2 1:: 12.OFl.O 9.4f1.3 In the presence of CaCL (1 mM), IBMX (20 /.LM) and TXAJPGH, receptor antagonists, platelet suspension was incubated with PGE, (50 nM) or PGD, (33.3 nM) for 1 min at 37 “C. The values represent means + SD of four experiments.
Change of cAMP level by arachidonic acid Fig.7 shows change of CAMP level by AA. As well known, AA-induced aggregation yielded a bell-shaped dose response curve (Fig.6). And cAMP level was elevated with the increasing concentration of AA, as already reported (3,7). Platelets were pretreated by indomethacin to exclude effects of TXA, formed from AA. Indomethacin (1 w M) inhibThe increase of CAMP level ited completely AA-induced aggregation. was apparently suppressed by the pretreatment., although the suppres-
Vol. 64, No. 6
ELEVATION OF CAMP LEVEL BY STA,
673
cAM1’ level of platelets by arachidonic acid. Af’l.cbr Lreafmenl. with 20 FM of IISMX, platelets stimulaLed by AA ( 0,O) for 1 min at 37°C. Indome0lacin (1 &MM) was added I min kJcf'Ore addition of AA (0). AggregaLion ( Cl) was measured wiLhoul f"kl~~S~~ Lreatments. Moan *SD was calculated from four separale experiments. SLiiLisLical analysis (control versus expose to AA) was by paired L Lest.: ‘p
I:ig.7
I realed
AA PJV In Lho same condition, ST&-induced cAMI’ ~lc?siori was noL complete. vat.ion was no1 afTect_ed by indomethacin (data noL shown). This is because TXA, synthesis by stimulation wi1.h TXAJl’GlI, agonists is negligible in sLored pla&?lets (10).
I)ISCUSSION I~f’l‘c~ct of TXA,/l’GIl, rcceplor agonisLs on cAMI” level of platelets were examined. The agonists (STA,, U-46619) elevated cAMP level of plaLclcLs. IHMX, an inhibiLor of phosphodiestzrase, enhanced Lhe CAMI’ DI)A, an anlagonist c!levaLion and inhibiLod STA?-induced aggregation. cyclase (AC), suppressed Lhe reduction of aggregation at Of adcnylaLc1 high c:oric:c?ritraLioris of ST&. These findings indicale that STA, elevates cAM1’ level Lhrough aclivation of AC and also indicate that Lhe rc>duction 01‘ aggregalion by high c:orlc:onLraLiorls of STA, is due to the c>lcvaLiorl oI‘ cAMt’ level. Murray et. al. (9) have dnmonsLraLed t.haL cAMI’ elevation by ilor)rost. was amnlif’ied bv Drier exDosure to U-46619 throunh acLivaLioyl of TXA/l’<511? receiLo;. In o;lr rcsulLs, cAMP level Gas elevaLed only by TXAJl’GFl~~ rccepLor agonists without activators of AC such as iloprosl. 11, is
known LhaL l’G12, l’Gl$ and I’Gl)? elevate plaLele1 cAMI’ do noL synthesize PGIz and I’GE,. And Watanabe ctL al. (14) have reported t.ha~ conversion of l’GlI, to I’GD, does not, occur in washed plat.ele&. I:urLhermore, reduction of aggregation a~ high concentralions of‘ STA:. was also observed in washed plaLelets t.reated by 33 GM of indomeLhacin (IO). Therefore, CAMP elevation by ST& is not_ due Lo formaLion of these proslaglandins by AA melabolism.
1C!\’(?1.
well
I%uL plaLc?leLs
TXAJI’GII, receptor is linked Lo phospholipase C (PI,C) via GTl’binding protein (1). I’I,C forms IP, and diacylgrycerol from phosphaLidyl inosiLol and the products mobilize cyLoplasmic Ca” and activale prot_ein kinase C (I’KC), respectively. cAMP elevaLion by STAz was 1101, aff’t?cLed by chelation of cyt,oplasmic Ca” with HAPTA and was ac:celeraLed by a I’KC inhibitor, sLaurosporinc. Furthermore, ONO-3708, which inhibilcd STAz-induced aggregation, did noL suppress the cAMP olevalion. These rcsulLs indicaLo that t,he elevation of cAMI’ occurcs
674
\ria sysLems
ELEVATION OF CAMP LEVEL BY STA,
except
of
TXA2/PGHr
receptor
linking
Lo
Vol. 64, No. 6 PIG.
Williams et al (13) have reported that PKC inhibits AC activity. agreement with the report., staurosporine enhanced cAMP elevation Therefore, the enhancement of cAMP elevation by TXAJl’GHT \Jy STAZ. anLagonisLs is due to that the antagonists suppress PKC activation through inhibition of STAz binding to the receptor linking to PLC. In
13-APA inhibited STAZ-induced r.AMP elevation in the condition the PKC effect using ruling out staurosporine, although ONO-3708 had no effect. And inhibitory effect of 13-APA on aggregation was weaker in high concentrations of STAI, compared with ONO-3708. This is because 13-APA inhibits STAz-induced aggregation by antagonizing while it probably enhances the STA, binding Lo PLC-linked receptor, aggregation by suppressing CAMP elevation. These results can not be explained if platelets have only one type of receptor against TXAJ l’GH2. It has been reported that platelet has single receptor against TXAJPGH, (12,15). However, some researchers (16,17) have reported subLype of the receptor. Our results indicate that platelets have aL least two types of receptor against TXAJPGH2, that is, a PLC-linked and an AC-linked receptor. receptor It seemes that ONO-3708 selectively binds LO the PLC-linked receptor, and STAz and 13-APA bind to not only the PLC-linked receptor but also the AC-linked reccplor. The AC-linked receptor may be PGIJPGE,or PGDz-receptor. To make clear whether the AC-linked receptor is against the PGs or not, offeeLs of TXA2/PGHz receptor antagonists on CAMP elevation were oxamined, because 13-APA inhibited CAMP elevation by STA,. cAMP elevation by 50 nM of PGE, or 33.3 nM of PGD, was about the same degree as that by 100 nM of STAz with PKC inhibitor. The increase by STA? was inhibited by 1 ,uM of 13-APA, but PGE,- or PGDz-induced elevation was not affecLed even by 100 KM of the antagonist. These results suggested that 13-APA has no effect on the PG receptors, and that inhibition of STA?-induced CAMP elevation by 13-APA is not due to antagonism of 13-APA Lo the PG receptors. Therefore, CAMP elevation by STAz is thought t.o occure through receptor(s) other than Lhe PG receptors. There are reports (3,7) that reduction of AA-induced aggregation by high concentrations of AA is due to elevation of CAMP level by AA ilself. And in Lhe report (7), hinder eL al. have demonstrated that ~~~~-~h~tcetdh~g~rega!.ion was inhibited by high concentrations r of AA inhibited aggregation wras noL restored by t0 FM of Although they have not shown the effect of indomethacin. indomeLhacin on CAMP lecel, their results seemed to be in conflict with our data that the CAMP elevation was inhibited bv indomethacin It is possible that AA itself’ elevates cAMP level, because (Fig.6). the suppression of cAMP elevation by indomethacin was not complele. We have already reported that of indomethacin with a high concentration (33 ,uM) has inhibitory effect on TXAJ PGHZ-induced aggregation Therefore, in their condition, the restoration of aggregation (IO). inhibitory effects of was probably not observed because of the indomethacin and CAMP elevation by AA itself. our results suggest that not only stable analogs of Therefore, TXAJPGH2 but also TXAz elevates platelet CAMP level through AC-linked TXAJPGH, receptor and that a part of inhibitory effect of AA is due to TXA, formed from AA.
Vol. 64, No. 6
ELEVATION
OF CAMP LEVEL BY STA,
675
ACKNOWLEDGEMENT help
The authors extend in this research.
their
gratitude
to
Dr. S. Sasakawa
for
her
Ii EI’EK ENCES 1. BRASS, I,. F. Regulation of‘ biological responses in platelets. In: Blood Cells and Arteries in Ilypcrtcnsion and Atheroslerosis. P. Meyer and F’.Marche (Ed.) N ew York: Raven Press, 1989, pp.59-92. 2.
HASHIMOTO, Y., NAITO, C., KUME, S., KATOJI., WATANABET., KAWAMUKA, M., TERAMOTO, T. and OKA, II. IIigh concentrations of arachidonic acid induce platelet aggregation and serotonin relcasc independent of prostaglandin endoperoxides and thromboxanc A,. Biochim. Biophys. & &l, 283-291, 1985.
3. KOWALSKA, exogenous stimulation
M. A., KAO, A. K. and DISA, J. IIigh concentrations of‘ arachidonatc inhibit calcium mobilization in Iatelcts of adenylatc cyclase. Biochcm. -J. -’ 253 255- 3 62, 1988.
by
4. SATO, T., IIASHIZUME, T., NAKAO, K., AKIBA. S. and FUJII, T. Platelet desensitization by arachidonic acid is assosiated with the suppression of cndoperoxidc/thromboxane AL binding to the membrane receptor. Biochim. Biophys. Acta 992, 168-173, 1989. .5 . IINDER, B. I,., CHEKNOFF, A., KAPI.AN, K. 1~.and GOODMAN, D. S. Release platelet-derived growth factor from human platelets by arachidonic acid. -Proc. Natl. --Acad. Sci. USA 2 76 4107-4111, 1979. 6. AHARONY, D., SMITH, J. B. and SILVER, M. J. Regulation induced platelet aggregation by the lipoxygenase peroxyeicosatetraenoic acid. Biochim. Biophys. Acta Studies 7. LINDER, B. I,. and GOODMAN, D. S. inhibition of platelet a gregation levels of’ arachidonate. B?ood 60, 436%%,
on the release 1982.
of
of arachidonat.eproduct, 12+ydro718, 193-200, 1982. mechanism induced
of by
the high
8. BEST. I,. C., McGUIRE, M. B., MARTIN, T. J., PRESTON, F. E. and KUSSELL, Ii. G. G. Effects of epoxymethano analogues of prostaglandin endoon release of 5-hydroxytryptamine and on peroxides on aggregation, the metabolism of 3’,5’-cyclic AMP and cyclic GMP in human platelets. Biochim. Biophys. & 583, 344-351, 1979. endoper9 . . MURKAY, R., SHIPP, E. and FITZGERALD, G. A. Prostaglandin oxide/TXA? receptor desensitization. Cross-talk with adenylate cyclase in human platelets. J. Biol. Chem. 265, 21670-21675, 1990. -~ IO. ISHIKAWA, Y., OGAWA, A. and SASAKAWA, on platelet activation by arachidonic 1989.
S. Effect of acid. Thromb.
cytoplasmic Res. ,54, 17-2$Il
11. KAIBUCHI, K., TAKAI, Y., SAWAMURA, M., HOSHIJIMA, M.,FUJIKURA, T. and INISHIZUKA, Y. Synergistic functions of protein phos horylation and calcium mobilization in platelet activation. J. Biol. CPIem. m, 6701 -~-6704,1983. 12. KATTELMAN, E. J., VENTON, I). L. and LE BRETON, G. C. Characterization intact human platelets and deterof 1146619 binding in unactivated, mination of binding site affinities of four TXA,/PGH, receptor antagonists (IS-APA, BM 13.177, ON0 3708 and SQ 29,548). Thromb. Res. 2,
676
ELEVATION OF CAMP LEVEL BY STA,
Vol. 64, No. 6
471-481, 1986. 13. WILLIAMS, K. A., MURPHY, W. and HASLAM, R. J. Effects of of protein kinase C on the agonist-induced stimulation tion of cyclic AMP formantion in intact human platelets. 243, 667-678, 1987.
activation and inhibiBiochem. J-
14. WATANABE. T., NARUMIYA, S., SHIMIZU. T. and HAYAISHI, 0. Characterior prosta landin Dz in human zation of the biosynthetic pathwa platelet-rich plasma. J. Biol. Chem. $57 , 14847-14g53, 1982. -15. POLLOCK, W. K., ARMSTRONG, R. A., BRYDON, L. J., JONES, R. L. and MACINTYRE, D. E. Thromboxane-induced phosphatidate formation in human platelets. Relationship to receptor occupant and to changes in cyLosolic free calcium. Blochem. J. 219, 833-842, l&4. 16. HUNG, S. C., GHALI, N. I., VENTON, D. L. binding of thromboxane A? antagonist platelet membranes. Biochlm. Biophys.
and LE BRETON, G. C. Specific 13-azaprostanoic acid to human Acta. 728, 171-178, 1983.
17. DORN II, G. W. Distinct platelet thromboxane Az/prostagrandin Hz receptor subty es. A radioli and binding study of human platelets. J. Clan. Invest. 8h , 1883-1891, 1689. --
BY
ELEVATION THHOMBOXANE
YOSHIHIDE The Japanese 4-l-31, Hiroo,
(Received
22.7.1991;
OF PLATELET CYCLIC AMP LEVEL Az/PROSTAGLANDIN H? RECEPTOR AGONISTS
ISHIKAWA and ATSUKO OGAWA Red Cross Central Blood Center Shibuya-ku, Tokyo 150, Japan
accepted
in revised form 1 .10.1991
by Editor A. Takada)
ABSTRACT Arachidonic acid (AA)or thromboxane Az/prostagIandin H, (TXA,/ PGH,) analog (STA? and U-46619)-induced aggregations yielded a bell-shaped dose-resnonse curve. The inhibitorv mechanism by high concentrations of the agonists was examined. STAz elevated CAMP level of platelet in a dose-dependent manner. And the aggregation was affected by metabolic inhibitors of CAMP. AA also rised CAMP level, and the rise was suppressed by indomethacin. These results indicate that the reduction of aggregation by high dose of the agonists is through CAMP elevation. The CAMP elevation was not suppressed by ruling out phospholipase C effects by chelation of cytoplasmic Ca”’ inhibition of protein kinase C (PKC). These results and suggest that the CAMP elevation is not due to activation of phospholipase C-linked TXAZ/PGH2 receptor. 13-APA, an antagonist of TXA2/PGH2 receptor, suppressed the CAMP elevation, although ONO-3708, another antagonist, had no effect. As to be expected effect of From this result, inhibitory 13-APA on high STA, level-induced aggregation was weaker than that of ONO-3708. The antagonists did not inhibit PGE,- or PGD2-induced CAMP elevation. suggest that platelet has adenylate These findings cyclase-linked TXA,/PGH, receptor.
INTRODUCTION Thromboxane AZ (TXA2) is a potent stimulator of platelet, and its binding to TXA,/PGH, receptor results in the formation of diacylglycerol and inositol 1,4,5-trisphosphate (IPB) through the activation of GTPbinding protein and phospholipase C (PLC)(l). It is known that dose-response curve for arachidonic acid (AA)-induced aggregation is biphasic (Z-4). The first phase is aggregatioEf deTp;;ding on TXA, synin spite of increase thesis (5). However, 2 synthesis with increasing AA dose, the aggregation yields a bell-shaped dose-response curve. There are several reports about the inhibitory mechanism by high concentrations of AA. For example, inhibitory effect of IipoxyKey words: Platelet aggregation, TXA,/PGH, receptor, STAz, Adenylate cyclase, CAMP
667
668
ELEVATION OF CAMP LEVEL BY STA,
genase products (6). perturbation elevation of cyclic AMP (CAMP) the cause of the inhibition.
Vol. 64, No. 6
of platelet membrane by AA (4) level by AA (3,7) are considered
and as
Best et al. (8) have demonstrated that synthetic analogs of prostaglandin endoperoxides, which caused platelet to aggregate, produced a small elevation of CAMP content in platelet. Murray et al. (9) have reported that U-46619 enhances iloprostor forskolin-induced Furthermore, cAMP elevation. we have already reported that aggregations induced by the TXAz/PGH, receptor agonists, STA, and U-46619, yield bell-shaped dose-response curves (10) as same as AA-induced aggregation. From these findings, we hypothesized that the inhibitory effect of high-dose of AA and TXA2/PGHz receptor agonists is due to elevation of CAMP level. To test this hypothesis, in this paper we have examined the change of CAMP level by these agonists and the mechanisms are discussed.
MATERIALS
AND
METHODS
Materials STAz ((+)-9,11-epithia-ll,lZ-methano-thromboxane A,) and ONO-3708 ((9, ll)(ll, 12)-dideoxa-9, a, 11a-dimethylmethano-11,12-methano-13,14dihydro-13-aza-14-oxo-15-cyclopenty1-16,17,18,19,20-pentanor-15-epithromboxane AZ) were gifts from Ono Pharmaceutical Co. (Osaka, Japan). 13-APA ((+)13-Azaprostanoic acid), U-46619 (15(S)-hydroxy-11,9-(epoxymethano)prosta-5%.13E-dicnoic acid), and Prostagrandin El and DZ (PGE,, PGDz) were obtained from Funakoshi (Osaka, Japan). DDA (2’,5’-dideoxyadenosine) was from Pharmacia ( Uppsala, Sweden) and IBMX (isobutylmethylxanthine) was from Sigma (St. Louis, USA). Staurosporine was from Kyowa Medex Co. Ltd. (Tokyo, Japan). BAPTA-AM (1,2-bis(o-aminophenoxy) -othane-N, N, N’, N’-tetraacetic acid) was from Dojindo Laboratories (Kumamoto, Japan). STA2, 13-APA, IBMX and staurosporine were dissolved in ethanol. DDA and ONO-3708 were dissolved in distilled water. BAI’TA-AM was dissolved in dimethyl sulfoxide. Preparation of platelets I’laLcletn (3.5 x lo5 cells/ u 1) in HEPES-buffered saline (135 mM NaCl, 5 mM -KCl, 10 mM HEPES, pH 7.4) ‘was prepared from 1 daystored platelet concentrate (PC) which was prepared by The Japanese lied Cross Central Blood Center. Treatment with inhibitors For studies of the effect of cytoplasmic Ca2’, platelet suspension was prepared from PC incubated with 100 ,uM of BAPTA-AM for 30 min at room temperature. For inhibition of CAMP metabolism, platelets were incubated with DDA or IBMX for 2 min at 37 “C. For the- urpose of inhibition-of protein kinase C, 500 nM of staurosporine was ad Z ed 2 min Antagonists of TXA,/PGH, receptor were before additi%n of agonist. added 1 min before stimulation of platelets. Measurement of platelet aggregation J STA,-, U-46619- or AA-induced anrrregation was _ measured in an aggregometer (NKK Hematracer 1, Nil; The aggregation ratio was determined at 2 min Isioscience Co., Japan). after the addition of agonists. Measurement of CAMP Platelets were incubated with TXA2/PGH2 receptor agonists, PGEl of 1 mM OF CaCh. The or PGD, for 1 min at 37°C in the presence adding trichloroacetic acid (TCA; final reaction was terminated by After removal of TCA with ether, CAMP concentration 6% (w/v)).
Vol. 64, No. 6
concentration kit (DuPont,
ELEVATION OF CAMP LEVEL BY STA,
of aqueous layer Wilmington, DE, USA).
was
determined
using
669
cAMP
[‘““I]
RIA-
RESULTS Relationship between cAMP elevation and aggregation by STA, As already reported (lo), STAz-induced aggregation exhibited a maximum value with about 50 nM of STA, (Fig.1, open circle). IBMX, an inhibited STA,-induced aggregation. inhibitor of phosphodiesterase, And DDA, an antagonist of adenylate cyclase (AC), enhanced the aggregation. Aggregation of DDA-treated platelet was hardly suppressed by a high concentration of STA,. As shown in Fig.2 in the presence of IBMX, cAMP level was apparently elevated by STA, with a concentration lower than that induces the maximum aggregation (see Fig.l), and the level was increased in a dose-dependent manner by STA,. Even in the absence of IBMX, CAMP level was elevated by a high concentration of STA,. Similar results were obtained with U-46619. U-46619-induced aggre-’ In the gation exhibited a maximum value with about 1 WM of U-46619. is a high concentration of presence of IBMX, 30~. of U-46619, which 11-46619, increased CAMP level by 2.42 + 0.56 pmol/lO” platelets (mean f SD, four experiments, pcO.01). The changes of aggregation shown in Fig.1 are thought to Ibe For confirmation of t-his idea, the due to the changes of CAMP level. relationship between cAMP level and aggregation by STA, was invcstiAs shown in Fig.3, CAMP level was increased by IBMX and degated.
loo-
s
80-
: z 605 bl cn 40a
STAZ (nM)
STAZ (nM)
Fig.1 (Left) Effects of CAMP metabolic inhibitors on STAz-induced aggregation. Platelets were incubated with DDA (200 MM; ?? ), IBMX (20 &MM;Cl) or without inhibitor ( 0) for 2 min before addition of STA,. Each point represents the mean value ? SD from three experiments. Fig.2 (Right) CAMP level of platelets stimulated by STA,. Platelet suspension was incubated with STAz for 1 min at 37 “C in the presence (11) or absence (0) of IBMX (20 rc~ M). Each point represents the mean value + SD from three experiments.
ELEVATION
670
loo-
a
OF CAMP LEVEL
-10
g 80-
loo-
s
BY STA,
Vol. 64, No. 6
b
-8 $
20 Oot,
HVIX WV
I 50
I 100
I 150
4% a 2o02 0 .I
DDA($4
Fig.3 Effects of cAMP metabolic inhibitors on CAMP elevation and After 2 min incubation with inhibitors, aggreaggregation by STAz. gation (open) and CAMP elevation (closed) induced by 100 nM (a) or 1000 nM of STAz (b) were measured. CAMP level of platelets treated by DDA (b) were determined in the presence of 20 ,uM of IBMX. The results are representative of four separate experiments.
”
-+ --
-
-+
-
-+
+
-
+
creased reverse
+
-+
Fig.4 Effects of inhibitors on STA,-induced CAMP elevation. Platelet suspension was prepared from platelet concentrate incubated with or without 100 hM of BAPTA-AM for 30 min at room temperature. Staurosporine (500 nM; Stauro.) was added 2 min before addition of STAz (0.1 y M). CAMP levels were determined in the presence of IBMX (20 KM). Mean + SD was calculated from four separate experiments.
STAz
Stauro. SAPTA-AM
by DDA in dose-dependent change of CAMP level.
manners.
And aggregation
showed
the
To determine whether STAz elevates CAMP level through PLC we studied the effects of second messengers released by activation, Protein kinase C activated by diacylglycerol was inhibited by PLC. by and rise of cytoplasmic Ca”’ by IP3 was diminished staurosporine, The concentrations of staurosporine and BAPTA inhibiBAPTA (Fig.4). of 47kDa (11) protein and elevation ted completely phospholylation of cytoplasmic Ca”, respectively, by 30 mu/ml of thrombin (data And chelation Staurosporine enhanced CAMP elevation. not shown). affect on STAz-induced CAMP BAPTA did not of cytoplasmic Ca” by elevation. The treatment by both BAPTA and staurosporine showed the If CAMP same results as the treatment with staurosporine alone. ought to elevation by STA, is through PLC activation, the elevation
Vol. 64, No. 6
loo-
ELEVATION OF CAMP LEVEL BY STA,
671
a
20-
A :“I _.;;
ol
J
10
100
1000
STAZ (nM)
,
,
,
10
100
1000
STAz (nM)
Fig.5 Effects of TXAJPGH, receptor antagonists on STAz-induced aggrePlatelets were incubated with 7.28 nM of ONO-3708 (a: ?? ), gation. 2.67 PM of 13-APA (b:O) or without antagonist (a,b:O) for 1 min at 37°C Each point represents the mean value ? SD of before addtion of STA,. three experiments. be suppressed plasmic Ca”.
by the inhibition But the elevation
PKC and the chelation of cytoof was enhanced by these treatment.
Effects
of TXA2/PGHz receptor antagonists ONO-3708 and 13-APA inhibited STAz-induced aggregation in doseFig.5 shows effects of the antagonists of dose dependent manners. which reduce the maximum aggregation (50 nM STAJ Lo abouL one-half. 13-APA concentration which was needed Lo inhibit STAz-induced aggregation was roughly two orders of magnitude higher than the concenLThis results seemed to be consistent with the ration of ONO-3708. demonstrated that the inhibreport by Kattelman et al. (12). They have itory potency of ONO-3708 for U-46619 binding was about two orders of ONO-3708 inhibited the aggremagnitude over the potency of 13-APA. gation by the high concentrations of STAz as well as that by the low But the inhibition by 13-APA was weaker in concentrations of STA,. For study of the difference of the high concentration range of STAZ. The antagonists CAMP levels were determined. inhibitory effects, The antagonists, however, alone had no effect on CAMP level (Fig.6). As PKC inhibits AC acLivity(l3), enhanced STAZ-induced CAMP elevation. PKC was inhibited by staurosporine to determine a net AC activity After treatment with induced by STAz and the antagonists. elevation of CAMP level was suppressed in a dosestaurosporine, dependent manner by 13-APA (Fig.G-b), although ONO-3708 had no effect (Fig.G-a). cAMP
elevation by PGE, and PGD, Activation of PGIJPGEIand PGD?-receptor results in CAMP eleIf STA, cross-reacts with these PG -receptors and if STA,vation. induced CAMP elevation is decreased by 13-APA antagonizing the PG receptors, the antagonist ought to decrease PGs-induced CAMP eleva.The 1’Gs were used with the concentrations which inhibit comLion. These concentrations pletely full aggregation induced by thrombin. of the PGs elevated CAMP level as the same extent as 0.1 ,uM of STA, (Fig.6) as shown in Table 1. Both ONO-3708 and 13-APA had no effect on CAMP elevation by the PGs.
ELEVATION OF CAMP LEVEL BY STA,
672
Vol. 64, No. 6
8-
6-
0
0.01
0
0.1
ONO-3708
1 13-APA
(ptvl)
10 (j&l)
Fig.6 c4MP levels of platelets treated by TXA2/PGH2 antagonists. After treatment with 20 ,uM of IBMX, platelets were incubated with (IQ) or without ( R) staurosporine (500 nM), and then stimulated by STA, (0.1 b M) for 1 min at 37 ‘C in the presence of antagonists. Open bar shows cAMP level of platelets incubated wil.h antagonists alone (a: ONO-3708, b:13-APA) for 2 min at 37 “C. Mean + SD was calculated from StatisLical analysis (conLrol@ in Fig.b) four separate experiments. versus expose to 13-APA) was by paired t test: ‘p
Effects of PGs-lnduced on
TXA,/PGH2
Antagonist ONO-3708
( FM) 0 0 . 1 1;::
13-APA
TABLE 1 receptor
antagonists
cAMP(pmol/lO”platelets) PGE, PGD, 11.4k1.7 8.9k2.1 10.7k3.5 9.2kl.l 10.6k2.4 9.8kO.9 11.623.0 8.2+1 .5
0 1
12.5k2.2 9.6k1.1 12.Ok3.3 9.7kl.l 10.9fl.O 8.9k1.2 1:: 12.OFl.O 9.4f1.3 In the presence of CaCL (1 mM), IBMX (20 /.LM) and TXAJPGH, receptor antagonists, platelet suspension was incubated with PGE, (50 nM) or PGD, (33.3 nM) for 1 min at 37 “C. The values represent means + SD of four experiments.
Change of cAMP level by arachidonic acid Fig.7 shows change of CAMP level by AA. As well known, AA-induced aggregation yielded a bell-shaped dose response curve (Fig.6). And cAMP level was elevated with the increasing concentration of AA, as already reported (3,7). Platelets were pretreated by indomethacin to exclude effects of TXA, formed from AA. Indomethacin (1 w M) inhibThe increase of CAMP level ited completely AA-induced aggregation. was apparently suppressed by the pretreatment., although the suppres-
Vol. 64, No. 6
ELEVATION OF CAMP LEVEL BY STA,
673
cAM1’ level of platelets by arachidonic acid. Af’l.cbr Lreafmenl. with 20 FM of IISMX, platelets stimulaLed by AA ( 0,O) for 1 min at 37°C. Indome0lacin (1 &MM) was added I min kJcf'Ore addition of AA (0). AggregaLion ( Cl) was measured wiLhoul f"kl~~S~~ Lreatments. Moan *SD was calculated from four separale experiments. SLiiLisLical analysis (control versus expose to AA) was by paired L Lest.: ‘p
I:ig.7
I realed
AA PJV In Lho same condition, ST&-induced cAMI’ ~lc?siori was noL complete. vat.ion was no1 afTect_ed by indomethacin (data noL shown). This is because TXA, synthesis by stimulation wi1.h TXAJl’GlI, agonists is negligible in sLored pla&?lets (10).
I)ISCUSSION I~f’l‘c~ct of TXA,/l’GIl, rcceplor agonisLs on cAMI” level of platelets were examined. The agonists (STA,, U-46619) elevated cAMP level of plaLclcLs. IHMX, an inhibiLor of phosphodiestzrase, enhanced Lhe CAMI’ DI)A, an anlagonist c!levaLion and inhibiLod STA?-induced aggregation. cyclase (AC), suppressed Lhe reduction of aggregation at Of adcnylaLc1 high c:oric:c?ritraLioris of ST&. These findings indicale that STA, elevates cAM1’ level Lhrough aclivation of AC and also indicate that Lhe rc>duction 01‘ aggregalion by high c:orlc:onLraLiorls of STA, is due to the c>lcvaLiorl oI‘ cAMt’ level. Murray et. al. (9) have dnmonsLraLed t.haL cAMI’ elevation by ilor)rost. was amnlif’ied bv Drier exDosure to U-46619 throunh acLivaLioyl of TXA/l’<511? receiLo;. In o;lr rcsulLs, cAMP level Gas elevaLed only by TXAJl’GFl~~ rccepLor agonists without activators of AC such as iloprosl. 11, is
known LhaL l’G12, l’Gl$ and I’Gl)? elevate plaLele1 cAMI’ do noL synthesize PGIz and I’GE,. And Watanabe ctL al. (14) have reported t.ha~ conversion of l’GlI, to I’GD, does not, occur in washed plat.ele&. I:urLhermore, reduction of aggregation a~ high concentralions of‘ STA:. was also observed in washed plaLelets t.reated by 33 GM of indomeLhacin (IO). Therefore, CAMP elevation by ST& is not_ due Lo formaLion of these proslaglandins by AA melabolism.
1C!\’(?1.
well
I%uL plaLc?leLs
TXAJI’GII, receptor is linked Lo phospholipase C (PI,C) via GTl’binding protein (1). I’I,C forms IP, and diacylgrycerol from phosphaLidyl inosiLol and the products mobilize cyLoplasmic Ca” and activale prot_ein kinase C (I’KC), respectively. cAMP elevaLion by STAz was 1101, aff’t?cLed by chelation of cyt,oplasmic Ca” with HAPTA and was ac:celeraLed by a I’KC inhibitor, sLaurosporinc. Furthermore, ONO-3708, which inhibilcd STAz-induced aggregation, did noL suppress the cAMP olevalion. These rcsulLs indicaLo that t,he elevation of cAMI’ occurcs
674
\ria sysLems
ELEVATION OF CAMP LEVEL BY STA,
except
of
TXA2/PGHr
receptor
linking
Lo
Vol. 64, No. 6 PIG.
Williams et al (13) have reported that PKC inhibits AC activity. agreement with the report., staurosporine enhanced cAMP elevation Therefore, the enhancement of cAMP elevation by TXAJl’GHT \Jy STAZ. anLagonisLs is due to that the antagonists suppress PKC activation through inhibition of STAz binding to the receptor linking to PLC. In
13-APA inhibited STAZ-induced r.AMP elevation in the condition the PKC effect using ruling out staurosporine, although ONO-3708 had no effect. And inhibitory effect of 13-APA on aggregation was weaker in high concentrations of STAI, compared with ONO-3708. This is because 13-APA inhibits STAz-induced aggregation by antagonizing while it probably enhances the STA, binding Lo PLC-linked receptor, aggregation by suppressing CAMP elevation. These results can not be explained if platelets have only one type of receptor against TXAJ l’GH2. It has been reported that platelet has single receptor against TXAJPGH, (12,15). However, some researchers (16,17) have reported subLype of the receptor. Our results indicate that platelets have aL least two types of receptor against TXAJPGH2, that is, a PLC-linked and an AC-linked receptor. receptor It seemes that ONO-3708 selectively binds LO the PLC-linked receptor, and STAz and 13-APA bind to not only the PLC-linked receptor but also the AC-linked reccplor. The AC-linked receptor may be PGIJPGE,or PGDz-receptor. To make clear whether the AC-linked receptor is against the PGs or not, offeeLs of TXA2/PGHz receptor antagonists on CAMP elevation were oxamined, because 13-APA inhibited CAMP elevation by STA,. cAMP elevation by 50 nM of PGE, or 33.3 nM of PGD, was about the same degree as that by 100 nM of STAz with PKC inhibitor. The increase by STA? was inhibited by 1 ,uM of 13-APA, but PGE,- or PGDz-induced elevation was not affecLed even by 100 KM of the antagonist. These results suggested that 13-APA has no effect on the PG receptors, and that inhibition of STA?-induced CAMP elevation by 13-APA is not due to antagonism of 13-APA Lo the PG receptors. Therefore, CAMP elevation by STAz is thought t.o occure through receptor(s) other than Lhe PG receptors. There are reports (3,7) that reduction of AA-induced aggregation by high concentrations of AA is due to elevation of CAMP level by AA ilself. And in Lhe report (7), hinder eL al. have demonstrated that ~~~~-~h~tcetdh~g~rega!.ion was inhibited by high concentrations r of AA inhibited aggregation wras noL restored by t0 FM of Although they have not shown the effect of indomethacin. indomeLhacin on CAMP lecel, their results seemed to be in conflict with our data that the CAMP elevation was inhibited bv indomethacin It is possible that AA itself’ elevates cAMP level, because (Fig.6). the suppression of cAMP elevation by indomethacin was not complele. We have already reported that of indomethacin with a high concentration (33 ,uM) has inhibitory effect on TXAJ PGHZ-induced aggregation Therefore, in their condition, the restoration of aggregation (IO). inhibitory effects of was probably not observed because of the indomethacin and CAMP elevation by AA itself. our results suggest that not only stable analogs of Therefore, TXAJPGH2 but also TXAz elevates platelet CAMP level through AC-linked TXAJPGH, receptor and that a part of inhibitory effect of AA is due to TXA, formed from AA.
Vol. 64, No. 6
ELEVATION
OF CAMP LEVEL BY STA,
675
ACKNOWLEDGEMENT help
The authors extend in this research.
their
gratitude
to
Dr. S. Sasakawa
for
her
Ii EI’EK ENCES 1. BRASS, I,. F. Regulation of‘ biological responses in platelets. In: Blood Cells and Arteries in Ilypcrtcnsion and Atheroslerosis. P. Meyer and F’.Marche (Ed.) N ew York: Raven Press, 1989, pp.59-92. 2.
HASHIMOTO, Y., NAITO, C., KUME, S., KATOJI., WATANABET., KAWAMUKA, M., TERAMOTO, T. and OKA, II. IIigh concentrations of arachidonic acid induce platelet aggregation and serotonin relcasc independent of prostaglandin endoperoxides and thromboxanc A,. Biochim. Biophys. & &l, 283-291, 1985.
3. KOWALSKA, exogenous stimulation
M. A., KAO, A. K. and DISA, J. IIigh concentrations of‘ arachidonatc inhibit calcium mobilization in Iatelcts of adenylatc cyclase. Biochcm. -J. -’ 253 255- 3 62, 1988.
by
4. SATO, T., IIASHIZUME, T., NAKAO, K., AKIBA. S. and FUJII, T. Platelet desensitization by arachidonic acid is assosiated with the suppression of cndoperoxidc/thromboxane AL binding to the membrane receptor. Biochim. Biophys. Acta 992, 168-173, 1989. .5 . IINDER, B. I,., CHEKNOFF, A., KAPI.AN, K. 1~.and GOODMAN, D. S. Release platelet-derived growth factor from human platelets by arachidonic acid. -Proc. Natl. --Acad. Sci. USA 2 76 4107-4111, 1979. 6. AHARONY, D., SMITH, J. B. and SILVER, M. J. Regulation induced platelet aggregation by the lipoxygenase peroxyeicosatetraenoic acid. Biochim. Biophys. Acta Studies 7. LINDER, B. I,. and GOODMAN, D. S. inhibition of platelet a gregation levels of’ arachidonate. B?ood 60, 436%%,
on the release 1982.
of
of arachidonat.eproduct, 12+ydro718, 193-200, 1982. mechanism induced
of by
the high
8. BEST. I,. C., McGUIRE, M. B., MARTIN, T. J., PRESTON, F. E. and KUSSELL, Ii. G. G. Effects of epoxymethano analogues of prostaglandin endoon release of 5-hydroxytryptamine and on peroxides on aggregation, the metabolism of 3’,5’-cyclic AMP and cyclic GMP in human platelets. Biochim. Biophys. & 583, 344-351, 1979. endoper9 . . MURKAY, R., SHIPP, E. and FITZGERALD, G. A. Prostaglandin oxide/TXA? receptor desensitization. Cross-talk with adenylate cyclase in human platelets. J. Biol. Chem. 265, 21670-21675, 1990. -~ IO. ISHIKAWA, Y., OGAWA, A. and SASAKAWA, on platelet activation by arachidonic 1989.
S. Effect of acid. Thromb.
cytoplasmic Res. ,54, 17-2$Il
11. KAIBUCHI, K., TAKAI, Y., SAWAMURA, M., HOSHIJIMA, M.,FUJIKURA, T. and INISHIZUKA, Y. Synergistic functions of protein phos horylation and calcium mobilization in platelet activation. J. Biol. CPIem. m, 6701 -~-6704,1983. 12. KATTELMAN, E. J., VENTON, I). L. and LE BRETON, G. C. Characterization intact human platelets and deterof 1146619 binding in unactivated, mination of binding site affinities of four TXA,/PGH, receptor antagonists (IS-APA, BM 13.177, ON0 3708 and SQ 29,548). Thromb. Res. 2,
676
ELEVATION OF CAMP LEVEL BY STA,
Vol. 64, No. 6
471-481, 1986. 13. WILLIAMS, K. A., MURPHY, W. and HASLAM, R. J. Effects of of protein kinase C on the agonist-induced stimulation tion of cyclic AMP formantion in intact human platelets. 243, 667-678, 1987.
activation and inhibiBiochem. J-
14. WATANABE. T., NARUMIYA, S., SHIMIZU. T. and HAYAISHI, 0. Characterior prosta landin Dz in human zation of the biosynthetic pathwa platelet-rich plasma. J. Biol. Chem. $57 , 14847-14g53, 1982. -15. POLLOCK, W. K., ARMSTRONG, R. A., BRYDON, L. J., JONES, R. L. and MACINTYRE, D. E. Thromboxane-induced phosphatidate formation in human platelets. Relationship to receptor occupant and to changes in cyLosolic free calcium. Blochem. J. 219, 833-842, l&4. 16. HUNG, S. C., GHALI, N. I., VENTON, D. L. binding of thromboxane A? antagonist platelet membranes. Biochlm. Biophys.
and LE BRETON, G. C. Specific 13-azaprostanoic acid to human Acta. 728, 171-178, 1983.
17. DORN II, G. W. Distinct platelet thromboxane Az/prostagrandin Hz receptor subty es. A radioli and binding study of human platelets. J. Clan. Invest. 8h , 1883-1891, 1689. --