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ABSTRACT |
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ABSTRACT
INTRODUCTION
METHODS
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As inhaled nitric oxide (iNO) may differently increase bleeding time (BT) and inhibit platelet aggregation in normal and lung-injured patients or experimental models, we studied the effects of iNO on hemostasis in presence and absence of an endotoxic lung injury in the rat. Eight hours after intratracheal administration of endotoxin (lipopolysaccharide [LPS]) or its solvent (phosphate-buffered solution [PBS]), four groups of rats were randomized according to the presence or absence of 15 ppm iNO added for an additional 10 h. We measured BT, ex vivo platelet aggregation, plasma fibrinogen, euglobulin clot lysis time (ECLT), and platelet and aortic cyclic guanosine 5'-monophosphate (cGMP) contents. Acute lung inflammation did not influence BT, but increased platelet aggregability, fibrinogen levels, and platelet and aortic cGMP. In control and endotoxic rats, iNO increased BT, reduced platelet aggregability, and increased platelet cGMP. iNO increased aortic cGMP only in healthy rats. ECLT was increased by LPS and unchanged with iNO. These results suggest that the extrapulmonary "systemic" effects induced by iNO on hemostasis were not strictly similar in healthy and LPS rats, inflammation inducing proper changes in coagulation parameters. However, iNO attenuated the procoagulant activity induced by acute lung inflammation, suggesting a potentially beneficial effect of this therapy.
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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In addition to the recently discovered biological properties of nitric oxide (NO), literature is increasing to describe the potential interest of inhaled NO (iNO) administration (1). iNO has been proved to be efficient in pulmonary hypertension and severe hypoxemic acute lung injury (2). In these indications, the vasodilating property of NO allows one to selectively reduce regional pulmonary vascular resistance, improving right ventricular afterload and/or ventilation/perfusion mismatching (2). In addition, NO may interfere with cells' functions such as polymorphonuclear neutrophils' (PMN) oxidative burst (3), mononuclear cells' cytotoxicity (4), and platelet functions (5). The latter property has been known for several years but its clinical impact remains debated although iNO may differently increase bleeding time and modulate platelet aggregation in healthy animals (6) and patients with acute lung injury (7).
The pathophysiology of acute pulmonary inflammation is a complex network of interactions between cells and mediators, among which platelet hyperaggregability plays an important role (8). Platelet aggregation may promote local microthrombosis, thus increasing pulmonary vascular resistance and contributing to the observed pulmonary hypertension. Because NO inhalation is proposed as a therapy in this clinical condition, the impact of acute pulmonary inflammation on platelet function and fibrinolysis was evaluated using a model of acute lung inflammation, induced by intratracheal endotoxin injection in the rat. Then, the effects of NO inhalation (studying all together the hemostatic, coagulation, and fibrinolytic systems) were measured in the presence or in the absence of 15 ppm NO inhalation. Finally, we measured platelet and aortic cyclic 5'-guanosine monophosphate (cGMP) contents to analyze whether cGMP might mediate the effects of iNO on platelets and on vascular wall.
Acute lung inflammation increased platelet aggregability and fibrinogen levels with a reduced fibrinolysis and increased platelet and aortic wall cGMP contents. NO inhalation increased bleeding time (BT), reduced platelet aggregability, decreased fibrinogen, and increased platelet cGMP contents. iNO also increased aortic cGMP contents in control, but not in endotoxic rats.
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INTRODUCTION
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Animal Preparation
Male CD rats weighing 300 ± 20 g (Charles River Laboratories, Saint-Aubin-Les-Elbeufs, France) were studied. The acute lung inflammation was induced by intratracheal endotoxin administration as previously described by Nelson and coworkers (9). Endotoxin from Escherichia coli (lipopolysaccharide [LPS] B strain O26:B6; Sigma Laboratories, St. Quentin Fallavier, France) was prepared for injection by resuspension in phosphate-buffered saline (PBS; Sigma Laboratories) to obtain a concentration of 1 mg/ml. After a brief ether anesthesia, the trachea was exposed after a midline cervical incision and 0.2 ml LPS was injected intratracheally through a 24-gauge catheter. Another group of rats underwent the same surgical procedure, but only the diluent of LPS, i.e., 0.2 ml of PBS, was injected into the trachea. LPS or PBS administration was immediately followed by 3 to 4 insufflations of 1 ml of air through the catheter and by rotating the animals to attempt to homogeneously distribute endotoxin or its diluent to the lungs. The experiments described in this article were performed in adherence to National Institutes of Health guidelines on the use of experimental animals. Approval of the animal use of the University of Lariboisière Hospital was obtained before initiating the experiments.
Gas Administration Procedure
After PBS or LPS administration, animals were immediately housed in specially designed Plexiglas boxes (Momeplast, Paris, France) with food and water available ad libitum during the observation period. These boxes were continuously ventilated with air at a flow rate of 2 L/ min. Nitric oxide at a fraction of 15 ppm (0.2 L/min from a 225 ppm tank of NO in nitrogen (N2) supplied by Air Liquide Santé, Paris, France), or a similar gas flow of 0.2 L/min pure N2 were added to the air flux via a separate conduit. NO fraction was measured by chemiluminescence (CLD 700 AL; Eco-Physics, Zürich, Switzerland) in gas samples obtained from an opposite outlet. The temperature in the box was maintained at 20-22° C and gases were administered at atmospheric pressure.
Description of the LPS-induced Lung Injury
In a previous series of animals, the kinetics of polymorphonuclear neutrophils (PMN) influx in the alveoli were studied at different times (before, 2, 3, 4, 6, 9, 18, and 70 h after intratracheal LPS administration). Based on the results obtained, the eighteenth hour after LPS injection was chosen as an adequate period because: (a) the early (3 h) afflux of inflammatory cells to the lungs was still present; (b) a significant lung injury developed; (c) this period would allow a relatively long period of iNO administration. Eighteen hours after LPS injection, rats were killed by pentobarbital overdose and exsanguinated by cardiac puncture. Lungs were removed en bloc, dissected free from extrapulmonary tissue for wet lung weight measurements and a bronchoalveolar lavage (BAL) was performed. Five 5-ml aliquots (25 ml total) of sterile pyrogen-free 0.9% NaCl at 4° C were instilled intratracheally and gently recovered. The recovered lavage fluid (usually > 85% of the injected saline) was filtered through gauze. Direct cell count using a Hemalog H1 (Technicon Instruments Corp., Tarrytown, NY) and cytospin preparations for differential cell counts were performed on cell monolayers stained with Wright-Giemsa solution. Finally, lungs were dried at 80° C in an oven and dry lung weights were measured.
Study Design
Eight hours after LPS injection, 15 ppm NO (LPS-NO) or a similar N2 flux (LPS-air) were added into the chamber during 10 additional hours (Figure 1). The healthy groups consisted of rats receiving PBS, the diluent of LPS (i.e., PBS-NO and PBS-air, respectively). Eighteen hours after PBS or LPS injection, animals were removed from their boxes for coagulation and fibrinolysis studies.
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Bleeding Time
BT was measured using a standard technique (10) in 48 unanesthetized animals. Briefly, the tip of the tail was transected with a razor blade and immediately immersed in a vertical position into a plastic tube filled with isotonic saline at 23° C. Observation period, made by two investigators, was extended up to 15 min for all rats. BT was assumed as the interval of time between transection and the total cessation of spontaneous bleeding for more than 1 min.
Platelet Studies and Fibrinogen Measurements
For platelet studies, 34 rats were anesthetized with 2 mg/kg pentobarbital intraperitoneally. The abdominal aorta was exposed after dissection and 10 ml of blood were collected into plastic tubes containing anticoagulant (1:9, 0.129 M trisodium citrate). Then, this citrated blood was immediately centrifuged at 160 g for 10 min at 12° C to obtain platelet-rich plasma (PRP). The remaining cellular suspension was further centrifuged at 1,700 g for 10 min at 18° C to obtain the platelet-poor plasma (PPP). Platelets from PRP were counted (T-890 Coulter; Coultronics Corp., Margency, France) and PRP was then diluted with autologous PPP for adjustment to a count of 700,000 platelets/ml in each sample.
Ex vivo PRP aggregometry was performed by stirring 400 µl of PRP equilibrated to 37° C in a recording aggregometer (Aggrometer; Chronolog Corp.), according to the method of Born and Cross (11). Aggregations were induced with adenosine diphosphate (ADP) at incremental concentrations (0.6, 1.2, and 2.4 µM) and recorded for 5 min after addition of the agonist. Aggregations were quantified as the maximal rate (velocity) and as the extent (intensity) of light transmittance in stimulated PRP, calibrated as 100% light transmission for PPP and 0% for nonstimulated PRP.
Plasmatic fibrinogen was determined in 19 rats by the chronometric method of von Clauss (Multifibren, Behring) according to a pool of rats' PPP calibrated for dry weight fibrinogen.
Platelet and Aortic Wall cGMP Concentrations
For platelet cGMP measurements, PRP of 29 rats were obtained by
centrifugation of arterial blood at 140 g for 10 min and platelets were
counted in each sample. One-milliliter samples of PRP were then centrifuged at 2,500 g for an additional minute to obtain a platelet pellet.
An aliquot of 1 ml of 6% trichloroacetic acid was added to each platelet pellet and vortexed for 1 min. Samples were then centrifuged at
9,000 g for 15 min, and the aqueous phase was stored at 
20° C (12).
For aortic cGMP measurements, 29 rat aortas were isolated, carefully dissected, removed and rapidly frozen in liquid nitrogen and
stored at 80° C. Frozen arteries were then cut into strips and homogenized in ice-cold 6% trichloroacetic acid with a Potter glass homogenizer at 4° C. The homogenate samples were centrifuged at 2,000 g for
15 min at 4° C. Supernatant fractions were extracted four times with
5 volumes of water-saturated diethyl ether, lyophilized, and assayed
for cGMP content by enzyme immunoassay (Amersham, les Ullis,
France). The assay was based on the competition between unlabeled
cGMP and a fixed quantity of peroxidase-labeled cGMP for a limited
number of binding sites on a cGMP-specific antibody. With fixed
amounts of antibody and peroxidase-labeled cGMP, the amount of
peroxidase-labeled ligand bound by the antibody was inversely proportional to the concentration of added unlabeled ligand. Platelet and
aortic cGMP were measured after acetylation of standards and samples to obtain higher sensitivity. The standard curves ranged from 2 to
512 femtomol (fmol) per well; platelet cGMP was expressed as pmole
per 109 platelets and arterial cGMP content as fmol/mg aorta.
Fibrinolysis
The fibrinolytic activity within plasma samples was assayed in vitro. In 20 rats, the euglobulin clot lysis time (ECLT) was assessed, based on the method of von Kaulla and Schultz (13) which mostly reflects tissue plasminogen activator (t-PA) activity. Briefly, blood containing trisodium citrate (3.15% wt/vol) in a ratio of 9:1 was centrifuged at 3,000 g and 4° C for 10 min to produce PPP. Distilled water was added (14 ml/ ml PPP) and the pH was adjusted to 5.9 by adding acetic acid. This procedure causes precipitation of the euglobulin fraction which contains t-PA and plasminogen, while plasminogen activator inhibitor (PAI) remains in the supernatant. After a second centrifugation at 2,000 g for 15 min, the supernatant was discarded and the euglobulin precipitate dissolved in 0.5 ml buffer containing calcium. After mixing carefully each tube, the fraction was then incubated at 37° C and the time for lysis to occur recorded.
Statistical Analysis
Values of lung weight and cellularity were analyzed using an unpaired t test. Values of coagulation parameters were first analyzed by 2-way analysis of variance for two between factors: presence or absence of the factor "inflammation" (i.e., LPS and PBS groups) and presence or absence of the factor "NO inhalation" (i.e., NO and air groups). This analysis allowed testing of the following hypotheses: (1) coagulation parameters are affected by NO inhalation; (2) coagulation parameters are affected by inflammation; (3) the effect of NO inhalation is influenced by the existence of an inflammation (i.e., a significant interaction is observed between these two factors). When interaction was significant, comparisons between NO-treated and air-treated rats were made separately for LPS- and PBS-treated rats by Student's t test. Due to a non-Gaussian distribution, statistical analysis was performed after a logarithmic transformation of the variables tested. For all tests, the significance level was fixed at 5%. Results are expressed as mean ± SEM.
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Description of the LPS-induced Lung Injury
After LPS administration, rats were still able to spontaneously breathe. Eighteen hours after intratracheal LPS administration, a significant lung edema was present, with a 50% increase in lung weights (Table 1). BAL cellularity of rats receiving PBS contained a predominance of macrophages, which accounted for > 99% of the total cells recovered (90 × 103 cells/ ml). These values were not different from those of normal healthy rats. Following intratracheal LPS administration, BAL cellularity increased in relation with an alveolar PMN infiltrate, starting at the third hour. Eighteen hours after LPS administration, total cell count of BAL fluid was 559 ± 99 cell/ mm3 with 85 ± 3% of PMN, a value that was not affected by NO inhalation (770 ± 99 cells/mm3 with 82 ± 3% of PMN).
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Bleeding Time
Bleeding time (BT) was 508 ± 58 s (ranging from 140 to 900 s) in healthy rats breathing air, and 407 ± 34 s (ranging from 280 to 610 s) in LPS animals breathing air (Figure 2). NO inhalation significantly increased (p < 0.0001) BT in healthy and LPS rats (753 ± 54 s and 878 ± 14 s, respectively, ranging from 500 to 900 s and 745 to 900 s). LPS administration had no influence on BT, regardless of the presence or absence of NO administration.
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Ex Vivo Platelet Aggregation
Platelet counts were comparable in the four groups of animals (Table 2). As shown in Figure 3, the addition of 0.6 µM ADP to the unstimulated PRP induced a reversible platelet aggregation. Significant differences between groups in platelet aggregation were exclusively observed with the lowest dose inducing a consistent response (0.6 µM ADP), whereas higher doses (1.2 and 2.4 µM) of ADP induced similar increases in platelet aggregation in all groups. The maximal intensity and velocity of platelet aggregation in response to 0.6 µM ADP was higher in LPS than in PBS groups (p < 0.0001). NO inhalation significantly decreased platelet aggregation (p < 0.005), but no significant statistical interaction was found between NO inhalation and inflammation using two-way ANOVA.
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p < 0.05 versus PBS-air and PBS-NO).
Fibrinogen
Plasma fibrinogen levels were higher in animals receiving intratracheal LPS administration compared with PBS animals (p < 0.01) (Figure 4). NO inhalation significantly decreased fibrinogen concentrations to a similar extent in both groups (p < 0.04), and no statistically significant interaction between NO inhalation and inflammation was found using two-way ANOVA.
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Platelet and Aortic cGMP levels
Platelet cGMP levels were higher in animals receiving LPS compared with PBS animals (p < 0.01) (Figure 5). NO inhalation significantly increased platelet cGMP (p < 0.0002), to a similar extent above their own control in both groups, and no significant effect between NO inhalation and inflammation was found.
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Aortic cGMP contents were higher in animals receiving LPS (p < 0.003) and in animals inhaling NO (p < 0.0008). However, analysis of variance demonstrated the existence of a significant interaction between NO inhalation and inflammation. This interaction observed between NO inhalation and the existence of a pulmonary inflammation did not yet concern pathophysiological interactions, but corresponded to the existence of a significant interaction between these two factors using 2-way ANOVA. Accordingly, aortic-wall cGMP contents were increased with inhaled NO in PBS animals (p < 0.0003), but were not further increased in LPS animals (p > 0.8).
Fibrinolysis
Euglobulin clot lysis time (ECLT) measured as an index of t-PA activity was significantly prolonged by intratracheal LPS administration (p < 0.02) (Figure 6). NO inhalation did not influence ECLT, but 2 way-ANOVA demonstrated the existence of a significant interaction between the effect of NO and the presence or absence of LPS administration (p < 0.02).
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From the pioneer work of Mellion and coworkers (5) demonstrating interactions between platelet function and NO donors, Högman and coworkers (6) have reported that inhaled NO (iNO) increased BT and decreased platelet aggregation, raising potential concerns in patients with bleeding tendencies. Bleeding time is determined by vascular (involving endothelial and smooth muscle cells), platelet, and fibrinolysis parameters. Because no previously published studies, to our knowledge, have performed a complete analysis of those main parameters involved in the hemostatic, coagulation, and fibrinolytic systems (6, 7, 14), we attempted to present a comprehensive analysis of the effects of iNO on these parameters. Moreover, these determinants of BT have been explored according to the inflammatory status of the lungs. The latter point is clinically relevant because the use of iNO as a therapy in severe hypoxemia concerns by definition an acutely inflamed lung tissue, associated at various degrees with a systemic inflammation (18). In this regard, intratracheal LPS administration was used to induce a predominantly compartmentalized inflammatory lung injury characterized by an increased lung wet weight by 50%, with an alveolar afflux of neutrophils that represented nearly 80% of the total cell population in the BAL. An associated systemic inflammatory response was also present as assessed by the changes in hemostatic, coagulation, and fibrinolytic parameters.
Previous reports of the effects of iNO on hemostasis have usually evidenced an increased BT and reduced platelet aggregation, although the fraction and duration of NO administered, and the underlying pathologic condition could have influenced the results observed. For example, Högman and coworkers described a prolongation of BT after 15 min NO inhalation in healthy rabbits (14) and human volunteers (6), but this group failed to exactly reproduce these initial results in healthy subjects, observing that BT only tended to increase following a short-term exposure to NO (15). In this latter study, BT was found to be significantly increased only after 55 min of exposure to NO, and despite increased plasma (and not platelet) cGMP levels, platelet aggregability was only little affected, with a considerable interindividual variability (15). In contrast to these studies performed in humans or animals with normal lungs, Samama and coworkers (7) reported in patients with acute respiratory distress syndrome an attenuation of platelet aggregation with iNO (measured with a specific technique to reduce the delay between blood drawing and platelet function study), but BT was unchanged, even though NO fractions up to 100 ppm were used. Whatever the cause of these discrepancies, these results and ours demonstrate that the existence of an acute pulmonary inflammation, resulting in a procoagulant activity (19, 20) can influence these "extrapulmonary" effects of inhaled NO.
If iNO increases plasma cGMP levels (15, 21, 22), suggesting a guanylate cyclase activation, the origin of such plasma cGMP response remained to be further investigated. In the present study, the observed increase in platelet cGMP with iNO suggested that this cell population might have been one of the numerous sources involved in plasma cGMP elevation previously described. This increased platelet cGMP during NO inhalation is consistent with numerous previous findings observed when NO donors were given in direct contact with the platelets or the endothelial cells in vivo (12) and in vitro (5). More recently, Nong and coworkers (23) observed that iNO could also reduce ex vivo collagen-induced platelet aggregation and attenuate the rise in pulmonary artery pressure caused by collagen-induced, platelet-mediated pulmonary thrombosis in rats. This antithrombotic activity of iNO was associated with a dose-dependent increase in intraplatelet cGMP levels (23). These observations suggest that the NO administered by inhalation was not totally fixed by hemoglobin, a sufficient part being able to diffuse and stimulate the formation of cGMP in the platelets.
In healthy animals, the increase in aortic wall cGMP contents with iNO suggested that iNO may also modify cells at a distance from the lungs. This observation has not been previously reported and suggests that iNO metabolic fate is not simple and may result from interactions of NO with biologic carriers and cells circulating in the lungs. Among these carriers, the sulfhydryl group of plasma proteins can combine with oxides of nitrogen under physiological conditions to form stable and biologically active NO adducts such as S-nitrosothiols, which possess certain properties similar to NO (24). These compounds, acting as a "NO reservoir," could in turn stimulate at a distance cGMP formation and can prolong BT, inhibit platelet aggregation, relax vascular smooth muscle cells, and activate fibrinolysis (24). In addition, NO liberated by platelets and other circulating cells such as neutrophils may also account for this effect. This sustained modulation of platelet and aortic function by NO inhalation may thus imply that despite its very short chemical half-life in the blood, its effective biologic half-life could be longer (25).
In addition, in the absence of iNO administration, an increase in platelet and aortic cGMP contents was observed with intratracheal LPS, suggesting the existence of a systemic response coupled with lung inflammation. This effect might have resulted from an enhanced endogenous NO synthesis. Although the origin of this NO production was not assessed, platelets and vascular cells possess a NO synthase which could be directly activated by LPS, or indirectly via interactions of these cells with endothelial, circulating, or resident inflammatory cells (26). For platelets, such a paradoxical increase in cGMP following aggregation has been first reported by Radomski and coworkers (26) as an evidence of a specific L-arginine-NO pathway. This effect could be interpreted as a "counter-regulatory" effect of endogenous NO in platelets on the inflammation-induced platelet-hyperaggregability by intratracheal LPS. Accordingly, the activation of platelet cGMP pathway has been reported to prevent the platelet adhesion receptors' GPIIb/IIIa expression and the membrane expression of P-selectin (27). This effect results in an inhibition of platelet aggregation and cooperation with inflammatory cells. For vascular wall cells, the increased aortic cGMP contents after intratracheal LPS administration is consistent with similar increases reported after intravenous LPS injection (28), during which reduced vascular contractile responses dependent upon an activation of the NO pathway have been described, especially in smooth muscle cells (29). In our study, compared with the elevated aortic cGMP contents following LPS, no further increases in these already elevated aortic cGMP levels were observed after NO inhalation. Although the discussion of this last point can only be speculative, interestingly, Kurrek and coworkers (30) observed that inducible NO synthase activity was not depressed by iNO added to animals receiving LPS, suggesting that the lack of further increase in aortic cGMP contents is not secondary to a downregulation of inducible NOS activity after iNO administration.
The fibrinolytic system is mainly governed by the vascular wall and participates in the global hemostasis. Tissue-type plasminogen activator (t-PA) and its inhibitor, the plasminogen activator inhibitor type 1 (PAI-1) are both released by endothelial cells, thus regulating endogenous fibrinolysis (31, 32). If there is an agreement that NO donors inhibit PAI-1 secretion (33), their effects on t-PA activity remain controversial, with reports of both increase (34) or decrease (35) in t-PA activity after administration of the NO-donor sodium nitroprusside. As judged by the unchanged ECLT, iNO had no effects on the fibrinolytic balance in healthy rats, whereas it partially attenuated the decreased fibrinolytic response observed in animals receiving intratracheal LPS. Accordingly, in healthy humans, intravenous administration of endotoxin has been reported to induce a biphasic response of the fibrinolytic system (36), with a 1-h early response associated with an increase in plasma t-PA, putatively to prevent fibrin deposition by plasmin generation. Three hours after LPS, t-PA activity ceased abruptly, with increased levels of PAI-1 activity, as a secondary compensatory mechanism of the initially increased fibrinolytic activity (36). In our study, the change in ECLT in animals with acute pulmonary inflammation receiving iNO can be interpreted as a modulation of this reaction by iNO. This global response can be speculatively related to a decrease of t-PA or is more probably related to an increase in PAI-1 secretion, and therefore to an inhibition of t-PA activity. However, the absence of differential measurements of t-PA and PAI in this model, or of a kinetic study does not allow one to hypothesize if iNO would restore t-PA activity or decrease PAI.
In conclusion, based on an experimental model of intratracheal LPS administration, the effects of NO inhalation on the hemostatic, coagulation, and fibrinolytic systems were analyzed in the presence and absence of an acute pulmonary inflammation. Acute pulmonary inflammation did not change bleeding time, but increased platelet aggregation, platelet and aortic cGMP contents, and decreased fibrinolysis. The "extrapulmonary" effects of iNO on hemostasis were not strictly similar in healthy and endotoxic rats, consistent with the proper changes in coagulation parameters induced by the acute pulmonary inflammation. iNO attenuated the procoagulant activity induced by the pulmonary inflammation, suggesting a potentially beneficial effect of this therapy. Although it is difficult to specifically study the fate of iNO, it significantly increased platelet cGMP contents in healthy and endotoxic rats. The nonuniform influence of iNO on aortic cGMP contents in endotoxic and healthy animals suggests that complex changes induced by both inflammatory reaction and NO administration govern the interactions existing between circulating and vascular wall cells.
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Correspondence and requests for reprints should be addressed to Didier Payen, M.D., Ph.D., Department of Anesthesiology and Critical Care Medicine, Lariboisière University Hospital, 2 Rue Ambroise Paré, 75010 Paris, France.
(Received in original form September 23, 1997 and in revised form April 6, 1998).
Acknowledgments: The authors thank Dr. Vicaut for his statistical advice.
Supported in part by a grant from the Délégation à la Recherche Clinique AP-HP 1990-1992 and the Direction de la Recherche et de l'Enseignement Doctoral contract 1990-1995.
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References |
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REFERENCES
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