Heart Vessels (2004) 19:3337 DOI 10.

1007/s00380-003-0729-5

Springer-Verlag 2004

ORIGINAL ARTICLE abovic Veronika Kloboves-Prevodnik Mis o S Dus an Keber

Effect of blood passage through the pulmonary circulation on brinolytic parameters

Received: April 22, 2003 / Accepted: July 11, 2003

Abstract The lung vasculature bed has a unique brinolytic potential, which has not yet been completely elucidated. We investigated the effect of blood passage through the pulmonary circulation on the values of brinolytic parameters in plasma. Forty-seven patients (16 women, 31 men, mean age 54 years, range 2167 years) who had undergone elective cardiac catheterization were included in the study. The blood samples were taken simultaneously from the right atrium and the left ventricle. The following brinolytic parameters were measured: tissue-type plasminogen (t-PA) antigen and activity, plasminogen activator inhibitor-1 (PAI-1) antigen and activity, and euglobulin clot lysis time (ECLT). No difference was found between the samples obtained from the right atrium and the left ventricle with respect to t-PA antigen: 8.1 (6.711.3) vs 8.4 (5.911.0) ng/ ml; t-PA activity: 92 (5680) vs 62 (32696) IU/ml; PAI-1 antigen: 8.4 (5.514.3) vs 8.7 (6.213.1) ng/ml; and ECLT: 5.5 (4.19.1) vs 5.6 (4.18.5) 1 000/min. In contrast, PAI activity decreased signicantly: 7.9 (5.810.3) vs 7.4 (6.0 10.4) IU/ml, P 0.01. Patients with and without pulmonary hypertension did not differ in any of measured parameters, either in the right atrium or in the left ventricle. These results show that under basal conditions brinolytic activity which is not attributed to t-PA is elevated in lung vasculature. Further, basal brinolytic activity in the lungs is not inuenced by pulmonary hypertension. Key words Pulmonary circulation Pulmonary hypertension Fibrinolysis Tissue-type plasminogen activator Plasminogen activator inhibitor

Introduction
Local brinolysis in the lungs is probably important for dissolving intravascular brin and permitting proper circulation through the lungs. The exact functioning of intravascular brinolysis in the lungs is as yet incompletely understood. Basically, intravascular brinolysis depends primarily on the relation between tissue-type plasminogen activator (t-PA) and the inhibitor of plasminogen activator (PAI-1).1,2 t-PA is secreted from endothelial cells, whereas PAI-1 is released from platelets and from endothelial cells.1,2 It is likely that local intravascular brinolytic activity, which reects the tissue values of brinolytic parameters, differs in different organs. It has been shown that the differences in organ concentrations might be due to differences in endothelial cell tissue density and/or regional differences in the rate of synthesis of t-PA.3,4 Lung vasculature might be specic in this regard. While brinolytic activity in lung parenchyma in humans has been studied extensively58 as well as the effect of exclusion of pulmonary circulation with additional circulation through extracorporeal circulation on brinolytic parameters,9,10 there are only limited data regarding the intravascular brinolytic activity in lung vasculature.11 We have investigated brinolytic activity in the pulmonary vasculature by simultaneous measurement of brinolytic parameters in the right atrium and left ventricle. Differences in the measured parameters at these particular points are inuenced solely by events in the pulmonary vasculature: the release or consumption of activators and inhibitors. Such an approach allows complete net release or consumption of brinolytic parameters in whole lung vasculature to be estimated.

V. Kloboves-Prevodnik Institute of Oncology, Ljubljana, Slovenia abovic M. S (*) Department of Vascular Diseases, Internal Clinic, University Medical Centre, Zalos ka 2, 1000 Ljubljana, Slovenia Tel. 386-1-522-8080; Fax 386-1-522-8070 e-mail: miso.sabovic@trnovo.kclj.si D. Keber Ministry of Health, Ljubljana, Slovenia

Materials and methods

Patients Forty-seven patients (16 women, 31 men, mean age 54 years, range 2167 years) were studied. All patients gave

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informed consent in accordance with local ethical committee approval. The reasons for elective catheterization were ischemic heart disease (n 8), valve disease (n 30), and ischemic heart disease together with valve disease (n 9). Ischemic heart disease was in a stable phase; patients with acute coronary syndrome were not included in the study. Patients did not have an acute illness or malign disease. Seven patients used warfarin, 12 used aspirin, and 2 used heparin. Cardiac catheterization and sampling Cardiac catheterization was performed between 8:30 and 13:30 h using a standard technique. Seven-French hemostat sheaths were inserted into the right femoral artery and vein under local anesthetic. Separate 7-F catheters were used for blood samples at the arterial and venous circulations. It was shown that these catheters do not induce endothelial damage, which would induce the release of t-PA and/or PAI-1.11 Samples were obtained 5 min after insertion of the catheters. Heparin and contrast were not administered until after blood samples were taken. Samples of 15 ml of blood were taken simultaneously from the right atrium (right heart catheterization) and the left ventricle (left heart catheterization). The rst 6 ml of blood was discarded. Fibrinolytic parameters Blood samples (9 ml) with 1 ml of citrate were centrifuged at 3 000 g for 15 min at 4C, and the supernatants snap frozen in liquid nitrogen and stored at 70C. t-PA and PAI-1 antigens were determined by enzyme-linked immunosorbent assay (Imulyse t-PA and Imulyse PAI-1, both from Biopool, Umea, Sweden). t-PA activity and PAI activity were determined by amidolytic assays (Spectrolyse/ brin, Biopool). Euglobulin clot lysis time (ECLT) was measured according to Buckell.12 Statistical methods Fibrinolytic parameters were not normally distributed. Paired dependent samples (obtained simultaneously from the right atrium and left ventricle) were compared using the Mann-Whitney U-test. Non-dependent samples (comparison of brinolytic parameters in patients with and without pulmonary hypertension) were analyzed by the Wilcoxon test. The 2 test was used to compare discrete variables. Correlations between the degree of pulmonary hypertension and t-PA antigen and PAI-1 antigen were tested using Pearsons coefcient. A P value of less than 0.05 was taken as statistically signicant.

Results
Differences in brinolytic parameters between the right atrium and left ventricle Samples were taken simultaneously from the right atrium and left ventricle. In the right atrium the following values were obtained. t-PA antigen: 8.1 (6.711.3) ng/ml; t-PA activity: 92 (5680) IU/ml; PAI-1 antigen: 8.4 (5.514.3) ng/ml; PAI-1 activity: 7.9 (5.810.3) IU/ml; ECLT: 5.5 (4.19.1) 1 000/min. In the left ventricle the following values were obtained. t-PA antigen: 8.4 (5.911.0) ng/ml; t-PA activity: 62 (32696) IU/ml; PAI-1 antigen: 8.7 (6.213.1) ng/ml; PAI1 activity: 7.4 (6.010.4) IU/ml; ECLT: 5.6 (4.18.5) 1 000/ min. No signicant differences between samples from the right atrium and left ventricle were observed for t-PA antigen (Fig. 1), t-PA activity, PAI-1 antigen, and ECLT. In contrast, PAI-1 activity was signicantly (P 0.01) lower in the left ventricle (Fig. 2). During passage through the pulmonary vasculature the measured parameters increased, decreased, or remained unchanged in the following numbers of studied persons: 16/14/17, 18/15/14, 14/18/15, 7/31/9, and 15/13/19, for t-PA antigen, t-PA activity, PAI antigen, PAI activity, and ECLT, respectively. The proportion of patients with decreased PAI activity after passage through the pulmonary vasculature versus patients with increased or unchanged PAI activity levels was signicant (P 0.05). All other comparisons were not signicant. Comparison of brinolytic parameters in patients without and with pulmonary hypertension Since diurnal variations could possibly inuence the comparison of the two groups, only patients (n 21) in whom

Fig. 1. Tissue-type plasminogen (t-PA) antigen in the right atrium and left ventricle. Points denote individual measurements; shadowed area denotes mean value

35 Fig. 2. Plasminogen activator inhibitor1 (PAI-1) activity in the right atrium and left ventricle. Points denote individual measurements; shadowed area denotes mean value

Fig. 3. Relation between t-PA antigen, obtained in the left ventricle, and degree of pulmonary hypertension (systolic pressure in pulmonary artery)

catheterization was performed between 8:30 and 10:30 h were used for analysis. The brinolytic parameters were compared in a group of patients without (pulmonary systolic pressure less than 30 mmHg; n 9) and a group with pulmonary hypertension (pulmonary systolic pressure more than 30 mmHg; n 12). Mean systolic pressures in the pulmonary artery were 27.1 2.6 vs 61.1 30.7 mmHg and mean diastolic pressures 9.2 2.5 vs 28.5 12.6 mmHg. No differences were observed in brinolytic parameters obtained in the left ventricle: t-PA antigen, 11.1 (8.313.4) vs 9.4 (5.212.0) ng/ml; t-PA activity, 58 (41568) vs 91 (9659) IU/ml; PAI-1 antigen, 9.8 (8.113.6) vs 12.2 (6.815.6) ng/ml; PAI-1 activity, 10.2 (6.314.7) vs 7.4 (6.69.5) IU/ml; ECLT, 5.0 (4.16.6) vs 4.9 (3.915.4) 1 000/min. Similarly, no differences were observed in brinolytic parameters obtained in the right atrium (data not shown). No correlation between the degree of pulmonary hypertension and t-PA antigen (Fig. 3) and PAI-1 antigen (data not shown), both measured in the left ventricle, was found.

Discussion
It is widely believed that the lung vasculature possesses a unique, high brinolytic potential. To investigate the brinolytic activity in lung vasculature under basal conditions we measured simultaneously brinolytic parameters (t-PA antigen and activity, PAI-1 antigen and activity, and ECLT) in the right atrium and left ventricle during left and right heart catheterization. Since t-PA is the main activator of the brinolytic system, it is important to nd out whether it is released continuously in the pulmonary vasculature, thereby increasing

basal brinolytic activity. This has been assumed by some authors, but not measured.13 In our cohort of patients no differences between pre- and post-lung plasma levels of t-PA antigen and t-PA activity were found. This suggests that, at least under basal conditions, t-PA is not released from the pulmonary endothelium in appreciable amounts. This nding is in contrast to the observation of elevated levels of t-PA antigen after passage of blood through the lungs in mice,14 but in agreement with the results of Jern et al., who found no release of t-PA in the lungs under basal conditions in pigs.15,16 It is possible that t-PA release in the lungs is species-specic. Furthermore, our results do not conrm the opposite assumption that t-PA is partially cleared from blood by binding to endothelial cells in the lung vasculature.17,18 Levels of PAI-1 antigen also did not change during passage through the lungs. This result indicates that PAI-1 is neither secreted by endothelial cells nor consumed or degraded in the lungs. During the passage of blood through the lungs, PAI-1 activity signicantly decreased. This result implies an increase in brinolytic activity in the lung vasculature that could not be attributed to a release of t-PA (since t-PA antigen levels remained unchanged) but rather to other unmeasured activator(s). Unchanged values of ECLT, a global test of brinolytic activity, during lung passage could be explained by the fact that ECLT is a less sensitive and specic test than PAI activity.19 It is known that PAI-1 activity is regulated by two activators: t-PA and urokinasetype plasminogen activator (u-PA).20 Thus, it seems very likely that the increased brinolytic activity in the lungs is due to the release of u-PA. This is unexpected, since it is well known that t-PA is implicated in brin degradation within the vasculature, whereas u-PA acts primarily in the context of cell surfaces to promote cell adhesion and migration through matrices and lysis of brin deposited at ex-

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travascular sites.21,22 However, several recent studies have shown that u-PA might also have an important role in intravascular brinolysis.23,24 Furthermore, u-PA and t-PA might act synergistically on thrombolysis, thereby achieving effective endogenous lysis at lower concentrations of both activators.25,26 Since it is known that various coagulation activation products are potent stimuli of acute t-PA release,27 one could speculate that in the lung vasculature u-PA is released under basal conditions, whereas t-PA is released after stimulus by brin. In regard to the mentioned hypothesis, our study is limited by the fact that we did not measure u-PA antigen. Anyway, our results conrm that under basal conditions brinolytic activity in the lung vasculature is elevated. Whether increased basal activity is further elevated by local and/or systemic stimuli remains unanswered, but it appears like to occur. To study the effect of pulmonary hypertension on the release of t-PA (and changes in other brinolytic parameters), we compared the groups with and without pulmonary hypertension. Elevated intravascular pressure, as occurs in pulmonary hypertension, might constitute a chronic stimulus on pulmonary endothelium. It could induce either an increased release of brinolytic parameters or the exhaustion of secretion. However, no difference in pre- and post-lung levels of t-PA antigen (and other brinolytic parameters) was found. This suggests that pulmonary hypertension does not signicantly inuence the release of t-PA in the lung endothelium under basal conditions. On the other hand, our result indicates that the brinolytic activity in the lungs of patients with pulmonary hypertension (patients with pulmonary hypertension due to pulmonary embolism were not included) is not impaired. This would be clinically relevant, since patients with pulmonary hypertension might, at least theoretically, have primary or secondary impaired brinolysis in the lungs. Our results do not conrm these assumptions. Analysis of correlations between the degree of pulmonary hypertension and both t-PA antigen and PAI-1 antigen did not show signicant relations. Overall, it appears that pulmonary hypertension does not inuence (basal) brinolytic activity in the lungs. In conclusion, the signicantly decreased PAI activity measured after blood passage through the lungs shows that under basal conditions brinolytic activity in the lung vasculature is elevated. This increase in brinolytic activity is not due to the release of t-PA, but to other unmeasured activator(s), of which u-PA is the most likely candidate. Pulmonary hypertension does not inuence the release of either t-PA or other brinolytic parameters.
Acknowledgments We are grateful to Andrej Cijan, MD, PhD (Department of Cardiology, University Medical Centre, Ljubljana, Slovenia) for providing blood samples obtained during heart catheterization, and to Gerard Doojewaard, PhD (Gaubius Laboratory, TNO-PG, Leiden, the Netherlands) for measuring PAI-1 antigen and activity.

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