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(Circulation Research. 2000;87:39.)
© 2000 American Heart Association, Inc.

Cellular Biology

ß₂-Adrenoceptors Activate Nitric Oxide Synthase in Human Platelets

Lindsay R. Queen, Biao Xu, Kazumi Horinouchi, Ian Fisher, Albert Ferro

From the Department of Clinical Pharmacology, Center for Cardiovascular Biology & Medicine, King’s College London, St Thomas’ Hospital, London, UK.

Correspondence to Lindsay R. Queen, Department of Clinical Pharmacology, Center for Cardiovascular Biology & Medicine, King’s College London, St Thomas’ Hospital, Lambeth Palace Road, London SE1 7EH, UK. E-mail lindsay.queen{at}kcl.ac.uk

	Abstract

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Abstract—Nitric oxide (NO), generated by platelets through stimulation of nitric oxide synthase (NOS), limits platelet adhesion and aggregation after a prothrombotic stimulus. Platelet ß-adrenoceptors (ßARs) mediate inhibition of aggregation, but no direct link has been shown between these receptors and platelet adhesion or NO production. We examined NOS activity in human platelets from the conversion of L-[³H]-arginine to L-[³H]-citrulline, after ßAR stimulation or cAMP elevation. Basal NOS activity was 0.11±0.03 pmol L-citrulline/10⁸ platelets. The ßAR agonist isoproterenol 1 µmol/L and the adenylyl cyclase activator forskolin 1 µmol/L each increased NOS activity, to 0.26±0.04 and 0.23±0.03 pmol L-citrulline/10⁸ platelets, respectively (P<0.01 for each). Both responses were abolished by the adenylyl cyclase inhibitor SQ22536 50 µmol/L. NOS activation by isoproterenol or forskolin was not associated with a change in intracellular Ca²⁺. In functional studies, isoproterenol inhibited U46619-induced platelet aggregation in a concentration-dependent manner, but this effect was not significantly diminished by NOS inhibition. In contrast, thrombin-stimulated platelet adhesion to cultured human umbilical vein endothelial cell monolayers was inhibited by isoproterenol, and this effect was abolished by NOS inhibition (1.3±0.2% versus 2.6±0.2% respectively; P<0.001). Effects of isoproterenol on NOS activity, platelet aggregation, and adhesion were mediated exclusively through ß₂ARs, as determined by coincubation with ßAR subtype-selective antagonists. We conclude that ß₂ARs activate platelet NOS by increasing cAMP, and that this activation is Ca²⁺-independent. ß₂ARs may contribute to modulation of platelet aggregation and adhesion to endothelium, and our findings suggest that activation of the L-arginine/NO system mediates the effects of ß₂ARs on adhesion but not aggregation. (Circ Res. 2000;87:39-44.)

Key Words: blood platelets • nitric oxide synthase • adrenoceptors • adenosine cyclic monophosphate

	Introduction

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Nitric oxide (NO) is synthesized from L-arginine by the enzyme nitric oxide synthase (NOS). NOS catalyzes the oxidation of the terminal guanidino nitrogen atom of L-arginine to form L-citrulline and NO.¹ Of the three isoforms of NOS identified to date, two have been found to be expressed in platelets, namely the endothelial (eNOS) and inducible (iNOS) types.²

NO inhibits platelet adhesion to vascular endothelium³ and platelet aggregation,⁴ through activation of soluble guanylyl cyclase and a consequent increase in cGMP. Indeed, NO generation by platelets may act as a negative feedback mechanism to regulate platelet aggregation after a proaggregatory stimulus.⁵ Furthermore, Freedman et al⁶ demonstrated that platelet-derived NO plays an important role in the regulation of platelet recruitment. Thus, platelet-derived NO may have important antithrombotic and antiatherogenic roles in vivo. It has been reported that patients with atherosclerosis have reduced eNOS expression and NO production by the vascular endothelium.⁷ These patients also exhibit an impaired inhibition of platelet aggregation,⁸ and this may be due partly to a reduction in NO generation by the endothelium but also to a decrease in NO production by platelets themselves. Indeed, reduced platelet NO generation has been shown in patients with acute coronary syndromes.⁹

ß-Adrenoceptors (ßARs) are present on human platelets and have been characterized to be of the ß₂ subtype as determined by radioligand binding.^{10 11 12} They are coupled to adenylyl cyclase and, when stimulated with isoproterenol, cause an increase in intracellular cAMP concentration,¹² which in turn causes inhibition of aggregation,¹³ although no data exist on the effect of ßARs on platelet adhesion. Furthermore, no direct link has been shown between ßAR stimulation and NO release in platelets. In contrast to the lack of such data in platelets, we have recently reported that ß₂AR stimulation or cAMP elevation by other means gives rise to NOS activation in human umbilical vein endothelial cells (HUVECs), and that ß₂AR-mediated vasorelaxation in human umbilical vein rings is endothelium- and NO-dependent.¹⁴ Isoproterenol-induced NOS activation in HUVECs occurs partly through the cAMP–protein kinase A pathway and partly through a tyrosine kinase–mediated mechanism.¹⁵

The main objective of the present study was to determine if ßAR stimulation or cAMP elevation can activate the L-arginine/NO pathway in human platelets. We also wished to determine whether the functional responses of platelets to ßAR stimulation could be explained, in part or in total, by NOS activation.

	Materials and Methods

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Preparation of Platelets
Sixty milliliters of venous blood, obtained from healthy subjects, was collected into trisodium citrate (0.38% final concentration). Platelet-rich plasma (PRP), prepared by centrifugation (800g for 8 minutes), was used for all aggregation and adhesion studies. Gel-filtered platelets, prepared from PRP as previously described,¹⁶ were used for all other studies. Platelet counts were measured using a Coulter counter.

Determination of NOS Activity
NOS activity in platelets was assessed by measuring the conversion of L-[³H]-arginine to L-[³H]-citrulline (method modified from Chen and Mehta¹⁷ ). Platelet suspensions were incubated with 1.0 µCi L-[³H]-arginine, in the absence or presence of N^G-monomethyl-L-arginine (L-NMMA) 100 µmol/L, CaCl₂ 1 mmol/L, or the cAMP inhibitor SQ22536 50 µmol/L^{18 19} (15 minutes, 37°C). Isoproterenol 1 µmol/L, forskolin 1 µmol/L, or vehicle was added and incubation continued for an additional 25 minutes. Platelet lysates were mixed with Dowex cation exchange resin (Na⁺ form), and L-[³H]-citrulline in the supernatant was measured by liquid scintillation counting. L-citrulline formation was calculated from the following equation:

(1)

where cpm is the cpm of the sample, and cpm_s is cpm in the standard (all standards contained 1 µCi L-[³H]-arginine, corresponding to 14 pmol). Results were corrected for number of platelets.

To determine the ßAR subtype selectivity of responses observed, incubations with isoproterenol were performed in the absence or presence of 300 nmol/L CGP20712A (a selective ß₁AR antagonist)²⁰ or 100 nmol/L ICI118551 (a selective ß₂AR antagonist).^{21 22}

Assessment of Changes in Cytoplasmic Ca²⁺
Platelets were loaded with fura 2-AM as previously described,¹⁶ then incubated with 1 µmol/L isoproterenol, 1 µmol/L forskolin, or 1 U/mL thrombin (as a positive control). Changes in cytoplasmic Ca²⁺ were examined as a function of time, from the ratio of emission at 510 nm after excitation at 340 and 380 nm, in an LS50 luminescence spectrometer.¹⁶

Measurement of Platelet Aggregation
Platelet aggregation was measured turbidimetrically using a Payton dual-channel aggregometer, as previously described.²³ PRP was incubated with phentolamine 10 µmol/L (a nonselective -adrenergic antagonist) for 30 seconds before addition of N^G-nitro-L-arginine methyl ester (L-NAME) 100 µmol/L, CGP20712A 300 nmol/L, ICI118551 100 nmol/L, or vehicle. After 60 seconds, isoproterenol (1 nmol/L to 10 µmol/L) or vehicle was added and incubated for an additional 60 seconds. Aggregation was stimulated with a predetermined, submaximal concentration of 15S-hydroxy-11,9-epoxymethanoprosta-5Z,13E-dienoic acid (U46619, concentration range 0.5 to 1.3 µmol/L). Results were expressed as the percentage of inhibition of aggregation compared with the control sample (U46619 alone).

Measurement of Platelet Adhesion
Platelets were labeled with indium-111-oxine as previously described.²⁴ Radiolabeled platelets were added to HUVEC monolayers in the presence of either L-NAME 100 µmol/L, CGP20712A 300 nmol/L, ICI118551 100 nmol/L, or vehicle and incubated for 5 minutes at 37°C. Isoproterenol 1 µmol/L or vehicle was added and incubation continued for 15 minutes. Platelet adhesion was stimulated by 3 mU/mL thrombin, and after an additional 15 minutes, HUVEC monolayers were washed to remove unattached platelets, solubilized, and radioactivity counted. The percentage of platelets bound was calculated from the following equation:

(2)

Statistical Analysis
All experiments were performed in triplicate, and the mean of each triplicate was used for further statistical analysis. Data were analyzed using repeated-measures ANOVA and expressed as mean±SEM. Statistical significance was taken as P<0.05 (two-sided).

An expanded Materials and Methods section is available online at http://www.circresaha.org.

	Results

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Platelet NOS Activity
NOS activity in platelets was evaluated from the conversion of L-[³H]-arginine to L-[³H]-citrulline. Basal L-citrulline production was 0.11±0.03 pmol/10⁸ platelets (n=6). Isoproterenol 1 µmol/L and forskolin 1 µmol/L each increased NOS activity, to 0.26±0.04 and 0.23±0.03 pmol L-citrulline/10⁸ platelets, respectively (n=6, P<0.01, for each).

Results for basal and isoproterenol- and forskolin-stimulated L-citrulline production were not different in the absence or presence of Ca²⁺ (1 mmol/L). Furthermore, SQ22536 50 µmol/L abolished the increase in L-citrulline seen with isoproterenol and forskolin (n=6, P<0.02 for each), although it had no effect on basal L-citrulline production (Figure 1). Coincubation with L-NMMA 100 µmol/L decreased the radioactivity found in the L-citrulline fraction in control incubations and prevented the increase in L-citrulline formation by isoproterenol 1 µmol/L (Table 1).

View larger version (18K): [in this window] [in a new window]   Figure 1. L-[3H]-citrulline production from L-[3H]-arginine in human platelets (n=6). Responses are shown to vehicle (basal), isoproterenol 1 µmol/L, and forskolin 1 µmol/L. L-citrulline formation was assessed in the absence or presence of CaCl2 1 mmol/L (-CaCl2 and +CaCl2, respectively) and in the absence or presence of SQ22536 50 µmol/L (-SQ22536 and +SQ22536, respectively). *P<0.01 vs corresponding basal values; P<0.02 vs +CaCl2 and -SQ22536 responses.

View this table: [in this window] [in a new window]   Table 1. [3H] Counts in the L-Citrulline Fraction, in the Absence and Presence of L-NMMA 100 µmol/L, after Incubation With Isoproterenol 1 µmol/L or Vehicle (Control)

To determine whether the ßAR-mediated increase in L-[³H]-citrulline production was mediated through ß₁ARs or ß₂ARs, we performed a larger series of experiments (n=25) with CGP20712A 300 nmol/L (a ß₁AR-selective antagonist)²⁰ or ICI118551 100 nmol/L (a ß₂AR-selective antagonist).^{21 22} Coincubation with CGP20712A did not affect L-[³H]-citrulline production in response to isoproterenol. By contrast, in the presence of ICI118551, isoproterenol did not increase L-[³H]-citrulline formation above control values, suggesting that the activation of NOS by isoproterenol was mediated exclusively through ß₂ARs (Figure 2).

View larger version (25K): [in this window] [in a new window]   Figure 2. L-[3H]-citrulline production from L-[3H]-arginine in human platelets, expressed as a percentage of change from basal (n=25). Responses are shown to isoproterenol 1 µmol/L, alone or in the presence of CGP20712A 300 nmol/L or ICI118551 100 nmol/L. *P<0.001 vs isoproterenol alone.

Cytoplasmic Ca²⁺ in Platelets
Because eNOS can be activated by an increase in cytoplasmic Ca²⁺, experiments were performed to determine whether ßAR stimulation or adenylyl cyclase activation increases intraplatelet Ca²⁺. Changes in intracellular Ca²⁺ were examined using platelets loaded with the fluorescent dye fura 2-AM (n=3). No change in intracellular Ca²⁺ was detected in platelets treated with isoproterenol 1 µmol/L or forskolin 1 µmol/L (Figures 3A and 3B). By contrast, intracellular Ca²⁺ increased in response to thrombin 1 U/mL, which was used as a positive control (Figure 3C).

View larger version (13K): [in this window] [in a new window]   Figure 3. Changes in intraplatelet Ca2+ levels (n=3) in response to isoproterenol 1 µmol/L (A), forskolin 1 µmol/L (B), and thrombin 1 U/mL (C), as determined by fura 2-AM fluorescence and expressed as the ratio of emission at 510 nm after excitation at 340 and 380 nm (R340/380). Tracings from a typical experiment are shown.

Platelet Aggregation
To determine the functional significance of ß₂AR-mediated NOS activation in platelets, experiments were performed to study the inhibition of U46619-induced platelet aggregation by isoproterenol and the effect of concomitant inhibition of NOS (n=11). Isoproterenol (1 nmol/L to 10 µmol/L) inhibited U46619-induced aggregation in a concentration-dependent manner. The response was not affected by coincubation with CGP20712A 300 nmol/L but was abolished by ICI118551 100 nmol/L, confirming that the effect of isoproterenol was mediated solely through ß₂ARs (Figure 4). Coincubation with L-NAME 100 µmol/L did not sig- nificantly alter the antiaggregatory effect of isoproterenol (Table 2).

View larger version (12K): [in this window] [in a new window]   Figure 4. Inhibition of U46619-induced platelet aggregation (n=11) by isoproterenol (concentration range 1 nmol/L to 10 µmol/L), alone or in the presence of CGP20712A 300 nmol/L or ICI118551 100 nmol/L. *P<0.001 vs isoproterenol alone.

View this table: [in this window] [in a new window]   Table 2. Effect of NOS Antagonism on Isoproterenol-Mediated Inhibition of Platelet Aggregation

Platelet Adhesion
Adhesion of indium-111–labeled platelets to HUVEC monolayers was measured in 12-well plates. In the absence of isoproterenol, thrombin 3 mU/mL resulted in 2.2±0.2% adhesion to HUVECs (n=7). Coincubation with isoproterenol 1 µmol/L decreased platelet adhesion to 1.3±0.2% (P<0.01 versus control). We found that L-NAME abolished this response to isoproterenol (Figure 5). CGP20712A 300 nmol/L did not affect the adhesion response to isoproterenol (1.5±0.3%, P>0.05 versus isoproterenol alone). However, in the presence of ICI118551 100 nmol/L, the inhibition of platelet adhesion by isoproterenol was abolished (2.5±0.2%, P<0.001 versus isoproterenol alone), confirming that the inhibitory effect of isoproterenol on platelet adhesion was mediated exclusively through ß₂ARs.

View larger version (12K): [in this window] [in a new window]   Figure 5. Effect of isoproterenol (n=7) on thrombin-induced platelet adhesion to HUVEC monolayers. Responses are shown to vehicle control and isoproterenol 1 µmol/L in the absence or presence of L-NAME 100 µmol/L. *P<0.01 vs control.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Platelets express two isoforms of NOS, namely eNOS and iNOS.² Under resting conditions, platelets generate NO,²⁵ which acts as a feedback mechanism to inhibit platelet adhesion and aggregation.⁴ In the present study, we investigated the possible role of ßARs in the regulation of platelet NOS activity and how this may be important in ßAR-mediated inhibition of platelet aggregation and adhesion to the endothelium.

ßARs on human platelets have been identified and characterized as being ß₂ARs in type,^{10 11 26} but the function of these receptors has not been fully elucidated. Studies have shown that stimulation of platelet ßARs increases intracellular cAMP and inhibits aggregation,^{12 13 27} although no data exist on the effects of ßARs on platelet adhesion. Our study provides the first direct evidence that ßAR stimulation activates platelet NOS. Stimulation of ßARs in platelets with isoproterenol elicited an increase in the conversion of L-arginine to L-citrulline, a response that was abolished by coincubation of the platelets with L-NMMA. The increased NOS activity was attributable solely to ß₂AR-subtype activation, because the response to isoproterenol was prevented by coincubation with the ß₂AR antagonist ICI118551 but not with the ß₁AR antagonist CGP20712A. Furthermore, increased NOS activity was also observed with forskolin, a direct activator of adenylyl cyclase, and the adenylyl cyclase inhibitor SQ22536 abolished the NOS responses to both isoproterenol and forskolin. This suggests that ß₂ARs activate NOS through an increase in cAMP. This contrasts with our recently reported findings in HUVECs, in which isoproterenol increased NOS activity partly through cAMP elevation and partly through a tyrosine kinase–mediated mechanism.¹⁵

The increased NOS activity observed in the presence of isoproterenol and forskolin was not accompanied by a measurable increase in intraplatelet Ca²⁺, as determined by fura 2-AM fluorescence. In addition, isoproterenol- and forskolin-mediated NOS activation was not different in the absence or presence of Ca²⁺ in the incubation medium. These results accord with previously published observations by Lantoine et al,²⁸ who demonstrated that stimulation of NO production in platelets with collagen was not associated with a corresponding increase in cytosolic Ca²⁺. These findings suggest that NOS stimulation in platelets need not involve Ca²⁺ influx and calmodulin activation. We have previously shown that ß₂ARs activate eNOS in HUVECs (the only NOS isoform present in these cells), and that this activation is similarly Ca²⁺-independent.¹⁴ Ca²⁺-independent activation of eNOS has also been observed in response to shear stress and can be ascribed to serine phosphorylation of eNOS by Akt, resulting in increased Ca²⁺ sensitivity.²⁹ An increase in eNOS sensitivity to Ca²⁺ may explain our present findings in platelets, although a possible role of iNOS cannot be excluded.

Isoproterenol caused a concentration-dependent inhibition of platelet aggregation through activation of ß₂ARs. These results are in agreement with previous studies,^{12 13} which have demonstrated that isoproterenol inhibits platelet aggregation through accumulation of cAMP. However, we found that NOS inhibition with L-NAME did not significantly affect the antiaggregatory action of isoproterenol. Although the maximal antiaggregatory effect (E_max) of isoproterenol was slightly lower in the presence of L-NAME, this was not significant. By contrast, ß₂AR activation inhibited thrombin-stimulated platelet adhesion to endothelial monolayers, and this effect was abolished by L-NAME. The precise mechanism by which ßAR-mediated NOS-dependent inhibition of adhesion occurs, with no observable change in platelet aggregation, remains to be elucidated. It is possible, for example, that differential effects are exerted on the expression or function of glycoprotein Ib compared with IIb/IIIa in our system; however, no published evidence exists on possible effects of ßAR stimulation or of NOS on these glycoproteins. Nevertheless, previous studies have shown that stimulation of NO in platelets inhibits their adhesion to intact endothelial monolayers.^{30 31} Our study provides the first direct evidence that ßARs inhibit platelet adhesion, and that they do so through generation of NO.

In our platelet adhesion studies, we examined the effect of isoproterenol on platelets in the presence of HUVECs. We have previously shown that ß₂AR stimulation activates the L-arginine/NO system in HUVECs.¹⁴ It is likely, therefore, that the NO generated in our system after ßAR stimulation derives from both the platelets and the endothelial cells. We cannot, from our experiments, define the precise contributions of ßAR-mediated NOS activation from platelets and HUVECs, separately, to the adhesion response. If we were to incubate platelets alone with isoproterenol, and subsequently to transfer the platelets to endothelial cells for the adhesion assays, it would be necessary to remove the isoproterenol before the transfer. By doing so, any platelet-generated NO (which is short-lived) would be removed concomitantly. Nevertheless, our data demonstrate that ßAR-mediated NOS-dependent inhibition of platelet adhesion to vascular endothelium is likely to be of physiological relevance. In vivo, catecholamines (derived from sympathetic stimulation) will act simultaneously on the vessel wall and on platelets within the lumen, which are in close proximity. This may be an important mechanism by which platelet adhesion is regulated and the antithrombotic properties of the vessel wall are maintained in vivo.

ß₂AR-mediated inhibition of adhesion and aggregation in platelets may partially offset the proaggregatory effects of AR stimulation in response to the endogenous catecholamines norepinephrine and epinephrine.³² In vivo, circulating plasma concentrations of epinephrine and norepinephrine range between 0.1 to 0.5 nmol/L and 0.3 to 3.0 nmol/L, respectively. These levels are much lower than the reported affinity of these catecholamines for ARs³³ and ßARs.¹³ However, in times of severe stress, for example, during myocardial infarction, circulating epinephrine and norepinephrine concentrations may reach considerably higher levels (10 nmol/L).³³ Such high catecholamine concentrations may contribute to the platelet aggregation seen in unstable angina and myocardial infarction through AR stimulation,^{34 35} and, at those concentrations, significant activation of ß₂ARs may also occur, which will potentially offset the AR-mediated effects. The precise mechanism whereby ß₂AR stimulation inhibits platelet aggregation remains to be elucidated. However, our results suggest that NO generation mediates the effects of ß₂AR stimulation on platelet adhesion to the vascular endothelium.

We have previously shown that ß₂AR stimulation in human vascular endothelial cells causes activation of the L-arginine/NO system.¹⁴ Although the present study reveals similar effects in platelets, additional studies will be needed to determine whether the responses in platelets correspond closely with those in the vascular endothelium. In disease states associated with endothelial dysfunction, such as hypercholesterolemia,³⁶ diabetes³⁷ and atherosclerosis,^{7 38} reduced NO production by both the endothelium and platelets may predispose to thrombotic events. Freedman et al⁹ showed that in patients with coronary atherosclerosis, platelet-derived NO production is impaired. Martina et al³⁹ reported that platelets from patients with type 1 and type 2 diabetes mellitus have reduced NOS activity. However, platelet ßAR function has not been determined in these conditions, and it remains to be seen whether these receptor responses are impaired in parallel, in endothelium and platelets.

In conclusion, we have shown that ßAR stimulation or cAMP elevation increases NOS activity in platelets, and that this occurs with no detectable change in intracellular Ca²⁺. In functional assays, ßAR stimulation causes inhibition of platelet aggregation and platelet adhesion to endothelial monolayers. ßAR-mediated platelet adhesion to vascular endothelial cells, but not platelet aggregation, is dependent on integrity of the L-arginine/NO system. Furthermore, all the measured responses to isoproterenol are mediated exclusively through ß₂AR-subtype activation. These results provide the first direct evidence that ß₂ARs can activate NOS in human platelets, through cAMP elevation. The physiological and pathophysiological relevance of these findings remains to be determined.

	Acknowledgments

 
This work was supported by a grant from the Grand Charity. We are grateful to the subjects who donated blood samples for this study.

Received April 3, 2000; accepted June 1, 2000.

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