Department of Internal Medicine, Justus-Liebig-University-Giessen, D-35385 Giessen, Germany
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Incorporation of pulmonary surfactant into
fibrin inhibits its plasmic degradation. In the present study we
investigated the influence of surfactant proteins (SP)-A, SP-B, and
SP-C on the fibrinolysis-inhibitory capacity of surfactant
phospholipids. Plasmin-induced fibrinolysis was quantified by means of
a 125I-fibrin plate assay, and surfactant incorporation
into polymerizing fibrin was analyzed by measuring the incorporation of
3H-labeled L--dipalmitoylphosphatidylcholine into the
insoluble clot material. Incorporation of a calf lung surfactant
extract (Alveofact) and an organic extract of natural rabbit large
surfactant aggregates (LSA) into a fibrin clot revealed a stronger
inhibitory effect on plasmic cleavage of this clot than a synthetic
phospholipid mixture (PLX) and unprocessed LSA. Reconstitution of PLX
with SP-B and SP-C increased, whereas reconstitution with SP-A
decreased, the fibrinolysis-inhibitory capacity of the phospholipids.
The SP-B effect was paralleled by an increased incorporation of
phospholipids into fibrin. We conclude that the inhibitory effect of
surfactant incorporation into polymerizing fibrin on its susceptibility
to plasmic cleavage is enhanced by SP-B and SP-C but reduced by SP-A. In the case of SP-B, increased phospholipid incorporation may underlie
this finding.
hyaline membrane; coagulation; pulmonary surfactant; surfactant protein; acute respiratory distress syndrome; fibrosis
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ALVEOLAR FIBRIN
FORMATION is a histopathological hallmark of the acute
respiratory distress syndrome and of various other acute or chronic
lung diseases (3, 8, 10, 17-19, 24, 25, 30).
Under inflammatory conditions, both alveolar macrophages and
alveolar epithelial cells may produce and shed significant amounts of
procoagulant activity, which can be almost exclusively attributed to
the extrinsic pathway enzyme tissue factor and factor VII (12,
17-19, 25, 31). Moreover, the fibrinolytic activity of the alveolar compartment is markedly impaired due to depressed urokinase (u-PA) activities and increased activities of the plasminogen activator inhibitor (PAI-I) and 2-antiplasmin (4,
18, 19). Fibrinogen, entering the alveolar space because of
increased endothelial and epithelial permeability, may thus rapidly be
converted to fibrin.
Polymerizing fibrin was noted to cause a severe impairment of surfactant function by incorporating hydrophobic surfactant constituents such as phospholipids and surfactant protein (SP)-B (29). The surfactant inhibitory capacity of polymerizing fibrin surpasses that of fibrinogen, soluble fibrin monomer, and albumin by more than two orders of magnitude, thus rendering fibrin formation the most effective surfactant inhibitory mechanism hitherto described for plasma proteins. Therefore, alveolar fibrin may thus well contribute to the impairment of gas exchange and lung mechanics in acute lung injury. Moreover, delayed clearance of fibrin may provide a provisional matrix for subsequent invasion by fibroblasts (1, 2, 5, 9, 11, 38). Surfactant impairment due to protein leakage, alveolar collapse, persistent "fibrin gluing" of opposed alveolar septae, and fibroblast invasion have been suggested as important sequelae in the pathogenesis of lung fibrosis, a process previously termed "collapse induration" (8). Lysis of such surfactant-incorporating fibrin clots, on the other hand, was noted to result in the liberation of intact surfactant material and marked improvement in surface tension properties (14). Timely dissolution of alveolar fibrin may thus represent an important feature for recovery from acute lung injury and prevention of fibrotic events.
Our group reported that surfactant-rich fibrin clots are less susceptible to proteolysis by plasmin, trypsin, or elastase (14). The present study was performed to further characterize the influence of the surfactant proteins on the fibrinolysis-inhibitory capacity of clot-embedded surfactant. In essence, this was found to be further enhanced by the hydrophobic proteins SP-B and SP-C, but reduced by SP-A. Thus these surfactant proteins apparently exert a differential influence on the kinetics of fibrin turnover in the alveolar space.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Calf lung surfactant extract (CLSE, Alveofact) was a generous
gift from Dr. H. Weller (Thomae, Biberach, Germany). Purified human
fibrinogen (>95% clottability, containing minor amounts of factor
XIII) was kindly provided by Prof. N. Heimburger (Behringwerke, Marburg, Germany). Human plasmin (specific activity 8 U/mg) was purchased from Boehringer Mannheim (Mannheim, Germany). Bovine thrombin
was from Behring (Behringwerke).
L--Dipalmitoylphosphatidylcholine (DPPC) and
phosphatidylglycerol (PG, derived from egg yolk) as well as
n-octyl-
-D-glucopyranoside were received from
Sigma (Munich, Germany). 125I-labeled human fibrinogen
(specific activity 200 µCi/mg fibrinogen, >90% clottability) and
dipalmitoylphosphatidyl (N-methyl-3H)choline
(specific activity 84.1 mCi/mg) were obtained from Amersham Buchler
(Braunschweig, Germany). Immuno-plates (Maxisorp) were purchased from
Nunc (Wiesbaden, Germany). All other chemicals were from Merck
(Darmstadt, Germany).
Determination of Phospholipid Concentration
Phospholipids were quantified by a colorimetric phosphorus assay as described by Rouser et al. (26). Aliquots of the samples were extracted according to Bligh and Dyer (6), and organic phases were taken for quantification. All measurements were performed in quadruple.Isolation of Surfactant Proteins
Dimeric SP-B and SP-C were isolated from rabbit lavage (New Zealand rabbits, 2.5-2.8 kg body wt) by means of LH60-chromatography [(36);Determination of Surfactant Protein Concentration
We determined the concentration of the purified surfactant proteins using a protein assay according to Bradford [SP-B, SP-C; (7)] or a commercially available bicinchoninic acid protein assay based on the Lowry principle [(23, 33); SP-A]. For determination of the filtrate recovery of SP-A, SP-B, and SP-C in the filter experiments as detailed below, a solid-phase adsorption ELISA (SP-B, SP-C) and a competitive ELISA protocol (SP-A) were used as described recently (20, 21, 28).Preparation of Surfactant Mixtures
Alveofact was delivered as lyophilisate and was resuspended with 150 mM NaCl containing 3 mM Ca2+ by brief sonication (Bandelin Sonopuls, Berlin, Germany; 50 W, 25 kHz, 1 min). Phospholipid concentration of the stock solution was measured and was adjusted to a final concentration of 50 mg/ml. We achieved supplementation of Alveofact with SP-A by adding increasing amounts of recombinant SP-A in 150 mM NaCl containing 3 mM Ca2+.A synthetic phospholipid mixture (PLX) was prepared by dissolving DPPC and PG in chloroform-methanol (2:1 vol/vol) at a ratio of 7:3 (wt/wt). After lyophilization by a vacuum centrifuge (Univap, Munich, Germany), the lipids were resuspended in 150 mM NaCl containing 3 mM Ca2+ by brief sonication (Bandelin Sonopuls; 50 W, 25 kHz, 1 min). Supplementation of this synthetic PLX with the surfactant proteins was achieved either by adding the hydrophobic SP-B and SP-C in chloroform-methanol to the DPPC/PG stock solution before lyophilization or by admixture of SP-A in saline after resuspension of the dried lipids with 150 mM NaCl containing 3 mM Ca2+ by brief sonication (50 W, 25 kHz, 1 min). The final phospholipid concentration of all preparations was adjusted to 50 mg/ml.
Bronchoalveolar lavages obtained from healthy New Zealand rabbits of either sex were cell depleted by centrifugation at 300 g (10 min, 4°C), and large surfactant aggregates (LSA) were isolated by high-speed centrifugation at 48,000 g. Aliquots of the 48,000 g pellet were SP-A depleted by chloroform-methanol extraction (6), and the organic phase was lyophilized and resuspended in 150 mM NaCl containing 3 mM Ca2+ (sonication as above). Both LSA preparations were analyzed for phospholipid content and adjusted to a final phospholipid concentration of 50 mg/ml. In addition, aliquots of the LSA stock solution were butanol extracted (32), and the aqueous phase was lyophilized and resuspended in the same volume of distilled water as initially taken for extraction. Corresponding volumes of this preparation compared with the native LSA were used in the fibrinolysis assay.
Fibrin Plate Assay
To address the influence of surfactant on fibrinolysis in dependence of the surfactant protein composition, we used a 125I-fibrin plate assay as previously described (34). All components were dissolved in 150 mM NaCl containing 3 mM Ca2+. First, unlabeled (5 mg/ml) and labeled fibrinogen were mixed with various natural and synthetic, surfactant protein-based surfactant preparations (0-30 mg/ml) and were introduced into the wells at a volume of 30 µl. All concentrations refer to this volume. Complete clotting, ascertained by preceding control experiments, was achieved by incubation with thrombin predissolved in 20 µl of buffer fluid (10 mU/ml, 37°C, 1.5 h). The plates were then spun at 1,485 g for 10 min (Univapo 150H centrifuge) for further consolidation of the clot material. Next, we carefully layered 200 µl of saline/Ca2+ or plasmin (1 U/ml) onto the surface of the clot and incubated it while gently vortexing (100×/min) the plates at 25°C. One hundred twenty minutes after the onset of protease or sham incubation, 150 µl of the supernatant were aspirated, and counts were measured in a Canberra PackardThe following protocols were employed.
Influence of a bovine surfactant extract (CLSE) vs. a synthetic, surfactant protein-free PLX on fibrinolysis. Increasing amounts of CLSE (Alveofact) or PLX (0-30 mg/ml) were admixed to the fibrinogen before clotting.
Influence of the native vs. the organic or aqueous fraction of rabbit LSA on fibrinolysis (native LSA, lipophilic and aqueous compounds). Increasing amounts (0-20 mg/ml) of native LSA or lipophilic or aqueous compounds of LSA were prepared as detailed in Preparation of Surfactant Mixtures and admixed to the fibrinogen before clotting.
Impact of surfactant proteins on the inhibition of fibrinolysis by a synthetic PLX. PLX (20 mg/ml), supplemented with increasing amounts (0.02-0.4 mg/ml, corresponding to 0.1-2% wt/wt, related to the lipids) of natural, dimeric rabbit SP-B, natural rabbit SP-C, and natural rabbit SP-A were added to the fibrinogen before clotting.
Impact of SP-A on the inhibition of fibrinolysis by a bovine surfactant extract (CLSE). CLSE (Alveofact, 20 mg/ml) supplemented with increasing amounts (0.1-0.2 mg/ml, corresponding to 0.5-1% wt/wt related to the lipids) of natural rabbit SP-A was added to the fibrinogen before clotting.
Impact of isolated surfactant proteins on the fibrinolysis in the absence of surfactant phospholipids. Natural rabbit SP-A, SP-B, and SP-C (0.2 mg/ml each) were added without any surfactant phospholipids to the fibrinogen before clotting.
Filter Experiments
Phospholipid and surfactant protein incorporation into fibrin was analyzed by means of filter experiments as recently described (29). For phospholipid incorporation, 3H-labeled DPPC was mixed with 2 mg/ml CLSE (Alveofact) or PLX, followed by the addition to increasing concentrations (0-4 mg/ml) of nonlabeled fibrinogen in 150 mM NaCl containing 3 mM Ca2+. In separate experiments, PLX was supplemented with increasing amounts of the purified surfactant proteins (0-2% wt/wt related to the lipids) before the addition of the tracer. After all mixtures were stirred rigorously and incubated for 1 h at 37°C, 10 mU/ml thrombin were added and further incubated for 1.5 h at 37°C. The aqueous phase was then separated from the insoluble clot material by centrifugation through a nylon gauze (pore size 150 µm, 170 g, 5 min). In alternative experiments, the DPPC label was omitted. Instead, 125I-labeled fibrinogen (200 µCi/mg fibrinogen) was added to the fibrinogen preparation to assess the influence of surfactant protein supplementation of PLX on the fibrin formation. 3H and 125I counts were determined in the filtrate and compared with the total activity provided in each individual experiment.The incorporation of SP-A, SP-B, and SP-C into fibrin was similarly assessed either in absence of any phospholipids or in presence of 2 mg/ml PLX. We used 0.02 mg/ml of surfactant protein (1% wt/wt related to lipids) throughout. SP-A, SP-B, and SP-C recoveries in the filtrates were determined by means of ELISA technique.
Statistics
All data are given as mean ± SE. The Mann-Whitney U-test was performed to assess the level of significance between the various experimental groups, which is indicated throughout the figures (+ or *P < 0.05, ++ or **P < 0.01, and +++ or ***P < 0.001). ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
As could be anticipated from the previous studies, incorporation
of a natural CLSE (Alveofact) into a fibrin matrix caused a
dose-dependent inhibition of plasmin-elicited fibrinolysis (Fig. 1). This held true also for the
surfactant protein-free synthetic PLX (Fig. 1), but PLX was clearly
less effective than CLSE. At the highest dosage used (30 mg/ml), CLSE
reduced the enzymatic release of split products from 37.1% (control,
absence of surfactant) to 5.2%, whereas the corresponding data was
14.4% for PLX.
|
Compared with PLX, LSA exerted similar inhibitory capacity with a
reduced enzymatic release of split products to 46% (LSA) and 43%
(PLX) of control at a phospholipid concentration of 20 mg/ml (Fig.
2). More prominent inhibitory capacity
was noted for the organic phase of LSA, which approximated that of CLSE
(38 and 41% of control at a phospholipid concentration of 10 mg/ml, respectively). In contrast, no inhibitory capacity was noted for corresponding amounts of aqueous LSA constituents.
|
Combining PLX with increasing amounts of hydrophilic vs. lipophilic
surfactant proteins exerted opposite effects on the fibrinolysis inhibition using this synthetic PLX (Fig.
3). Although the addition of natural
rabbit SP-A (0.2-2%, wt/wt related to lipid) almost completely
antagonized the inhibitory effect of 20 mg/ml PLX on fibrinolysis, the
supplementation of PLX with corresponding concentrations of natural
rabbit SP-B or SP-C resulted in a pronounced augmentation of the
fibrinolysis inhibitory properties. In this respect, SP-C and SP-B were
similarly effective. Moreover, natural rabbit SP-A (0.5-1%, wt/wt
related to lipid) was capable of reversing the inhibitory effect of
CLSE on plasmin-induced fibrinolysis (see Fig.
4). In contrast, the addition of the
surfactant proteins in the absence of phospholipids (0.2 mg/ml,
corresponding to 1% in Fig. 3) did not exert any influence on the
cleavage kinetics (control, 36.6%; +SP-B, 36.9%; +SP-C, 39.3%;
+SP-A, 37.8%)
|
|
As assessed by filter studies, addition of neither 2 mg/ml PLX alone
nor PLX supplemented with SP-A, SP-B, or SP-C (1% each) exerted any
effect on the clot formation (control, 95.1%; PLX, 96.5%; PLX plus
SP-A, 95.7%; PLX plus SP-B, 91.7%; PLX plus SP-C, 93.3%).
Approximately 95% of the 125I-label remained within the
insoluble clot material, suggesting complete coagulation and excluding
significant dissociation between label and fibrinogen. When assessing
the phospholipid incorporation using 3H-labeled DPPC, we
observed marked differences between PLX and CLSE. As depicted in Fig.
5, generation of 4 mg/ml fibrin in the presence of 2 mg/ml surfactant resulted in ~45% incorporation of
3H-labeled DPPC in the case of PLX but ~70%
incorporation in the case of CLSE. Reconstitution of PLX with natural
rabbit SP-B, SP-C, or SP-A exerted markedly different effects on the
incorporation rate of this synthetic phospholipid mixture (Fig.
6). Although SP-A and SP-C displayed only
minor efficacy, an admixture of SP-B dose dependently increased the
incorporation rate of PLX. In the presence of 2% SP-B, the percentage
of PLX incorporated into the arising clot increased to ~70% compared
with ~35% in the absence of SP-B, thus approaching incorporation
rates observed for CLSE (containing 1.7 ± 0.3% SP-B).
Accordingly, an increased phospholipid incorporation and enhanced
potency for fibrinolysis inhibition were noted to be closely correlated
for PLX with varying SP-B content (Fig.
7). In line with this finding, we
observed that SP-B itself was substantially incorporated into
polymerizing fibrin in both the presence and absence of phospholipids,
whereas neither SP-A or SP-C showed a significant degree of binding to
fibrin columns (Fig. 8).
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In accordance with previous studies, surfactant incorporation into polymerizing fibrin dose dependently inhibited the susceptibility of this clot to subsequent fibrinolysis by plasmin. We noted a major impact of the surfactant proteins employed in concentrations as encountered under in vivo conditions. The hydrophobic surfactant proteins SP-B and SP-C augmented, but SP-A reduced, the fibrinolysis-inhibitory capacity of the surfactant lipids, as 1) the natural CLSE (Alveofact) containing both SP-C and SP-B was more effective than the surfactant protein-free PLX, 2) the organic solvent extract of rabbit lavage LSA was more effective than the native LSA containing all surfactant proteins, 3) the inhibitory capacity of PLX was dose dependently enhanced on reconstitution with SP-B or SP-C but reduced on reconstitution with SP-A, and 4) the inhibitory capacity of CLSE was reduced on reconstitution with SP-A.
As demonstrated in our previous study (14), the fibrinolysis-inhibitory capacity of surfactant phospholipids is linked to their effective incorporation into a growing fibrin clot: when administered after termination of the clot generation, this effect of the surfactant lipids is fully lost. Hydrophobic-hydrophobic interactions between the phospholipids and lipophilic clusters well known to exist within the fibrin matrix have been suggested as underlying mechanism (14). Interposition of phospholipids between the fibrin fibers, possibly with membranous tapering of single fibrin strands, is assumed to hamper the access of plasmin to its binding sites within the fibrin molecule and thereby retarding its enzymatic cleavage. This view was also supported by the finding that the three-dimensional structure and the physiological properties of the surfactant lipid-rich fibrin differ greatly from those of normal fibrin (15). A direct interference of surfactant phospholipids with plasmin as an alternative mechanism was excluded as plasmin lysis of preformed fibrin was not affected by these lipids (14).
Several mechanisms may underlie the current observation that the fibrinolysis-inhibitory capacity of surfactant lipids is markedly enhanced by SP-B and SP-C, but reduced by SP-A.
Direct Influence of Surfactant Proteins on Fibrinolysis or Fibrin Clot Formation
When tested in the absence of surfactant phospholipids, none of the surfactant proteins exerted any effect on the plasmic cleavage of fibrin. This is in line with the previous observation that surfactant proteins do not interfere with the cleavage of fibrinogen by plasmin in vitro (13). Moreover, the percentage of fibrin(ogen) incorporated into the growing fibrin clot was not affected by the surfactant proteins either in the absence or presence of surfactant phospholipids, with numbers consistently ranging >90%. Thus there is no evidence that the surfactant proteins per se exert a direct influence on the process of fibrin clot formation or the plasmin-induced fibrinolysis.Influence of Surfactant Proteins on Phospholipid Incorporation
The inhibition of fibrinolysis is known to be dependent on the dose of surfactant present during the process of fibrin polymerization (29). Thus any alteration of the extent of surfactant incorporation into the growing fibrin clots could influence the resistance of this clot to plasmic cleavage. SP-B was, indeed, found to greatly enhance the percentage of phospholipid incorporation, from ~35% in the absence, to ~75% in the presence, of 2% SP-B under the given experimental conditions. Accordingly, a close correlation between the increase in phospholipid incorporation and the resistance to plasmic cleavage was noted for the SP-B-enriched PLX. It is in line with this observation that, both in the presence and absence of any phospholipids, SP-B itself was largely incorporated (>70%) into the growing clot material. Although not well established at the present time, SP-B might thus facilitate phospholipid incorporation into polymerizing fibrin by its binding to both surfactant phospholipids (well established) and distinct moieties within the fibrin lattice, thereby resulting in enhanced "anchoring" of the phospholipids in the fibrin clot and increasing the resistance to plasmic cleavage.Alteration of the Macromolecular Phospholipid Arrangement
In contrast to SP-B, SP-A and SP-C exerted no significant influence on the extent of phospholipid incorporation into the polymerizing fibrin; nevertheless, SP-A markedly enhanced, and SP-C decreased, its susceptibility to plasmic lysis and organically extracted LSA, which differs from native LSA mostly by the absence of SP-A and which showed markedly elevated fibrinolysis-inhibitory capacity. Alteration of the macromolecular phospholipid arrangement within the fibrin lattice offers the most feasible explanation for this observation. Because SP-A promotes lipid mixing, reconstitution of PLX with SP-A results in the formation of large multilamellar aggregates, whereas reconstitution with either SP-B or SP-C yields discoidal particles often associated with each other in vertical columns like stacked coins or in flat sheets of subunits arranged in a loose hexagonal array (37). If this also holds true for the architecture of phospholipids within the aqueous solvent channels of the fibrin matrix, a highly complex phospholipid arrangement due to SP-A might result in a rather nonhomogenous distribution of the phospholipid material, and a more membranous or discoidal arrangement of phospholipids due to SP-C might effect a more continuous tapering of the fibrin fibers, with disparate impact on the access of plasmin to its fibrin binding sites. SP-B might induce similar changes on the phospholipid architecture as SP-C but may be of minor importance in terms of fibrinolysis inhibition compared with the SP-B-induced increase in phospholipid incorporation rate. It remains currently open whether addition of SP-A to clinically used organic surfactant extracts may yield reduced inhibition of fibrinolysis on incorporation of these therapeutic surfactants into fibrin polymers formed in vivo under conditions of acute lung injury.Conclusions
We found that the hydrophobic surfactant proteins SP-B and SP-C further decreased, whereas SP-A increased, the susceptibility of a fibrin clot, generated in the presence of surfactant phospholipids, to plasmic cleavage. The most likely explanation for this observation is a differential impact of these surfactant proteins on the phospholipid arrangement within the fibrin matrix, thereby further inhibiting (SP-B, SP-C) or facilitating (SP-A) the access of the protease to its binding and cleavage sites on the fibrin strands. In addition, SP-B was found to augment the percentage of phospholipids incorporated into a growing fibrin clot, further adding to the resistance of the surfactant-phospholipid-rich clot to plasmic cleavage. In view of the profound disturbances of the alveolar hemostatic balance under conditions of acute and chronic inflammatory lung disease, including fibrinogen leakage and changes in alveolar surfactant protein concentrations, it is conceivable that such changes could have a major influence on the kinetics of fibrin turnover in the alveolar space. The differential impact of hydrophobic vs. hydrophilic surfactant proteins may influence therapeutic approaches with surfactant replacement, when the aim is to achieve timely dissolution of fibrin within the alveolar compartment to prevent loss of chronically atelectatic areas. ![]() |
ACKNOWLEDGEMENTS |
---|
This study was supported by the Deutsche Forschungsgemeinschaft (SFB 547 "cardiopulmonary vascular system" project B5).
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: A. Günther, Dept. of Internal Medicine, Med. Klinik II, Justus-Liebig-Univ., Klinikstr. 36, D-35385 Giessen, Germany (E-mail: andreas.guenther{at}innere.med.uni-giessen.de).
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.
September 6, 2002;10.1152/ajplung.00037.2002
Received 23 January 2002; accepted in final form 30 August 2002.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Akiyama, SK,
Hasegawa E,
Hasegawa T,
and
Yamada KM.
The interaction of fibronectin fragments with fibroblastic cells.
J Biol Chem
260:
13256-13260,
1985
2.
Aota, ST,
Nagai T,
Olden K,
Akiyama SK,
and
Yamada KM.
Fibronectin and integrins in cell adhesion and migration.
Biochem Soc Trans
19:
830-835,
1991[ISI][Medline].
3.
Bachofen, M,
and
Weibel EF.
Structural alterations of lung parenchyma in the adult respiratory distress syndrome.
Clin Chest Med
3:
35-56,
1982[ISI][Medline].
4.
Bertozzi, P,
Astedt B,
Zenzius L,
Lynch K,
LeMaire F,
Zapol W,
and
Chapman HA.
Depressed bronchoalveolar urokinase activity in patients with adult respiratory distress syndrome.
N Engl J Med
322:
890-897,
1990[Abstract].
5.
Bittermann, PB,
Rennard SI,
Adelberg S,
and
Crystal RG.
Role of fibronectin as a growth factor for fibroblasts.
J Cell Biol
97:
1925-1932,
1983[Abstract].
6.
Bligh, EG,
and
Dyer WJ.
A rapid method of total lipid extraction and purification.
Can J Biochem
37:
911-917,
1959[ISI].
7.
Bradford, MM.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal Biochem
72:
248-254,
1976[ISI][Medline].
8.
Burkhardt, A.
Alveolitis and collapse in the pathogenesis of pulmonary fibrosis.
Am Rev Respir Dis
140:
513-524,
1989[ISI][Medline].
9.
Campbell, EJ,
Senior RM,
and
Welgus HG.
Extracellular matrix injury during lung inflammation.
Chest
92:
161-167,
1987[ISI][Medline].
10.
Chapman, HA,
Allen CL,
and
Stone L.
Abnormalities in pathways of alveolar fibrin turnover among patients with interstitial lung disease.
Am Rev Respir Dis
133:
437-443,
1986[ISI][Medline].
11.
Couchman, JR,
Austria MR,
and
Woods A.
Fibronectin-cell interactions.
J Invest Dermatol
94:
7S-14S,
1990[Abstract].
12.
Gross, TJ,
Simon RH,
and
Sitrin RG.
Tissue factor procoagulant expression by rat alveolar epithelial cells.
Am J Respir Cell Mol Biol
6:
397-403,
1992[ISI][Medline].
13.
Günther, A,
Bleyl H,
and
Seeger W.
Apoprotein-based synthetic surfactants inhibit plasmic cleavage of fibrinogen in vitro.
Am J Physiol Lung Cell Mol Physiol
265:
L186-L192,
1993
14.
Günther, A,
Kalinowski M,
Elssner A,
and
Seeger W.
Clot-embedded natural surfactant: kinetics of fibrinolysis and surface activity.
Am J Physiol Lung Cell Mol Physiol
267:
L618-L624,
1994
15.
Günther, A,
Kalinowski M,
Rosseau S,
and
Seeger W.
Surfactant incorporation markedly alters mechanical properties of a fibrin clot.
Am J Respir Cell Mol Biol
9:
213-220,
1995.
16.
Haagsman, HP,
Sargeant T,
Hauschka PV,
Benson BJ,
and
Hawgood S.
Binding of calcium to SP-A, a surfactant-associated protein.
Biochemistry
29:
8894-8900,
1990[ISI][Medline].
17.
Idell, S,
Gonzalez K,
Bradford H,
MacArthur CK,
Fein AM,
Maunder RJ,
Garcia JG,
Griffith DE,
Weiland J,
and
Martin TR.
Procoagulant activity in bronchoalveolar lavage in the adult respiratory distress syndrome. Contribution of tissue factor associated with factor VII.
Am Rev Respir Dis
136:
1466-1474,
1987[ISI][Medline].
18.
Idell, S,
James KK,
Levin EG,
Schwartz BS,
Manchanda N,
Maunder RJ,
Martin TR,
McLarty J,
and
Fair DS.
Local abnormalities in coagulation and fibrinolytic pathways.
J Clin Invest
84:
695-705,
1989[ISI][Medline].
19.
Idell, S,
Koenig KB,
Fair DS,
Martin TR,
McLarty J,
and
Maunder RJ.
Serial abnormalities of fibrin turnover in evolving adult respiratory distress syndrome.
Am J Physiol Lung Cell Mol Physiol
261:
L240-L248,
1991
20.
Krämer, HJ,
Schmidt R,
Günther A,
Becker G,
Suzuki Y,
and
Seeger W.
ELISA technique for quantification of surfactant protein B (SP-B) in bronchoalveolar lavage fluid.
Am J Respir Crit Care Med
152:
1540-1544,
1995[Abstract].
21.
Kuroki, Y,
Fukada Y,
Takahashi H,
and
Akino T.
Monoclonal antibodies against human pulmonary surfactant apoproteins: specificity and application in immunoassays.
Biochim Biophys Acta
836:
201-209,
1985[ISI][Medline].
22.
Kuroki, Y,
Mason RJ,
and
Voelker DR.
Alveolar type II cells express a high-affinity receptor for pulmonary surfactant protein.
Proc Natl Acad Sci USA
85:
5566-5570,
1988[Abstract].
23.
Lowry, OH,
Rosebrough NJ,
Farr AL,
and
Randall RJ.
Protein measurement with the folin phenol reagent.
J Biol Chem
193:
265-275,
1951
24.
McDonald, JA.
The yin and yang of fibrin in the airways.
N Engl J Med
13:
929-931,
1992.
25.
Nakstad, B,
Lyberg T,
Skjonsberg OH,
and
Boye NP.
Local activation of the coagulation and fibrinolysis systems in lung disease.
Thromb Res
57:
827-838,
1990[ISI][Medline].
26.
Rouser, G,
Fleischer S,
and
Yamamoto A.
Two-dimensional thin layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots.
Lipids
5:
494-496,
1970[ISI][Medline].
27.
Schägger, H,
and
von Jagow G.
Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa.
Anal Biochem
166:
368-379,
1987[ISI][Medline].
28.
Schmidt, R,
Steinhilber W,
Ruppert C,
Daum C,
Grimminger F,
Seeger W,
and
Günther A.
An ELISA technique for quantification of surfactant apoprotein (SP)-C in bronchoalveolar lavage fluid.
Am J Respir Crit Care Med
165:
1-5,
2002
29.
Seeger, W,
Elssner A,
Günther A,
Krämer HJ,
and
Kalinowski HO.
Lung surfactant phospholipids associate with polymerizing fibrin: loss of surface activity.
Am J Respir Cell Mol Biol
9:
213-220,
1993[ISI][Medline].
30.
Seeger, W,
Günther A,
Walmrath HD,
Grimminger F,
and
Lasch HG.
Alveolar surfactant and adult respiratory distress syndrome. Pathogenetic role and therapeutic prospects.
Clin Investig
71:
177-190,
1993[ISI][Medline].
31.
Seeger, W,
Hübel J,
Klapettek K,
Pison U,
Obertacke U,
Joka T,
and
Roka L.
Procoagulant activity in bronchoalveolar lavage of severely traumatized patients-relation to the development of acute respiratory distress.
Thromb Res
61:
53-64,
1991[ISI][Medline].
32.
Sigrist, H,
Sigrist-Nelson K,
and
Gitler C.
Single-phase butanol extraction: a new tool for proteolipid isolation.
Biochem Biophys Res Commun
74:
178-184,
1977[ISI][Medline].
33.
Smith, PK,
Krohn RI,
Hermanson GT,
Mallia AK,
Gartner FH,
Provenzano MD,
Fujimoto EK,
Goeke NM,
Olson BJ,
and
Klenk DC.
Measurement of protein using bicinchoninic acid.
Anal Biochem
150:
76-85,
1985[ISI][Medline].
34.
Unkeless, JC,
Gordon S,
and
Reich E.
Secretin of plasminogen activator by stimulated macrophages.
J Exp Med
139:
834-850,
1974[ISI][Medline].
35.
Van Iwaarden, JF,
van Berkhout FT,
Whitsett JA,
Oosting RS,
and
van Golde LMG
A novel procedure for the rapid isolation of surfactant protein A with retention of its alveolar-macrophage-stimulating properties.
Biochem J
309:
551-555,
1995[ISI][Medline].
36.
Warr, RG,
Hawgood S,
Buckley DI,
Crisp TM,
Schilling J,
Benson BJ,
Ballard PL,
Clements JA,
and
White RT.
Low molecular weight human pulmonary surfactant protein (SP5): isolation, characterization, and cDNA and amino acid sequences.
Proc Natl Acad Sci USA
84:
7915-7919,
1987[Abstract].
37.
Williams, MC,
Hawgood S,
and
Hamilton RL.
Changes in lipid structure produced by surfactant proteins SP-A, SP-B, and SP-C.
Am J Respir Cell Mol Biol
5:
41-50,
1991[ISI][Medline].
38.
Yamada, KM.
Adhesive recognition sequences.
J Biol Chem
266:
12809-12812,
1991
|
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |