Nitric Oxide Inhalation Inhibits Platelet Aggregation and Platelet-Mediated Pulmonary Thrombosis in Rats
Abstract Endothelium-derived nitric oxide (NO) inhibits in vitro platelet aggregation via a cGMP-dependent mechanism. The effect of inhaled NO on platelet-mediated pulmonary thrombosis following intravenous thrombotic challenge with collagen was examined in rats and compared with the effect of G4120, a cyclic Arg-Gly-Asp–containing synthetic pentapeptide that binds to the platelet glycoprotein IIb/IIIa receptor. Intraplatelet cGMP dose-dependently increased from 39±6 fmol/108 platelets in control to 46±6, 68±13, and 81±13 fmol/108 platelets after inhalation with 20, 40, and 80 ppm NO, respectively (P<.05 for 40 and 80 ppm). Ex vivo platelet aggregation of platelet-rich plasma induced by 1 μg/mL collagen was reduced from 75±4% in control rats to 22±10% and 20±7% in rats ventilated with 40 and 80 ppm NO, respectively, and to 30±9% in G4120-treated rats (each P<.05 versus control). Circulating platelet counts 3 minutes after collagen injection were significantly higher in the inhaled NO and G4120 groups compared with control rats (250 000±18 000 and 223 000±10 000/μL versus 160 000±18 000/μL, each P<.05). The rise in pulmonary arterial pressure after collagen injection was significantly reduced in NO- and G4120-treated rats (26±1 and 27±1 versus 32±1 mm Hg in control rats, each P<.05). The number of pulmonary resistance vessels containing platelet thrombi was significantly smaller after inhaled NO and G4120 treatment compared with control (56±3% and 50±3% versus 68±3%, respectively; P<.05). Thus, NO inhalation reduces in vivo activation of circulating platelets and platelet-rich thrombosis in thromboembolic pulmonary hypertension. Inhalation of NO may be useful in cardiovascular diseases associated with platelet activation.
Endothelium-derived NO inhibits platelet adhesion and aggregation via a cGMP-dependent mechanism.1 2 NO activates soluble guanylate cyclase, which catalyzes the formation of cGMP from GTP.2 The amount of NO available for the regulation of platelet function primarily depends on the synthesis and release of this free radical from vascular endothelium. Decreased formation of NO by dysfunctional endothelium can lead directly to platelet-rich thrombosis, which has been implicated in the pathogenesis of atherosclerosis, primary pulmonary hypertension, and acute respiratory distress syndrome.3 4 5 6
Thrombosis is the most commonly encountered obstructive process in pulmonary arteries, leading to pulmonary hypertension with significant morbidity and mortality.5 7 8 9 Pulmonary thrombi, whether embolic in origin or developed in situ, may occlude a large portion of the pulmonary arterial bed and increase pulmonary artery pressure and pulmonary vascular resistance.8 9 Platelet activation is an important factor leading to thrombosis, and pharmacological interventions aimed at reducing platelet activation may reduce pulmonary thrombosis and subsequent pulmonary hypertension.10 11 Anticoagulant therapy indeed significantly improves survival in primary pulmonary hypertension patients, who frequently present with in situ thrombosis.12
In experimental animal models, increased concentrations of NO reduce platelet aggregation, adhesion, and platelet-rich thrombus formation following endothelial injury.13 14 In these models, increased levels of NO were obtained by intravenous administration of an NO donor or by stimulation of endogenous NO synthesis or release with l-arginine or endotoxin,14 15 which may, however, be associated with systemic side effects.13 14 16
Inhaled NO gas is a safe and selective pulmonary vasodilator in patients with pulmonary hypertension.17 18 NO readily diffuses across the alveolar wall to neighboring precapillary pulmonary vascular smooth muscle cells but is rapidly inactivated in the capillary bed by hemoglobin, thereby reducing potential systemic hypotensive effects.19 NO inhalation also significantly decreased platelet-mediated coronary artery occlusion in dogs20 and reduced neointimal formation in response to peripheral vascular injury in rats,21 suggesting an effect of inhaled NO on circulating platelets and/or leukocytes.
In the present study, we evaluated the effect of inhaled NO on ex vivo collagen-induced platelet aggregation and intraplatelet cGMP levels and on in vivo antithrombotic activity in a rat model of platelet-mediated pulmonary thrombosis. To compare the antithrombotic effects of inhaled NO with conventional pharmacological antiplatelet therapy, a separate group of rats was treated with G4120, a cyclic RGD–containing synthetic pentapeptide that binds to the platelet GPIIb/IIIa receptor.22 The same variables were measured in the ex vivo and in vivo study protocols.
Materials and Methods
Male Wistar rats (330 to 360 g) were anesthetized with pentobarbital 50 mg/kg IP, orally intubated, and mechanically ventilated with room air or a mixture of air with different concentrations of NO (tidal volume of 7 mL/kg and respiratory rate of 60/min) following institutional guidelines for animal experimentation. During mechanical ventilation, arterial blood gases were analyzed on a blood gas system 288 (CIBA Corning). NO was released from a NO tank containing 800 ppm, mixed with room air (3 L/min), and delivered to the animals via a rodent ventilator (model 683, Harvard Apparatus). NO and NO2 concentrations were monitored continuously (Inhaled NO Therapy Monitor, Bedfont Scientific Ltd). NO flow was adjusted to obtain NO concentrations of 20, 40, and 80 ppm. Generation of NO2 from NO and O2 was <2 ppm at the highest NO concentrations administered.
Ex Vivo Platelet Aggregation Studies
To examine the effects of inhaled NO on platelet activation, groups of 6 rats were ventilated with 20, 40, or 80 ppm NO. After 2 hours, 4 mL blood was taken from the carotid artery in a heparin-containing syringe (0.4 mL of heparin [1000 IU/mL]). To exclude an effect of mechanical ventilation on platelet aggregation, 6 rats were ventilated with room air for 2 hours, and blood samples were collected for platelet aggregation studies. The antiplatelet activity of inhaled NO was compared with the antithrombotic properties of a platelet GPIIb/IIIa antagonistic peptide G4120. Three rats were ventilated with room air and received a bolus injection of G4120 (3 mg/kg IV). Five minutes after the injection of G4120, blood samples were collected for platelet aggregation studies.
Blood samples were processed immediately after withdrawal by centrifugation at 900g for 10 minutes at room temperature to obtain PRP. The platelet count in PRP, which varied from 600 000/μL to 900 000/μL, was adjusted to 500 000/μL by dilution with PPP, obtained by centrifugation at 3000g for 15 minutes. Platelet aggregation was induced by rotating microtiter plates containing 50 μL PRP for 5 minutes at 37°C with various concentrations of collagen (0, 0.5, 1, and 2 μg/mL). Formaldehyde (200 μL, 1%) was added to arrest aggregation, and 200 μL of the platelet suspension was transferred to a second microtiter plate in which aggregation was determined from light scattering at 620 nm in a microtiter plate reader (EAR 400AT, SLT-Lab Instruments). Aggregation was expressed as percent increase of light transmission in PRP. PPP was used as the standard for 100% light transmission.
Effects of Inhaled NO on Intraplatelet cGMP Level
Groups of 4 rats were intubated and ventilated for 2 hours with room air (control) or 20, 40, or 80 ppm NO, respectively. A 2.5F catheter was introduced in the right carotid artery for blood pressure measurements and withdrawal of 4 mL blood in a syringe containing 0.4 mL of 75 mmol/L EDTA and 40 μL of 5 mmol/L isobutyl methylxanthine. Blood was centrifuged at 900g for 10 minutes at room temperature to obtain PRP. PRP was centrifuged at 1500g for 5 minutes, the plasma was removed, and the platelet pellet was resuspended in 0.2 mL of 4 mmol/L EDTA.
For cGMP measurements, samples were sonicated, and cGMP was extracted in ice-cold 50% trichloroacetic acid (pH 4.0) and quantified by a commercial radioimmunoassay after acetylation, according to the manufacturer’s instructions (Amersham Life Science).
In Vivo Thrombosis Model
A previously described murine model of pulmonary platelet thromboembolism23 24 was used with minor modifications. After 2 hours of mechanical ventilation with room air (control) or 80 ppm NO, in groups of 6 rats, a silastic catheter (inner diameter, 0.30 mm; outer diameter, 0.64 mm) was introduced, via the right jugular vein, into the pulmonary artery for pulmonary artery pressure measurements. Collagen (2.5 mL/kg of 250 μg/mL) was injected into the jugular vein, and the changes in pulmonary artery pressure were recorded continuously to monitor the hemodynamic consequences of collagen-induced pulmonary thrombosis. Similar measurements were obtained from 6 animals that received a bolus injection of G4120 (3 mg/kg IV) 5 minutes before collagen challenge.
Platelet counts were carried out before and 3 minutes after injection of collagen. Whole blood (1 mL) was collected from the carotid cannula and anticoagulated with heparin (10% [vol/vol] of 1000 IU/mL). After mixing thoroughly, platelets were counted automatically on a cell-DYN 1300 (Abbott Laboratories).
Animals were killed 10 minutes after the injection of collagen. The chest was opened, the pulmonary vessels were perfused with saline at a pressure of 80 cm H2O, and the lungs were instilled with 10% formalin via the trachea. The trachea was ligated and removed together with the lungs and immersed in 10% formalin for 24 hours. Two transverse sections through the right lower lobe and the left lower lobe were paraffin-embedded and cut into 7-μm sections. The presence of platelet-rich thrombi in pulmonary vessels was determined via immunohistochemical staining using a murine monoclonal antibody, G28E5, raised in our laboratory with human GPIbα, which cross-reacted with rat GPIbα. After overnight incubation at 4°C with G28E5 (10 μg/mL), sections were exposed to normal goat serum to block nonspecific sites and incubated with a secondary goat anti-mouse antibody conjugated with horseradish peroxidase (1/100, Dako A/S). Bound antibody was visualized with diaminobenzidine and examined by light microscopy. For every experimental condition, two sections from each lobe were examined, and at least 20 microscopic fields were studied at ×400 magnification. The total number of identifiable small lung vessels with a diameter <100 μm was counted, and the percentage of vessels filled with platelet-rich thrombi was determined. All sections were encoded and analyzed by investigators blinded to the experimental treatment.
All values are given as mean±SEM. ANOVA and subsequent multiple comparison using Fisher’s test were used to determine differences between groups. Paired Student’s t tests were used when appropriate. Significance in all cases was defined as two-sided P<.05.
Arterial blood gases were similar in control rats and in rats ventilated with room air or 80 ppm NO (Pao2, 96±3 mm Hg; Paco2, 39±2 mm Hg; pH 7.40±0.02). Inhalation of different concentrations of NO for 2 hours did not affect systolic blood pressure (153±3 mm Hg after 80 ppm NO versus 154±4 mm Hg in the control group).
Inhaled NO exerted a dose-dependent effect on intraplatelet cGMP levels. Platelet cGMP levels 2 hours after inhalation of 40 and 80 ppm NO were significantly increased compared with control values (68±13 and 81±13 fmol/108 platelets, respectively, versus 39±6 fmol/108 platelets; P<.05; Fig 1⇓). Ex vivo platelet aggregation with various concentrations of collagen was identical between spontaneously breathing rats and rats ventilated with room air (data not shown). Inhalation with 40 and 80 ppm NO inhibited ex vivo platelet aggregation induced with 0.5, 1, and 2 μg/mL collagen (from 37±6%, 75±4%, and 97±2% in control to 5±2%, 22±10%, and 62±9% after 40 ppm NO and to 6±4%, 20±7%, and 65±11% after 80 ppm NO, respectively; Fig 2⇓). Inhibition was significant at all collagen concentrations studied. At 80 ppm, NO aggregation was not further reduced, suggesting a plateau at 40 ppm NO. Bolus injection of the platelet GPIIb/IIIa antagonistic peptide G4120 reduced ex vivo platelet aggregation to similar degrees as found with 40 and 80 ppm NO (13±4%, 30±9%, and 51±10% with 0.5, 1, and 2 μg/mL collagen, respectively; Fig 2⇓).
During mechanical ventilation, baseline mPAP before the thrombotic challenge was 16±0.2 mm Hg in control animals and 17±0.2 mm Hg in G4120-treated animals. mPAP rapidly increased after intravenous injection of 250 μg/mL collagen, suggesting that collagen-induced platelet aggregation caused pulmonary thrombosis during the passage of platelets through the pulmonary circulation. Because of fluctuations in mPAP during the first 2 minutes, mPAP was measured after 3 minutes, when the pressure had stabilized. mPAP rose to 32±1 mm Hg in control rats but was significantly lower in rats treated with 80 ppm NO or G4120 (26±1 and 27±1 mm Hg, respectively; P<0.05; Fig 3⇓).
Baseline circulating platelet count was 590 000±13 000/μL in control rats (n=6), 620 000±19 000/μL in rats inhaling 80 ppm NO (n=6, P=NS), and 624 000±2/μL in rats treated with G4120 (n=5, P=NS). Three minutes after the intravenous collagen challenge, the platelet count dropped in control animals by 74±3% to 160 000±18 000/μL. The decrease in circulating platelets in animals ventilated with 80 ppm NO and animals treated with G4120 was significantly smaller than that in control animals (250 000±18 000/μL, a 57±2% reduction, and 223 000±10 000/μL, a 64±1% reduction, respectively; P<.05 versus control).
Three rats in the control group, but none in the NO group or G4120 group, died within 10 minutes after collagen challenge. Survival after collagen challenge in this model constitutes an important marker for the efficiency of antithrombotic agents,23 24 although the small group sizes do not allow statistical analysis of mortality.
To investigate whether observed differences in mPAP and circulating platelet count correlated with a protective effect of NO against intrapulmonary thrombosis, specific immunochemical staining of platelets was performed on lung sections from control, NO-treated, and G4120-treated rats using a monoclonal anti-GPIbα antibody (Fig 4a⇓). Vessels filled with platelet thrombi generally had a diameter <75 μm, whereas larger-sized vessels were often filled with red blood cell aggregates, which did not stain with the anti-GPIbα antibody (Fig 4b⇓). In lung sections from control animals injected with collagen, 68±3% of small pulmonary vessels were totally or partially (>50% of lumen) occluded by platelet-rich thrombi. In NO-treated and G4120-treated animals, the number of occluded pulmonary vessels was significantly reduced (56±3% and 50±3%, respectively; P<.05 versus control).
In the present study, inhalation of NO gas was found to significantly reduce ex vivo collagen-induced platelet aggregation and to attenuate the rise in the pulmonary artery pressure caused by collagen-induced platelet-mediated pulmonary thrombosis. NO-treated rats had less platelet thrombi in small pulmonary vessels and higher residual circulating platelet counts than did the control animals. The antithrombotic activity of inhaled NO was similar to effects obtained with the platelet GPIIb/IIIa antagonistic peptide G4120, suggesting a direct action of inhaled NO on circulating platelet activation. Intraplatelet cGMP levels increased dose-dependently with inhaled NO concentrations, suggesting that the effects on platelet function were most likely mediated by a cGMP-dependent mechanism.
Endothelial cells constitutively release NO, an important regulator of vessel tone and vascular homeostasis through its effect on platelet and smooth muscle cell function. Incubation of human PRP with NO donors in vitro inhibited platelet aggregation induced by ADP, collagen, and thrombin,25 26 27 and administration of NO donor compounds or nitrovasodilators in vivo resulted in transient increases in NO concentration and inhibition of platelet function.13 14 16 In a rabbit thrombosis model consisting of an external constrictor around endothelium-denuded carotid arteries, local infusion of a solution of NO abolished cyclic flow reductions due to recurrent platelet aggregation.13 However, cyclic flow reductions were restored spontaneously within 10 minutes after cessation of NO infusion.
Inhalation of NO gas, which allows continuous administration of NO without systemic hypotensive side effects, can also affect platelet function.28 In the present study, 40 or 80 ppm but not 20 ppm of inhaled NO inhibited platelet aggregation and did not affect systolic blood pressure. The effect of inhaled NO on platelet aggregation was found to be associated with concentration-dependent changes in intraplatelet cGMP levels. The platelet cGMP signal transduction system indeed functions as a negative-feedback mechanism regulating the physiological activation of platelets. Sodium nitroprusside, a direct NO donor compound, has a concentration-dependent effect on platelet guanylate cyclase activity in vitro, and the resulting rise in cGMP causes a time-dependent disaggregation.16
Inhaled NO is also a selective pulmonary vasodilator and effectively reduces pulmonary hypertension.17 18 The vasodilatory action of NO most likely does not contribute to the reduction of pulmonary artery pressure in the present study, because animals ventilated with 80 ppm NO or room air had normal baseline pulmonary pressures before the thrombotic challenge. Moreover, collagen, which is often used to test the antiaggregatory efficacy,23 24 has no vasoconstrictor properties. However, we cannot exclude that after collagen-induced platelet activation, platelet-release products, including thromboxane A2 and serotonin, secondarily affect pulmonary vascular tone.29 Therefore, we tested in this model the efficacy of a platelet GPIIb/IIIa antagonistic peptide, which has no direct effects on pulmonary vascular tone. The synthetic peptide inhibits the binding of fibrinogen via the RGD recognition sequence to the platelet GPIIb/IIIa receptor, an essential step of collagen-induced platelet aggregation,22 but does not block the interaction of vasoactive substances with the vessel wall. High concentrations of G4120, a cyclic RGD–containing pentapeptide (3 mg/kg IV bolus), inhibit ex vivo platelet aggregation in rat PRP to the same extent as inhalation of 80 ppm NO. The effects on pulmonary artery pressure rise and on pulmonary platelet-rich thrombosis were also similar, suggesting that in this experimental model, inhaled NO predominantly acts by modulating platelet function.
It has previously been demonstrated that neointimal lesion formation in balloon-injured rat carotid arteries is significantly inhibited by sustained NO inhalation.21 Mitogens released from activated platelets are important in initiating migration of cells into the intima. The present study shows that inhaled NO significantly inhibits platelet activation and adhesion and suggests that modulation of circulating platelet function with NO gas may account for previously reported effects on neointimal formation. Moreover, it has been shown that NO is produced in human platelets and that changes in intraplatelet NO production have important physiological and pathophysiological implications.30 Impaired intraplatelet NO production has been observed in patients with coronary atherosclerosis, and this impairment is associated with increased platelet aggregation,31 especially during acute coronary syndromes. Our findings suggest that inhaled NO might compensate for reduced intraplatelet NO production.
In conclusion, NO inhalation significantly reduces circulating platelet activation without systemic side effects. Inhalation of NO may therefore be useful in cardiovascular diseases characterized by platelet activation, such as primary pulmonary hypertension, acute respiratory distress syndrome, unstable angina, or recurrent angina after myocardial infarction. Whether administration and dosing of inhaled NO can be achieved in a fashion that is safe and beneficial to these patients remains to be determined.
Selected Abbreviations and Acronyms
|G4120||=||l-cysteine, N-(mercaptoacetyl)-d-tyrosyl-l-arginylglycyl-l-α-aspartyl-cyclic (1-5) sulfide, 5-oxide|
|GPIbα, GPIIb/IIIa||=||glycoprotein Ibα and IIb/IIIa|
|mPAP||=||mean pulmonary artery pressure|
This study was supported by the National Fund for Scientific Research (NFWO to Dr Janssens) and by an interuniversity grant from the Belgian government (IUAP No. P4/34 to Dr Hoylaerts). Dr Janssens is the recipient of a chair financed by Zeneca Pharmaceuticals Inc.
- Received March 13, 1997.
- Accepted July 23, 1997.
- © 1997 American Heart Association, Inc.
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