Inhaled Nitric Oxide Increases Coronary Artery Patency After Thrombolysis
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.
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
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-NOx 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 F2α; 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.
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-NOx 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.
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.
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.
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).
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.
Systemic arterial pressure and left atrial pressure did not differ during the three observation periods or among the four groups of animals (Table 3⇓).
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⇓).
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%).
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 Systemic14 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 thrombolysis14 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 NG-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 (1.5×10−7 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 Ho¨gman 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.
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|
|ppm||=||parts per million|
|TPA||=||tissue plasminogen activator|
This study was supported by a grant from the Socie´te´ de Re´animation de Langue Franc¸aise (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.
- Copyright © 1996 by American Heart Association
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