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RESEARCH ARTICLE
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INHALED NEBULIZED NITRITE DECREASES PLATELET ACTIVITY IN HEALTHY
VOLUNTEERS
Tipparat Parakaw1,2, Kran Suknantha1, Pornpun Vivithanaporn1, Supeenun
Unchern1, Nathawut Sibmooh1, Sirada Srihirun2*
1
Department of Pharmacology, Faculty of Science, Mahidol University, Bangkok,
Thailand, 10400
2
Department of Pharmacology, Faculty of Dentistry, Mahidol University, Bangkok,
Thailand, 10400
Abstract
Nitrite, a nitric oxide (NO) metabolite, inhibits platelet activation through
its conversion to NO. In this study, we investigated the effect of inhaled nebulized
nitrite on human platelets ex vivo. Seven healthy volunteers were administered
37.5-mg sodium nitrite by inhaled nebulization. Levels of platelet activation before
and immediately after inhalation were measured by aggregometry and flow
cytometry using P-selectin, and activated glycoprotein aGPIIb/IIIa expression in
response to adenosine 5’ diphosphate (ADP) and U46619, a thromboxane A2
(TXA2) agonist. After nebulization, the nitrite level in whole blood increased from
0.15  0.03 to 1.7  0.23 M. Nitrite decreased platelet aggregation and P-selectin
expression induced by ADP. However, nitrite had no effect on ADP-induced
aGPIIb/IIIa expression and U46619 induced P-selectin and aGPIIb/IIIa expression.
In conclusion, inhaled nitrite decreases platelet activation, and may have potential
to use in patients with platelet hyperactivation.
KEYWORDS: PLATELET, NITRITE, NITRIC OXIDE, INHALATION.
Introduction
Nitric oxide (NO) is an endogenous vasodilator and platelet inhibitor1,2. In
circulation, NO is constitutively synthesized by endothelial nitric oxide synthase
(eNOS). In platelets, NO increases cGMP, which further causes deactivation of
glycoprotein IIb/IIIa (GPIIb/IIIa) receptors and P-selectin expression on platelet
surface leading to platelet inhibition3. NO is also generated from nitrite anion (NO2) by reductase activity of heme-containing proteins such as deoxyhemoglobin4.
Because NO is unstable, nitrite which is a more stable metabolite of NO, has been
investigated for an alternative treatment. Our previous in vitro study demonstrated
the antiplatelet activity of nitrite in the presence of erythrocytes under hypoxic
condition5.
Nitrite inhalation has been entered phase II trial for treatment of pulmonary
hypertension6. In phase I clinical trial, nitrite reached peak concentration time
(Tmax) immediately after inhalation and had half-life (t1/2) of 35 minutes7. The
Address correspondence and reprint request to: Sirada Srihirun Department of Pharmacology,
Faculty of Dentistry, Mahidol University E-mail address: Sirada.srh@mahidol.ac.th.
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maximum tolerated dose was 90 mg. The major adverse effect was transient
asymptomatic orthostasis and no serious adverse effect was reported.
In this study, we aimed to determine the effect of inhaled sodium nitrite ex
vivo at dose of 37.5 mg (almost half of maximum tolerated dose) on platelet
activity in healthy subjects. The levels of platelet activation before and after nitrite
inhalation were measured by aggregometry and flow cytometry.
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Materials and Methods
Sodium nitrite (NaNO2) nebulizer was prepared by Faculty of
Pharmaceutical Science, Chulalongkorn University. Sodium nitrite was
administrated to volunteers by Beurer Inhalator Nebuliser IH25/1 model (Exeter,
NH, USA). Adenosine 5’ diphosphate (ADP) was purchased from Sigma (St Louis,
MO, USA). U46619 were purchased from Calbiochem (Billerica, MA, USA).
Sodium nitrite, ADP and U46619 were dissolved in PBS pH 7.4.
Monoclonal antibodies: Fluorescein isothiocyanate (FITC)-labeled antihuman PAC-1, Phycoerythrin (PE)-labeled anti-human CD62P and Phycoerythrin
cyanine5 (PEcy5)-labeled anti-human CD42b were purchased from BD bioscience
(San Jose, CA, USA).
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Blood sample collection
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The venous blood was collected using 3.8% sodium citrate as an anticoagulant. Whole blood was 2-fold diluted with phosphate buffer saline (PBS) for
platelet aggregation measured by an aggregometer (Chronolog, Havertown, PA,
USA). Whole blood was 10-fold diluted with PBS for measurement of platelet
surface markers by FC500 flow cytometer (Cytomics FC 500, Beckman coulter,
Brea, CA, USA). For measurement of nitrite in blood, the whole blood was mixed
immediately with the nitrite-stabilizing solution containing 0.8 M potassium
hexacyanoferrate (III), 10 mM N-ethylmaleimide (NEM), and 5% nonidet P-40
(NP40) dissolved in nitrite-free deionized water. The preserved samples were
stored at -80 C and the measurement was done within 2 weeks.
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The effect of inhaled nitrite on platelet aggregation
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Citrated (3.8%) whole blood was collected before 37.5-mg nitrite inhalation
(baseline measurement) and immediately after inhalation. Platelet aggregation
induced by 20 M ADP was measured for 6 minutes by impedance aggregometry.
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The effect of inhaled nitrite on expression of platelet surface markers
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Subjects
This study was approved by Ramathibodi Hospital Ethics Committee (ID
03-56-27). Seven healthy volunteers with 31.7±1.6 years of age who signed
informed consent enrolled in the study. Subjects who had history of smoking, heart
disease, hemoglobinopathy, platelet function disorders, asthma, or COPD were
excluded from this study.
Diluted whole blood was incubated with PEcy5-labeled CD42b, FITC-
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labeled PAC-1 and PE-labeled CD62P and platelet agonists either ADP (20 μM) or
U46619 (20 μM) for 10 minutes at room temperature. Then, samples were fixed
with 1% paraformaldehyde and kept at 4 C. The platelet population was identified
by light scatter and anti-CD42b antibody. Percentage of platelets positively
expressed P-selectin and aGPIIb/IIIa were calculated from 10,000 events positive
for CD42b.
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Measurement of nitrite in blood
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The nitrite levels in whole blood were measured by tri-iodide based
chemiluminescence assay. Whole blood samples were mixed with methanol and
centrifuged at 14,000 g for 5 minutes. Supernatant was injected into a purge vessel
containing tri-iodide solution and the nitrite levels were measured by a
chemiluminescence NO analyzer (Eco medics CLD88, Duernten, Switzerland).
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Statistical analysis
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Data processing and statistical analysis were analyzed by GraphPad Prism ®
version 4 (GraphPad software Inc., San Diego, CA, USA). Data represents mean ±
SEM. Wilcoxon matched-pairs signed rank test was used to compare with
acceptable P-value < 0.05.
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Results
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Whole blood nitrite
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Whole blood samples from healthy volunteers were collected before and
immediately after nitrite inhalation. The levels of nitrite were determined by
chemiluminescence NO analyzer. After inhalation of 37.5-mg nitrite, whole blood
nitrite levels increased from 0.15  0.03 to 1.70  0.23 M. (figure 1).
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Figure 1: Nitrite levels in whole blood before and immediately after
inhalation. Whole blood was collected in nitrite-stabilizing solution before and
after inhalation. Tri-iodide based chemiluminescence was used to measure the level
of nitrite in whole blood. Data are mean  SEM (n=7). *P-value < 0.05 (MannWhitney test)
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The effect of inhaled nitrite on platelet aggregation
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The levels of platelet aggregation before and after inhalation were measured
ex vivo by impedance aggregometry. After nitrite inhalation, platelet aggregation
induced by ADP decreased significantly (P = 0.02 by Wilcoxon matched-pairs
signed rank test). Platelet aggregation decreased from 10.73 (7.10 to 11.67) to 9.27
(6.10 to 10.49) ohm; median (interquartile range) (figure 2).
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The effect of inhaled nitrite on P-selectin and aGPIIb/IIIa expression
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P-selectin and aGPIIb/IIIa expression before and after inhalation was
induced by 20 μM ADP and U46619, and determined by flow cytometry. After
inhalation, P-selectin expression induced by ADP significantly decreased (P = 0.02
by Wilcoxon matched-pairs signed rank test). The percentage of ADP-induced Pselectin expression before and after nitrite inhalation was 30.65 (17.68-39.40) and
24.63 (17.17-37.80), respectively; median (interquartile range) (figure 3A).
However, inhalation of nitrite had no effect on ADP-induced aGPIIb/IIIa
expression and U46619-induced P-selectin and aGPIIb/IIIa expression (figure 3BD).
Platelet aggregation (W)
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Before nitrite inhalation
After nitrite inhalation
Figure 2: The effect of inhaled nitrite on platelet aggregation induced by ADP.
Whole blood samples were collected before (baseline) and after nitrite inhalation.
Platelet aggregation in whole blood was induced by 20 μM ADP and measured by
impedance aggregometry. *P < 0.05 tested by Wilcoxon matched-pairs signed rank
test (n = 7).
Discussion
Our results demonstrated the inhibitory effect of inhaled nitrite on platelet
activity ex vivo. The aggregometry showed that inhaled nitrite reduced platelet
aggregation induced by ADP. In addition, the expression of P-selectin induced by
ADP decreased after nitrite inhalation. However, the expressions of aGPIIb/IIIa
induced by ADP, P-selectin and aGPIIb/IIIa induced by U46619 were not changed.
Inhalation of 37.5-mg nitrite increased whole blood nitrite to the maximum
concentration of 1.7 M, which was lower than the level reported elsewhere
(estimated plasma nitrite of 4.5 uM at the same dose)7. This may result from the
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efficiency of nebulizer. However, the plasma nitrite of 0.4-0.6 μM was shown to
decrease platelet activation8.
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Figure 3: The effect of inhaled nitrite on P-selectin and aGPIIb/IIIa
expression. Platelets in 10-fold diluted whole blood samples were stimulated by 20
μM ADP or U46619. ADP-induced P-selectin expression (A), ADP-induced
aGPIIb/IIIa expression (B), U46619-induced P-selectin expression, (C) and
U46619-induced aGPIIb/IIIa expression (D) were measured by flow cytometry. *P
< 0.05 tested by Wilcoxon matched-pairs signed rank test (n = 7).
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Our ex vivo study demonstrated that nitrite inhalation decreased ADPinduced platelet aggregation and P-selectin expression, which is consistent with the
previous in vitro anti-platelet effect of nitrite reported by our group5,9. On the
contrary, inhaled nitrite could not inhibit ADP-induced aGPIIb/IIIa expression. Our
results showed that ADP induced almost 100% of aGPIIb/IIIa expression. It is
possible that the nitrite level in this study may not be enough to inhibit the effect of
ADP on aGPIIb/IIIa expression. An ex vivo study showed that 65 g/ml aspirin has
less inhibitory effect on the expression of aGPIIb/IIIa than the inhibitory effect on
the expression of P-selectin (36.5% versus 81%)10. Therefore, aGPIIb/IIIa
expression may be more difficult to inhibit than P-selectin. However, inhaled nitrite
had no effect on U46619-induced P-selectin and aGPIIb/IIIa expression with
unknown reason. Therefore, these results should be further confirmed in larger
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number of subjects.
The anti-platelet effect of nitrite may arise from nitrite reduction to NO.
Heme-containing proteins such as deoxyhemoglobin may be responsible for nitrite
reduction to NO11. cGMP, a marker of NO, in platelets increased after incubation
with nitrite in vitro12. Therefore, the anti-platelet mechanism of inhaled nitrite ex
vivo needs further investigation.
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Acknowledgements
The authors would like to thank the volunteers who participated in this
research. We thank Miss Thanaporn Sriwantana, Mr. Piyadon Sathavorasmith,
Miss Jirada Kaewchuchuen and Miss Porntip Tomong for blood sample collection.
We thank Prof. Garnpimon Ritthidej for preparation of nitrite suspension for
inhalation and Department of Molecular Tropical Medicine and Genetics, Faculty
of Tropical Medicine, Mahidol University for the use of flow cytometry
instrument. This work was supported by National Research Council of Thailand
and DPST Research grant 024/2557.
Conclusion
Inhaled nitrite decreases platelet aggregation and P-selectin expression
induced by ADP ex vivo. The results support the idea that inhaled nitrite may have
potential benefit for treat patients with platelet hyperaggregation. However, the
effect of inhaled nitrite on platelets needs further studies in a larger group of
population.
References
1. Samama CM, Diaby M, Fellahi JL, Mdhafar A, Eyraud D, Arock M, et al.
Inhibition of platelet aggregation by inhaled nitric oxide in patients with acute
respiratory distress syndrome. Anesthesiology. 1995;83(1):56-65.
2. George TN, Johnson KJ, Bates JN, Segar JL. The effect of inhaled nitric oxide
therapy on bleeding time and platelet aggregation in neonates. The Journal of
pediatrics. 1998;132(4):731-4.
3. Irwin C, Roberts W, Naseem KM. Nitric oxide inhibits platelet adhesion to
collagen through cGMP-dependent and independent mechanisms: the potential
role for S-nitrosylation. Platelets. 2009;20(7):478-86.
4. Kim-Shapiro DB, Gladwin MT. Mechanisms of nitrite bioactivation. Nitric
oxide : biology and chemistry / official journal of the Nitric Oxide Society.
2014;38:58-68.
5. Srihirun S, Sriwantana T, Unchern S, Kittikool D, Noulsri E, Pattanapanyasat K,
et al. Platelet inhibition by nitrite is dependent on erythrocytes and
deoxygenation. PloS one. 2012;7(1):e30380.
6. A service of the U.S. National Institute of Health [internet}. 2015 [cited 2015
July 26]. Available from: http://clinicaltrails.gov
7. Rix PJ, Vick A, Attkins NJ, Barker GE, Bott AW, Alcorn H, Jr., et al.
Pharmacokinetics, pharmacodynamics, safety, and tolerability of nebulized
sodium nitrite (AIR001) following repeat-dose inhalation in healthy subjects.
Clinical pharmacokinetics. 2015;54(3):261-72.
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8. Webb AJ, Patel N, Loukogeorgakis S, Okorie M, Aboud Z, Misra S, et al. Acute
blood pressure lowering, vasoprotective and anti-platelet properties of dietary
nitrate via bioconversion to nitrite. Hypertension. 2008;51(3):784-90
9. Akrawinthawong K, Park JW, Piknova B, Sibmooh N, Fucharoen S, Schechter
AN. A Flow Cytometric Analysis of the Inhibition of Platelet Reactivity Due to
Nitrite Reduction by Deoxygenated Erythrocytes. PloS one. 2014;9(3)
10. Le Guyader A, Pacheco G, Seaver N, Davis-Gorman G, Copeland J,
McDonagh PF. Inhibition of platelet GPIIb-IIIa and P-selectin expression by
aspirin is impaired by stress hyperglycemia. Journal of diabetes and its
complications. 2009;23(1):65-70.
11. Kim-Shapiro DB, Gladwin MT. Mechanisms of nitrite bioactivation. Nitric
oxide : biology and chemistry / official journal of the Nitric Oxide Society.
2014;38:58-68.
12. Borgognone A, Loka T, Chimen M, Rainger E, Feelisch M, Watson S, et al.
Nitrite is a cGMP generator in isolated platelets. BMC Pharmacology and
Toxicology. 2015;16(Suppl 1):A65.