Download Exosite-dependent regulation of factor VIIIa by

HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Exosite-dependent regulation of factor VIIIa by activated protein C
Chandrashekhara Manithody, Philip J. Fay, and Alireza R. Rezaie
Activated protein C (APC) is a natural
anticoagulant serine protease in plasma
that down-regulates the coagulation cascade by degrading cofactors Va and VIIIa
by limited proteolysis. Recent results have
indicated that basic residues of 2 surface
loops known as the 39-loop (Lys37-Lys39)
and the Ca2ⴙ-binding 70-80–loop (Arg74
and Arg75) are critical for the anticoagulant function of APC. Kinetics of factor Va
degradation by APC mutants in purified
systems have demonstrated that basic
residues of these loops are involved in
determination of the cleavage specificity
of the Arg506 scissile bond on the A2
domain of factor Va. In this study, we
characterized the properties of the same
exosite mutants of APC with respect to
their ability to interact with factor VIIIa.
Time course of the factor VIIIa degradation by APC mutants suggested that the
same basic residues of APC are also
critical for recognition and degradation of
factor VIIIa. Sodium dodecyl sulfate–
polyacrylamide gel electrophoresis (SDSPAGE) of the factor VIIIa cleavage reactions revealed that these residues are
involved in determination of the specificity of both A1 and A2 subunits in factor
VIIIa, thus facilitating the cleavages of
both Arg336 and Arg562 scissile bonds in
the cofactor. (Blood. 2003;101:4802-4807)
© 2003 by The American Society of Hematology
Introduction
Protein C is a multidomain vitamin K–dependent serine protease
zymogen in plasma that, upon activation by the complex of
thrombin and thrombomodulin, down-regulates the coagulation
cascade by degrading cofactors Va and VIIIa by limited proteolysis.1-3 Activated protein C (APC) circulates in plasma as a light and
heavy chain molecule held together by a single disulfide bond.4
Similar to other vitamin K–dependent coagulation proteases, the
N-terminal light chain of APC contains the noncatalytic ␥-carboxyglutamic domain and 2 epidermal growth factor–like domains.5
The catalytic domain of APC with a trypsin-like primary specificity
pocket is located on the C-terminal heavy chain of the molecule.6
Structural data have indicated that the catalytic domain of APC
contains several positively charged residues that are clustered at a
region on the right side of the active site pocket.6 These residues are
conserved at the same 3-dimensional location as those on the
fibrinogen-binding exosite of thrombin.6,7 The basic residues of this
region in APC are clustered on 3 conserved surface loops, known in
the chymotrypsinogen numbering system7 as the 39-loop (Lys37Lys39), 60-loop (Lys62 and Lys63), and 70-80–loop (Arg74, Arg75,
and Lys78). Earlier structure-function studies have demonstrated
that basic residues of all 3 loops are involved in the interaction of
APC with heparin8; however, only basic residues of the 39- and
70-80–loops are required for the anticoagulant function of the
protease.9,10 In addition to these loops, the autolysis loop (148loop) of APC is also highly basic, with 5 Lys/Arg between residues
143 to 154. Previous mutagenesis studies have indicated that these
residues are also critical for the anticoagulant function of APC.10
Factors Va and VIIIa are 2 essential procoagulant cofactors of
the extrinsic and intrinsic pathways of the coagulation cascade,
respectively.11-13 Both cofactors are homologous glycoproteins,
synthesized as single chain precursors with domain structures
A1-A2-B-A3-C1-C2.14-17 During proteolytic activation by either
thrombin or factor Xa, the B domain from both cofactors is released
and the resulting A1-A2 heavy chain remains noncovalently
associated with the A3-C1-C2 light chain in a divalent cationdependent manner.17-20 While A1-A2 domains in factor Va remain
contiguous, the cleavage at Arg372 of factor VIII by thrombin
transforms the heavy chain of the cofactor into 2 separate
subunits.21 The A1 subunit of factor VIIIa retains a stable metal-ion–
dependent linkage with the A3-C1-C2 subunit, whereas the A2
subunit remains weakly associated with the dimer through electrostatic interactions.22 The inactivation of human factor Va by APC
involves 2 cleavages at Arg306 and Arg506 sites, located on the A1
and A2 domains of the cofactor, respectively.3,23 It has been
demonstrated that APC cleaves these 2 sites in a sequential kinetic
order, with an initial cleavage occurring rapidly at the Arg506 site
followed by a slower cleavage occurring at the Arg306 site.3 While
the cleavage of the Arg506 site is independent of a cofactor, the
cleavage of the Arg306 site is accelerated approximately 20-fold by
protein S on negatively charged membrane surfaces.24 In the case
of factor VIIIa, APC also cleaves the cofactor at the homologous
sites Arg336 and Arg562, located on the A1 and A2 subunits,
respectively.21,25 Unlike factor Va, however, in which a single
cleavage at Arg506 site only partially inactivates the cofactor,3,24 the
APC cleavage of either site in factor VIIIa leads to a near-complete
inactivation of the cofactor.26 In the case of factor VIIIa, protein S
From the Edward A. Doisy Department of Biochemistry and Molecular Biology,
St Louis University School of Medicine, MO; and the Department of
Biochemistry and Biophysics, University of Rochester School of Medicine, NY.
Reprints: Alireza R. Rezaie, Department of Biochemistry and Molecular
Biology, St Louis University School of Medicine, 1402 S Grand Blvd, St Louis,
MO 63104; e-mail: [email protected].
Submitted January 15, 2003; accepted February 7, 2003. Prepublished online as
Blood First Edition Paper, February 20, 2003; DOI 10.1182/blood-2003-01-0126.
Supported by grants awarded by the National Heart, Lung, and Blood Institute
of the National Institutes of Health (HL 62565 and HL 68571 to A.R.R.; and HL
30616 and HL 38199 to P.J.F.).
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The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2003 by The American Society of Hematology
BLOOD, 15 JUNE 2003 䡠 VOLUME 101, NUMBER 12
BLOOD, 15 JUNE 2003 䡠 VOLUME 101, NUMBER 12
has a modest cofactor effect toward the cleavage of the Arg336 site
(⬃ 2-fold), however, it enhances the cleavage rate of the Arg562 site
approximately 5-fold.27
APC is a highly specific enzyme with cofactors Va and VIIIa
being the only known substrates for the protease in plasma.2 The
exact mechanism by which APC interacts with these cofactors is
not known in detail. In the case of factor Va, results from several
laboratories have indicated that basic residues of the 39-loop
(Lys37-Lys39) and 70-80–loop (particularly Arg74 and Arg75) play
critical roles in determination of the cleavage specificity of the
Arg506 site, without influencing the cleavage of the Arg306 site.8-10
In the case of factor VIIIa, however, the role of these basic residues
in determination of the cleavage specificity of APC has not been
studied. Thus, we addressed this question in this study by
examining the time course of inactivation of human factor VIIIa by
the exosite mutants of APC in both the absence and presence of
protein S. Inactivation kinetics (IXa-mediated factor Xa generation
in the presence of factor VIIIa) and sodium dodecyl sulfate–
polyacrylamide gel electrophoresis (SDS-PAGE) analysis of cleavage reactions both indicated that the same basic residues of APC,
which are involved in a protein S–independent interaction with
factor Va, are also involved in interaction of APC with factor VIIIa.
However, unlike the critical role of these residues in the selective
cleavage of the A2 domain of factor Va, these residues were found
to be involved in determination of the specificity of cleavage at
both Arg336 and Arg562 scissile bonds of the cofactor.
Materials and methods
Construction and expression of recombinant proteins
Construction, expression, and purification of wild-type and mutant protein C
derivatives in which the 3 basic residues of the 39-loop (Lys37-Lys39) were
substituted with the identical sequence of thrombin (Pro-Gln-Glu) in a triple
mutant (Lys-Lys-Lys/Pro-Gln-Glu) and basic residues of the 60-loop
(Lys62, Lys63, and Arg67) and 70-80–loop (Arg74, Arg75, and Lys78) were
individually substituted with Ala (Lys62Ala, Lys63Ala, Arg76Ala, Arg74Ala,
Arg75Ala, and Lys78Ala) have been described previously.8,28 The wildtype and mutant protein C derivatives were all expressed in HEK293 cells
using the pRC/RSV expression vector (Invitrogen, San Diego, CA) as
described.8 All protein C derivatives were purified by immunoaffinity
chromatography using the Ca2⫹-dependent monoclonal antibody, HPC4,
linked to Affi-Gel 10 (Bio-Rad, Hercules, CA) as described.8
Human plasma proteins factor Va, protein S, and factor IXa were
purchased from Haematologic Technologies (Essex Junction, VT). Recombinant factor VIII preparations were gifts from Bayer (Berkeley, CA).
Factor VIII was activated with thrombin and resulting factor VIIIa was
purified on CM-Sepharose column (Pharmacia, Piscataway, NJ) as described.29 The A1/A3-C1-C2 dimer was isolated from thrombin-treated
factor VIIIa using a Mono S column (Pharmacia) and residual A2 subunit
was removed by an anti-A2 subunit monoclonal antibody coupled to
Affi-Gel 10 as described.30 The A1 subunit was isolated from thrombintreated factor VIIIa heavy chain by fast-protein liquid chromatography
using a combination of Hi-Trap Heparin (Pharmacia) and Mono Q columns
(Pharmacia) as described.31 Recombinant proteins factor X,32 thrombin,33
and protein C inhibitor8 were prepared by cited methods. Phospholipid
vesicles containing 80% phosphatidylcholine and 20% phosphatidylserine
(PC/PS) were prepared as described.34 The chromogenic substrates, Spectrozyme PCa (SpPCa) and Spectrozyme FXa (SpFXa) were purchased from
American Diagnostica (Greenwich, CT).
Protein C activation
Following purification on the HPC4 immunoaffinity column, 1 mg of each
protein C derivative was incubated with thrombin (100 ␮g) in 0.1 M NaCl,
APC REGULATION OF FACTOR VIIIa
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0.02 M Tris (tris(hydroxymethyl)aminomethane)–HCl (pH 7.4; Trisbuffered saline [TBS]) containing 5 mM EDTA (ethylenediaminetetraacetic
acid) for 2 hours at 37°C. Activated protein C derivatives were separated
from thrombin by fast-protein liquid chromatography using a Mono Q
column developed with 40-mL linear gradient from 0.1 to 1.0 M NaCl,
0.02 M Tris-HCl (pH 7.4) as described.8 Partially and fully ␥-carboxylated
APC derivatives were eluted from the Mono Q column as 2 distinct peaks at
approximately 0.3 M and approximately 0.4 M NaCl, respectively, as
described.8 The APC concentrations were determined from the absorbance
at 280 nm assuming a molecular weight of 56 200 and extinction coefficient
1% ) of 14.5,35 by an amidolytic activity assay using SpPCa, and by
(E1cm
stoichiometric titration of enzymes with known concentrations of protein C
inhibitor as described previously.8
Factor VIIIa degradation assay
Factor VIIIa inactivation by APC derivatives was evaluated using a 3-stage
coupled assay as described.36 Briefly, in the first stage, factor VIIIa (1 nM)
was incubated with wild-type or mutant APC (2.5 nM) on PC/PS vesicles
(50 ␮M) at room temperature in TBS containing 5 mM Ca2⫹, 1 mg/mL
bovine serum albumin (BSA), and 0.1% PEG 8000. In the second stage, at
different time intervals (0-20 minutes), the remaining factor VIIIa activity
was determined in an intrinsic Xase assay from the factor VIIIa–dependent
factor X activation by factor IXa as described.27,36 Factor X activation with
the intrinsic Xase complex was carried out for one minute with excess
factor X (0.5 ␮M) and saturating factor IXa (20 nM) on PC/PS (25 ␮M)
vesicles at room temperature. In the third stage, the remaining activity of
factor VIIIa was determined from a decrease in the rate of factor Xa
generation as monitored by an amidolytic activity assay using 200
␮M SpFXa.
SDS-PAGE and Western blotting
SDS-PAGE analysis of the cleavage reactions was carried out for both
factor VIIIa (A1/A2/A3-C1-C2) and the A1/A3-C1-C2 dimer as described.27 Factor VIIIa (250 nM) or the A1/A3-C1-C2 dimer (250 nM) was
incubated with each APC derivative (40 nM) on PC/PS vesicles (100 ␮M)
in the absence or presence of saturating protein S (250 nM) for 5 minutes at
37°C in TBS containing 5 mM Ca2⫹ and 0.1% PEG 8000. The cleavage
reactions were stopped by addition of 5 ⫻ reducing sample buffer followed
by boiling for 5 minutes. In a Bio-Rad minigel system, 10-␮L samples of
the cleavage reactions were analyzed by a 10% SDS-PAGE using the
methods of Laemmli. Following electrophoresis for one hour at 200 V,
proteins were transferred to nitrocellulose membranes using a semidry
Bio-Rad minute-transblot apparatus at 15 V for 30 minutes in a buffer
containing 48 mM Tris, 39 mM glycine, 20% MetOH, and 0.0375% SDS
(pH 9.2). The membranes were blocked with TBS containing 2 mg/mL
BSA for 2 hours. They were then incubated with either one of the 2
monoclonal antibodies, the 58.12 anti-A1 subunit (a gift from Bayer) or the
R8B12 anti-A2 subunit (Green Mountain Antibodies, Burlington, VT),
followed by goat antimouse horseradish peroxidase–conjugated secondary
antibody as described.27 The signals on blots were developed by enhanced
chemiluminescence (ECL) system using luminol as the substrate. Blots
were exposed to films and the band densities were scanned by Scan
Analysis software (Scion Image Beta 4.02; Scion, Frederick, MD).
SDS-PAGE analysis of the A1 subunit cleavage reaction was carried out
as described in the previous paragraph with the exception that the A1
subunit (2.5 ␮M) was incubated with APC derivatives (250 nM) on PC/PS
vesicles (100 ␮M) for 2 to 4 hours at 37°C.
Results
Expression and purification of recombinant proteins
Expression, purification, and activation of wild-type and mutant
protein C derivatives by thrombin have been described previously.8,28 The amidolytic activity and the anticoagulant function of
these APC mutants have been evaluated by both clotting and factor
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MANITHODY et al
Figure 1. APC degradation of factor VIIIa in the absence and presence of
protein S. (A) Human factor VIIIa (1 nM) was incubated with wild-type or mutant APC
derivatives (2.5 nM each) on 25 ␮M PC/PS vesicles in TBS containing 1 mg/mL BSA,
0.1% PEG 8000, and 5 mM Ca2⫹. At the indicated time intervals, the remaining factor
VIIIa activity was determined from the rate of factor Xa generation by the intrinsic
Xase complex composed of 20 nM factor IXa, 500 nM factor X, and 25 ␮M PC/PS as
described in “Materials and methods.” (B) Procedures were the same as those
described in panel A except that the inactivation assay was carried out in the
presence of human protein S (250 nM). The symbols for both panels are as follows:
wild type (E), Lys-Lys-Lys/Pro-Gln-Glu (F), Lys62Ala (䡺), Lys63Ala (f), Arg67Ala
(‚), Arg74Ala (Œ), Arg75Ala (ƒ), and Lys78Ala (). The symbols for the wild-type
APC (E) are masked by those of the Lys62Ala mutant (䡺) because of similar
degradation rates for both proteases.
Va degradation assays in several previous studies.8-10 Results have
demonstrated that, with the exception of the Arg67Ala mutant, all
other APC mutants exhibited normal amidolytic activities.17 In the
case of Arg67Ala, in addition to the proteolytic activity, the
amidolytic activity of the mutant has also been impaired.17 Thus, it
is possible that mutagenesis of this residue has lead to a global
alteration of the APC structure. Although we have included results
with this mutant in the figures, we cannot draw any firm conclusion
as to whether or not Arg67 contributes to the specificity of APC
interaction with factor VIIIa. Since all other mutants are activated
normally by thrombin and have normal amidolytic activities, they
have most likely folded properly, and, therefore, changes in the
specificity of these mutants in factor VIIIa recognition can be
attributed to mutant residues with a relatively high degree of
certainty. The rationale for substituting Lys37-Lys39 of APC with
corresponding residues of thrombin was based on the observation
that these 2 proteases have the greatest structural similarities
among the coagulation proteases and that such substitution may
minimally affect the folding of the mutant proteins. However, the
presence of a proline (Pro) in this sequence could lead to local
structural constraints on the loop that may not be present in the
wild-type protease. Thus, results with this mutant should be
interpreted with this limitation in mind.
Inactivation of factor VIIIa by APC mutants
Previously, plasma-based activated partial thromboplastin time
(aPTT) assays have established that basic residues of the 39-loop
(Lys37-Lys39) and 70-80–loop (particularly Arg74 and Arg75) are
important for the anticoagulant function of APC.8-10 Factor Va
degradation assays in purified systems have indicated that these
residues are involved in recognition and cleavage of the Arg506
scissile bond, which is located on the A2 subunit of the cofactor.9,10
As shown in Figure 1, factor VIIIa degradation assays revealed that
the same basic residues of APC are also involved in recognition and
efficient inactivation of factor VIIIa in both the absence and
presence of protein S. In the absence of protein S, wild-type APC
and the Lys62Ala, Lys63Ala, and Lys78Ala mutants inactivated
50% of the cofactor function of factor VIIIa in approximately 15
minutes (Figure 1A). Under the same experimental conditions,
however, the time required to inactivate 50% of the cofactor
activity of factor VIIIa was prolonged 2-fold (⬃ 30 minutes) for
both Arg74Ala and Arg75Ala mutants, and 3- to 4-fold for the
Arg67Ala and Lys-Lys-Lys/Pro-Gln-Glu mutants (45-60 minutes).
BLOOD, 15 JUNE 2003 䡠 VOLUME 101, NUMBER 12
The catalytic efficiency of APC derivatives toward factor VIIIa in
the presence of protein S was improved (Figure 1B). Protein S,
however, did not restore the catalytic defects of the APC mutants
since the rate of factor VIIIa inactivation by the mutants was
impaired nearly to the same extent. APC inactivated 50% of the
cofactor activity of factor VIIIa in approximately 1.5 to 2 minutes
in the presence of protein S. Similar to results in the absence of
protein S, minimum impairments in the catalytic efficiency of
Lys62Ala, Lys63Ala, and Lys78Ala mutants were observed in the
presence of protein S. However, the catalytic efficiencies of
Arg74Ala and Arg75Ala mutants were moderately and those of
Lys-Lys-Lys/Pro-Gln-Glu and Arg67Ala mutants were severely
impaired (50% inactivation of the cofactor activity occurring in 4-5
minutes and 20 minutes, respectively; Figure 1B). These results
indicate that protein S can promote the rate of factor VIIIa
inactivation approximately 5- to 10-fold with all APC derivatives,
which is consistent with the literature.27 The results further suggest
that, similar to inactivation of factor Va, the basic residues of the
39-loop (Lys37-Lys39) and 70-80–loop (Arg74 and Arg75) are
recognition sites for interaction with factor VIIIa independent of
protein S.
SDS-PAGE and Western blotting of factor VIIIa
cleavage reactions
The APC cleavage of factor VIIIa at either Arg336 or Arg562, located
on the A1 and A2 subunits, respectively, can result in the
inactivation of the cofactor.27 To determine whether either cleavage
was selectively affected by the APC mutagenesis, samples of the
factor VIIIa cleavage reactions (5-minute time points) were run on
10% SDS polyacrylamide gels and transferred to nitrocellulose
membranes for Western blotting. Detection used monoclonal
antibodies specific for either the A1 (58.12) or the A2 (R8B12)
subunit of factor VIIIa. Moreover, in addition to intact factor VIIIa
trimer (A1/A2/A3-C1-C2), the APC cleavage reactions of the
A1/A3-C1-C2 dimer and isolated A1 subunit were also analyzed by
these procedures. Wild-type APC was capable of cleaving both the
A1 (Figure 2A) and A2 (Figure 2B) subunits of factor VIIIa, though
the cleavage of the A1 subunit occurred with a significantly higher
rate. In agreement with the kinetic data, catalytic activities of
Lys62Ala, Lys63Ala, and Lys78Ala mutants were minimally
affected as they cleaved both the A1 and A2 subunits with catalytic
efficiencies that appeared nearly comparable with those of the
wild-type APC. However, the catalytic efficiencies of both Arg74Ala
and Arg75Ala mutants were moderately impaired, while that of the
Lys-Lys-Lys/Pro-Gln-Glu mutant was severely impaired. Scans of
the band intensities of cleavages by these mutants are presented in
Figure 2. SDS-PAGE and Western blotting of factor VIIIa cleavage reactions.
Human factor VIIIa (250 nM) was incubated with APC derivatives (40 nM) on 100 ␮M
PC/PS vesicles in 5 mM Ca2⫹ for 5 minutes at 37°C. Following electrophoresis,
proteins were transferred to nitrocellulose membranes and Western blotted by either
the 58.12 anti-A1 subunit monoclonal antibody (A) or by the R8B12 anti-A2 subunit
monoclonal antibody (B).
BLOOD, 15 JUNE 2003 䡠 VOLUME 101, NUMBER 12
Figure 3. Apparent rate values were estimated using the experimental conditions described in the legend of Figure 2. Wild-type APC
cleaved the A1 subunit with an apparent rate (0.46 minutes⫺1) that
was 46-fold higher than that for cleavage by the Lys-Lys-Lys/ProGln-Glu mutant (0.01 minutes⫺1). It should be noted that these
values may be underestimated since the rate of cleavage by the
wild-type APC was determined from a cleavage reaction in which
approximately 37% of the cofactor was cleaved in 5 minutes
(Figure 3A). The apparent rate of A1 subunit cleavage by the
Arg74Ala (0.11 minutes⫺1) and Arg75Ala (0.25 minutes⫺1) mutants was impaired approximately 4- and approximately 2-fold,
respectively. The apparent rate of cleavage by the Lys62Ala (0.32
minutes⫺1) and Lys63Ala (0.40 minutes⫺1) mutants was minimally
affected and the rate of cleavage by Lys78Ala mutant was slightly
improved (0.50 minutes⫺1). The catalytic properties of APC
mutants toward the cleavage of the A2 subunit were nearly a mirror
image of the cleavage reactions observed with the A1 subunit, with
the exception that all APC forms cleaved the A2 subunit approximately 2-fold slower (Figures 2B,3B).
The Western blot analysis and the scan of the band intensities
for cleavage reactions using the A1/A3-C1-C2 dimer yielded
essentially identical results (Figure 4A, Western blot only). In this
case, however, the cleavage of the A1 subunit by the wild-type APC
was slightly improved, the cleavage by the Lys62Ala mutant was
slightly impaired, and no cleavage reaction was observed for either
the Arg67Ala or the Lys-Lys-Lys/Pro-Gln-Glu mutant. These
observations suggest that the presence of the A2 subunit may have
some conformational effect on the A1 subunit in the trimeric
cofactor, and thus its absence in the A1/A3-C1-C2 dimer could lead
to subtle alterations in the specificity of APC derivatives to cleave
this substrate. This hypothesis is consistent with earlier observations.27 The Western blot of the isolated A1 subunit is shown in
Figure 4B. Although APC cleaved A1 in complex with A3-C1-C2
at a rate that was comparable with the cleavage rate of the intact
trimeric cofactor (compare Figures 2A and 4A), it cleaved the A1
subunit in the absence of A3-C1-C2 with a dramatically impaired
APC REGULATION OF FACTOR VIIIa
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Figure 4. SDS-PAGE and Western blotting of the A1/A3-C1-C2 dimeric subunit
and isolated A1 subunit cleavage reactions. (A) A1/A3-C1-C2 subunit (250 nM)
was incubated with APC derivatives (40 nM) on 100 ␮M PC/PS vesicles in 5 mM Ca2⫹
for 5 minutes at 37°C. Following electrophoresis, proteins were transferred to
nitrocellulose membranes and Western blotted by the 58.12 anti-A1 subunit monoclonal antibody. (B) Procedures were the same as those described in panel A except that
the Western blot is derived from the incubation of A1 subunit (2500 nM) with APC
derivatives (250 nM) on 100 ␮M PC/PS vesicles in 5 mM Ca2⫹ for 4 hours at 37°C.
efficiency. As indicated in “Materials and methods” and in the
Figure 4B legend, in order to observe a quantitatively comparable
cleavage product, the concentration of the APC and the A1 subunit
had to be increased 6- to 10-fold and the incubation time had to be
increased substantially (4 hours at 37°C). These conditions were
due in large part to kinetic parameter (Km) considerations, since
the isolated A1 subunit does not associate with phospholipid
surfaces. Similar to cleavage of the trimeric and dimeric substrates,
the catalytic efficiency of the Lys-Lys-Lys/Pro-Gln-Glu and
Arg67Ala mutants toward the Arg336 scissile bond was dramatically
impaired (Figure 4B). The rate of cleavage of this bond by other
mutants was impaired less than 2-fold. An interesting observation,
however, was the finding that, in addition to the Arg336 cleavage
site, APC slowly cleaved the A1 subunit at an alternative site. The
size of the new cleavage product appeared to be approximately 30
to 35 kDa. Noting that the A1 subunit contains 372 residues with an
apparent molecular weight of 54 kDa, it is likely that APC mutants
cleave after one of several Arg residues located between residues
200 and 250. Cleavage at this site was not detected using the
dimeric and trimeric substrates, suggesting the association of A1
with A3-C1-C2 may alter the presentation of this scissile bond to
the enzyme active site. With the exception of Arg75Ala and
Lys78Ala, the mutants recognized the secondary site of the A1
subunit with relatively high efficiency. This was evidenced by the
observation that the band intensity of the alternative cleavage
product was nearly comparable with the band intensity of the main
product generated from cleavage at Arg336. This was particularly
true with the Lys62Ala and Lys63Ala mutants (Figure 4B, lanes
4-5). Thus, the interaction of basic residues of this exosite with a
complementary site of the A1 subunit not only participates in the
efficient cleavage of the Arg336 scissile bond but also appears to
confer a restricted specificity for APC hindering its interaction with
other nonspecific sites on the cofactor.
Discussion
Figure 3. Scans of the band intensities of factor VIIIa cleavage reactions. Data
are derived from the Western blot analysis by the 58.12 anti-A1 subunit (A) and
R8B12 anti-A2 subunit monoclonal antibodies (B) as shown in Figure 2.
Basic residues of the 3 surface loops including 39-loop (Lys37Lys39), 60-loop (Lys62, Lys63, and Arg67), and 70-80–loop (Arg74,
Arg75, and Lys78) form a basic exosite on the catalytic domain of
APC that is 3-dimensionally homologous to exosite 1 of thrombin.6,7 The location and relative orientation of these residues in the
crystal structure of the catalytic domain of APC are shown in
Figure 5.6 Previous studies have indicated that, similar to thrombin,
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MANITHODY et al
Figure 5. The space-filling model of the catalytic domain of the 4-carboxyglutamic acid (Gla)–domainless APC–D-Phe-Pro-Arg chloromethylketone (PPACK)
complex. The relative 3-dimensional locations of the basic residues of 3 surface
loops 39, 60, and 70-80 are shown in cyan. Shown in red are 2 of the catalytic triad
residues (His57 and Ser195).
the interaction of basic residues of this exosite with natural
substrates, cofactors, and inhibitors plays an important role in
determination of specificity and the catalytic function of APC.8-10
Both plasma-based clotting and factor Va degradation assays in the
purified system have indicated that all of these residues with the
exception of the 2 basic residues of the 60-loop (Lys62 and Lys63)
are required for the anticoagulant function of APC.8-10 APC
inactivates factor Va by a sequential cleavage first at the Arg506 and
then at the Arg306 scissile bonds.3 Studies in the purified system
have demonstrated that basic residues of APC are primarily critical
for the cleavage specificity of the Arg506 scissile bond on the A2
domain of factor Va without any effect on the cleavage specificity
of the Arg306 scissile bond that is located on a loop in the boundary
of the A1 and A2 domains of the cofactor.37 The results of the
current study suggest that the same basic residues of APC are also
involved in interaction and degradation of factor VIIIa. However,
unlike the degradation of factor Va, these residues appear to be
critical for the cleavage specificity of both the Arg336 and Arg562
scissile bonds located on the A1 and A2 subunits of factor VIIIa,
respectively.
The sites on the cofactor subunits that interact with the basic exosite
of APC have not been identified. However, an interactive site for APC
has been reported on the light chain of both factors Va and VIIIa.38 This
site has been mapped to the C-terminal end of the A3 domain in both
cofactors.38 This was evidenced by the observation that the peptide
HAGMSTLFIV corresponding to residues 2009-2018 of factor VIII
inhibited the anticoagulant activity of APC and its inactivation of factor
VIIIa in the purified system.38 A similar sequence derived from the
C-terminal end of the A3 domain of factor V exhibited similar properties
and inhibited the APC inactivation of factor Va. It is known that the C2
domain of the light chain of both factor Va and VIIIa binds on the
phospholipid surface. Noting that the putative inhibitory peptide is
located on the membrane proximal A3 domain of the cofactor,39 it is
likely that it interacts with the light chain of APC, but not the catalytic
domain of the protease on the heavy chain. The fact that this peptide is
not acidic and contains several hydrophobic residues is also consistent
with this hypothesis. On the other hand, both of the A1 and A2 subunits
of factor VIIIa contain acidic C-terminal sequences that might potentially provide contact sites for the basic exosite of APC.17 In support of
this hypothesis, the catalytic function of the APC mutants in degradation
of the trimeric factor VIIIa, dimeric A1/A3-C1-C2, and the isolated A1
subunit was similarly affected. This indicates that an interactive site on
the cofactor is located within the A1 domain. It is interesting to note that
previous studies have indicated that the same C-terminal sequence of the
BLOOD, 15 JUNE 2003 䡠 VOLUME 101, NUMBER 12
A1 subunit may also provide an interactive site for factor X.30 It has been
shown that the association of the substrate factor X with the intrinsic
Xase complex confers a protective effect for factor VIIIa against
inactivation by APC, specifically blocking the cleavage at the A1 site.27
Thus, the A1 subunit of the cofactor may have overlapping binding sites
for both factor X and APC on the C-terminal acidic domain. Furthermore, recent data also support the association of factor Xa with this
region as a step leading to cleavages within the A1 subunit.40 On the
other hand, the A2 subunit of factor VIIIa shows an extended interactive
surface for factor IXa.41 Factor IXa also demonstrates protection of
factor VIIIa from APC, specifically blocking cleavage at the A2 site
(Arg562).27 Blocking of the cleavage at this site likely results from factor
IXa interacting at the 558-565–loop,42 thereby masking the susceptible
bond. It is not known whether additional protective effects are derived
from overlapping interactive sites on A2 for the 2 proteases.
The observation that APC mutants cleaved the A1 subunit at an
alternative site with similar efficiency to that of the Arg336 site suggests
that in addition to mediating the effective cleavages of the Arg336 and
Arg562 scissile bonds, the basic residues of APC impede the protease
interaction with a nonspecific site on the A1 subunit of the cofactor. The
observed cleavage at a secondary site on the A1 subunit by the Lys62Ala
and Lys63Ala mutants was of particular interest since these mutants
exhibited nearly normal activities in both clotting and factors Va and
VIIIa degradation assays, apparently suggesting that these residues may
be dispensable for the anticoagulant function of APC.9,10 However, the
results of this study demonstrate that all basic residues of this exosite are
critical for determination of the specificity of factor VIIIa degradation,
albeit with a lack of impairment in the catalytic function of some
mutants in functional assays. This is a good example of how coagulation
proteases in general and APC in particular select their macromolecular
substrates and inhibitors with a high degree of specificity.
APC also reacts very slowly with plasma inhibitors, and thus
has a long half-life of 27 minutes in circulation.43 Protein C
inhibitor, ␣1-antitrypsin, and the plasminogen activator inhibitor-1
(PAI-1)–vitronectin complex are thought to be primarily responsible for regulation of the APC activity in circulation.28,44 We
recently demonstrated that the basic residues of the 39-loop
(Lys37-Lys39) are also required for the protease interaction with
PAI-1.28 PAI-1 has 2 acidic glutamic acid (Glu) residues at the P4⬘
and P5⬘ sites of the reactive site loop. Previous results have
indicated that the interaction of basic residues of the 39-loops of the
plasminogen activators with the P4⬘ and/or P5⬘ residues of PAI-1 is
responsible for the rapid neutralization of the profibrinolytic
enzymes by the serpin.45 The same appears to be true for the
APC–PAI-1 reaction since it has been shown that the reactivity of
the Lys-Lys-Lys/Pro-Gln-Glu mutant of APC with the serpin is
dramatically impaired.28 Interestingly, examination of the acidic
C-terminal domain of the A1 subunit of factor VIIIa at the primed
site of the Arg336 scissile bond indicated that both P5⬘ and P6⬘
residues are also Glu,15 one or both of which can interact with the
basic 39-loop of APC. Unlike the Arg336 site on the A1 subunit, the
sequence surrounding the Arg562 scissile bond from the P3-P6⬘ site
(DQRGNQIMS) on the A2 subunit contains no acidic residues at
the primed site, but instead a nonoptimal aspartic acid (Asp)
residue at the P3 site of the cleavage site. We previously demonstrated that the presence of an acidic residue at the P3 site of
substrates is inhibitory for interaction with APC because of its
possible collision with an acidic Glu residue at position 192 of the
catalytic pocket.46 The scan of band intensities of the factor VIIIa
cleavage reactions showed that APC cleaves the Arg336 scissile
bond approximately 2-fold faster than the Arg562 scissile bonds.
Thus, differences in the structure of residues surrounding the 2
BLOOD, 15 JUNE 2003 䡠 VOLUME 101, NUMBER 12
APC REGULATION OF FACTOR VIIIa
cleavage sites on the A1 and A2 subunits of factor VIIIa may
account for their differential reactivity with APC.
In summary, results of this study suggest that the basic residues
of all 3 surface loops (39, 60, and 70-80) in APC are critical for
determination of factor VIIIa specificity. The interactions of these
residues with factor VIIIa are critical for the cleavage specificity of
both Arg336 and Arg562 scissile bonds, located on the A1 and A2
subunits, respectively. It is known that the binding of both
procofactors V and VIII on exosite 1 of thrombin is also required
for their rapid activation by thrombin.47 Further studies are needed
to determine whether the sites on procofactors that interact with
4807
exosite 1 of thrombin are the same sites on activated cofactors that
interact with the homologous exosite of APC.
Acknowledgments
We thank Dr Lisa Regan at Bayer for the generous gifts of
recombinant factor VIII. We also acknowledge Julie Dill and Jan
Freas for excellent technical assistance in preparation of factor
VIIIa substrates.
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