1 The Medical Research Council
Group in Developmental Lung Biology and
2 Respiratory Research
Division, Respiratory
distress syndrome is characterized by fibrin deposition in the lung.
Fibrin adversely affects surfactant function and stimulates
proliferation of fibroblasts. There is evidence that these properties
may be important to the development of bronchopulmonary dysplasia.
Despite successful initial treatment of neonatal respiratory distress
syndrome with surfactant, the incidence of bronchopulmonary dysplasia
has not decreased. In previous studies, it has been demonstrated that
rat fetal distal lung epithelium (FDLE) possesses both procoagulant and
anticoagulant properties. In this report, we have demonstrated (using
factor VII-deficient plasma) that tissue factor is expressed on the
FDLE surface and promotes thrombin generation. To regulate thrombin
within this procoagulant environment, we have developed a novel
anticoagulant, antithrombin-heparin covalent complex (ATH) that can be
retained within the lung after intrapulmonary instillation. We have
demonstrated that ATH was superior to antithrombin plus standard
heparin in suppressing thrombin generation
(P < 0.001) and prothrombin
consumption (P < 0.01) in
recalcified defibrinated plasma on the surface of FDLE. Further studies
with ATH in vivo need to be performed.
tissue factor; respiratory distress syndrome; bronchopulmonary
dysplasia; anticoagulant
RESPIRATORY DISTRESS SYNDROME (RDS) is a common
condition in premature infants and is characterized by increased
permeability, pulmonary edema, and fibrin deposition within the lung's
intravascular, interstitial, and intra-alveolar spaces (2, 12, 14).
Although surfactant therapy has been used with success in decreasing
mortality associated with neonatal RDS, surfactant has failed to reduce the incidence of bronchopulmonary dysplasia (BPD) (20, 21, 23, 43),
suggesting that other mechanisms are important. Some studies (9, 11,
37) suggested that fibrin deposition may contribute to the severity of
RDS as well as to the development of BPD. Fibrin monomer, produced by
thrombin proteolysis of fibrinogen, leads to impaired surfactant
function (31, 41), increased permeability, and further fibrin
formation. Fibrin stimulates fibroblast proliferation in the lung (10,
11), which may contribute to the pathogenesis of BPD.
Regulation of thrombin, a key enzyme in coagulation, is critically
important to the generation of fibrin. One can hypothesize that if
thrombin activity within the alveolar space can be downregulated, fibrin deposition can be decreased and can potentially reduce the
incidence and severity of BPD. Heparin has been used as an anticoagulant for both the treatment and prophylaxis of intravascular thrombosis. However, when heparin was directly instilled into the lung,
it was not retained within the lung (19). Furthermore, in the absence
of antithrombin (AT), heparin would have no AT activity within the
lung. Although plasma proteins can diffuse into the lung during RDS,
these molecules may not all reach the alveolar space in the same
relative proportions, such that the amount of AT present for
interaction with heparin may still be insufficient. This scenario is
likely because it has been shown that there is increased binding of AT
to the subendothelial extracellular matrix compared with fibrinogen
(45) due to the presence of proteoheparan sulfate molecules (18). To
obviate these difficulties, we developed a novel AT-heparin covalent
complex (ATH) with high specific AT activity. Our studies showed that
ATH is retained within the rabbit lung for at least 48 h and that ATH
extracted from the lung 48 h after its administration still retained AT activity (8).
Rat fetal distal lung epithelium (FDLE) provides an in vitro system
that has been used to study epithelial cell regulation of thrombin
(29). Previously, our laboratory (1) has shown that the FDLE surface is
procoagulant and promotes thrombin generation in plasma as well as
secreting factor VII (FVII)-dependent tissue factor (TF) activity.
However, FDLE also expresses glycosaminoglycans that have AT catalytic
activities (29) and may promote thrombin inhibition through plasma
inhibitors bound to receptors on the cell surface (1). Thus, given the
complicated balance of coagulant factors expressed by FDLE, it would be
advantageous to regulate thrombin generation by a controlled
administration of an anticoagulant such as ATH. Although we have shown
that active [anti-factor Xa (FXa)] ATH could be recovered
from the rabbit lung 48 h after administration, it was not determined
whether ATH could inhibit thrombin generation on the alveolar surface.
Because the AT and heparin components of ATH cannot dissociate, its
pharmacokinetics may be different compared with free AT plus heparin
due to altered binding to the FDLE plasma membrane or endocytosis.
Also, the mechanism by which ATH could affect thrombin generation may
be complicated because it has been shown that ATH has direct AT
activity as well as the ability to catalyze inhibition of thrombin by
exogenous AT (8). For these reasons, it was necessary to investigate the potential effect of ATH on thrombin generation on FDLE in vitro
before proceeding to more complicated in vivo models of lung injury.
In this study, we have provided further proof that thrombin generation
on the surface of the FDLE was TF dependent. We also showed that
thrombin generation on the surface of FDLE could be suppressed by
anticoagulants and that ATH was more efficient than either standard
heparin (SH) or SH+AT. Some of the possible mechanisms by which ATH
inhibits thrombin generation on FDLE are discussed.
Cell culture. FDLE was isolated from
fetal rats and grown in primary culture according to methods previously
described in detail (30). In brief, lungs from fetal rats (Wistar,
Charles River) of 20-day gestational age (term = 22 days) were removed, and epithelial cells were isolated and separated from fibroblasts by
differential adherence. Cells were plated out in 24-well Nunclon plastic plates (GIBCO BRL) at 2 × 106 cells/well. FDLE was
grown in a 37°C incubator with a 95% humidified room air-5%
CO2 atmosphere. Previous analyses
have shown that the cells cultured by this method were >90% type II
epithelial with Thrombin generation. All plasmas used
were from adult human donors. Thrombin generation studies were
conducted with control pooled plasma from healthy adults or
FVII-deficient plasma (Instrumentation Laboratory, Lexington,
MA). FVII levels were The method for measuring thrombin generation on FDLE has been reported
previously (29). Reactions were done on FDLE monolayers in 24-well
plates that were placed on a metal block on a Thermolyne dri-bath set at 37°C. After the plates were removed
from the incubator atmosphere and placed on the heated block in room
air, thrombin generation was not carried out until the pH of the
culture medium (measured by testing aliquots on litmus paper) had
increased to 7.2 but not >7.6. Each well of confluent FDLE monolayer
was washed with 2 × 1 ml of buffer (0.036 M sodium acetate, 0.036 M sodium diethylbarbiturate, and 0.145 M NaCl, pH 7.40) and was then
incubated for 3 min with 100 µl of buffer and 200 µl of
defibrinated plasma (prepared by incubating 500 µl of plasma with 15 µl of 6 U Ancrod/ml buffer at 37°C for 10 min, winding out the
clot, and winding out any further clot formed after incubating on ice
for another 10 min). A clock was started as 100 µl of 0.04 M
CaCl2 in buffer were added, and at
various times, 25-µl aliquots of the reaction mixture on the surface
of the FDLE were removed and mixed with 475 µl of 0.005 M
Na2-EDTA on ice. Twenty-five
microliters of each EDTA sample were then mixed with 775 µl of
0.00016 M S-2238 (KabiVitum, Stockholm, Sweden) in buffer and heated at
37°C for 10 min before termination of the amidolytic reaction by
the addition of 200 µl of 50% acetic acid. The absorbance at 405 nm
was measured, and the concentration of thrombin was determined by
comparing results to a standard curve generated with thrombin in
S-2238. EDTA samples were also used to measure the concentrations of
prothrombin, thrombin-AT (TAT) complexes, and thrombin-heparin cofactor
II (IIa-HCII) complexes. Because thrombin bound to
TF dependence of thrombin generation on
FDLE. To verify that thrombin generation on the surface
of FDLE was TF dependent, FVII-depleted plasma was used, and the
results were compared with experiments using control plasma. To
investigate the degree to which the intrinsic pathway participates in
any thrombin generated with FVII-depleted plasma, the following
experiments were done. Thrombin and
IIa- Suppression of FDLE promoted thrombin generation by
ATH and other anticoagulants. To compare the AT
activity of ATH [>98% free of starting AT and heparin prepared
as described previously (8)] with that of SH (grade I-A, sodium
salt; Sigma, Mississauga, ON) and that of AT (single band on SDS-PAGE
and >90% functional AT activity; Bayer, Mississauga, ON)+SH, each
anticoagulant was diluted in buffer and incubated with defibrinated
control plasma on the surface of FDLE. Equivalent amounts, by mass, of
AT and/or SH were used in each group. The heparin and AT
concentrations in the recalcified reaction mixtures were 0.128 and
0.505 µg/ml, respectively. Thrombin generation assays were measured
in the presence of FDLE for buffer alone, ATH in buffer, AT+SH in
buffer, and SH in buffer.
Prothrombin consumption. The EDTA time
samples were used to determine prothrombin consumption during the
experiments. Prothrombin concentrations were measured at each time
point during the thrombin generation experiments with a commercially
available ELISA (Affinity Biologicals). Control plasma with a known
prothrombin concentration was used as a standard.
Thrombin inhibition (thrombin-inhibitor
complexes). The EDTA time samples were used to
determine the TAT and IIa-HCII complexes formed during the experiments.
TATs were measured with an ELISA kit (Affinity Biologicals). Purified
TATs (Affinity Biologicals) placed in control plasma were used as
standards. It was verified that thrombin-ATH (IIa-ATH) could be
detected by the ELISA kit from analyses of the reaction of human
thrombin in rabbit plasma. Only IIa-ATH gave a positive result because
rabbit AT could not be detected by the anti-human AT antibody. IIa-HCII
complex was measured with an ELISA kit (Affinity Biologicals). Purified
IIa-HCII complexes (Affinity Biologicals) placed in control plasma were used as standards.
Statistics. Results are reported as
means ± SE unless otherwise indicated. Comparisons among different
groups were made by repeated-measures ANOVA. For time-course
experiments, repeated-measures ANOVA over time was compared among
groups. On finding significance with ANOVA, the ATH group was then
compared with the AT+SH group. Unpaired Student's
t-test was used when only two groups
were compared. The rate of prothrombin consumption was obtained by
calculating the slope over the first 4 min using linear regression.
Values were considered statistically different for
P values < 0.05.
Influence of FDLE on thrombin generation in control
plasma and FVII-deficient plasma. In control plasma,
free thrombin was generated rapidly, with peak activities achieved 4 min after the addition of calcium. However, in FVII-deficient plasma,
peak thrombin generation was both delayed and decreased (Fig.
1). The total amount of free thrombin
differed significantly between control plasma and FVII-deficient plasma
(P < 0.001). These data confirm that
TF is present on the FDLE, which accelerates thrombin generation because the absence of FVII in the plasma delayed and decreased free
thrombin activity. To determine whether the intrinsic pathway was
involved in the generation of thrombin when FVII was absent, experiments were carried out with an anti-human FXI antibody to block
FXI activation. Thrombin generation in control plasma with calcium+TF
on a plastic surface was slightly delayed when anti-FXI antibody was
present in the reaction mixture (Fig.
2A).
When FVII-deficient plasma was reacted with calcium+TF on plastic, FXI
antibody caused thrombin generation to be significantly decreased and
delayed (Fig. 2B). These effects
were not seen if nonimmune IgG was used as a control. The reaction
between FVII-deficient plasma+calcium and added anti-human FXI antibody
on an FDLE surface resulted in no detectable generation of thrombin
after 32 min. These data verify that thrombin generation in
FVII-deficient plasma was likely enhanced by FXI activation through the
intrinsic pathway.
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
5% fibroblast contamination (30). All studies were
completed on the cells in primary culture before the first passage and
within 3 days of harvest.
1% according to the manufacturer. Plasma from
adults was obtained by mixing whole blood with 3.8% sodium citrate (9 parts blood to 1 part citrate) followed by centrifugation for 20 min at
3,000 g to obtain supernatant plasmas.
2-macroglobulin (
2M) retains activity against
small substrates (5), the contribution of
thrombin-
2M
(IIa-
2M) to total thrombin
activity was measured with a previously described method (29). In this
case, the same method as the one described above was used for total
thrombin except that 50 µl of the reaction mixture were taken at each
time point and incubated with 0.007 ml of 0.15 M NaCl containing 0.5 U
SH and 0.084 U AT (to inhibit any free thrombin) for 1 min on ice
before mixing 25 µl of the incubate with 475 µl of
Na2-EDTA. Any thrombin activity
measured was due only to
2M-bound thrombin. Subtraction of the
IIa-
2M activity
from the total thrombin activity gave the amount of free
thrombin. In one set of experiments, FXa generation was estimated with
the same procedure as that above for thrombin generation except that
S-2222 (KabiVitum) was used as the substrate.
2M generation were
carried out in plastic tubes at 37°C as described in
Thrombin generation
except that 135 µl of defibrinated plasma + 15 µl of buffer
[containing, in some cases, either 4 µl of 49.9 mg anti-human
factor XI (FXI) goat IgG/ml or 11.6 µl of 16.96 mg nonimmune goat
IgG/ml (both from Affinity Biologicals, Hamilton, ON)] were
mixed with 33 µl of TF reagent (Thromborel S human
thromboplastin PT reagent; Behringwerke, Marburg, Germany) + 117 µl
of 0.04 M CaCl2 in buffer at
0-min time. The effect of the anti-FXI antibody on the thrombin
generation in FVII-deficient plasma on FDLE was also investigated.
Attenuation of the production of free thrombin by the blockage of FXI
activation would indicate that contact activation of the intrinsic
pathway was involved in thrombin generation in the absence of FVII
(extrinsic pathway).
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
View larger version (15K):
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Fig. 1.
Effect of factor VII on plasma thrombin generation in factor
VII-deficient plasma ( ) and control plasma (
) on fetal distal
lung epithelium surfaces. Data are means ± SE. Factor VII-deficient
plasma generated significantly less thrombin compared with control
plasma, P < 0.001.
View larger version (15K):
[in a new window]
Fig. 2.
Effect of anti-factor XI antibody on thrombin generation in plasma with
added tissue factor. Thrombin generation was measured in either control
plasma (A) or factor VII-deficient
plasma (B) on a plastic surface in
presence of tissue factor and either buffer ( ), nonimmune goat IgG
(
), or anti-human factor XI IgG (
). Generation of thrombin was
slightly delayed by anti-factor XI antibody in control plasma and
significantly decreased and delayed by anti-factor XI antibody in
factor VII-deficient plasma.
Influence of FDLE on prothrombin consumption in control plasma and FVII-deficient plasma. The pattern of prothrombin consumption during thrombin-generation experiments differed dramatically between control plasma and FVII-deficient plasma. In control plasma, after the addition of calcium, prothrombin was rapidly consumed by 4 min (65% depleted), which coincided with the peak of thrombin generated (Fig. 3). However, in FVII-deficient plasma, there was no significant consumption of prothrombin from 0.5 to 4 min, and a significant decrease in prothrombin was not apparent until 8 min (Fig. 3). The concentration of prothrombin remaining at the end of the experiments was negligible in control plasma, whereas the concentration of prothrombin remaining in FVII-deficient plasma was ~30%. These results were consistent with the delay and decreased amounts of thrombin generated in the FVII-deficient plasma compared with the control plasma and demonstrate the effect of FVII (TF) on prothrombin consumption.
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Thrombin-inhibitor complex formation in control plasma and FVII-deficient plasma. Thrombin-inhibitor complex formation was significantly less in the FVII-deficient plasma compared with the control plasma (P = 0.02; Table 1). In addition, the appearance of peak concentrations of TAT and IIa-HCII complexes was delayed in the FVII-deficient plasma compared with the control plasma. These results were in agreement with the decreased conversion of prothrombin to thrombin in FVII-deficient plasma.
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Suppression of thrombin generation on FDLE by ATH. The amount of thrombin generated on the surface of FDLE was statistically different (P < 0.001) among all four groups (ATH, AT+SH, SH, and buffer). ATH suppressed thrombin generation to a greater extent than did AT+SH (P < 0.001; Fig. 4). Significant amounts of free thrombin were generated 1-2 min after the start of the reaction in the AT+SH, SH, and buffer groups, whereas no thrombin activity was detected until 4 min in the ATH group. The maximum amount of thrombin generated occurred at 4 min for the AT+SH, SH, and buffer groups and at 4-8 min for the ATH group. ATH decreased the peak amount of thrombin generated by 80% (Fig. 4). An attempt was made in one set of experiments to measure free FXa generation on the FDLE. A small peak of activity against FXa substrate (S-2222) was observed at 1 min in buffer, which was inhibited by either ATH, SH, or AT+SH.
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Prothrombin consumption on FDLE in the presence of ATH. Because peak amounts of thrombin occurred after 4 min, we compared the rate of prothrombin consumption between the ATH group and the AT+SH group up to that time point. The rate of prothrombin consumption was significantly slower in the presence of ATH compared with AT+SH (P < 0.01; Fig. 5). The concentrations of prothrombin left at 32 min in the presence of ATH were significantly increased compared with AT+SH (P < 0.01; Fig. 5).
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Thrombin-inhibitor complexes in the presence of ATH. Concentrations of thrombin-inhibitor complexes were significantly less in the ATH group compared with the AT+SH group (P < 0.05; Table 2). Peak TAT was achieved earlier in the ATH group (4-8 min) compared with the AT+SH group (8 min) and the SH or buffer group (>8 min). However, peak concentrations of IIa-HCII complex occurred at the same rate for all groups (4 min).
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DISCUSSION |
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The failure of surfactant to reduce the incidence of BPD in premature infants has stimulated the investigation of other potentially responsible pathological mechanisms contributing to neonatal RDS and subsequent BPD. Fibrin deposition in the lung, a pathological feature of neonatal RDS, promotes fibroblast proliferation that contributes to the development of BPD (15). Fibrin is formed after activation of the coagulation system, generation of thrombin, and thrombin proteolysis of fibrinogen (6). Previous work (1, 16) has shown that FDLE promotes the generation of thrombin and that the TF pathway may be important. Our results show that the generation of thrombin in FVII-deficient plasma on the surface of FDLE is impaired compared with that in control plasma (Fig. 1). These data provide further evidence for the importance of the TF pathway in the generation of thrombin on the apical FDLE surface. Our results also show that a novel inhibitor of thrombin, ATH, more effectively inhibits thrombin generation on the surface of FDLE than either SH alone or AT and SH in combination (Fig. 4).
Neonatal RDS is a complex disease that likely has multiple pathological mechanisms contributing to its severity and subsequent development of BPD (15). The presence of fibrin in the lung in neonatal RDS, fibrin monomer inhibition of surfactant function (31, 41), and fibrin promotion of fibroblast proliferation (10, 11) all support the concept that fibrin contributes to the severity of both neonatal RDS and BPD. Early use of dexamethasone and surfactant reduces the incidence of BPD in very low birth weight infants (20, 21, 23, 43). Dexamethasone reduces vascular permeability that decreases protein leakage into the alveolar space (42). Preventing leakage of coagulation proteins, particularly fibrinogen, into the alveolar space will decrease fibrin formation, which may be beneficial in neonatal RDS.
The mechanism(s) by which fibrin is formed in the lung is important to understand in order to develop strategies to inhibit this process. Although blood coagulation can theoretically be initiated by the contact system or by exposure of blood to TF, the latter is the physiologically important activation pathway (4, 26). In this pathway, activated FVII (FVIIa) binds to TF and subsequently activates factor X (25) as well as other feedback loops (27, 34, 35). FXa in combination with activated factor V, calcium, phospholipid, and prothrombin form the prothrombinase complex that converts prothrombin to thrombin (24). TF, an extracellular lipoprotein bound to the membranes of cells that synthesize it (17), functions as a cell-surface receptor for FVIIa (34). TF is produced by several cell types and is present in almost all tissues. It has been shown that rat adult lung epithelial cells express TF (16). Therefore, it is likely that TF is important for thrombin generation on the surface of FDLE and contributes to fibrin formation.
To provide further evidence regarding the importance of TF in the
generation of thrombin on the surface of FDLE, we compared FVII-depleted plasma with control plasma (which contained physiological amounts of FVII). The amount of thrombin generated (Fig. 1) and prothrombin consumed (Fig. 3) was significantly decreased in
FVII-depleted plasma compared with control plasma. A significant amount
of the thrombin generated in FVII-deficient plasma was likely due to activation reactions in the intrinsic pathway because addition of
anti-FXI antibody caused the appearance of free thrombin to be both
delayed and decreased (Fig. 2). Prothrombin consumption was also
delayed in FVII-deficient plasma relative to control plasma (Fig. 3),
which agreed with the delay in appearance of peak TAT and IIa-HCII
complex formation (Table 1). The initial decrease in prothrombin from 0 to 0.5 min was probably due to denatured prothrombin adsorbed onto cell
surfaces because there was no thrombin generated during that period of
time. At times 4 min, very little prothrombin remained in the normal
control plasma for conversion to free thrombin, whereas
prothrombin-to-thrombin conversion was slow throughout the experiments
in FVII-deficient plasma. Because little prothrombin remained in the
normal plasma at
4 min, all of the thrombin that could be generated
was available for conversion to inhibitor complexes, which reduced the
free thrombin concentrations observed at
8 min to the levels detected in the depleted plasma. Reduction in thrombin generation and
prothrombin consumption in the absence of FVII shows that the TF
pathway is the predominant pathway by which thrombin is generated on
the surface of FDLE.
Another factor related to TF that may be involved in thrombin generation on FDLE is the TF pathway inhibitor (TFPI). TFPI inhibits FXa directly by a calcium-independent binding of FXa through the second Kunitz domain of TFPI (13). Also, TFPI can inhibit FVIIa activity by a calcium-dependent mechanism that involves binding of the first Kunitz domain of TFPI to FVIIa either by interaction of TFPI-FXa complexes with FVIIa-TF complexes (13) or by interaction of TFPI with FXa-FVIIa-TF complexes (7). In vivo, TFPI is produced in endothelial cells (3, 44), but it is unclear whether TFPI is made by other cells (38). Although TFPI has been detected in lavage fluid from adult patients that are at risk for RDS, as well as those with RDS (36), it has not been shown whether alveolar epithelial cells produce the inhibitor. Intravascularly, TFPI is found either bound to the vessel wall (50-80% of total), circulating in plasma, or in platelets (38). TFPI in the plasma phase exists in concentrations of 50-150 ng/ml (28), but >85% is present as lipoprotein complexes with low anticoagulant activity (22). During RDS, increased lung permeability may allow for access of plasma TFPI to the alveolar space, but it is unclear how much of free plasma TFPI may appear in the lung fluid because mesenchymal and epithelial cell TFPI binding is largely unknown. Nevertheless, immunodepletion studies in rabbits have shown that TFPI is necessary to prevent disseminated intravascular coagulation elicited by low doses of TF (39). Further experiments are required to determine the possible involvement of TFPI in thrombin generation on FDLE surfaces.
Effective inhibition of thrombin generation or thrombin itself by an
anticoagulant offers a potential therapeutic modality for neonatal RDS
in addition to surfactant. Although an anticoagulant could be
administered systemically, the concentrations within the alveolar space
would likely be low, particularly before significant damage of the
lung. Another route of administration is directly into the lung, with
the goal of preventing thrombin generation on the surface of FDLE
before significant lung damage. Physiologically, there are several
processes by which thrombin formation is regulated. Therapeutically,
heparin and related compounds are the most frequently used agents to
regulate thrombin. Heparin is a heterogeneous compound with an average
molecular weight of 15,000 and a range of
3,000-30,000. Heparin contains a pentasaccharide sequence that
binds to the inhibitor AT, converting it into a rapid inhibitor of many
serine proteases including thrombin (33). Heparin has been administered previously into the lung in adults and was found to be rapidly absorbed
(19). Thus the direct administration of heparin alone into the lung is
not likely a viable option for neonatal RDS. Purified AT is available
and could be administered into the lung; however, AT is a relatively
weak inhibitor of thrombin in the absence of heparin (33). We have
previously produced a covalent conjugate of AT to heparin and called it
ATH. In the absence of a cellular surface, Chan et al. (8)
have shown that ATH has significantly greater AT activity compared with
a combination of SH and AT that are not covalently linked. However, the
effect of ATH on the mechanisms involved in thrombin generation were not studied. Unlike assays that measure inactivation of a limited amount of exogenous thrombin, inhibition of thrombin generation relies
on the effects of the anticoagulant on inhibition of feedback activation of the coagulation cascade, as well as on catalytic capacity
(32). It was unclear whether the concentration of ATH used in this
study (8 nM) would be sufficient to significantly prevent feedback
activation by direct, noncatalytic reaction with the initial thrombin
(or FXa) generated, and the level of ATH catalytic activity (0.1
U/ml) employed has been shown previously, in experiments with heparin,
to give only modest effects on thrombin generation (40). Furthermore,
although it has been demonstrated that ATH remains in the lungs of
rabbits for at least 48 h, with high anti-FXa activity and essentially
no leakage into the systemic circulation (8), the effect of FDLE on the
capacity of ATH to inhibit thrombin generation was not determined. If
ATH were sequestered by FDLE to a significant degree (either by binding to the plasma membrane or by endocytosis), the AT activities could have
been diminished. Thus, although ATH had many properties that suggested
that it might be an anticoagulant that could be administered prophylactically into the lungs of neonates with RDS, it was necessary to investigate thrombin generation on FDLE in the presence of ATH
before proceeding to an animal model of neonatal RDS.
Our results showed that ATH was considerably more effective in regulating thrombin generation in the presence of FDLE compared with AT+SH (Figs. 4 and 5, Table 2). Because it has been shown previously that the direct inhibition of thrombin by ATH is extremely rapid compared with that of AT+SH (8), the delay in the appearance of free thrombin with ATH (Fig. 4) may be due to a rapid inhibition of the thrombin formed initially, which would block thrombin-mediated feedback activation of the coagulation cascade (32). This hypothesis is in agreement with the observation that although TAT concentrations were lower with ATH, peak TAT formation occurred earlier compared with the other groups (Table 2). The superior AT activity may be due to the conjugation of AT to heparin because one equilibrium step in the inhibition of thrombin by AT and heparin was not required with ATH. This property may be particularly important for the potential effectiveness of ATH in the intra-alveolar space. Experiments are underway to test the superior activity of ATH against thrombin generation on lung epithelium in a rat damaged lung model.
In summary, we have shown that the TF pathway plays a significant role in the generation of thrombin on the surface of rat FDLE. A novel ATH was shown to effectively suppress thrombin generation on FDLE, with superior activity compared with those of AT and heparin.
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ACKNOWLEDGEMENTS |
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We thank Sue Smith for technical assistance in animal care and LuAnn Brooker for assistance in preparation of the manuscript.
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FOOTNOTES |
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This work was supported by Project 7 from the Medical Research Council of Canada Group in Developmental Lung Biology.
M. Andrew holds a Career Investigator Award from the Heart and Stroke Foundation of Canada. A. Chan holds a Research Fellowship Award from the Research Institute at the Hospital for Sick Children, Toronto, Canada.
Address for reprint requests: M. Andrew, Hamilton Civic Hospitals Research Centre, Henderson General Division, 711 Concession St., Hamilton, Ontario, Canada L8V 1C3.
Received 6 August 1997; accepted in final form 2 March 1998.
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