|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 12 (June 15), 1998:
pp. 4561-4571
Transgenically Produced Human Antithrombin: Structural and Functional
Comparison to Human Plasma-Derived Antithrombin
By
Tim Edmunds,
Scott M. Van Patten,
Julie Pollock,
Eric Hanson,
Richard Bernasconi,
Elizabeth Higgins,
Partha Manavalan,
Carol Ziomek,
Harry Meade,
John M. McPherson, and
Edward S. Cole
From the Cell and Protein Therapeutics Department, Genzyme Corp, and
Genzyme Transgenics Corp, Framingham, MA.
 |
ABSTRACT |
Recombinant human antithrombin (rhAT) produced in transgenic goat
milk was purified to greater than 99%. The specific activity of the
rhAT was identical to human plasma-derived AT (phAT) in an in vitro
thrombin inhibition assay. However, rhAT had a fourfold higher affinity
for heparin than phAT. The rhAT was analyzed and compared with phAT by
reverse phase high-performance liquid chromatography, circular
dichroism, fluorophore-assisted carbohydrate electrophoresis (FACE),
amino acid sequence, and liquid chromatography/mass spectrography peptide mapping. Based on these analyses, rhAT was determined to be
structurally identical to phAT except for differences in glycosylation.
Oligomannose structures were found on the Asn 155 site of the
transgenic protein, whereas only complex structures were observed on
the plasma protein. RhAT contained a GalNAc for galactose substitution
on some N-linked oligosaccharides, as well as a high degree of
fucosylation. RhAT was less sialylated than phAT and contained both
N-acetylneuraminic and N-glycolylneuraminic acid. We postulate that the
increase in affinity for heparin found with rhAT resulted from the
presence of oligomannose-type structures on the Asn 155 glycosylation
site and differences in sialylation.
| |
INTRODUCTION |
ANTITHROMBIN (AT) is a serine protease
inhibitor that inhibits thrombin and factor Xa and, to a lesser extent,
factors IXa, XIa, XIIa, tPA, urokinase, trypsin, plasmin, and
kallikrein.1-4 Human AT, which is synthesized in the liver,
is normally present in plasma at levels of 14 to 20 mg/dL.5,6 It has a molecular weight of approximately 58,000 Da and contains 432 amino acids, three disulfide bridges, and four
carbohydrate side chains, which account for 15% of the total
mass.7,8 Decreased levels of AT may be found in the serum
of individuals who have either a hereditary deficiency of AT or an
acquired deficiency, which can result from a number of pathological
conditions.3
AT circulates in a form with low inhibitory activity.9,10
The addition of heparin increases the inhibitory capacity of AT for
thrombin by 1,000- to 5,000-fold or greater11-13 The AT binds to a well defined pentasaccharide on the heparin
chain.13-16 The interaction between AT and thrombin then
produces a tightly bound TAT (thrombin:antithrombin)
complex17 that is essentially irreversible18
and cleared quickly from circulation. Therapeutic use of heparin
as an anticoagulant works through activation of endogenous AT.
Patients whose AT levels are very low can become refractory to heparin
therapy.19,20 Although circulating AT provides an
anticoagulant effect with exogenously administered heparin, the heparan
sulfate glycosaminoglycans on the endothelial layer lining the blood
vessels may be the physiological site of action for circulating
AT.21-24
We have previously shown that human proteins can be made in the milk of
transgenic goats25,26 and that transgenic production of
large quantities of recombinant protein is feasible.27 We have now produced recombinant human antithrombin (rhAT) in this production mode focusing on large-scale production. The current yearly
production of plasma-derived AT is estimated at approximately 100 kg
worldwide. Because AT is glycosylated7 and glycosylation is
important for half-life and sometimes function28 of
glycoproteins, a mammalian expression system was favored for
recombinant production of this glycoprotein for therapeutic use.
Assuming the highest level of recombinant protein production in
mammalian cell culture is 100 mg/L of culture media and 50% recovery
through the purification process, 2,000,000 L of culture media would be
required to produce 100 kg of purified rhAT. At 2 g/L expression level
in goat's milk, 100,000 L of milk from approximately 150 goats would
be sufficient, assuming similar recovery. We present here the initial
characterization of human AT transgenically produced in the milk of
goats (rhAT) and its comparison with human plasma-derived AT (phAT).
| |
MATERIALS AND METHODS |
Production of the transgenic animal.
Transgenic goats expressing rhAT in their milk, under control of the
 -casein promoter, were made essentially as described previously.27 The goats were developed in collaboration
with Dr Karl Ebert of Tuft's University School of Veterinary Medicine (Boston, MA). The goat -casein gene was cloned as previously described by Roberts et al.29 The coding region between
exons 2 and 7 was removed and replaced with an Xho I
restriction site.27,30 The human AT cDNA was derived from a
modified pBAT6 plasmid,31 which allows excision of the
cDNA as an Xho I to Sal I fragment. The Xho
I/Sal I human AT cDNA was ligated between exons 2 and 7 of the
goat -casein gene to form a goat -casein-human AT cDNA transgene.
Embryos were collected, microinjected, and transferred to recipient
female goats as previously described.27 A founder
(F0) transgenic goat, a male was identified by analyzing
genomic DNA from both a sample of ear tissue and blood by polymerase
chain reaction (PCR)32 and by Southern blot
analysis.27 This founder male was bred to nontransgenic
females and produced transgenic female and male offspring. Transmission
of the transgene was analyzed in genomic DNA isolated from blood by PCR
and Southern blot analysis. The milk from two of the (F1)
transgenic female goats was pooled and used in this study.
Purification.
Milk from AT transgenic goats was stored frozen at  40°C
before use. For purification, milk was thawed, proprietary chemicals were added to enhance filtration, and the milk was clarified through a
500-kD tangential flow membrane filtration unit (AG Technology Corp,
Needham, MA). The permeate from the filter was loaded directly onto a
Heparin Hyper-D (Biosepra, Marlborough, MA) affinity chromatography column. The column was equilibrated in 40 mmol/L sodium phosphate, 135 mmol/L NaCl, pH 6.9, washed with 20 mmol/L sodium phosphate, 400 mmol/L
NaCl, pH 7.0 and eluted with 20 mmol/L sodium phosphate, 2.5 mol/L
NaCl, pH 7.0.
The heparin column eluate was concentrated and diafiltered
against 20 mmol/L sodium phosphate, 65 mmol/L NaCl, pH 7.0 by
ultrafiltration to reduce the conductivity. This solution was applied
to an ANX Sepharose Fast Flow column (Pharmacia, Piscataway, NJ)
equilibrated with 20 mmol/L sodium phosphate, 65 mmol/L NaCl, pH 7.0. The column was washed with equilibration buffer and eluted with 20 mmol/L sodium phosphate, 320 mmol/L NaCl, pH 6.7. The anion exchange eluate was adjusted to 1.26 mol/L citrate by addition of sodium citrate
and loaded onto a Methyl HyperD column (Biosepra) equilibrated with
1.26 mol/L sodium citrate, 7.7 mmol/L citric acid, pH 7.0. The column
was washed with equilibration buffer and eluted with 0.9 mol/L sodium
citrate, 5.5 mmol/L citric acid, pH 7.0. The rhAT was then concentrated
to 25 mg/mL by tangential flow ultrafiltration and formulated.
Analytical methods.
A commercially available (Thrombate; Miles Inc, Elkart, IN) preparation
of human plasma AT was used for comparison with rhAT. The lyophilized
product (Lot # 03B002 B) was reconstituted with 10 mL of
high-performance liquid chromatography (HPLC) grade water and aliquots
(53.3 IU/mL) frozen at 80°C. Goat plasma AT was isolated
from goat plasma by an adaptation of the methods shown for the
recombinant AT.
The expression level of the AT in the milk was determined by
using a two-stage colorimetric endpoint assay in a microplate format,
which measures the degree of thrombin inhibition. This assay is a
modified AT/heparin cofactor assay.33,34 In this method, AT
was added to an excess of porcine heparin (Grade I-A #H3393; Sigma, St
Louis, MO) followed by the addition of a constant amount of thrombin.
After a period of incubation, thrombin activity was measured by the
addition of a thrombin-specific chromogenic substrate (S2238,
Chromogenics, Mölndal, Sweden).
Inhibitory activity of purified rhAT and phAT was measured with
respect to thrombin in a similar manner by using the S2238 substrate
and an excess of heparin (55.5 nmol/L). The concentration of thrombin
(Calbiochem #605190, La Jolla, CA) was 243 mU/mL for this assay, with a
substrate concentration of 337 µmol/L. Inhibitory activity with
respect to factor Xa was measured in an equivalent assay, but with the
substitution of factor Xa (Calbiochem #233526; 6.46 mU/mL in the assay)
for thrombin, and using the S2765 substrate from Kabi. In both assays
the enzyme was added to a mixture of AT and heparin for a 10-minute
preincubation at 37°C before the initiation of the reaction by
addition of substrate. Reaction volumes were 0.6 mL, and all reactions
were stopped after a period of 10 minutes by the addition of glacial
acetic acid. The reaction rate was shown to be linear for this
10-minute period. Heparin cofactor activation of thrombin inhibition
was measured using multiple subsaturating concentrations of heparin in
the assays with AT at 3 nmol/L.
Total protein was determined by using a modified Bradford protein assay
(Pierce, Rockford, IL) with bovine serum albumin as standard. The
concentration of the purified protein was confirmed by amino acid
analysis as previously described.35 Recovery of rhAT after
each purification step was determined by a rapid reverse-phase HPLC
(RP-HPLC) chromatography method performed by using a Hewlett Packard (Wilmington, DE) 1100 series HPLC equipped with
detection at 214 nm. Samples were analyzed on a POROS R2/H column (2.1 × 30 mm; Perseptive Biosystems, Framingham, MA) equilibrated with 0.1% trifluoro acetic acid (TFA) in HPLC grade water at a flow rate of
2.0 mL/min. rhAT was eluted from the column by using 0.08% TFA in
acetonitrile. Test sample concentrations were determined by rhAT peak
area comparisons to a standard quantitated by amino acid analysis.
Polyacrylamide gel electrophoresis was performed according to the
Laemmli method36 by using gradient gels (10% to 20%)
(Integrated Separations Systems, Natick, MA). Gels were stained with
silver by the method of Morrissey.37 Purity was determined
by scanning the gels with an LKB Model 2202 laser
densitometer (LKB Inst, Uppsala, Sweden). BIORAD molecular weight
standards were used for molecular weight estimation. Western blot
analysis was performed by using a modification of the method of
Burnette.38 The labeling antibody was affinity purified
sheep anti-hAT-HRP (SeroTec, Oxford, UK). Development was
with the enhanced chemiluminescence (ECL) system (Amersham, Princeton,
NJ).
Protein purity was assessed by RP-HPLC on a Hewlett Packard 1090 HPLC equipped with photodiode array detection. Samples were diluted to
80 µg/mL in 0.1% TFA and 250 µL aliquots analyzed on a Vydac C4
column (2.1 × 250 mm) equilibrated in 0.1% TFA. The column was
developed with a complex gradient at a flow rate of 0.3 mL/min. Solvent
A was 0.1% TFA and solvent B was 0.08% TFA in acetonitrile; 0 to 2 minutes = 0% solvent B; 2 to 22 minutes = 0% to 40% solvent B; 22 to
27 minutes = 40% to 45% solvent B; 27 to 34.5 minutes = 45% to 60%
solvent B; 34.5 to 40 minutes = 60% to 90% solvent B; and 40 to 42 minutes = 90% solvent B. Peaks were integrated at 215 nm and a purity
value was calculated.
For peptide mapping, samples of AT were reduced and
pyridylethylated and the modified protein was desalted by RP-HPLC on a C4 column. Digestions of both the native and reduced/pyridylethylated protein were performed with Lys-C protease at pH 8.5 at an
enzyme:substrate ratio of 1:50 for 18 hours at 37°C. Resultant
digests were quenched and peptide mapping was performed on a Vydac C8
reverse phase column (2.1 × 150 mm). Peptides were eluted in an
acetonitrile/TFA gradient.
For LC/MS analysis, eluant from the chromatography column was
introduced directly into the electrospray interface of the mass spectrometer and spectra acquired over m/z range of 200 to 4,000 at a
3-second scan rate. Electrospray mass spectrometry was performed on a
Finnigan TSQ 700 triple quadrupole mass spectrometer equipped with a
Finnigan Electrospray source. Chromatography was performed on a Michrom
UMA HPLC at a flow rate of 50 µL/min.
Amino terminal sequence analysis was performed by using an Applied
Biosystems (Foster City, CA) 477 sequencer.
Phenylthiohydantoin (PTH) amino acid analysis was performed with an
on-line Applied Biosystems 120A PTH analyzer equipped with an Applied
Biosystems 2.1 × 220-mm PTH C-18 column.
Monosaccharide analysis was performed according to the method of
Hardy et al39 by using a Dionex HPLC
(Sunnyvale, CA) equipped with a pulsed amperometric detector. Sialic
acid determination was performed by using the thiobarbituric acid
method as modified by Powell and Hart40 by using a Hewlett
Packard 1090 HPLC. Monosaccharide standards were purchased from
Phanstiel Laboratories Inc (Waukegan, IL).
Oligosaccharide content was examined by using the fluorophore-assisted
carbohydrate electrophoresis (FACE) system (Glyko, Novato, CA) and LC/MS analysis of glycopeptides. FACE was performed on
oligosaccharides released from glycoproteins by using N-Glycanase (Genzyme, Framingham, MA). Proteins were denatured with 0.15% sodium
dodecyl sulfate (SDS), 65mmol/L -mercaptoethanol at 100°C for 5 minutes. SDS binding was displaced with 1.2% Nonidet-40 after cooling
on ice for 5 minutes. N-Glycanase was added at 150 U/mg AT and samples
incubated overnight at 37°C. The released oligosaccharides were
labeled overnight at 37°C with the fluorescent label 8, aminonaphthalene-1,3,6-trisulfonate (ANTS) by using reagents and
protocol included in the Oligosaccharide Labelling Reagent Pack
(Glyko). Labeled samples were loaded onto precast oligosaccharide profiling gels from Glyko. Gels were run at 15 mA/gel for 85 minutes. Gels were imaged by using an SE1000 Imager (Glyko) and quantitated by
using FACE Analytical Software (Glyko).
Heparin binding of purified AT was assessed by fractionation on a
TSK-Gel Heparin 5PW affinity column (5 mm internal diameter [ID] × 50 mm; TosoHaas, Montgomeryville, PA) on a Hewlett Packard 1050 HPLC. Samples were applied to the column equilibrated in 50 mmol/L
Tris-Cl, 10 mmol/L sodium citrate, pH 7.4 at a flow rate of 0.5 mL/min.
The column was washed for 10 minutes with equilibration buffer and
eluted with a linear gradient of 0 to 3 mol/L NaCl in equilibration
buffer over 20 minutes. Protein was monitored by absorbance at 215 nm.
Absorbance of a blank run was subtracted to give the final
chromatograms.
Heparin binding of AT was also assessed by measuring the change in
fluorescence of Trp according to Fan et al.41 Samples of AT
were diluted to 20 nmol/L in 20 mmol/L sodium phosphate, 100 mmol/L
NaCl, 100 µmol/L EDTA, pH 7.4 containing 0.1% PEG-4000 (Fluka, Buchs, Switzerland). Fluorescence was measured on
a Spex FluoroMax fluorometer (Spex Industries, Edison,
NJ) by using an excitation wavelength of 280 nmol/L (5-nm bandwidth)
and an emission wavelength of 340 nmol/L (10-nm bandwidth). Heparin
(Sigma Grade I-A #H3393; porcine) was added sequentially in 1-µL
aliquots to up to 16 µL total added volume.
Circular dichroism spectroscopy was performed on a Jasco J-720
spectropolarimeter (Jasco Inc, Easton, MD). The instrument was
calibrated with (+)-10-camphorsulfonic acid. Measurements were made at
room temperature by using cylindrical cuvettes with path lengths 0.01 cm (far ultraviolet, 170 to 260 nm) and 1.0 cm (near ultraviolet, 250 to 360 nm). Data analysis was performed on the average of at least 45 scans. The average spectrum was smoothed by Fourier transformation and
corrected for the baseline. The sample was in 133 mmol/L glycine, 135 mmol/L NaCl, 10 mmol/L sodium citrate buffer at pH 7.0.
| |
RESULTS |
Expression of rhAT.
A graphic presentation of rhAT expression in transgenic goat milk
during a full lactation cycle is shown in
Fig 1. The rhAT expression level remained
relatively constant throughout the full lactation cycle, which lasted
over 300 days with a fairly constant yield throughout the lactation.
The purification process (see Materials and Methods) provided a
cumulative yield of greater than 50% (Table 1) with
high step recoveries at all stages during the process.
View larger version (15K):
[in this window]
[in a new window]
| Fig 1.
Milk production and rhAT expression level of a typical
lactation cycle from one female goat. ( ), milk volume in L/d; ( ), rhAT level in g/d.
|
|
Purity.
The purity of the rhAT was greater than 99% and was at least
equivalent to that observed for the commercial phAT as judged by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Fig 2A). No protein bands other than AT (determined by
Western blot; Fig 2B) were evident with silver staining at a high
protein load (Fig 2A). The higher molecular weight bands in all cases appeared to be multimers of AT with the major oligomeric form in the
phAT having the apparent molecular weight of a dimer. Based on this
gel, the rhAT has a lower level of oligomerization than the phAT.
Purity as assessed by RP-HPLC was determined to be greater than 99%
(Fig 3). The chromatographic profile and retention time of the rhAT was similar to the plasma-derived protein. Analysis of the
leading shoulder areas (seen in both samples) by peptide mapping
coupled with mass spectrometry, identified these peaks as AT molecules
that are partially oxidized (data not shown).
View larger version (71K):
[in this window]
[in a new window]
| Fig 2.
SDS-PAGE gel (A) and Western blot (B) of rhAT (lane 1),
rhAT assay standard (lane 2), and phAT (lane 3). Molecular weight markers are shown on the silver stained gel (A). Twenty micrograms of
protein were applied to each sample lane (lanes 1, 2, and 3). The
SDS-PAGE gel (A) was developed with the Morrissey silver stain, and the
Western blot (B) was developed with a sheep antihuman AT-HRP antibody
(SeroTec) and color development was the ECL system (Amersham).
|
|
View larger version (13K):
[in this window]
[in a new window]
| Fig 3.
RP-HPLC chromatograms showing the purity of rhAT (A) and
phAT (B). Chromatography was performed with 20 µg of protein applied to a Vydac C4 column (2.1 × 250 mm) with a series of linear gradients (see Materials and Methods). Detection was by absorbance at 215 nm.
|
|
Assessment of activity and heparin binding.
The transgenically produced rhAT was found to have a specific activity
of 6 IU/mg protein, equivalent to that of the phAT, by using an assay
that measured inhibition of thrombin in an excess of heparin. The
inhibitory capacity of rhAT and phAT in both thrombin and factor Xa
activity assays was examined (Fig 4). Equivalent inhibition was observed with both the major known targets of
AT.
View larger version (17K):
[in this window]
[in a new window]
| Fig 4.
Comparative inhibition of thrombin (circles) and factor
Xa (inverted triangles) by rhAT ( and  ) and phAT ( and  )
with a saturating concentration of heparin.
|
|
Heparin cofactor activation of rhAT was examined by varying the amount
of heparin used in inhibition assays of either thrombin or factor Xa
(Fig
5). These assays showed that rhAT requires a lower concentration of
heparin than phAT for inhibition of both enzymes. The heparin
concentrations required for half-maximal inhibition of thrombin by rhAT
and phAT were 0.045 nmol/L and 0.151 nmol/L, respectively. For
inhibition of factor Xa, these values were 0.71 nmol/L and 2.22 nmol/L
for rhAT and phAT. In both cases, rhAT required a threefold to fourfold
lower concentration of heparin for activation of the inhibitor.
View larger version (12K):
[in this window]
[in a new window]
| Fig 5.
Heparin cofactor activation assays measured by using
increasing concentrations of heparin. (A) Activation of thrombin
inhibition. (B) Activation of factor Xa inhibition. ( ), rhAT; (),
phAT.
|
|
Analysis of the binding characteristics of each AT was also examined
with a solid-phase heparin affinity column
(Fig 6). The complexity observed in the
elution profile (Fig 6A) was caused by both the heterogeneous
population of heparin used to make the column and the presence of the
higher affinity -isoform of AT, which lacks glycosylation at Asn
135. The  isomer eluted as two peaks, at 21 and approximately 25 minutes for rhAT and 20 and approximately 24 minutes for phAT. The
-AT form also eluted as two peaks at 24 and 28.5 minutes for rhAT
and 23 and 28 minutes for phAT. Both the major and minor peaks
associated with rhAT required a higher salt concentration for elution
compared with the coordinate peaks of phAT indicating a higher heparin
affinity for all forms of the transgenically produced AT. Because the
first -isoform peak coeluted with the second -isoform peak,
quantitation of the forms from this method was not possible.
Examination of the thrombin inhibitory activity across the elution
pattern of the rhAT (Fig 6B) indicated that all of the fractions
detected as having protein also had activity.
View larger version (16K):
[in this window]
[in a new window]
| Fig 6.
Chromatography of rhAT and phAT on a TSK-Gel Heparin-5PW
affinity column. Sample load was 175 µg (A) and 25 µg (B). AT was eluted with a 0- to 3-mol/L NaCl gradient in equilibration buffer with
detection at 215 nm. For (A), the dotted line represents phAT and the
solid line represents rhAT. For (B), the solid line represents rhAT
protein and the circles represent thrombin inhibition activity. The
difference in retention times between panels resulted from a 10-minute
wash preceding the elution gradient in (A) that was not used in (B).
|
|
Examination of heparin binding affinity by using the Trp fluorescence
assay41 provided additional evidence that the two molecules
differ in their affinity for heparin (Fig
7). A fourfold higher affinity for heparin was observed with the rhAT
when compared with the phAT. The calculated binding constants
(Kd values) were 10.3 nmol/L and 41.2 nmol/L for rhAT and
phAT, respectively. However, the Fmax (fluorescence at
saturating heparin) values were indistinguishable for rhAT and phAT.
Dissociation constants were calculated by using nonlinear least squares
analysis as described by Fan et al41 assuming a mean
molecular weight of 13,500 D for heparin.42
View larger version (16K):
[in this window]
[in a new window]
| Fig 7.
The binding of AT to heparin was analyzed by protein
fluorescence enhancement.41 rhAT () and phAT () were
diluted to 20 nmol/L in 20 mmol/L sodium phosphate, 100 mmol/L NaCl,
100 µmol/L EDTA, 0.1% PEG, and pH 7.4. Heparin was added in 1-µL
aliquots, and fluorescence was measured with excitation at 280 nm and
emission at 340 nm.
|
|
Structural characterization.
A detailed biochemical characterization was performed in an effort to
determine the basis for the increased heparin affinity in the
transgenic protein. N-terminal sequence analysis confirmed that the
rhAT had the correct N-terminal sequence. A minor sequence lacking the
first two residues and beginning with serine was observed in both the
rhAT and phAT, with a slightly lower percentage (8% v 13%) of
this form present in the rhAT. The reduced and pyridylethylated peptide
map of rhAT was essentially identical to that of phAT (Fig 8). The only differences observed were
in the regions of the three glycopeptides (K8, K15, and K18). The
peptide containing the Asn 135 glycosylation site (K12) is a tripeptide
and was not retained on the chromatographic column used. The
differences observed in the glycopeptides were caused by the
increased glycosylation heterogeneity present in the transgenic
protein.
View larger version (33K):
[in this window]
[in a new window]
| Fig 8.
Lys C peptide maps of reduced and alkylated rhAT (A) and
phAT (B). The K# designation corresponds to the peptide produced by Lys
C digestion as shown in Table 2.
|
|
The primary sequence of rhAT was confirmed by on-line LC/MS analysis of
an endoproteinase Lys-C digest. By using this procedure, 27 of the 35 theoretical Lys-C peptides were identified
(Table 2). Seven of the eight unidentified
peptides had masses of less than 375 D, which would result in a doubly
charged ion below the 200 mass/charge ratio (m/z) lower
mass range used in these studies. The eighth peptide (K26) was observed
as an incomplete cleavage product because of the presence of a proline
C-terminal to the lysine. The only post-translational modifications
detected were at the known N-glycosylation sites with no evidence of
any other modifications on either the rhAT or the phAT.
View this table:
[in this window]
[in a new window]
|
Table 2.
Lys C Peptide Number, the Residues in Each Peptide
With Each Theoretical Mass, and the Masses Observed for rhAT and
phAT by LC/MS
|
|
Peptide mapping under nonreducing conditions (data not shown) confirmed
that the three disulfide linkages occurred between cysteines 8 to 128, 21 to 95, and 247 to 430 as reported previously.43 The
conformation of rhAT was further analyzed by CD
spectroscopy (Fig 9). The AT far
ultraviolet circular dichroism (CD) spectrum was similar for both
proteins and was characterized by two negative bands at 220 to 222 nm
and 210 nm and an intense positive band around 193 nm. The two
negative minima and a positive maximum were indicative of the presence
of both -helix and sheet in the protein, which was consistent
with the crystal structure data of phAT.44 The near
ultraviolet spectrum of both proteins was also similar with a
number of positive and negative peaks between 260 nm and 300 nm (Fig
9B). These data were in excellent agreement with the previously
published spectra of AT derived from human and bovine
plasma.45
View larger version (18K):
[in this window]
[in a new window]
| Fig 9.
Circular dichroism spectra of rhAT (solid line) and phAT
(broken line) in the far ultraviolet (A) and near ultraviolet (B) with
(+) and without () heparin. The molar ratio between protein and
heparin was 1:7. Molar ellipticity is expressed in deg × cm2 × dmol  1.
|
|
We also monitored changes in the CD of AT in the presence of a
saturating level of heparin (Fig 9B). In the far ultraviolet spectrum,
the addition of heparin produced little change (data not shown), which
suggested that secondary structures were not altered. However, the near
ultraviolet spectrum showed a dramatic increase in band intensity
across the whole region when heparin was added. This marked enhancement
in chiral absorption can be attributed mainly to the perturbation of
buried and exposed tryptophan residues. This CD result also indicated
that for the same protein concentration, the increase in CD intensity
was higher for rhAT than for phAT, which again indicated that the
heparin affinity of the rhAT was higher than that of phAT.
Carbohydrate analyses.
The oligosaccharide structures at all four N-linked glycosylation sites
on phAT have been reported to be similar and to contain primarily
biantennary disialylated complex oligosaccharides without fucose.7 The monosaccharide composition obtained for phAT
(Table 3) was consistent with such a
glycosylation pattern. The monosaccharide composition obtained for rhAT
indicated that the glycosylation was significantly different from that
of phAT. The key differences were the presence of fucose and GalNAc, a
higher level of mannose, a lower level of galactose and sialic acid. No
evidence of O-linked glycosylation was observed during LC/MS analysis
of the rhAT peptide map, which suggested that the GalNAc is present on
N-linked oligosaccharides. The increased level of mannose also
suggested the presence of oligomannose structures on the recombinant
protein.
The glycosylation heterogeneity of phAT and rhAT was further analyzed
by LC/MS analysis of the individual glycopeptides
(Fig 10). By using the LC/MS technique,
the monosaccharide composition of the oligosaccharide structures
present at each site were deduced by subtracting the amino acid mass
from the total mass of the glycopeptide. The predominant
oligosaccharide observed at each site is presented in
Table 4. The glycopeptide containing the Asn 135 site of both phAT and rhAT could not be analyzed in this manner
because of its poor retention on the chromatography column. Other LC/MS
data indicated that the Asn 135 site of rhAT was greater than 80%
glycosylated with the complex oligosaccharide types noted below (data
not shown). In humans, AT lacking glycosylation at the Asn 135 site
(the -isoform) was measured at 5% to 15% of the total AT found in
plasma.46,47
View larger version (25K):
[in this window]
[in a new window]
| Fig 10.
The mass spectrum obtained for the glycopeptides
containing Asn 96, Asn 155, and Asn 192 for rhAT (A, B, and C) and phAT
(D, E, and F), respectively.
|
|
For all three phAT glycopeptides analyzed, the major oligosaccharide
species had a mass of 2,206 to 2,207 D. This mass corresponded to a
monosaccharide composition of HexNAc4, Hex5,NANA2, which was
consistent with a biantennary disialylated structure. This assessment
was in agreement with the published data of Franzen et al.7
In contrast, the mass spectra obtained for the rhAT glycopeptides are
more complex. The mass spectrum obtained for the glycopeptide containing the Asn 96 site (K8) of rhAT (Fig 10A) had a major peak at
1,326.5 m/z corresponding to the 3+ charge state of the glycopeptide with a fucosylated biantennary monosialylated oligosaccharide structure. The peak at 995.4 m/z corresponded to the 4+ charge state of
the same species. The peak at 1,423.5 m/z corresponded to the
fucosylated biantennary disialylated oligosaccharide. Additional minor
peaks were observed surrounding the major peaks, which could be
accounted for by monosaccharide substitutions. The substitution of an
N-glycolyl neuraminic acid (NGNA) for a N-acetyl neuraminic acid (NANA)
resulted in a 16-D mass increase, and the substitution of a HexNAc
(GalNAc) for Hex (Gal) resulted in a 42-D mass increase. The presence
of N-glycolyl neuraminic acid was confirmed by chromatographic analysis. The GalNAc/Gal substitutions accounted for the lower level of
galactose and presence of GalNAc in the monosaccharide compositional
analysis of rhAT. The spectrum for the phAT Asn 96 glycopeptide (Fig
10D) was less complex with the two major peaks at 1,375 and 1,031.3 m/z
corresponding to the 3+ and 4+ charge states of the glycopeptide
containing a biantennary disialylated structure.
The mass spectrum of rhAT Asn 155 containing glycopeptide (K15) (Fig
10B), was more heterogeneous than that of the Asn 96 site. This
spectrum was dominated by two series of peaks, one centered around the
m/z of 1,779.4 (2+ charge) and one around m/z 1,187 (3+ charge) and corresponded to a series of masses
differing by 162 D representing oligomannose-type oligosaccharides
ranging from mannose 3 to mannose 9. The peak with an m/z value of
1,352.1 (3+ charge) corresponded to the peptide Asn 155 with a hybrid oligosaccharide structure. The peaks with m/z values of
735 and 863 arose from earlier eluting peptides K33 and K34,
respectively. In contrast the phAT spectrum for this glycopeptide (Fig
10E) had a single main peak with an m/z value of 1,462.8, again
corresponding to a glycopeptide containing a biantennary disialylated
oligosaccharide structure. Additionally, treatment of rhAT with endoH
to remove oligomannose-type oligosaccharides produced a shift in the
peptide map peak containing Asn 155 (K15) (data not shown).
The predominant oligosaccharide at the Asn 192 glycosylation site of
rhAT (K18) (Fig 10C) was a fucosylated biantennary monosialylated structure giving an m/z of 899.0 for the 4+ and 1,348.0 for
the 3+ charge states. The series of peaks around m/z
1,006.6 corresponded to fucosylated biantennary disialylated
structures. The heterogeneity observed around these peaks also arose
from the monosaccharide substitutions described for the Asn 96 glycopeptide (above). The phAT spectrum (Fig 10F) contained two major
peaks at an m/z of 947.3 and 1,420.6 corresponding to the
3+ and 2+ charge states of the glycopeptide
containing a biantennary disialylated oligosaccharide structure.
FACE analysis of oligosaccharides released from rhAT and phAT confirmed
that the predominant oligosaccharide present on the recombinant protein
was a monosialylated biantennary fucosylated structure
(Fig 11, lane 4). Fucose on the rhAT
oligosaccharides made the oligosaccharide slightly larger (slower
mobility) than the nonfucoslyated form. The lack of a single sialic
acid retarded the mobility of charged oligosaccharides (Fig 11, lane 4)
in which the upper major band was identified as the monosialylated and the lower band disialylated. Adding sialic acid to rhAT with
sialyltransferase resulted in a FACE gel pattern with shift in
intensity from the upper to the lower band (data not shown). The FACE
analysis also showed the increased oligosaccharide heterogeneity
present on rhAT compared with both phAT and goat plasma AT although the
method did not clearly resolve the oligomannose structures in the rhAT sample. That the FACE profile of goat AT was similar to that of phAT
suggested that the increased fucosylation and heterogeneity, as well as
the presence of oligomannose structures, was a result of expression in
the mammary gland and did not arise because of goat/human glycosylation
differences.
View larger version (114K):
[in this window]
[in a new window]
| Fig 11.
FACE analysis of phAT (lane 2), goat plasma AT (lane 3),
and rhAT (lane 4) N-glycanase released oligosaccharides. Lane 1 contains dextran polymers of varying length beginning with a trimer at the bottom of the gel and increasing by one sugar for each band to the
top. Lane 5 contains a series of standard oligosaccharides (Dionex
Corp) from bottom to top: disialylated biantennary, trisialylated triantennary, asialo biantennary, asialo triantennary, and
asialotetraantennary complex oligosaccharides.
|
|
| |
DISCUSSION |
Production of rhAT in the mammary gland of transgenic goats has
provided a means for high level (>1 g/L) expression of this therapeutic protein. This report provides the first detailed structural and functional analysis of a human therapeutic protein produced in the
milk of a transgenic animal to be used in human clinical studies. In
fact, rhAT was successfully evaluated in phase I and phase II human
clinical studies designed to test the ability of rhAT to maintain
hemostasis during coronary artery bypass graft surgery.
The rhAT isolated from the milk of transgenic goats was
indistinguishable from phAT with the exception of its heparin-binding affinity and the nature of its glycosylation. The rhAT and phAT exhibited equivalent activity in in vitro thrombin and factor Xa
inhibition assays and were structurally comparable in RP-HPLC binding
characteristics, LC/MS peptide mapping profiles (except for the
glycopeptides) and near and far ultraviolet circular dichroism spectra.
The major differences in glycosylation noted were that the rhAT was
less sialylated, more fucosylated, and contained GalNAc for Gal
substitutions and some NGNA as well as NANA as terminal
sugars when compared with phAT.
These differences in glycosylation between the rhAT and phAT are
presumed responsible for the difference in heparin binding. Previous
reports by Zettlmeissl et al48 described site mutation experiments on individual glycosylation sites of rhAT produced in
Chinese hamster ovary (CHO) cells, which resulted in higher affinity
for heparin when compared with phAT. A current report,49 using similar techniques, corroborated the above study and further showed that the presence of glycosylation on the Asn 155 site was
responsible for generating the lower affinity glycoform of the fully
glycosylated AT made in CHO cells. The nature of the oligosaccharides
present was not determined. In another study, Garone et
al50 reported that fucosylation at the Asn 155 site reduced
the heparin affinity of a variant of rhAT made in baby hamster kidney
cells in which Asn 135 was mutated to Gln 135. Neither the phAT nor the
rhAT examined here had fucosylated oligosaccharides at the Asn 155 site. The main difference observed between phAT and rhAT at Asn 155 was
in the nature of the glycosylation: phAT had a charged oligosaccharide
and rhAT had a noncharged oligosaccharide. Because of the different
expression systems used and differences in glycosylation at sites other
than the Asn 155 site, it is difficult to directly compare the
results we have obtained with these other studies. However, it is
apparent that glycosylation differences at the Asn 155 site are
important in determining the overall heparin affinity of AT.
The data presented above indicated that both the and isoforms
of rhAT bound more tightly to heparin than the and isoforms of
phAT. Because all of the sites of the form are fully glycosylated, the increase in heparin affinity observed must have been caused by the
nature of the glycosylation present not the degree of site occupancy.
As noted above, rhAT exhibited an overall lower level of sialylation
than phAT and a very different glycoform distribution at the Asn 155 site. Initial studies suggested that removal of sialic acid from phAT
resulted in a higher affinity for heparin (data not shown). The
prevention of glycosylation on the individual sites increased heparin
affinity48,49 purportedly by reducing steric hindrance to
heparin binding. Alternatively, the prevention of glycosylation by site
mutation also prevented potential sialylation, which could exert a
charge repulsion effect with negatively charged heparin sulfate.
Whether the absence of sialic acid on specific glycosylation sites
would impart any enhancement of heparin binding will require further
study.
The fact that transgenically produced rhAT had a higher heparin
affinity may have relevance to its clinical use. The importance of an
increased affinity of AT for heparin in vivo may lie in the fact that
heparin and heparin-like glycosaminoglycans are receptors for AT and
thrombin and that these glycosaminoglycans are found in the vascular
endothelium and extravascular spaces, not circulating in the blood
stream. AT is activated by binding to heparan sulfate
proteoglycans on endothelial cells,17,21,51,52 and
clearance of AT from blood occurs by redistribution into the extravascular space and binding to the endothelial
surfaces.24,51,53
The control of thrombin activity locally may be the initial step in
preventing disseminated intravascular coagulation (DIC) by controlling
the occurrence of coagulatable sites in blood vessels and
organs.21 Thrombin was implicated in stimulating the
vascular endothelium to synthesize prostacyclin,54 tissue
plasminogen activator,55 and tissue plasminogen activator
inhibitor56 besides its activation of coagulation. AT was
shown to inhibit thrombin-induced prostacyclin induction in HUVEC
cells.57 Thrombin was also found to enhance the adhesion of
neutrophil leukocytes to endothelial cells.58,59
A recent study showed that AT at high dose, without added heparin, was
an effective and safe therapy for disseminated intravascular coagulation with organ failure in four children.60 Thus,
enhancing the ability of AT to inhibit thrombin locally may provide a
better way to control not just coagulation but other thrombin-induced events. rhAT, with its higher affinity for heparin, could provide more
AT at sites of insult or inflammation to help control DIC and other
events stimulated by the presence of thrombin. Studies to ascertain
differences in the biodistribution and efficacy because of increased
heparin affinity are in progress.
| |
FOOTNOTES |
Submitted July 9, 1997;
accepted January 5, 1998.
Address reprint requests to Edward S. Cole, PhD, Cell and Protein
Therapeutics Department, Genzyme Corp, 1 Mountain Rd, Framingham, MA
01701-9322.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Paul DiTullio, currently of Midas Biologicals
Inc, (Grafton, MA) for his extensive efforts in making
this program possible. We also thank Karen Albee, Karen Lee, and Robert
Segalla of the Structural Protein Chemistry Group for
providing expert analytical support.
| |
REFERENCES |
1.
Travis J,
Salvesen GS:
Human plasma proteinase inhibitors.
Annu Rev Biochem
52:655,
1983[Medline]
[Order article via Infotrieve]
2.
Menache D:
Antithrombin III: Introduction.
Semin Hematol
28:1,
1991[Medline]
[Order article via Infotrieve]
3.
Menache D,
Grossman B,
Jackson C:
Antithrombin III: Physiology, deficiency, and replacement therapy.
Transfusion
32:580,
1992[Medline]
[Order article via Infotrieve]
4.
Lahiri B,
Bagdasarian A,
Mitchell B,
Talamo RC,
Colman RW,
Rosenberg RD:
Antithrombin-Heparin Cofactor: An inhibitor of plasma kallikrein.
Arch Biochem Biophys
175:737,
1976[Medline]
[Order article via Infotrieve]
5.
Rosenberg RD,
Bauer KA,
Marcum JA:
Antithrombin III, The heparin antithrombin system.
Rev Hematol
2:351,
1986
6.
Murano G,
Williams L,
Andersson M:
Some properties of antithrombin III and its concentration in human plasma.
Thromb Res
18:259,
1980[Medline]
[Order article via Infotrieve]
7.
Franzen LE,
Svensson S,
Larm O:
Structural studies on the carbohydrate portion of human antithrombin III.
J Biol Chem
255:5090,
1980[Abstract/Free Full Text]
8.
Magnusson S:
Primary structure of antithrombin III (Heparin Cofactor). Partial homology between 1 antitrypsin and antithrombin-III
, in Collen DW,
Wiman B,
Verstraete M
(eds):
The Physiological Inhibitions of Blood Coagulation and Fibrinolysis.
Amsterdam, The Netherlands, Elsevier/North-Holland Biomedical Press
, 1979
, p 43
9.
Pike R,
Potempa J,
Skinner R,
Fitton H,
McGraw W,
Travis J,
Owen M,
Jin L,
Carrell R:
Heparin-dependent modification of the reactive center arginine of antithrombin and consequent increase in heparin binding affinity.
J Biol Chem
272:19652,
1997[Abstract/Free Full Text]
10.
Ersdal-Badju E,
Lu A,
Zuo Y,
Picard V,
Bock S:
Identification of the antithrombin III heparin binding site.
J Biol Chem
272:19393,
1997[Abstract/Free Full Text]
11.
Rosenberg RD:
Role of heparin and heparinlike molecules in thrombosis and atherosclerosis.
Fed Proc
44:404,
1985[Medline]
[Order article via Infotrieve]
12.
Danielsson A:
Antithrombin and related inhibitors of coagulation proteinases
, in Barrett AJ,
Salvesen GS
(eds):
Proteinase Inhibitors, vol 17.
Amsterdam, The Netherlands, Elsevier Science Publishers (Biomedical Division)
, 1986
, p 489
13.
Olson ST,
Bjork I,
Sheffer R,
Craig PA,
Shore JD,
Choay J:
Role of the antithrombin-binding pentasaccharide in heparin acceleration of antithrombin-proteinase reactions. Resolution of the antithrombin conformational change contribution to heparin rate enhancement.
J Biol Chem
267:12528,
1992[Abstract/Free Full Text]
14.
Choay J,
Lormeau JC,
Petitou M,
Sinay P,
Fareed J:
Structural studies on a biologically active hexasaccharide obtained from heparin.
Ann NY Acad Sci
370:644,
1981[Medline]
[Order article via Infotrieve]
15.
Choay J,
Petitou M,
Lormeau J-C,
Sinay P,
Casu B,
Gatti G:
Structure activity relationship in heparin: A synthetic pentasaccharide sequence with high affinity for antithrombin III and eliciting high anti factor Xa activity.
Biochem Biophys Res Commun
116:492,
1983[Medline]
[Order article via Infotrieve]
16.
Olson ST,
Bjork I:
Predominant contribution of surface approximation to the mechanism of heparin acceleration of the antithrombin-thrombin reaction. Elucidation from salt concentration effects.
J Biol Chem
266:6353,
1991[Abstract/Free Full Text]
17.
Bauer KA,
Rosenberg RD:
Role of antithrombin III as a regulator of in vivo coagulation.
Semin Hematol
28:10,
1991[Medline]
[Order article via Infotrieve]
18.
Carrell R,
Skinner R,
Jin L,
Abrahams J:
Structural mobility of antithrombin and its modulation by heparin.
Thromb Haemost
78:516,
1997[Medline]
[Order article via Infotrieve]
19.
Hashimoto K,
Yamagishi M,
Sasaki T,
Nakano M,
Kurosawa H:
Heparin and Antithrombin III levels during Cardiopulmonary bypass: Correlation with subclinical plasma coagulation.
Ann Thorac Surg
58:799,
1994[Abstract]
20.
Cloyd GM,
D'Ambra MN,
Akins CW:
Diminished anticoagulant response to heparin in patients undergoing coronary artery bypass grafting.
J Thorac Cardiovasc Surg
94:535,
1987[Abstract]
21. (suppl)
Rosenberg RD:
Biochemistry of heparin antithrombin interactions, and the physiologic role of this natural anticoagulant mechanism.
Am J Med
87:2S,
1989[Medline]
[Order article via Infotrieve]
22.
Mertens G,
Cassiman J-J,
Van den Berghe H,
Vermylen J,
David G:
Cell surface heparan sulfate proteoglycans from human vascular endothelial cells.
J Biol Chem
267:20435,
1992[Abstract/Free Full Text]
23.
Akai T,
Kaji T,
Hayakawa Y,
Hayashi T,
Sakuragawa N:
Antithrombin modulates the effect of thrombin in the metabolism of glycosaminoglycans in cultured endothelial cells.
Thrombosis Res
62:707,
1991[Medline]
[Order article via Infotrieve]
24.
de-Agostini AI,
Watkins SC,
Slayter HS,
Youssoufian H,
Rosenberg RD:
Localization of anticoagulantly active heparan sulfate proteoglycans in vascular endothelium: Antithrombin binding on cultured endothelial cells and perfused rat aorta.
J Cell Biol
111:1293,
1990[Abstract/Free Full Text]
25.
Ebert KM,
Selgrath JP,
DiTullio P,
Denman J,
Smith TE,
Memon MA,
Schindler JE,
Monastersky GM,
Vitale JA,
Gordon C:
Transgenic production of a variant of human tissue-type plasminogen activator in goat milk: Generation of transgenic goats and analysis of expression.
Bio/Technology
9:835,
1991[Medline]
[Order article via Infotrieve]
26.
Denman J,
Hayes M,
O'Day C,
Edmunds T,
Bartlett C,
Hirani S,
Ebert KM,
Gordon K,
McPherson JM:
Transgenic expression of a variant of human tissue-type plasminogen activator in goat milk: Purification and characterization of the recombinant enzyme.
Bio/Technology
9:839,
1991[Medline]
[Order article via Infotrieve]
27.
Ebert KM,
DiTullio P,
Barry CA,
Schindler JE,
Ayres SL,
Smith TE,
Pellerin LJ,
Meade HM,
Denman J,
Roberts B:
Induction of human tissue plasminogen activator in the mammary gland of transgenic goats.
Bio/Technology
12:699,
1994[Medline]
[Order article via Infotrieve]
28.
Varki A:
Biological roles of oligosaccharides: All of the theories are correct.
Glycobiology
3:97,
1993[Abstract/Free Full Text]
29.
Roberts B,
DiTullio P,
Vitale J,
Hehir K,
Gordon K:
Cloning of the goat-casein encoding gene and expression in transgenic mice.
Gene
121:255,
1992[Medline]
[Order article via Infotrieve]
30.
DiTullio P,
Cheng SH,
Marshal J,
Gregory RJ,
Ebert KM,
Meade HM,
Smith AE:
Production of cystic fibrosis transmembrane regulator in the milk of transgenic mice.
Bio/Technology
10:74,
1992[Medline]
[Order article via Infotrieve]
31.
Broker M,
Ragg H,
Karges HE:
Expression of human antithrombin III in Saccharomyces cerevisiae and Schizosaccharomyces pombe.
Biochim Biophys Acta
908:203,
1987[Medline]
[Order article via Infotrieve]
32.
Gould SJ,
Subramani S,
Scheffler IE:
Use of the DNA polymerase chain reaction for homology probing: Isolation of partial cDNA or genomic clones encoding the iron-sulfur protein succinate dehydrogenase from several species.
Proc Natl Acad Sci USA
86:1934,
1989[Abstract/Free Full Text]
33.
Abildgaard U,
Lie M,
Odegard OR:
Antithrombin (heparin cofactor) assay with "new" chromogenic substrates (S-2238 and Chromozym TH).
Thromb Res
11:549,
1977[Medline]
[Order article via Infotrieve]
34.
Abildgaard U:
Methodological considerations on antithrombin determination.
Acta Chir Scand Suppl
509:97,
1982[Medline]
[Order article via Infotrieve]
35.
Cole ES,
Nichols EH,
Lauziere K,
Edmunds T,
McPherson JM:
Characterization of the microheterogeneity of recombinant primate prolactin: Implications for posttranslational modifications of the hormone in vivo.
Endocrinology
129:2639,
1991[Abstract/Free Full Text]
36.
Laemmli UK:
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:680,
1970[Medline]
[Order article via Infotrieve]
37.
Morrissey JH:
Silver stain for proteins in polyacrylamide gels: A modified procedure with enhanced uniform sensitivity.
Anal Biochem
117:307,
1981[Medline]
[Order article via Infotrieve]
38.
Burnette WN:
"Western Blotting." Electrophoretic transfer of proteins from SDS-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A.
Anal Biochem
170:195,
1981
39.
Hardy MR,
Townsend RR,
Lee YC:
Monosaccharide analysis of glycoconjugates by anion exchange chromatography with pulsed amperometric detection.
Anal Biochem
170:54,
1988[Medline]
[Order article via Infotrieve]
40.
Powell LD,
Hart GW:
Quantitation of picomole levels of N-acetyl- and N-glycolylneuraminic acids by a HPLC-adaptation of the thiobarbituric acid assay.
Anal Biochem
157:179,
1986[Medline]
[Order article via Infotrieve]
41.
Fan B,
Crews BC,
Turko IV,
Choay J,
Zettlmeissl G,
Gettins P:
Heterogeneity of recombinant human antithrombin III expressed in baby hamster kidney cells. Effect of glycosylation differences on heparin binding and structure.
J Biol Chem
268:17588,
1993[Abstract/Free Full Text]
42.
Knobloch J,
Shaklee P:
Absolute molecular weight distribution of low-molecular weight heparins by size-exclusion chromatography with multiangle laser light scattering detection.
Anal Biochem
245:231,
1997[Medline]
[Order article via Infotrieve]
43.
Carrell RW,
Stein PE,
Fermi G,
Wardell MR:
Biological implications of a 3A structure of dimeric antithrombin.
Structure
2:257,
1994[Abstract/Free Full Text]
44.
Schreuder HA,
de-Boer B,
Dijkema R,
Mulders J,
Theunissen HJ,
Grootenhuis PD,
Hol WG:
The intact and cleaved human antithrombin III complex as a model for serpin-proteinase interactions.
Nat Struct Biol
1:48,
1994[Medline]
[Order article via Infotrieve]
45.
Nordenman B,
Nystrom C,
Bjork I:
The size and shape of human and bovine antithrombin III.
Eur J Biochem
78:195,
1977[Medline]
[Order article via Infotrieve]
46.
Frebelius S,
Isaksson S,
Swedenborg J:
Thrombin inhibition by antithrombin III on the subendothelium is explained by the isoform AT beta.
Arterioscler Thromb Vasc Biol
16:1292,
1996[Abstract/Free Full Text]
47.
Peterson CB,
Blackburn MN:
Isolation and characterization of an antithrombin III variant with reduced carbohydrate content and enhanced heparin binding.
J Biol Chem
260:610,
1985[Abstract/Free Full Text]
48.
Zettlmeissl G,
Conradt HS,
Nimtz M,
Haigwood N,
Paques EP:
Influence of glycosylation on the functional properties of human therapeutic plasma proteins.
GBF Monogr Ser
15:259,
1991
49.
Olson S,
Frances-Chmura A,
Swanson R,
Bjork I,
Zettlmeissl G:
Effect of individual carbohydrate chains of recombinant antithrombin on heparin affinity and on the generation of glycoforms differing in heparin affinity.
Arch Biochem Biophys
341:212,
1997[Medline]
[Order article via Infotrieve]
50.
Garone L,
Edmunds T,
Hanson E,
Bernasconi R,
Huntington JA,
Meagher JL,
Fan B,
Gettins PGW:
Antithrombin-heparin affinity reduced by fucosylation of carbohydrate at asparagine 155.
Biochemistry
35:8881,
1996[Medline]
[Order article via Infotrieve]
51.
Hatton MWC,
Moar SL,
Richardson M:
On the interaction of rabbit antithrombin III with the luminal surface of the normal and de-endothelialized rabbit thoracic aorta in vitro.
Blood
67:878,
1986[Abstract/Free Full Text]
52.
Hatton MWC,
Moar SL,
Richardson M:
Evidence that rabbit 125I-antithrombin III binds to proteoheparan sulphate at the subendothelium of the rabbit aorta in vitro.
Blood Vessels
25:12,
1988[Medline]
[Order article via Infotrieve]
53. (suppl)
Pizzo SV:
Serpin receptor 1: A hepatic receptor that mediates the clearance of antithrombin III-proteinase complexes.
Am J Med
87:10S,
1989[Medline]
[Order article via Infotrieve]
54.
Weksler BB,
Ley CW,
Jaffe EA:
Stimulation of endothelial cell prostacyclin production by thrombin, trypsin and theionophore A23187.
J Clin Invest
62:923,
1978
55.
Levin EG,
Loskutoff PJ:
Cultured bovine endothelial cells produce both urokinase and tissue-type plasminogen activators.
J Cell Biol
94:631,
1982[Abstract/Free Full Text]
56.
Gelehter TJ,
Sznycer-Laszuk R:
Thrombin induction of plasminogen activator-inhibitor in cultured human endothelial cells.
J Clin Invest
77:165,
1986
57.
Kaji T,
Itoh F,
Hayakawa Y,
Oguma Y,
Sakurgawa N:
Interaction of thrombin with heparin cofactor II and antithrombin III on prostacyclin production by cultured endothelial cells.
Thromb Res
56:99,
1989[Medline]
[Order article via Infotrieve]
58.
Toothill VJ,
Van Mourik JS,
Niewenhuis hK,
Metzelaar MJ,
Pearson JD:
Characterization of the enhanced adhesion of neutrophil leukocytes to thrombin-stimulated endothelial cells.
J Immunol
145:283,
1990[Abstract]
59.
Moser R,
Groscurth P,
Fehr J:
Promotion of transendothelial neutrophil passage by human thrombin.
J Cell Science
96:737,
1990[Abstract/Free Full Text]
60.
Fuse S,
Tomita H,
Yoshida M,
Hori T,
Igarashi C,
Fujita S:
High dose of intravenous antithrombin III without heparin in the treatment of disseminated intravascular coagulation and organ failure in four children.
Am J Hematol
53:18,
1996[Medline]
[Order article via Infotrieve]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
G.-C. Gil, W. H Velander, and K. E Van Cott
Analysis of the N-glycans of recombinant human Factor IX purified from transgenic pig milk
Glycobiology,
July 1, 2008;
18(7):
526 - 539.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. D. Spiess
Treating Heparin Resistance With Antithrombin or Fresh Frozen Plasma
Ann. Thorac. Surg.,
June 1, 2008;
85(6):
2153 - 2160.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Koles, P. H. C. van Berkel, F. R. Pieper, J. H. Nuijens, M. L.M. Mannesse, J. F.G. Vliegenthart, and J. P. Kamerling
N- and O-glycans of recombinant human C1 inhibitor expressed in the milk of transgenic rabbits
Glycobiology,
January 1, 2004;
14(1):
51 - 64.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Cao, A. Lundwall, V. Gadaleanu, H. Lilja, and A. Bjartell
Anti-Thrombin Is Expressed in the Benign Prostatic Epithelium and in Prostate Cancer and Is Capable of Forming Complexes with Prostate-Specific Antigen and Human Glandular Kallikrein 2
Am. J. Pathol.,
December 1, 2002;
161(6):
2053 - 2063.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. C. Reggio, A. N. James, H. L. Green, W. G. Gavin, E. Behboodi, Y. Echelard, and R. A. Godke
Cloned Transgenic Offspring Resulting from Somatic Cell Nuclear Transfer in the Goat: Oocytes Derived from Both Follicle-Stimulating Hormone-Stimulated and Nonstimulated Abattoir-Derived Ovaries
Biol Reprod,
November 1, 2001;
65(5):
1528 - 1533.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. C. Minnema, A. C. K. Chang, P. M. Jansen, Y. T. P. Lubbers, B. M. Pratt, B. G. Whittaker, F. B. Taylor, C. E. Hack, and B. Friedman
Recombinant human antithrombin III improves survival and attenuates inflammatory responses in baboons lethally challenged with Escherichia coli
Blood,
February 15, 2000;
95(4):
1117 - 1123.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. M. Van Patten, E. Hanson, R. Bernasconi, K. Zhang, P. Manavalan, E. S. Cole, J. M. McPherson, and T. Edmunds
Oxidation of Methionine Residues in Antithrombin. EFFECTS ON BIOLOGICAL ACTIVITY AND HEPARIN BINDING
J. Biol. Chem.,
April 9, 1999;
274(15):
10268 - 10276.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Abstract
Introduction
Abstract