From the Department of Anesthesiology and Department of Cardiology (C.B.,
K.P.), University of Heidelberg, Germany.
Correspondence to André Gries, MD, Department of Anesthesiology, University of Heidelberg, Im Neuenheimer Feld 110, D-69120 Heidelberg, Germany. E-mail andre_gries{at}krzmail.krz.uni-heidelberg.de
Methods and Results—In 30 healthy volunteers, platelet-rich
plasma was incubated with an air/5% CO2 mixture containing
0, 100, 450, and 884 ppm inhaled NO. ADP- and collagen-induced
platelet aggregation, the membrane expression of P-selectin, and
the binding of fibrinogen to the platelet glycoprotein
(GP) IIb/IIIa receptor were determined before (t0) and
during the 240 minutes of incubation. In addition, eight patients
suffering from severe adult respiratory distress syndrome (ARDS) were
investigated before and 120 minutes after the beginning of
administration of 10 ppm inhaled NO. In vitro, NO led to a
dose-dependent inhibition of both ADP-induced (3±3% at 884 ppm versus
70±6% at 0 ppm after 240 minutes; P<.001) and
collagen-induced (13±5% versus 62±5%; P<.01)
platelet aggregation. Furthermore, P-selectin expression (36±7%
of t0 value; P<.01) and fibrinogen binding
(33±11%; P<.01) were inhibited. In patients with
ARDS, after two who did not respond to NO inhalation with an
improvement in oxygenation had been excluded, an
increase in plasma cGMP, prolongation of in vitro bleeding time, and
inhibition of platelet aggregation and P-selectin expression were
observed, and fibrinogen binding was also inhibited (19±7% versus
30±8%; P<.05).
Conclusions—NO-dependent inhibition of platelet aggregation
may be caused by a decrease in fibrinogen binding to the platelet
GP IIb/IIIa receptor.
Although several NO-releasing compounds have been investigated and the
platelet inhibitory effect has been shown to be dose
dependent in vitro and in vivo,12 13 14 15 16 it is
currently unknown whether the inhibitory effect of inhaled
NO on platelet function is also dose related. Furthermore, the
mechanisms by which inhaled NO may affect platelet function are not
completely understood. Inhibition of fibrinogen binding to the
platelet membrane via an increase in intracellular cGMP
concentration has been postulated to be responsible for the inhibition
of platelet adhesion and aggregation.2 3 17
However, fibrinogen binding to human platelets during NO inhalation
has not been investigated in detail. The present study was
performed to investigate the dose dependency of platelet inhibition
in vitro and in patients with ARDS by use of platelet aggregation
studies, determination of in vitro bleeding time, and flow cytometry to
elucidate the mechanism by which inhaled NO affects platelet
function.
Ten minutes after gas administration was begun, 0, 100, 450, and 884
ppm NO were added for 240 minutes. NO was supplied by Messer Griesheim
at an original gas concentration of 884 ppm NO in
N2. Gas samples for measurement of the
concentrations of NO and NO2 were taken
continuously at the inlet and outlet tubes and analyzed by
chemiluminescence
(NO/NO2/NOx
analyzer CLD 700 AL, Zellweger-Ecco).
Determination of Platelet Aggregation
Determination of P-Selectin Expression and Fibrinogen
Binding
NO Gas in Older Volunteers
50 ppm NO In Vitro
Inhaled NO in Patients With ARDS
All patients were sedated with intravenous midazolam
(Dormicum; Hoffmann-LaRoche) and fentanyl (Janssen) and mechanically
ventilated to ensure an arterial carbon dioxide tension
(PaCO2) between 40 and 50 mm Hg
(EVITA 2, Dräger). In all patients, hemodynamic
measurements were performed with a pulmonary artery catheter
(93–631-5.5F, Baxter Healthcare Corp) and a radial
arterial catheter (20 gauge; Abbocath-T, Abbott), and data
were continuously displayed on a multichannel oscillograph (Vicom
SMU-612, Hellige). After the patients had been selected, 10 ppm inhaled
NO (Messer Griesheim) were administered with a commercially supplied
administration unit connected to the ventilator (NOdomo, Dräger).
Inspiratory and expiratory concentrations of NO and
NO2 were analyzed continuously at the
proximal end of the endotracheal tube with the
NO/NO2/NOx analyzer
(Zellweger-Ecco).
To analyze platelet function, the first 10 mL of
arterial blood was discarded, and thereafter samples were
carefully withdrawn into plastic tubes (Monovette) before
(t0) and 120 minutes after
(t120) NO administration was initiated.
Determination of In Vitro Bleeding Time
Determination of Platelet Aggregation
Determination of P-Selectin Expression and Fibrinogen
Binding
Measurement of Platelet Count, White Blood Cell Count, and
Plasma cGMP
Statistical Analysis
In vitro administration of 100 ppm NO led to a significant inhibition
of P-selectin expression at t240 (76±12% versus
t0 value, P<.05). During incubation
with 884 ppm NO, P-selectin expression was inhibited at
t60, t120,
t180, and t240 (77±12%,
P<.05; 73±13%, P<.05; 67±23%,
P<.01; and 36±7%, P<.01, respectively) (Fig 4
NO-induced dose-related inhibition of platelet aggregation,
P-selectin expression, and fibrinogen binding was also observed in the
older study population (Table 1
Incubation with 50 ppm NO also led to a significant inhibition of
ADP-induced platelet aggregation in vitro. In addition, a slight
inhibition in P-selectin expression and a significant inhibition in
fibrinogen binding were observed (Table 2
Inhaled NO in Patients With ARDS
In addition to their vasodilatory properties, systemically administered
NO-releasing compounds have been shown to inhibit platelet function
in vitro and in vivo,1 3 24 and their
antithrombotic effect is proposed to be
beneficial.25 The inhibition of platelet
function may be explained by an activation of the soluble
guanylate cyclase in the platelet cytosol, leading to
an increase in cGMP levels.17 Increased cGMP
levels in platelets can induce a decrease in intracellular calcium
ion concentration, which may contribute to an inhibition of fibrinogen
binding to the GP IIb/IIIa receptor on the surface of the platelet
membrane, which mediates platelet
aggregation.2 3 17 26 Furthermore, an increase in
intracellular cGMP levels at the early stage of aggregation has been
proposed, which could be further enhanced by relatively low
concentrations of NO and finally lead to platelet
disaggregation.14 In the present study, we
were not able to determine intracellular cGMP levels. However, NO
therapy in patients with ARDS led to a significant increase in plasma
cGMP levels, which have also been demonstrated to reflect NO-induced
guanylate cyclase activation in
vivo.27 28 29
The inhibition of platelet aggregation was reported to be dependent
on the NO concentration in vitro and in healthy
volunteers.12 13 14 15 16 17 Moreover, the antiplatelet
effect was persistent despite a hemodynamic tolerance
during nitroglycerin therapy in
pigs.30 Conversely, bleeding time was shortened
after systemic inhibition of NO
production.31 The results of the
present study are in accordance with these findings, demonstrating
a dose-dependent inhibition of both ADP- and collagen-induced
platelet aggregation during NO administration in vitro. In
comparison, in another study, 200 and 400 ppm inhaled NO were found to
significantly inhibit ADP-induced maximal platelet
aggregation.32 When collagen was used to induce
platelet aggregation, however, this inhibition was observed later
and to a lesser extent, as also reported by other
investigators.10 33 34 This may be explained by
the relatively high collagen concentration that was used in the
aggregation studies. Lower concentrations of collagen or the use of
epinephrine, which led to good results in the in vitro bleeding
time measurements, might have revealed a comparable platelet
inhibitory effect.
In the present study, inhibition of ADP-induced platelet
aggregation and prolongation of in vitro bleeding time were also
observed during inhalation of 10 ppm NO in vivo. A prolongation of
bleeding time during NO administration was observed in
rabbits,8 whereas in rats, no change in bleeding
time was observed at 80 ppm NO.35 In healthy
volunteers, 30 ppm inhaled NO also prolonged bleeding
time9 without an effect on filter aggregometry
findings.28 Other investigators observed no
effect of inhaled NO either on ß-thromboglobulin
or on thromboxane B2
levels.36 Recently, it was shown that 3 to 30 ppm
inhaled NO inhibits platelet aggregation without prolonging
bleeding time in patients with ARDS.10 In
addition, platelet activation was abolished during NO
administration in pigs after acute pulmonary
embolism.11 In accordance with our findings, most
of these studies demonstrated inhaled NO to have an
inhibitory effect on platelet function. Different
findings with regard to bleeding time and platelet aggregation may
be explained by the different methods and study populations that were
used10 11 18 23 28 and are therefore not
necessarily contradictory.
In contrast to the in vitro findings, we cannot say whether the
inhibition of platelet function by inhaled NO is also dose
dependent in vivo, because only one NO concentration was administered.
The maximal inhibition of platelet aggregation was already achieved
at 3 ppm NO during inhalation of 1 to 100 ppm NO in a comparable study
population.10 In healthy volunteers inhaling 30
ppm NO, no further prolongation of bleeding time was observed at 80
ppm.28 In comparison with these studies, no
definitive dose-related effect of inhaled NO on platelet function
could be found in animals,8 11 and further
studies are necessary to investigate a dose relation in a larger study
population.
In activated platelets, P-selectin is translocated to the
platelet surface from
A decrease in platelet fibrinogen binding was observed after
administration of various NO-releasing compounds in vitro and in
vivo.26 43 44 Furthermore, the increase in GP
IIb/IIIa receptor expression was abolished during administration of
S-nitrosoglutathione and nitroglycerin in
patients with unstable angina and acute myocardial
infarction21 and during
percutaneous transluminal coronary
angioplasty.38 The results of the present
study are comparable to these findings, because the binding of
fibrinogen to the GP IIb/IIIa receptor was inhibited in vitro and
during NO inhalation in patients with ARDS. As with the results of the
aggregation studies, this inhibition was dose dependent in vitro.
Moreover, in the present study, a significant correlation between
aggregation ratio and fibrinogen-binding ratio was found, suggesting
that the prolongation of the in vitro bleeding time and the inhibition
of platelet aggregation are most likely due to the inhibition of
fibrinogen binding to the platelet surface GP IIb/IIIa, which is
required for platelet adhesion and
aggregation.2 3 Because we were not able to
measure the amounts of GP IIb/IIIa receptors on
platelets,45 we cannot say whether inhaled NO
influences the structure or the number of expressed GP IIb/IIIa
receptors or both. Nevertheless, recent studies suggest that NO may
inhibit the number of expressed GP IIb/IIIa receptors in vitro and in
vivo.21 38 46 47 Thus, the expression of GP
IIb/IIIa receptors may also be influenced by inhaled NO, which may
contribute, at least in part, to the inhibition of fibrinogen binding,
as demonstrated in the present study.
Although the in vitro model seems to be useful because it was possible
to obtain mechanical platelet activation by means of gas
bubbles48 and a filter membrane was used to
imitate the alveolar membrane, the in vitro findings cannot be
completely extrapolated to the clinical setting. In the present
study, relatively high concentrations of NO were used in vitro.
Nevertheless, our results seem to be relevant, because a significant
inhibition of platelet aggregation and fibrinogen binding was
already observed at 50 and 100 ppm, concentrations that have been used
in patients.7 10 Furthermore, PRP was used,
whereas in the lung, NO comes into contact with hemoglobin, which is
known to inhibit its effects.5 7 On the other
hand, the inhibitory effect on platelet function may be
more pronounced in the pulmonary vessels, where inhaled NO may
improve the microcirculation, because NO inhalation decreased
platelet sequestration in the lungs during extracorporeal
circulation in pigs.41 In the present study,
however, the inhibition of platelet aggregation and fibrinogen
binding in vivo supports the in vitro findings.
In conclusion, the results from the present study provide new
evidence of an inhibitory effect of inhaled NO on
platelet function. In fact, the inhibition of platelet adhesion
and aggregation seems to be mediated via an inhibition of fibrinogen
binding to the platelet membrane. The antiaggregatory effect could
be beneficial: in a canine model of platelet-mediated reocclusion
after thrombolysis, inhaled NO improved the
coronary artery patency ratio,32 and
platelet activation was inhibited after pulmonary embolism
in pigs.11
Because of its positive effect on hemodynamics and its
antiplatelet action, the administration of inhaled NO in critically
ill patients may be a beneficial and attractive adjunct to
anticoagulation therapy. The present study provides a rational
basis for investigating the efficacy of inhaled NO as an
antiplatelet agent in further clinical studies.
Received August 29, 1997;
revision received October 28, 1997;
accepted December 5, 1997.
2.
Ivanova K, Schaefer M, Drummer C, Gerzer R. Effects of
nitric oxide-containing compounds on increases in cytosolic ionized
Ca2+ and on aggregation of human platelets.
Eur J Pharmacol. 1993;244:37–47.[Medline]
[Order article via Infotrieve]
3.
Loscalzo J. Antiplatelet and antithrombotic
effects of organic nitrates. Am J Cardiol. 1992;70:18B–22B.[Medline]
[Order article via Infotrieve]
4.
Roberts JD, Polaner DM, Lang P, Zapol WM. Inhaled
nitric oxide in persistent pulmonary hypertension of the
newborn. Lancet. 1992;340:818–819.[Medline]
[Order article via Infotrieve]
5.
Rossaint R, Falke KJ, Lopez F, Slama K, Pison U, Zapol
WM. Inhaled nitric oxide in adult respiratory distress syndrome.
N Engl J Med. 1993;328:399–405.
6.
Sellden H, Winberg P, Gustaffson LE, Lundell B,
Böök K, Frostell CG. Inhalation of nitric oxide reduced
pulmonary hypertension after cardiac surgery in a 3.2 kg
infant. Anesthesiology. 1993;78:577–580.[Medline]
[Order article via Infotrieve]
7.
Pearl RG. Inhaled nitric oxide: the past, the
present, and the future. Anesthesiology. 1993;78:413–416.[Medline]
[Order article via Infotrieve]
8.
Högman 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]
9.
Högman M, Frostell C, Arnberg H, Hedenstierna G.
Bleeding time prolongation and NO inhalation. Lancet. 1993;341:1664–1665. Letter.[Medline]
[Order article via Infotrieve]
10.
Samama CM, Diaby M, Fellahi JL, Mdhafar A, Eyraud D,
Arock M, Guillosson JJ, Coriat P, Rouby JJ. Inhibition of
platelet aggregation by inhaled nitric oxide in patients with acute
respiratory distress syndrome. Anesthesiology. 1995;83:56–65.[Medline]
[Order article via Infotrieve]
11.
Gries A, Böttiger BW, Dörsam J, Bauer H,
Weimann J, Bode C, Martin E, Motsch J. Inhaled nitric oxide
inhibits platelet aggregation following pulmonary embolism
in pigs. Anesthesiology. 1997;86:387–393.[Medline]
[Order article via Infotrieve]
12.
Radomski MW, Palmer RMJ, Moncada S. Comparative
pharmacology of endothelium-derived relaxing factor,
nitric oxide and prostacyclin in platelets. Br J
Pharmacol. 1987;92:181–187.[Medline]
[Order article via Infotrieve]
13.
Amano M, Takahashi M, Kosaka T, Kinoshita M.
Differential inhibition of platelet aggregation and calcium
mobilization by nitroglycerin and stabilized nitric
oxide. J Cardiovasc Pharmacol. 1994;24:860–866.[Medline]
[Order article via Infotrieve]
14.
Chirkov YY, Naujalis JI, Barber S, Sage RE, Gove DW,
Brealey JK, Horowitz JD. Reversal of human platelet aggregation by
low concentrations of nitroglycerin in vitro in normal
subjects. Am J Cardiol. 1992;70:802–806.[Medline]
[Order article via Infotrieve]
15.
Karlberg KE, Torfgard K, Ahlner J, Sylvén C.
Dose-dependent effect of intravenous
nitroglycerin on platelet aggregation, and
correlation with plasma glyceryl dinitrate concentration in healthy
men. Am J Cardiol. 1992;69:802–805.[Medline]
[Order article via Infotrieve]
16.
Harris SN, Rinder CS, Rinder HM, Tracey JB, Smith BR,
Hines R. Nitroprusside inhibition of platelet function is transient
and reversible by catecholamine priming.
Anesthesiology. 1995;83:1145–1152.[Medline]
[Order article via Infotrieve]
17.
Bassenge E. Antiplatelet effects of
endothelium-derived relaxing factor and nitric oxide
donors. Eur Heart J. 1991;12(suppl E):12–15.
18.
Born GVR. Aggregation of blood platelets by
adenosine diphosphate and its reversal. Nature. 1962;194:927–929.[Medline]
[Order article via Infotrieve]
19.
Gawaz MP, Dobos G, Späth M, Schollmeyer P,
Gurland HJ, Mujais SK. Impaired function of platelet membrane
glycoprotein IIb-IIIa in end stage renal disease.
J Am Soc Nephrol. 1994;5:36–46.[Abstract]
20.
Kestin AS, Ellis PA, Barnard MR, Errichetti A, Rosner
BA, Michelson AD. Effect of strenuous exercise on platelet
activation state and reactivity. Circulation. 1993;88:1502–1511.
21.
Langford EJ, Wainwright RJ, Martin JF. Platelet
activation in acute myocardial infarction and unstable angina is
inhibited by nitric oxide donors. Arterioscler Thromb Vasc
Biol. 1996;16:51–55.
22.
Bernard GR, Artigas A, Brigham KL, Carlet J, Falke K,
Hudson L, Lamy M, Legall JR, Morris A, Spragg R, and the Consensus
Committee. The American-European Consensus Conference on ARDS:
definitions, mechanisms, relevant outcomes, and clinical trial
coordination. Am J Respir Crit Care Med. 1994;149:818–824.[Abstract]
23.
Kratzer MAA, Born GVR. Simulation of primary hemostasis
in vitro. Hemostasis. 1985;15:357–362.
24.
Moncada S, Palmer MJ, Higgs EA. Nitric oxide:
physiology, pathophysiology, and pharmacology. Pharmacol
Rev. 1991;43:108–142.
25.
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]
26.
Michelson AD, Benoit SE, Furman MI, Breckwoldt WL,
Rohrer MJ, Barnard MR, Loscalzo J. Effects of nitric oxide/EDRF on
platelet surface glycoproteins. Am J
Physiol. 1996;270:H1640–H1648.
27.
Rovira I, Chen TY, Winkler M, Kawai N, Bloch KD, Zapol
WM. Effects of inhaled nitric oxide on pulmonary
hemodynamics and gas exchange in an ovine model of
ARDS. J Appl Physiol. 1994;76:345–355.
28.
Albert J, Wallen NH, Broijersen A, Frostell C, Hjemdahl
P. Effects of inhaled nitric oxide compared with aspirin on
platelet function in vivo in healthy subjects. Clin Sci
(Colch). 1996;91:225–231.[Medline]
[Order article via Infotrieve]
29.
Roberts JD Jr, Chen TY, Kawai N, Wain J, Dupuy P,
Shimouchi A, Bloch K, Polaner D, Zapol WM. Inhaled nitric oxide
reverses pulmonary vasoconstriction in the hypoxic and acidotic
newborn lamb. Circ Res. 1993;72:246–254.
30.
Hébert D, Xiang JX, Lam JYT. Persistent
inhibition of platelets during continuous
nitroglycerin therapy despite
hemodynamic tolerance. Circulation. 1997;95:1308–1313.
31.
Simon DI, Stamler JS, Loh E, Loscalzo J, Francis SA,
Creager MA. Effect of nitric oxide synthase inhibition on bleeding time
in humans. J Cardiovasc Pharmacol. 1995;26:339–342.[Medline]
[Order article via Infotrieve]
32.
Adrie C, Bloch KD, Moreno PR, Hurford WE, Guerrero JL,
Holt R, Zapol WM, Gold HK, Semigran MJ. Inhaled nitric oxide
increases coronary artery patency after
thrombolysis. Circulation. 1996;94:1919–1926.
33.
Vallance P, Benjamin N, Collier J. The effect of
endothelium-derived nitric oxide on ex vivo whole blood
aggregation in man. Eur J Clin Pharmacol. 1992;42:37–41.[Medline]
[Order article via Infotrieve]
34.
Shernan S, Fox J, Body S, Morse D, Magnami B. Inhaled
nitric oxide does not inhibit platelets in healthy human
volunteers. Anesthesiology. 1996;85:A139. Abstract.
35.
Lee JS, Adrie C, Jacob HJ, Roberts JD Jr, Zapol WM,
Bloch KD. Chronic inhalation of nitric oxide inhibits
neointimal formation after balloon-induced
arterial injury. Circ Res. 1996;78:337–342.
36.
Krejcy K, Schmetterer L, Kastner J, Nieszpaur-Los M,
Monitzer B, Schutz W, Eichler HG, Kyrle PA. Role of nitric oxide in
hemostatic system activation in vivo in humans. Arterioscler
Thromb Vasc Biol. 1995;15:2063–2067.
37.
Murohara T, Parkinson SJ, Waldmann SA, Lefer AM.
Inhibition of nitric oxide biosynthesis promotes P-selectin expression
in platelets: role of protein kinase C. Arterioscler Thromb
Vasc Biol. 1995;15:2068–2075.
38.
Langford EJ, Brown AS, Wainwright RJ, de Belder AJ,
Thomas MR, Smith REA, Martin JF, Moncada S. Inhibition of
platelet activity by S-nitrosoglutathione during coronary
angioplasty. Lancet. 1994;344:1458–1460.[Medline]
[Order article via Infotrieve]
39.
Lees C, Langford E, Brown AS, de Belder A, Pickles A,
Martin JF, Campbell G. The effects of S-nitrosoglutathione on
platelet activation, hypertension, and uterine and fetal
Doppler in severe preeclampsia. Obstet Gynecol. 1996;88:14–19.[Medline]
[Order article via Infotrieve]
40.
Davenpeck KL, Gauthier TW, Lefer AM. Inhibition of
endothelial-derived nitric oxide promotes P-selectin
expression and actions in the rat microcirculation.
Gastroenterology. 1994;107:1050–1058.[Medline]
[Order article via Infotrieve]
41.
Malmros C, Blomquist S, Dahm P, Martensson L,
Thörne J. Nitric oxide inhalation decreases pulmonary
platelet and neutrophil sequestration during extracorporeal
circulation in the pig. Crit Care Med. 1996;24:845–849.[Medline]
[Order article via Infotrieve]
42.
Bacha EA, Herve P, Murakami S, Chapelier A, Mazmanian
GM, de Montpreville V, Detruit H, Libert JM, Dartevelle P. Lasting
beneficial effect of short-term inhaled nitric oxide on graft function
after lung transplantation. J Thorac Cardiovasc Surg. 1996;112:590–598.
43.
Salas E, Moro MA, Askew S, Hodson HF, Butler AR,
Radomski MW, Moncada S. Comparative pharmacology of analogues of
S-nitroso-N-acetyl-DL-penicillamine on human platelets.
Br J Pharmacol. 1994;112:1071–1076.[Medline]
[Order article via Infotrieve]
44.
Simon DI, Stamler JS, Jaraki O, Keaney JF, Osborne JA,
Francis SA, Singel DJ, Loscalzo J. Antiplatelet properties of
protein S-nitrosothiols derived from nitric oxide and
endothelium-derived relaxing factor. Arterioscler
Thromb. 1993;13:791–799.
45.
Wagner CL, Mascelli MA, Neblock DS, Weisman HF, Coller
BS, Jordan RE. Analysis of GPIIb/IIIa receptor number by
quantification of 7E3 binding to human platelets. Blood. 1996;88:907–914.
46.
Lechi C, Andrioli G, Gaino S, Tommasoli R, Zuliani V,
Ortolani R, Degan M, Benoni G, Bellavite P, Lechi A, Minuz P. The
antiplatelet effect of a new nitroderivate of acetylic acid: an in
vitro study of inhibition on the early phase of platelet activation
and on TXA2 production. Thromb Haemost. 1996;76:791–798.[Medline]
[Order article via Infotrieve]
47.
Keh D, Gerlach M, Kurer I, Seiler S, Kerner T, Falke
KJ, Gerlach H. The effects of nitric oxide (NO) on platelet
membrane receptor expression during activation with human
alpha-thrombin. Blood Coagul Fibrinolysis. 1996;7:615–624.[Medline]
[Order article via Infotrieve]
48.
Pickles DM, Ogston D, MacDonald AG. Effects of gas
bubbling and other forms of convection on platelets in vitro.
J Appl Physiol. 1989;67:1250–1255.
© 1998 American Heart Association, Inc.
Clinical Investigation and Reports
Inhaled Nitric Oxide Inhibits Human Platelet Aggregation, P-Selectin Expression, and Fibrinogen Binding In Vitro and In Vivo
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Background—Recent data suggest
that inhaled NO can inhibit platelet aggregation. This study
investigates whether inhaled NO affects the expression level and
avidity of platelet membrane receptors that mediate platelet
adhesion and aggregation.
Key Words: platelets • P-selectin • fibrinogen • nitric oxide • respiration
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
Nitric oxide
has been found to relax vascular smooth muscle and to inhibit
platelet adhesion and platelet aggregation in
vitro.1 2 3 Inhaled NO decreases elevated
pulmonary arterial pressure and pulmonary
vascular resistance and improves oxygenation. This
principle has been used successfully to treat ARDS, persistent
pulmonary hypertension of the newborn, and pulmonary
hypertension in patients who have undergone cardiac
surgery.4 5 6 Despite rapid inactivation of
inhaled NO by hemoglobin and high selectivity for the pulmonary
vascular system,5 7 recent data suggest that
inhaled NO also has systemic effects on hemostasis by inhibiting
platelet function. During NO inhalation, bleeding time was
prolonged in animals and in healthy volunteers, and platelet
aggregation was inhibited in patients with
ARDS.8 9 10 In addition, inhibition of
platelet aggregation was also observed during NO inhalation after
acute massive pulmonary embolism in
pigs.11
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
NO Gas In Vitro
Platelet Preparation and NO Administration
After approval from the ethics committee and informed consent
from the study subjects had been obtained, venous blood was drawn from
an antecubital vein in 30 healthy volunteers (26±5 years old) who had
taken no drugs that could affect platelet function during the
previous 14 days. After the first 10 mL had been discarded, samples
were carefully drawn into plastic tubes containing 3.8% sodium citrate
(Monovette, Sarstedt). Samples were centrifuged immediately at
150g for 10 minutes to obtain PRP and subsequently at
2500g for 10 minutes to obtain platelet-poor plasma. The
platelet concentration was standardized to 300/nL by addition of
platelet-poor plasma to PRP. Aliquots of 10 mL PRP were put into a
tube consisting of a dialysis membrane (Spectra/Por, Spectrum) with a
molecular weight cutoff at 1000 daltons. A central venous catheter
(Certo 358, Braun) was placed into the tube to allow PRP sampling
during the study period. The tube was fixed in a larger glass container
filled with Tyrode's solution. Temperature was maintained at 37°C in
a water bath. An air/oxygen mixture
(FiO2=0.3) with 5%
CO2 was administered to the buffer at a flow rate
of 1.5 L/min. The indirect method of administering gas to PRP was
chosen to prevent mechanical platelet activation by gas bubbles
(Fig 1
).
View larger version (48K):
[in a new window]
Figure 1. Experimental setting. Tube containing PRP (A)
consists of a dialysis membrane with a molecular weight cutoff at 1000
daltons. It is fixed in a larger, gas-light container (B) filled with
Tyrode's solution. A central venous catheter (C) was used to take PRP
samples during study. Temperature was maintained with a warming water
bath (D). Air/oxygen/carbon dioxide/NO mixture was administered to
buffer via inlet tube (E). Gas samples from E and from outlet tube (F)
were taken to determine NO concentrations.
To determine platelet aggregation, PRP samples were
carefully withdrawn from the central venous catheter at the beginning
(t0) and after 20 (t20), 40
(t40), and every 60 minutes of NO administration
(t60, t120,
t180, and t240) for an
overall time of 240 minutes. ADP-induced (Mölab; FC, 5
µmol/L) and collagen-induced (Mölab; FC, 0.19 mg/mL)
platelet aggregation were measured in a four-channel platelet
amplifier (PAP-4, Biodata Corp) at 37°C according to the method
described by Born.18 Each test was carried out in
duplicate, and the mean value of both measurements was recorded.
Aggregation was quantified by measurement of the maximal extent of
light transmission (maximal aggregation, measured as percentage).
To determine P-selectin expression and fibrinogen binding to the
platelet GP IIb/IIIa receptor, additional PRP samples were
carefully taken from the tube containing PRP at
t0, t60,
t120, t180, and
t240 during administration of 0, 100, and 884 ppm
NO. There were no centrifugation or vortexing steps, so
that in vitro platelet activation was avoided. To determine
P-selectin expression, PRP was diluted in HEPES solution, and to
measure fibrinogen binding, in Tyrode's buffer (Roth). P-selectin and
fibrinogen were measured after stimulation with ADP (Mölab, FC,
2 µmol/L). The samples were incubated with the specific
antibodies CD62P CloneAC1.2 (Becton Dickinson) to determine P-selectin
expression and anti-human fibrinogen FITC-conjugated chicken antibody
(Biopool) to determine fibrinogen binding for 20 minutes in the dark at
22°C. After fixation with Cellfix (Becton Dickinson), the samples
were measured in a flow cytometer within 6 hours. The platelet
population was analyzed at a low flow rate and identified on
the basis of forward and sideways scatter characteristics. For each
sample, 10 000 platelets were collected. Data were
analyzed with LYSIS II software (FACScan, Becton
Dickinson).19 20 21
Because of the age imbalance between the normal volunteers in
the in vitro study and the ARDS patients, PRP from five additional
volunteers (55 to 65 years old) was incubated with 0, 100, and 884 ppm
NO. Platelet aggregation, P-selectin expression, and fibrinogen
binding were investigated as described above.
The NO concentrations used in the in vitro investigation were
relatively high (100 to 884 ppm). To investigate a lower NO
concentration, PRP from five additional volunteers was incubated with
50 ppm for 240 minutes. Platelet aggregation, P-selectin
expression, and fibrinogen binding were investigated at
t180 and t240 as described
above.
Patients and NO Administration
After approval by the ethics committee and informed consent from
the relatives had been obtained, eight surgical intensive care patients
(64±4 years old; seven men and one woman) diagnosed with ARDS who
fulfilled the criteria of ARDS according to the consensus
conference22 were investigated. Two patients had
been admitted with multiple trauma, four had undergone coronary
revascularization, major vascular surgery had been
performed in one, and one had developed ARDS after major abdominal
surgery. Exclusion criteria were suspected or confirmed intracranial
hemorrhage, leukopenia, and a previous history of severe
chronic kidney, liver, or lung disease.
With a platelet function analyzer (PFA 100, Dade),
the in vitro bleeding time was determined according to the following
method, described by Kratzer and Born23 : At a
constant negative pressure, samples of citrated whole blood (800 µL)
are suctioned through a small capillary and a filter membrane with a
diameter of 150 µm. The filter membrane is covered with collagen
and soaked with epinephrine. During movement through the
capillary, platelets adhere and aggregate at the filter membrane,
diminishing blood flow until it stops. The total time in seconds of the
blood flow is called in vitro bleeding time and is measured
electronically.
As described above, whole blood (8 mL) was centrifuged
at 500g for 5 minutes and subsequently at 2500g
for 10 minutes to prepare PRP, and ADP-induced platelet aggregation
(FC, 5 µmol/L) was measured. Because of the short half-life of
NO and its rapid inactivation after contact with hemoglobin, all
samples were centrifuged immediately and measured within 30
minutes after sampling.
To determine P-selectin expression and fibrinogen binding to the
platelet membrane in patients during NO inhalation, whole-blood
samples (20 µL) were measured as described above. In these patients,
P-selectin expression was determined without (basal P-selectin) and
after activation with ADP (FC, 2 µmol/L).
Platelet count and white blood cell count were measured with
a Coulter Counter STKS (Coulter Electronics). Arterial
plasma samples that had been stored deep frozen (-80°C) until
measurement were used to determine plasma cGMP levels with a
commercially supplied enzyme immunoassay used according to the
manufacturer's guidelines (Immunodiagnostik).
All results are given as mean±SEM. In addition, the values are
also given as a percentage of the baseline value
(t0, given as 100%) to demonstrate the relative
changes during the time course of P-selectin expression and fibrinogen
binding. Statistical analysis was performed with one-way ANOVA
for repeated measurements, followed by the Scheffé test to
demonstrate changes in platelet aggregation, P-selectin expression,
and fibrinogen binding and to analyze the differences between
the two groups. For additional data analysis, the
Wilcoxon signed rank test for paired samples was used in the
ARDS patients. To analyze whether the effect of NO on
platelet function was due to changes in fibrinogen binding, the
changes in aggregation (aggregation ratio) and in fibrinogen binding
(fibrinogen-binding ratio) during incubation with 884 ppm NO were
correlated by the Pearson test. A value of P<.05 was
considered to be statistically significant.
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
NO Gas In Vitro
In the control group, ADP-induced platelet aggregation did not
change during the study (72±5% at t0, 73±7%
at t20, 67±7% at t40,
68±6% at t60, 65±5% at
t120, 67±7% at t180, and
70±6% at t240). During administration of 100
ppm NO, ADP-induced platelet aggregation was inhibited, reaching
statistical significance at time points t120
(46±7%, P<.05), t180 (41±7%,
P<.05), and t240 (26±4%,
P<.01). Administration of 450 ppm NO significantly
inhibited ADP-induced platelet aggregation at
t60 (43±10%, P<.05),
t120 (32±7%, P<.01),
t180 (23±7%, P<.01), and
t240 (12±4%, P<.001). The highest
NO concentration (884 ppm) resulted in the most pronounced inhibition
of platelet aggregation: 37±5% at t20
(P<.01), 38±6% at t40
(P<.01), 29±5% at t60
(P<.001), 15±5% at t120
(P<.001), 9±5% at t180
(P<.001), and 3±3% at t240
(P<.001) (Fig 2). In
accordance with these findings, collagen-induced platelet
aggregation also was dose-dependently inhibited during NO
administration, reaching statistical significance during administration
of 450 and 884 ppm NO. During administration of 450 ppm (884 ppm) at
t120, t180, and
t240, collagen-induced platelet aggregation
was 49±8% (55±3%, P<.05), 45±6% (37±3%,
P<.01), and 25±10% (13±5%, P<.001),
respectively (Fig 3).
View larger version (18K):
[in a new window]
Figure 2. ADP-induced platelet aggregation (FC, 5
µmol/L) at 0, 20, 40, 60, 120, 180, and 240 minutes. NO was
administered in vitro at 0 (diamonds; control), 100 (squares), 450
(circles), and 884 (triangles) ppm (mean±SEM; *P<.05,
P<.01, 
P<.001 vs control).
View larger version (15K):
[in a new window]
Figure 3. Collagen-induced platelet aggregation (FC,
0.19 mg/dL) at 0, 20, 40, 60, 120, 180, and 240 minutes. NO was
administered in vitro at 0 (diamonds; control), 100 (squares), 450
(circles), and 884 (triangles) ppm in vitro (mean±SEM;
*P<.05, P<.01,
P<.001 vs control).). Furthermore, in accordance with the
other findings, NO administration to PRP resulted in a significant
inhibition of platelet fibrinogen binding to the GP IIb/IIIa
receptor. Compared with t0 values, during
administration of 450 ppm (884 ppm) NO, fibrinogen binding was 73±17%
at t120 (66±16%, P<.05), 57±30%
at t180 (51±15%, P<.01), and
48±28% at t240 (33±11%, P<.01)
(Fig 5). Moreover, a significant
correlation between the aggregation ratio and the fibrinogen-binding
ratio was found during NO incubation (Fig 6).
View larger version (13K):
[in a new window]
Figure 4. P-selectin expression after activation with ADP
(FC, 2 µmol/L) as a percentage of baseline value at 60, 120,
180, and 240 minutes. NO was administered in vitro at 100 (open bars)
and 884 (solid bars) ppm (mean±SEM; *P<.05,
P<.01 vs control).
View larger version (13K):
[in a new window]
Figure 5. Fibrinogen binding to the GP IIb/IIIa receptor
after activation with ADP (FC, 2 µmol/L) as a percentage of
baseline value at 60, 120, 180, and 240 minutes. NO was administered in
vitro at 100 (open bars) and 884 (solid bars) ppm NO (mean±SEM;
*P<.05, P<.01 vs
t0).
View larger version (13K):
[in a new window]
Figure 6. Correlation between change in platelet
aggregation (aggregation ratio) and change in fibrinogen binding to GP
IIb/IIIa receptor (fibrinogen-binding ratio) during administration of
884 ppm NO in vitro (statistical analysis: Pearson test,
R=.86, P<.0001). ). In
comparison with the younger population, no statistically significant
differences were observed.
View this table:
[in a new window]
Table 1. ADP- and Collagen-Induced Platelet Aggregation
and ADP-Activated P-Selectin Expression and Fibrinogen Binding
to the GP IIb/IIIa Receptor as a Percentage of Baseline Values at 60,
120, 180, and 240 Minutes in Older Volunteers (55 to 65 Years Old) ).
View this table:
[in a new window]
Table 2. ADP-Induced Maximal Platelet Aggregation (FC=5
µmol/L), ADP-Stimulated P-Selectin Expression and Fibrinogen Binding
(FC=2 µmol/L) as a Percentage of Baseline Values 180 and 240 Minutes
After the Beginning of NO Administration (50 ppm) In Vitro
Two patients suffering from ARDS who had undergone cardiac surgery
did not respond to NO inhalation with an improvement in
oxygenation. NO was withdrawn in these patients, and
they were excluded from the study. In the remaining six patients, the
in vitro bleeding time was prolonged (99±13 seconds at
t120 versus 71±11 seconds at
t0, P<.05) and ADP-induced
platelet aggregation was inhibited (19±3% versus 26±2%,
P<.05) during inhalation of 10 ppm NO. In addition, NO
inhalation significantly inhibited both basal and ADP-stimulated
P-selectin expression: 6.7±1.7% versus 10.1±2.2%
(P<.05) and 43±6% versus 57±7% (P<.05),
respectively. In accordance with these findings, the fibrinogen binding
to the platelet GP IIb/IIIa receptor was also inhibited during NO
inhalation in patients with ARDS (19±7% versus 30±8%,
P<.05). In addition, NO therapy led to a significant
increase in plasma cGMP levels (8.1±1.6 versus 4.4±1.2 ng/mL,
P<.05) but had no influence on platelet and white blood
cell count (Table 3).
View this table:
[in a new window]
Table 3. Platelet Count, White Blood Cell Count, Plasma
cGMP, In Vitro Bleeding Time, ADP-Induced Platelet Aggregation,
Basal and ADP-Stimulated P-Selectin Expression, and ADP-Stimulated
Fibrinogen Binding in Patients With ARDS Before (0) and 120 Minutes
After Start of Administration of 10 ppm Inhaled NO
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
The present study demonstrates that inhaled NO inhibits
platelet aggregation, P-selectin expression, and fibrinogen binding
to the GP IIb/IIIa receptor of human platelets in vitro in a
dose-dependent manner. These results could be substantiated in a
clinical situation: besides prolonging the in vitro bleeding time, NO
inhalation in patients with ARDS led to an inhibition of platelet
aggregation, P-selectin expression, and fibrinogen binding. 
-granules.20
Inhibition of NO synthesis led to an increase in P-selectin
expression.37 Furthermore, the administration of
NO-releasing compounds inhibited P-selectin expression in
vitro16 26 38 and in several clinical
conditions.21 38 39 In keeping with the data from
the aggregation study, dose-dependent inhibition of P-selectin
expression in ADP-activated platelets was observed in the
present study during NO administration in vitro. In patients with
ARDS, basal and ADP-activated P-selectin expression were also
inhibited during NO inhalation, reflecting its platelet
inhibitory effect. P-selectin also mediates leukocyte and
endothelial interaction, which was significantly
affected by NO in vivo.40 In the present
study, the platelet and white blood cell counts remained stable
during NO administration, most likely excluding a relevant leukocyte or
platelet sequestration.41 42
Selected Abbreviations and Acronyms
ARDS
=
acute respiratory distress syndrome
FC
=
final concentration
GP
=
glycoprotein
PRP
=
platelet-rich plasma
Footnotes
Presented in part at the annual meeting of the American Society of Anesthesiologists, New Orleans, La, October 18–23, 1996, and the German Congress of Anesthesiology, Hamburg, Germany, April 23–26, 1997, and published in abstract form (Anesthesiology. 1995;85[suppl]:A1144).
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
1.
Radomski MW, Palmer RMJ, Moncada S.
Endogenous nitric oxide inhibits human platelet
adhesion to vascular endothelium.
Lancet. 1987;305:1057–1058.
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