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Blood, Vol. 96 No. 2 (July 15), 2000:
pp. 585-593
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
The autolysis loop of activated protein C interacts with factor Va
and differentiates between the Arg506 and Arg306 cleavage
sites
Andrew J. Gale,
Mary J. Heeb, and
John H. Griffin
From the Departments of Molecular and Experimental Medicine and of
Vascular Biology, The Scripps Research Institute, La Jolla, CA.
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Abstract |
The anticoagulant human plasma serine protease, activated protein C
(APC), inactivates blood coagulation factors Va (FVa) and VIIIa. The
so-called autolysis loop of APC (residues 301-316, equivalent to
chymotrypsin [CHT] residues 142-153) has been hypothesized to bind
FVa. In this study, site-directed mutagenesis was used to probe the
role of the charged residues in this loop in interactions between APC
and FVa. Residues Arg306 (147 CHT), Glu307, Lys308, Glu309, Lys311,
Arg312, and Arg314 were each individually, or in selected combinations,
mutated to Ala. The purified recombinant protein C mutants were
characterized using activated partial thromboplastin time (APTT)
clotting assays and FVa inactivation assays. Mutants 306A, 308A, 311A,
312A, and 314A had mildly reduced anticoagulant activity. Based on FVa
inactivation assays and APTT assays using purified Gln506-FVa and
plasma containing Gln506-FV, it appeared that these mutants were
primarily impaired for cleavage of FVa at Arg506. Studies of the
quadruple APC mutant (306A, 311A, 312A, and 314A) suggested that the
autolysis loop provides for up to 15-fold discrimination of the Arg506
cleavage site relative to the Arg306 cleavage site. This study shows
that the loop on APC of residues 306 to 314 defines an FVa binding site
and accounts for much of the difference in cleavage rates at the 2 major cleavage sites in FVa.
(Blood. 2000;96:585-593)
© 2000 by The American Society of Hematology.
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Introduction |
Protein C is a vitamin K-dependent zymogen of a plasma
serine protease.1 When activated by the
thrombin-thrombomodulin complex2,3 protein C acts as an
anticoagulant, in concert with its cofactor protein S, on the surface
of phospholipid membranes by inactivation of the blood coagulation
factors Va (FVa) and VIIIa.4-6 The importance of activated
protein C (APC) is illustrated by an increased risk of venous
thromboembolism associated with heterozygous protein C
deficiency.7 Furthermore, homozygous protein C
deficiency is responsible for severe and generalized thrombotic
disease.8,9
Inactivation of FVa, the primary substrate of APC, is a complex
process. APC cleaves FVa at 3 locations, Arg506, Arg306, and Arg679.
The significance of cleavage at Arg679 is not known, but the cleavages
at Arg506 and Arg306 are primarily responsible for inactivation of FVa.
Cleavage at Arg506 partially inactivates FVa, whereas cleavage at
Arg306 completely inactivates FVa. In the absence of the cofactor
protein S, cleavage of Arg506 is about 20-fold faster than cleavage at
Arg306.10-12 But protein S accelerates cleavage of Arg306
to a rate close to that of cleavage at Arg506.13
Fisher et al14 built a molecular model of the serine
protease domain of APC based on the known crystal structures of
homologous serine proteases. Inspection of this model revealed a
concentration of basic residues in the lower "east" quadrant of
the molecule when the model was in the standard orientation for serine
proteases. Subsequently, a crystal structure of a truncated version of
APC confirmed the presence of this basic exosite,15 which
is generally in a position similar to that of the anion binding exosite
I of thrombin, an exosite that is involved in protein-protein
interactions. Several basic residues in the autolysis loop of APC are
part of this basic exosite. The autolysis loop of APC (residues
301-316, equivalent to chymotrypsin [CHT] residues 142-153) contains
an insert of 4 residues relative to chymotrypsin and has 5 basic and 2 acidic residues. A synthetic peptide with sequence overlapping part of
this autolysis loop inhibits the APC inactivation of FVa, thus
implicating this region in FVa binding.16
Given the presence of so many charged residues and the inhibition of
FVa inactivation by this synthetic peptide, we decided to create
recombinant mutant APC molecules with individual mutations of the
charged residues of the autolysis loop to investigate the role of this
loop in the interaction of APC with FVa. We mutated the residues
Arg306, Glu307, Lys308, Glu309, Lys311, Arg312, and Arg314 to Ala. We
also made a mutant with both Arg306 and Arg312 mutated to alanines
(306/312AA) and a mutant with Arg306, Lys311, Arg312, and Arg314 all
mutated to alanines (306-314AAAA). These purified mutant proteins were
analyzed for their anticoagulant activity and for their ability to
inactivate FVa.
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Materials and methods |
Proteins and reagents
Factor Va and Q506-FVa were prepared as described.12,17
FXa was purchased from Enzyme Research Laboratories (South
Bend, IN). Phospholipid vesicles (80% phosphatidylcholine, 20%
phosphatidylserine) were prepared as described.18 The
chromogenic substrate
L-pyroglutamyl-L-prolyl-L-argininyl-p-nitroanilide hydrochloride (S-2366) was purchased from Chromogenix
(Franklin, OH). The chromogenic substrate H-D-lysyl
(g-Cbo)-prolyl-argininyl-p-nitroanilide (Spectrozyme PCa) was purchased
from American Diagnostica (Greenwich, CT). The chromogenic substrate
CBS 34-47 was purchased from American Bioproducts (Parsippany, NJ).
Normal human citrate-anticoagulated plasma was purchased from Precision
Biologicals (Dartmouth, Novia Scotia). Factor V-deficient plasma was
purchased from George King Bio-Medical, Inc (Overland Park, KS).
Biotinylated goat antirabbit-IgG, streptavidin alkaline phosphatase
conjugate, nitroblue tetrazolium chloride (NBT), and
5-bromo-4-chloro-3'-indolylphosphate-p-toluidine salt
(BCIP) were purchased from Pierce (Rockford, IL). Polyvinylidene fluoride membrane was purchased from Millipore (Bedford, MA).
Expression and purification of recombinant protein C
Mutant protein C expression vectors were constructed and purified
recombinant protein C was prepared as described.19,20 Briefly, crude recombinant protein C was loaded on a fast-flow Q-Sepharose column in 50 mmol/L Tris, 150 mmol/L NaCl, pH 7.4. Then
protein C was eluted with 50 mmol/L Tris, 120 mmol/L NaCl, and 15 mmol/L CaCl2. This protocol selects for properly
 -carboxylated protein C because undercarboxylated protein C is not
eluted by CaCl2.21 Some preparations of
recombinant protein C were purified further by chromatography on a
calcium-dependent sheep polyclonal antiprotein C antibody-Sepharose
column.22 The protein C was loaded onto the column in 50 mmol/L Tris, pH 7.4, 10 mmol/L CaCl2, 1 mol/L NaCl, 0.02%
Tween 20, and 0.05% NaN3. After washing the column with
this buffer, protein C was eluted with 50 mmol/L Tris, pH 7.4, 10 mmol/L EDTA, 0.1 mol/L NaCl, 0.02% Tween 20, and 0.05% NaN3. The protein C-containing fractions were pooled and
dialyzed into HBS (50 mmol/L HEPES, pH 7.4, 150 mmol/L NaCl) for
storage at  80°C. Final concentration of protein C was
determined using the Asserachrom Protein C ELISA from American Bioproducts.
Functional assays
Protein C was activated by thrombin. Protein C in HBS plus 2 mmol/L
EDTA and 0.5% bovine serum albumin (BSA), at a concentration of 50 µg/mL, was incubated for 2.5 hours with 5 µg/mL thrombin at
37°C followed by the addition of 1.1 U hirudin per unit of thrombin
to inactivate the thrombin. Controls were done in amidolytic assays,
activated partial thromboplastin time (APTT) clotting assays, and FVa
inactivation assays to verify that the thrombin and hirudin used had no
effect on these assays. Wild-type APC and the mutants 306/312AA and
306-314AAAA were quantitated using an active site titration adapted
from Chase and Shaw23 using APC at approximately 8 µmol/L
in HBS and p-nitrophenol-guanidino benzoate at 0.1 mmol/L with an
extinction coefficient for p-nitrophenol of 9890 M-1
cm-1 calculated for the observed pH.
Amidolytic activity of each APC was measured using the chromogenic
substrate S-2366 (0.83 mmol/L) in TBS, 0.5% BSA, 0.02%
NaN3, pH 8.2 and an EL312 Microplate Bio-Kinetics reader
(Bio-Tek Instruments, Winooski, VT).18 Alternatively the
substrate Spectrozyme PCa was used in HBS, 0.5% BSA, 5 mmol/L CaCl2, 0.1 mmol/L MnCl2, pH 7.4. Concentrations
of single site APC mutants were calibrated relative to wild-type APC
based on amidolytic activity for the substrate S2366. By varying
chromogenic substrate concentration from 1.43 to 0.0446 mmol/L we
derived Km and
kcat values using Eadie-Hofstee plots.
For APTT clotting assays, 50 µL of plasma was mixed with 50 µL of
APTT reagent (Platelin LS; Organon Technika Corp, Durham, NC) and
preincubated at 37°C for 3 minutes. Then 2 µL of APC was added
followed by 50 µL of HBS, 0.5% BSA, and 25 mmol/L CaCl2. The clotting time was recorded using an ST4 coagulometer (Diagnostica Stago, Asnieres, France).
Inactivation of FVa was measured as follows. A mixture of 1 nmol/L FVa
with 25 µmol/L phospholipid vesicles was made in 50 mmol/L HEPES, pH
7.4, 100 mmol/L NaCl, 0.5% BSA, 5 mmol/L CaCl2, 0.1 mmol/L
MnCl2. Inactivation was initiated by the addition of APC.
Aliquots of 1 µL were removed at time points and added to 40 µL of 1.25 nmol/L factor Xa with 25 µmol/L phospholipid
vesicles, followed by 10 µL of 3 µmol/L prothrombin
(final concentrations: 1 nmol/L FXa, 20 pmol/L FVa, 25 µmol/L
phospholipid vesicles, and 0.6 µmol/L prothrombin). After 2.5 minutes
a 15-µL aliquot of this mixture was quenched by addition to 55 µL
HBS containing 10 mmol/L EDTA, 0.5% BSA, pH 8.2. Chromogenic substrate
CBS 34-47 was added and the rate of thrombin formation was assessed by
measuring the change in absorbance at 405 nm as described earlier.
Curve fitting of these pseudo-first-order time courses of FVa
inactivation was done according to Nicolaes and
colleagues11 using the following equation:
Vat is the cofactor activity determined at time t.
Vao is the cofactor activity determined before APC is
added. B is the relative cofactor activity of factor Vaint,
the partially inactivated Va cleaved only at Arg506 (expressed as a
fraction of the cofactor activity of intact FVa).
k506 is the apparent second-order rate
constant for cleavage at Arg506 in native FVa.
k306 is the apparent second-order rate
constant for cleavage at Arg306 in FVaint and
k'306 is the apparent second-order
rate constant for cleavage at Arg306 in intact FVa.
k'306 was determined from the
inactivation time courses of Q506-FVa fit to a single exponential and
was taken as a constant in the above equation. Data were fit to the
equations using nonlinear least-squares regression analysis.
These apparent second-order rate constants in units of M-1 s-1 are equivalent to
kcat/Km,
so the change in transition-state stabilization energy
( GT ) can be calculated using the
equation:
in
which R is the gas constant and T is the absolute
temperature.24
Plasma inactivation of APC and the inactivation of APC by the serpin
inhibitors, protein C inhibitor (PCI), and  1-antitrypsin were measured essentially according to Heeb and
coworkers.25 For Western blots of FVa inactivated by APC,
20 nmol/L FVa was incubated with the indicated concentrations of
wild-type APC or 306-314AAAA-APC in the buffer described above.
Aliquots were removed at the indicated time points and boiled in sodium
dodecyl sulfate (SDS) sample buffer containing 10 mmol/L EDTA, then
electrophoresed in a 4% to 12% Bis-Tris gradient gel (Novex, San
Diego, CA) and transferred to PVDF membrane. FVa and FVa cleavage
products were monitored by Western blotting with a polyclonal antibody
against the human FVa heavy chain26 and a monoclonal
antibody against the human FVa heavy chain (Enzyme Research
Laboratory).12
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Results |
Production and characterization of protein C mutants
Wild-type and mutant recombinant protein C was recovered at levels
varying from 0.1 to 1.5 mg/mL of conditioned media. After purification
the concentrations of the recombinant protein C were determined by
absorbance at 280 nm and by enzyme-linked immunosorbent assay (ELISA).
From ELISA and silver-stained SDS-polyacrylamide gel electrophoresis
(SDS-PAGE) we estimated purity to range from about 75% to 95% (data
not shown). These silver-stained gels also showed no changes in
apparent molecular weight for the mutants, indicating that the
mutations had no apparent effect on N-linked glycosylation. The lowest
purity levels were observed for the proteins that were recovered at the
lowest levels (307A, 308A, and 309A protein C mutants). All the protein
C was activated with thrombin as described. All the mutants except
306-314AAAA had Km values for S2366 that
were indistinguishable from that of wild-type and the
Km for 306-314AAAA was increased by about
10% (data not shown).
Anticoagulant activity of single-point mutants
The anticoagulant activity of the single-point mutant APCs was
measured in an APTT assay with normal human plasma (Figure 1). In this clotting assay the mutant
306A-APC had significantly reduced activity. Mutant 307A-APC had near
wild-type activity; 308A-APC had a moderate reduction in activity; and
309A-APC had somewhat increased anticoagulant activity (Figure 1A).
Figure 1B shows that mutants 311A-APC, 312A-APC, and 314A-APC all had a
significantly reduced level of anticoagulant activity.
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| Fig 1.
Anticoagulant activity of APC single-point mutants.
The anticoagulant activity of the autolysis loop single mutants was
tested in an APTT assay as described in "Materials and methods"
over a range of APC concentration from 1.6 to 6.3 nmol/L. (A) APC:
wild-type (+), 306A ( ), 307A ( ), 308A ( ), 309A ( ). (B) APC:
wild-type (+), 311A (), 312A (), 314A (). All curves are
averaged from 4 to 8 individual experiments.
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Inactivation of FVa by single-point mutants
Inactivation of FVa was tested in a purified system over a time
course as described in "Materials and methods." Results are presented in 2 panels with inactivation curves for wild-type APC, 306A-APC, 307A-APC, 308A-APC, and 309A-APC in Figure
2A and inactivation curves for wild-type
APC, 311A-APC, 312A-APC, and 314A-APC in Figure 2B. The inactivation
curves were biphasic on a log scale. Investigators at several
laboratories have postulated that this is due to rapid cleavage at
Arg506 resulting in a FVa reaction intermediate that retains partial
cofactor activity in prothrombin activation and that this partially
active form is then fully inactivated by a slower cleavage at
Arg306.10,11,26,27 These inactivation time courses are
pseudo-first-order time courses because they were performed under
first-order conditions, that is, when the inactivation rate was
directly proportional to the residual concentration of FVa because the
FVa concentration was below the Km for the
inactivation cleavages. Therefore, rate constants for cleavage at
Arg506 and for cleavage at Arg306 in the partially inactive form were
determined by fitting the data to the biphasic exponential equation
described in "Materials and methods" according to Nicolaes and
colleagues.11 This analysis yielded second-order rate
constants in units of M-1 s-1 that
are equivalent to
kcat/Km
for the reaction of APC with the respective cleavage site. In the
equation that was used, the value of k306
represents the second-order rate of cleavage at Arg306 in the
intermediate form of FVa that is already partially inactivated by
Arg506 cleavage. Cleavage at Arg506 is represented by the second-order
rate constant k506. A value for the
fraction of remaining FVa activity following cleavage at Arg506 was
first determined, using wild-type APC, to be 0.66 and was then fixed
for all further line fits. A value for the apparent second-order rate
of cleavage at Arg306 in the absence of Arg506 cleavage was determined
in the time course of inactivation of Q506-FVa, which was fit to a
single exponential equation. This value
(k'306) was determined for each
mutant and the wild-type APC using purified Q506-FVa (Table
1) and was fixed in the equation used to
fit the inactivation time courses of wild-type FVa.
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| Fig 2.
Inactivation of Factor Va by APC single-point mutants.
FVa was incubated at a concentration of 1 nmol/L with 50 pmol/L
wild-type or mutant APC and inactivation was followed over time as
described in "Materials and methods." (A) APC: wild-type (+),
306A (), 307A (), 308A (), 309A (). (B) APC: wild-type (+),
311A (), 312A (), 314A (). Data points shown are averages of
between 3 to 6 experiments for each APC. Standard deviations for all
the data points shown averaged ± 3.4%. Error bars are shown for
wild-type APC in panel B for illustrative purposes. Other error bars
are left out because many of the curves are so close to each other.
Curves were fit to the equation in "Materials and methods"
according to Nicolaes and colleagues.l11 Coefficient of
determination (r2) values were 0.995 or greater for all the
curves except for that of 314A-APC (0.977).
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In Figure 2A and Table 1, 307A-APC, 308A-APC, and 309A-APC appeared
very similar to wild-type in their cleavage rates for both Arg506 and
Arg306. In this assay 307A-APC had slightly decreased activity toward
Arg506 but a slightly increased activity toward Arg306 (Table 1). The
mutants 308A-APC and 309A-APC had values very close to that of
wild-type, but 308A-APC had somewhat higher activity toward Arg306 than
wild-type. This is in contrast to the results from APTT assays in which
308A-APC had somewhat decreased activity and 309A-APC had activity
greater than wild-type. In Figure 2A, 306A-APC was distinct from the
other mutants because it had significantly decreased activity toward
Arg506 with a cleavage rate of 8.8 × 107
M-1 s-1. However, 306A-APC was
only slightly decreased in activity toward Arg306 relative to wild-type
with a cleavage rate of 6.7 × 106 M-1 s-1 (Table 1). In the case of 306A-APC the
final FVa activity at the end of the time course was only slightly
greater than that of the FVa inactivated by wild-type APC. This
illustrated that in this prothrombinase assay, final FVa activity
(below 60%) was largely dictated by cleavage at Arg306. In contrast,
anticoagulant activity in APTT assays correlated better with the rate
of cleavage at Arg506.
The FVa inactivation time courses for mutants 311A-APC, 312A-APC, and
314A-APC are shown in Figure 3B. All 3 had
similar inactivation time courses with reduced cleavage rates for both
Arg506 and Arg306 in FVa as shown in Table 1. Table 1 also shows
cleavage rates for the mutant APCs for Arg306 of purified Q506-FVa. All
of the APCs cleaved Arg306 in Q506-FVa with a second-order rate
constant that was about 20% to 50% less than the rate of cleavage of
Arg306 following Arg506 cleavage in wild-type FVa. The mutants
307A-APC, 308A-APC, and 309A-APC had essentially wild-type activity
toward the Arg306 cleavage site in Q506-FVa. The mutants 306A-APC,
311A-APC, and 312A-APC had 65% to 70% of the activity of wild-type
APC toward the Arg306 cleavage site in Q506-FVa. Finally, the mutant
314A-APC had about 40% of the activity of wild-type APC toward the
Arg306 cleavage site in Q506-FVa. Table 1 illustrates that cleavage at
Arg506 was more sensitive to mutation in the autolysis loop than
cleavage at Arg306. For example, mutation of Arg306 in APC to Ala
reduced k506 by 2.8-fold but only reduced
k306 by 1.1-fold and
k'306 by 1.5-fold.
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| Fig 3.
Anticoagulant activity of APC single-point mutants
relative to wild-type APC in normal human plasma versus homozygous
Q506-FV plasma.
(A) Normal human plasma. (B) Q506-FV plasma. APTT assays were done
using plasma mixtures containing 10% normal plasma or 10% Q506-FV
plasma and 90% FV-deficient plasma. The percentage of wild-type
activity for each APC mutant was calculated as follows. The wild-type
APC dose-response data were fit to a line and then that equation was
used to calculate a relative amount of wild-type APC for the respective
clotting time values for each mutant. For each experiment, values from
2 to 3 concentration points for each mutant were averaged together to
get a percent of wild-type APC activity for that experiment. The
results of 2 to 3 experiments were averaged together and the SDs are
shown as error bars.
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Clotting assays with Q506-FV plasma
We assessed cleavage at Arg306 in FVa based on APTT clotting assays
using homozygous Q506-FV human plasma. Figure 3 illustrates the
reduction in activity of the mutant APCs relative to wild-type in both
normal plasma (Figure 3A) and Q506-FV plasma (Figure 3B). These APTT
assays were done using plasma mixtures that were 90% FV-deficient
plasma and 10% either normal or Q506-FV plasma. This equalized the
quantities of all the coagulation factors other than FV coming from the
normal and Q506-FV plasmas. Furthermore, because the resulting plasma
mixtures had only 10% of the usual FV levels, the sensitivity of the
assay toward APC was increased. In Figure 3 it is clear that
mutagenesis in the autolysis loop caused less reduction of the
anticoagulant activity of APC toward the Arg306 cleavage site (Q506-FV
plasma) compared with cleavage at both sites (normal plasma). Most
notably, 306A-APC had 45% of normal anticoagulant activity in normal
human plasma but 97% of normal anticoagulant activity in Q506-FV
plasma (Figure 3).
Double and quadruple mutants
Given the apparent reduction in cleavage site specificity for the
single site mutants, 306A-APC, 311A-APC, 312A-APC, and 314A-APC, we
decided to make selected combined mutants to determine the cumulative
effects of these mutations. The assumption was that the effects of the
individual mutants might be additive. The APC mutants constructed were
a double mutant of R306A and R312A (306/312AA) and a quadruple mutant
of R306A, K311A, R312A, and R314A (306-314AAAA). Figure
4 shows results of APTT assays using normal
and Q506-FV plasma. Both of the mutants had significantly reduced
anticoagulant activity relative to recombinant wild-type APC in normal
plasma where 306/312AA-APC had 17% activity and 306-314AAAA-APC had
1.6% of wild-type activity (Figure 4A). In contrast both mutants had relatively high activity in Q506-FV plasma where 306/312AA-APC had 75%
activity and 306-314AAAA-APC had 28% activity (Figure 4B). This is a
4.4-fold difference in activity for 306/312AA-APC and an 18-fold
difference in activity for 306-314AAAA-APC.
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| Fig 4.
Anticoagulant activity of mutants 306/312AA-APC and
306-314AAAA-APC versus wild-type APC.
(A) Normal human plasma: wild-type APC (+), 306A/312AA-APC (),
306A-314AAAA-APC (). (B) Q506-FV human plasma: wild-type APC (+),
306A/312AA-APC (), 306A-314AAAA-APC (). APTT assays were
performed using plasma mixtures containing 10% normal human plasma or
10% Q506-FV human plasma as described above. Data shown are the
average of 4 experiments for each line and SDs are shown as error
bars.
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To confirm that loss of anticoagulant activity in APTT assays was not
caused by an increased neutralization of these mutants by plasma
serpins, we followed the rate of inactivation of wild-type APC and the
least active mutant, 306-314AAAA-APC, in plasma. The half-life of the
mutant 306-314AAAA-APC in plasma was not shorter than the normal
half-life (20 minutes) of wild-type APC (data not shown). Thus, the
more than 98% reduction of anticoagulant activity of 306-314AAAA-APC
in plasma APTT assays was not due to more rapid neutralization by
plasma serpins.
The activity of these mutants was also tested in the FVa inactivation
assay with purified components. The rate of cleavage of Arg306 in the
absence of any possible Arg506 cleavage was determined using purified
Q506-FVa. Rates of cleavage at Arg506 and cleavage at Arg306 following
Arg506 cleavage were determined using normal FVa. Apparent second-order
rate constants for these cleavages show remarkable differences in
effects of the mutations on k506 versus
k306. Mutant 306-314AAAA-APC cleaved Arg506
at a rate that was reduced 71-fold relative to wild-type APC (Table 1).
However, 306-314AAAA-APC cleaved Arg306 at a rate that was reduced
6.4-fold relative to wild-type APC following Arg506 cleavage (k306) and 4.7-fold in Q506-FVa
(k'306). From these apparent
second-order rate constants we were able to calculate the change in
transition-state stabilization energy
(GT) for each of the mutants
relative to wild-type (Table 2). Changes in
free energy for both 306/312AA-APC and 306-314AAAA-APC were nearly
additive for both cleavage sites. As implied by the APTT results,
mutation of 4 residues in the autolysis loop had a much larger effect
on Arg506 cleavage than on Arg306 cleavage, such that these 4 residues
appear to provide for 11- to 17-fold of the discrimination of the
Arg506 cleavage site over the Arg306 cleavage site.
To rule out any possibility that reduction in activity of the APC
mutants was merely due to a defect in phospholipid binding, we studied
FVa inactivation using wild-type APC and the least active mutant,
306-314AAAA-APC, in the absence of phospholipid vesicles. Within 6 minutes wild-type APC inactivated FVa to about 60% activity, at which
point the activity leveled off because cleavage at Arg306 requires
phospholipid.28 FVa inactivation by the 306-314AAAA-APC
mutant was greatly reduced relative to wild-type and was so slow that
the activity of FVa was not appreciably decreased during the course of
the assay (30 minutes, data not shown). Hence, the 306-314AAAA-APC
mutant had the same reduction in activity involving cleavage at Arg506
in both the presence and absence of phospholipid vesicles, thus
verifying that reduction in activity for the APC mutant was not a
result of defective phospholipid binding.
To confirm that the decreases in anticoagulant activity of the APC
mutants were not due to extensive disruption of the active site, the
Km and kcat for
the chromogenic substrate Spectrozyme PCa were determined for wild-type
APC and the least active mutant, 306-314AAAA-APC, in the same
conditions in which the FVa inactivation assays were performed. The
catalytic efficiency (kcat/Km)
of wild-type APC toward Spectrozyme PCa was 1.3 × 105 M-1 s-1 and the catalytic efficiency of 306-314AAAA was
0.98 × 105 M-1 s-1 (Table 3). Thus, compared with
wild-type APC, 306-314AAAA-APC retained 75% activity toward the
chromogenic substrate Spectrozyme PCa, whereas it exhibited only 1.6%
anticoagulant activity. A similar modest reduction in catalytic
efficiency was seen with the substrate S2366 (data not shown). We also
compared the rates of reaction of purified plasma serpins, PCI, and
1-antitrypsin, with wild-type and
306-314AAAA-APC.25 For reaction of PCI with wild-type APC
and 306-314AAAA-APC the second-order rate constants were 480 M-1 s-1 and 350 M-1
s-1, respectively, and for 1-antitrypsin
they were 0.84 M-1 s-1 and 0.47 M-1
s-1, respectively. The reductions for
306-314AAAA-APC in serpin reactivity were comparable to the reductions
seen in chromogenic substrate reactivity and are minor relative to the
effect on cleavage of Arg506 in FVa. Thus, we can make the assumption
that mutation-induced changes in APC activity toward FVa are mainly due
to altered interactions specific for the APC-FVa complex, rather than
due to alterations in the conformation of the immediate active site
that would have nonspecific effects on any substrate.
Western blots of FVa inactivation
To verify that these cleavage rates calculated from FVa inactivation
curves do correspond to cleavages at Arg506 and Arg306 in the FV heavy
chain, we performed SDS-PAGE analysis of aliquots of FVa inactivated by
wild-type APC and 306-314AAAA-APC. Figure 5A shows the time course of inactivation of
FVa by wild-type APC. The gel was blotted with anti-FV heavy chain
antibodies. The observed pattern of cleavage is typical of published
results.10,11,26 A band at about 75 kd and a triplet of
bands at 26 to 28 kd, which appear early in the time course, were the
result of cleavage at Arg506. Over a longer period of time the 75 kd
band disappeared, as a band at about 45 kd appeared that was the
fragment (residues 1-306) that results from cleavage at Arg306.
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| Fig 5.
Western blot of FVa inactivation by wild-type APC and
306-314AAAA-APC.
(A) Wild-type APC, 200 pmol/L. (B) 306-314AAAA-APC, 2 nmol/L. FVa at a
concentration of 20 nmol/L was incubated in 50 mmol/L HEPES, pH 7.4, 100 mmol/L NaCl, 5 mmol/L CaCl2, 0.1 mmol/L
MnCl2, 0.1% BSA with 25 µmol/L phospholipid vesicles.
APC was added and aliquots were removed at 1 through 40 minutes. The
Western blot was developed as described in "Materials and
methods." Molecular weight standards are indicated on the left in
kilodalton units. On the right fragments of the FVa heavy chain are
labeled. HC = intact FVa heavy chain.
|
|
In contrast to the wild-type APC cleavage pattern, a blot of a time
course of FVa cleavage by the mutant 306-314AAAA-APC shows a very
different pattern (Figure 5B). This FVa inactivation reaction was done
with the mutant APC at a 10 times higher level than the wild-type APC
reaction to obtain similar levels of remaining intact FVa. In this
blot, one can see that the band at 45 kd due to Arg306 cleavage
appeared early in the reaction time course and at higher levels than
the 75-kd band due to Arg506 cleavage alone. In Figure 5B the faint
75-kd band disappeared soon afterward and never accumulated to the
level that it did in the reaction with wild-type. This was presumably
due to subsequent, relatively fast cleavage at Arg306 in the 75-kd
fragment comprising residues 1 to 506. However, the 26- to 28-kd
triplet band that was from the fragment 506 to 709 was still present
and levels of the band remained fairly constant over the time course.
This pattern of inactivation was very similar to that seen when protein
S is added to a FVa inactivation assay, which is known to increase the
cleavage rate at Arg306 about 18-fold.13
Thus, it appears that significant amounts of intact FVa heavy chain are
cleaved first at Arg306 by the quadruple mutant APC directly resulting
in production of the 1 to 306 fragment and the 307 to 709 fragment. The
FVa heavy chain that is cleaved first at Arg506 by the 306-314AAAA-APC
is rapidly cleaved further at Arg306 so that little of the 1 to 506 fragment ever accumulates.
| |
Discussion |
Systematic mutational analysis of a protein-protein interaction is
an established method for characterizing the molecular details of that
interaction. However, it is important that the effects of the
substitution made can be limited to the interaction under analysis. The
ideal substitution is a nondisruptive deletion of a side chain that
removes the chemical group involved in a specific interaction but has
no other effects on the structure of the protein.29 The
technique of charged-to-alanine scanning mutagenesis has proven
effective in isolating specific interactions.30 Given the
exposed and flexible nature of the autolysis loop in the 3-dimensional
structure of APC,14,15 we reasoned that mutation of all
charged residues to alanine would be unlikely to have large global
structural effects on protein C. Analysis of the kinetics of cleavage
of 2 different amidolytic tripeptide substrates and analysis of
inactivation by 2 serpin inhibitors showed minor effects of these
mutations in APC on these interactions of various small molecules and
macromolecules with the active site of APC. The effect of the most
severely affected APC mutation (306-314AAAA) on reactivity with
amidolytic tripeptide substrates is a reduction in activity to about
70% to 75% of wild-type. Similarly the effect of the mutation
306-314AAAA on APC reactivity with the serpins PCI and
1-antitrypsin is a reduction in the second-order rate constants of inactivation to 56% to 73% of wild-type. In contrast, the cleavage rate for Arg506 in FVa by the mutant 306-314AAAA-APC is
reduced to 1.4% of the wild-type APC; that is, wild-type APC is
70-fold more active than the tetra-Ala mutant against FVa but only
about 1.7-fold more active with serpins. Hence, the effects on FVa
inactivation of these mutations are mainly the result of specific
interactions of FVa with the autolysis loop rather than the result of
major structural perturbations of the APC active site. In a somewhat
parallel finding, the thrombin derivative -thrombin, which is
cleaved in its homologous autolysis loop, has only slightly reduced
activity toward chromogenic substrates but a dramatic decrease (greater
than 90%) in its ability to clot fibrinogen. 31
If the sites of 2 mutations in a protein that interacts with another
protein are thermodynamically independent of each another, then the
free energy changes observed for the 2 individual mutants should be
additive in a double mutant of those 2 sites.24,32,33 We
analyzed the double mutant 306/312AA-APC and the quadruple mutant
306-314AAAA-APC and found that for both the Arg506 and the Arg306
cleavages the GT values were very
close to being additive of the GT
values for the individual mutants. In both cases though, the GT values for the combined mutants
were slightly less than the added values of the individual mutants.
This is not surprising because all the mutants are adjacent to each
other on the autolysis loop. The result of this additivity of free
energy is that the relative effect of the combined mutations on
apparent second-order cleavage rate constants for Arg506 versus Arg306
is multiplied. For the 4 single-point mutants that had reduced
activity, the changes in free energy for the 2 different cleavage sites
in FVa, Arg506 and Arg306, were not the same. The
GT values for the 2 cleavages were
uniformly smaller for the Arg306 cleavage than for the Arg506 cleavage.
Therefore, k506 is 33-fold greater than
k306 and 54-fold greater than k'306 for wild-type APC but only
2.9-fold and 3.6-fold greater than these 2 rate constants,
respectively, for 306-314AAAA-APC (Table 1). This is essentially an 11- to 15-fold reduction in discrimination between the Arg506 cleavage site
and the Arg306 cleavage site.
This reduction in preference for the Arg506 cleavage relative to the
Arg306 cleavage is closely reflected in the APTT clotting assays
(Figures 3 and 4). Clearly the complexity of the many reactions in
plasma does not allow us to monitor unambiguously FVa inactivation by
APC in a plasma-based clotting assay. However, the close correlation of
results between APTT assays and FVa inactivation assays in purified
mixtures suggests that APTT assays are primarily measuring relative FVa
inactivation rates. In normal human plasma, APC anticoagulant activity
determined by prolongation of the APTT seems to be primarily sensitive
to cleavage at Arg506.12,34 Therefore, the effect of these
mutations on APC prolongation of clotting times in normal human plasma
is probably primarily reflecting a reduction in the cleavage rate at
Arg506. Thus, the effects in APTT assays with normal human plasma can
be compared to the calculated second-order rate constants for cleavage
at Arg506 (k506). The effects in APTT
assays with Q506-FV plasma can be compared to the second-order rate
constants for cleavage at Arg306 in the absence of Arg506 cleavage
(k'306). For example, in the case of
306-314AAAA-APC, the correlations in activity are striking. In the
normal human plasma APTT assay, wild-type APC has 63 times the activity
of the mutant 306-314AAAA-APC, whereas k506
for FVa inactivation is correspondingly 71-fold greater for wild-type
APC than for 306-314AAAA-APC. In the Q506-FV plasma APTT assay,
wild-type APC has only 3.6-fold greater anticoagulant activity than the
mutant 306-314AAAA-APC, whereas k'306
for Q506-FVa inactivation is correspondingly 4.7-fold greater for
wild-type APC than for 306-314AAAA-APC. Thus, when the quadruple mutant
is compared with wild-type APC, the APTT assay implies a 17-fold
reduction in discrimination between Arg506 and Arg306 and the FVa
inactivation assay shows a 15-fold reduction in discrimination between
Arg506 and Arg306. Therefore, the APTT assays for APC activity also
support the conclusion that the autolysis loop contributes to the
discrimination of the 2 cleavage sites on FVa by APC.
Kinetic analyses of the cleavages of normal FVa and Q506-FVa by normal
APC performed by Nicolaes et al11 demonstrated that the
primary difference between cleavage at Arg506 and Arg306 was on the
Km for the interaction of APC with the
respective cleavage sites (wild-type FVa,
Km = 20 nmol/L; Q506-FVa, Km = 196 nmol/L). Therefore, it is likely
that the main effect of mutations in the autolysis loop of APC is on
the Km values for the 2 substrates.
Although our experimentally determined second-order rate constants
should be equivalent to
kcat/Km for the reactions of APC with the 2 cleavage sites, we did not independently determine kcat and
Km so this was not conclusively determined
in our study.
The serine protease domain of APC contains a cluster of positively
charged residues made up of residues in the autolysis loop as well as
the loop from 225 to 235 (Ca++ ion binding loop) and the
loop of residues from 191 to 193. If individual basic residues Arg222
and Arg352 are also included, at least 13 Arg and Lys residues may be
included in this basic exosite on the surface of the APC protease
domain.14,15 Thrombin contains a similar basic exosite,
anion binding exosite I, in this region on its surface. Among the blood
coagulation serine proteases, thrombin is most closely related to
protein C.35 In thrombin this positive exosite is involved
in binding to fibrinogen, hirudin, and thrombomodulin.36 In
APC this basic exosite was proposed to be involved in heparin
binding14 and has also been implicated in FVa binding by
studies involving inhibition of FVa inactivation by a synthetic peptide
with the sequence of residues 311 to 325 of APC,16 which
partially represents the autolysis loop. Our results directly
demonstrate that this basic exosite in APC is important for FVa binding.
An insertion in the autolysis loop of 4 residues is present in human,
monkey, mouse, and rat protein C but is absent in dog, cat, goat,
horse, and bovine protein C. However, in the autolysis loop of the
known protein Cs, the sequence KRNR is absolutely conserved and an Arg
or Lys residue homologous to Arg306 in human protein C is
conserved.37 Thrombin is the only other serine protease
that has an insertion in this autolysis loop, but there is little
sequence homology between thrombin and protein C in this loop. It is
likely that the interactions of the autolysis loop of protein C with
FVa are not with residues directly adjacent to the FVa cleavage sites
on the primed side of the FVa cleavage sites, that is, with residues
507 to 511 or 307 to 311 of FVa. Rather, the APC autolysis loop likely
interacts with surface exosites of FVa that complement the basic
exosite of APC. Fisher and coworkers14 modeled the Arg506
cleavage site loop of FVa into a model of APC and proposed contacts
with APC as distant as the P4' residue of FVa (Arg510) but did
not propose any specific interactions of FVa with the APC autolysis
loop. The similar anion binding exosite I of thrombin binds to regions
of fibrinogen that are distant from the fibrinopeptide cleavage
site.38
It is not necessary that each of the basic residues in the autolysis
loop be involved in a specific salt bridge with a corresponding acidic
residue in FVa. The crystal structure of thrombin in complex with
hirudin showed that, although the C-terminal tail of hirudin contains 5 negatively charged residues, only 2 of them, Asp55 and Glu58, make
specific salt bridge contacts with thrombin.39,40 However,
all 5 of the negatively charged residues in this C-terminal region of
hirudin made similar contributions to binding energy. This can be
explained by the interaction of these charges with the general positive
electrostatic potential of thrombin resulting in stabilization of the
complex and positive effects on the kinetics of complex formation due
to "electrostatic steering."41 In support of this
concept a 3-dimensional model of a complex of FVa and APC bound to the
Arg506 cleavage site shows a significant number of negatively charged
residues on the surface of FVa in close proximity to the positive
exosite of APC (Pellequer et al, in preparation). Therefore, we
speculate that differences in the negative electrostatic surface
potential surrounding the 2 FVa cleavage sites at Arg506 and Arg306 may
result in differing complementary acidic exosites on FVa that are
responsible for the discrimination of the basic exosite on APC toward
the 2 FVa cleavage sites via an electrostatic steering effect. Future
experimentation will further characterize the contributions of other
residues in the basic exosite on APC as well as the complementary
contributions of specific residues in FVa near the 2 main cleavage
sites, Arg506 and Arg306.
| |
Footnotes |
Submitted December 15, 1999; accepted March 13, 2000.
Supported in part by NIH grants R37HL52246 and R01HL21544. A.J.G. is a
fellow of the Leukemia & Lymphoma Society. M.J.H. is partly supported
by a grant from the American Heart Association.
Reprints: John H. Griffin, Department of Molecular and
Experimental Medicine, MEM-180, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037; e-mail:
jgriffin{at}scripps.edu.
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.
| |
References |
1.
Stenflo JA.
A new vitamin K-dependent protein: purification from bovine plasma and preliminary characterization.
J Biol Chem.
1976;251:355-363[Abstract/Free Full Text].
2.
Kisiel W.
Human plasma protein C. Isolation, characterization and mechanism of activation by -thrombin.
J Clin Invest.
1979;64:761-769.
3.
Esmon CT, Owen WG.
Identification of an endothelial cell cofactor for thrombin-catalyzed activation of protein C.
Proc Natl Acad Sci U S A.
1981;78:2249-2252[Abstract/Free Full Text].
4.
Kisiel W, Canfield WM, Ericsson LH, Davie EW.
Anticoagulant properties of bovine plasma protein C following activation by thrombin.
Biochemistry.
1977;16:5824-5831[Medline]
[Order article via Infotrieve].
5.
Walker FJ, Sexton PW, Esmon CT.
The inhibition of blood coagulation by activated protein C through the selective inactivation of activated factor V.
Biochim Biophys Acta.
1979;571:333-342[Medline]
[Order article via Infotrieve].
6.
Marlar RA, Kleiss AJ, Griffin JH.
Mechanism of action of human activated protein C, a thrombin-dependent anticoagulant enzyme.
Blood.
1982;59:1067-1072[Free Full Text].
7.
Griffin JH, Evatt B, Zimmerman TS, Kleiss AJ, Wideman C.
Deficiency of protein C in congenital thrombotic disease.
J Clin Invest.
1981;68:1370-1373.
8.
Branson HE, Katz J, Marble R, Griffin JH.
Inherited protein C deficiency and a coumarin-responsive chronic relapsing purpura fulminans syndrome in a neonate.
Lancet.
1983;2:1165-1168[Medline]
[Order article via Infotrieve].
9.
Seligsohn U, Berger A, Abend M, et al.
Homozygous protein C deficiency manifested by massive venous thrombosis in the newborn.
N Engl J Med.
1984;310:559-562[Abstract].
10.
Kalafatis M, Rand MD, Mann KG.
The mechanism of inactivation of human factor V and human factor Va by activated protein C.
J Biol Chem.
1994;269:31869-31880[Abstract/Free Full Text].
11.
Nicolaes GAF, Tans G, Thomassen MCLGD, et al.
Peptide bond cleavages and loss of functional activity during inactivation of factor Va and factor VaR506Q by activated protein C.
J Biol Chem.
1995;270:21158-21166[Abstract/Free Full Text].
12.
Heeb MJ, Kojima Y, Greengard J, Griffin JH.
Activated protein C resistance: molecular mechanisms based on studies using purified Gln506-factor V.
Blood.
1995;85:3405-3411[Abstract/Free Full Text].
13.
Rosing J, Hoekema L, Nicolaes GAF, et al.
Effects of protein S and factor Xa on peptide bond cleavages during inactivation of factor Va and factor VaR506Q by activated protein C.
J Biol Chem.
1995;270:27852-27858[Abstract/Free Full Text].
14.
Fisher CL, Greengard JS, Griffin JH.
Models of the serine protease domain of the human antithrombotic plasma factor activated protein C and its zymogen.
Protein Sci.
1994;3:588-599[Medline]
[Order article via Infotrieve].
15.
Mather T, Oganessyan V, Hof P, et al.
The 2.8 Å crystal structure of Gla-domainless activated protein C.
EMBO J.
1996;15:6822-6831[Medline]
[Order article via Infotrieve].
16.
Mesters RM, Heeb MJ, Griffin JH.
Interactions and inhibition of blood coagulation factor Va involving residues 311-325 of activated protein C.
Protein Sci.
1993;2:1482-1489[Medline]
[Order article via Infotrieve].
17.
Heeb MJ, Mesters RM, Tans G, Rosing J, Griffin JH.
Binding of protein S to factor Va associated with inhibition of prothrombinase that is independent of activated protein C.
J Biol Chem.
1993;268:2872-2877[Abstract/Free Full Text].
18.
Mesters RM, Houghten RA, Griffin JH.
Identification of a sequence of human activated protein C (residues 390-404) essential for its anticoagulant activity.
J Biol Chem.
1991;266:24514-24519[Abstract/Free Full Text].
19.
Zhang L, Castellino FJ.
A gamma-carboxyglutamic acid variant (gamma6D, gamma7D) of human activated protein C displays greatly reduced activity as an anticoagulant.
Biochemistry
1990;29:10828-10834[Medline]
[Order article via Infotrieve].
20.
Gale AJ, Sun X, Heeb MJ, Griffin JH.
Nonenzymatic anticoagulant activity of the mutant serine protease Ser360Ala-activated protein C mediated by factor Va.
Protein Sci.
1997;6:132-140[Medline]
[Order article via Infotrieve].
21.
Yan SCB, Pazzano P, Chao YB, et al.
Characterization and novel purification of recombinant human protein C from three mammalian cell lines.
Biotechnology.
1990;8:655-661[Medline]
[Order article via Infotrieve].
22.
Gruber A, Harker LA, Hanson SR, Kelly AB, Griffin JH.
Antithrombotic effects of combining activated protein C and urokinase in non-human primates.
Circulation.
1991;84:2454-2462[Abstract/Free Full Text].
23.
Chase T, Shaw E.
p-Nitrophenyl-p'-guanidinobenzoate HCl: a new active site titrant for trypsin.
Biochem Biophys Res Commun.
1967;29:508-514[Medline]
[Order article via Infotrieve].
24.
Wells JA.
Additivity of mutational effects in proteins.
Biochemistry.
1990;29:8509-8517[Medline]
[Order article via Infotrieve].
25.
Heeb MJ, Bischoff R, Courtney M, Griffin JH.
Inhibition of activated protein C by recominbinant 1antitrypsin variants with substitution of arginine or leucine for methionine 358.
J Biol Chem.
1990;265:2365-2369[Abstract/Free Full Text].
26.
Heeb MJ, Rehemtulla A, Moussalli M, Kojima Y, Kaufman RJ.
Importance of individual activated protein C cleavage site regions in coagulation factor V for factor Va inactivation and for factor Xa activation.
Eur J Biochem.
1999;260:64-75[Medline]
[Order article via Infotrieve].
27.
Kalafatis M, Bertina RM, Rand MD, Mann KG.
Characterization of the molecular defect in factor VR506Q.
J Biol Chem.
1995;270:4053-4057[Abstract/Free Full Text].
28.
Kalafatis M, Mann KG.
Role of the membrane in the inactivation of factor Va by activated protein C.
J Biol Chem.
1993;268:27246-27257[Abstract/Free Full Text].
29.
Fersht AR, Leatherbarrow RJ, Wells TN.
Structure-activity relationships in engineered proteins: analysis of use of binding energy by linear free energy relationships.
Biochemistry.
1987;26:6030-6038[Medline]
[Order article via Infotrieve].
30.
Wells JA.
Systematic mutational analyses of protein-protein interfaces.
Methods Enzymol.
1991;202:390-411[Medline]
[Order article via Infotrieve].
31.
Elion J, Boissel JP, Le Bonniec B, et al.
Proteolytic derivatives of thrombin.
Ann N Y Acad Sci.
1986;485:16-26[Medline]
[Order article via Infotrieve].
32.
Carter PJ, Winter G, Wilkinson AJ, Fersht AR.
The use of double mutants to detect structural changes in the active site of the tyrosyl-tRNA synthetase (Bacillus stearothermophilus).
Cell.
1984;38:835-840[Medline]
[Order article via Infotrieve].
33.
Di Cera E.
Site-specific analysis of mutational effects in proteins.
Adv Protein Chem.
1998;51:59-119[Medline]
[Order article via Infotrieve].
34.
Kalafatis M, Mann KG.
Factor VLeiden and thrombophilia.
Arterioscler Thromb Vasc Biol.
1997;17:620-627[Free Full Text].
35.
Patthy L.
Evolution of the proteases of blood coagulation and fibrinolysis by assembly from modules.
Cell.
1985;41:657-663[Medline]
[Order article via Infotrieve].
36.
Bode W, Turk D, Karshikov A.
The refined 1.9-A X-ray crystal structure of D-Phe-Pro-Arg chloromethylketone-inhibited human alpha-thrombin: structure analysis, overall structure, electrostatic properties, detailed active-site geometry, and structure-function relationships.
Protein Sci.
1992;1:426-471[Medline]
[Order article via Infotrieve].
37.
Murakawa M, Okamura T, Kamura T, Kuroiwa M, Harada M, Niho Y.
A comparative study of partial primary structures of the catalytic region of mammalian protein C.
Br J Haematol.
1994;86:590-600[Medline]
[Order article via Infotrieve].
38.
Stubbs MT, Bode W.
A player of many parts: the spotlight falls on thrombin's structure.
Thromb Res.
1993;69:1-58[Medline]
[Order article via Infotrieve].
39.
Grutter MG, Priestle JP, Rahuel J, et al.
Crystal structure of the thrombin-hirudin complex: a novel mode of serine protease inhibition.
EMBO J.
1990;9:2361-2365[Medline]
[Order article via Infotrieve].
40.
Rydel TJ, Ravichandran KG, Tulinsky A, et al.
The structure of a complex of recombinant hirudin and human alpha- thrombin.
Science.
1990;249:277-280[Abstract/Free Full Text].
41.
Karshikov A, Bode W, Tulinsky A, Stone SR.
Electrostatic interactions in the association of proteins: an analysis of the thrombin-hirudin complex.
Protein Sci.
1992;1:727-735[Medline]
[Order article via Infotrieve].
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Top
Abstract
Introduction
