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Heart Vessels (2004) 19:33–37 DOI 10.1007/s00380-003-0729-5

© Springer-Verlag 2004

ORIGINAL ARTICLE

ORIGINAL ARTICLE

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Veronika Kloboves-Prevodnik · Misˇo S abovicˇ

Dusˇan Keber

Effect of blood passage through the pulmonary circulation on fibrinolytic parameters

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

Abstract The lung vasculature bed has a unique fibrinolytic potential, which has not yet been completely elucidated. We investigated the effect of blood passage through the pulmo- nary circulation on the values of fibrinolytic parameters in plasma. Forty-seven patients (16 women, 31 men, mean age 54 years, range 21–67 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 fibrinolytic 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 (6.7–11.3) vs 8.4 (5.9–11.0)ng/ ml; t-PA activity: 92 (5–680) vs 62 (32–696)IU/ml; PAI-1 antigen: 8.4 (5.5–14.3) vs 8.7 (6.2–13.1)ng/ml; and ECLT:

5.5 (4.1–9.1) vs 5.6 (4.1–8.5) 1000/min. In contrast, PAI activity decreased significantly: 7.9 (5.8–10.3) 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 fibrinolytic activity which is not attributed to t-PA is elevated in lung vascula- ture. Further, basal fibrinolytic activity in the lungs is not influenced by pulmonary hypertension.

Key words Pulmonary circulation · Pulmonary hyper- tension · Fibrinolysis · Tissue-type plasminogen activator · Plasminogen activator inhibitor

V. Kloboves-Prevodnik

Institute of Oncology, Ljubljana, Slovenia

 

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M.

S abovicˇ (*)

Department of Vascular Diseases, Internal Clinic, University Medical Centre, Zalosˇka 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

Introduction

Local fibrinolysis in the lungs is probably important for dissolving intravascular fibrin and permitting proper circula- tion through the lungs. The exact functioning of intravascu- lar fibrinolysis in the lungs is as yet incompletely understood. Basically, intravascular fibrinolysis 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 fibrinolytic activity, which reflects the tissue values of fibrinolytic 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 syn- thesis of t-PA. 3,4 Lung vasculature might be specific in this regard. While fibrinolytic activity in lung parenchyma in humans has been studied extensively 58 as well as the effect of exclusion of pulmonary circulation with additional circula- tion through extracorporeal circulation on fibrinolytic parameters, 9,10 there are only limited data regarding the intravascular fibrinolytic activity in lung vasculature. 11 We have investigated fibrinolytic activity in the pulmo- nary vasculature by simultaneous measurement of fibrin- olytic parameters in the right atrium and left ventricle. Differences in the measured parameters at these particular points are influenced 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 fibrinolytic parameters in whole lung vascu- lature to be estimated.

Materials and methods

Patients

Forty-seven patients (16 women, 31 men, mean age 54 years, range 21–67 years) were studied. All patients gave

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informed consent in accordance with local ethical commit- tee 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 dis- ease. Seven patients used warfarin, 12 used aspirin, and 2 used heparin.

Cardiac catheterization and sampling

Cardiac catheterization was performed between 8:30 and 13:30h 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 dam- age, which would induce the release of t-PA and/or PAI-1. 11 Samples were obtained 5min after insertion of the cath- eters. Heparin and contrast were not administered until after blood samples were taken. Samples of 15ml of blood were taken simultaneously from the right atrium (right heart catheterization) and the left ventricle (left heart cath- eterization). The first 6ml of blood was discarded.

Fibrinolytic parameters

Blood samples (9ml) with 1ml of citrate were centrifuged at 3 000 g for 15min at 4°C, and the supernatants snap frozen in liquid nitrogen and stored at 70°C. t-PA and PAI-1 antigens were determined by enzyme-linked im- munosorbent 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/ fibrin, 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 (compari- son of fibrinolytic 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 hyperten- sion and t-PA antigen and PAI-1 antigen were tested using Pearson’s coefficient. A P value of less than 0.05 was taken as statistically significant.

Results

Differences in fibrinolytic 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 (6.7–11.3)ng/ml; t-PA ac- tivity: 92 (5–680)IU/ml; PAI-1 antigen: 8.4 (5.5–14.3)ng/ml; PAI-1 activity: 7.9 (5.8–10.3)IU/ml; ECLT: 5.5 (4.1–9.1) 1 000/min. In the left ventricle the following values were obtained. t-PA antigen: 8.4 (5.9–11.0) ng/ml; t-PA activity:

62 (32–696) IU/ml; PAI-1 antigen: 8.7 (6.2–13.1)ng/ml; PAI- 1 activity: 7.4 (6.0–10.4) IU/ml; ECLT: 5.6 (4.1–8.5) 1000/ min. No significant differences between samples from the right atrium and left ventricle were observed for t-PA anti- gen (Fig. 1), t-PA activity, PAI-1 antigen, and ECLT. In contrast, PAI-1 activity was significantly (P 0.01) lower in the left ventricle (Fig. 2). During passage through the pul- monary vasculature the measured parameters increased, decreased, or remained unchanged in the following num- bers 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 significant (P 0.05). All other comparisons were not significant.

Comparison of fibrinolytic parameters in patients without and with pulmonary hypertension

Since diurnal variations could possibly influence the com- parison 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

in the right atrium and left ventricle. Points denote individual measurements; shadowed area denotes mean value

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

individual measurements; shadowed area denotes mean value catheterization was performed between 8:30 and 10:30h were

catheterization was performed between 8:30 and 10:30h were used for analysis. The fibrinolytic parameters were compared in a group of patients without (pulmonary sys- tolic pressure less than 30mmHg; n 9) and a group with pulmonary hypertension (pulmonary systolic pressure more than 30mmHg; n 12). Mean systolic pressures in the pulmonary artery were 27.1 2.6 vs 61.1 30.7mmHg and mean diastolic pressures 9.2 2.5 vs 28.5 12.6mmHg. No differences were observed in fibrinolytic parameters obtained in the left ventricle: t-PA antigen, 11.1 (8.3–13.4) vs 9.4 (5.2–12.0) ng/ml; t-PA activity, 58 (41–568) vs 91 (9–659)IU/ml; PAI-1 antigen, 9.8 (8.1–13.6) vs 12.2 (6.8–15.6)ng/ml; PAI-1 activity, 10.2 (6.3–14.7) vs 7.4 (6.6–9.5) IU/ml; ECLT, 5.0 (4.1–6.6) vs 4.9 (3.9–15.4) 1 000/min. Similarly, no differences were observed in fibrinolytic parameters obtained in the right atrium (data not shown). No correlation between the degree of pulmonary hyper- tension 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 fibrinolytic potential. To investigate the fibrin- olytic activity in lung vasculature under basal conditions we measured simultaneously fibrinolytic parameters (t-PA an- tigen 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 fibrinolytic sys- tem, it is important to find out whether it is released con- tinuously in the pulmonary vasculature, thereby increasing

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in the pulmonary vasculature, thereby increasing 35 Fig. 3. Relation between t-PA antigen, obtained in the

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

basal fibrinolytic 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 finding 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-specific. Furthermore, our results do not confirm 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 indi- cates 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 significantly decreased. This result implies an in- crease in fibrinolytic 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 fibrinolytic activity, during lung passage could be explained by the fact that ECLT is a less sensitive and specific test than PAI activity. 19 It is known that PAI-1 activity is regulated by two activators: t-PA and urokinase- type plasminogen activator (u-PA). 20 Thus, it seems very likely that the increased fibrinolytic 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 fibrin degradation within the vasculature, whereas u-PA acts primarily in the context of cell surfaces to promote cell adhesion and migra- tion through matrices and lysis of fibrin 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 intra- vascular fibrinolysis. 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 re- lease, 27 one could speculate that in the lung vasculature u-PA is released under basal conditions, whereas t-PA is released after stimulus by fibrin. In regard to the mentioned hypothesis, our study is limited by the fact that we did not measure u-PA antigen. Anyway, our results confirm that under basal conditions fibrinolytic activity in the lung vascu- lature is elevated. Whether increased basal activity is fur- ther 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 fibrinolytic param- eters), we compared the groups with and without pul- monary hypertension. Elevated intravascular pressure, as occurs in pulmonary hypertension, might constitute a chronic stimulus on pulmonary endothelium. It could in- duce either an increased release of fibrinolytic parameters or the exhaustion of secretion. However, no difference in pre- and post-lung levels of t-PA antigen (and other fibrin- olytic parameters) was found. This suggests that pulmonary hypertension does not significantly influence the release of t-PA in the lung endothelium under basal conditions. On the other hand, our result indicates that the fibrinolytic activity in the lungs of patients with pulmonary hyper- tension (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 fibrinolysis in the lungs. Our results do not confirm these assumptions. Analysis of corre- lations between the degree of pulmonary hypertension and both t-PA antigen and PAI-1 antigen did not show significant relations. Overall, it appears that pulmonary hy- pertension does not influence (basal) fibrinolytic activity in the lungs. In conclusion, the significantly decreased PAI activity measured after blood passage through the lungs shows that under basal conditions fibrinolytic activity in the lung vascu- lature is elevated. This increase in fibrinolytic 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 influence the release of either t-PA or other fibrinolytic 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 cathe- terization, and to Gerard Doojewaard, PhD (Gaubius Laboratory, TNO-PG, Leiden, the Netherlands) for measuring PAI-1 antigen and activity.

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