3Division of Pulmonary and Critical Care Medicine, Departments of 1Pediatrics and 5Pathology, University of Alabama, Birmingham, Alabama 35294; 2Division of Allergy, Pulmonary, and Critical Care Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232; and 4Cardiovascular Research Institute, University of California at San Francisco, San Francisco, California 94143
Submitted 16 September 2002 ; accepted in final form 12 February 2003
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ABSTRACT |
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fibrinolysis; prognosis
ALI is characterized by the deposition of fibrin in the alveolar space.
Deposition of fibrin in the alveolar space is the net result of an alteration
in the balance of coagulation and fibrinolytic proteases [plasmin and
urokinase type plasminogen activators (u-PA)] and antiproteases [plasminogen
activator inhibitor-1 (PAI-1) and 2-antiplasmin] and the
availability of plasma-derived fibrinogen
(31,
35). Upon fibrinogen influx
into the alveolar compartment, coagulation/fibrinolysis
proteases/antiproteases act to generate insoluble cross-linked fibrin and
soluble plasmic-cleavage fragments
(13,
14,
22,
25,
35). The significance of
fibrin turnover in sepsis is underscored by the recent trial demonstrating an
improvement in mortality upon administration of the anticoagulant, activated
protein C (3,
21). Although a fraction of
these patients had sepsis-related ALI, the utility of activated protein C in
ALI, sepsis related or not, remains to be determined
(21).
Several previous studies have demonstrated increased levels of PAI-1 in BAL obtained from patients with ALI, evidence that there is a suppression of the normal profibrinolytic environment in the lung (4, 23). However, PAI-1 antigen levels in the BAL are undetectable in many patients and most controls. Animal models of bleomycin- or hyperoxia-induced lung injury demonstrate a pathogenic role for PAI-1 since genetic deletion of PAI-1 has been shown to be protective against pulmonary fibrosis or lethality, respectively (1, 10).
We hypothesized that the levels of PAI-1 in plasma or undiluted edema fluid obtained within hours of intubation would be significantly higher in patients with pulmonary edema from ALI compared with those with HYDRO. We also hypothesized that the magnitude of elevation in PAI-1 antigen would have important prognostic implications in patients with ALI. Last, to understand the effects of altered fibrinolysis on fibrin turnover, we characterized and quantified the fibrinogen-related molecules in pulmonary edema fluid.
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METHODS |
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Patients in the ALI group were further segregated according to the presence or absence of sepsis or pneumonia. Sepsis was defined as a temperature of >38.3°C or <35°C during the 48 h preceding the diagnosis of ALI, a systolic blood pressure of <100 mmHg or >30 mmHg below baseline for >1 h during the 48 h preceding the diagnosis of ALI, and a positive culture. Pneumonia was defined as the presence of infiltrates on the chest radiograph and positive respiratory tract microbiology. Patients with both pneumonia and sepsis were included in the sepsis group. The simplified acute physiology score (SAPS II) was calculated for each patient as described (28).
PAI-1 antigen assay. PAI-1 antigen levels were measured by sandwich ELISA (American Diagnostica, Greenwich, CT). This assay detects both active and latent forms of PAI-1 but is insensitive to PAI-2. The protocol was performed essentially as recommended by the manufacturer and as published (27, 34). Samples were incubated overnight in microplate wells precoated with capture antibody (murine monoclonal antibody to human PAI-1). The signals were detected with a secondary biotinylated antibody that recognizes the bound PAI-1 molecules using streptavidin-conjugated horseradish peroxidase (HRP)-dependent cleavage of the tetramethylbenzidine substrate in an end point format. PAI-1 levels were quantified by measuring the absorbance of the wells at 450 nm (MR 5000 Dynatech Microplate Reader) and comparing the values with those of a standard curve using known quantities of recombinant human PAI-1.
PAI-1 activity assay. PAI-1 antigen levels were measured by microplate tissue type plasminogen activators (t-PA) inhibition assay according to the manufacturer's instructions and as published (Biopool, Ventura, CA) (27, 34). Briefly, samples or active PAI-1 standards were added to microplate wells precoated with immobilized active t-PA. The wells were then incubated [30 min at room temperature (RT)] with an HRP-linked monoclonal anti-PAI-1 antibody, MoAb-12A4, and washed. The HRP substrate reaction was stopped after 5 min at RT with 1.6 M sulfuric acid. PAI-1 activity levels were quantified by measuring the absorbance of the wells at 490 nm (MR 5000 Dynatech Microplate Reader) and comparing the values with those of a standard curve using known quantities of active human PAI-1.
Quantification and characterization of fibrin(ogen) fragments. The antigenic levels of soluble plasmin degradation product of cross-linked fibrin, D-dimer, were measured by sandwich ELISA according to the manufacturer's instructions (Biopool). The samples and standards were added to microplate wells precoated with monoclonal antibody (MoAb-8D3). After HRP-linked anti-D-dimer Fab fragments were added, the plate was incubated (30 min at RT) and washed, and the HRP reaction was carried out (15 min at RT) with O-Phenylene-diamine substrate and stopped by the addition of 1.6 M sulfuric acid. D-dimer antigen levels were quantified by measuring the absorbance of the wells at 490 nm (MR 5000 Dynatech Microplate Reader) and comparing the values with those of a standard curve using known quantities of purified human D-dimer. Western blot analysis for fibrin (ogen) fragments was performed under reducing and nonreducing conditions as published (18, 35).
Outcome variables. Two outcome variables were assessed by chart review. Hospital mortality was determined for the period immediately after intubation and initiation of mechanical ventilation. Severity of lung injury was assessed by the number of days alive with unassisted ventilation over the 28-day period immediately after intubation and mechanical ventilation, a valuable end point in clinical lung injury (39).
Statistics. The continuous variables were compared by means of the
unpaired Student's t-test or the Mann-Whitney's U-test if
the variables were not normally distributed
(50). All categorical values
were compared by means of a Fisher's exact test
(50). Statistical significance
was accepted at P 0.05.
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RESULTS |
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Plasma and pulmonary edema fluid PAI-1 antigen levels are increased in ALI. To determine whether PAI-1 antigen levels discriminate between ALI and HYDRO, we measured the PAI-1 antigen level in plasma and edema fluid by ELISA in both patient groups. Plasma PAI-1 antigen was more than threefold higher in the ALI group compared with the HYDRO group (means ± SD, ALI, 1,265 ± 1,340 ng/ml; HYDRO, 388 ± 258 ng/ml; P = 0.002; Mann-Whitney's U-test; Fig. 1). Edema fluid PAI-1 antigen was >11-fold higher in the ALI group compared with the HYDRO group (means ± SD, ALI, 6,421 ± 8,333 ng/ml; HYDRO, 587 ± 951 ng/ml; P < 0.001; Fig. 1).
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A receiver operator curve (ROC) was constructed to assess the sensitivity and specificity of the PAI-1 antigen level in pulmonary edema fluid as a discriminator between ALI and HYDRO. The area under the ROC was 0.91 [95% confidence interval (CI), 0.830.99], and the inflection point of the curve was at a PAI-1 antigen value of 700 ng/ml (Fig. 2). At this level, PAI-1 antigen discriminated between ALI and HYDRO with a sensitivity of 90%, a specificity of 85%, a positive predictive value of 87%, and a negative predictive value of 87% (P < 0.000001). Patients with PAI-1 antigen values >700 ng/ml were 40-fold more likely to have ALI than hydrostatic edema (Fig. 2). Plasma levels of PAI-1 were less discriminating, with an area under the curve of 0.75 (95% CI, 0.6160.897; Fig. 2).
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Evidence for intrapulmonary PAI-1 production. As evidence for significant pulmonary production of PAI-1 in ALI, we calculated the ratio of PAI-1 antigen in the edema fluid to that of the simultaneously drawn plasma sample. There was eightfold more PAI-1 antigen in edema fluid compared with plasma in the ALI group (Fig. 3), whereas the ratio of total protein concentration in edema fluid to plasma was equal to 1 (1 ± 0.2). Furthermore, there were approximately equal amounts of PAI-1 antigen in the edema fluid and plasma in the HYDRO group (means ± SD, ALI, 8 ± 7; HYDRO, 1.5 ± 2; P < 0.001; Fig. 3).
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PAI-1 antigen has prognostic significance in ALI. To determine whether an elevated PAI-1 antigen level identifies patients with ALI with a poor outcome, we compared the PAI-1 antigen level in plasma and edema fluid with hospital mortality and with the number of days of unassisted ventilation. First, demographic and physiological parameters including age, smoking status, gender, lung injury score, edema fluid/plasma total protein ratio, PAO2 - PaO2, PaO2/FIO2 ratio, tidal volume, or tidal volume/kg body wt did not associate with mortality in either group. The presence of sepsis carries a higher risk for mortality (2.2-fold relative risk, P = 0.03), and a higher SAPS II score trended with mortality (>55, 1.6-fold relative risk, P = 0.08) only in the ALI group.
Plasma PAI-1 antigen values were approximately fivefold higher in patients with ALI with a poor outcome (died or <5 days of unassisted ventilation) compared with those with a better outcome (survived or had >5 days of unassisted ventilation; Figs. 4 and 5). A plasma PAI-1 antigen value of 640 ng/ml was at the upper limit of two standard deviations above the mean (95% CI) in patients with ALI that survived. With the use of this threshold, the positive predictive value of a plasma PAI-1 antigen level >640 ng/ml was 100% for mortality (P = 0.005, Fisher's exact test) and fewer than 5 days of unassisted ventilation (P = 0.005, Fisher's exact test; Tables 2 and 3). The edema fluid PAI-1 was threefold higher in patients with ALI with a good outcome compared with those with a poor outcome (Figs. 4 and 5). A similar analysis revealed that an edema fluid PAI-1 antigen value of 6,200 ng/ml was at the upper limit of two standard deviations above the mean (95% CI) in patients with ALI who survived. With the use of this threshold, the positive predictive value for an edema fluid PAI-1 antigen level of >6,200 ng/ml was 100% for both mortality (P = 0.023, Fisher's exact test) and for fewer than 5 days of unassisted ventilation (P = 0.023, Fisher's exact test). These post hoc assessments of the data show that elevation of PAI-1 antigen in both plasma and edema fluid, within hours of intubation, carry significant negative prognostic implications in patients with clinical ALI. In contrast, a higher PAI-1 antigen level in plasma, edema fluid, or the edema fluid/plasma ratio of PAI-1 did not associate with mortality in the group with HYDRO.
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PAI-1 activity levels in pulmonary edema fluid are higher in ALI. To determine the physiological relevance of our observations regarding PAI-1 antigen to alveolar fibrinolytic activity, we measured PAI-1 activity in plasma and edema fluid from patients in both groups by its ability to neutralize immobilized t-PA. There was a 13-fold greater PAI-1 activity in plasma in the patients with ALI compared with HYDRO (means ± SD; HYDRO, 8.9 ± 15 IU/ml; ALI, 120 ± 265 IU/ml; P = 0.04, Mann-Whitney's U-test). Similarly, there was a 17-fold greater PAI-1 activity in the edema fluid of patients with ALI compared with those with hydrostatic edema (means ± SD; HYDRO, 8 ± 9 IU/ml; ALI, 136 ± 244 IU/ml; P = 0.0001, Mann-Whitney's U-test). Concordant with the PAI-1 antigen findings, there was a 17-fold greater PAI-1 activity in edema fluid compared with plasma in the ALI patients, indicating that the locally increased PAI-1 in edema fluid is largely active. These data demonstrate a large, compartmentalized inhibition of fibrinolytic activity in the alveoli of patients with early ALI.
Levels of plasmin-degradation product of cross-linked fibrin (D-dimer)
were elevated in both ALI and hydrostatic edema groups. D-dimer and
D-dimer/E complexes are the major soluble products of plasmin proteolysis
(fibrinolysis) of cross-linked fibrin, whereas D-monomer is a plasmin
proteolytic product of fibrinogen and/or noncross-linked fibrin
(35). The relationship between
the cause of pulmonary edema and D-dimer levels was determined by measuring
D-dimer antigen in plasma and edema fluid from both patient groups by ELISA.
Given the vast differences in PAI-1 levels (e.g., fibrinolytic inhibition),
the D-dimer levels in both plasma and edema fluid were surprisingly similar
among the two patient groups (median, 2575%; plasma - HYDRO, 1,757, 718
- 4,370 ng/ml, ALI, 1,653, 985 - 2,546 ng/ml; P = 0.53; edema fluid -
HYDRO, 9,400, 4,800 - 24,650 ng/ml, ALI, 11,000, 4,100 - 36,575; P =
0.88). Furthermore, there was an 17-fold greater concentration of D-dimer
antigen in edema fluid compared with plasma in both groups (HYDRO -21 and ALI
-14; P = 0.6), consistent with a compartmentalization of fibrin
turnover in the alveoli in both groups. There was no discernable relationship
between survival and D-dimer antigen (edema fluid, plasma, or edema
fluid/plasma ratio) in either patient group.
Although the quantity of D-dimer antigen by ELISA was similar among the groups, analysis of the soluble fibrin(ogen)-derived components by Western blot analysis revealed important differences between ALI and HYDRO fluid (Fig. 6). In both groups, the major products comigrated with plasmin-derived fragments of fibrin(ogen) and confirmed the D-dimer ELISA data. However, there was a sevenfold greater proportion of D-monomer (80100 kDa; fibrinogen/noncross-linked fibrin-derived fragments) relative to D-dimer (180 kDa; cross-linked fibrin-derived fragment) in ALI than in HYDRO (ALI, 1.4 ± 0.8; HYDRO, 0.2 ± 0.3; P = 0.001; Fig. 6). Thus a sevenfold greater proportion of the plasmin-cleaved fibrin(ogen) fragments was derived from fibrinogen/noncross-linked fibrin in the ALI group.
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Effect of sepsis on PAI-1 antigen levels in ALI. Previous reports have documented an increase in plasma PAI-1 in populations of patients with sepsis (43). Among patients with ALI, the level of PAI-1 antigen in plasma was approximately fourfold greater in those who had sepsis as an underlying condition compared with those who did not have sepsis (ALI with sepsis, 1,479 ± 1,435 ng/ml; ALI without sepsis, 369 ± 165 ng/ml; P = 0.02, Mann-Whitney's U-test; Fig. 7). In contrast, pulmonary edema fluid levels of PAI-1 antigen were clearly overlapping in septic and nonseptic patients with ALI (median, 2575%; ALI with sepsis, 2,920, 1,47410,025 ng/ml; ALI without sepsis, 3,916, 2,6964,648 ng/ml; P = 0.7, Mann-Whitney's U-test). These data indicate that PAI-1 levels in the plasma compartment, but not the pulmonary alveolar compartment, are affected by the septic state.
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DISCUSSION |
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Whereas patients with sepsis-related ALI have higher levels of PAI-1 antigen in plasma, as previously reported (43), there was no difference in the edema fluid levels of PAI-1 antigen in patients with and without sepsis. This suggests that the elevation of PAI-1 in the alveolar compartment was a consequence of local, rather than systemic, factors. The correlation of PAI-1 in edema fluid with the clinically assessed degree of lung injury (mechanical ventilation requirement) and its increase in the alveolar compartment compared with plasma point toward the extent of intrapulmonary injury as the dominant factor in determining the level of PAI-1 in the alveolar compartment. In contrast, the edema fluid/plasma ratio of PAI-1 in HYDRO was close to 1, indicating little or no intrapulmonary PAI-1 production.
Although endothelial cells, epithelial cells, fibroblasts, and macrophages
produce PAI-1 in a regulated manner in vitro, no observations regarding the
cell of origin are available in humans with early lung injury. This is due to
a lack of availability of appropriately timed biopsy material for study.
However, prior work in patients later in the course of lung injury reveals
high levels of PAI-1 mRNA in alveolar macrophages
(6). In vitro studies have also
identified several potent cytokine regulators of macrophage PAI-1 production
with direct relevance to the pathobiology of ALI. These include IL-1, IL-10,
TNF-, LPS, and the matrix glycosaminoglycan hyaluronan
(6,
20). Likewise, in a model of
hemorrhagic shock, increased alveolar fibrin deposition was shown to be
related to stimulation of macrophages by TNF-
(12). Experimental lung injury
models also indicate a high level of PAI-1 expression in fibroblasts during
the later phases of lung injury in which transforming growth factor
(TGF)-
and fibrin fragments can upregulate PAI-1 expression in lung
fibroblasts (18,
26,
31,
32,
36). Thus there are multiple
cell type-specific PAI-1 mediators present and active in excess, which can,
alone or in concert, mediate the induction of PAI-1 in the alveolar
compartment in ALI.
One of the strengths of this study compared with previous studies is that we were able to measure a discrete PAI-1 antigen value in all patients and, therefore, make quantitative and predictive conclusions. A comparison with prior studies was performed by normalizing to the total protein to account for dilutional effects of the BAL procedure. Interestingly, we still found PAI-1 antigen levels in ALI edema fluid to be 3-fold and 30-fold higher than that previously reported in BAL performed within the first few days after intubation (17, 24). This difference is likely time related, since the elevations in PAI-1 antigen in BAL in ALI normalize over the first 1014 days after intubation (17, 24). In contrast, the edema fluid PAI-1 antigen/total protein ratio of hydrostatic control patients was similar to that seen in the BAL from healthy controls, suggesting that the alveolar fibrinolytic balance is not altered by hydrostatic edema (17).
Alveolar compartment fibrin(ogen)-related molecules are generated as a consequence of exudation of plasma-derived fibrin(ogen) and the action of several proteases. D-dimer, a unique product of plasmin action on cross-linked fibrin, is several logs higher in BAL from patients with ALI compared with healthy controls (14). To our surprise, D-dimer levels, as measured by ELISA in edema fluid, were similar in ALI and hydrostatic edema. However, a much greater fraction of the total alveolar fibrin(ogen) burden in ALI patients was due to D-monomer, a product of plasmin action on fibrinogen/noncross-linked fibrin. Furthermore, the histological hallmark of ALI, namely, insoluble fibrinrich hyaline membranes, does not develop in hydrostatic edema. Together, these data suggest that the vast majority of fibrinogen in the alveolar compartment in hydrostatic edema is cross-linked by thrombin/transglutaminases and solubilized by plasmin, thereby generating D-dimer and related fragments. In contrast, in ALI, there is massive extravasation of plasma-derived fibrin(ogen), which overwhelms the thrombin/transglutaminase capacity to generate cross-linked fibrin (i.e., incomplete coagulation), and only a minor fraction of the total fibrin is solubilized by plasmin (i.e., incomplete fibrinolysis). The incomplete fibrinolysis in ALI can be partly ascribed to the elevated, locally produced, alveolar compartment PAI-1 activity that slows the activation of the zymogen plasminogen to plasmin.
We have identified an early elevation in PAI-1 antigen as a clinical marker for both the underlying etiology and prognosis of early ALI. The question of its pathophysiological importance and the mechanism of its effect on lung injury and repair remain unresolved. Fortunately, animal models have contributed to our understanding of the pathophysiological importance of PAI-1 in lung injury and repair. Rodent models of lung injury due to hyperoxia and intratracheal bleomycin show a clear detrimental effect of PAI-1 in terms of mortality and subsequent fibrosis (1, 10). In a similar vein, manipulation of the alveolar fibrinolytic activity with an adenovirus encoding the human u-PA, PAI-1's target proteinase, exhibited a small, protective effect toward bleomycin-induced lung injury (41). From these data, it seems that PAI-1 plays a pivotal role in the progression of ALI and the development of pulmonary fibrosis in this species.
Of less certainty is the mechanism of PAI-1's detrimental effect on lung
injury and repair. This is critically important because PAI-1 has both
protease inhibitor-dependent and -independent effects on cell adhesion,
migration, and intracellular signaling as well as effects on proteolysis that
are directed pericellularly and on bulk proteolysis of the fibrinous alveolar
provisional matrix (5,
35,
42,
49). It has been well
established that alveolar fibrin is a prominent component of the surfactant
dysfunction in early lung injury and may play a role in repair
(16,
40). However, recent data
suggest that PAI-1 exerts its effect on bleomycin lung injury independent of
the presence of fibrin in the injured alveolus
(19). Furthermore, through
inhibition of target proteinases in a matrix-directed fashion, PAI-1 can
affect the activation and matrix release of several growth factors, including
basic fibroblast growth factor, TGF-, and hepatocyte growth factor
(5,
11). The mechanism of PAI-1's
effects in lung injury remains an area of active basic research.
This study has some limitations. Because no biopsy material was available from our patients, we were unable to investigate the cellular source(s) of PAI-1. Furthermore, the ALI patients were from a mixed population of sepsis-related and nonsepsis-related medical and surgical intensive care unit populations with a relatively high overall mortality rate. The 95% CI for mortality rate in our study overlaps that of some prospective outcome-based studies and prior studies of ALI patients with aspirable pulmonary edema fluid (5070%) (9, 30, 45, 51). However, the mortality rate is higher than other, more recent, outcome-based or therapeutic trials (29, 44). We acknowledge the possibility that patients who have aspirable edema fluid may have more severe lung injury and a higher mortality rate than the general population with ALI. This possibility does not diminish the prognostic significance of high PAI-1 levels in this group. Clearly, our findings should be prospectively extended to a larger group of ALI patients, including those with milder forms of lung injury and/or those with nonaspirable edema fluid. Unexpectedly, the presence of sepsis did not influence the alveolar compartment PAI-1 or D-dimer findings, thus providing further support for the importance of intrapulmonary inflammation in governing alveolar compartment fibrinolytic alterations.
In conclusion, elevated levels of PAI-1 antigen in both plasma and pulmonary edema fluid are higher in ALI than in hydrostatic edema. Furthermore, these elevated levels of plasma and edema fluid PAI-1 antigen predict a higher mortality and fewer days of unassisted ventilation in patients with ALI. Together with the fibrin fragment results, the data indicate that fibrin turnover in early lung injury is a consequence of rapid fibrinogen influx and fractional fibrinolytic inhibition.
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ACKNOWLEDGMENTS |
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P. Prabhakaran is a Dixon Fellow in Pediatric Research
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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