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Published ahead of print on July 29, 2004, doi:10.1165/rcmb.2004-0162OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. 552-558, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2004-0162OC

Localization of Plasminogen Activator Activity within Normal and Injured Lungs by In Situ Zymography

Teruaki Nishiuma, Thomas H. Sisson, Natalya Subbotina and Richard H. Simon

Pulmonary and Critical Care Medicine Division, Department of Internal Medicine, University of Michigan School of Medicine, Ann Arbor, Michigan

Address correspondence to: Richard H. Simon, Pulmonary and Critical Care Medicine Division, Department of Internal Medicine, University of Michigan School of Medicine, Ann Arbor, MI 48109. E-mail: richsimo{at}umich.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
During inflammatory lung injury, the fibrinolytic activity that is normally present within bronchoalveolar lavage (BAL) fluid (BALF) is often suppressed due to increased levels of inhibitors, including plasminogen activator inhibitor (PAI)-1. Despite this suppression, BALF frequently contains fibrin degradation products, indicating persistence of fibrinolytic activity within the lung. To address this discrepancy and determine the sites where plasminogen activation is occurring, we developed an in situ zymographic technique for frozen sections of lung tissue that localizes plasminogen activator activity at the cellular level. After validating the method using enzyme inhibitors and mice with genetic manipulations of their plasminogen system genes, we applied the technique to lungs of normal and bleomycin-exposed mice. In normal mice, plasminogen activator activity was localized to bronchial epithelial cells, cells of the alveolar walls, and alveolar macrophages. After bleomycin exposure, in situ zymography showed that, despite loss of fibrinolytic activity within BALF, abundant enzymatic activity was associated with aggregates of inflammatory cells. PAI-1–deficient mice that are protected from bleomycin-induced fibrosis had preserved plasminogen activator activity in BALF and increased tissue activity, as determined by in situ zymography. We conclude that analysis of BALF does not adequately reflect the fibrinolytic activity that persists within microenvironments of the lung during inflammation.

Abbreviations: 7-amino-3-trifluromethylcoumarine, AFC • bronchoalveolar lavage, BAL • BAL fluid, BALF • differential interference contrast, DIC • inhibition constant, Ki • plasminogen activator inhibitor-1, PAI-1 • mice genetically deficient in PAI-1, PAI-1–/– • phosphate-buffered saline, PBS • tissue-type plasminogen activator, tPA • urokinase-type plasminogen activator, uPA • mice genetically deficient in uPA, uPA–/–


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Bronchoalveolar lavage (BAL) fluid (BALF) from normal human or animal lungs has net fibrinolytic activity due to the presence of urokinase-type plasminogen activator (uPA) (1–5). This activity is sufficient to clear fibrin that forms when plasma is introduced into the normal alveolar space. However, during lung injury that occurs in both human diseases and animal models of human disease, the fibrinolytic activity within the BALF is often suppressed due to increased levels of inhibitors, including plasminogen activator inhibitor (PAI)-1 (1–5). Despite suppression of plasminogen activator activity, the BALF from injured lungs often contains high levels of fibrin degradation products, indicating that both fibrin formation and breakdown have occurred (5–8). Apparently, there continue to be microenvironments within the lung where plasminogen is activated and fibrin is degraded despite the lack of fibrinolytic activity in lavage fluid. Where these microenvironments are located within the lung are not known.

The level of plasminogen activation within the lung is an important determinant of the extent of fibrosis that follows pulmonary injury. Enhancement of plasminogen activation reduces fibrosis and inhibition increases it. For example, mice genetically deficient in PAI-1 or mice with enhanced lung uPA activity accumulate less collagen after bleomycin-induced lung injury (9–14). Conversely, mice with decreased fibrinolysis due to an overexpressing PAI-1 transgene, or to deletion of plasminogen genes, develop increased fibrosis (10, 15). A parallel relationship is seen in humans, where individuals with nonspecific interstitial pneumonia are more likely than a control population to have a particular allele at a PAI-1 promoter polymorphic site that causes increased PAI-1 expression (16).

Because of the importance of uPA and PAI-1 in determining the extent of pulmonary fibrosis, we desired to determine precisely where within the normal and injured lung the plasminogen system is active. To address this issue, we developed and validated a tissue zymographic method that localizes plasminogen activator activity within frozen sections of lung tissue with resolution to the cellular level. With this technique, we were able to locate where the plasminogen system is activated within the lung in mice having various genetic manipulations and after bleomycin-induced lung injury. These data were then compared with measurements of plasminogen activator activity within BALF.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Human uPA, human glu-plasminogen, and 2,7-bis-(4-amidinobenzylidene)-cycloheptanone-1-one dihydrochloride ("tPA-stop"), an inhibitor of tissue-type plasminogen activator (tPA), were purchased from American Diagnostica (Stamford, CT). The tPA-stop has an inhibition constant (Ki) for tPA of 0.035 µM, whereas the Ki's for uPA and plasmin are 100- and 200-times higher, respectively (manufacturer-supplied data). B428, a uPA inhibitor, was generously supplied by Eisai Research Institute (Andover, MA). Its Ki for uPA is 0.53 µM, whereas the Ki's for tPA and plasmin are > 300- and > 1000-times higher, respectively (17). Plasminogen-depleted human plasma fibrinogen was purchased from Calbiochem (San Diego, CA).

Animals
Mice genetically deficient in either PAI-1 (PAI-1–/–) or uPA (uPA–/–) were originally engineered by P. Carmeliet (18, 19). The mice were subsequently backcrossed to a C57BL/6 background for greater than eight generations. Wild-type C57BL/6 mice were purchased from Charles River Laboratories (Wilmington, MA). Mice with doxycycline-inducible lung-specific uPA overexpression were obtained as previously described (13).

Bleomycin Administration
Bleomycin-induced lung fibrosis was induced in mice as previously described (10, 13). Briefly, age- and weight-matched groups of mice were anesthetized with intraperitoneal pentobarbital and their tracheas were exposed by neck incisions. Bleomycin (Nippon Kayaku Co., Tokyo, Japan), at the dose of 2.5 U/kg in 50 µl of phosphate-buffered saline (PBS), or PBS alone as a control, was introduced intratracheally through a 27-gauge needle. The neck incision was closed with a sterile metal clip and the animals were allowed to recover.

Plasminogen Activator Activity in BALF
After killing by CO2 asphyxiation, the trachea of each mouse was cannulated and the chest opened. BAL was performed using a 1 ml aliquot of sterile PBS. The BALF was centrifuged at 3,000 rpm for 5 min at 4°C and the supernatant recovered for analyses. To measure plasminogen activator activity, a fibrin clot lysis assay was used (20). Briefly, 50 µl of BALF was mixed in wells of a 96-well plate with 50 µl plasminogen-depleted human plasma fibrinogen (2 µM final concentration) and 50 µl human glu-plasminogen (0.5 µM final concentration). Human thrombin (0.2 U/ml, Sigma-Aldrich Co., St. Louis, MO) in 50 µl aliquots was added to each well, plates were incubated at 37°C, and the absorbance at 405 nm was monitored. After thrombin addition, light absorbance at 405 nm increased rapidly as the fibrinogen polymerized (Figure 1A). In wells containing plasminogen activator activity, the absorbance would return to baseline at a dose-dependent rate. Reagent human uPA was used to generate a standard curve with the rate of fibrinolysis recorded as the time required for 50% of turbidity to be lost (Figure 1B).



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  		Figure 1. Plasminogen activator activity assay. (A) After addition of thrombin to a solution of fibrinogen and plasminogen within wells of 96-well plates, a fibrin gel is formed that is detectable by an increase in turbidity, as measured by increased light absorption at 405 nm. In the absence of plasminogen activators, the fibrin matrix remains stable for > 9 h (open squares). Inclusion of reagent uPA in the fibrin clots causes a progressive lysis of the matrix in a dose-dependent fashion as monitored by a reduction in absorbance at 405 nm. The family of curves (closed squares) represents successive 1:1 dilutions of uPA from 250 mIU (left-most curve) to 7.8 mIU (right-most curve). (B) The time required for a 50% loss of turbidity (t1/2) was used as a measure of fibrinolytic rate. A standard curve was constructed relating the t1/2 to the amount of reagent uPA measured in mIU that was added to the sample.

 
Tissue Specimen Preparation
At 7 and 14 d after intratracheal bleomycin or PBS administration, animals were killed by CO2 asphyxiation. Each mouse's trachea was cannulated with an 18-gauge needle and the chest opened. The lungs were distended to total lung capacity with Tissue-Tek OCT compound (Sakura Finetek USA, Inc., Torrance, CA), removed en bloc, and frozen at –80°C. Cryosections were cut at 7 µm thickness and transferred to glass slides.

In Situ Zymography Analysis
To assess plasminogen activator activity in the lung tissue, a fluorogenic substrate specific for plasmin D-Val-Leu-Lys-(7-amino-3-trifluromethylcoumarine) was purchased from Enzyme Systems Products (AFC-081; Livermore, CA) (21). After cleavage by plasmin, the 7-amino-3-trifluromethylcoumarine (AFC) fluoresces with an excitation maximum at 400 nm and emission maximum at 505 nm. The fluorogenic substrate was dissolved at 10 mg/ml in dimethyl sulfoxide and stored at –20°C. For in situ analyses, the substrate was diluted to a final concentration of 25 µg/ml in 50 mM Tris-HCl, pH 7.5, 50 µg/ml plasminogen, and reagents to generate a 10% polyacrylamide gel. After addition of ammonium persulfate to the mixture, the solution was immediately overlaid onto the frozen tissue sections, covered with a glass coverslip, and maintained for 15 min at 4°C, during which time the polyacrylamide solidified. The slides were then incubated at 37°C for 1 h and visualized with an Eclipse E600 Nikon microscope. Each section was surveyed using reflected light differential interference contrast (DIC) optics to locate informative areas (i.e., those containing the structures of interest or regions of bleomycin-induced injury). Images were captured using DIC optics and a Spot RT Slider camera and software (Diagnostic Instruments, Sterling Heights, MI) and stored digitally for subsequent analysis. The fluorescent emission of the same field was then captured using a Nikon UV-2E/C filter cube. Because the fluorescence of AFC is progressively quenched on exposure to UV light, the tissue sections were not prescreened with fluorescent optics, and each field was exposed to UV light only long enough to capture the image. The fluorescent images were converted to gray scale by image processing in which the blue fluorescence from the cleaved AFC was transformed to appear white against a black, fluorescence-negative background (Adobe Photoshop, San Jose, California). All panels that were used to compare fluorescent intensities were captured using identical camera settings and processed digitally in an identical manner. In several samples, the coverslips were removed after obtaining the fluorescent images using DIC and fluorescent optics, after which the sections were stained with hematoxylin. Previous image capture of areas of these tissue sections were repeated using standard transmitted light optics.

Statistical Analysis
Results are presented as mean ± SEM. Group means are compared for statistically significant differences (P < 0.05) using analysis of variance with the Newman-Keuls multiple comparison test for post hoc pairwise comparisons (Prizm; GraphPad Software, Inc., San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Plasminogen Activator Activity In BALF
Plasminogen activator activity contained in BALF obtained from mice 7 d after intratracheal administration of PBS or bleomycin was measured. When plasminogen was omitted from the fibrinogen/thrombin mixture, no fibrinolytic activity was detected in any of the BALFs, indicating that the assay was detecting plasminogen activator activity. BALF from PBS-treated wild-type and PAI-1–/– mice contained low but detectable levels of plasminogen activator activity, with the activity in lavage fluid from PAI-1–/– mice being higher than that from wild-type mice (P < 0.01) (Figure 2). After bleomycin administration, the plasminogen activator activity within BALF from wild-type mice was totally suppressed. This is in contrast to the effect of bleomycin on PAI-1–/– mice, in which the plasminogen activator activity was easily detectable, at levels higher, in fact, than that found in PBS-treated wild-type or PAI-1–/– mice (P < 0.001).



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  		Figure 2. Plasminogen activator activity within BALF. Wild-type (WT) or PAI-1–/– mice were administered PBS or bleomycin (Bleo) intratracheally and then killed 7 d later. BALF was obtained and the plasminogen activator activity measured using the fibrin degradation assay (see MATERIALS AND METHODS and Figure 1). Individual data points and means ± SEM are displayed.

 
Localization of Plasminogen Activator Activity within the Normal Lung
Using in situ zymography, lung sections from normal mice were examined for plasminogen activator activity (Figure 3). The highest fluorescence intensity was seen associated with bronchial epithelium (thin arrows). Slightly less intense fluorescence emanated from cells that constituted the alveolar walls. These cells were frequently located in the corners or junction points of the alveoli (arrowheads). Alveolar macrophages identified by being free within the alveolar space also showed fluorescent activity (thick arrows). The arterioles adjacent to bronchioles fluoresced less intensely (asterisk).



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  		Figure 3. Localization of plasminogen activator activity by in situ zymography. Frozen sections of lung tissue from wild-type mice were overlaid with polyacrylamide-containing plasminogen and the fluorogenic plasmin substrate as detailed in MATERIALS AND METHODS. Representative DIC (left) and fluorescent (right) microscopic images are displayed. Prominent activity is found in bronchial lining cells (thin arrows) and in scattered cells of the alveolar walls. Activity is present, although weaker, in the walls of bronchiolar arterioles (asterisk). The insets display a positive fluorescing alveolar macrophage (thick arrow) and an alveolar wall cell (arrowhead).

 
Specificity of Reaction Product for uPA
Compared with the level of fluorescence seen in positive control sections incubated with fluorogenic substrate and plasminogen (Figure 4A), fluorescence was markedly diminished in the absence of plasminogen (Figure 4D). This indicates that the fluorescence was measuring plasminogen activator activity. Aprotinin, an antiprotease that inhibits plasmin activity but not uPA or tPA, blocked fluorescence generation (Figure 4G). The uPA inhibitor B428 at 20 µM decreased the intensity of fluorescence (Figure 4E) compared with its positive control (Figure 4B). The effect of amiloride, another inhibitor of uPA but not of tPA, could not be tested because it fluoresces with excitation and emission spectra that overlap those of AFC, the cleavage product of our fluorogenic substrate. An inhibitor of tPA, 2,7-bis-(4-amidinobenzylidene)-cycloheptanone-1-one ("tPA-stop"), which is 100-fold more inhibitory for tPA than for uPA, caused little reduction in tissue fluorescence (Figure 4F) at 1 µM compared with its control condition (Figure 4C). These results demonstrate that the fluorescence generated by the in situ zymography of lung tissue is predominantly due to uPA activity.



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  		Figure 4. Specificity of uPA activity assessed by in situ zymography. The factors responsible for the generation of fluorescence were assessed using lung sections from wild-type mice. Each panel in the top row (A–C) is a representative photomicrograph, showing the generation of fluorescence under the positive control condition (fluorogenic substrate plus plasminogen). These are used for comparisons with the panels directly below (D–G) where each column represents a separate experiment. The omission of plasminogen (D) or the addition of 10 µg/ml of the plasmin inhibitor aprotinin (G) greatly reduced the generation of fluorescence. Fluorescence was also reduced by addition of 20 µM B428 (E), an inhibitor of uPA but not tPA. However, addition of 1 µM of the tPA inhibitor, tPA-stop, caused little change in fluorescent intensity (F).

 
Fluorescence Intensity of Sections from Genetically Manipulated Mice
To further validate that the in situ zymography was a reflection of uPA activity, we studied lung tissue obtained from mice having various genetic manipulations of the plasminogen system. The fluorescence generated by lung tissue from mice genetically deficient in uPA (uPA–/–) (Figure 5C) was considerably less than that of wild-type mice (Figure 5A). The residual activity observed in uPA–/– mice was totally inhibited by tPA-stop (data not shown). Tissue from mice homozygous for targeted deletion of their PAI-1 gene (PAI-1–/–) had increased fluorescent intensity (Figure 5B). The tissue of transgenic mice expressing a lung-specific uPA transgene (Figure 5D) had a higher level of fluorescent intensity than that of wild-type mice. These results indicate that the intensity of fluorescence generated during in situ zymography varied in accordance with the predicted level of plasminogen activation in the lungs of these genetically manipulated mice.



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  		Figure 5. In situ zymography of lung sections from genetically manipulated mice. Representative photomicrographs are displayed of the fluorescence generated by tissue sections of (A) wild-type, (B) PAI-1–/–, (C) uPA–/–, and (D) uPA-overexpressing mice.

 
Localization of Plasminogen Activator Activity in Lungs of Mice after Intratracheal Bleomycin Administration
As reported above, plasminogen activator activity within BALF of mice receiving bleomycin was abolished in wild-type mice but increased in PAI-1–/– mice. To localize the plasminogen activator activity in the lung after bleomycin administration, in situ zymography was performed on lung tissue obtained 7 and 14 d after intratracheal bleomycin administration. In wild-type mice, cellular infiltrates were present 7 d after bleomycin (Figures 6A, 6C, and 6D) but were not present in untreated (Figure 3) or PBS-treated (data not shown) mice. In addition to the bronchial lining cells, the cells showing prominent fluorescence were alveolar macrophages (arrows) and cells within the alveolar walls (arrow heads) within areas of apparent bleomycin-induced injury. Analysis of lung tissue from PAI-1–/– mice taken 7 d after bleomycin exposure (Figure 6B) also demonstrated fluorescing cellular aggregates in injured areas, the intensity of which appeared even greater than that of aggregates present in wild-type mice. The fluorescent intensity of cells within areas of injured lung was greater than that of uninjured regions within the same lung (Figures 6E and 6F). This was particularly apparent in areas overlying cellular aggregates, which fluoresced intensely and likely represent inflammatory cells (Figure 6E).



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  		Figure 6. In situ zymography of lung sections from wild-type and PAI-1–/– mice 7 d after exposure to bleomycin. Representative photomicrographs are displayed from sections of bleomycin-exposed wild-type (A, C–F) and PAI-1–/– (B) mice using fluorescent (A–C, E-F) and bright-field (D, hematoxylin-stained) optics. Areas containing fluorescent macrophages (arrows) and cells within alveolar walls (arrowheads) are seen. (E and F) Photomicrographs of lung tissue from the same wild-type animal are compared showing a region demonstrating bleomycin-induced injury (E) and an unaffected region from the same animal (F), taken with the same camera settings and processed identically.

 
At 14 d after bleomycin treatment, brightly fluorescing aggregates of inflammatory cells continued to be observable in lungs of wild-type mice (Figures 7A and 7B; arrows) and, much less commonly, in PAI-1–/– mice (Figures 7C and 7D). Also seen in tissue from wild-type mice were regions of amorphous material that fluoresced with a granular pattern (Figure 7B; arrowheads).



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  		Figure 7. In situ zymography of lung sections from wild-type and PAI-1–/– mice 14 d after exposure to bleomycin. Representative photomicrographs are displayed using DIC (A, C) and fluorescent (B, D) optics of lung sections from bleomycin-exposed wild-type (A, B) and PAI-1–/– (C, D) mice. Areas of fluorescent cellular aggregates (arrows) are present in lungs from mice of both genotypes although more prominent in the wild-type mice. In addition, wild-type mice had areas of granular fluorescing material (arrowheads) that were rarely seen in PAI-1–/– mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The in situ zymographic method we used for these studies provides a level of information not previously available from other analytical techniques. BAL studies have reported that the fibrinolytic activity present in normal subjects is often suppressed in patients with the adult respiratory distress syndrome or chronic interstitial lung diseases (1, 3, 4). These and subsequent studies showed that the decrease in plasminogen activator activity in lavage fluid was due to an increase in enzyme inhibitors, particularly PAI-1. Similar results were found in studies of BALF in animal models of acute or chronic lung injury (2, 22). In agreement with these previous reports, we found that bleomycin administration suppressed plasminogen activator activity in BALF from wild-type mice, but the activity was preserved in fluid from PAI-1–/– mice. This finding is particularly revealing because we have shown that PAI-1–/– mice are relatively protected from bleomycin-induced lung injury (10, 12).

Although these studies of BALF have been instrumental in focusing attention on the role of the plasminogen system in lung injury and repair, the lavage method has its limitations. In particular, it provides an assessment of only one compartment of the lung; namely, the airway/alveolar lining fluid. The method inherently has another shortcoming in that it mixes together lining fluid from all regions within the lung, thereby obscuring local differences. Furthermore, it ignores the contribution that cell surface phenomena play in proteolysis within the alveolar space (see below).

Techniques other than BAL have been used to characterize the plasminogen system within the lung. Levels of mRNA transcripts for components of the plasminogen activation system have been measured in lung homogenates from experimental animals (22, 23). Using Northern analysis, Olman and colleagues reported that mRNAs for uPA, tPA, and PAI-1 increased within 4 d of bleomycin administration in mice (22). They found by in situ hybridization that the PAI-1 transcripts increased early in areas of active alveolitis and that both uPA and PAI-1 were increased later in areas of active fibroproliferation. Other investigators have used immunohistochemistry to locate fibrinolytic enzymes and inhibitors within the lung (23–28). Although informative, these techniques do not assess whether the balance between activators and inhibitors will allow plasminogen activation.

To more precisely localize plasminogen activation within the lung, investigators have used in situ zymography. Smokovitis and Astrup reported that cryosections of normal lung tissue would degrade an overlying fibrin gel that contained plasminogen (29). The resolution of their technique was not sufficient to assign the proteolytic activity to particular regions within the lung. Using a similar approach, Olman and colleagues noted that sections of lung tissue would digest a plasminogen-containing fibrin matrix in areas of large blood vessels in a tPA-dependent fashion and, in areas of alveolar tissue, by a uPA-dependent mechanism (22). However, when applied to lung tissue from bleomycin-exposed mice, the resolution of the method did not allow specific assessment of normal versus inflamed or fibrotic lung tissue.

To circumvent the limitation of these previously described methods, we used a fluorogenic plasmin substrate embedded in polyacrylamide for in situ zymography. To validate that the method appropriately reflects plasminogen activator activity, we first demonstrated that the generation of fluorescent product required the presence of plasminogen. Second, we found that the level of fluorescence was suppressed by inhibitors of plasmin, uPA, and, to a much lesser extent, tPA. However, it should be noted that the in situ zymographic conditions favor uPA detection because fibrin or fibrin fragments, which would increase the activity of tPA, were not added. Finally, we found that the level of fluorescence of lung tissue obtained from genetically manipulated mice varied in accord with the balance of uPA and PAI-1. Specifically, fluorescence was decreased in uPA–/– mice and was increased in both PAI-1–/– mice and mice expressing a uPA transgene.

The in situ zymography method we adapted for the current work demonstrated that the balance of plasminogen activators and inhibitors in normal lung permits net plasminogen activation that is particularly prominent in bronchial epithelium and in cells lining the alveolar walls. Slightly less activity is apparent in alveolar macrophages and overlying peribronchiolar arterioles. After exposure to bleomycin, intense plasminogen activator activity is localized to areas of lung injury. Of note, this prominent activity is manifested at a time when the plasminogen activator activity within BALF is completely suppressed. This discrepancy between BALF and in situ zymography can be ascribed to the importance of the cellular surface in plasminogen activation. In particular, it is known that uPA binding to its cell surface receptor brings the enzyme into close proximity with cell surface–bound plasminogen. This juxtaposition provides a kinetically favorable configuration that accelerates plasminogen activation (30). Furthermore, when bound to cellular surfaces, plasmin is relatively protected from inactivation by its major inhibitor {alpha}2-antiplasmin (31). Both uPA and its receptor are known to be expressed on alveolar macrophages (32) and on alveolar epithelial cells (33–35). In addition, we have previously reported that the binding of uPA to its receptor on alveolar epithelial cells accelerates fibrin clearance from the alveolar space (36).

In summary, we have successfully used an in situ zymographic technique to demonstrate that plasminogen activation with the lungs of bleomycin-treated mice is preserved on the surfaces of inflammatory cells and is likely responsible for the reported presence of fibrin degradation products within BALF during inflammation. We have also shown that suppression of PAI-1 by genetic deletion leads to accentuation of plasminogen activation within BALF and injured lung tissue. Finally, our results emphasize that assessment of plasminogen activator activity by analyzing BALF or lung homogenates is incomplete. These techniques fail to preserve local relationships between enzymes and their inhibitors. Furthermore, they downplay the important contribution of cell surfaces or solid-phase events where enzyme activity can be preserved despite the presence of soluble inhibitors.


   Acknowledgments
 
The authors thank Galina Kuznetsov, Ph.D., Eisai Research Institute, Andover, MA, for supplying B428. This study was supported by grants K08-HL-04434 and P50-HL-56402 from the National Institutes of Health.


   Footnotes
 
Conflict of Interest Statement: T.N. has no declared conflicts of interest; T.H.S. has no declared conflicts of interest; N.S. has no declared conflicts of interest; and R.H.S. has no declared conflicts of interest.

Received in original form May 13, 2004

Received in final form July 15, 2004


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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