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Am J Physiol Heart Circ Physiol 273: H1854-H1859, 1997;
0363-6135/97 $5.00
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Vol. 273, Issue 4, H1854-H1859, October 1997

Cyclooxygenase inhibition decreases nitric oxide synthase activity in human platelets

Liying Chen, Mina N. Salafranca, and Jawahar L. Mehta

Departments of Medicine and Pharmacology, University of Florida and Veterans Affairs Medical Center, Gainesville, Florida 32610-0277

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Activity of both nitric oxide (NO) synthase (NOS) and cyclooxygenase (COX) plays an important role in the regulation of platelet function. NO has been shown to directly activate COX. This study was designed to determine whether products of the COX pathway in turn regulate NOS activity. Human platelets were incubated with aspirin, indomethacin, the selective thromboxane A2 synthase inhibitor U-63557A, or the prostaglandin H2-thromboxane A2-receptor blocker SQ-29548 for 1 h at 37°C. Multiple indexes of the activity of the L-arginine-NO pathway and changes in cytosolic Ca2+ concentration ([Ca2+]i) were measured in platelets. Both aspirin and indomethacin decreased NOS activity, measured as the conversion of L-arginine to L-citrulline and nitrite (+nitrate) formation, in platelets in a concentration-dependent fashion. Aspirin also decreased guanosine 3',5'-cyclic monophosphate accumulation in platelets. The NOS inhibitory effects of these aspirin and indomethacin effects were reversed by coincubation with the thromboxane A2 analog U-46619 or an excess of CaCl2. Incubation of COX inhibitors with platelets was associated with significant reductions in basal as well as thrombin-stimulated [Ca2+]i, and the reduction in [Ca2+]i was reversed by U-46619. Incubation of platelets with U-63557A and SQ-29548 resulted in inhibitory effects on NOS activity qualitatively similar to those of COX inhibitors. The effects of COX inhibitors or U-63557A were not associated with a change in NOS protein expression in platelets. These data suggest that NOS activity in human platelets is inhibited by COX inhibitors, mediated, at least in part, via suppression of thromboxane A2 and [Ca2+]i mobilization in platelets.

aspirin; indomethacin; thromboxane A2

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

PLATELETS POSSESS an active cyclooxygenase (COX) enzyme that catalyzes the formation of thromboxane A2 and other eicosanoids from arachidonic acid. Platelets have also been reported to synthesize nitric oxide (NO) from L-arginine (19, 20). Mehta et al. (11) identified a calcium-dependent endothelial-type constitutive NO synthase (NOS) isoform using reverse transcription-polymerase chain reaction in human platelets. They also found that the classic inhibitors for NO synthesis, NG-nitro-L-arginine methyl ester and Nomega -nitro-L-arginine, significantly decrease L-arginine (the substrate of NOS) uptake in intact platelets and NOS activity in intact platelets and platelet cytosol (2, 3, 11). Both COX and NOS pathways regulate platelet function. In addition to the important role of their products in platelet function, COX and NOS also share a number of similarities. Eicosanoids and NO are paracrine modulators of platelet function and mediate intracellular signaling via cyclic nucleotides [adenosine 3',5'-cyclic monophosphate or guanosine 3',5'-cyclic monophosphate (cGMP)]. Thromboxane A2 induces platelet aggregation, whereas NO inhibits it (13, 15-17). Both NOS and COX enzymes require heme as a cofactor (6, 22) and have constitutive and cytokine-inducible forms. NO binds to the heme-Fe2+ prosthetic group of the soluble guanylate cyclase, leading to its activation, with a consequent increase in the levels of cGMP.

NO has been shown to stimulate COX activity, leading to the simultaneous release of mediators such as prostaglandin (PG) E2 in macrophages and renal tissues (7, 21). Thus a close interaction between the products of NOS and COX inhibition has become evident. However, the effects of COX activation on the L-arginine-NO pathway has not been elucidated, especially in platelets. In the present study, we demonstrate regulation of NOS activity by COX inhibition in human platelets.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. L-[2,3,4,5-3H]arginine (69 Ci/mmol; 1 Ci = 37 GBq; 1.0 mCi/ml) and the RPN 2108 ECL Western blotting analysis system were obtained from Amersham Life Science. A cGMP enzyme-linked immunosorbent assay (ELISA) kit was obtained from Cayman Chemical. Mouse monoclonal anti-human endothelial or rat brain NOS antibodies were obtained from Transduction Laboratories. A 10-kDa protein ladder (10,000-200,000 Da) was obtained from Life Technologies. Indomethacin was obtained from Merck. The selective thromboxane A2 synthase inhibitor U-63557A (12) and the thromboxane A2-endoperoxide analog U-46619 were obtained from Upjohn. The thromboxane A2-endoperoxide-receptor blocker SQ-29548 was a gift from Bristol-Myers, Squibb. All other chemicals were purchased from Sigma Chemical.

Platelet preparation and treatment. Peripheral venous blood was collected in 3.8% sodium citrate from normal healthy volunteers who had not taken any drugs in the previous 10 days. The blood was centrifuged at 800 revolutions/min (rpm) for 10 min at room temperature to obtain platelet-rich plasma (PRP). The PRP was incubated with aspirin (0.3-6 mM), indomethacin (10-100 µM), or U-63557A (10 µM) or SQ-29548 (1 µM) for 1 h at 37°C. After incubation, the PRP was centrifuged for 10 min at 3,000 rpm in the presence of 10% ACD buffer (0.8% citric acid, 2.2% sodium citrate, and 2.4% dextrose) to obtain the platelet pellet. The platelet pellet was then washed with calcium-free Tyrode buffer (composition in mM: 137 NaCl, 2.7 KCl, 1.0 MgCl2, 0.35 NaH2PO4, 11.9 NaHCO3, and 5.5 glucose; pH 6.5) in the presence of ACD buffer. The platelets were then resuspended in the appropriate buffer at 2-3 × 108 cells/ml. These washed platelets were not activated during the process of washing, as evident from the preserved aggregatory response to thrombin.

Determination of L-citrulline production. The time course of L-arginine uptake in the platelets was measured in preliminary experiments, and the maximal uptake of L-arginine was observed at 30 min. Therefore, to examine the effect of COX inhibitors on NOS activity, the platelets were treated with different agents and incubated with L-[3H]arginine (average count 1,000,000 disintegrations/min) plus cold L-arginine (0.5 mM) in 1 ml of NO buffer [in mM: 25 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 140 NaCl, 5.4 KCl, 1 CaCl2 and MgCl2; pH 7.4] with or without U-46619 (60 nM) or various combinations of the agents for 30 min. The reaction was stopped with 1 ml of cold buffer (composition in mM: 25 HEPES, 118 NaCl, 4.7 KCl, 1.18 KH2PO4, 24.8 NaHCO3, 5 Nomega -nitro-L-arginine, and 4 EDTA; pH 5.5), and each tube was washed twice. The platelet pellet was disrupted by adding 1 ml of 0.3 M HClO4 and neutralized with 65 µl of 3 M K2CO3. Aliquots of the cell suspensions were then applied to 2-ml columns of Dowex AG 50W-X8 (Na+ form), which were eluted with 6 ml of distilled water (11). L-[3H]citrulline in the eluent was quantitated by liquid scintillation spectroscopy. NOS activity was expressed as the formation of L-citrulline in picomoles per 2 × 107 platelets (3).

To investigate the regulation of NOS activity by Ca2+, platelets (2 × 107 cells) treated with buffer or COX inhibitors were incubated with L-[3H]arginine plus unlabeled L-arginine for 30 min in 1 ml of NO buffer containing different concentrations of CaCl2 (0-5 mM) with or without ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) at 37°C. NOS activity was also measured in platelets treated with different concentrations of the Ca2+-channel blocker verapamil (0.1-10 µg/ml) for 1 h at 37°C. L-Citrulline formation was used as an index of NOS activity (3).

Determination of nitrite (+nitrate) in platelet supernatants. Platelet nitrite (+nitrate) level was measured by a fluorometric assay, with modification of the method of Damiani and Burini (4). Platelets (5 × 107 cells/ml) treated with different agents were incubated with L-arginine (1 mM) in 1 ml of NO buffer for 1 h at 37°C. The reaction was stopped by centrifuging the sample at 3,000 rpm for 15 min at 4°C. After centrifugation, supernatants of the platelets were collected. Each aliquot was incubated with 20 mU of nitrate reductase in the presence of 1.44 mM NADPH for 1 h at 37°C, thereby reducing nitrate to nitrite. The reaction was terminated by the addition of 1 ml of distilled water followed by the addition of 200 µl of 2,3-diaminonaphthalene (0.05 mg/ml) to form the fluorescent product 1-(H)-naphthotriazole under acidic conditions. After 15 min of incubation, this reaction was terminated with 100 µl of 2.8 N NaOH. Relative fluorescence intensities in different samples were measured with a Perkin-Elmer MPF44A spectrofluorimeter with an excitation wavelength of 365 nm and an emission wavelength of 405 nm. Nitrite (+nitrate) concentration was determined with sodium nitrite as the standard. In control experiments, we looked for interference in the assay by aspirin, indomethacin, and U-63557A, and no inhibitory effect was identified in multiple control experiments.

Determination of cGMP levels in platelets. PRP (3 × 108 platelets/ml) aliquots were incubated with buffer or aspirin (0.3-6 mM) for 1 h at 37°C. Thereafter, 0.5 ml of trichloroacetic acid (TCA; final concentration 10%) was added to the PRP aliquots. After centrifugation at 3,000 rpm for 15 min, TCA was extracted five times from the supernatant with water-saturated ether. The aqueous phase was dried under a stream of nitrogen and resuspended in 1.5 ml of phosphate buffer. cGMP levels were measured by ELISA (variability between duplicate values < 10%). The values of cGMP in platelet-poor plasma were subtracted, and the results were expressed as femtomoles per 3 × 108 platelets.

Determination of cytosolic Ca2+ concentration in platelets. The washed platelets were incubated at 37°C in the presence of 2 µM fura 2-acetoxymethyl ester for 45 min. To study the regulation of Ca2+ influx by exogenous calcium, the cytosolic Ca2+ concentration ([Ca2+]i) in the platelets was measured under varying extracellular concentrations of Ca2+ (0-5 mM). To measure the effects of COX and thromboxane synthase inhibitors on Ca2+ influx, the platelets were suspended in physiological saline (in mM: 25 HEPES, 118 NaCl, 4.7 KCl, 1 CaCl2, 1.18 MgSO4, 1.18 KH2PO4, and 10 glucose; pH 7.4) at a concentration of 2 × 108 cells/ml and incubated with aspirin (60-600 µM), indomethacin (2-20 µM), or U-63557A (10 µM) for 15 min before the addition of agonists (10 µM ADP, 2.5 U/ml of thrombin, or 0.6 µM U-46619). The platelet suspensions were continuously stirred with a magnetic stirrer. [Ca2+]i was monitored with a microcomputer-controlled SLM Aminco spectrofluorometer (SLM Instruments). Excitation wavelengths were changed every 2 s between 340 and 380 nm, and emission light was detected at 500 nm at both excitation wavelengths. Maximal and minimal fluorescence was determined in each sample by adding 10% Triton X-100 followed by 2 mM MnCl2.

Determination of thromboxane B2 in platelets. PRP (3 × 108 platelets/ml) aliquots were incubated with buffer, aspirin (0.3-6 mM), or indomethacin (10-100 µM) for 1 h at 37°C. Thereafter, 0.5 ml of TCA (final concentration 10%) was added to the PRP aliquots. After centrifugation at 3,000 rpm for 15 min, TCA was extracted five times from the supernatant with water-saturated ether. The aqueous phase was dried under a stream of nitrogen and resuspended in 1.5 ml of phosphate buffer. Thromboxane B2 levels were measured by ELISA (variability between duplicate values < 10%). The values of thromboxane B2 in platelet-poor plasma were subtracted, and the results are expressed as nanograms per 3 × 108 platelets.

Western analysis. PRP was incubated with aspirin, indomethacin, the thromboxane A2 synthase inhibitor, or the thromboxane A2 analog for 1-3 h. After incubation, the platelets were isolated from the PRP, washed, and lysed with lysis buffer [1% sodium dodecyl sulfate, 0.5% Triton X-100, and 10 mM tris(hydroxymethyl)aminomethane · HCl; pH 7.4] supplemented with protease inhibitors and centrifuged at 30,000 rpm for 60 min at 4°C. The cytosolic protein from different platelet aliquots (10 µg/lane) was separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a Bio-Rad Mini-Protean Cell, transferred to nitrocellulose filters (Amersham Life Science), and then immunoblotted with a mouse monoclonal antibody against human endothelial NOS peptide sequence 1030-1209 at a 1:250 dilution. A mouse anti-rat brain NOS monoclonal antibody was used as a negative control. Anti-mouse horseradish peroxidase-conjugated antibody was used as a secondary antibody at a 1:2,500 dilution. The blots were detected with the enhanced chemiluminescence method (ECL Western blot kit) (3).

Statistics. All data are based on at least three experiments and are expressed as means ± SE. Statistical analyses were performed with analysis of variance or Student's t-tests (paired or unpaired data), as appropriate. A P value < 0.05 was considered significant.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

[Ca2+]i and NOS activity in intact human platelets. Platelet [Ca2+]i increased as exogenous Ca2+ was increased from 0 to 5 mM (from 43 ± 5 nM in the Ca2+-free buffer to 130 ± 25 nM in the presence of 5 mM CaCl2; Fig. 1). NOS activity in the platelets, measured as L-citrulline formation, was maximal in the presence of 0.5 mM L-arginine and increased as exogenous Ca2+ was increased, with a peak value at 2 mM (Fig. 1). Higher concentrations of Ca2+ did not cause a further increase in NOS activity, indicating that the Ca2+ dependence of NOS activity in platelets is maximal at physiological concentrations of extracellular Ca2+. NOS activity was about one-fifth in Ca2+-free buffer compared with that in Ca2+-rich buffer (8.9 ± 1.3 vs. 43.0 ± 9.2 pmol/2 × 107 platelets; P < 0.01). Furthermore, the addition of EGTA (1.5 mM) in Ca2+-free buffer resulted in an additional 25% decrease in NOS activity. As additional evidence for the Ca2+-dependent nature of NOS activity in platelets, verapamil decreased NOS activity in a concentration-dependent manner. Verapamil at 0.1 µg/ml decreased NOS activity by 29% (L-citrulline formation 38.5 ± 1.4 to 27.3 ± 1.2 pmol/2 × 107 cells; P < 0.05), whereas a higher concentration of verapamil (10 µg/ml) resulted in an ~50% decrease in NOS activity (19.9 ± 0.8 pmol/2 × 107 cells).


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  		Fig. 1.   Nitric oxide synthase (NOS) activity in platelets expressed as formation of L-citrulline (A) and cytosolic Ca2+ concentration ([Ca2+]i; B) in presence of different concentrations of extracellular Ca2+. Note an almost linear rise in [Ca2+]i with increase in exogenous Ca2+, but NOS activity is maximal in presence of 1-2 mM extracellular Ca2+.

COX inhibitors and NOS activity. Both aspirin and indomethacin inhibited L-citrulline formation in platelets in a concentration-dependent fashion. These COX inhibitors also inhibited nitrite (+nitrate) production in platelets. A low concentration of aspirin (<0.3 mM) had only a modest effect (data not shown), whereas the highest concentration (6 mM) almost totally blocked nitrite (+nitrate) production. These effects of COX inhibitors were reversed by the addition of either exogenous Ca2+ or U-46619. Neither exogenous Ca2+ nor U-46619 per se significantly increased L-citrulline or nitrite (+nitrate) formation. Incubation of platelets with U-63557A and SQ-29548 caused qualitatively similar changes as aspirin or indomethacin. These data are summarized in Table 1.

                              
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  		Table 1.   Modulation of NOS activity by ASA, Indo, and U-63557A

Aspirin and platelet cytosolic cGMP. As additional evidence for the inhibition of NO production, aspirin elicited a concentration-related decrease in platelet cGMP content. The low concentration of aspirin (0.3 mM) decreased cGMP content from 550 ± 35 to 50 ± 6 fmol/3 × 108 cells (P < 0.01), whereas the higher concentration of aspirin (0.6 mM) resulted in undetectable cGMP levels in the platelets.

COX inhibitors and changes in [Ca2+]i. The [Ca2+]i in resting platelets was 120 ± 16 nM, in keeping with previous reports indicating that platelets were not activated during the preparation. Pretreatment of platelets with aspirin or indomethacin elicited a marked decrease in the resting platelet [Ca2+]i levels (Fig. 2, Table 2). Both aspirin and indomethacin also markedly inhibited the thrombin- or ADP-mediated rise in [Ca2+]i, whereas the U-46619-induced peak in [Ca2+]i was only modestly, but significantly, attenuated (Fig. 2). To investigate the role of thromboxane A2 in the mobilization of [Ca2+]i, platelets were treated with the thromboxane A2 synthase inhibitor U-63557A. U-63557A decreased resting platelet [Ca2+]i by ~20% and attenuated the rise in [Ca2+]i induced by thrombin and ADP by 26 and 50%, respectively (P < 0.02). As expected, U-63557A did not block the rise in [Ca2+]i induced by U-46619, but [Ca2+]i levels declined rapidly thereafter to resting levels (Fig. 2).


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  		Fig. 2.   Changes in [Ca2+]i in fura 2-loaded human platelets in response to thrombin, U-46619, or ADP. Buffer, aspirin (600 µM), indomethacin (20 µM), or selective thromboxane A2 synthetase inhibitor U-63557A (10 µM) was added 20 min before addition of these agonists. Fluorescence tracings are representative of 4 separate experiments. Note that resting [Ca2+]i was decreased by both aspirin and indomethacin in fura 2-loaded platelets.

                              
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  		Table 2.   Effects of ASA, Indo, and U-63557A on thrombin, U-46619, or ADP-stimulated [Ca2+]i

After treatment with the Ca2+ chelater EGTA, thrombin caused a small increase in [Ca2+]i (66 ± 7 to 116 ± 11 nM), indicating the release of intracellular Ca2+. However, in the presence of aspirin and EGTA, the small rise in [Ca2+]i induced by thrombin was totally abolished, suggesting that aspirin inhibits not only Ca2+ influx but also intracellular Ca2+ release induced by thrombin (Fig. 3).


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  		Fig. 3.   Changes in [Ca2+]i in fura 2-loaded human platelets in response to thrombin in presence of 10 mM EGTA. Aspirin (600 µM) was added 20 min before EGTA. Fluorescence tracings are representative of duplicate determinations from 2 separate experiments. Note decrease in resting platelet [Ca2+]i by aspirin.

Aspirin, indomethacin, and platelet thromboxane B2. As direct evidence for the inhibition of thromboxane A2 production, aspirin elicited a concentration-related decrease in platelet thromboxane A2 production. The low concentration of aspirin (0.3 mM) decreased the thromboxane B2 content by 55% (from 0.46 ± 0.02 to 0.22 ± 0.02 ng/3 × 108 cells; P < 0.01; n = 3 experiments), whereas the highest concentration of aspirin (6 mM) resulted in an undetectable thromboxane B2 level in the platelet suspension. Indomethacin likewise decreased platelet thromboxane A2 production, so that the 100 µg/ml concentration decreased thromboxane B2 levels by >95% in all experiments.

Western analysis. Western blot analyses of platelet proteins were performed with a mouse anti-human endothelial-type constitutive NOS monoclonal antibody. Immunoblotting consistently identified a band with an estimated molecular mass of 140 kDa in platelets. There was no evidence of brain-type NOS in the platelets. In all analyses, the endothelial-type NOS protein level was not changed in platelets treated with either aspirin, indomethacin, or U-63557A or U-46619. Results of a representative experiment are shown in Fig. 4.


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  		Fig. 4.   Western analysis of platelets with a mouse monoclonal antibody against human endothelial-type NOS from control (lane B), aspirin-treated (6 mM; lane C), indomethacin-treated (100 µM; lane D), U-63557A-treated (10 µM; lane E), and U-46619-treated platelets (60 nM; lane F). Lane A, endothelial cell cytosol. An equal amount of platelet protein (10-20 µg) was loaded onto each lane. Constitutive NOS protein in both endothelial cells and platelets has a molecular mass of ~140 kDa. This Western analysis is representative of 3 separate analyses in which platelets were incubated with aspirin, indomethacin, U-63557A, or U-46619 for 3 h.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Our study demonstrates that inhibition of COX by two different agents, aspirin and indomethacin, markedly attenuates NOS activity as determined directly by the measurement of L-citrulline and nitrite (+nitrate) formation and indirectly by the measurement of cGMP accumulation in platelets. The inhibitory effects of aspirin and indomethacin on NOS activity were reversed by exogenous Ca2+ as well as by U-46619, a functional thromboxane A2 mimetic. The selective thromboxane A2 synthase inhibitor U-63557A and the thromboxane A2-endoperoxide-receptor blocker SQ-29548 exerted qualitatively similar effects as aspirin and indomethacin on NOS activity. These observations suggest that the effects of these COX inhibitors are mediated, at least in part, via thromboxane A2 inhibition. Western analysis showed that aspirin or indomethacin does not change NOS protein level in platelets, suggesting that inhibition of NOS by aspirin and indomethacin is not mediated by a decrease in NOS expression at the transcriptional level or posttranscriptional destabilization of this protein.

Arachidonic acid, the substrate for COX, fulfills several functions to be considered a classic stimulator of platelets by increasing [Ca2+]i (1). Our observations relative to the inhibition of COX activity and reduction in the rise of platelet [Ca2+]i induced by thrombin or ADP by aspirin and indomethacin are in accordance with these data (1, 8, 17, 18). We now show that aspirin and indomethacin decrease platelet [Ca2+]i by the inhibition of Ca2+ influx (Fig. 2) as well as Ca2+ release from intracellular storage granules (Fig. 3). Notably, these agents reduced resting platelet [Ca2+]i levels by 20-40%. Concurrent with the decrease in [Ca2+]i, we found that aspirin and indomethacin at high concentrations almost totally inhibited the formation of thromboxane A2 in resting platelets. These findings suggest that COX activation and the subsequent release of thromboxane A2 in platelets are very important, but not unique, regulators of Ca2+ influx and intracellular release in platelets. This phenomenon may also explain the dissociation of the inhibitory effects of COX inhibitors from NOS activity. Furthermore, there appeared to be a direct relationship between the inhibition of NOS activity and platelet [Ca2+]i levels. The selective thromboxane A2 synthase inhibitor U-63557A also decreased platelet [Ca2+]i levels and NOS activity by a similar magnitude as aspirin and indomethacin. These observations imply a regulatory role for platelet [Ca2+]i levels in the activation of the L-arginine-NO pathway in platelets, which may, in part, be regulated by thromboxane A2 formation.

Radomski et al. (19, 20) demonstrated that platelet NOS is of the endothelial constitutive type, and this was confirmed in a previous study from our laboratory with reverse transcription-polymerase chain reaction (11). The platelet L-arginine-NO pathway is perhaps important in the regulation of platelet function in the basal and stimulated states (11). Because the platelet constitutive NOS is of the endothelial type, it is not surprising that NOS activity in platelets is regulated by Ca2+ (Fig. 1). In this context, it is also not unexpected that aspirin and indomethacin, both of which reduce [Ca2+]i, decrease platelet NOS activity and that this inhibition of NOS can be reversed by exogenous Ca2+ or U-46619.

Although [Ca2+]i increased linearly in response to a rise in exogenous Ca2+ (0-5 mM), the NOS activity was maximal when the platelets were incubated with a physiological concentration of Ca2+ (approx 2 mM). This dissociation between [Ca2+]i and NOS activity indicates that NOS activity is maximal in platelets incubated with physiological concentrations of extracellular Ca2+ (Fig. 1).


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  		Fig. 5.   A proposed interaction between NOS and cyclooxygenase (COX) activity and a central role for [Ca2+]i in this interaction. Solid lines, positive effect; dotted lines, inhibitory effect. Acetyl salicylic acid (ASA) and indomethacin (Indo) inhibit COX, reduce [Ca2+]i, and thereby decrease constitutive NOS (cNOS) activity in platelets. Selective thromboxane A2 (TxA2) synthase blocker U-63557A also decreases NOS activity, most probably by reducing [Ca2+]i. PGG2 and PGH2, prostaglandin G2 and H2, respectively. Stimulatory effect of nitric oxide (NO) on COX activity has been previously shown by Salvemini et al. (20).

Thromboxane A2 is a major product of COX activation in human platelets and functions via the second messenger Ca2+ (1). To gain further insight into the mechanism of the effect of aspirin and indomethacin on NOS activity, we measured platelet [Ca2+]i and NOS activity in response to the selective thromboxane synthesis blocker U-63557A. This agent alone caused a modest decrease in the resting platelet [Ca2+]i as well as in the formation of L-citrulline and nitrite (+nitrate). The effects of U-63557A were qualitatively similar to those of aspirin and indomethacin (Fig. 2, Tables 1 and 2). In human platelets, arachidonic acid is converted by COX to PG endoperoxides, which serve as the substrate for the synthesis of thromboxane A2, PGE2, and PGF2alpha . Because PG endoperoxides, PGF2alpha , and PGE2 also increase [Ca2+]i (14), it is possible that the formation of these prostanoids increases after selective thromboxane A2 synthase inhibition by U-63557A, which may be the reason why U-63557A did not more significantly affect platelet [Ca2+]i and NOS activity. Indeed, our study shows that there is a greater decrease in NOS activity in the thromboxane A2-endoperoxide-receptor blocker SQ-29548-treated platelets than in the U-63557A-treated platelets.

The magnitude of the inhibitory effect of aspirin and indomethacin on nitrite (+nitrate) production was greater than that on L-citrulline production (Table 1). It has been proposed that the oxidation of NO by reactive oxygen species results in the formation of peroxynitrite, which predominantly decomposes to nitrate (9). Therefore, the formation and measurement of nitrite (+nitrate) depends not only on NOS activity but also on the availability of oxygen reactive species. COX inhibitors have been shown to inhibit formation of superoxide anions (5). These phenomena may have a bearing on our observation of the larger decrease in nitrite (+nitrate) formation than in L-citrulline formation by aspirin or indomethacin. It is possible that the COX inhibitors directly interfere with the formation of citrulline, but this aspect cannot be evaluated from the present studies.

It may have been ideal to show a direct suppressive effect of COX inhibitors on the activity of isolated NOS from platelets. However, in preliminary studies, we were not able to show such an effect in the platelet cytosol, suggesting the critical need for intact COX and intracellular Ca2+ equilibrium for the effect of COX inhibitors on NOS activity to be manifested. Nonetheless, there was clear evidence for the inhibitory effect of aspirin, indomethacin, and U-63557A on NOS activity in intact cells based on multiple approaches, including L-citrilline formation, nitrite (+nitrate) levels, and cGMP accumulation.

NO, an important physiological signal, downregulates platelet function (19, 20). Taking into consideration its potent antiplatelet effects, one might expect that aspirin would increase NO formation. NO has been shown to stimulate COX activity, leading to simultaneous release of mediators from the COX pathway (7, 21), which can activate platelets. Platelet activation induced by arachidonic acid metabolites is initialized by the rise in [Ca2+]i (23). Our results indeed suggest that the antiplatelet effects of the COX inhibitors aspirin and indomethacin are associated with a reduction in [Ca2+]i. Furthermore, COX inhibitors decrease superoxide anion production (5, 12), which may cause a diminished rate of NO degradation (10). In our studies, the effects of the selective thromboxane A2 synthase inhibitor U-63557A and the thromboxane A2-endoperoxide-receptor blocker SQ-29548 on NOS activity and [Ca2+]i were qualitatively similar to those of aspirin and indomethacin. Because [Ca2+]i is a key regulator of NOS activity, it is not surprising that a reduction in [Ca2+]i by aspirin, indomethacin, or the selective thromboxane A2 synthase inhibitor U-63557A results in decreased NOS activity. Thus mobilization of [Ca2+]i is a key step in the regulation of platelet function by different agents. In this regard, we postulate that the inhibition of NOS by COX inhibitors may serve as a regulatory step in the interaction between the COX and NOS pathways, and the changes in [Ca2+]i are critical in this interaction. Our model to illustrate these interactions is shown in Fig. 5. The eventual effect of different agents on platelet function will depend on the modulation of different steps.

    ACKNOWLEDGEMENTS

This study was supported by a Merit Review Grant from the Department of Veterans Affairs (J. L. Mehta).

    FOOTNOTES

Address for reprint requests: J. L. Mehta, Univ. of Florida College of Medicine, PO Box 100277, JHMHC, Gainesville, FL 32610-0277.

Received 2 April 1997; accepted in final form 16 June 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Heart Circ Physiol 273(4):H1854-H1859
0363-6135/97 $5.00 Copyright © 1997 the American Physiological Society


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