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Antithrombin-111-Hamilton, Ala 382 to Thr: An Antithrombin-I11 Variant That
Acts as a Substrate But Not an Inhibitor of a-Thrombin and Factor Xa
By Richard C. Austin, Richard A. Rachubinski, Fred A. Ofosu, and Morris A. Blajchman
Antithrombin-Ill-Hamilton has been shown t o be a structural
variant of antithrombin-Ill (AT-Ill) with normal heparin affinity but impaired protease inhibitory activity. The molecular defect of AT-Ill-Hamilton is the substitution of Thr for Ala
at amino acid residue 382. The plasma of affected individuals
contains approximately equal quantities of normal AT-Ill and
AT-Ill-Hamilton. When AT-Ill was isolated from the plasma of
the propositus by heparin-Sepharose chromatography, it
had identical mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) t o normal plasmaderived AT-Ill, under both reducing and nonreducing conditions. However, the AT-Ill-Hamilton species, separated from
the propositus’ normal AT-Ill by a combination of heparinSepharose and thrombin-Sepharose chromatography, had
increased mobility on reductive SDS-PAGE compared with
AT-Ill from the propositus isolated by heparin-Sepharose
chromatography alone. Under nonreducing conditions this
AT-Ill-Hamilton species had decreased mobility compared
with AT-Ill from the propositus (or normal AT-Ill] isolated
only by heparin-Sepharose chromatography. When incu-
bated with either human a-thrombin or human factor Xa, this
AT-Ill-Hamilton species was unreactive. Approximately 50%
of the AT-Ill from the propositus isolated by heparinSepharose chromatography, when incubated with either
human a-thrombin or factor Xa, did not form complex but
was cleaved, presumably at the reactive center Arg393Ser394. To further substantiate the biological behavior of this
variant, AT-Ill-Hamilton polypeptides were synthesized in a
cell-free system. This recombinantly produced AT-Ill-Hamilton, when incubated with either human a-thrombin or factor
Xa, was cleaved by both these proteases, but did not show
any complex formation. The results indicate that AT-IllHamilton does not form a stable covalent inhibitory complex
with these serine proteases but can be cleaved at the
reactive center. Thus, the inhibition of serine proteases by
their natural inhibitors (the serpins) involves at least two
separate, but interrelated events; hydrolysis at the reactive
center followed by complex formation. AT-Ill-Hamilton is
capable of only the first of these events.
o 1991by The American Society of Hematology.
H
phosphorylase b; 66 Kd, bovine serum albumin (BSA); 45 Kd, egg
albumin; 36 Kd, rabbit muscle glyceraldehyde-3-phosphate dehydrogenase; and 29 Kd, bovine erythrocyte carbonic anhydrase. All
other chemicals and reagents were of the highest quality available.
Uh4AN ANTITHROMBIN-I11 (AT-111) is a member
of a family of serine protease inhibitors that are
referred to as the serpins.’B2AT-I11 is the major physiological inhibitor of thrombin as well as the other serine
proteases of the coagulation cascade. Protease inactivation
by AT-I11 is through the formation of a 1:l stoichiometric
covalent inhibitory complex between the serine protease
involved and the reactive center Arg393-Ser394 of AT-III.’~*
The physiological importance of AT-111 is clearly demonstrated by individuals with hereditary AT-111 deficiency
who often suffer from recurrent thromboembolic disease.'^^
One such variant, AT-111-Hamilton, was previously described having normal heparin affinity but impaired protease inhibitory activity.’ The molecular defect in the
AT-111-Hamilton allele was shown to be a substitution of
Thr for Ala at amino acid position 382. In this report, we
demonstrate that the AT-111-Hamilton gene product acts as
a substrate for both a-thrombin and factor Xa, but not as an
inhibitor of these proteases.
MATERIALS AND METHODS
Materials
The AT-I11cDNA used in these studies was generously provided
by Dr E.V. Prochownik (University of Michigan, Ann Arbor, MI).
Human a-thrombin ( > 3,000 NIH Uimg; > 99% active) was kindly
provided by Dr J. Fenton (New York State Division of Biologicals,
Albany, NY). Human factor Xa (0.82 mg/mL, >95% active) was
prepared as previously described.6 The cell-free expression vector
pGEM-3Zf( +) and rabbit reticulocyte lysate (RRL) were purchased from Promega-Biotec (Toronto, Ontario, Canada). D-phenylalanyl-L-propyl-L-argininechloromethyl ketone (PPACK)
(> 99% pure; showing full biological activity) was purchased from
Calbiochem (Mississauga, Ontario, Canada). a-(’*P)dATP (3,000
Ciimmol) was purchased from Amersham (Oakville, Ontario,
Canada) and L-(3sS)methionine( > 1,OOO Ciimmol translation grade)
was purchased from New England Nuclear (Lachine, Quebec,
Canada). The molecular weight protein markers (Kd) were purchased from Sigma (St Louis, MO) and are: 97 Kd, rabbit muscle
Blood, Vol77, No 10 (May 15), 1991: pp 2185-2189
Methods
Isolation and characterization ofplasma-derivedAT-111-Hamilton.
AT-111 from the propositus (containing approximately equal quantities of both normal AT-I11 and AT-111-Hamilton) was isolated
from plasma by heparin-Sepharose chromatography using standard porcine heparin (> 150 USP Uimg; Sigma) coupled to
cyanogen bromide-Sepharose. AT-111-Hamilton was separated
from the normal AT-111 of the propositus by thrombin-Sepharose
chromatography.’ Specifically, 5 p,g of plasma-derived normal
AT-I11 (2.9 mgimL) or AT-I11 from the propositus (3.7 mg/mL),
previously isolated by heparin-Sepharose chromatography, were
resuspended separately in 100 pL of 0.15 mol/L NaCI, 0.02 moliL
Tris-HCI, pH 7.5, and applied to 1 vol of thrombin-Sepharose
~~~~~~~
From the Canadian Red Cross Blood Transfusion Service and the
Departments of Biochemistry and Pathology, McMaster University,
Hamilton, Ontario, Canada.
Submitted August 14, 1990; accepted January 18, 1991.
Supported by a grant (HAM-04-89)from the Canadian Red Cross
Society R&D Fund. R.C.A. is the recipient of a Studentship from the
Heart and Stroke Foundation of Ontario.
A preliminaiy report of this work waspresented at the Thirty-Second
Annual Meeting of the American Society of Hematology, Boston, MA,
December 1990 and has appeared in abstract form in Blood 76:411a,
I990 (suppl 1).
Address reprint requests to M A . Blajchman, MD, FRCP(C),
Department of Pathology, Room 2N31, McMaster University Medical
Centre, 1200 Main St W, Hamilton, Ontario L8N 325 Canada.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1991 by The American Society of Hematology.
0006-4971/91l7710-0008$3.00l0
2185
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AUSTIN ET AL
2186
Mr
1
2
3
4
5
2
M , 1
97-
3
4
5
97-
66-
66I
45-
45-
36-
36-
29-
A
29-
B
Fig 1. SDS-PAGE analysis of plasma-derived normal AT-Ill, AT-Ill from the propositus, and AT-Ill-Hamilton on a 10% t o 15% gradient
polyacrylamide gel under reducing (A) and nonreducing (B) conditions. Normal AT-Ill and AT-Ill from the propositus were isolated by
heparin-Sepharose chromatography while AT-Ill-Hamilton was separated from normal AT-Ill of the propositus by sequential heparin-Sepharose
and thrombin-Sepharose chromatography (see Materials and Methods). Lane 1 contains normal AT-Ill. Lane 2 contains AT-Ill from the propositus.
Lane 3 contains purified AT-Ill-Hamilton. Lane 4 contains an artificial mixture of normal AT-Ill and purified AT-Ill-Hamilton. Lane 5 contains normal
AT-Ill after chromatography on thrombin-Sepharose. The polyacrylamide gel was stained with Coomassie brilliant blue t o visualize the protein
bands. Mr, molecular weight protein markers (Kd). The arrow indicates the migration position of normal AT-Ill and AT-Ill from the propositus. The
arrowhead represents the migration position of the AT-Ill-Hamilton species after thrombin-Sepharose chromatography.
beads (50% capacity) washed in the same buffer. After 60 minutes
the thrombin-Sepharose beads were pelleted by brief centrifugation and the supernatant removed. Ten microliters of the supernatant was then analyzed by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) under both reducing and nonreducing conditions. The protein bands were visualized by staining
the gel with Coomassie brilliant blue (Sigma). Purified AT-IIIHamilton, normal AT-111, and AT-I11 from the propositus were
each analyzed for their ability to form covalent complexes with
human a-thrombin and human factor Xa. Each AT-I11 preparation
was diluted in 0.15 m o l b NaCI, 0.02 mol/L Tris-HCI, pH 7.5, and
incubated at 37°C in the presence of either human a-thrombin or
human factor Xa. At specific time points the reactions were
terminated by the addition of PPACK to a final concentration of 5
pmolb. The reaction mixtures were then analyzed by SDS-PAGE
under reducing conditions (with dithiothreitol) and nonreducing
conditions using 10% to 15% polyacrylamide gradient gels?'
Under reducing conditions, both a-thrombin and factor Xa separate into a and p forms.
Site-directed mutagenesis to form the AT-III-Hamilton mutation.
Escherichia coli DH5aF' cells containing the recombinant vector
pGEM-3Zf( +)-AT-III,.43z were infected with M13K07 helper
phage to yield single-stranded DNA for site-directed mutagenesis."' Site-directed mutagenesis was performed by the method of
Taylor et all' using the Amersham mutagenesis kit. The AT-IIIHamilton variant was synthesized using the mutant oligonucleotide
primer 5'-GCAGTGAAACAGCTGCAAG-3', which contains a
single base substitution that converts Ala382 (GCA) to Thr (ACA).
Authenticity of the mutation was confirmed by double-stranded
DNA sequencing using the chemically modified T7 DNA polymerase method of Tabor and Richardson." A combination of DNA
sequencing and a homology search of the mutant oligonucleotide
primer against the entire AT-I11 cDNA sequence confirmed that
the site of mutation was only at amino acid position 382 of AT-111.
Expression and characterization of the cell-freederived AT-IIIHamilton. Cell-free transcription-translation was performed as
previously described by Austin et aI.l3The ability of ("S)methioninelabeled cell-free-derived normal AT-I11 and AT-111-Hamilton
polypeptides to bind to heparin was assessed by heparin-Sepharose
chromatography as described previo~sly.'~
Cell-free-derived normal AT-I11 and AT-111-Hamilton polypeptides were each analyzed
by SDS-PAGE for their ability to form covalent complexes with
human a-thrombin and human factor Xa. The translation mixture
containing cell-free-derived AT-I11 was diluted twofold in 0.15
mol/L NaCI, 0.02 mol/L Tris-HC1, pH 7.5, and dialyzed overnight at
4°C in the same buffer. A 100-fold molar excess of human
a-thrombin or factor Xa to cell-free-derived normal AT-I11 or
1
2
3
4
5
6
7
8
-97
AT-TAT-
-66
-45
-36
T-
-29
A
.97
AT-TAT-
66
45
.36
T-29
B
0
15"
1'
2'
5'
10'
20'
40'
Fig 2. Time-dependent formation of plasma-derived AT-IIIthrombin complexes. A 2:l molar ratio of normal AT-Ill (A) or AT-Ill
from the propositus (B) t o *thrombin was incubated at 37°C in 0.15
mol/L NaCI, 0.02 mol/L Tris-HCI, pH 7.5.At 15 seconds, 1,2,5,10,20,
and 40 minutes, respectively (Lanes 2 through 8). aliquots of the
reaction mixture were analyzed by SDS-PAGE on a 10% t o 15%
gradient polyacrylamide gel under reducing conditions. AT-Ill (AT);
a-thrombin (T); AT-Ill-thrombin complexes (AT-T). The arrowhead
indicates the migration position of the thrombin-dependent cleaved
AT-Ill.
~
~
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2187
ANTITHROMBIN-Ill-HAMILTON
AT-111-Hamilton was then added to the translation mixture prewarmed to 37°C with or without normal plasma AT-111. The
reactions were then terminated at specific times by the addition of
5 pnol/L PPACK. The radiolabeled protein bands were then
analyzed by SDS-PAGE, under reducing conditions, followed by
fluorography. Densitometric scanning of the autoradiograms was
performed using the Hoefer GS300 transmittanceheflectancescanning densitometer and the GS350 data system (San Francisco,
CA).
RESULTS
Characterizationof Plasma-DerivedA T-IIl
AT-I11 isolated from the plasmas of both a normal
subject and the propositus (containing approximately equal
quantities of normal AT-I11 and AT-111-Hamilton) by
elution from heparin-Sepharose showed identical mobilities on SDS-PAGE under both reducing and nonreducing
conditions (Fig 1, lanes 1 and 2). However, AT-IIIHamilton separated from the normal AT-I11 of the propositus by sequential heparin-Sepharose and thrombinSepharose chromatography had increased mobility on SDSPAGE under reducing conditions (Fig lA, lane 3) when
compared with normal AT-I11 (Fig lA, lane 1) and AT-111
from the propositus (Fig lA, lane 2). Under reducing
conditions, purified AT-111-Hamilton showed evidence for
a smaller polypeptide that migrated with the tracking dye
(data not shown). This smaller polypeptide most likely
represents the cleaved portion of AT-I11 from Ser394Lys432. However, under nonreducing conditions AT-IIIHamilton ran with decreased mobility (Fig lB, lane 3) when
compared with normal AT-I11 (Fig lB, lane 1) or AT-I11
from the propositus (Fig lB, lane 2). In addition, the
smaller polypeptide of AT-111 was not seen. An artificial
mixture of normal AT-I11 and pnrified AT-111-Hamilton
was readily separable by SDS-PAGE under reducing and
nonreducing conditions (Fig 1, lane 4). When normal
AT-111 was chromatographed on thrombin-Sepharose the
majority bound to the a-thrombin on the column and could
not be detected by SDS-PAGE (Fig 1, lane 5). These results
strongly indicate that proteolysis of the AT-111-Hamilton
had occurred, presumably at the reactive center Arg393Ser394, when applied to and then eluted from the thrombinSepharose column. Thus, the proteolysis of AT-IIIHamilton results in an AT-I11 species consisting of a heavy
chain (amino acids 1-393) and a light chain (amino acids
394-432) linked by a disulfide bond between Cys247Cys432. Under reducing conditions these two chains separate, causing increased mobility on SDS-PAGE compared
with normal AT-111. However, under nonreducing conditions the heavy and light chains are still connected, slowing
its mobility through the gel.
Plasma-DerivedA T-III-Thrombin Complex Formation
Incubation of a 2:l molar ratio of human a-thrombin to
the heparin-Sepharose and thrombin-Sepharose purified
AT-111-Hamilton, for up to 40 minutes, with or without
heparin, showed no AT-111-thrombin complex formation or
change in mobility of AT-111-Hamilton on SDS-PAGE
(data not shown). AT-I11 from the propositus at a 2 1 molar
ratio to a-thrombin showed approximately 40% to 50%
AT-111-thrombin complex formation. The remainder of the
AT-111 underwent thrombin-dependent cleavage (Fig 2B).
The addition of heparin at a concentration of 0.2 U/mL
increased the rate of reaction but did not increase the
amount of complex formation between AT-111 and a-thrombin (data not shown). In addition, uncomplexed a-thrombin
was still evident even after 40 minutes. In contrast, when a
2:l molar ratio of normal AT-I11 to a-thrombin was used,
all of the a-thrombin was complexed by normal AT-I11 after
2 minutes (Fig 2A). Although the majority of normal AT-I11
was found complexed with a-thrombin, there was a minor
amount that shows thrombin-dependent cleavage (Fig 2A).
Nevertheless, there is still a striking difference between the
two AT-I11 samples with respect to their ability to react
with a-thrombin. Similar results were obtained when normal AT-I11 and AT-111 from the propositus were incubated
with purified factor Xa, except that the reaction rate was
slower (Fig 3).
Cell-Free-DerivedA T-III-Thrombin Complex Formation
From the specific activity of the ("S)methionine and the
predicted amino acid sequence of AT-111, a minimum
concentration of AT-IIIl.432of approximately 300 ng/mL of
RRL was synthesized, which corresponds to a concentration of approximately 6 nmol/L. Both the cell-free-derived
1
2
3
4
5
6
7
~.
-8-
~
- 97
AT-Xa-
-66
AT-45
-36
Xa-
-29
A
-97
AT-Xa-
-66
AT-45
-36
Xa -
' -29
B
0
15"
1'
2'
5'
10'
20'
40.
Fig 3. Time-dependent formation of plasma-derived AT-IIIfactor-Xa complexes. A 2:l molar ratio of normal AT-Ill (A) or AT-Ill
from the propositus (6) to factor Xa was incubated at 37°C in 0.15
mol/L NaCI, 0.02 mol/L Tris-HCI, pH 7.5. At specific time points (lanes
2 through 8). aliquots of the reaction mixtures were analyzed by
SDS-PAGE on a 10% polyacrylamide gel under reducing conditions.
AT-Ill (AT); activated factor X (Xa); AT-Ill-activated factor X complexes (AT-Xa). The arrowhead indicates the migration position of the
factor Xa-dependent cleaved AT-Ill.
,
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2188
AUSTIN ET AL
normal AT-I11 and AT-111-Hamilton polypeptides showed
identical mobilities on SDS-PAGE under reducing conditions. Cell-free-derived normal AT-I11 and AT-III-Hamilton had very similar elution patterns off heparin-Sepharose
(Fig 4), suggesting that the substitution of Thr for Ala at
amino acid position 382 does not alter heparin binding. The
results in Fig 5 A show that, when incubated with a-thrombin, cell-free-derived normal AT-I11 formed a covalent
complex with a-thrombin. Densitometric scanning of the
autoradiogram indicates that approximately 15% of cellfree-derived normal AT-111 forms complex with a-thrombin at 2 minutes. Complex formation was also evident even
after the addition of plasma AT-I11 at a 2:l or 1:l molar
ratio to a-thrombin (data not shown). In addition, we also
observed the appearance of an a-thrombin-dependent
43-Kd AT-I11 polypeptide (Fig 5A) and a smaller 4-Kd
polypeptide (not shown). When the cell-free-derived AT111-Hamilton variant was incubated with a-thrombin, there
was no detectable complex formation ( < 1% at 2 minutes).
However, the majority of cell-free-derived AT-III-Hamilton ( > 90% after 40 minutes) was cleaved by a-thrombin,
presumably at the reactive center, to show a 43-kD polypeptide (Fig 5B) and a smaller 4-kD polypeptide (not shown on
the gels). Similar results were obtained when cell-freederived AT-111-Hamilton was incubated with human factor
Xa (data not shown).
DISCUSSION
This study was undertaken to determine what effect the
Ala to Thr substitution at position 382 had on the ability of
:I
Percent Binding
20
0
0.2
0.4
0.8
0.8
1
1.2
1.4
1.8
1.8
2
NaCl Concentration (M)
Fig 4. Heparin-Sepharose chromatography of cell-free-derived
normal AT-Ill and AT-Ill-Hamilton. Cell-free translation mixtures containing (”S)methionine-labeled AT-Ill polypeptides were diluted twofold in 0.1 mol/L NaCI, 0.02 mol/L Tris-HCI, pH 7.5, dialyzed overnight
at 4°C in the same buffer, and applied t o heparin-Sepharose. Following incubation for 60 minutes at room temperature, cell-free-derived
AT-Ill polypeptides were eluted from heparin-Sepharosein 0.02 mol/L
Tris-HCI, pH 7.5, containing 0.1 mol/L to 2.0 mol/L NaCl (0.1 mol/L
increments t o 1.0 mol/L). The fractions were then analyzed by
SDS-PAGE on a 12% polyacrylamide gel under reducing conditions,
followed by fluorography. The percentage of bound and unbound
AT-Ill polypeptides chromatographed on heparin-Sepharose was
determined by densitometric scanning of the autoradiograms. Heparin affinity profile of cell-free-derived normal AT-Ill (0)and cell-freederived AT-Ill-Hamilton (0).
66
45
36
A
29
66
45
36
B
29
0
15”
1’
2’
5’
10’
20’
40’
Fig 5. Time-dependent formation of cell-free-derived AT-IIIthrombin complexes. (”S)Methionine-labeled RRL translation mixtures containing either cell-free-derived normal AT-Ill (A) or cell-freederived AT-Ill-Hamilton (E) were diluted twofold in 0.15 mol/L NaCI,
0.02 mol/L Tris-HCI, pH 7.5, dialyzed overnight in the same buffer, and
incubated with a 100-fold molar excess of a-thrombin at 37°C. At
specific time points (lanes 2 through 8) aliquots of the reaction
mixture were analyzed by SDS-PAGE on a 10% polyacrylamide gel
under reducing conditions followed by fluorography. Abbreviations
are the same as those used in Fig 2.
AT-111-Hamilton to interact with a-thrombin and factor
Xa. AT-I11 from the propositus contains approximately
equal quantities of normal AT-I11 and AT-111-Hamilton.
The electrophoretic mobility of AT-111-Hamilton, before
thrombin-Sepharose chromatography, was identical to normal AT-111. Purified AT-111-Hamilton, prepared by a combination of heparin-Sepharose and thrombin-Sepharose
chr~matography,~,’
had increased mobility on SDS-PAGE
under reducing conditions and decreased mobility under
nonreducing conditions, when compared with normal ATI11 and AT-I11 from the propositus purified by heparinSepharose chromatography only. The results indicate that
proteolytic cleavage of AT-111-Hamilton occurs during
thrombin-Sepharose chromatography, presumably at the
reactive center Arg393-Ser394, but does not result in the
formation of a stable covalent inhibitory complex.
Under reducing conditions the disulfide bond between
Cys247 and Cys430 of the purified AT-111-Hamilton would
be eliminated, resulting in two AT-I11 fragments; one from
His1 to Arg393 (which migrates slightly faster than normal
AT-111) and the other from Ser394 to Lys432 (which
migrates with the tracking dye). However, under nonreducing conditions this disulfide bond should be intact, resulting
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
ANTITHROMBIN-Ill-HAMILTON
in a partially unfolded molecule with decreased mobility on
SDS-PAGE. In addition, purified AT-111-Hamilton did not
form a stable covalent inhibitory complex when incubated
with a-thrombin for up to 40 minutes. This result would be
expected because the stressed conformation of the reactive
loop presumed essential for inhibitory activity14would have
been eliminated by proteolytic cleavage of the reactive
center during thrombin-Sepharose chromatography, thus
producing a relaxed, inactive form of AT-111. AT-111 from
the propositus (isolated by heparin-Sepharose chromatography), which contained approximately equal quantities of
both normal AT-I11 and uncleaved AT-111-Hamilton,
showed only 40% to 50% complex formation in the presence of thrombin or factor Xa. This result is expected
because the AT-111-Hamiltonvariant does not form a stable
covalent complex with a-thrombin or factor Xa. Consequently, the AT-111-Hamilton was cleaved in an a-thrombin- or factor Xa-dependent manner, presumably at the
reactive center Arg393-Ser394. This result indicates that
the Ala to Thr substitution at position 382 results in an
AT-I11 species that can act as a substrate for appropriate
serine proteases but cannot complex with these proteases.
This hypothesis was substantiated using cell-free-derived
AT-111-Hamilton that could be cleaved by both a-thrombin
and factor Xa but could not complex with these two serine
proteases. In contrast, cell-free-derived normal AT-I11
showed complex formation with both a-thrombin and
factor Xa and is consistent with previous observation^.'^ In
2189
addition, both cell-free-derived normal AT-I11 and AT-IIIHamilton had identical elution patterns off heparinSepharose, which supports previous observations for plasmaderived AT-I11 that the Ala to Thr substitution at position
382 in AT-111-Hamilton does not affect heparin affinity.5
These results thus extend previous data indicating that the
cell-free approach together with site-directed mutagenesis
can be used to explore structural-functional relationships of
AT-111.
These observations, together with the results shown for
AT-111-Charle~ille,'~AT-III-Cambridge,I6 and AT-IIISudbury," all of which contain the substitution of Ala to
Pro at position 384, strongly suggest that substitutions at or
near Ala 382 alter the orientation of the reactive loop such
that the mutant AT-I11 acts as a substrate but not an
inhibitor of a-thrombin and factor Xa. Furthermore, it
would appear that the inhibition of serine proteases by their
natural inhibitors (the serpins), involves at least two separate but interrelated events; hydrolysis by the protease at
the reactive center followed by complex formation. Clearly,
AT-111-Hamilton is capable of only the first of these events.
ACKNOWLEDGMENT
We thank Dr F. Fernandez-Rachubinski for preparing the
thrombin-Sepharose and Leslie Bardossy for isolating AT-I11 from
the propositus. We also thank Dr W.P. Sheffield for helpful
discussions.
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Identification of the ATIII Charleville as an Ala 384 Pro mutation.
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PM, Larrieu MJ: Purification and further characterization of
16. Perry DJ, Harper PL, Fairham S, Daly M, Carrell R W
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1991 77: 2185-2189
Antithrombin-III-Hamilton, Ala 382 to Thr: an antithrombin-III variant
that acts as a substrate but not an inhibitor of alpha-thrombin and
factor Xa
RC Austin, RA Rachubinski, FA Ofosu and MA Blajchman
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