Catalytic consumption of nitric oxide by prostaglandin H synthase-1 regulates platelet function.

Nitric oxide (( small middle dot)NO) plays a central role in vascular homeostasis via regulation of smooth muscle relaxation and platelet aggregation. Although mechanisms for ( small middle dot)NO formation are well known, removal pathways are less well characterized, particularly in cells that respond to ( small middle dot)NO through activation of soluble guanylate cyclase. Herein, we report that ( small middle dot)NO is catalytically consumed by prostaglandin H synthase-1 (PGHS-1) through acting as a reducing peroxidase substrate. With purified ovine PGHS-1, ( small middle dot)NO consumption requires peroxide (LOOH or H(2)O(2)), with a K(m)( (app)) for 15(S)hydroperoxyeicosatetraenoic acid (HPETE) of 3. 27 +/- 0.35 microm. During this, 2 mol ( small middle dot)NO are consumed per mol HPETE, and loss of HPETE hydroperoxy group occurs with retention of the conjugated diene spectrum. Hydroperoxide-stimulated ( small middle dot)NO consumption requires heme incorporation, is not inhibited by indomethacin, and is further stimulated by the reducing peroxidase substrate, phenol. PGHS-1-dependent ( small middle dot)NO consumption also occurs during arachidonate, thrombin, or activation of platelets (1-2 microm.min(-1) for typical plasma platelet concentrations) and prevents ( small middle dot)NO stimulation of platelet soluble guanylate cyclase. Platelet sensitivity to ( small middle dot)NO as an inhibitor of aggregation is greater using a platelet-activating stimulus () that does not cause ( small middle dot)NO consumption, indicating that this mechanism overcomes the anti-aggregatory effects of ( small middle dot)NO. Catalytic consumption of ( small middle dot)NO during eicosanoid synthesis thus represents both a novel proaggregatory function for PGHS-1 and a regulated mechanism for vascular ( small middle dot)NO removal.

In the vasculature, strict control of nitric oxide ( ⅐ NO) bioactivity is essential for both maintaining vascular tone and inhibiting platelet aggregation. Reaction with oxyhemoglobin (oxyHb) 1 is generally viewed to be the major fate of ⅐ NO gen-erated in the vasculature. However, recent work has shown that erythrocyte sequestration of hemoglobin, combined with flow, decreases oxyHb reaction with ⅐ NO and thus renders it less effective at antagonizing ⅐ NO bioactivity in the vessel lumen (1,2). Also, the biological half-life of ⅐ NO, when determined under oxyHb-free conditions (0.1-3 s), is far shorter than expected rates of ⅐ NO autooxidation (3,4). Both of these observations indicate that cell-dependent catalytic scavenging reactions will play a role in regulating ⅐ NO signaling.
Under pathological conditions of vascular dysfunction, accelerated ⅐ NO loss is often observed (5)(6)(7). In hypertensive states, a role for superoxide (O 2 . ) reacting with ⅐ NO to form peroxynitrite (ONOO Ϫ ) accounts for removal of a proportion of ⅐ NO. However, this is by no means the only mechanism for ⅐ NO removal because it is incompletely restored by O 2 . scavengers (7). Nitric oxide consumption by bacterial flavohemoglobins forms NO 3 Ϫ via reaction with the oxy form (8 -10) and is a proposed defense against "nitrosative stress." Recently, rabbit and human 15-lipoxygenases were also found to catalytically consume ⅐ NO, through reaction with an enzyme-bound lipid peroxyl radical intermediate (E red -LOO ⅐ ) (11), indicating that mammalian cells also possess catalytic mechanisms for ⅐ NO removal.
In this report, a second mammalian enzyme, prostaglandin H synthase-1 (PGHS-1) is shown to also catalyze ⅐ NO consumption in both purified enzyme preparations and intact platelets. In platelets, the reaction of PGHS-1 with ⅐ NO led to inhibition of soluble guanylate cyclase, causing platelets to lose responsiveness to the antiaggregatory effects of ⅐ NO. Because platelet-derived ⅐ NO serves a central role in preventing aggregation following activation of soluble guanylate cyclase, these observations indicate that, in addition to the generation of eicosanoids, catalytic consumption of ⅐ NO by PGHS-1 can also contribute to its proaggregatory activity.

EXPERIMENTAL PROCEDURES
Materials-15(S)Hydroperoxyeicosatetraenoic acid (15(S)HPETE), prostaglandin H 2 (PGH 2 ), and U46619 were obtained from Cayman Chemical (Ann Arbor, MI). Arachidonic acid was from Sigma. 15(S)HPETE and PGH 2 were stored at Ϫ80°C in the dark under N 2 . Arachidonic acid and U46619 were stored at Ϫ20°C in the dark. Before use, the sodium salts of arachidonate or 15(S)HPETE were prepared by dilution with 10% methanol, 0.01 M NaOH in water. U46619 and PGH 2 were used as ethanolic solutions. All fatty acids were prepared from stocks on the day of the experiment, stored on ice, and discarded after use. Unless otherwise stated, all other chemicals were from Sigma.
Platelet Aggregation-Aggregation of washed platelets (2 ϫ 10 8 ⅐ml Ϫ1 ) was monitored using a PAP4 optical aggregometer (Bio/Data Corp.). Typical aggregation responses to 0.04 unit/ml thrombin (bovine; Sigma) were 70 -80%, and white blood cell contamination was less than 0.1%. For each platelet isolate, the minimum fatty acid concentration required for maximal aggregation was determined. Because albumin was routinely included to preserve functional responses, fatty acid concentrations of 40 -100 M were typically required for maximal aggregation (15). For comparison of ⅐ NO inhibition of aggregation using either arachidonate or U46619, varying amounts of S-nitrosoglutathione (GSNO, synthesized as described in Ref. 16) were added to washed platelets, with 1 mM CaCl 2 , 1 mM N -nitro-D-arginine methyl ester (L-NAME) 20 s prior to addition of agonist, and the effect on aggregation response was determined.
Nitric Oxide Consumption-Anaerobic solutions of 1.9 mM ⅐ NO were prepared by equilibrating ⅐ NO gas in argon-saturated deionized water. Any ⅐ NO 2 present was eliminated by first bubbling ⅐ NO through 1 M NaOH. Nitric oxide was measured electrochemically using a ⅐ NO sensor (Iso-NO, WPI Inc., Sarasota, FL). Electrode response calibration was performed by measuring ⅐ NO liberated from S-nitroso-N-acetyl-D,L-penicillamine (Alexis Corp, San Diego, CA) in the presence of 0. For all k obs values determined, the square of the Pearson product moment correlation coefficient (r) for the slope of the replotted data was always greater than 0.9, confirming that the reaction followed first order kinetics (Table I). Where ⅐ NO loss on addition of enzyme/substrates was linear, the slope of the initial rate of ⅐ NO disappearance was determined. For all ⅐ NO uptake rates and first order rate constants, data are the means Ϯ S.D. (n ϭ 3).
For measurement of ⅐ NO consumption by purified PGHS-1, a bolus of ⅐ NO (3. Once electrode response had stabilized, PGHS-1 (56 nM), apoPGHS-1 (56 nM), or hematin (112 nM) was added, and the rates of ⅐ NO consumption were recorded. For measurement of ⅐ NO consumption by platelets, ⅐ NO (3.8 M) was added to 0.5 ml of Tyrode's buffer containing platelets and 1 mM CaCl 2 , in the chamber of the ⅐ NO electrode, at 37°C with stirring. L-NAME (500 M) was added to inhibit platelet generation of ⅐ NO during assays. Once the response had stabilized, arachidonate or A23187 was added, and ⅐ NO removal rates were monitored. In some experiments, platelets were preincubated with 20 M indomethacin for 10 min at 37°C before ⅐ NO addition. Where used, PGH 2 (30 M) was added along with arachidonate. For measurement of ⅐ NO consumption by PRP, ⅐ NO was added to 0.5 ml PRP at 37°C with stirring. In some experiments, thrombin (0.04 unit/ml) was added 10 s before ⅐ NO.
Hydroperoxide and Conjugated Diene Analysis-Repeated additions of ⅐ NO (3.8 M each) and PGHS-1 (50 nM each), were made to 0.5 ml of 100 mM phosphate buffer, pH 7.4 containing 22 M 15(S)HPETE. Following each addition of ⅐ NO and PGHS-1, rates of ⅐ NO depletion were monitored electrochemically, until levels approached 0 M, at which point the next ⅐ NO additions was made. When ⅐ NO uptake rates slowed, because of PGHS-1 self-inactivation, additional enzyme was added. Finally, when PGHS-1 addition no longer caused ⅐ NO consumption (i.e. upon depletion of the 22 M 15(S)HPETE), aliquots were removed for hydroperoxide analysis. At this point, 250 nM PGHS-1 and 49.4 M ⅐ NO had been added. For hydroperoxide analysis, 100 l of sample was added to 50 l of assay reagent (5 mg of leukomethylene blue, 8 ml of dimethylformamide, 1.4 g of Triton X-100, 5.5 mg of hemoglobin in 100 ml of 0.05 M potassium phosphate buffer, pH 5.0), and 620 nm absorbance was measured (18). For spectral analysis, PGHS-1 (56 nM) with or without 19 M ⅐ NO was added to 1 ml of 100 mM phosphate buffer, pH 7.4, containing 7.2 M HPETE and incubated for 6 min at room temperature. Conjugated dienes were determined spectroscopically.

Characterization of ⅐ NO Loss in Reaction Systems-Nitric
oxide decay in aerobic phosphate buffer followed first order kinetics with a rate constant (k obs ) of 8.6 Ϯ 1.6 ϫ 10 Ϫ3 s Ϫ1 . Although aerobic oxidation of ⅐ NO is second order overall (17), at the low ⅐ NO concentrations used in this study alternative first order reactions predominate that include gas phase diffu- a For these samples, rates of ⅐ NO loss at 3.8 M were determined using the calculated first order rate constant (k obs ) for each sample. For all others, initial linear rates of ⅐ NO loss on addition of PGHS-1 were determined (n ϭ 3, means Ϯ S.D.). sion and ⅐ NO consumption by the electrode. Using this k obs , rates of background ⅐ NO loss at any concentration could be determined (i.e. at 3.8 M, ⅐ NO loss was 1.96 Ϯ 0.3 M⅐min Ϫ1 ).
Nitric Oxide Is Consumed by Arachidonate or A23187-activated Platelets-Rates of ⅐ NO consumption by washed platelets (8 ϫ 10 8 ml Ϫ1 ) were not significantly increased over background and followed first order kinetics (k obs ϭ 8.0 Ϯ 0.2 ϫ 10 Ϫ3 s Ϫ1 ), indicating that cell-dependent ⅐ NO uptake does not occur in resting platelets. Upon arachidonate addition, increased ⅐ NO consumption immediately occurred (0.40 Ϯ 0.04 nmol⅐min Ϫ1 ⅐10 8 platelets Ϫ1 , after background correction; n ϭ 3, means Ϯ SD) (Fig. 1a). Fatty acid concentrations of 40 -100 M were required for maximal aggregation because of the albumin added to maintain platelet functional responses, thus reducing the potency of exogenously added fatty acids as platelet agonists (15). For each platelet preparation, the minimum fatty acid concentration required for maximal aggregation was first determined and utilized thereafter. Addition of CuZn superoxide dismutase (6000 units/ml) was without effect (not shown), however, ⅐ NO consumption was completely inhibited by the PGHS-1 inhibitor, indomethacin (20 M ; Fig. 1a). Rates of ⅐ NO uptake decreased over 1-1.5 min, paralleling decreasing rates of arachidonate-dependent oxygen consumption (not shown), an index of platelet PGHS-1 activity (19,20). Addition of the PGHS-1 product, PGH 2 (30 M), and arachidonate in concert to indomethacin-treated platelets did not stimulate ⅐ NO uptake, revealing that PGHS-1 activity, rather than secondary reactions of PGHS-1 products (e.g. through PGH 2 metabolism by thromboxane synthase), was responsible for ⅐ NO consumption (Fig. 1a).
Removal of ⅐ NO by PGHS-1 Prevents Activation of Platelet Soluble Guanylate Cyclase-To elucidate the impact of ⅐ NO consumption by PGHS-1 during platelet aggregation, platelet cGMP was determined following addition of exogenous ⅐ NO (500 nM) and activation of platelets by arachidonate. Significant suppression of ⅐ NO-stimulated soluble guanylate cyclase activity occurred following arachidonate activation of platelets and upon addition of oxyHb to scavenge ⅐ NO (Fig. 2). The thromboxane A 2 agonist U46619, which activates platelet aggregation downstream of PGHS-1 without causing ⅐ NO uptake (not shown), did not suppress stimulation of platelet cGMP generation (Fig. 2). These data indicate that PGHS-1-mediated ⅐ NO consumption inhibits ⅐ NO activation of soluble guanylate cyclase.
Platelet Responses to ⅐ NO Are Greater with PGHS-1-independent Stimuli-To determine whether catalytic ⅐ NO consumption influences platelet function, the sensitivity of platelets to ⅐ NO-mediated inhibition of aggregation was evaluated following activation with arachidonate and the thromboxane A 2 mimetic, U46619, that does not suppress ⅐ NO-dependent cGMP generation (Fig. 2). Both agonists trigger platelet aggregation via thromboxane receptor activation, the phosphorylation of which is partially responsible for inhibition of aggregation by ⅐ NO (21). GSNO was utilized to provide a continuous source of ⅐ NO, and L-NAME was added to prevent platelet ⅐ NO generation. The requirement for ⅐ NO in the platelet inhibitory effects of GSNO was indicated by full restoration of aggregation upon inclusion of 3 M oxyHb (not shown). Concentrations of GSNO (0.5-2.0 M) that had no effect on arachidonate-induced platelet aggregation fully inhibited aggregation induced by U46619 (Fig. 3). For partial inhibition of U46619-induced aggregation, only 1-20 nM GSNO was required, whereas up to 50 M GSNO did not significantly impact arachidonate-induced aggregation (not shown). These data implicate PGHS-1dependent ⅐ NO consumption in the regulation of platelet responses to ⅐ NO.

Nitric Oxide Is Consumed during Thrombin-induced Platelet
Aggregation-For experiments utilizing thrombin, PRP was utilized since it is a more physiological platelet preparation (e.g. containing plasma-derived constituents such as albumin and lipoproteins). Nitric oxide or cGMP analogs inhibit thrombin receptor signaling, including activation of platelet arachidonate release (22)(23)(24). To ensure that ⅐ NO did not block arachidonate release, the order of agonist addition was reversed, with thrombin added to PRP 10 s prior to ⅐ NO, and the peak of detectable ⅐ NO then determined (Fig. 4B). When ⅐ NO was added to PRP in the absence of agonists, the initial concentration of detectable ⅐ NO was consistently lower than when ⅐ NO was added to phosphate-buffered saline (a decrease of 1.1 Ϯ 0.6 M, n ϭ 3 different PRP preparations, not shown). This may result from reaction of ⅐ NO with contaminating oxyHb or the possible formation of S-nitrosothiols upon addition of ⅐ NO to plasma (25). However, following ⅐ NO addition to 0.04 unit/ml thrombin-activated PRP, a further and significant decrease in peak ⅐ NO concentration was observed (representing a decrease of 0.8 Ϯ 0.17 M, n ϭ 3 different donors; Fig. 4, a and  b). This response was fully inhibited by indomethacin, indicating a requirement for PGHS-1 activity, and suppressed generation of cGMP by platelet soluble guanylate cyclase (Fig. 4, a  and c). These data indicate that thrombin-stimulated PGHS-1 attenuates the antiaggregatory effects of ⅐ NO in platelets.
Following platelet activation by collagen (4 g/ml), indomethacin-insensitive but superoxide dismutase-inhibitable ⅐ NO consumption was observed (not shown). This was expected because collagen stimulates platelet superoxide (O 2 . ) generation through an uncharacterized pathway, and O 2 . reacts with ⅐ NO at almost diffusion limited rates (26 -28). Finally, ⅐ NO consumption was not stimulated in PRP by ADP (5 M) (not shown), possibly because of ADP being relatively less potent than thrombin (29,30).
Hydroperoxides Stimulate Nitric Oxide Consumption by Purified Ovine Prostaglandin H Synthase-1-To determine the mechanism(s) by which platelet PGHS-1 could consume ⅐ NO, purified PGHS-1 was utilized. In the absence of substrate, heme-reconstituted holoenzyme did not consume ⅐ NO (Fig. 5a) Table 1; and data not shown). 15(S)HPETE was the more potent co-substrate, because it is more efficiently reduced by the peroxidase activity of PGHS-1, having a K m (app) of 3.27 Ϯ 0.35 M (Fig. 6a). Hydroperoxide-stimulated ⅐ NO consumption by PGHS-1 required heme reconstitution (Fig. 5b) and was not inhibited by indomethacin. These data indicate that ⅐ NO is consumed by acting as a reducing cosubstrate for PGHS-1 peroxidase activity. The reducing substrate phenol did not stimulate PGHS-1-dependent ⅐ NO consumption (not shown), excluding a reaction of ⅐ NO with the ferric heme. However, phenol stimulated rates of both H 2 O 2 and 15(S)HPETE-dependent ⅐ NO consumption by PGHS-1, most likely through reaction of ⅐ NO with phenoxyl radicals generated via one-electron oxidation of phenol by PGHS-1 compounds 1 and 2 ( Fig. 5b and data not shown).
To further probe the mechanism of PGHS-1-dependent ⅐ NO consumption, the fate of added 15(S)HPETE was examined. Following incubation with PGHS-1 and ⅐ NO, 92% of the hydroperoxide substituent of 15(S)HPETE became undetectable, with retention of the characteristic conjugated diene spectrum (Fig. 6, b and c). The stoichiometry of ⅐ NO consumed per mol 15(S)HPETE added was 2.14 Ϯ 0.2 (r ϭ 0.98) (Fig. 6d), consistent with ⅐ NO acting as a reducing substrate for PGHS-1 reduction of LOOH to LOH (Scheme 1). DISCUSSION This study shows that PGHS-1 consumes ⅐ NO via peroxidasedependent mechanisms and that ⅐ NO consumption by platelet PGHS-1 attenuates the antiaggregatory actions of ⅐ NO. This controlled catalytic mechanism for ⅐ NO consumption and the consequent inhibition of ⅐ NO signaling also reveals a concerted proaggregatory function of PGHS-1, the generation of eicosanoid products, and the catalytic consumption of ⅐ NO.
Prostaglandin H synthase is a heme enzyme that catalyzes the initial steps of arachidonate oxidation required for eicosanoid synthesis. PGHS exhibits two distinct activities, a cyclooxygenase activity utilizing a tyrosyl radical intermediate to convert arachidonate to PGG 2 and a heme-dependent peroxidase which reduces PGG 2 to PGH 2 . Although both activities are situated on separate sites on the PGHS protein, cross-over reactions do take place (31)(32)(33)(34). For example, oxidation of the heme by peroxides (e.g. PGG 2 ) is required to form PGHS compound 1, which in turn activates the cyclooxygenase activity by oxidizing the catalytic tyrosine, forming the tyrosyl radical (35).
Nitric oxide interacts with purified PGHS in multiple ways. First, reaction of ⅐ NO with the resting ferric heme to yield a nitrosyl complex occurs with equilibrium dissociation constant (K d ) of 0.92 mM (36). Because ⅐ NO concentrations do not approach mM levels in vivo, this complex is not expected biologically. Second, ⅐ NO can terminate the catalytic tyrosyl radical, forming 3-nitrosotyrosine, which rearranges to form 3-nitrotyrosine (37,38). Although this reaction has allowed identification of the catalytic tyrosine residue as Tyr 385 , it does not appear to play a role under biological conditions, because it is effectively suppressed by arachidonate (39). Finally, ⅐ NO can act as a reducing peroxidase substrate for PGHS-1, promoting reduction of 5-phenyl-4-pentenyl-hydroperoxide (40). Although rate constants are not known for this reaction, ⅐ NO reacts with compounds 1 and 2 of horseradish peroxidase, at rates of 10 5 -10 6 M Ϫ1 ⅐s Ϫ1 (41,42). The present data are consistent with this mechanism mediating ⅐ NO consumption, because purified PGHS-1 catalytically consumed ⅐ NO in the presence of hydroperoxide, although in intact platelets, additional mechanisms may be operative. Unlike purified PGHS-1, ⅐ NO uptake by platelets was sensitive to the cyclooxygenase inhibitor indomethacin. In these cells, peroxidase activity will require prior generation of the hydroperoxide, PGG 2 , generated by PGHS-1 through indomethacin-sensitive cyclooxygenase turnover.
To identify pathways of cellular ⅐ NO consumption, platelets were utilized because they both generate and respond to ⅐ NO. Nitric oxide blocks thrombin signaling in platelets via cGMPdependent inhibition of phosphoinositide3-kinase and PLA 2 and phosphorylation of the thromboxane receptor (21)(22)(23)(24). Therefore, to explore the influence of thrombin on PGHS-1-dependent ⅐ NO uptake, thrombin was added to platelets immediately before ⅐ NO. In vivo, thrombin activation of platelets and thromboxane synthesis occurs and is elevated in vascular diseases including sickle cell anemia and atherosclerosis. Because ⅐ NO is a central inhibitor of platelet-endothelial interactions in vivo, both the accelerated generation of thromboxane and exposure of platelets to ⅐ NO will occur together in the vasculature.
Generation of ⅐ NO by platelets in response to pro-aggregatory agonists acts as a negative feedback mechanism because aggregation is inhibited by ⅐ NO (43)(44)(45). Platelet activation stimulates endogenous ⅐ NO generation rates of between 0.004 and 1.35 nmol⅐min Ϫ1 10 8 platelets Ϫ1 (43,44,46). Herein, platelet ⅐ NO consumption of 0.1-0.4 nmol⅐min Ϫ1 ⅐10 8 platelets Ϫ1 or 0.8 Ϯ 0.17 M by thrombin-activated PRP indicates that platelet ⅐ NO removal will significantly impair ⅐ NO-dependent signaling during aggregation. In support of this precept, activation of platelets by thrombin or arachidonate was found to significantly (i) suppress ⅐ NO-dependent platelet cGMP generation and (ii) decrease the sensitivity of platelets to the antiaggregatory effects of ⅐ NO.
In summary, PGHS-1 consumes ⅐ NO via utilization as a reducing peroxidase substrate. Also, PGHS-1-dependent consumption of ⅐ NO during platelet aggregation inhibits cGMP generation and decreases platelet sensitivity to the antiaggregatory effects of ⅐ NO. The activation of PGHS-1 and subsequent generation of proaggregatory thromboxane A 2 plays a central role in platelet aggregation. Therefore, PGHS-1 consumption of ⅐ NO is important because it (i) represents a novel proaggregatory function for PGHS-1 by serving as a regulatable biological sink for ⅐ NO and (ii) demonstrates a controlled catalytic pathway for ⅐ NO consumption by vascular cells.