© 1995 American Heart Association, Inc.
Articles |
Role of Nitric Oxide in Hemostatic System Activation In Vivo in Humans
From the Department of Clinical Pharmacology (K.K., L.S., J.K., B.M., H.-G.E.), Institute of Pharmacology (K.K., W.S.), Department of Internal Medicine I, Division of Hematology and Hemostaseology (M.N.-L., P.A.K.), University of Vienna, Vienna, Austria.
Correspondence to Kurt Krejcy, MD, Department of Clinical Pharmacology, Vienna University Hospital, Allgemeines Krankenhaus Wien, A-1090 Wien, Währinger Gürtel 18-20, Austria.
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Abstract |
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Abstract NO is a potent inhibitor of in vitro platelet aggregation and adhesion. In view of possible future widespread use of NO in pulmonary and cardiovascular diseases, we investigated the role of NO in hemostatic system activation in vivo in humans. Sixteen healthy male volunteers (age range, 22 to 33 years) received either NO by inhalation (50 ppm over 30 minutes; n=8) or the NO synthase inhibitor NG-monomethyl L-arginine (L-NMMA 3 mg/kg body weight IV over 5 minutes; n=8). ß-Thromboglobulin (ß-TG), an indicator of platelet activity; prothrombin fragment 1+2 (F1+2), an index of coagulation activation; and thromboxane B2 (TxB2), a measure of platelet prostaglandin synthesis, were determined in blood samples obtained from bleeding-time incisions ("shed blood") at baseline and after administration of the respective drug. In addition, ß-TG and F1+2 were also determined in venous blood. To verify the systemic effects of the drugs, methemoglobin and plasma nitrites/nitrates were measured in the NO group, and cardiac output and exhaled NO were measured in the L-NMMA group. Compared with baseline, methemoglobin and plasma nitrates increased by 73±12% (P=.006) and 60±9% (P<.001), respectively, following NO inhalation. L-NMMA infusion resulted in decreases in both cardiac output (by 16±2%; P<.001) and exhaled NO (by 54±7%; P<.001). NO inhalation or L-NMMA infusion had no significant effect on ß-TG, F1+2, and TxB2 levels in shed blood. No significant changes in platelet counts and levels of coagulation activation markers were found in venous blood after drug administration. In contrast to the in vitro data, neither inhalation of NO nor inhibition of NO synthesis by infusion of L-NMMA had a major impact on hemostatic system activation in vivo in healthy individuals.
Key Words: NO inhalation • L-NMMA • bleeding time • hemostatic system activation
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Introduction |
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NO is a potent vasodilator1 2 and inhibitor of platelet aggregation in response to a variety of stimuli, such as ADP, arachidonic acid, collagen, and thrombin.3 Furthermore, NO impairs adhesion of blood platelets to collagen fibrils, the endothelial cell matrix, and monolayers.4 5 In animals, inhalation of NO results in prolongation of the bleeding time6 ; inhibition of NO synthesis by L-NMMA shortens the prolonged bleeding time in uremic rats.7 Data obtained in vitro or from animal experiments, however, do not necessarily reflect the mechanisms relevant to hemostatic system activation in vivo in humans.
NO inhalation has recently been shown to be clinically effective in patients with pulmonary hypertension and acute respiratory distress syndrome.8 Because NO is expected to be routinely applied in a number of clinical situations in the near future, it is important to know whether NO can be given safely to patients with regard to its potential inhibitory effects on hemostasis.
There is only limited information on the impact of NO on the hemostatic system in humans. In a small number of healthy volunteers, Högman et al9 found prolongation of the bleeding time during NO inhalation; in a subsequent study, however, this effect of NO could not be duplicated.10 Simon et al11 reported a shortening of the bleeding time after administration of L-NMMA.
In healthy subjects, we have studied the effects of NO inhalation and inhibition of NO synthesis by L-NMMA on hemostasis by use of a technique that allows investigation of the mechanism(s) that lead to plug formation under conditions that closely resemble the in vivo situation.12 13 14 15 16 17 18 This method consists of standardized injury of the microvasculature (bleeding-time incision) and subsequent measurement of specific indicators of platelet function. (ß-TG, as an overall index of platelet activation), of platelet prostaglandin synthesis (TxB2, the stable metabolite of TxA2), and of coagulation activation (F1+2, a measure of the action of factor Xa on thrombin)
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Subjects
Sixteen healthy male volunteers who were nonsmokers and drug-free for at least 3 weeks before the study were selected as study participants. Eight subjects (median age, 30 years; age range, 26 to 33; body mass index, 23.3±0.8 kg/m2) received NO as an inhalant, and the remaining 8 subjects (median age, 24 years; age range, 22 to 31; body mass index, 22.7±0.6 kg/m2) received an intravenous infusion of L-NMMA. The study was approved by the Ethics Committee of the Vienna University School of Medicine. Written informed consent was obtained from all volunteers prior to inclusion in the study.
Study Protocol
NO Inhalation
After the subjects had rested for 15 minutes in a sitting
position, inhalation of 50 ppm NO was started. NO was administered by
mask inhalation through an open nonrebreathing system with a
two-way valve. Subjects were instructed to continue breathing
normally. The inspired gas was a mixture of O2 and
N2 to produce an FIO2
of .21. Volumetrically calibrated flowmeters were used to add NO mixed
with N2 to obtain a final concentration of 50 ppm NO at
constant FIO2. NO was obtained from
AGA as a mixture of 1000 ppm in N2. The concentration of NO
in the inspired gas was monitored throughout the inhalation period by a
chemiluminescence detector.
Determination of MetHb in capillary blood, blood sampling, and determination of bleeding time were performed before and 20 minutes after NO inhalation was started. Blood pressure was measured every 10 minutes throughout the study period.
L-NMMA Injection
After the subjects had rested for 15 minutes in a sitting
position, L-NMMA (3 mg/kg body weight) was administered
intravenously over 5 minutes. Doppler ultrasound
measurement of CO, measurement of NO content in exhaled air, blood
sampling, and determination of bleeding time were performed before drug
administration and 10 minutes after L-NMMA infusion had
ended. Blood pressure was measured every 2 minutes throughout the study
period.
Bleeding Time
Template bleeding time was determined as described by Mielke et
al.19 A sphygmomanometer cuff was placed on the upper arm
and inflated to 40 mm Hg. Two incisions 3 mm apart, each 5 mm long and
1 mm deep, were made with a disposable standard device (Simplate II,
Organon Teknika) on the lateral volar aspect of the forearm, parallel
to the antecubital crease. The procedure was performed by the same
investigator each time.
Sampling of Blood Emerging From Bleeding-Time Incisions
(`Shed Blood')
Shed blood was collected as described previously.13
For determination of ß-TG, TxB2, and
F1+2, blood was collected at 15-second intervals
directly from the edge of the skin wound into ice-cooled Eppendorf
tubes containing 20 µL anticoagulant mixture, consisting of 3.8%
(vol/vol) sodium citrate, 100 mmol/L EDTA, 30 µmol/L
indomethacin (Sigma Chemical Co), 1000 U/mL aprotinin
(Trasylol, Bayer AG), and 1500 U/mL sodium heparin (Immuno AG).
Shed-blood samples were collected in four 1-minute aliquots
(minutes 1 through 4). The tubes were immediately centrifuged
at 12 000g for 2 minutes. The supernatants were removed,
frozen, and stored at -80°C until assayed.
Sampling of Venous Blood
Venous blood was collected by sterile puncture of an antecubital
vein with a 19-gauge needle. For measurement of PT, APTT,
F1+2, and fibrinogen, blood was drawn into tubes
containing 1/10 volume of 3.8% (vol/vol) sodium citrate. For
determination of plasma nitrite/nitrate, blood was collected into tubes
containing lithium heparin. For determination of ß-TG, special tubes
supplied by Amersham International were used; the samples were
immediately centrifuged at 2000g for 20 minutes. The
supernatants were removed, frozen, and stored at -80°C until
assayed.
Assays
PT, APTT, fibrinogen, and platelet counts were measured by
routine laboratory procedures. ß-TG was measured with the use of a
commercially available RIA (ß-TG RIA Kit, Amersham International).
TxB2 was determined by an RIA using
[5,6,8,9,11,12,14,15-3H(N)]TxB2 (New England
Nuclear) as a tracer. Authentic TxB2 was obtained from
Advanced Magnetics Inc. The rabbit anti-TxB2 antibody was
provided by D.J. Westwick, Department of Pharmacology, Royal College of
Surgeons, London, UK. F1+2 was measured by an
enzyme-linked immunosorbent assay technique, using a commercially
available procedure (Enzygnost F1+2, Behringwerke
AG). MetHb was measured photometrically by means of an automatic blood
gas system (AVL). Plasma nitrite/nitrate concentrations were determined
colorimetrically after reaction with the Griess
reagent, as described in detail by Roth et al.20
Determination of Exhaled NO
Exhaled NO was measured with a chemiluminescence detector
(nitrogen oxides analyzer model 8840, Monitor Labs Inc)
connected to a strip-chart recorder. Calibration of the
instrument was performed with certified gases (300 ppb NO in
N2, AGA) diluted by precision flowmeters. The
subjects were instructed to fully inflate their lungs, hold their
breath for 10 seconds, and then exhale for 10 seconds into a
polytetrafluoroethylene tube. Some of the
exhaled air (1000 mL/min) was allowed to enter the inlet port. Three
consecutive readings were made under nasal occlusion at each
measurement point. The end-expiratory values from the
strip-chart recorder readings were used to ensure that inspired
NO from the ambient air did not distort the results. Detector response
was linear from 0 to 100 ppm, and the detection limit was 2 ppb. This
method of quantifying the degree of NO synthesis has been used
previously.21
Noninvasive Measurement of Systemic
Hemodynamics
Systolic and diastolic blood pressures were
measured on the upper arm by an automated oscillometric device. CO was
determined by a 3.25-MHz probe with a pulsed Doppler device (CFM
750, Vingmed Sound) positioned at the level of the aortic valve. A
detailed description of the time course of hemodynamic
changes that were observed in response to L-NMMA in the
present study is given elsewhere.22
Data Analysis
To quantify the formation of ß-TG, TxB2,
and F1+2 in shed blood, concentrations of the respective
activation markers were measured in four 1-minute aliquots. The area
under the concentration-versus-time curve was calculated
(minutes 1 through 4) by the trapezoidal rule and regarded as the
measure of mediator production in the microcirculation.
Student's t test and the Wilcoxon signed-rank
test were used for normally and nonnormally distributed data,
respectively, to compare baseline values with those measured during NO
inhalation or after administration of L-NMMA. Data are
presented as mean±SEM. Statistical significance was set at
P<.05.
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Results |
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NO Inhalation
The levels of hemostatic system activation markers in shed and venous blood samples are given in Table 1
. ß-TG,
TxB2, and F1+2 levels in shed blood and ß-TG
and F1+2 levels in venous blood before NO inhalation were
similar to those found after exposure to NO. We consider the small but
statistically significant increase in ß-TG in shed blood after NO
inhalation as a consequence of statistical chance rather than as a
biologically relevant effect of NO. As shown in Table 2,
neither PT, APTT, fibrinogen, platelet count, nor bleeding time was
affected by NO inhalation.
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MetHb formation increased from 0.63±0.03% at baseline to 1.08±05% (P=.006) during NO inhalation, an increase of 73±12%. Plasma levels of nitrates increased from 30.4±5.8 to 46.3±7.1 µmol/L (P<.001), an increase of 60±9%. No significant change in plasma nitrites was observed (0.81±0.07 versus 0.84±0.06 µmol/L, P>.05).
Systolic and diastolic blood pressures as well as heart rate remained unchanged in response to NO inhalation. The baseline values and those determined 20 minutes after beginning the NO inhalation were 129±2 mm Hg versus 123±3 mm Hg for systolic blood pressure, 78±2 versus 74±2 mm Hg for diastolic blood pressure, and 69±4 versus 72±4 beats per minute for heart rate.
Infusion of L-NMMA
As shown in Table 3, infusion of L-NMMA
(3 mg/kg body weight) did not cause any significant changes in ß-TG,
TxB2, and F1+2 levels in shed blood and did not
affect ß-TG and F1+2 levels in venous blood. Bleeding
times, platelet counts, PT, APTT, and fibrinogen concentrations
(Table 2
) were also unaffected by infusion of
L-NMMA.
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The concentration of NO in exhaled air was 44.7±9.6 ppb at baseline and 23.7±6.8 ppb following L-NMMA infusion (P<.001), corresponding to a 53.6±6.9% decrease. L-NMMA induced only a small, significant decrease in plasma nitrates (40.7±7.5 µmol/L versus 36.4±7.6 µmol/L, P=.01) but no significant change in plasma nitrites (0.79±0.19 µmol/L versus 0.63 µmol/L, P=.42).
Infusion of L-NMMA did not cause significant changes in systolic and diastolic blood pressures or heart rate. The baseline values and those determined 10 minutes after discontinuation of the L-NMMA infusion were 125±5 versus 122±2 mm Hg for systolic blood pressure, 65±3 versus 71±3 mm Hg for diastolic blood pressure, and 70±3 versus 65±4 beats per minute for heart rate. However, CO decreased significantly, from 6.8±0.7 L/min at baseline to 5.9±0.5 L/min (P<.001) 10 minutes after discontinuation of the L-NMMA infusion.
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Discussion |
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The present study clearly demonstrates that inhalation of NO has no major impact on hemostasis in healthy subjects, because the levels of platelet- and coagulation-specific activation markers, ie, ß-TG, TxB2, and F1+2, were unaffected by exposure to NO. These findings were noted when the coagulation system was in a resting state, ie, when only small amounts of thrombin are generated as occurs in venous blood, and after activation of the clotting cascade, ie, when large amounts of thrombin are generated in response to injury of the microvasculature.
In contrast to our in vivo findings, NO is a potent inhibitor of platelet function in vitro. NO has been shown to inhibit platelet aggregation3 and adhesion.4 5 NO elevates cGMP levels in platelets,23 thereby decreasing the number of platelet-bound fibrinogen molecules and inhibiting intracellular calcium flux and platelet secretion.24 25
One reason for the discrepancy between the in vivo and in vitro findings may very well be the rapid inactivation of NO by Hb that occurs under in vivo circumstances.26 Our observation of a 70% increase in MetHb after 20 minutes of NO inhalation strongly suggests substantial absorption of NO by red blood cells. An additional major pathway of inhaled NO involves its conversion to nitrate,26 as reflected by the 60% increase in plasma nitrate content in the present study. Whereas the antiplatelet and vasorelaxant effects of NO are mediated via elevated levels of cGMP, the NO sensitivity of vascular smooth muscle cells is postulated to be much higher than that of platelets.27 28 Thus, one may speculate that the platelet concentration of NO that is achieved during therapeutic NO inhalation is not high enough to inhibit platelet function.
In the present study, the NO dose selected has been shown to effectively dilate the pulmonary arteries of patients with acute respiratory distress syndrome and pulmonary hypertension.8 Because NO is rapidly inactivated by Hb,26 29 inhaled NO acts selectively on the pulmonary vasculature without affecting systemic blood pressure and systemic vascular resistance.30 There is substantial evidence that organic nitrates, which are ultimately converted to NO, have potent platelet-inhibiting effects in vitro in addition to their vasodilatory properties.31 32 Interestingly, in humans the antiplatelet effects of nitroglycerin are not apparent without concomitant hemodynamic changes.32 These data corroborate our findings that inhalation of a dose of NO that does not influence systemic hemodynamics also does not affect platelet function in healthy human subjects.
In the second part of our study, we infused 3 mg/kg body weight of the NO synthase inhibitor L-NMMA into healthy individuals. We selected this experimental approach for two reasons: (1) to further investigate the role of NO in hemostatic system activation by inhibiting endogenous NO production and (2) in view of a possible clinical application of L-NMMA as an antagonist of enhanced NO production in patients with septicemia.33 In accordance with animal data34 in the present study, only a small decrease in plasma nitrate levels was observed after NO synthase inhibition. However, the systemic effect of L-NMMA was demonstrable not only by the significant decrease in CO but also by a marked reduction in NO concentration in exhaled air, which is regarded as a means of directly quantifying the effects of NO donors and NO synthesis inhibitors on endogenous NO production.21 35 In contrast, we saw no effect of L-NMMA on either ß-TG, TxB2, and F1+2 levels in shed blood or ß-TG and F1+2 levels in venous blood, indicating that inhibition of basal NO synthesis does not affect hemostatic system activation in vivo, irrespective of the state of activation.
Under in vitro conditions, L-arginine analogues like L-NMMA have been shown to potentiate platelet aggregation in response to arachidonic acid, ADP, and thrombin,36 whereas similar concentrations of L-NMMA did not affect generation of platelet-specific activation markers in our in vivo study. One explanation for this discrepancy may be that platelet behavior in vitro is studied in the absence of the vascular endothelium, whereas platelet function in vivo is likely to be affected by platelet antiaggregatory agents, such as NO and prostacyclin, that are released from the vasculature.36 Thus, it can be assumed that counterregulatory mechanisms may be operative under in vivo conditions and thereby mask the direct effect of NO synthesis inhibition on platelets.
Our inability to detect an effect of L-NMMA on hemostatic system activation in healthy subjects is in concert with animal data showing normalization of bleeding time in uremic rats following L-NMMA infusion, whereas no effect of L-NMMA has been observed in healthy animals.7 It is conceivable that NO synthase inhibitors may exert their effects on platelet function under pathological conditions that are accompanied by an enhanced synthesis of endogenous NO34 rather than in a physiological setting wherein endogenous NO production is not stimulated.
In summary, neither NO inhalation nor NO synthesis inhibition by infusion of L-NMMA has a major impact on hemostatic system activation in vivo in healthy volunteers. Our data warrant further studies on hemostatic system activation in patients undergoing NO inhalation or NO synthase inhibition therapy.
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Received April 19, 1995; accepted August 14, 1995.
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References |
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