Changes in the alveolar hemostatic balance in severe pneumonia were compared with those in the acute respiratory distress syndrome (ARDS). Analysis was performed in bronchoalveolar lavage fluids (BALF) of patients with ARDS triggered by nonpulmonary underlying events in the absence of lung infection (ARDS; n = 25), pneumonia demanding mechanical ventilation (PNEU-vent; n = 114), spontaneously breathing patients with pneumonia (PNEU-spon; n = 40), and ARDS in combination with lung infection (ARDS + PNEU; n = 43); comparison with healthy control subjects (n = 35) was performed. In all groups of patients, BALF total procoagulant activity was increased by nearly two orders of magnitude, being largely attributable to the tissue factor pathway of coagulation. Concomitantly, markedly reduced overall fibrinolytic capacity (fibrin plate assay) was noted in the lavage fluids of all patients. BALF levels of urokinase-type plasminogen activator were significantly reduced throughout, whereas the lavage concentrations of tissue-type plasminogen activator did not differ from those in control subjects. In addition, markedly enhanced levels of plasminogen activator- inhibitor I and α2-antiplasmin were noted in ARDS, ARDS + PNEU, and PNEU-vent, but not in PNEU-spon. In all groups of patients, the changes in the lavage enzymatic activities were paralleled by manifold increased BALF concentrations of fibrinopeptide A and D-dimer, reflecting in vivo coagulation processes. Within the overall number of patients with pneumonia, changes in the alveolar hemostatic balance were more prominent in alveolar and interstitial pneumonia than in bronchopneumonia. Acute inflammatory lung injury, whether triggered by nonpulmonary systemic events or primary lung infection, is thus consistently characterized by both enhanced procoagulant and depressed fibrinolytic activities in the alveolar lining layer, with the appearance of fibrin formation in this compartment. Profile and extent of changes in severe pneumonia demanding respirator therapy are virtually identical to those in ARDS, whereas somewhat less prominent alterations of the alveolar hemostatic balance are noted in spontaneously breathing patients with pneumonia. Günther A, Mosavi P, Heinemann S, Ruppert C, Muth H, Markart P, Grimminger F, Walmrath D, Temmesfeld-Wollbrück B, Seeger W. Alveolar fibrin formation caused by enhanced procoagulant and depressed fibrinolytic capacities in severe pneumonia: comparison with the acute respiratory distress syndrome.
Intraalveolar fibrin deposition, commonly described as hyaline membrane formation, is a hallmark of many acute inflammatory lung diseases (1). Such fibrin formation may exert beneficial effects in the gas exchange area by sealing leakage sites where the capillary endothelial and in particular the alveolar epithelial barrier are compromised, e.g., at locations of denuded alveolar epithelium, and by providing a primary matrix for wound repair in this region. On the other hand, alveolar clotting processes may also be harmful if overwhelming and persistent. Neutrophils and fibroblasts may be activated by thrombin (2), fibrinopeptides (3), and fibrin degradation products (4, 5), and the latter may even compromise endothelial monolayer integrity (6, 7). Surfactant components may be incorporated into polymerizing fibrin, with subsequent loss of surface activity and alveolar instability (8), as well as altered mechanical properties and reduced susceptibility towards fibrinolysis of the arising fibrin matrix (9, 10). In addition to favoring alveolar collapse and thereby shunt-flow, such events may play a major pathogenetic role for the rapid onset of lung fibrosis in acute and widespread inflammatory lung injury (11).
Changes in the alveolar hemostatic balance have yet been addressed in the acute respiratory distress syndrome (ARDS), mostly because of primary nonpulmonary events such as sepsis or polytrauma as underlying etiology (12-16). In this prototype of acute lung injury, an increase in tissue factor (TF), factor VII (F VII), and plasminogen activator inhibitor I (PAI-I) and a decrease in urokinase-type plasminogen activator (u-PA) activities were consistently noted (12-16). These changes occurred early in the course of ARDS and were shown to persist for several days (13, 14).
Intraalveolar fibrin formation has also long been known to be morphologically detectable in severe pneumonia (11); however, the underlying changes in coagulation and fibrinolysis pathways have not yet been disclosed. In most cases of pneumonia, such alveolar fibrin deposits obviously represent transitory abnormalities, with complete dissolution and restitutio ad integrum of the delicate lung gas-exchange barrier, but they may be related to scarring events in prolonged courses of lung infection. In addition, severe pneumonia is now recognized as an increasingly frequent antecedent of ARDS (17), and the American-European consensus conference definition of ARDS includes patients with primary and secondary pneumonia once the criteria of severity of gas exchange abnormalities and diffuse bilateral lung involvement are met (18).
Against this background the current study was undertaken to characterize the changes in the alveolar hemostatic balance by investigating bronchoalveolar lavage fluid in patients with severe primary pneumonia, whether spontaneously breathing or demanding mechanical ventilation, in comparison with those with ARDS triggered by nonpulmonary underlying events. Different categories of pneumonia were investigated, and in addition studies were performed in patients presenting with ARDS in combination with lung infection. In essence, markedly enhanced alveolar procoagulant activity, almost exclusively attributable to the tissue factor pathway, and fibrin formation were noted in all categories of patients with lung infection, with changes in patients with pneumonia demanding respirator therapy being quantitatively comparable with those in ARDS. Concomitantly, overall alveolar fibrinolytic capacities were significantly depressed with decreased u-PA and increased PAI-I and α2-antiplasmin levels. We conclude that a shift of the alveolar hemostatic balance to the procoagulant side with regional fibrin formation is a common feature of severe pneumonia and ARDS developing independent of lung infection.
The following antibodies and antigen standards were used to establish ELISA procedures for quantification of u-PA, t-PA, PAI-I, and α2-AP and to quantify tissue factor activity: Polyclonal antihuman u-PA (no. 398), polyclonal antihuman α2-AP (no. 361), monoclonal antihuman tissue factor (no. 4509), human melanoma PAI-I (no. 105), human α2-AP (no. 4030), human single chain u-PA (no. 107), and monoclonal anti human α2-AP (no. 3612) from American Diagnostica (Greenwich, CT); biotinylated, monoclonal antihuman u-PA (U-6-B), biotinylated, monoclonal antihuman t-PA (T-I-B) biotinylated, monoclonal antihuman PAI-I (I-2-B), and monoclonal antihuman PAI-I (I-1-005) from Monozyme (Hoersholm, Denmark); human single (no. 102101) or two- chain (no. 102201) t-PA and polyclonal antihuman t-PA (no. 105201) from Biopool (Umea, Sweden); biotinylated, monoclonal antimouse IgG from Amersham (Braunschweig, Germany). For ELISA, the following buffer preparations were used. Coating buffer: 1.59 g Na2CO3, 2.93 g NaHCO3 ad 1,000 ml at pH 9.6; Washing buffer: 29.3 g NaCl, 0.2 g KH2PO4, 1.15 g Na2HPO4 × 2 H2O, ad 1,000 ml at pH 7.2; Dilution buffer: 1% (wt/vol) bovine serum albumin (BSA) (Paesel and Lorei, Frankfurt, Germany) in washing buffer. Substrate buffer: 8.2 g CH3COONa × 3 H2O, 6.9 g NaH2PO4 × H2O ad 1,000 ml (pH, 4.2). Phosphate-buffered saline (PBS): 8 g NaCl, 2.9 g Na2HPO4 × 12 H2O, 0.2 g KCl, 0.2 g KH2PO4 ad 1,000 ml (pH, 7.4). Avidin-biotin-horseradish-peroxidase (AB-complex) was purchased from Dakopatts GmbH (Hamburg, Germany), 2.2′-azino-di(3-ethylbenzthiazolinsulfonat, ABTS) from Boehringer GmbH (Mannheim, Germany).
For coagulation assays, a normal human plasma pool was prepared by mixing 15 plasma samples obtained from the antecubital vein of healthy subjects. The plasma pool was aliquoted and stored at −80° C. Human-factor-VII deficient plasma and purified, human factor VII (specific activity, 1,000 U/mg protein) were purchased from Sigma (Munich, Germany). A human tissue factor preparation (Thromborel) and human u-PA (Actosolv) were obtained from Behring (Marburg, Germany). ELISA kits for determination of fibrinopeptide A (FP-A) and D-dimer, lyophilized bovine thrombin (specific activity, 1,250 U/mg) and human plasmin (specific activity, 8 U/mg) were from Boehringer Mannheim (Mannheim, Germany). 125I-labeled human fibrinogen (specific activity, 200 μCi/mg) was supplied by Amersham (Braunschweig, Germany) and purified human fibrinogen (purity of > 95%, with trace amounts of plasminogen and F XIII) was a generous gift of Prof. N. Heimburger (Behring, Marburg, Germany). A protein determination kit (BCA) was received from Pierce (via Bender& Hohbein, Munich, Germany). All other chemicals were from Merck (Darmstadt, Germany).
All patients included in this study were recruited from the Division of Respiratory and Critical Care Medicine, Department of Internal Medicine, Justus-Liebig-University Giessen and the nearby Klinik für Lungenerkrankungen, Waldhof-Elgershausen. The project was approved by the local ethics committee, and informed consent was obtained from either the patient or the closest relatives.
Definition of categories. In total, 222 patients 15 to 75 yr of age were investigated and compared with 35 healthy volunteers without a history of cardiac or pulmonary disease (control subjects). Demographic data are given in Table 1. Classification of the patients in different categories was performed as recently published (19). There was no overlap between these main categories. General exclusion criteria for all groups included proven or suspected malignancy of the lung, chronic obstructive or interstitial lung diseases, recent head trauma, stroke, or subarachnoidal hemorrhage.
|Control Subjects (n = 35)||Patients with ARDS (n = 25)||Patients with ARDS+PNEU (n = 43)||Patients with PNEU-vent (n = 114)||Patients with PNEU-spon (n = 40)|
|Age, yr||42.4 ± 3.2||51.3 ± 3.2||46.7 ± 2.3||55.9 ± 1.4||52.9 ± 2.3|
|Never smoker, n||35||18||25||76||24|
|Current smoker, n||4||2||9||4|
|PaO2 /Fi O2 , mm Hg||410 ± 23||181 ± 17†||177 ± 15†||192 ± 11†||217 ± 36†|
|Neutrophils, %||2.5 ± 0.4||46.5 ± 6.4†||31.7 ± 5.3†||34.9 ± 2.2†||19.7 ± 4.0†|
|Alveolar macrophages, %||93.1 ± 0.8||50.74 ± 6.4†||59.0 ± 5.0†||58.8 ± 27.4†||68.3 ± 3.9†|
|Lymphocytes, %||5.4 ± 0.8||3.9 ± 0.8||8.4 ± 2.5||5.9 ± 0.7||9.7 ± 2.2|
|Protein, mg/ml||0.069 ± 0.008||0.920 ± 0.263†||0.518 ± 0.085†||0.432 ± 68†||0.340 ± 0.071†|
|PTT, s||34 ± 2.5||34.6 ± 4.1||46.9 ± 3.5||45.6 ± 2||37.3 ± 2.4|
Severe pneumonia demanding mechanical ventilation (PNEU-vent). In total, 114 patients with a clinical history of severe primary lung infection necessitating respirator therapy were examined. Diagnostic criteria were fever, tachycardia, dyspnea, typical auscultatory findings, characteristic chest roentgenograms. Microbiologic identification of pathogens in the lower respiratory tract by bronchoscopy was mandatory for inclusion of these patients. As a result, patients with a history and clinical picture suggestive of pneumonia but without proof of the microbiologic agent were excluded from the study. Patients with concomitant left ventricular failure, as diagnosed by elevated pulmonary capillary wedge pressures above 16 mm Hg (right heart catherization performed in all patients) were not included in this group. For further characterization of the prothrombotic and fibrinolytic factors in dependence of the severity of radiologic changes, a subgroup consisting of patients with unilateral, lobar infiltrates was analyzed (PNEU-vent lobar).
Pneumonia in spontaneously breathing subjects (PNEU-spon). In total, 40 patients fulfilling the same criteria as listed above but not mechanically ventilated were included in this group.
If unequivocally attributable, based on radiologic criteria, ventilated or spontaneously breathing patients with PNEU were ascribed to subgroups of bronchopneumonia (patchy peribronchially centered consolidation; BroPNEU; n = 40), alveolar pneumonia (homogenous, sharply demarcated consolidations with air bronchogram; AlvPNEU; n = 61) and interstitial pneumonia (reticular pattern/ground glass opacities throughout the lung; IntPNEU; n = 38). The remaining patients could not be ascribed to one of these subgroups.
ARDS in the absence of primary lung infection (25 patients). For diagnosis of ARDS, the recently proposed criteria of the American-European Consensus Conference on ARDS (18) had to be fulfilled; however, patients with primary or secondary lung infection were excluded in this group. Criteria included PaO2 /Fi O2 < 200 mm Hg, diffuse and bilateral alveolar infiltrates on chest radiograph, pulmonary capillary wedge pressures < 16 mm Hg, and the presence of a typical initiating nonpulmonary catastrophic event such as sepsis, pancreatitis, or polytrauma.
ARDS in combination with pneumonia (ARDS+PNEU). In total, 43 patients were included, according to two subgroups. (1) Mechanically ventilated patients with the initial clinical diagnosis of severe pneumonia with circumscript lung infiltrates in whom sequential chest radiographs displayed rapid, diffuse, and more or less homogenous bilateral spreading of infiltrates, imposing a typical radiographic pattern of ARDS. (2) Patients initially classified as having ARDS, with underlying events differing from pneumonia, who acquired secondary (nosocomial) pneumonia within < 72 h (infection confirmed by microbiologic identification of pathogens in BALF).
Except for the spontaneously breathing patients with pneumonia (PNEU-spon), all patients required mechanical ventilation; Fi O2 and respirator settings, including positive end-expiratory pressure (PEEP), were chosen according to the requirements of gas exchange abnormalities. PaO2 /Fi O2 values did not substantially differ among the various groups treated by respirator therapy, but they were slightly higher in the spontaneously breathing patients with pneumonia (Table 1). As stated above, proof of the responsible microbiologic agent was mandatory in cases of pneumonia; an overview of the responsible agents is depicted in Table 2. Antibiotic drugs were applied depending on microbiologic findings. General therapeutic approaches included intravenous volume substitution and low-dose heparin application without significant prolongation of partial thromboplastin time (PTT) (see Table 1). Vasoactive or inotropic drugs were administered according to the patient's hemodynamic variables as determined by right heart catheterization. Flexible bronchoscopy and lavage were primarily performed for diagnostic purposes (e.g., microbiologic investigation). Therefore, the timing of BAL was coupled to the clinical necessities and diagnostic steps and did not follow a strictly time-matched protocol, but was performed within the first 4 d after onset of disease. In order to assess the influence of the duration of mechanical ventilation on the thrombotic and fibrinolytic factors, all mechanically ventilated patients (including those with ARDS, ARDS+PNEU, or PNEU-vent) were subcategorized into a group of 1 to 2 d and a group of 3 to 4 d of mechanical ventilation until lavage (ARDS or PNEU-vent Day 1-2 and ARDS or PNEU-vent Day 3-4). No analysis of sequential lavages was performed.
|Coagulase negative staphylococci||28|
|Streptococci (incl. S. pneumoniae)||45|
|Other Gram+ bacteria||1|
|Other Gram− bacteria||22|
Bronchoscopy was performed with a fiberoptic bronchoscope through the endotracheal tube. Ten 20-ml aliquots of sterile saline were infused into one segment of the lingula or the right middle lobe and removed by gentle suction (recovery, 55 to 70%). In patients with pneumonia, the lung with predominant infection was examined. Lavage fluids were filtered through sterile gauze, collected on ice, and immediately centrifuged at 200 × g (for 10 min at 4° C) to sediment cellular material. Staining and counting of the pelleted cells were performed as routine technique. Supernatant aliquots were frozen in liquid nitrogen and stored at −85° C for subsequent measurements. In parallel to BAL sampling, blood samples were taken for performance of global coagulation assays.
The sandwich ELISA technique was applied in these assays. In brief, catching antibodies were diluted 1:166 (polyclonal anti-u-PA), 1:250 (monoclonal anti-PAI-I), and 1:500 (polyclonal anti-t-PA and polyclonal anti-α2-AP) in coating buffer and were transferred to the wells at a volume of 100 μl. After 12 h at 4° C, plates were washed three times with washing buffer and free binding sites were blocked by incubation with 200 μl dilution buffer for 30 min. Subsequent to three further washing steps, standard or samples were applied at a volume of 100 μl in duplicate (1-h incubation). Upon triple washing, the targeting antibodies were transferred to the plates in a volume of 100 μl and at dilutions of 1:250 (biotinylated, monoclonal anti-t-PA), 1:500 (biotinylated, monoclonal anti-u-PA, and biotinylated, monoclonal anti-PAI-I) and 1:1,000 (nonbiotinylated, monoclonal anti-α2-AP-1). After 1 h, plates were again washed three times. The α2-AP plates were then incubated with a biotinylated, monoclonal antimouse Ig at a dilution of 1:1,000 (1 h, 100 μl/well) and again washed three times with washing buffer. In all assays, enhancement of the signal/noise ratio was achieved by a 2-h incubation period with AB-complex (200 μl/well, diluted as recommended by the manufacturer). Upon triple wash, coloring of the plates was obtained after application of ABTS in substrate buffer (20 mg in 30 ml substrate buffer and 10 μl 30% H2O2). Absorbance was then read at 405 nm using an ELISA reader (SLT, Overrath, Germany), with computer-based generation of calibration curves and automatic calculation of concentration.
Separate control experiments ascertained that with these ELISA techniques (1) single and two-chain u-PA as well as PAI-bound u-PA are equally well detected, (2) free PAI-I and PAI-I complexed with u-PA or t-PA are detected to the same extent, (3) α2-AP quantification is independent of its binding to plasmin.
For all assays, spiking experiments employing 12 BALF of different underlying diseases were performed for evaluation of test validity under conditions of BALF analysis. Excellent recovery rates were noted thoughout, with slopes near 1 and regression coefficients of 0.999834 (PAI-I), 0.98029 (α2-AP), 0.99976 (t-PA), and 0.99714 (u-PA). Upon use of an internal BALF standard at different days, low interassay variability was noted, as evident from the low SE values; data were 8.01 ± 0.18 (u-PA; n = 19), 10.17 ± 0.99 (α2-AP; n = 20), 8.87 ± 0.66 (PAI-I; n = 14), and 7.72 ± 0.08 (t-PA; n = 4) (all values are mean ± SE and are given in ng/ml). The detection limit of these ELISA procedures was found to range at ≈ 0.1 ng/ml.
These proteins were measured using commercially available ELISA kits. The assays were performed strictly as recommended by the supplier.
Procoagulant activity was assessed by means of a bead coagulometer as previously described (15). In brief, 80 μl plasma and 80 μl diethylbarbiturate acetate buffer solution and 40 μl citrated BALF (adjusted to a constant phospholipid concentration of 10 μg/ml) or purified tissue factor (Thromborel, diluted serially) were incubated for 3 min at 37° C and clotting was initialized upon addition of 80 μl CaCl3 (0.025 M). The procoagulant activity of the sample was calculated on the basis of a computer-based standard curve and is given in arbitrary units. Tissue factor activity was assessed in parallel experiments employing an inhibitory antibody against human tissue factor, which was added to the BALF samples at a final concentration of 1 μg/ml at 37° C 30 min prior to starting the recalcification assay. The net procoagulant activity minus the activity in the presence of this antibody is regarded to present tissue factor activity, given in percent of total procoagulant activity.
Factor VII activity was assessed by means of F VII-deficient plasma. In brief, 50 μl deficient plasma was mixed with 50 μl citrated BALF and incubated for 60 s at 37° C. Coagulation was then started by addition of 100 μl of a calcium-containing tissue factor preparation (Thromborel). Clotting times were converted into units on the basis of a computer-based standard curve obtained with purified FVII.
Proteolysis of fibrin clots was assayed by use of the fibrin plate assay as previously reported (9). The final volume in each well was 150 μl. All concentrations refer to this final volume, and all components were dissolved in 3 mM Ca2+ containing saline. First, unlabeled (6 mg/ml) and labeled (≈ 0.2 nCi/ml) fibrinogen were introduced into the wells in a volume of 30 μl. Complete clotting was achieved by incubation, with thrombin predissolved in 20 μl buffer fluid (0.02 U/ml at 37° C for 1.5 h). Next, 100 μl PBS or noncitrated BALF was carefully layered onto the surface of the clot, and incubation was performed for 72 h at room temperature. Then 75 μl of the supernatant fluid were aspirated and counts were measured using a Canberry Packard γ-counter. Quench-corrected radioactivity in the supernatant was related to the initially provided radioactivity and is given in percentage. Nonenzymatic release of labeled compounds into the supernatant, as assessed by incubating the clots with PBS only, never exceeded 10% of the initially provided activity and was subtracted from the activity as obtained for the BALF samples in order to obtain net enzymatic release of split products. Pilot experiments showed that the human fibrinogen preparation used per se contained a sufficient amount of plasminogen to enable plasminogen activator-based fibrinolysis since (1) addition of purified u-PA to this assay system resulted in a dose-dependent release of split products and (2) additional admixture of 25 μg/ml human plasminogen to the fibrinogen prior to clotting did not further enhance the BALF-elicited liberation of scission products, as ascertained in control experiments with 15 different BALF samples.
Colorimetric assays were employed as described previously (19). All experiments were performed in duplicate.
Because of the shortage of lavage material it was not possible to perform all tests in all samples, which is the reason for differing total numbers among the various assays. All data are presented as mean ± SE. Analysis of statistical differences between the main groups (ARDS, ARDS+PNEU, PNEU-vent, and PNEU-spon.) or the PNEU subgroups (BroPNEU, AlvPNEU, IntPNEU) and control was performed by testing principal significant diversity first (Kruskal-Wallis H-test), followed by comparison with a nonparameteric test (Mann-Whitney U-test). Significance level was set at p < 0.05.
All patients with ARDS or pneumonia or a combination of both displayed marked changes in BALF cellularity, characterized by an increase in the percentage of neutrophils and a concomitant decrease in the percentage of alveolar macrophages (Table 1). This was paralleled by plasma protein leakage into the alveolar compartment, with ≈ 10-fold elevated protein concentrations in BALF samples from patients with ARDS, and ≈ 5-fold increased BALF protein levels in both mechanically ventilated and spontaneously breathing patients with pneumonia.
A manifold and highly significant increase in BALF procoagulant activity was observed in all groups of patients (Figure 1). Procoagulant activity (PCA) data were increased by nearly two orders of magnitude in all patients with ARDS and in patients with pneumonia demanding respirator therapy, with somewhat less increased values in spontaneously breathing patients with pneumonia. Within the group of patients with PNEU, PCA levels were lower in BroPNEU than in AlvPNEU and IntPNEU. All PCA was shown to be nearly exclusively exerted via the extrinsic coagulation pathway, as preincubation with an antihuman tissue factor consistently suppressed > 95% of activity in control subjects and the different groups of patients (Table 3). In addition, F VII activity was shown to be significantly elevated in all patients with ARDS and PNEU, with somewhat more prominent changes in ARDS and PNEU- vent as compared with PNEU-spon (Table 3). As can be assumed on the basis of these findings, the BALF levels of fibrinopeptide A (Table 3) markedly surpassed control values in all patients with ARDS and pneumonia.
|F VII, mU/ml||18.9 ± 9||140.7 ± 36‡||135 ± 47‡||87.8 ± 13‡||52.3 ± 11†|
|n = 35||n = 25||n = 43||n = 114||n = 40|
|TF, % of total PCA||99.9 ± 0.2||95.9 ± 4.3||99.8 ± 0.6||98.4 ± 2.2||98.5 ± 4.6|
|n = 35||n = 25||n = 43||n = 114||n = 40|
|FP-A, ng/ml||1.49 ± 0.3||6.3 ± 1.6†||5.1 ± 1.5†||6.6 ± 1.2‡||5.9 ± 1.3‡|
|n = 35||n = 25||n = 43||n = 114||n = 40|
|tPA, pg/ml||578 ± 86||626 ± 79||784 ± 124||713 ± 88||731 ± 151|
|n = 10||n = 12||n = 20||n = 30||n = 18|
|PAI-1/u-PA ratio||6.1 ± 1.7||38.7 ± 12§||76.1 ± 26§||34.6 ± 7§||6.6 ± 2|
|n = 13||n = 13||n = 18||n = 47||n = 12|
Opposite to the changes in PCA, the overall BALF fibrinolytic activity was greatly reduced in all groups of patients (Figure 2). The most prominent suppression was noted in patients with ARDS either in absence or presence of lung infection, but a significant reduction of fibrinolytic activity was also observed in spontaneously breathing and mechanically ventilated patients with pneumonia. Within the PNEU subgroups, loss of fibrinolytic capacity was more prominent in alveolar and interstitial pneumonia than in bronchopneumonia. In accordance with these findings, BALF u-PA antigen levels were significantly reduced in all patients in comparison with that in control subjects (Figure 3). In contrast, t-PA levels in the lavage samples of patients with ARDS and pneumonia did not differ from those in control subjects (Table 3). The BALF concentrations of both plasminogen activator inhibitor-1 (PAI-1) and α2-antiplasmin (α2-AP) were elevated by approximately one order of magnitude in patients with ARDS and in patients with pneumonia needing mechanical ventilation, but were only slightly or virtually not increased in the PNEU-spon group (Figures 4 and 5). As a consequence, the PAI-1/u-PA ratio was found to range at 5- to 10-fold elevated levels in ARDS, PNEU-vent and ARDS+PNEU, but was virtually normal in spontaneously breathing patients with pneumonia (Table 3). Among the different PNEU subgroups, again patients with alveolar pneumonia displayed the most pronounced changes in BALF PAI-1 and α2-AP levels.
BALF D-dimer levels, indicating overall alveolar fibrin turnover, were increased by nearly two orders of magnitude in all patients with ARDS and in mechanically ventilated patients with pneumonia (Figure 6). Data were somewhat less, but still highly significantly elevated, in the spontaneously breathing patients with pneumonia. Among the subgroups of patients with pneumonia, the lavage D-dimer concentrations in AlvPNEU and IntPNEU surpassed those in BroPNEU.
Concerning the PNEU-vent group, the above described changes of the coagulant and fibrinolytic factors were not dependent on the magnitude of the radiologic changes. When analyzing the data obtained from those patients with PNEU-vent and unilateral, lobar infiltrates, similar alterations of the coagulant and fibrinolytic factors were evident (with the lavage being performed in the radiologically suspective lung region) (Table 4). Similarly, there was no difference in the severity of changes, when data obtained from all ventilated patients (ARDS, ARDS+PNEU, PNEU-vent) were analyzed in dependence of the duration of ventilation until BAL (Day 1-2 versus Day 3-4) (see Table 4). Only D-dimer levels seemed to be higher in the group of patients being lavaged within the first 2 d. However, one has to keep in mind that sequential lavages were not performed and that direct comparibility of these data is thus difficult.
|PCA (U/ml )||F VII (mU/ml )||FP-A (ng/ml )||D-dimer (ng/ml )||Fibr. Act. (% release)||u-PA (ng/ml )||t-PA (ng/ml )||α2-AP (ng/ml )||PAI-1 (ng/ml )|
|Control||0.42 ± 0.01||18.9 ± 9.0||1.5 ± 0.3||25 ± 7.5||25.8 ± 2.9||1.37 ± 0.12||0.60 ± 0.08||0.7 ± 0.1||5.5 ± 0.7|
|PNEU-vent (all)||18.6 ± 4.7||87.7 ± 13||6.6 ± 1.2||740 ± 128||16.4 ± 1.3||0.77 ± 0.07||0.71 ± 0.0||9.6 ± 1.6||20.9 ± 4.1|
|PNEUlobar-vent||17.4 ± 6.9||106.0 ± 26||5.8 ± 1.1||802 ± 285||15.8 ± 2.4||0.63 ± 0.09||0.91 ± 0.2||9.6 ± 2.8||38.7 ± 11.7|
|ARDS or PNEU-|
|vent Day 1-2||23.6 ± 5.8||123.9 ± 18||7.2 ± 1.3||1,250 ± 268||15.3 ± 1.5||0.98 ± 0.16||0.73 ± 0.15||9.2 ± 1.5||34.8 ± 8.9|
|ARDS or PNEU-|
|vent Day 3-4||18.5 ± 4.9||117.2 ± 19||5.2 ± 1.2||513 ± 115||12.6 ± 1.3||0.71 ± 0.06||0.70 ± 0.05||10.5 ± 1.9||25.6 ± 5.3|
In the present study, undertaken on 222 patients with acute respiratory failure, marked disturbances in the alveolar hemostatic balance were noted. Patients with ARDS triggered by nonpulmonary underlying events were characterized by dramatically increased procoagulant activity in the alveolar lining layer, largely attributable to the tissue factor pathway, and a concomitant depression of overall fibrinolytic activities with decrease in u-PA and increase in PAI-1 and α2-AP levels. Interestingly, qualitatively and quantitatively virtually identical changes were noted when ARDS was combined with lung infection and in patients with severe pneumonia demanding mechanical ventilation regardless of the magnitude of infiltrates. Moreover, a corresponding profile of altered alveolar hemostatic balance was observed in spontaneously breathing patients with pneumonia, however, both enhancement of procoagulant and depression of fibrinolytic activities were less prominent in this category. Within the subgroups of patients with pneumonia, alterations in alveolar and interstitial pneumonia surpassed those in bronchopneumonia. Acute inflammatory lung injury, whether triggered by nonpulmonary systemic events or primary lung infection, is thus consistently characterized by a marked shift of the alveolar hemostatic balance to the procoagulant side, with the appearance of alveolar fibrin formation and fibrin degradation products.
All data in patients with ARDS and pneumonia were compared with those in a group of healthy volunteers, the mean age of which was lower than that in the various categories of respiratory failure. It is, however, highly improbable that such variation in age might account for the differences in BALF procoagulant and fibrinolytic properties between the control subjects and the patients with ARDS and/or severe lung infection. First, a detailed analysis of age-dependency of the various variables of coagulation and (anti-)fibrinolysis in the lavage fluids within the groups of patients with pneumonia and ARDS and within the healthy volunteers did not forward any evidence for such age dependency. And second, the differences between control subjects and the various categories of patients with pneumonia and ARDS reached nearly two orders of magnitude, in dependence on the variable under investigation, which is hardly imaginable to be based on the rather small difference in mean age.
The current study supports previous investigations in finding markedly increased procoagulant activity in the alveolar lining layer in patients with ARDS triggered by extrapulmonary underlying events such as sepsis, multiple trauma, and pancreatitis (12-16). A more specific analysis of the PCA increase clearly demonstrated that this was virtually fully attributable to an activation of the tissue factor pathway, operating in companion with F VII. Such elevation of lavage procoagulant enzyme activity may well result from an activation of alveolar macrophages. Even under physiologic conditions, these macrophages are known to express, synthesize, and particulate significant amounts of tissue factor and F VII and to assemble the prothrombin activator complex on their surface (20-23), and such activity is markedly enhanced in response to inflammatory stimuli (24). In addition, alveolar epithelial type II cells and lung fibroblasts are known to synthesize tissue factor under baseline conditions, again augmented in a proinflammatory environment (25-30).
Extending the results of previous investigations, the present study shows that a corresponding increase in tissue factor- related procoagulant activity is observed in the alveolar lining layer of patients with severe primary pneumonia demanding mechanical ventilation. Interestingly, both the data of increased PCA units and augmented lavage fibrinopeptide A levels, indicating thrombin activity in this compartment, were virtually identical in the patients with ARDS and those with pneumonia. When analyzing a subgroup of the PNEUvent patients displaying lobar, unilateral infiltrates, similar changes in the procoagulant and fibinolytic parameters as compared with all patients with pneumonia needing mechanical ventilation were encountered. Moreover, exactly corresponding data were again obtained from patients classified as having both ARDS and lung infection. Finally, the same profile of changes was observed in patients with pneumonia who suffered from respiratory failure as evident from the reduced PaO2 /Fi O2 ratio but were still spontaneously breathing, with moderately lower data of PCA, F VII, and FP-A as compared with patients with ARDS and pneumonia receiving respirator therapy. These findings clearly demonstrate that (1) it is not the respirator therapy per se that forwards such marked alteration of procoagulant activity, but the underlying diseases responsible for these changes, and (2) primary systemic events such as those underlying the development of ARDS in the current study and primary lung infection evidently provoke a comparable response of tissue-factor-related procoagulant state in the bronchoalveolar compartment. These observations are thus in line with the finding of enhanced lavage procoagulant activity in HIV positive patients with Pneumocystis carinii pneumonia (31).
In companion with the activation of the tissue factor pathway, a marked reduction of overall fibrinolytic activity was noted in the alveolar lining layer of patients with ARDS, with depressed u-PA and augmented PAI-1 and α2-AP levels being suggested as main contributors to this lowering in lytic capacity. This finding supports previous obervations in lavage fluids of patients with ARDS with respect to the reduction in u-PA (12, 13, 16) and the increase in PAI-1 (12, 13, 16). Interestingly, qualitatively and quantitatively nearly identical changes as noted in ARDS were again observed in patients with primary lung infection needing respirator therapy, and in patients with ARDS in combination with pneumonia. Moreover, a corresponding decrease in u-PA was also found in the spontaneously breathing patients with pneumonia, but there was no significant increase in plasminogen activator and plasmin inhibitor capacity in these patients. In none of the categories of respiratory failure presently studied was the depression of the u-PA, the predominant alveolar space plasminogen activator, followed by any change in the levels of tissue-type plasminogen activator. These observations on the BALF fibrinolytic profile thus again support the notion that primary systemic events, such as those triggering ARDS in this study, and primary lung infection by different microbial agents provoke a comparable response of altered alveolar lining layer hemostatic balance. As u-PA is the predominant plasminogen activator of alveolar macrophages when investigated in vitro (33, 34), known to be downregulated under conditions of inflammatory stress, whereas such circumstances enhance macrophage PAI-1 synthesis (32-34), the macrophages of the alveolar space again offer as major contributors to the noted changes in fibrinolytic profile in ARDS and severe penumonia. In addition, synthesis of u-PA and PAI-1 has also been reported for lung alveolar epithelial cells type II and lung fibroblasts (27– 30), which may thus act in concert with altered alveolar macrophage activity to forward changes in overall fibrinolytic capacity in the alveolar lining layer in acutely inflamed lungs. The cellular source of t-PA in the lining layer is not yet fully established, but the obvious discrepancy between u-PA and t-PA regulation may suggest different cellular origin.
Within the subgroups of patients with pneumonia, both enhancement of procoagulant and depression of fibrinolytic capacities were more prominent in patients categorized as alveolar and interstitial pneumonia than as compared to bronchopneumonia, and this difference was also reflected by the lower D-dimer levels in the latter subgroup. As such differential analysis of alveolar hemostatic balance for various categories of pneumonia has hitherto not been performed, either clinically or experimentally, no comparison with data in the literature can be undertaken. The results are, however, consistent with current thinking on the pathogenesis in these types of pneumonia. In bronchopneumonia the infectious process is first located in the bronchial compartment, with only secondary involvement of the alveolar and interstitial space, whereas the opposite is true for alveolar and interstitial pneumonia. As the lavage procedure, caused by the quantitative predominance of the alveolar surface in comparison with the bronchial surface, preferentially addresses changes in the alveolar as compared with the bronchial lining layer, changes in the BALF coagulation components in alveolar and interstitial pneumonia may well supervene those in bronchopneumonia.
Is it appropiate to assume that the findings in the lavage fluid supernatant correctly reflect the alveolar hemostatic balance and the alveolar fibrin turnover in vivo? This question is particularly relevant with respect to the predominant procoagulant factor, the tissue factor, which is primarily membrane-bound and thus cell-associated (35). However, shedding of tissue factor into the extracellular lining layer by alveolar macrophages and epithelial cells has been noted (26), and a correlation of membrane expression and shedding of TF has been reported for this compartment (24). U-PA and PAI-1 are well known to be released by cultured alveolar epithelial cells and macrophages (25, 32). Thus, there is good evidence to assume that the present analysis of cell-free lavage supernatant will reflect local procoagulant and fibrinolytic activities in the alveolar lining layer, even when membrane-bound components are not directly addressed by this approach. This notion is fully supported by the results obtained on lavage D-dimer levels. The appearance of this fibrin scission product demands preceding in vivo coagulation and fibrinolysis processes, and its elevation by nearly two orders of magnitude in patients with ARDS, severe pneumonia, or a combination of both underscores the fact that pronounced coagulation abnormalities indeed occurred in the alveolar compartment of the patients under investigation.
The upregulation of the tissue-factor-related pathway of coagulation in the alveolar compartment, with concomitant inhibition of regional fibrinolysis and the appearance of alveolar fibrin, may well contribute to the lung functional impairment in ARDS and severe pneumonia. Fibrin has been shown to be a potent inhibitor of surfactant function by incorporation of hydrophobic surfactant components (8-10), which results in alveolar collapse and thereby shunt flow, a characteristic feature of gas exchange abnormalities in acute pneumonia and ARDS (36, 37). Moreover, although representing a precondition for secondary reparative processes, persistent alveolar deposition of a fibrin matrix might promote and localize invasion by fibroblasts (24, 28, 32, 33). Such events may then be followed by irreversible loss of alveolar spaces because of scarring processes (concept of alveolar collapse induration) (11), known to start within a few days under conditions of ongoing ARDS or pneumonic infiltration.
In conclusion, pronounced disturbances of the alveolar hemostatic balance were noted in patients with severe pneumonia, both spontaneously breathing and needing mechanical ventilation, comparable to those in ARDS triggered by nonpulmonary underlying events. A marked increase in regional procoagulant activity, largely attributable to the tissue factor pathway, was paralleled by a depression of overall fibrinolytic activity in the alveolar lining layer, with decreased u-PA (all patients with pneumonia) and increased PAI-1 and α2-AP levels (patients with pneumonia receiving respirator therapy) as prominent features. These changes in BALF enzymatic activity were paralleled by the appearance of coagulation products in the lavage fluid such as fibrinopeptide A and D-dimer, reflecting preceding in vivo clotting processes in the alveolar compartment. A marked shift of the alveolar hemostatic balance to the procoagulant side is thus a consistent feature of acute inflammatory lung injury, whether triggered by nonpulmonary systemic events or by primary lung infection.
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