(Received for publication, February 28, 1997, and in revised form, May 19, 1997)
From the Research Institute of the Hospital for Sick
Children, Toronto, Ontario M5G 1X8, the § Hamilton Civic
Hospitals Research Centre, Hamilton, Ontario L8V 1C3, and the
¶ Medical Research Council Group in Developmental Lung
Biology, Respiratory Research Division, Department of Pediatrics,
University of Toronto Hospital for Sick Children's Research
Institute, Ontario M5G 1X8, Canada
Although heparin has been used clinically for
prophylaxis and treatment of thrombosis, it has suffered from problems
such as short duration within compartments in vivo that
require long term anticoagulation. A covalent antithrombin-heparin
complex has been produced with high anticoagulant activity and a long half-life relative to heparin. The product had high anti-factor Xa and
antithrombin activities compared with noncovalent mixtures of
antithrombin and heparin (861 and 753 units/mg versus 209 and 198 units/mg, respectively). Reaction with thrombin was rapid with
bimolecular and second order rate constants of 1.3 × 109 M1 s
1 and
3.1 × 109 M
1
s
1, respectively. The intravenous half-life of the
complex in rabbits was 2.6 h as compared with 0.32 h for
similar loads of heparin. Subcutaneous injection of
antithrombin-heparin resulted in plasma levels (peaking at 24-30 h)
that were still detectable 96 h post-injection. Given the
increased lifetime in these vascular and intravascular spaces, use of
the covalent complex in the lung was investigated. Activity of
antithrombin-heparin instilled into rabbit lungs remained for 48 h
with no detection of any complex systemically. Thus, this highly active
agent has features required for pulmonary sequestration as a possible
treatment for thrombotic diseases such as respiratory distress
syndrome.
Fibrin deposition in the intravascular system causes significant morbidity in the form of deep vein thrombosis, pulmonary embolism, stroke, and myocardial infarction. Heparin is an anticoagulant that, when administered systemically, is effective for both the prevention and treatment of intravascular thromboembolic events (1). Fibrin deposition also occurs outside the intravascular system in a variety of organs and contributes to the morbidity of several diseases (2, 3). One example is neonatal respiratory distress syndrome (RDS),1 which is characterized by fibrin deposition in the extravascular, interstitial, and intra-alveolar spaces (4, 5). Neonatal RDS is a complex disease that probably has multiple pathologic mechanisms contributing to its severity, and the subsequent development of the chronic lung disease, bronchopulmonary dysplasis (BPD) (6-8). The potential importance of fibrin to the severity of neonatal RDS and BPD is supported by the pathologic presence of fibrin in the lung in neonatal RDS, fibrin monomer inhibition of surfactant function (7), and fibrin promotion of fibroblast proliferation (6). The potential role of anticoagulants such as heparin for the prevention of extravascular fibrin deposition in neonatal RDS has not been assessed previously.
A significant limitation for the potential benefit of anticoagulants in most disease states characterized by extravascular fibrin deposition is that anticoagulants cannot be administered locally. One exception is the lung, where anticoagulants can be administered directly into the airways. Heparin has been administered to adult patients via the lung for the purposes of systemic anticoagulation for intravascular thrombosis (9). Unfortunately, heparin itself is not an ideal anticoagulant to administer into the lung in newborns with RDS because of heparin's rapid entry into the intravascular system and its dependence on antithrombin (AT) for activity, which would probably not be present in the intra-alveolar space in the early stages of neonatal RDS. Although purified AT is available, it is a relatively poor inhibitor of thrombin in the absence of heparin (10).
Heparin covalently linked to AT would probably be retained in the lung because of its size. If its anticoagulant activities were retained, heparin complexed to AT could function locally as an anticoagulant. Heparin has previously been covalently linked to AT for the purposes of prolonging its half-life when administered intravascularly (11, 12). The major difficulty encountered in earlier attempts to conjugate AT to heparin was that the introduction of reactive groups for linkage, on AT or heparin, led to complexes with reduced anticoagulant activities. Additionally, the presence of excess unblocked linkage groups in the products could have had unwanted interactions with other molecules or surfaces in vivo.
Recently, we described the synthesis of a unique covalent complex
(ATH), formed from AT and standard commercial heparin, which required
no modification of either the protein or glycosaminoglycan (GAG) prior
to conjugation.2 Production
of ATH was accomplished by taking advantage of the fact that a
subpopulation of heparin molecules contain aldose termini and the
concept that, during prolonged incubations, a Schiff base may form
spontaneously between AT lysyl -amino and heparin aldose aldehyde
groups. Amadori rearrangement could then occur, if there were a free
hydroxyl group on C2 of the terminal sugar, or the Schiff
base could be stabilized by mild reduction. In this report, we
characterize the purification, physicochemical properties, in
vitro activity, and in vivo pharmacokinetics of the ATH
complex. Since it was likely that the high molecular size of ATH would
allow for its sequestration in extravascular spaces, retention of the
complex in the lung, as a possible treatment for neonatal RDS, was
investigated.
All chemicals were of analytical grade. AT was obtained from Bayer. Heparin was obtained from Leo Laboratories Canada Ltd. or Sigma (sodium salt, grade I-A, from porcine intestinal mucosa). High affinity heparin, purified from commercial (standard) heparin, was a kind gift from Dr. Edward Young (Hamilton Civic Hospitals Research Centre, Hamilton, ON, Canada). Heparinase (heparin lyase, EC 4.2.2.7) was from ICN, Costa Mesa, CA. Factor Xa and thrombin(IIa) were obtained from Enzyme Research Laboratories Inc. Phenylalanyl-prolyl-arginyl-thrombin (FPR-thrombin; active site-blocked thrombin) was generated from reaction of thrombin and D-phenylalanyl-prolyl-arginyl chloromethyl ketone (Calbiochem).
Complex PreparationProduction of ATH has been described previously.2 The relative quantities of AT and heparin to be used in the incubations were determined from an estimate of the fraction of heparin molecules containing both a pentasaccharide (13) and an aldose (14) at their termini (Table I). In brief, a covalent complex between AT and heparin was prepared by incubating 0.5-3 mg/ml AT with 50-70 mg/ml heparin in buffer (0.3 M phosphate, 1 M NaCl, pH 8.0; or 0.02 M phosphate, 0.15 M NaCl, pH 7.3) for 3-14 days at 40 °C. Subsequently, 0.1 ml of 0.5 M NaBH3CN in buffer/ml of reaction mixture was added at 37 °C and incubated for an additional 5 h, allowing reduction of any Schiff base not stabilized by Amadori rearrangement.
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Subsequently the covalent ATH product was purified by gel filtration on Sephadex G-200 (Pharmacia, Uppsala, Sweden) using 2 M NaCl for elution. A high molecular weight fraction containing covalent ATH complex was isolated and was essentially void of free AT. The ATH fraction was further purified by electrophoresis on a 7.5% polyacrylamide gel at pH 8.8, using nondenaturing conditions. The section of gel that contained only the ATH complex was cut out and the ATH eluted by incubation in buffer (3.0 g/liter Tris, 14.4 g/liter glycine, pH 8.8) at 23 °C. Purification of ATH was also accomplished on butyl-agarose in one step. Adjustment of the (NH4)2SO4 concentration from 2.5 M to 1.8 M allowed pure ATH to be eluted from the beads while AT remained bound. Large quantities of ATH were purified from the reaction mixture by a two-step procedure involving hydrophobic chromatography on butyl-agarose (Sigma) followed by anion exchange chromatography on DEAE-Sepharose Fast Flow (Pharmacia, Uppsala, Sweden). In 2.5 M (NH4)2SO4, 0.02 M phosphate, pH 7.0, ATH and AT bound to butyl-agarose beads while heparin did not. The bound material was eluted with 0.02 M phosphate, pH 7.0, and dialyzed against 0.01 M Tris·HCl, pH 8.0 buffer, then mixed with pre-equilibrated DEAE Sepharose beads. After washing the beads with 0.2 M NaCl in pH 8.0 buffer to remove bound AT, ATH was eluted with 2 M NaCl in pH 8.0 buffer. The purified ATH was concentrated at 4 °C by dialysis, with a Mr 12,000-14,000 cut-off, under nitrogen pressure (1 atmosphere). All further analyses were carried out using ATH purified by the two-step (butyl-agarose/DEAE) procedure.
Analysis of ATH ComplexThe ATH complex was subjected to heparin degradation by incubation at 37 °C with 0.01 unit/ml heparinase, 0.001 M CaCl2, 0.001 M sodium acetate, 0.15 M NaCl. Protease digestion of the ATH complex involved reaction at 37 °C for 96 h in 0.5 M Tris·HCl, pH 8.0, with the general protease P-5147 (Sigma), which contained no heparin-degrading activity. 2 mg/ml P-5147 was added initially, followed by additions of another 2 mg/ml every 24 h. Following SDS-PAGE as per Laemmli (15), the GAG portion of ATH was stained sequentially with alcian blue and silver (16). The AT portion of ATH was stained using Coomassie Blue. To estimate the molar ratio of AT to H in ATH, SDS-PAGE gels of heparinase-treated ATH (with multiple AT standards), and protease-treated ATH (with multiple samples of known heparin mass), were prepared. They were stained for protein and GAG, respectively. The relative mass of the ATH components was determined by comparing laser densitometry readings against the similarly quantitated standards. The AT to heparin molar ratio was calculated using a molecular weight of 59,000 for AT and an average molecular weight of 15,000 for heparin. Western immunoblots of ATH employed standard techniques (17) with a sheep anti-human AT antibody (Affinity Biologicals, Hamilton, Ontario, Canada). Anti-factor Xa and antithrombin activities were determined on an ACL300 using kits (Stachrom anti-Xa, Diagnostica Stago, Asnières, France; IL test anti-IIa, Instrumentation Laboratory, Milano, Italy). The method for measuring activity involved incubation of the sample with excess enzyme (factor Xa or thrombin) for 30 s in buffer (containing, in some cases, exogenous AT), followed by addition of a chromagenic peptide substrate to determine the residual activity. The amount of enzyme activity inhibited during the reaction was compared with a standard curve, constructed from reactions with plasma samples containing standard heparin (from Diagnostica Stago) of known concentrations (units/ml). The units/ml of the sample was divided by the mg/ml of heparin present to obtain specific activities for the compound. To check that antithrombin activity was due to covalent binding of thrombin, SDS-PAGE of ATH reacted with 125I-thrombin (labeled by chloramine T; Ref. 18) was performed followed by autoradiography. Fluorescence intensity measurements of buffered (phosphate-buffered saline) solutions of ATH, AT + heparin, and ATH + exogenous heparin were made using a Perkin-Elmer LS50B luminescence spectrometer. AT protein concentrations in rabbit plasma and bronchoalveolar lavage (BAL) samples were measured using an antibody that did not cross-react with rabbit AT (Affinity Biologicals).
Kinetics of Thrombin InhibitionThe methods used to determine the bimolecular and second order rate constants were the same as those used by Hoylaerts et al. (10). Constants for the hypothetical kinetic mechanism shown below (Reaction 1) were determined.
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Rabbits were injected either
intravenously or subcutaneously with different amounts of ATH, AT, H,
or AT + H in 1-2 ml of 0.15 M NaCl, 0.02 M
phosphate, pH 7.3. At predetermined times, 0.5-ml samples of blood were
taken into 3.8% sodium citrate (volume blood:citrate = 9:1),
centrifuged at 3000 × g for 15 min, and the
supernatant plasma stored at 60 °C. Plasma samples were analyzed for anti-factor Xa activity and AT protein concentration as described previously. Half-lives of the various species were calculated by
nonlinear least squares fitting to single- or double-exponential equations.
The biodistribution of ATH, after introduction into the rabbit lung, was determined. Anesthetized rabbits were orally intubated (polypropylene endotracheal tubes). ATH in 0.15 M NaCl (0.5-1.5 ml/kg) was instilled sequentially into each lung through the tube by alternating lateral positioning of the rabbit. In a subsample of animals, immediate suction was applied to the tube to retrieve the instilled fluid. All rabbits were extubated and recovered from anesthesia. Blood was obtained over time for plasma samples as described above. After 48 h, the rabbits were again anesthetized and intubated, and BAL was performed with 2 ml of 0.15 M NaCl.
Fig.
1A shows the SDS-PAGE of
fractions collected during gel filtration on Sephadex G-200 under high
ionic strength conditions of the reaction mixture of AT plus H
following incubation. Polydisperse high molecular mass ATH complexes
separated in early fractions followed by a mixture of unseparated lower
molecular mass ATH, unreacted AT (approximately 59 kDa), and free
heparin, as identified by sequential staining for protein and GAG.
Butyl-agarose chromatography of the ATH reaction mixture separated ATH
complex from the beads at 2.0-1.75 M
(NH4)2SO4 (Fig. 1B). At
(NH4)2SO4 concentrations of 1.6 M or lower, significant amounts of unreacted AT were eluted
along with the remaining ATH. Butyl-agarose followed by DEAE-Sepharose
chromatography was able to separate all of the ATH complex (Fig.
2, lane 2). Laser
densitometric analyses of SDS-PAGE gels, stained for either protein or
GAG, showed that ATH purified using butyl-agarose and DEAE-Sepharose was >99% and >95% free of AT and heparin contamination,
respectively. If the purified ATH was chromatographed on Sephadex
G-200, followed by densitometric analysis of SDS-PAGE gels (stained for
GAG) of the fractions, it was determined that <4% of the GAG was
present as free heparin.
Physicochemical Properties
The AT to H ratio in the ATH
complex is 0.9 or approximately 1 to 1. The molecular mass of ATH
complexes ranged from 69 to 100 kDa. An analysis of the purified
product and its constituent components following enzyme degradation is
shown in Fig. 2. Subsequent gel filtration of the heparin from protease
degraded ATH complex showed it to have slightly larger average
molecular mass (18 kDa) compared with the starting heparin (15 kDa).
Heparinase-degraded ATH complex appears as one to three blurred bands,
migrating at slightly higher molecular mass compared with starting AT
(Fig. 2, lane 4) due to the one to three disaccharide units
that remained linked to the AT despite heparinase. This was not seen
with heparinase treatments of noncovalent mixtures of AT and heparin
(Fig. 2, lane 6). Western immunoblots of ATH, and ATH
pretreated with heparinase, shown in Fig.
3, provide further evidence that ATH
contains AT, covalently linked to heparin, which is released only after
heparin degradation.
In Vitro Anticoagulant Activity
Incubation of
125I-labeled thrombin with ATH complex, followed by
SDS-PAGE and autoradiography, demonstrated ATH-thrombin complex formation (Fig. 4). Titration of ATH with
purified factor Xa or thrombin showed that >98% of the molecules were
active. Thus, 0.98-1 mol of either factor Xa or thrombin were
inhibited for each mole of ATH used. Furthermore, anti-factor Xa assays
of fractions from Sephadex G-200 chromatography of ATH demonstrated
that a significant amount of the conjugate (10%) had high activity
(>200 units/mg of heparin) and contained a low molecular mass heparin component (
2000 to
4000 Da, determined by gel filtration of the
heparin released by protease treatment). Relative fluorescence intensity measurements of 100 nM ATH showed it to be
elevated compared with 100 nM AT. Addition of a
5-fold
molar excess of heparin (saturating amounts) was required to obtain a
similar fluorescence with AT, while addition of exogenous heparin to
the ATH solution had no effect.
Specific anti-factor Xa and antithrombin activities of ATH are shown in Table II. When exogenous AT was added, a more than 15-fold increase in capacity of ATH to inhibit either factor Xa or thrombin was observed. Thus the ATH compound had catalytic in addition to noncatalytic activities. Additionally, the activities of ATH, when exogenous AT was present, were approximately 4 times and 2 times greater than the corresponding values for standard heparin and high affinity standard heparin (HASH), respectively. This result indicated that, during ATH formation, AT had selected for heparin molecules enriched with AT binding sites (and thus anticoagulant activities) compared with heparin prepared by affinity chromatography (HASH).
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In vitro kinetics of the reaction of ATH with thrombin are
shown in Fig. 5. The bimolecular rate
constant and second order rate constants obtained for ATH inhibition of
thrombin were 1.3 × 109 ± 2 × 108
M1 s
1 (n = 3)
and 3.1 × 109 ± 4 × 108
M
1 s
1 (n = 3 k
1 determinations) respectively. In
comparison, the bimolecular rate constant for noncovalent mixtures of
AT + standard heparin was 1 × 108
M
1 s
1. The addition of
exogenous heparin at molar concentrations greater than 5 times the
[ATH] inhibited the thrombin + ATH reaction (Fig. 6).
In Vivo Pharmacokinetics
The intravascular clearance of human
AT, heparin, noncovalent mixtures of AT + heparin, and ATH from plasma
after intravenous injection into rabbits are shown in Fig.
7. Heparin with or without coinjection of
AT was rapidly cleared. The clearance of AT followed an exponential
decay pattern. If the plasma curves of heparin, heparin coinjected with
AT, AT, and ATH were all analyzed as single-exponential decays (simple
two-compartment model) half-lives of 0.32, 0.41, 13, and 2.6 h,
respectively, were obtained. Use of a double-exponential model for AT
and ATH gives half-lives for the phase of 69 and 13 h,
respectively. The plasma clearance of ATH more closely resembled AT
than heparin in the rabbit model.
Subcutaneous injection into rabbits of ATH, AT, H, and AT + H showed
ATH reached peak plasma concentration at 24-30 h post-injection, with
significant amounts observed up to 96 h post-injection (Fig. 8). Heparin was rapidly absorbed, with
peak activity by 1 h, and disappearance by 4 h. AT
pharmacokinetics were similar to ATH; however, the maximum
concentration of ATH in plasma after subcutaneous injection was only
3-5% of that for AT.
Results from tracheal instillation of ATH are shown in Fig.
9. Approximately 50% of the anti-factor
Xa activity of the starting fluid could be recovered in fluid extracted
immediately after instillation (Fig. 9A). A similar amount
of ATH was detected by mass (Fig. 9B). BAL taken 48 h
after the introduction of ATH, still contained measurable anti-factor
Xa activity (2% of instillation fluid) and human AT (
3% of
instillation fluid) in all animals, regardless of whether initial
suction had been performed. Plasma samples taken throughout the lung
experiments revealed that, in all animals, no detectable ATH (either by
anti-factor Xa assay or AT enzyme-linked immunosorbent assay) was
present in the circulation.
Fibrin deposition in the lung is a consistent feature of neonatal RDS (4, 5). A variety of observations suggest that fibrin may be contributing both to the severity of neonatal RDS and subsequent development of BPD (6, 8, 19). The local administration of an anticoagulant that could prevent fibrin formation in the lung, but not be absorbed systemically, could be beneficial. The properties of an anticoagulant that may be successful locally within the alveolar space include a large molecular weight, to prevent systemic absorption, and the capacity to inhibit thrombin in the absence of any other components of the coagulation system. To develop such an agent, we explored the possibility of conjugating heparin to the natural thrombin inhibitor, AT. Taking advantage of unique properties of AT and heparin, the complex ATH was formed. ATH was of large molecular weight, was retained within the lung when administered locally, and had potent antithrombin activity.
Heparin is a highly sulfonated polymer containing repeating uronic
acid-glucosamine disaccharides (20). Endogenously, heparin is produced
and secreted by mast cells in a proteoglycan form containing long
heparin GAG chains linked glycosidically to serine residues of a core
protein (21). Commercially, heparin is prepared from intestinal mucosa
or lung as a degradation product consisting of GAG molecules that have
no polypeptide attached. Most of these heparin GAG fragments are still
linked to a serine, but approximately 10% have a free aldose terminus
(14). The free aldose aldehyde group was used to produce a novel
covalent complex of AT and heparin with high anti-factor Xa/IIa
activities and a prolonged half-life when administered intravenously or
locally in the lung. Purification of the ATH conjugate was optimally
achieved by a two-step procedure consisting of hydrophobic
chromatography (butyl-agarose) followed by anion exchange
chromatography (DEAE-Sepharose). The final product contained AT and
heparin linked together by either Amadori rearrangement or reduction of
the Schiff base between an AT lysyl -amino group and a heparin
aldose aldehyde group. In the ATH complex, the AT to heparin molar
ratio was close to 1:1. Due to heparin's polydispersity, the molecular
mass ranged from 69 to 100 kDa. ATH exhibited characteristics of a
covalent conjugate, which showed no signs of dissociation on SDS-PAGE
after heating at 100 °C in
-mercaptoethanol- and detergent-containing buffer (Fig. 2). The average molecular mass of the
heparin component of ATH was slightly higher than the starting heparin
(18 kDa compared to 15 kDa), indicating that AT had selected for larger
heparin molecules during conjugation.
Since complex formation could occur by simple incubation of unmodified
AT and heparin, it was not surprising that a high activity compound was
produced. Under these conditions, binding of heparin by AT would
initially occur via non-covalent interactions with high affinity
binding sites (pentasaccharide sequences; Ref. 22). When a heparin
molecule was bound to AT via a pentasaccharide sequence that was in
close proximity to an aldose terminus on the heparin molecule, covalent
attachment occurred. Evidence for this mechanism is apparent by the
fact that preparations contained highly active ATH molecules where the
pentasaccharide sequence was close to the AT component due to the short
heparin chain length (2000 to
4000 Da). AT in the conjugate had
an elevated fluorescence characteristic of the active conformation
(23), probably due to direct interaction with the heparin in the
complex, since similar mixtures of AT + heparin had significantly
reduced values.
The specific anti-factor Xa and antithrombin activities of ATH were
found to be significantly and unexpectedly increased, compared with
either standard heparin or HASH, when exogenous AT was added (Table
II). The anti-factor Xa activity of ATH is comparable to the value
obtained by Rosenberg et al. (24) for a subfraction of
commercial heparin, which contained two AT binding sites/molecule. If
the heparin moiety in ATH contained, on the average, more than 1 AT
binding pentasaccharide/molecule, this may explain how the conjugated
heparin could catalyze inhibition by exogenous AT while still being
linked to the AT at the aldose terminus. Selection for the very small
percentage of multi-pentasaccharide, very high affinity, heparin
present in standard heparin (1-3%; Ref. 24) was possible, given
the long incubations with a high heparin:AT molar ratio
(200:1).
Kinetically, the velocity of reaction with thrombin was about 1 order
of magnitude faster for ATH than for AT + standard heparin. The second
order rate constant of 3.1 × 109
M1 s
1 is one of the highest
ever reported and is probably diffusion rate-limited (25). There are
several potential reasons for the very high rate of reaction. Unlike
noncovalent mixtures of AT and heparin, there is no initial binding of
serpin and GAG required, which is the rate-determining step (26). In
addition, AT may have selected for heparins with higher average anionic
density, which would enhance the interactions of thrombin with ATH
since its binding is a charge-dependent phenomenon (27).
All of the heparin molecules in ATH contain the high affinity AT
binding site, and, since the average heparin chain length in the
conjugate was greater than the starting heparin, thrombin would have a
shorter mean path distance for contacting the inhibitor complex. The
geometry of heparin in the ATH complex was optimal, since addition of
exogenous heparin inhibited the reaction with thrombin (Fig. 6).
ATH has several important differences compared with previous covalent
AT-heparin complexes. AT conjugated to nitrous acid-treated heparin via
reduction of the Schiff base between a lysyl -amino and
anhydromannose aldehyde had significantly lower anti-factor Xa activity
(140 units/mg of heparin) and essentially no anti-thrombin activity
(28). Joining of AT to heparin by hexamethylenediamine spacer arms
between AT lysyl
-amino groups and heparin uronic acid carboxyl
groups resulted in a heterogeneous product composed of molecules with
variable numbers of AT joined to heparin chains, as well as significant
amounts of unreacted linkage groups remaining on the complex (12).
Attachment of hexamethylenediamine linkage groups to high affinity
heparin reduced the specific anti-factor Xa activity from 250 units/mg
to 162 units/mg, and addition of exogenous heparin to the spacer arm
bonded complex doubled the rate of inhibition of Xa (12), indicating
that significant amounts of the modified heparin had lost activity. In
contrast, added heparin had no effect on the reaction of ATH with Xa
(data not shown). The second order rate constant determined for
reaction of the hexamethylenediamine-linked AT and heparin with
thrombin was 6.7 × 108 M
1
s
1 (10). This is 4-5 times lower than that for ATH
reported here using the same method of measurement. Covalent bonding of
AT and heparin using other methods (29, 30) has given products with decreased activity.
The clearance of ATH, when administered intravenously, was significantly decreased compared with free heparin. The intravenous half-life of ATH was at least 8 times longer than that for heparin, and its disappearance profile approached that of human AT in the rabbit (Fig. 7). Given the increased size of ATH compared with heparin, its elimination via glomerular filtration through the kidneys would be limited. It is possible that the covalently linked AT in ATH may have also have inhibited binding of the heparin component with cell surface receptors involved in clearance. Although subcutaneous administration resulted in prolonged in vivo activity, the bioavailability was greatly reduced (Fig. 8). The poor absorption of ATH into the blood stream, from a subcutaneous injection, suggested that it might be retained in the lung and, if so, may have a role as an anticoagulant for neonatal RDS.
RDS is characterized by increased lung permeability, which leads to intra-alveolar coagulation and the formation of a hyaline membranes that contain fibrin (4, 5). The hyaline membrane inhibits gas exchange (8), causes recruitment of fibroblasts resulting in fibrosis (6), and assists in the evolution toward BPD (19). As thrombin inhibitors are not likely to be present in consistently high levels in the air space, it would be highly useful to have a permanently activated AT molecule available to prevent thrombin-mediated fibrin deposition.
Results from the introduction of ATH into the lungs of rabbits showed that significant activity, as well as AT concentration, could be recovered in BAL taken 48 h after the start of the experiment (Fig. 9). Heparin delivered intratracheally in dogs is readily lost from the lungs into the circulation, with plasma values peaking at 6 h (9). Conversely, ATH was retained in the lungs for over 48 h without detectable ATH in plasma by a highly sensitive AT enzyme-linked immunosorbent assay. Thus, ATH is retained with high antithrombin activity in the lung following local instillation. These properties are critically important to prevent fibrin deposition in the lung. The potential benefits of ATH in neonatal RDS warrant evaluation in an animal model and potentially in newborns.
We gratefully acknowledge Sue Smith for technical assistance with the animal experiments. We also thank Alan Stafford for carrying out the fluorescence experiments.