Thrombotic complications of pulmonary circulation occur in patients with chronic obstructive pulmonary disease (COPD). In the present study, we sought to evaluate in vivo platelet activation through the measurement of 11-dehydro-thromboxane (Tx) B2, TxA2 major metabolite in the urine, in 29 patients with COPD, compared with 29 sex- and age-matched healthy subjects. The urinary excretion of 11-dehydro-TxB2 was significantly higher in patients with COPD than in control subjects: median (range), 753 (277–4,409) and 275 (129–612) pg/mg creatinine, respectively; p < 0.0001). Moreover, 11-dehydro-TxB2 excretion was inversely related with arterial oxygen tension (rho = − 0.46; p = 0.0145). In five of the 29 patients a short-term therapeutic course with oxygen supplementation induced a significant decrease of urinary 11-dehydro-TxB2 excretion: median range, 941 (452–2,640) to 445 (166–1,560) pg/mg creatinine. Moreover, selective inhibition of platelet cyclooxygenase activity by low-dose aspirin was associated with more than 90% inhibition of thromboxane metabolite excretion, demonstrating its being of platelet origin. Plasma levels of prothrombin fragment F1 + 2 were higher in patients than in control subjects (2.6 ± 1.5 versus 0.9 ± 0.4 nM, p = 0.0001). No relation between 11-dehydro-TxB2 excretion and plasma F1 + 2 levels was found. We conclude that platelet TxA2 biosynthesis is enhanced in patients with COPD and may be influenced by arterial oxygen tension changes. Davı̀ G, Basili S, Vieri M, Cipollone F, Santarone S, Alessandri C, Gazzaniga PP, Cordova C, Violi F, Chronic Obstructive Bronchitis and Haemostasis Study Group. Enhanced thromboxane biosynthesis in patients with chronic obstructive pulmonary disease.
Clinical and autopsy studies provide evidence that the clinical history of patients with chronic obstructive pulmonary disease (COPD) may be complicated by thrombotic events occurring in the pulmonary vessels (1, 2). The mechanism leading to thrombosis in patients with COPD is still uncertain. Previous ex vivo studies in patients with COPD have demonstrated increased platelet sensitivity to a variety of agonists and elevated plasma levels of β-thromboglobulin (3-7). Even if these studies suggest the occurrence of platelet “hyperactivity” in patients with COPD, artefacts related to the evaluation of platelet function ex vivo render uncertain whether these findings actually reflect the occurrence of in vivo platelet activation (8).
The rate of thromboxane biosynthesis in vivo, as reflected by the urinary excretion of its major enzymatic metabolites (9, 10), represents an index of platelet activation in vivo with a favorable signal-to-noise ratio, by virtue of this eicosanoid being synthesized and released by platelets in response to a variety of agonists and in turn inducing platelet aggregation. Until now, urinary excretion of 11-dehydro-thromboxane (Tx) B2, a major enzymatic metabolite of TxA2, has been studied in a small cohort of patients with COPD by Christman and coworkers (11). These investigators did not find any difference in comparison with control subjects, but three of nine patients with COPD had increased urinary 11-dehydro-thromboxane B2 excretion.
The aims of this study were (1) to evaluate the rate of thromboxane biosynthesis in a larger group of patients with COPD than previously reported; (2) to gain insights into the pathophysiologic mechanisms involved in thromboxane-dependent platelet activation; (3) to analyze the relationship between hypoxia and urinary excretion of 11-dehydro-thromboxane B2.
Twenty-nine consecutive outpatients 51 to 77 yr of age (seven women and 22 men, seven current smokers and 17 ex-smokers), with chronic bronchitis and irreversible airway obstruction, and 29 healthy subjects 51 to 77 yr of age (seven women and 22 men, six current smokers and 18 ex-smokers), were studied on several occasions between May 1992 and October 1994. Informed consent was obtained from each subject.
A diagnosis of COPD was established on the basis of clinical history, physical examination, chest radiographs, electrocardiographic and echocardiographic evidences of right heart strain, arterial blood gas analysis, and physiologic ventilation tests. The inclusion criteria were a ratio FEV1 to FVC of less than 70% of the predicted value and a hematocrit value lower than 50%. Patients with acute inflammatory disease, chronic asthma, episodes of previous pulmonary embolism, metabolic acidosis, immunologic disease, cancer, history of venous or arterial thrombosis, hypertension, peripheral vascular disease, diabetes mellitus, and renal disease were excluded.
The standard treatment consisted of cardiac glycosides (n = 12), salbutamol (n = 15), and aminophylline (n = 25); six patients received occasional oxygen supplementation after exercise or at night (1 to 2 L/min by 0.24 Venturi's mask).
None of the subjects enrolled in the study had received any drug known to interfere with the coagulation system or platelet function during the previous month.
The individual characteristics of the patients with COPD are detailed in Table 1.
|Patient No.||Age (yr)||Sex||Smoking||PaO2 (mm Hg)||PaCO2 (mm Hg)||FEV1(L)||F1+2 (nM)||U-11-dehydro-TxB2(pg/mg creatinine)|
In the first study, a cross-sectional comparison of thromboxane metabolite excretion and prothrombin fragment F1+2 plasma levels was performed between patients and control subjects. Both patients and control subjects were studied on an outpatient basis and fasted overnight before the study. Patients were not given oxygen therapy in the previous 12 h, and, if smokers, had not smoked in the previous 12 h. After a 20-min rest period, a blood sample was drawn from the radial artery by a heparinized syringe and immediately used for blood gas analysis; another blood sample was taken from the antecubital vein to study clotting system. Urine was collected during the 12 h preceding blood sampling; the samples were frozen immediately and kept at −20° C until extraction.
A second study was designed to examine the effect of a short-term oxygen treatment on urinary 11-dehydro-TxB2 excretion. Five of the 29 patients (four male and one female; three ex-smokers; 66 to 74 yr of age) with a PaO2 of 61 ± 14 mm Hg and a PaCO2 of 48 ± 18 mm Hg were given oxygen therapy (1 to 2 L/min for 15 h daily by 0.24 Venturi's mask); urine was collected before and after 15 d of oxygen treatment. All patients were instructed to maintain their standard therapy during this phase of the study.
A third study was designed to evaluate the relative contribution of platelets to the excretion of 11-dehydro-TxB2. For this purpose five patients 51 to 70 yr of age (two female, three male) were given aspirin (Bayer S.p.A., Milan, Italy) at a dose of 100 mg/d for 7 d, and 12-h urine collections were obtained immediately before and on the seventh day of treatment. The five patients were selected on the basis of (1) no contraindication to aspirin therapy and (2) willingness to participate in the study. In comparison with the general COPD population these patients showed similar values of PaO2 : median (range), 55 (51–83) mm Hg; FEV1: 0.96 (0.80–1.40) L; and PaCO2 : 43 (38–52) mm Hg.
The studies were approved by the ethical committee board of our Institution.
Arterial blood samples were analyzed for gas tension on a Radiometer ABL3 (Detta, Copenhagen, Denmark).
The FEV1 and FVC were measured with a dry spirometer.
Plasma F1+2 concentration was measured by an enzyme immunoassay (Enzygnost F1+2; Behringwerke AG, Marburg, Germany), according to the manufacturer's instructions. In 20 healthy subjects matched for sex and age the mean ± standard deviation was 0.81 ± 0.42 mM (range, 0.35 to 1.8). Intraassay and interassay coefficients of variation were 8 and 9%, respectively.
Measurement of urinary 11-dehydro-thromboxane B2 was performed by a previously described and validated radioimmunoassay method (10).
Statistical analysis was performed by chi-square statistics or Fisher's exact test (if n ⩽ 5) for independence and by unpaired t test. When necessary appropriate nonparametric tests were employed. Spearman's rank correlation test was used to study the different correlations. The effect of oxygen therapy on metabolite excretion was analyzed with Wilcoxon's signed-rank test. Data are presented as mean ± SD and 95% confidence limits (95% CL). Median and range are given for urinary 11-dehydro-thromboxane B2 because it shows appreciably skewed distribution. Only two-tailed probabilities were used for testing statistical significance; p values lower than 0.05 were regarded as statistically significant (12). All calculations were made with the computer program Stat-View II (Abacus Concepts, Berkeley, CA).
Clinical and laboratory characteristics of the patients with COPD are shown in Table 1. Five never smoked, seven were current smokers (more than 10 cigarettes daily), and 17 stopped smoking 2 to 4 yr before the study. PaO2 was 68 ± 13 mm Hg (95% CL, 66–74), six (21%) patients had PaO2 values below or equal to 55 mm Hg (definition of resting hypoxemia). Mean blood PaCO2 was 43 ± 9 mm Hg (95% CL, 39 to 47). FEV1 was inversely correlated with PaCO2 (rho = −0.68, p < 0.0003) and directly with PaO2 (rho = 0.35, p < 0.06).
Urinary 11-dehydro-TXB2 excretion was significantly higher in patients with COPD than in matched control subjects: median (range), 753 (277– 4,409) versus 275 (129–612) pg/mg creatinine; p < 0.0001 (Figure 1). In 24 (83%) of the 29 patients, metabolite excretion was more than 2 SD above the normal mean (539 pg/mg creatinine) (Figure 1). After excluding smokers and ex-smokers, patients with COPD (n = 5) had values of urinary 11-dehydro-TxB2 excretion significantly higher than those of the five control subjects: median (range), 642 (305–4,407) versus 254 (236–418) pg/mg creatinine, respectively (p < 0.03).
Among patients with COPD, never-smokers (n = 5), ex-smokers (n = 17), and current-smokers (n = 7) had similar amounts of excretion: median (range), 642 (305–4,407), 753 (277–4,409), and 807 (585–1,591) pg/mg creatinine, respectively (p > 0.05). The six patients who were given occasional oxygen supplementation, had a mean PaO2 of 67.5 ± 14 mm Hg, and urinary 11-dehydro-TxB2 excretion was similar to that observed in the whole population: median (range), 656 (277– 1,592) pg/mg creatinine (p > 0.05).
In five patients 66 to 74 yr of age (four male and one female) the reproducibility of 11-dehydro-thromboxane B2 excretion was assessed by obtaining an additional urine sample 1 mo later. Throughout this period the patients, whose clinical, therapeutic, and spirometric characteristics remained stable, did not show significant changes of urinary 11-dehydro-TxB2 excretion: median (range), 850 (345–1,760) versus 643 (257–2,012) pg/mg creatinine (p = 0.34), suggesting that their usual treatment did not affect urinary 11-dehydro-TxB2 levels.
In no patient was 11-dehydro-TxB2 excretion correlated with PaCO2 (rho = 0.14, p > 0.05) and FEV1 (rho = 0.13, p > 0.05). Conversely, a statistically significant inverse correlation between PaO2 and urinary 11-dehydro-TxB2 levels was found (rho = −0.46, p = 0.0145) (Figure 2). Also, in patients with resting hypoxemia (PaO2 below or equal to 55 mm Hg) urinary 11- dehydro-TxB2 excretion levels were higher than in patients (n = 23) with PaO2 > 55 mm Hg: median (range), 1,676 (807– 4,409) versus 670 (277–1,992) pg/mg creatinine (p < 0.002).
In patients (n = 5) undergoing short-term oxygen treatment urinary 11-dehydro-TxB2 excretion rate decreased significantly (−41%, p < 0.05) at the end of treatment period: from median (range), 941 (452–2,640) to 445 (166–1,560) pg/mg creatinine (Figure 3). Post-treatment PaO2 values were quite similar to those observed at the entry in the study: median (range), 62 (47–79) versus 71 (49–80) mm Hg (p = 0.181).
Plasma levels of fragment F1+2 were significantly higher in patients with COPD than in control subjects (2.6 ± 1.5 versus 1.1 ± 0.2 nM, p = 0.0001). Twenty-five patients with COPD (86%) had values of F1+2 above 1.54 nM (mean, +2 SD of control subjects). Plasma F1+2 was not correlated with 11- dehydro-TxB2 (rho = −0.12, p = 0.526), PaO2 (rho = −0.04, p = 0.844), or PaCO2 (rho = 0.01, p = 0.942).
To characterize the enhanced excretion of 11-dehydro-TxB2 in patients with COPD as being from platelet or nonplatelet origin, we evaluated the short-term effect of a platelet-selective regimen of aspirin therapy (100 mg/d for 7 d) on the degree of suppression of metabolite excretion in five patients. Before aspirin administration, the amount of 11-dehydro-TxB2 excretion averaged 1,095 ± 636 pg/mg creatinine. After 1 wk of aspirin administration, metabolite excretion was significantly reduced to 79 ± 65 pg/mg creatinine (Figure 4). This finding is consistent with the cumulative nature of acetylation of platelet PGG/H synthase and inhibition of thromboxane biosynthesis by low-dose aspirin in healthy subjects (13, 14).
In patients with COPD, thromboembolic events occur in about 28% of those with chronic airway obstruction. This percentage is higher when COPD is complicated by congestive heart failure (2).
Recently, Jousilahti and coworkers (15) showed that patients with chronic bronchitis, which is one of the major causes of COPD, are at high risk of coronary disease, independent of other known risk factors such as age, smoking, serum cholesterol, and systolic blood pressure. The significant association between COPD and in vivo platelet activation found in our study gives an explanation of this increased risk of thrombosis. In fact, the enhanced thromboxane biosynthesis in patients with COPD is likely to reflect in vivo platelet activation, in view of the capacity of low-dose aspirin to selectively acetylate platelet PGG/H synthase in a cumulative fashion upon repeated daily dosing (13, 14). The finding of approximately 90% suppression in 11-dehydro-TxB2 excretion after a 1-wk administration of low-dose aspirin is consistent with platelets representing a major source of enhanced TxA2 production (Figure 4).
Because patients with COPD are usually smokers and smoking is associated with increased thromboxane biosynthesis (16) a possible explanation of our finding could be that the increased thromboxane biosynthesis is not due to COPD per se but to the smoking habit of patients. However, we did not find differences in thromboxane biosynthesis in smoking and ex-smoking patients with COPD.
We also examined whether increased thromboxane biosynthesis may be, at least in part, a consequence of hypoxia. In fact, a previous in vitro study demonstrated that platelets under conditions of hypoxia have increased sensitivity and synthesize greater amounts of thromboxane B2 in response to several agonists (17). Moreover in animals and human beings exposed to simulated or real altitude platelet hyperaggregability, increased thromboxane production and platelet β-thromboglobulin release have been demonstrated (18-20). These findings suggest that hypoxia may play a role in inducing platelet activation inasmuch as we found that hypoxia is associated with an increased thromboxane biosynthesis and PaO2 is inversely correlated with urinary 11-dehydro-TxB2 levels. This link is strengthened by the oxygen administration study. Our data are in agreement with previous findings showing in patients with COPD a shortened platelet half-life increased after oxygen administration (5, 21-23).
The mechanism responsible for the association between hypoxemia and increased thromboxane biosynthesis is not clear. Hypoxia might per se induce metabolic changes on the platelet membrane, leading to increased activation of the arachidonic acid pathway. In fact, in vitro reduction of oxygen content is associated with spontaneous platelet aggregation and increased platelet thromboxane A2 synthesis (17). Alternatively, hypoxia might indirectly affect platelet function by virtue of its capability to induce endothelial perturbation and clotting activation. Signs of endothelial activation have been found in subjects exposed to altitude (24, 25). Moreover, in vitro hypoxia increases procoagulant activity of endothelial cells with reduced thrombomodulin expression and with activation of factor X (26). The shift of endothelial function towards a procoagulant state could explain the prothombotic state observed in COPD (6, 27), with an increased thrombin generation, which in turn induces enhanced platelet thromboxane formation (28). The lack of correlation between F1+2 and urinary 11-dehydro-TxB2 levels does not exclude this hypothesis because of different half-lives of F1+2 and 11-dehydro-TxB2 (90 min and 1 to 3 min, respectively) and different procedures to measure F1+2 (plasma levels) and 11-dehydro-TxB2 (urinary excretion).
In conclusion, this study demonstrated that an increased thromboxane biosynthesis is present in a large percentage of patients with COPD. Hypoxia may play a role, inducing directly or indirectly thromboxane-dependent platelet activation. This finding gives new insight into the mechanism(s) responsible for thrombotic vascular complications in COPD and offers a rationale for testing the potential usefulness of antiplatelet drugs in this clinical setting.
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The Chronic Obstructive Bronchitis and Haemostasis (C.O.B.H.) Study Group comprised the following: Institute of Medical Therapy, University of Rome I, Italy: N. De Luca, M.D., L. Coppotelli, M.D., M. Paradiso, M.D., A. Bellomo, M.D., and A. Belogi, M.D. Experimental Medicine Department, University of Rome I, Italy: P. Ferroni, M.D., and F. Pulcinelli, Ph.D. Cattedra di Ematalogia, Università di Chieti “G. D'Annunzio,” Italy: S. Roccaforte, M.D., and T. Antidormi, M.D.