From the Department of Clinical Chemistry, Lund
University, The Wallenberg Laboratory, MAS, SE-205 02 Malmö,
Sweden and § INSERM U428, University of Paris V, 4 Avenue de
L'Observatoire, F-750 06 Paris, France
Received for publication, April 9, 2001
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ABSTRACT |
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Proteolytic inactivation of activated factor V
(FVa) by activated protein C (APC) is a key reaction in the regulation
of hemostasis. We now demonstrate the importance of a positive cluster
in loop 37 of the serine protease (SP) domain of APC for the
degradation of FVa. Lysine residues in APC at positions 37, 38, and 39 form a secondary binding site for FVa, which is important for
cleavage of FVa at Arg-506 while having no effect on Arg-306 cleavage. In contrast, topological neighbors Lys-62, Lys-63, and Arg-74 in APC
appear of minor importance in FVa degradation. This demonstrates that secondary binding exosites of APC specifically guide the proteolytic action of APC, resulting in a more favorable degradation of
the 506-507 peptide bond as compared with the 306-307 bond.
The protein C pathway provides a major anticoagulant mechanism,
which is important for regulation of hemostasis (1-3). The key
component in this pathway is the vitamin K-dependent
protein C, which circulates in plasma as an inactive zymogen to a
serine protease (4). Protein C is composed of a light chain (containing a Computer analysis of the x-ray structure of Gla-domainless APC (9),
theoretical screening of its molecular surface (10) and some
experimental data (11-13) have indicated the importance of an
electropositive region within the SP domain of APC for protein-protein interaction. Thus, a cluster of basic residues located on loops 37, 60, and 70 was shown to be involved in direct interactions with
thrombomodulin during the activation process of protein C and with
heparin, the protein C inhibitor (PCI) and In the present study, we have examined the role of positively charged
amino acids in loops 37, 60, and 70 of APC in the degradation of FVa.
The basic residues in loop 37 were found to form a substrate-binding exosite for FVa that is important during cleavage of Arg-506, whereas
they have virtually no effect on the FVa Arg-306 cleavage rate.
Construction, Expression, and Purification of Recombinant Human
Protein C Variants--
Recombinant protein C variants were created by
polymerase chain reaction-based site-directed mutagenesis, expressed in
HU293 cells (ATCC CRL-1573), purified and activated following
procedures previously described (13, 15). Similar expression levels
(2.7-6.7 mg/liter) and amidolytic activities (Km
and kcat) of wt and APC variants demonstrated
the functional integrity of the proteins (13).
Prothrombinase Assay--
Time courses of FVa inactivation
by APC were determined by following the loss of FVa cofactor activity
as a function of time (16). Therefore, 0.8 nM
plasma-purified human FVa was incubated with 0.17-0.8 nM
wt or APC variants in the absence or presence of 500 nM
protein S and 25 µM phospholipid vesicles (10/90
dioleoylphosphatidylserine (DOPS)/dioleoylphosphatidylcholine (DOPC)
M/M). The remaining cofactor activity of FVa was determined by
quantification of the rate of FXa-catalyzed prothrombin activation, as
described previously (16).
Kinetic Data Analysis--
Km and
Vmax values were obtained from Lineweaver-Burk
plots and kcat was calculated from the
Michaelis-Menten equation (17, 18). Rate constants for APC-catalyzed
Arg-506 and Arg-306 cleavage were calculated using a method described
previously (16). Briefly, data from time courses of FVa inactivation
were fitted to an equation describing the APC-catalyzed inactivation of
FVa using non-linear least squares regression analysis (16). This equation describes a model for inactivation where FVa is initially randomly cleaved at either Arg-306 or Arg-506. Cleavage at Arg-306 leads to a complete loss of FVa cofactor activity, whereas cleavage at
Arg-506 results in the formation of a reaction intermediate having
~40% cofactor activity compared with the uncleaved cofactor. Further
cleavage of this intermediate at Arg-306 is necessary to abolish
completely FVa cofactor activity.
Western Blot Analysis of FVa Inactivation by APC--
To
identify fragment generation during the APC-catalyzed cleavage of FVa
plasma-derived human FVa was incubated with wt APC or the APC variants
in the presence of phospholipid vesicles. At various time points,
aliquots were removed, subjected to a 10% SDS-polyacrylamide gel
electrophoresis under reducing conditions and analyzed by Western
blotting (15) using a polyclonal antibody directed against a peptide
located carboxyl-terminal to the Arg-306 cleavage site in the heavy
chain of human FVa.
Insights into Structure-Function Relationships Involved in the
Cleavage of FVa by APC--
In a recently created model of the three
A-domains of FV, we observed that several positively charged residues
are located around Arg-506 and Arg-306 (19). These positive charges
could guide and orient FVa into the relatively electronegative active site of APC during the initial approach while repulsing the positively charged loops of the enzyme. We also noted that Arg-506 is located on a
well defined loop-like structure, not protruding significantly outside
the molecular surface, which could insert into the catalytic cleft of
APC after some structural changes. In addition, we predicted the
Arg-306 segment to adopt either an Anticoagulant Response of APC Variants in Normal Human
Plasma--
The effect of different APC variants on the APTT of normal
human plasma was tested (Fig. 1). Because
of the anticoagulant property of wt APC, a
concentration-dependent prolongation of the clotting time
was detected. The two APC variants K37S/K38Q/K39Q and
K37S/K38Q/K39Q/K62N/K63D showed reduced anticoagulant response, whereas
APC variants R74Q and K62N/K63D expressed only slight reduction of
anticoagulant response toward normal human plasma compared with that of
wt APC. These results suggested the cluster of three lysines in loop 37 to be important for the interaction between APC and FV/FVa and/or
FVIIIa.
Loop 37 Variants and the Selective Influence on the Arg-506
Cleavage in FVa--
To elucidate the effect of the APC variants on
the degradation of FVa, a system with purified components was used. In
this experiment, FVa was incubated with APC in the presence of
negatively charged phospholipid vesicles and the remaining FVa activity
determined in a PTase assay. A typical inactivation time of FVa by wt
APC appears biphasic (Fig. 2), in
agreement with the results on record (5). Under these conditions, this
biphasic shape is due to the APC-catalyzed rapid cleavage at Arg-506,
yielding a partially active intermediate that is subsequently fully
inactivated by the ~20-fold slower cleavage at Arg-306. The FVa
inhibition curves obtained with the two APC variants K37S/K38Q/K39Q and
K37S/K38Q/K39Q/K62N/K63D were distinctly different from that obtained
with wt APC (Fig. 2). Both APC variants were considerably less
efficient in inhibiting FVa than wt APC, and the generated curves were
monophasic. In contrast, curves obtained with K62N/K63D and R74Q
resembled those obtained with the wt APC (data not shown). Using the
experimental results, apparent second order rate constants of 4.97 × 107 M Fragment Generation during APC-catalyzed Cleavage of FVa--
To
further analyze the effects on the cleavage of the different FVa
cleavage sites, the fragmentation pattern generated during APC cleavage
was analyzed by Western blotting (Fig.
3). During degradation of FVa by wt APC,
Arg-506 is cleaved much faster than Arg-306. As a consequence, a 75-kDa
fragment (residues 1-506) is transiently observed, whereas the 60-kDa
fragment comprising residues 307-709 is not observed with Western
blotting using an antibody raised against a peptide located
COOH-terminal of the 306 cleavage site (Fig. 3A). In
contrast, the 30-kDa fragment comprising residues 307-506 is readily
observed on the blots already early during the reaction. It should be
noticed that for practical reason, the FVa concentration used in this
experiment is higher than of the experiment illustrated in Fig. 2,
which explains the faster cleavage at 306 (16). The APC variant
K37S/K38Q/K39Q/K62N/K63D yielded a different degradation pattern (Fig.
3B). In this case, a transient 60-kDa fragment was observed
and in addition, the 30-kDa fragment was generated more slowly than in
the case of wt APC. The obtained pattern is compatible with a reduction
in the rate of cleavage at Arg-506 and with an unaffected cleavage at
Arg-306, thus supporting the conclusions from the functional analysis
of Fig. 2.
FVa Degradation Catalyzed by APC in the Presence of Protein
S--
The activity of APC toward FVa is stimulated by the
non-enzymatic cofactor, protein S. The exact mechanism of this
stimulation is still not defined. It is known that protein S
specifically accelerates the APC-induced FVa cleavage at position
Arg-306 (20-fold) and relocates the active site of APC closer to the
membrane surface (21-23). Protein S interacts directly with residues
493-506 in FVa (24) and therefore could stimulate the APC-induced FVa
Arg-306 cleavage and/or protect the FVa Arg-506 region from APC
degradation. To evaluate the effect of protein S on the interaction of
the APC variants with FVa, time courses for APC-catalyzed FVa
inactivation were performed in the presence of protein S. The
inactivation rates for Arg-506 were unaffected by the presence of
protein S, whereas with respect to the Arg-306 cleavage, all variants
were stimulated to the same degree by protein S (data not shown). This demonstrates that these mutations in the SP domain of APC have no
influence on the cofactor activity of protein S in the APC-catalyzed cleavage of Arg-306 in FVa.
In conclusion, our results indicate that positively charged amino acids
Lys-37, Lys-38, and Lys-39, located on loop 37 of APC, are
indispensable for the APC-mediated cleavage at Arg-506 in FVa but not
for the cleavage at position Arg-306. In contrast, amino acids Lys-62,
Lys-63 in loop 60, and/or amino acid Arg-74 in the so-called
calcium-binding loop (loop 70) play no or only a minor role in the
Arg-506 and Arg-306 cleavages. Because loops 37 and 60 form one side of
the wall lining the catalytic cleft, we can conclude that only loop 37 constitutes a secondary binding exosite for FVa critical for
appropriate orientation of APC toward FVa and that this applies only
for the Arg-506 site. At a structural level, loop 37 could indeed
interact with FVa residues Asp-577 and Asp-578 as suggested by
Pellequer (20) or with other surrounding negatively charged residues,
yet to be defined experimentally. Interestingly, the autolysis loop of
APC (loop 148) is suggested to play a similar role in the degradation
of FVa as the now reported loop 37, discriminating between the 306 and
506 cleavages (8). This suggests that the 506 segment is in sandwich
between loop 37 and loop 148 of APC and that these two regions, but not
residues 62, 63, and 74 of APC contact FVa directly. In contrast, all
these residues play minor roles for the cleavage at Arg-306. These
results also support preliminary docking experiments of the Arg-306
site into APC, in which it is observed that the FV Arg-306 loop segment protrudes far outside the molecular surface of FVa as compared with the
506 loop. As such, when Arg-306 enters the S1 pocket of APC, at least
the loops 37, 60, 70, and 148 of APC make no clear contact with FVa.
The absence of favorable electrostatic guidance for cleavage at Arg-306
could partly explain the much slower inactivation rate at this site
when compared with cleavage at Arg-506. The results now presented
provide important information about the orientation of FVa in the
active site of APC. Further definition of the interface will require
the production of several FV variants able to complement the mutations
in APC. A very accurate three-dimensional model of this important
macromolecular complex confirmed by experimental data should soon
emerge from such investigation and shed new light onto key
antithrombotic and anti-inflammatory reactions in the human body.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-carboxyglutamatic acid
(Gla)1 domain and two
epidermal growth factor (EGF)-like domains), which is linked by a
disulfide bridge to the heavy chain that comprises the activation
peptide and the serine protease (SP) domain. After activation by the
thrombin-thrombomodulin complex on endothelial cell surfaces, activated
protein C (APC) regulates blood coagulation by cleaving and inhibiting
the activated forms of factor V (FVa) and factor VIII (FVIIIa). FVa and
FVIIIa are membrane-bound cofactors to factor Xa (FXa) and factor IXa
(FIXa) in the prothrombinase (PTase) and tenase complex, respectively.
FV/FVa and FVIIIa are the only known physiological substrates for APC.
Until now, the structural background for this narrow substrate
specificity is not clearly understood (5). Peptide inhibition studies
showed the involvement of the region 149-154 (chymotrypsinogen
numbering)2 within the SP
domain of APC in FVa binding (6). Furthermore, recently published loop
grafting experiments and site-directed mutagenesis data confirm that
residues in loop 148 play an important role in the kinetic properties
of APC and as such could contact directly FVa during the APC-catalyzed
inactivation (7, 8).
1-antitrypsin (7, 13,
14).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-helical conformation or a long,
potentially not well structured loop, which protrudes relatively far
outside the molecular surface. Whereas the orientation of the Arg-506
and Arg-306 regions in the active site of APC are not easily
envisioned, the Arg-306 site is certainly the most difficult to
predict. A preliminary model of the FVa-APC complex has recently been
reported (20) thereby helping to analyze this interaction. Despite this
progress, the exact definition of residues in contact is still not
clear because several parameters such as the relative orientation of
the proteins with regard to the membrane plane, the organization of the
FV domains and the conformation of the FVa loops 306 and 506 are
ill-defined. As a consequence, we observe different sets of atomic
interactions between FVa and APC depending on which structure of loop
506 or 306 is used.3 For
instance, docking Arg-506 into the active site of APC suggests several
possible contact regions between the enzyme and the substrate around
APC loop 37. In contrast, the same experiments performed for the
Arg-306 site do not clearly highlight important contacts between FVa
and the loops lining the catalytic cleft of APC. In this study, we have
performed site-directed mutagenesis of APC to define which residues in
the loop 37 area of the enzyme interact with FVa. This information will
help determine and refine the orientation of the Arg-506 and Arg-306
sites in the catalytic cleft of APC. Such data are indeed essential for
both functional characterization of this important macromolecular
complex and structural understanding of the enzymatic reactions. To
define rationally the location and type of substitution to introduce in
protein C, we analyzed the structures of several serine proteases, including PC sequences from different species (data not shown). We
decided to produce five PC variants with amino acid exchanges at
positions 37, 38, 39, 60a, 61, 62, 63, and 74 to probe the region of interest.
View larger version (17K):
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Fig. 1.
Anticoagulant effects of wt APC and APC
variants on normal human plasma. The activated partial
thromboplastin time (APTT) of normal human plasma was
determined in the presence of wt and APC variants. The proteins were
incubated with 50 µl of APTT reagent containing purified
phospholipids with colloidal silica as contact activator (Chromogenix)
for 200 s. The clotting reaction was initiated by addition of 50 µl of 25 mM calcium chloride, and clot formation was
measured in a KC 10 coagulometer (Amelung AG Lemgo). Wt APC ( ),
K62N/K63D (
), K37S/K38Q/K39Q (
),
K37S/K38Q/K39Q/K62N/K63D (
), R74Q (
). Each data point
represents the mean value of six independently performed experiments.
Error bars represent mean ± S.D.
1 s
1 and
1.18 × 106 M
1
s
1 were calculated for the wt-APC-catalyzed Arg-506 and
Arg-306 cleavages, respectively (Table
I). Similar rates for both cleavage sites
were determined for the variants K62N/K63D and R74Q. In contrast, both
variants K37S/K38Q/K39Q and K37S/K38Q/K39Q/K62N/K63D showed
significantly reduced rate constants for Arg-506 cleavage (3.41 × 106 and 4.03 × 106, respectively) while
Arg-306 cleavage rates were similar to the rates obtained for the wt
APC (Table I). These results suggest a 12- to 15-fold specific
reduction in the rate of Arg-506 cleavage as a result of the loop 37 mutagenesis while the Arg-306 cleavage rate was unaffected by these
mutations.
View larger version (15K):
[in a new window]
Fig. 2.
Illustration of the effect of APC variants on
human FVa inactivation. To characterize the APC variants by the
APC-catalyzed FVa inactivation, time courses for the inactivation of
plasma-derived human FVa by 0.8 nM wt or variant APCs were
performed, and rate constants for Arg-506 and Arg-306 cleavage were
calculated as described previously (16). Because of the different
velocities of FVa Arg-506 and Arg-306 cleavage, wt APC ( ) induces a
biphasic FVa inactivation time course. The curves of variant
K37S/K38Q/K39Q (
) and variant K37S/K38Q/K39Q/K62N/K63D (
) are
more monophasic in shape. The figure represents one of three
independent experiments. The calculated kinetic constants are given in
Table I.
Apparent second order rate constants for APC-mediated cleavage of
Arg-306 and Arg-506 in FVa
View larger version (35K):
[in a new window]
Fig. 3.
Western blot analysis of FVa inactivation
catalyzed by wt and APC variants. To identify fragments generated
during APC-catalyzed cleavage of FVa, plasma-derived human FVa was
incubated with 1 nM wt APC (A) or variant
K37S/K38Q/K39Q/K62N/K63D (B), 25 µM of a
phospholipid mixture (DOPS/DOPC in a ratio of 10:90 mol/mol) at
37 °C in 25 mM Hepes, 150 mM NaCl, 5 mM CaCl2, and 5 mg/ml bovine serum albumin.
Aliquots were removed after 0, 1, 2.5, 5, 10, 15, 30, 45, and 60 min
and subjected to SDS-polyacrylamide gel electrophoresis. The proteins
were transferred to a polyvinylidene difluoride membrane, and fragments
were detected with a polyclonal antibody raised against a peptide
located carboxyl-terminal to the Arg-306 cleavage site in the heavy
chain of human FVa. An alkaline-phosphatase-conjugated swine
anti-rabbit antibody (No. 306 DAKO) was used as secondary
antibody.
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FOOTNOTES |
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* This work was supported by grants from the Swedish Medical Research Council, a Senior Investigator's grant from the Foundation for Strategic Research, and a grant from La Fondation pour La Recherche Médicale.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 46 40 33 15 01; Fax: 46 40 33 70 44; E-mail: bjorn.dahlback@klkemi.mas.lu.se.
Present address: Cardiovascular Research Institute Maastricht,
Dept. of Biochemistry, Maastricht University, P.O. Box 616, 6200 MD
Maastricht, The Netherlands.
Published, JBC Papers in Press, April 17, 2001, DOI 10.1074/jbc.M103138200
2 The numbering of amino acid positions in protein C corresponds to the chymotrypsinogen nomenclature.
3 B. Villoutreix, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
Gla, -carboxyglutamatic acid;
APC, activated protein C;
wt, wild type;
FV, factor V;
FVa, activated factor V;
FVIII, factor VIII;
PCI, protein
C inhibitor;
SP, serine protease;
a, activated;
PTase, prothrombinase;
DOPS, dioleoylphosphatidylserine;
DOPC, dioleoylphospatidylcholine;
APTT, activated partial thromboplastin time.
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