This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Adrie, C. Articles by Semigran, M. J. Search for Related Content PubMed PubMed Citation Articles by Adrie, C. Articles by Semigran, M. J. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB NITRIC OXIDE

(Circulation. 1996;94:1919-1926.)
© 1996 American Heart Association, Inc.

Articles

Inhaled Nitric Oxide Increases Coronary Artery Patency After Thrombolysis

Christophe Adrie, MD; Kenneth D. Bloch, MD; Pedro R. Moreno, MD; William E. Hurford, MD; J. Luis Guerrero, BS; Robert Holt, BS; Warren M. Zapol, MD; Herman K. Gold, MD; Marc J. Semigran, MD

the Department of Anesthesia (C.A., W.E.H., W.M.Z.), the Cardiac Unit (K.D.B., P.R.M., J.L.G., R.H., H.K.G., M.J.S.), and Cardiovascular Research Center (K.D.B.) of the Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Mass.

Correspondence to Marc J. Semigran, MD, Cardiac Unit, Massachusetts General Hospital, Bulfinch 211, Boston, MA 02114. E-mail semigran@olorin.mgh.harvard.edu.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background Nitric oxide (NO) and nitrosovasodilators that release NO inhibit platelet aggregation. The antithrombotic effect of intravenously infused nitrosovasodilators is usually accompanied by systemic vasodilation. Inhaled NO is a pulmonary vasodilator that does not produce systemic hemodynamic effects. This study examines the antithrombotic effect of inhaled NO in a canine model of platelet-mediated coronary artery reocclusion after thrombolysis.

Methods and Results In 25 anesthetized dogs, a segment of the left anterior descending coronary artery was traumatized and a high-grade stenosis created. Thrombus was injected at this site, and tissue plasminogen activator was administered, producing cyclic flow variations (CFVs) in 24 of 25 dogs. CFV frequency was unchanged in dogs not breathing NO but decreased by 35±9% (P<.05) and 53±7% (P<.01) while dogs breathed 20 and 80 parts per million (ppm) NO, respectively. The coronary artery patency ratio (fraction of time during which the coronary artery was patent; CAPR) was unchanged in dogs not treated with NO but increased from 51±7% to 64±8% while breathing 20 ppm NO (P<.01) and from 49±3% to 75±7% while breathing 80 ppm NO (P<.01). The increased CAPR during 80 ppm NO administration persisted during a 45-minute posttreatment period (70±7%, P<.05 versus baseline). NO inhalation did not change systemic hemodynamics. In a pharmacological model of coronary vasoconstriction, inhaled NO did not reverse the effect of the thromboxane A₂ agonist U-46619. In vitro ADP-induced platelet aggregation was inhibited by NO gas.

Conclusions Inhaled NO at concentrations of 20 and 80 ppm increases coronary patency and decreases CFV frequency in a canine model of platelet-mediated coronary reocclusion after thrombolysis without producing systemic hemodynamic effects.

Key Words: nitric oxide • thrombolysis • thrombus

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Activation of platelet adhesion and aggregation is believed to play an important role in the pathogenesis of atherosclerosis as well as in the development of ischemic coronary artery syndromes including unstable angina and myocardial infarction.^{1 2} This hypothesis is supported by the effectiveness of inhibitors of platelet activation such as aspirin, ticlopidine, and agents that block platelet GPIIb/IIIa receptors in the treatment of ischemic heart disease.^{3 4 5} Aspirin improves the effectiveness of streptokinase therapy for acute myocardial infarction⁴ by inhibiting platelet-mediated reocclusion,³ and the c7E3 antibody decreases ischemic complications after coronary angioplasty.⁵

A canine model of coronary artery thrombosis with superimposed endothelial damage and high-grade stenosis has been developed that mimics the platelet-mediated cyclic reocclusion observed after thrombolytic therapy.^{6 7} Antiplatelet agents such as the c7E3 monoclonal antibody⁶ and kistrin,⁷ which improve coronary patency in this animal model, have been shown to be effective in the treatment of clinical thrombotic syndromes⁵ or are under clinical investigation.

NO is an endothelium-derived relaxation factor that is produced from L-arginine by NO synthase.⁸ Both NO and nitrosovasodilators that release NO activate soluble guanylate cyclase, leading to rapid increases in platelet cGMP and inhibition of platelet aggregation.^{9 10 11 12 13} Intravenous infusions of NTG and SNP are known to inhibit platelet thrombus formation in a canine model of coronary stenosis; however, their beneficial effect is limited by systemic vasodilation and hypotension.^{14 15}

Inhaled NO diffuses directly from alveoli to pulmonary vascular smooth muscle, inducing pulmonary vasodilation without systemic hemodynamic effects.^{16 17 18 19} NO is rapidly inactivated by combination with hemoglobin,²⁰ and it was initially believed that the effects of inhaled NO would not extend beyond the lungs.²¹ Nonetheless, it has recently been observed that inhaled NO modestly prolongs the bleeding time in rabbits and humans.^{22 23} This observation suggests that platelets transiting the pulmonary circulation could be altered by NO. To investigate the ability of inhaled NO to inhibit platelet aggregation in vivo, we studied the effect of breathing low levels of NO gas on coronary artery patency in a canine model of coronary artery reocclusion after thrombosis and thrombolysis.^{6 7}

	Methods

Top Abstract Introduction Methods Results Discussion References

 
These investigations were performed with approval from the Subcommittee on Research Animal Care of the Massachusetts General Hospital.

Preparation of Canine Coronary Artery Model of Reocclusion After Thrombosis and Thrombolysis
Twenty-five adult mongrel dogs (weight, 20 to 25 kg) of either sex were anesthetized with 30 mg/kg IV pentobarbital. Supplemental pentobarbital was given as required to maintain general anesthesia. The dogs were intubated and mechanically ventilated. The inspired O₂ fraction (FIO₂) was adjusted between 0.21 and 0.35 with the use of an oximeter (Hudson Ventronics) to maintain the arterial blood oxygen tension between 80 and 100 mm Hg, as determined by pulse oximetry (Nellcor).

After cannulation of the femoral vein and artery, a left thoracotomy was performed in the fifth intercostal space, and the pericardium was opened and suspended to create a pericardial cradle. A 2-mm internal diameter polyvinyl catheter was placed in the left atrium for hemodynamic monitoring. A 2.5-cm segment of the LAD coronary artery distal to the first diagonal branch was isolated. A 0.7-mm internal diameter catheter was inserted into a side branch of the isolated LAD coronary artery segment, and any other side branches were ligated. A 3-0 Flexon wire was sutured to the epicardium distal to the isolated LAD segment for ECG monitoring. One milliliter of blood was sampled for later use in forming a thrombus. An ultrasonic flow probe (T101, Transonic Systems) was placed on the proximal portion of the artery. A 2-mm-wide plastic wire tie (Mass Gas and Electric Supply) was progressively constricted around the distal end of the arterial segment to reduce blood flow to 50±10% of baseline. A previous angiographic study has shown that this constriction decreases luminal diameter by >90%.⁶

The isolated LAD coronary artery was traumatized by four consecutive external compressions with a blunt forceps over 3 to 5 seconds to damage the endothelium and promote thrombus adherence. Before thrombus formation, lidocaine (75 mg bolus followed by a constant intravenous infusion of 1 mg/min) was administered for prophylaxis of arrhythmias. Snare occluders distal to the probe and proximal to the constriction site were applied, and a mixture of 0.1 mL thrombin (100 units/mL; Thrombinar, Armour Pharmaceutical) and 0.3 mL of previously sampled blood was injected through the side branch catheter into the emptied and isolated coronary artery segment to induce thrombus formation. After 10 minutes, the proximal snare was released, and 2 minutes later, the distal snare was released. Twenty minutes after thrombus formation, a 75 units/kg bolus of heparin was administered intravenously and followed by a continuous infusion of 50 units/kg per hour. After a 30-minute period of stable occlusion, a 0.45 mg/kg bolus of TPA (Genentech) was administered intravenously at 15-minute intervals until reflow through the thrombosed coronary artery was achieved or a maximum of four boluses had been administered. This procedure induces alternating periods of recanalization and reocclusion. Reocclusion was defined as occurring at any time after recanalization when coronary blood flow was <25% of that observed after creation of the initial stenosis (baseline flow).⁶ A CFV was defined as reocclusion of the artery after the occurrence of a spontaneous increase to >25% of baseline flow. The coronary artery patency ratio was defined as the fraction of an observation period during which the coronary artery was patent. Animals were excluded from further study in the event of (1) failure to reperfuse, (2) reperfusion without reocclusion, (3) less than 3 cycles occurring during the first 45-minute observation period, or (4) death before the end of the first observation period.

NO Delivery System
NO gas (800 ppm by volume NO in nitrogen, Airco) was mixed with room air with the use of a standard oxygen blender (Bird Blender) and titrated with varying quantities of oxygen to maintain a constant FIO₂ just before delivery to the ventilator. Expired gas was scavenged by continuous aspiration of the expiratory limb of the ventilator. The inspired NO level was continuously monitored by a chemiluminescence NO-NO_x analyzer (model 14A, Thermo Environmental Instruments).²⁴

Measurement of Coronary Flow
Continuous measurements of coronary blood flow, systemic arterial pressure, and left atrial pressure as well as the epicardial ECG were made while dogs breathed at an FIO₂ of 0.3±0.1 (mean±SEM) for 45 minutes (baseline period). The animals were then divided into four groups of six dogs. Measurements were continued through a 45-minute treatment period during which group A inhaled 0 ppm NO, group B inhaled 20 ppm NO, group C inhaled 80 ppm NO, and group D inhaled 200 ppm NO. NO administration was then discontinued, and animals were observed for a posttreatment 45-minute period. Arterial blood samples were withdrawn at the end of each period to measure hemoglobin, methemoglobin, and platelet concentrations. ACTs were performed within 2 hours after beginning the heparin infusion with the use of a Hemochron 800 dual-well coagulation timer and FTCA 510 reaction tubes (International Technidyne).²⁵

Effects of Inhaled NO on Coronary Flow in a Pharmacological Model of Coronary Artery Vasoconstriction
In a separate group of five dogs, the effect of inhaled NO on coronary blood flow in the presence of pharmacological vasoconstriction was studied. The dogs were anesthetized with 30 mg/kg IV pentobarbital, intubated, and ventilated at an FIO₂ of 0.21 to 0.35. A left thoracotomy was performed, and the heart was suspended in a pericardial cradle as described above. Catheters for monitoring left atrial and femoral arterial pressure were placed. A segment of the LAD coronary artery was dissected free from the surrounding tissue, and a side branch was cannulated with a 0.7-mm polyvinyl catheter. The ultrasonic probe was placed distal to the catheter. Coronary flow and arterial pressure were measured for a 45-minute baseline period, during a 45-minute period of inhalation of 80 ppm NO, and for 45 minutes after the inhalation of NO. Hemodynamic and coronary flow measurements were then made after 5 minutes of sequential infusion of the NO donor SNP at 1 µg/kg per minute and the thromboxane A₂ agonist U-46619 (9,11-dideoxy-9-epoxymethano-prostaglandin F₂; Sigma Chemical Co) at 10 nmol/min through the intracoronary catheter. The dose of U-46619 was chosen to decrease coronary blood flow by 20% to 25%, on the basis of prior studies in our laboratory.²⁶ The U-46619 infusion continued as dogs inhaled 80 ppm NO. Hemodynamic measurements were repeated after 5 minutes, and the NO was stopped with continuation of the U-46619 infusion. A 1 µg/kg per minute infusion of SNP through the intracoronary catheter was resumed, and hemodynamic and coronary flow measurements were repeated after 5 minutes.

Bleeding Time
In a separate group of four dogs, the bleeding time response to inhaled NO was studied. After the dogs were anesthetized with 30 mg/kg IV pentobarbital and intubated, NO (0, 20, 80, and 200 ppm) was inhaled in sequential 45-minute periods. Template bleeding times were performed using an automated, spring-loaded device (Simplate-II, General Diagnostics) on both the ventral aspect of the tongue²⁷ and the shaved forearm²⁸ at the conclusion of each period.

In Vitro Platelet Aggregation
Thirty milliliters of blood from an additional eight dogs was collected in 0.01 mol/L sodium citrate and centrifuged at 370g for 5 minutes at room temperature to prepare platelet-rich plasma (PRP) and then at 1200g for 20 minutes to prepare platelet-poor plasma (PPP). PRP was exposed only to plastic containers or, during aggregometry, to siliconized glassware. The platelet concentration (Thrombocounter C, Coulter Electronics) of PRP was adjusted by dilution with autologous PPP to obtain levels of 300 000/mm³±10%. PRP was then aliquoted into cuvettes incubated at 37°C with magnetic stirring (1000 rpm) in a dual-channel aggregometer (model 440, Chrono-Log Corp) for 3 minutes. Light transmission was continuously recorded on a Chrono-Log recorder (model 707). The cuvette was sealed with a rubber cap, and two 19-gauge needles were placed through this cap to deliver a mixture of NO gas, air, and oxygen above the PRP. The inlet needle was connected via a flowmeter (model 603, Matheson) to a gas reservoir, into which a mixture of NO gas, air, and oxygen was delivered. The FIO₂ was maintained constant at 0.21, with varying quantities of oxygen added throughout the experiment. The NO concentration was continuously monitored with the chemiluminescence NO-NO_x analyzer. A needle valve allowed regulation of the gas flow over the sample to 40 mL/min. Positive pressure was maintained in the system by submerging the distal end of the outlet tubing beneath 5 cm of water.

Aggregation was induced by ADP, which was titrated before the beginning of the experiment to induce optimal platelet aggregation. The same dose of ADP was administered throughout the experiment. ADP-induced platelet aggregometry studies were performed after exposing the test cuvette containing 450 µL PRP for 10 minutes to 20, 80, 200, and 400 ppm NO in a random order. Gas administration was continued during the measurement of ADP-induced aggregation. Control ADP-induced platelet aggregation studies without NO were performed at the beginning and at the end of the experiment to assess the stability of the PRP preparation. All experiments were completed within 4 hours of blood collection.

Data Analysis
All results are expressed as mean±SEM. The significance of differences in the frequency of CFVs, the coronary artery patency ratio, the bleeding time, or any of the hemodynamic variables during or after treatment with inhaled NO, SNP, or U-46619 was determined by ANOVA followed by Dunnett's multiple comparison test. The significance of both the effect of NO gas on in vitro platelet aggregation and the dose-response relationship was determined by two-way ANOVA followed by Newman-Keuls multiple comparison test. A value of P<.05 was considered significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Canine Coronary Artery Model of Reocclusion After Thrombosis and Thrombolysis
The characteristics of the canine coronary artery model of reocclusion after thrombosis and thrombolysis are summarized in Table 1. The external constrictor reduced LAD blood flow by 53±2%, from 22±2 to 10±1 mL/min. The median number of TPA boluses required to obtain reperfusion after thrombolysis was 2, with a range of 1 to 4. During the baseline period, there was no difference in poststenotic blood flow, the number of TPA boluses, ACT, the frequency of CFVs, or coronary artery patency ratio between any of the four groups (Table 1). Cyclic reflow and reocclusion were accompanied by ECG evidence of myocardial injury (Fig 1) and occurred in all the animals except one, in which reocclusion did not occur; this animal was excluded from further study. Despite an infusion of lidocaine, ventricular fibrillation occurred in five dogs before the baseline period and was successfully treated by DC electrical defibrillation. There was no recurrence of arrhythmias during the baseline, NO inhalation, or posttreatment periods, and no dog died before conclusion of the study.

View this table: [in this window] [in a new window]   Table 1. Characteristics of the Canine Coronary Artery Model of Reocclusion After Thrombosis and Thrombolysis

View larger version (74K): [in this window] [in a new window]   Figure 1. Representative occlusion/reperfusion cycle during the baseline period (45 minutes) before NO administration. ECG indicates epicardial ECG; CBF, left anterior descending coronary artery blood flow; LAP, left atrial pressure; and SAP, systemic arterial pressure (phasic and mean). Note the change of ST elevation during the occlusion/reperfusion cycle.

Effects of Inhaled NO on Coronary Patency After Thrombolysis and Reocclusion
To assess the effects of inhaled NO on coronary artery patency in this model, we measured both the frequency of CFVs and the coronary artery patency ratio. While the frequency of CFVs occurring in each of the three 45-minute periods was unchanged in dogs that did not inhale NO (group A), it decreased by 35±9% during the inhalation of 20 ppm NO (group B, P<.05) and by 53±7% during the inhalation of 80 ppm NO (group C, P<.01). The decrease in the frequency of CFVs persisted during the posttreatment period in the group C dogs (58±8%, P<.01). There was no change in the frequency of CFVs during inhalation of 200 ppm NO (group D).

The coronary artery patency ratio (Fig 2) did not change during the three periods in animals that did not inhale NO (group A). In dogs breathing 20 ppm (group B), the patency ratio increased from 51±7% to 64±8% (P<.01). In dogs breathing 80 ppm (group C), the patency ratio increased from 49±3% to 75±7% (P<.01), and this increase persisted during the 45-minute posttreatment period (70±7%, P<.05). There was no change in the patency ratio in dogs breathing 200 ppm NO (group D). During both the treatment period and the posttreatment period, the coronary artery patency ratio differed between group A (0 ppm) and the pooled observations of groups B, C, and D (P<.005).

View larger version (49K): [in this window] [in a new window]   Figure 2. Coronary artery patency ratio during baseline, treatment, and posttreatment periods. During the treatment period, animals inhaled 0, 20, 80, or 200 ppm NO. Values are mean±SEM. *P<.05 vs baseline period; **P<.01 vs baseline period. n=6 dogs for each group.

Neither the platelet count nor the hemoglobin concentration changed during the treatment or posttreatment periods in any of the groups of animals (Table 2). Methemoglobin content, as a fraction of total hemoglobin, increased from 0.002±0.001 to 0.011±0.004 (P<.05) in group D animals during 200 ppm NO inhalation and remained elevated at 0.009±0.003 during the posttreatment period (P<.05 versus baseline; Table 2). There was no change in methemoglobin levels in the animals breathing lower concentrations of NO.

View this table: [in this window] [in a new window]   Table 2. Hematological Values at Baseline, During NO Inhalation, and During the Posttreatment Period

Systemic arterial pressure and left atrial pressure did not differ during the three observation periods or among the four groups of animals (Table 3).

View this table: [in this window] [in a new window]   Table 3. Hemodynamic Values at Baseline, During NO Inhalation, and During the Posttreatment Period

Effects of Inhaled NO in a Pharmacological Model of Coronary Artery Vasoconstriction
To assess the effects of inhaled NO on coronary vasomotor tone, we measured coronary blood flow during the inhalation of 80 ppm NO and compared the ability of inhaled NO to reverse the vasoconstrictor effects of the thromboxane A₂ agonist U-46619 with that of the intra-arterial infusion of the nitrosovasodilator SNP. Neither mean (99±7% of control) nor peak diastolic (99±5% of control) coronary blood flow was changed by the inhalation of 80 ppm NO for a 45-minute treatment period. The intra-arterial infusion of 1 µg/kg per minute SNP increased mean and peak diastolic coronary flow to 198±35% and 178±22% of control, respectively (P<.05 for both) (Fig 3), despite a decrease in mean arterial pressure to 93±2% of control (P<.05). The intra-arterial infusion of 10 nmol/min U-46619 decreased mean and peak diastolic coronary flow to 80±5% and 71±6% of control, respectively (P<.05 for both). During U-46619 infusion, the inhalation of 80 ppm NO did not reverse coronary vasoconstriction, but coronary flow did increase during the simultaneous infusion of 1 µg/kg per minute SNP (Fig 3).

View larger version (14K): [in this window] [in a new window]   Figure 3. Changes in mean (A) and peak diastolic (B) coronary flow during intracoronary infusion of 1 µg/kg per minute SNP; intracoronary infusion of 10 nmol/min U-46619; simultaneous inhalation of 80 ppm NO and infusion of 10 nmol/min U-46619; and simultaneous infusions of 1 µg/kg per minute SNP and 10 nmol/min U-46619. Values are mean±SEM. *P<.05 vs baseline period. n=5 dogs.

Effects of Inhaled NO on Bleeding Time
In four additional dogs, the bleeding times measured at baseline on the forearm and on the ventral aspect of the tongue were 218±91 seconds and 213±37 seconds, respectively. There was no effect of inhaling 20, 80, or 200 ppm NO on either forearm or tongue bleeding times.

Effects of NO Gas on ADP-Induced Platelet Aggregation In Vitro
Adding 21±2 µmol/L ADP to the PRP induced maximal platelet aggregation. The addition of NO to the gas mixture above the PRP inhibited ADP-induced platelet aggregation in a dose-dependent manner (P<.001) (Figs 4 and 5). Two hundred and 400 ppm NO inhibited maximal platelet aggregation (P<.002, P<.001), while there was no effect of either 20 or 80 ppm NO. The ADP-induced maximal increase in light transmission in the control aggregation curves performed at the beginning and end of each study did not differ (69±6% versus 69±4%).

View larger version (11K): [in this window] [in a new window]   Figure 4. Representative ADP-induced platelet aggregation curves with or without (control) exposure to 400 ppm NO in air.

View larger version (54K): [in this window] [in a new window]   Figure 5. Dose response of the effect of NO gas on platelet aggregation as characterized by % maximal increase in light transmission induced by ADP. Exposure to a gas mixture containing NO decreased platelet aggregation in a dose-dependent manner (P<.001). There was a significant difference between the mean of the control group and the mean of the pooled NO groups (P<.001). The % increase in light transmission observed in the two control aggregation curves at the beginning and end of the experiment was similar, attesting to the stability of the platelet aggregation response. Values are mean±SEM. *P=.002; **P<.001 vs control aggregation curves. n=8 dogs.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Platelet adhesion and aggregation play an important role in the progression of acute coronary ischemic syndromes,^{1 2} including thrombotic occlusion after thrombolysis⁴ and angioplasty.²⁹ Systemic^{14 15 30 31} and local³² administration of NO donor compounds have been shown to exert inhibitory effects on platelet aggregation, but their efficacy is limited by systemic hypotension or the need for intra-arterial catheterization. Inhaled NO is a selective pulmonary vasodilator,^{16 19} which was recently found to modestly prolong the bleeding time in rabbits and humans.^{22 23} We hypothesized that inhaled NO would be an effective inhibitor of platelet aggregation and would decrease coronary artery reocclusion after thrombolysis without producing systemic hemodynamic effects.

We selected a canine model of platelet-mediated coronary thrombosis resembling that occurring in patients with acute myocardial infarction who are treated with thrombolytic therapy.^{6 7} In this model of thrombosis superimposed on endothelial injury and arterial stenosis, thrombolysis with TPA induces alternating periods of arterial occlusion and reperfusion, accompanied by ECG evidence of myocardial injury (Fig 1). Pathological studies have shown that the coronary reocclusion occurring in this model is caused by platelet-rich thrombus.⁶ We observed a decrease in the frequency of CFVs during the inhalation of 20 and 80 ppm NO. Since the frequency of CFVs is lower in this model than in models of platelet-mediated coronary occlusion that do not utilize preformed thrombus followed by thrombolysis^{14 31} and a complete cessation of coronary reocclusion was not observed, we also measured the fractional duration of coronary patency during each treatment period to provide a more sensitive and specific assessment of myocardial perfusion. We observed during inhalation of 20 and 80 ppm NO that the fractional duration of coronary artery patency increased. Furthermore, both the decrease in frequency of CFVs and the increase in coronary artery patency ratio were sustained for at least 45 minutes after the administration of 80 ppm NO was discontinued, well beyond the expected half-life of this molecule in biological fluids.⁸

Other investigators have demonstrated an effect of NO donors on platelet-mediated arterial thrombosis. In a model of canine carotid artery injury induced by an electric current, Werns et al³⁰ observed that high infusion rates of NTG (10 µg/kg per minute) lowered both the incidence of primary thrombosis and of reocclusion after thrombolysis. Folts and colleagues demonstrated in injured, severely stenotic canine coronary arteries that infusion of NTG¹⁴ or SNP¹⁵ decreased the frequency of CFVs. However, the antithrombotic effect of NO donors reported in both of these models was associated with a decrease in systemic blood pressure. Yao et al³¹ observed that the inhibition of endogenous NO production with the NO synthase inhibitor N^G-monomethyl-L-arginine induced CFVs in injured, moderately stenotic canine coronary arteries and that these flow reductions ceased after the intravenous administration of the substrate for NO synthesis, L-arginine. The administration of L-arginine was accompanied by systemic vasodilation and a 22% reduction in mean aortic pressure.

Systemic vasodilation in patients with acute ischemic coronary syndromes could cause reflex tachycardia that would increase myocardial oxygen consumption and worsen myocardial ischemia. To avoid the systemic hypotension previously observed with intravenous NO donors, Golino et al³² studied the effects of intra-arterial infusion of solutions containing either dissolved NO gas or the NO donor compound S-nitroso-cysteine at the site of injured stenotic rabbit carotid arteries. While CFVs were abolished and arterial pressure remained unchanged in this study, intra-arterial catheterization was necessary to achieve the local concentration of NO (1.5x10^-7 mol/L) necessary to observe an antithrombotic effect. In the present study of inhaled NO on coronary reocclusion after thrombolysis, we found a decrease in CFVs and an increase in coronary patency. Administration of NO to achieve this antithrombotic effect was not accompanied by systemic hypotension, nor did it require intravascular catheterization.

Platelet deposition in animal models of endothelial injury and coronary stenosis has been observed to lead to epicardial coronary vasoconstriction mediated by TXA₂ and serotonin.³³ We considered the possibility that the increase in coronary patency observed in dogs breathing NO could have been due to a direct effect on the coronary vascular smooth muscle. Breathing NO did not affect flow in the uninjured coronary artery, nor did it reverse the coronary vasoconstrictor effect of U-46619 (a TXA₂ mimetic). In contrast, intracoronary administration of the NO donor compound SNP both increased flow and reversed the vasoconstrictor effect of U-46619 despite a decrease in systemic arterial pressure. Inhaled NO does not appear to directly dilate the coronary vasculature, which is consistent with our hypothesis that the hemodynamic effects of inhaled NO do not extend beyond the pulmonary circulation. An antithrombotic effect of breathing NO appears to be the best explanation for the improvement in coronary patency observed in this model of platelet-mediated coronary thrombosis.

To further examine the mechanism of the antithrombotic effect of inhaled NO, we investigated the ability of NO gas to prolong the bleeding time in vivo and inhibit platelet aggregation in vitro. In contrast to the studies of Hogman et al²³ in rabbits, we did not measure any effect of inhaled NO on the bleeding time in dogs. However, we did observe that NO gas exerts an antiaggregatory effect in vitro at concentrations similar to those we administered in vivo. A dissociation between antithrombotic and bleeding time effects has been previously observed with the antithrombotic agent TP9201. This cyclic peptide derivative binds to the platelet GPIIb/IIIa fibrinogen receptor,³⁴ inhibits occlusion in a canine femoral artery platelet-mediated thrombosis model, and decreases ADP-induced aggregation of both hamster and canine platelets in vitro without altering the bleeding time.²⁸ The mechanism for the differential effect of these agents at sites of bleeding and sites of thrombosis is unknown but may relate to the presence of areas of endothelial injury in the models of intra-arterial thrombosis. This endothelial injury produces an intense proaggregatory effect on platelets¹ and may be the setting in which the platelet inhibitory activity of inhaled NO and TP9201 is observable.

The persistence of CFVs in our model during NO inhalation suggests that the antithrombotic potency of inhaled NO is less than that of the c7E3 antibody and kistrin, both of which abolished CFVs.^{6 7} Aspirin does not affect CFVs in this model³⁵ yet is a clinically effective antithrombotic agent.⁴ Moreover, because inhaled NO does not prolong the bleeding time, it may offer an advantage over more potent antithrombotic agents, whose use is frequently complicated by the need for transfusion.⁵ Combination of inhaled NO with an antithrombotic agent that affects a different pathway of platelet activation may permit a reduction in the dosage and hence the toxicity of that agent.

The observations that inhaled NO attenuated coronary reocclusion in a model of platelet-mediated thrombosis after thrombolysis and that NO gas inhibited ADP-induced platelet aggregation provide evidence that inhaled NO has a systemic effect on platelet function. Since NO is rapidly inactivated in the presence of hemoglobin,²⁰ it is likely that inhaled NO affects platelets as they transit the pulmonary circulation. An alternative explanation for the antithrombotic effect of inhaled NO is that inhaled NO may react with substances present in blood to form adducts with longer half-lives than NO itself. These adducts could release NO at the site of thrombosis to mediate an antithrombotic effect. However, when NO adducts synthesized in vitro were administered intravenously, they produced antiplatelet effects that appeared to correlate with their hypotensive effects.²⁷ We did not observe a vasodilator effect of breathing NO on systemic blood pressure or on coronary flow, making it unlikely that the antithrombotic effect is attributable to NO adduct formation.

In vivo, we observed a significant antithrombotic effect of inhaled NO at doses of 20 and 80 ppm, but there was no statistically significant effect while breathing 200 ppm. Animal and clinical studies have shown that NO can interact with superoxide anion (O₂^-) present in lung tissue to form the potent oxidant peroxynitrite (ONOO^-),^{36 37} which blocks the stimulatory effect of NO on soluble guanylate cyclase³⁸ and has a proaggregatory effect on human platelets.³⁹ It is possible that when high concentrations of NO are inhaled, peroxynitrite could accumulate in sufficient quantity to have a proaggregatory effect. NO can also inactivate enzymes containing ferrous iron,⁴⁰ the inhibition of which could alter cellular metabolism and therefore might have a proaggregatory effect on platelets. Further studies are required to determine the dose of inhaled NO that produces the maximal antithrombotic effect in humans.

Conclusions
Inhaled NO decreased CFVs and increased coronary artery patency in a canine model of platelet-mediated coronary reocclusion after thrombolysis. This antithrombotic effect of inhaled NO was not associated with any systemic hemodynamic effects. NO gas also inhibited ADP-induced platelet aggregation in vitro. These observations suggest that inhaled NO may be a safe, effective antithrombotic therapy for acute coronary ischemic syndromes.

	Selected Abbreviations and Acronyms

 

ACT = activated clotting time CFV = cyclic flow variation LAD = left anterior descending NO = nitric oxide NTG = nitroglycerin ppm = parts per million SNP = sodium nitroprusside TPA = tissue plasminogen activator

	Acknowledgments

 
This study was supported by a grant from the Societe de Reanimation de Langue Francaise (C.A.) and USPHS grants HL-45895 (K.D.B.) and HL-42397 (W.M.Z.). The authors thank Michael Cheng and Dr Alan Zaslavsky for statistical assistance and Tracy Swizzero and Dr Mona Hirani for technical assistance.

Received February 7, 1996; revision received April 24, 1996; accepted May 1, 1996.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Fuster V, Badimon L, Badimon JJ, Chesebro JH. The pathogenesis of coronary artery disease and the acute coronary syndrome, part 1. N Engl J Med. 1992;326:242-250.[Medline] [Order article via Infotrieve]
   
2. Fuster V, Badimon L, Badimon JJ, Chesebro JH. The pathogenesis of coronary artery disease and the acute coronary syndrome, part 2. N Engl J Med. 1992;326:310-318.[Medline] [Order article via Infotrieve]
   
3. Coller BS. Platelets and thrombolytic therapy. N Engl J Med. 1990;322:33-42.[Medline] [Order article via Infotrieve]
   
4. ISIS-2 (Second International Study of Infarct Survival) Collaborative Group. Randomized trial of intravenous streptokinase, oral aspirin, both or neither among 17,187 cases of suspected acute myocardial infarction: ISIS-2. Lancet. 1988;2:349-360.[Medline] [Order article via Infotrieve]
   
5. The EPIC Investigators. Use of a monoclonal antibody directed against the platelet glycoprotein IIb/IIIa receptor in high-risk coronary angioplasty. N Engl J Med. 1994;330:956-961.[Abstract/Free Full Text]
   
6. Gold HK, Yasuda T, Jang IK, Guerrero L, Fallon JT, Leinbach RC, Collen D. Animal models for arterial thrombolysis and prevention of reocclusion: erythrocyte-rich versus platelet-rich thrombus. Circulation. 1991;83(suppl IV):IV-26-IV-40.
   
7. Yasuda T, Gold HK, Leinbach RC, Yaoita H, Fallon JT, Guerrero L, Napier MA, Bunting S, Collen D. Kistrin, a polypeptide platelet GPIIb/IIIa receptor antagonist, enhances and sustains coronary arterial thrombolysis with recombinant tissue-type plasminogen activator in a canine preparation. Circulation. 1990;83:1038-1047.[Abstract/Free Full Text]
   
8. Ignarro LJ. Biological actions and properties of endothelium-derived nitric oxide formed and released from artery and vein. Circ Res. 1989;65:1-21.[Free Full Text]
   
9. Holzmann S. Endothelium-induced relaxation by acetylcholine associated with large rises in cGMP in coronary arterial strips. J Cyclic Nucl Res. 1982;8:409-419.
   
10. Ignarro LJ, Burke TM, Wood KS, Wolin MS, Kadowitz PJ. Association between cyclic GMP accumulation and acetylcholine-elicited relaxation of bovine intrapulmonary artery. J Pharmacol Exp Ther. 1983;228:682-690.[Abstract/Free Full Text]
    
11. Radowski MW, Palmer RMJ, Moncada S. An L-arginine/nitric oxide pathway present in human platelets regulates aggregation. Proc Natl Acad Sci U S A. 1990;87:5193-5197.[Abstract/Free Full Text]
    
12. Mellion BT, Ignarro LJ, Ohlstein EH, Pontecorvo EG, Hyman AL, Kadowitz PJ. Inhibition of human platelet aggregation by S-nitrosothiols: heme-dependent activation of soluble guanylate cyclase and stimulation of cyclic GMP accumulation. Mol Pharmacol. 1983;23:653-664.[Abstract]
    
13. Mellion BT, Ignarro LJ, Ohlstein EH, Pontecorvo EG, Hyman AL, Kadowitz PJ. Evidence for the inhibitory role of guanosine 3',5'-monophosphate in ADP-induced human platelet aggregation in the presence of nitric oxide and related vasodilators. Blood. 1981;57:946-955.[Free Full Text]
    
14. Folts JD, Stamler J, Loscalzo J. Intravenous nitroglycerin infusion inhibits cyclic blood flow responses caused by periodic platelet thrombus formation in stenosed canine coronary arteries. Circulation. 1991;83:2122-2127.[Abstract/Free Full Text]
    
15. Rovin JD, Stamler JS, Loscalzo J, Folts JD. Sodium nitroprusside, an endothelium-derived relaxing factor congener, increases platelet cyclic GMP levels and inhibits epinephrine-exacerbated in vivo platelet thrombus formation in stenosed canine coronary arteries. J Cardiovasc Pharmacol. 1993;22:626-631.[Medline] [Order article via Infotrieve]
    
16. Frostell C, Fratacci MD, Wain JC, Jones R, Zapol WM. Inhaled nitric oxide: a selective pulmonary vasodilator reversing hypoxic pulmonary vasoconstriction. Circulation. 1991;83:2038-2047.[Abstract/Free Full Text]
    
17. Pepke-Zaba J, Higenbottam TW, Dinh-Xuan AT, Stone D, Walwork J. Inhaled nitric oxide as a cause of selective vasodilation in pulmonary hypertension. Lancet. 1991;338:1173-1174.[Medline] [Order article via Infotrieve]
    
18. Rossaint R, Falke KJ, Lopez F, Slama K, Pison U, Zapol WM. Inhaled nitric oxide for the adult respiratory distress syndrome. N Engl J Med. 1993;328:399-405.[Abstract/Free Full Text]
    
19. Semigran MJ, Cockrill BA, Kacmarek R, Thompson BT, Zapol M, Dec GM, Fifer MA. Hemodynamic effects of inhaled nitric oxide in heart failure. J Am Coll Cardiol. 1994;24:982-988.[Abstract]
    
20. Cassoly R, Gibson Q. Conformation, co-operativity and ligand binding in human hemoglobin. J Mol Biol. 1975;91:301-313.[Medline] [Order article via Infotrieve]
    
21. Rimar S, Gillis N. Selective pulmonary vasodilation by inhaled nitric oxide is due to hemoglobin inactivation. Circulation. 1993;88:2884-2887.[Abstract/Free Full Text]
    
22. Hogman M, Frostell C, Arnberg H, Sandhagen B, Hedenstierna G. Bleeding time prolongation and nitric oxide inhalation. Lancet. 1993;341:1664-1665.[Medline] [Order article via Infotrieve]
    
23. Hogman M, Frostell C, Arnberg H, Sandhagen B, Hedenstierna G. Prolonged bleeding time during nitric oxide inhalation in the rabbit. Acta Physiol Scand. 1994;151:125-129.[Medline] [Order article via Infotrieve]
    
24. Fontjin A, Sabadell AJ, Ronco RJ. Homogeneous chemiluminescent measurement of nitric oxide with ozone. Anal Chem. 1970;42:575-579.
    
25. Hatterley PG. Activated coagulation time of whole blood. JAMA. 1966;196:436-440.[Medline] [Order article via Infotrieve]
    
26. Michelassi F, Castorena G, Hill RD, Lowenstein E, Watkins WD, Petkau AJ, Zapol WM. Effects of leukotrienes B₄ and C₄ on coronary circulation and myocardial contractility. Surgery. 1983;94:267-274.[Medline] [Order article via Infotrieve]
    
27. Keaney JF, Simon DI, Stamler JS, Jaraki O, Scharfstein J, Vita JA, Loscalzo J. NO forms an adduct with serum albumin that has endothelium-derived relaxing factor-like properties. J Clin Invest. 1993;91:1582-1589.
    
28. Collen D, Lu HR, Stassen JM, Vreys I, Yasuda T, Bunting S, Gold HK. Antithrombotic effects and bleeding time prolongation with synthetic platelet GPIIb/IIIa inhibitors in animal models of platelet-mediated thrombosis. Thromb Haemost. 1994;71:95-102.[Medline] [Order article via Infotrieve]
    
29. Schwartz L, Bourassa MG, Lesperance J, Aldridge HE, Kazim F, Salvatori VA, Henderson M, Bonan R, David PR. Aspirin and dipyridamole in the prevention of restenosis after percutaneous transluminal coronary angioplasty. N Engl J Med. 1988;318:1714-1719.[Abstract]
    
30. Werns SW, Rote WE, Davis JH, Guevara T, Lucchesi BR. Nitroglycerin inhibits experimental thrombosis and reocclusion after thrombolysis. Am Heart J. 1994;127:727-737.[Medline] [Order article via Infotrieve]
    
31. Yao SK, Ober JC, Krishnaswami A, Ferguson JJ, Anderson HV, Golino P, Buja LM, Willerson JT. Endogenous nitric oxide protects against platelet aggregation and cyclic flow variations in stenosed and endothelium-injured arteries. Circulation. 1992;86:1302-1309.[Abstract/Free Full Text]
    
32. Golino P, Cappelli-Bigazzi M, Ambrosio G, Ragni M, Russolillo E, Condorelli M, Chiariello M. Endothelium-derived relaxing factor modulates platelet aggregation in an in vivo model of recurrent platelet activation. Circ Res. 1992;71:1447-1456.[Abstract/Free Full Text]
    
33. Golino P, Ashton JH, Buja LM, Rosolowsky M, Taylor AL, McNatt J, Campbell WB, Willerson JT. Local platelet activation causes vasoconstriction of large epicardial canine coronary arteries in vivo. Circulation. 1989:79:154-166.
    
34. Tschopp JF, Bell DJ, Bunting S. Novel RGD-based glycoprotein IIb/IIIa receptor antagonists as antithrombotics. Blood. 1992;80(suppl I):320a. Abstract.
    
35. Yasuda T, Gold HK, Yaoita H, Leinbach RC, Guerrero JL, Jang IK, Holt R, Fallon JT, Collen D. Comparative effects of aspirin, a synthetic thrombin inhibitor and a monoclonal antiplatelet glycoprotein IIb/IIIa antibody on coronary artery reperfusion, reocclusion and bleeding with recombinant tissue-type plasminogen activator in a canine preparation. J Am Coll Cardiol. 1990;16:714-722.[Abstract]
    
36. Mulligan MS, Hevel JM, Marletta MA, Ward PA. Tissue injury caused by deposition of immune complexes is L-arginine dependent. Proc Natl Acad Sci U S A. 1991;88:6338-6342.[Abstract/Free Full Text]
    
37. Kooy NW, Royall JA, Ye YZ, Kelly DR, Beckman JS. Evidence for in vivo peroxynitrite production in human acute lung injury. Am J Respir Crit Care Med. 1995;151:1250-1254.[Abstract]
    
38. Mayer B, Klatt P, Werner ER, Schmidt K. Kinetics and mechanism of tetrahydrobiopterin-induced oxidation of nitric oxide. J Biol Chem. 1995;270:655-659.[Abstract/Free Full Text]
    
39. Moro MA, Darley-Usmar VM, Goodwin DA, Read NG, Zamora-Pino R, Feelisch M, Radomski MW, Moncada S. Paradoxical fate and biological action of peroxynitrite on human platelets. Proc Natl Acad Sci U S A. 1994;91:6702-6706.[Abstract/Free Full Text]
    
40. Kim YM, Bergonia HA, Muller C, Pitt BR, Watkins WD, Lancaster JR. Loss and degradation of enzyme-bound heme induced by cellular nitric oxide synthesis. J Biol Chem. 1995;270:5710-5713.[Abstract/Free Full Text]
    

This article has been cited by other articles:

				X. Liu, Y. Huang, P. Pokreisz, P. Vermeersch, G. Marsboom, M. Swinnen, E. Verbeken, J. Santos, M. Pellens, H. Gillijns, et al. Nitric Oxide Inhalation Improves Microvascular Flow and Decreases Infarction Size After Myocardial Ischemia and Reperfusion J. Am. Coll. Cardiol., August 21, 2007; 50(8): 808 - 817. [Abstract] [Full Text] [PDF]

				K. D. Bloch, F. Ichinose, J. D. Roberts Jr., and W. M. Zapol Inhaled NO as a therapeutic agent Cardiovasc Res, July 15, 2007; 75(2): 339 - 348. [Abstract] [Full Text] [PDF]

				R. Hataishi, A. C. Rodrigues, T. G. Neilan, J. G. Morgan, E. Buys, S. Shiva, R. Tambouret, D. S. Jassal, M. J. Raher, E. Furutani, et al. Inhaled nitric oxide decreases infarction size and improves left ventricular function in a murine model of myocardial ischemia-reperfusion injury Am J Physiol Heart Circ Physiol, July 1, 2006; 291(1): H379 - H384. [Abstract] [Full Text] [PDF]

				R. Hataishi, W. M. Zapol, K. D. Bloch, and F. Ichinose Inhaled nitric oxide does not reduce systemic vascular resistance in mice Am J Physiol Heart Circ Physiol, May 1, 2006; 290(5): H1826 - H1829. [Abstract] [Full Text] [PDF]

				G. D. Lewis, C. Witzke, P. Colon-Hernandez, J. L. Guerrero, K. D. Bloch, and M. J. Semigran Sildenafil Improves Coronary Artery Patency in a Canine Model of Platelet-Mediated Cyclic Coronary Occlusion After Thrombolysis J. Am. Coll. Cardiol., April 4, 2006; 47(7): 1471 - 1477. [Abstract] [Full Text] [PDF]

				T. J. McMahon and A. Doctor Extrapulmonary effects of inhaled nitric oxide: role of reversible s-nitrosylation of erythrocytic hemoglobin. Proceedings of the ATS, January 1, 2006; 3(2): 153 - 160. [Abstract] [Full Text] [PDF]

				J. Gianetti, P. Del Sarto, S. Bevilacqua, C. Vassalle, R. De Filippis, M. Kacila, P. A. Farneti, A. Clerico, M. Glauber, and A. Biagini Supplemental nitric oxide and its effect on myocardial injury and function in patients undergoing cardiac surgery with extracorporeal circulation J. Thorac. Cardiovasc. Surg., January 1, 2004; 127(1): 44 - 50. [Abstract] [Full Text] [PDF]

				T. Wang, D. El Kebir, and G. Blaise Inhaled nitric oxide in 2003: a review of its mechanisms of action: [L'inhalation de monoxyde d'azote en 2003 : une revue de ses mecanismes et de son action] Can J Anesth, October 1, 2003; 50(8): 839 - 846. [Abstract] [Full Text] [PDF]

				M. Beghetti, C. Sparling, P. N. Cox, D. Stephens, and I. Adatia Inhaled NO inhibits platelet aggregation and elevates plasma but not intraplatelet cGMP in healthy human volunteers Am J Physiol Heart Circ Physiol, July 11, 2003; 285(2): H637 - H642. [Abstract] [Full Text] [PDF]

				U. Schmidt, R. O. Han, T. G. DiSalvo, J. L. Guerrero, H. K. Gold, W. M. Zapol, K. D. Bloch, and M. J. Semigran Cessation of platelet-mediated cyclic canine coronary occlusion after thrombolysis by combining nitric oxide inhalation with phosphodiesterase-5 inhibition J. Am. Coll. Cardiol., June 1, 2001; 37(7): 1981 - 1988. [Abstract] [Full Text] [PDF]

				S. Massberg, G. Enders, F. C. d. M. Matos, L. I. D. Tomic, R. Leiderer, S. Eisenmenger, K. Messmer, and F. Krombach Fibrinogen Deposition at the Postischemic Vessel Wall Promotes Platelet Adhesion During Ischemia-Reperfusion In Vivo Blood, December 1, 1999; 94(11): 3829 - 3838. [Abstract] [Full Text] [PDF]

				K. Hata, P. Whittaker, R. A. Kloner, and K. Przyklenk Brief Myocardial Ischemia Attenuates Platelet Thrombosis in Remote, Damaged, and Stenotic Carotid Arteries Circulation, August 24, 1999; 100(8): 843 - 848. [Abstract] [Full Text] [PDF]

				C. S Hayward, R. P Kelly, and P. S Macdonald Inhaled nitric oxide in cardiology practice Cardiovasc Res, August 15, 1999; 43(3): 628 - 638. [Abstract] [Full Text] [PDF]

				S. Massberg, M. Sausbier, P. Klatt, M. Bauer, A. Pfeifer, W. Siess, R. Fassler, P. Ruth, F. Krombach, and F. Hofmann Increased Adhesion and Aggregation of Platelets Lacking Cyclic Guanosine 3',5'-Monophosphate Kinase I J. Exp. Med., April 19, 1999; 189(8): 1255 - 1264. [Abstract] [Full Text] [PDF]

				M. M. Bednar and C. E. Gross Antiplatelet Therapy in Acute Cerebral Ischemia Stroke, April 1, 1999; 30(4): 887 - 893. [Abstract] [Full Text] [PDF]