Tài liệu Báo cáo khoa học: Altered inactivation pathway of factor Va by activated protein C in the presence of heparin doc

Altered inactivation pathway of factor Va by activated protein C
in the presence of heparin
Gerry A. F. Nicolaes
1
, Kristoffer W. Sørensen
1,2
, Ute Friedrich
2,3
, Guido Tans
1
, Jan Rosing
1
, Ludovic Autin
4
,
Bjo¨ rn Dahlba¨ck
2
and Bruno O. Villoutreix
4
1
Department of Biochemistry, Cardiovascular Research Institute Maastricht, the Netherlands;
2
Department of Clinical Chemistry,
University Hospital, Malmo
¨
, Sweden;
3
Immunochemistry Department, Novo Nordisk A/S, Gentofte, Denmark;
4
INSERM U428,
University of Paris V, France

Inactivation of factor Va (FVa) by activated protein C
(APC) is a predominant mechanism in the down-regulation
of thrombin generation. In normal FVa, APC-mediated
inactivation occurs after cleavage at Arg306 (with corres-
ponding rate constant k¢
306
) or after cleavage at Arg506
(k
506
) and subsequent cleavage at Arg306 (k
306
). We have
studied the influence of heparin on APC-catalyzed FVa
inactivation by kinetic analysis of the time courses of inac-
tivation. Peptide bond cleavage was identified by Western
blotting using FV-specific antibodies. In normal FVa, un-
fractionated heparin (UFH) was found to inhibit cleavage at
Arg506 in a dose-dependent manner. Maximal inhibition of
k
506
by UFH was 12-fold, with the secondary cleavage at
Arg306 (k
306
) being virtually unaffected. In contrast, UFH
stimulated the initial cleavage at Arg306 (k¢
306
)two-to
threefold. Low molecular weight heparin (FragminÒ)had
the same effects on the rate constants of FVa inactivation as
UFH, but pentasaccharide did not inhibit FVa inactivation.

Analysis of these data in the context of the 3D structures of
APC and FVa and of simulated APC–heparin and FVa–
APC complexes suggests that the heparin-binding loops 37
and 70 in APC complement electronegative areas sur-
rounding the Arg506 site, with additional contributions
from APC loop 148. Fewer contacts are observed between
APC and the region around the Arg306 site in FVa. The
modeling and experimental data suggest that heparin, when
bound to APC, prevents optimal docking of APC at Arg506
and promotes association between FVa and APC at position
Arg306.
Keywords: coagulation; factor V; heparin; protein C; protein
docking.
Activated factor V (FVa) is an essential cofactor in the
prothrombin-activating complex, stimulating the activity
of membrane-bound factor Xa (FXa) more than
100 000-fold [1,2]. Hence, FVa is an ideal target for
the regulation of thrombin formation [3]. Downregula-
tion of FVa activity is achieved through proteolysis
mainly mediated by the anticoagulant protein C pathway
(reviewed in [4,5]). Protein C is composed of a heavy and
a light chain held together by a single disulfide bond [6].
The light chain contains the c-carboxyglutamic acid
(Gla)-rich domain and two epidermal growth factor-like
domains [7]. The heavy chain comprises a short activa-
tion peptide and a serine protease (SP) domain which
contains the active site of the enzyme. Activated protein
C (APC), the product of a thrombin–thrombomodulin-
catalyzed activation of the zymogen protein C, proteo-
lytically inactivates the coagulation cofactors, FVa and

FVIIIa [8], in reactions stimulated by the APC cofactor
protein S.
FVa consists of a 105 kDa heavy (A1 and A2 domains)
and a 71–74-kDa light (A3, C1, and C2 domains) chain
which are noncovalently associated.
During APC-catalyzed inactivation of FVa, the heavy
chain of FVa is cleaved at three sites: Arg306, Arg506 and
Arg679 [9]. The cleavages at Arg306 and Arg506 appear to
be crucial for inactivation, but the cleavage at Arg679 is
probably less important [10]. The cleavage at Arg506 is
kinetically favored over that at Arg306 and results in the
formation of an inactivation intermediate (FVa
int
), which
retains partial FVa cofactor activity owing to its ability to
bind FXa, albeit with lower affinity [10]. The FVa activity is
lost after cleavage at Arg306. In carriers of the common
FV
Leiden
mutation, in whom the Arg506 has been replaced
by a Gln, inactivation occurs via the slow Arg306 cleavage
(reviewed in [11]). Cleavage at Arg306 results in a large
reduction in FXa affinity and also the dissociation of
the A2 domain, the two processes ultimately rendering
FVa inactive as a cofactor of FXa [12,13]. APC-
catalyzed inactivation of FVa is modulated by other plasma
components. Thus, the nonenzymatic cofactor protein S
Correspondence to G. A. F. Nicolaes, Department of Biochemistry,
Cardiovascular Research Institute Maastricht, Maastricht,
the Netherlands. Fax: + 31 43 3884159, Tel.: + 31 43 3881539,

E-mail:
Abbreviations: APC, activated protein C; DEGR-FXa, 1,5-DNS-
GGACK-factor Xa; DOPS, 1,2 dioleoyl-sn-glycero-3-phosphoserine;
DOPC, 1,2 dioleoyl-sn-glycero-3-phosphocholine; FV, coagulation
factor V; FVa, activated FV; FVa
2
, the FVa isoform lacking glyco-
sylation at Asn2181; FVIII, factor VIII; Gla, c-carboxyglutamic acid;
SP, serine protease; UFH, unfractionated heparin.
Note: Numbering of amino-acid positions in protein C corresponds to
the chymotrypsinogen nomenclature.
(Received 29 January 2004, revised 30 March 2004,
accepted 4 May 2004)
Eur. J. Biochem. 271, 2724–2736 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04201.x
promotes cleavage at Arg306, whereas FXa specifically
blocks the cleavage at Arg506 [14].
It has recently been shown that basic residues in two
surface loops (37 and 70) in the SP domain of APC
(chymotrypsinogen nomenclature) form an extended bind-
ing site for FVa [15–17]. In addition, APC loop 148 also
plays a role in FVa degradation [18,19]. Loop 60 is probably
less important as mutagenesis of positive residues in this
loop did not affect inactivation of FVa by APC [15,16].
Heparin is an important regulator of APC activity,
promoting the interaction between APC and one of its
inhibitors, the serpin protein C inhibitor (PCI) [17]. This is
probably mediated via a template mechanism, for which
binding of heparin to basic residues in three of the four
surface loops in APC (60, 37 and 70) is crucial [17,20,21].
Interestingly, heparin has also been reported to stimulate

APC-catalyzed inactivation of intact FV,but not FVa [22,23].
We performed a detailed kinetic analysis of the influence
of heparin on the inactivation of FVa by APC and found a
specific heparin-mediated inhibition of the cleavage at
Arg506, whereas cleavage at Arg306 was mildly stimulated.
Structural analysis strongly suggested that the heparin-
binding loops 37, 60 and 70 in APC could complement
electronegative areas surrounding the Arg506 site of FVa
indicating that electrostatic interactions between regions of
FVa and APC could be critical for the formation of the
APC–FVa complex which is involved in the cleavage at
position Arg506. These electrostatic interactions are inhib-
ited by heparin when heparin is bound to the electropositive
cluster on loops 37, 60 and 70 located at one edge of the SP
domain of APC. Our data further suggest that heparin can
potentially bridge APC to exosites around Arg306, thereby
facilitating cleavage at position Arg306.
Materials and Methods
Proteins and reagents
Human FV and FV
Leiden
were purified from the plasma
of a normal individual and an individual homozygous for
the FV Arg506Gln mutation, and FVa
2
was prepared as
described [24]. Throughout the work presented here FVa
2
was used. Factor Xa, a-thrombin, protein S, human
activated protein C and prothrombin were purchased

from Kordia Laboratory Supplies (Leiden, the Nether-
lands). All coagulation factors were of human origin
unless otherwise stated. 1,5-DNS-GGACK-factor Xa
(DEGR-FXa) was prepared as described previously [14].
The monoclonal antibody AHV 5146 was purchased from
Haematologic Technologies (Essex Junction, VT, USA).
Unfractionated heparin (UFH) and low molecular weight
heparin (FragminÒ) were obtained from Leo (Ballerup,
Denmark); 1 IUÆmL
)1
UFH contains % 5.7 lg UF-
HÆmL
)1
[23]. Pentasaccharide was from Sanofi-Re
´
cherche
(Montpellier, France). Phospholipid vesicles [10% 1,2
dioleoyl-sn-glycero-3-phosphoserine (DOPS), 90% 1,2
dioleoyl-sn-glycero-3-phosphocholine (DOPC), mol/mol]
were prepared as described [25]. The chromogenic sub-
strates S-2366 and S-2238 were obtained from Chromo-
genix (Milano, Italy), and biotrace
TM
poly(vinylidene
difluoride) transfer membranes from Pall Gelman Labor-
atory (Ann Arbor, MI, USA).
Expression and purification of recombinant human
protein C
Recombinant protein C variants K37S/K38Q/K39Q
(37-loop mutant), K62N/K63D (60-loop mutant),

K37S/K38Q/K39Q/K62N/K63D (37+60-loop mutant)
were created by PCR-based site-directed mutagenesis of
the eukaryotic expression vector pGT-hyg (Eli Lilly),
expressed in 293 cells (CRL-1573; ATCC), purified, and
characterized as described previously [21,26].
Assay of FVa
FVa activity was determined by quantification of the rate of
FXa-catalyzed prothrombin activation, as described previ-
ously [10]. Briefly, in a reaction mixture that contained
0.5 l
M
prothrombin, a limiting amount of FVa (83 p
M
FVa), 5 n
M
FXa, 40 l
M
phospholipids (10 : 90 DOPS/
DOPC, mol/mol), 0.5 mgÆmL
)1
ovalbumin, and 2 m
M
CaCl
2
, prothrombin activation was allowed for 1 min at
37 °C. The amount of prothrombin activated was then
determined using S-2238 [2].
APC-catalyzed inactivation of FVa
Time courses of FVa inactivation by APC were determined
by following the loss of FXa cofactor activity of FVa in the

prothrombinase complex as a function of time. Routinely,
0.8 n
M
plasma-derived human FVa or FVa
Leiden
was prein-
cubatedwith25l
M
phospholipid vesicles (10 : 90 DOPS/
DOPC, mol/mol) in the absence or presence of protein S
(200 n
M
) and/or heparin (0.01–25 IUÆmL
)1
)in25m
M
Hepes buffer (pH 7.5), containing 150 m
M
NaCl, 3 m
M
CaCl
2
,and5 mgÆmL
)1
BSA, for 5 min at 37 °C. Inactivation
was started by adding wild-type APC or APC variants, and
the progressive loss of FVa was monitored for up to 20 min
by transfer of aliquots to the FVa assay described above.
Analysis of kinetic data
Rate constants for APC-catalyzed Arg506 and Arg306

cleavage were obtained as described previously by fitting
the time courses of FVa inactivation to a random-order,
two-cleavage model using nonlinear least-squares analysis
[10]. In this model, FVa can be randomly cleaved at
either Arg306 or Arg506. Cleavage at Arg306, with an
apparent second-order rate constant of k¢
306
,resultsin
complete loss of FVa cofactor activity (FVa
i
; pathway 1,
Eqn 1). Alternatively, initial cleavage at Arg506, with a
corresponding rate constant of k
506
, results in a reaction
intermediate (FVa
int
)with% 40% residual cofactor activ-
ity, which must be further cleaved at Arg306 (k
306
)in
order to completely abolish FVa cofactor activity (path-
way 2, Eqn 2).
Factor Va
k
0
306
À!
Factor Va
i

ð1Þ
Factor Va
k
506
À!
Factor Va
int
k
306
À!
Factor Va
i
ð2Þ
In wild-type FVa, in which cleavage at Arg506 is % 20-fold
faster than cleavage at Arg306, the major part (% 95%) of
Ó FEBS 2004 Heparin and APC-catalyzed inactivation of FVa (Eur. J. Biochem. 271) 2725
FVa is inactivated via pathway 2. To reliably determine
k¢
306
, single exponential inactivation time courses were
determined for FVa
Leiden
, representing cleavage at Arg306
only and the k¢
306
values obtained were used in the fits for
normal FVa. We have verified our previous findings [10]
that the APC-catalysed FVa inactivation time courses were
second-order throughout, i.e. were directly proportional to
FVa and APC concentrations between 0 and 1.5 n

M
FVa
and between 0.05 and 5 n
M
APC. Thus, the second-order
rate constants obtained by this method can be directly
compared within the given ranges of FVa and APC.
Statistical analysis using the StatGraphics Plus for Windows
package was performed to determine kinetic parameter
significance.
Western blot analysis of FVa inactivation by APC
Human FVa (10 n
M
), phospholipid vesicles (25 l
M
)and
wild-type APC were incubated at 37 °Cin25m
M
Hepes
(pH 7.5), containing 150 m
M
NaCl, 3 m
M
CaCl
2
,and
5mgÆmL
)1
BSA in the absence and presence of 25 IUÆmL
)1

UFH. Aliquots of 20 lL were removed at various time
points, and subjected to SDS/PAGE (7.5% gel) under
reducing conditions. After transfer to poly(vinylidene diflu-
oride) membranes, heavy chain fragments were visualized
using a monoclonal antibody (AHV 5146) directed against
the FVa heavy chain.
Electrostatic potentials for APC and FVa A domains
The 3D structure of Gla-domainless APC [27] and the
homology model for the three A domains of FVa [28] (co-
ordinate file at were
investigated using the programs
INSIGHTII
,
BIOPOLYMER
AND DELPHI
(Accelrys, San Diego, CA, USA). Electrostatic
potentials were computed with DelPhi (reviewed in [29])
using a standard set of formal charges. The standard
protocol was applied. The volume inside the APC or FVa
molecular surface was assigned a dielectric constant of 4 and
the outside volume was given a value of 80.
Docking heparin-like molecules on to APC
Three different methods were used to dock heparin and
APC. The first method follows the script reported by
Fernandez-Recio and coworkers [30] as integrated in the
modeling package
ICM
(Molsoft LLC, San Diego, CA,
USA). This first protocol included a pseudo-Brownian
rigid-body docking, an extended force field, and a soft

interaction energy function precalculated on a grid. To
validate the docking protocol, it was first applied to three
different experimental protein–heparin complexes deposited
at the Protein Data Bank (PDB) files [31], PDB code 1bfc,
1azx, 1e0o [32–34]. These proteins were energy-minimized
to allow relaxation of side chains, and to simplify calcula-
tions, we used a negatively charged polypeptide to mimic
heparin. Negatively charged groups were moved with the
simulation software Discover (Accelrys) in order to repro-
duce the overall positioning in space of charges and overall
shape, as observed on the NMR/modeled structure of
heparin (file 1hpn [35]).
Amino acids known to be part of the heparin-binding site
in these three crystallized protein–heparin complexes were
given as starting point for the docking search [30]. At least
one conformation of the simulated protein–peptide com-
plexes among the lowest five docking energy scores repro-
duced accurately the X-ray crystal structures of the
equivalent complexes. Therefore, the above docking proto-
col combined with partial knowledge of the binding site for
heparin at the surface of APC as defined by several
mutagenesis studies [17,18,21] was deemed appropriate to
reasonably predict the overall orientation of a peptide
mimicking heparin at the surface of APC.
The second approach involved flexible docking of
heparin, PDB files 1hpn [35] and 1e0o [34], onto a rigid
APC structure using the ICM package.
Finally, a structure-based virtual screening approach
(reviewed in [36]) was used to dock short sugar molecules in
the APC loop 37 area. The heparin (PDB file 1hpn) was

shortened, and 10 trisaccharides were generated. These
sugar molecules were considered rigid during the docking
but in order to consider some conformational flexibility, 13
conformers for each molecule were generated and all
structures stored in a single data file. A shape-based
Gaussian docking function as integrated in the program
FRED
was used to position the short sugar molecules at the
surface of APC [37].
Partial docking of APC on to FVa
The X-ray crystal structure of APC (with modifications in
loop 148, see below) and a model structure for the three A
domains of FVa (with some modifications at the Arg506
and Arg306 sites, see below) were used in two different
automated docking procedures. Rigid-body-docking calcu-
lations with soft potentials were performed with the ICM
package, as described [30]. Alternatively, we used the
approach reported by Norel et al. [38]. In docking with
ICM, FVa Arg506 or Arg306 was given as starting point for
the search, whereas for the method of Norel et al. the entire
surfaces of both interacting molecules were investigated and
as such the search was not restricted to known binding
regions (i.e. at the Arg306 or Arg506 site).
In all experimentally known complexes of serine prote-
ases/macromolecular inhibitors/substrates, the peptide
bond to be cleaved tends to be located on a loop structure
that protrudes far outside the molecular surface, either
because this loop is indeed ill-structured or because its
conformation has to change during the interaction with
proteases. A loop structure in FVa including Arg506 was

predicted from the X-ray crystal structure of ceruloplasmin
and does not project significantly outside the surface of
FVa. Numerous initial conformations (2000–20 000) were
generated for residues 500–510 using the loop prediction
program of Xiang et al. [39]. Several runs were performed,
and the 10 best-energy conformations were kept. From
these structures, two residues before and after Arg506 were
template-forced to partially adopt the conformation of the
serpin reactive loop as present in the PDB file 1l99 [40].
Similarly, the FVa loop that contains Arg306 was built
again (residues 302–320) and several conformations were
generated. Because it is known that APC loop 148 plays a
2726 G. A. F. Nicolaes et al.(Eur. J. Biochem. 271) Ó FEBS 2004
role in FVa inactivation [19,26] and as this loop is not
well defined in the PDB file 1aut, the APC loop includ-
ing residues 145–153 was rebuilt using the approach of
Xiang et al., and the 10 lowest-energy conformations were
selected.
One structure of FVa with the Arg506 loop sufficiently
solvent-exposed to allow interactions with APC was selected
for the docking procedures. Docking simulations were
performed with the two best ranked structures for the FVa
Arg306 loop and an APC model in which the loop 148 tends
to be covering the active site (one of the lowest-energy
conformations).
For final optimization of the docking procedure, it was
necessary to include experimentally obtained data. Inter-
active docking was consequently performed starting from
the best theoretical complexes (i.e. the complexes that have
orientation compatible with key experimental/structural/

theoretical data) using as guidelines the relative orientation
of a protease associated with a substrate/inhibitor as seen in
the X-ray crystal structure of a Michaelis serpin–protease
complex (file 1l99 [40]) to optimize further the positioning of
the molecules.
Results
APC-catalyzed inactivation of FVa in the presence
of heparin
Heparin strongly influenced the time course of FVa
inactivation by wild-type APC (Fig. 1). In the absence of
heparin (Fig. 1A, open circles) the typical biphasic inacti-
vation curve was obtained for normal FVa (see also [10]).
This is indicative of rapid cleavage at Arg506, which yields a
partially active reaction intermediate that is subsequently
fully inactivated on cleavage at Arg306. In the presence of
25 IUÆmL
)1
heparin, FVa inactivation was significantly
reduced (Fig. 1A, closed circles). The shape of the curve
suggested strong impairment of the fast first phase (Arg506
cleavage) of the reaction without influence on the second
phase (Arg306 cleavage). A direct effect of heparin in the
FVa assay was excluded because preincubation of FVa with
25 IUÆmL
)1
UFH or direct addition of heparin (1 IUÆmL
)1
)
to the FVa assay mixture resulted in assay outcomes that
were identical with those obtained in the absence of heparin

(data not shown).
To gain insights into which rate constants (k
506
and k¢
306
in intact FVa and k
306
in FVa
int
) were influenced by heparin,
two additional experiments were performed. In the first,
shown in Fig. 1B, FVa was incubated for 5 min with APC
to allow completion of the first phase of the reaction, i.e.
cleavage at Arg506 with all FVa activity converted into
FVa
int
. Thereafter, the reaction volume was divided into
two equal parts and transferred into new reaction tubes
containing either heparin or buffer. Monitoring of FVa
activity in these two tubes was continued for another
25 min, during which time the loss of FVa activity in the
absence of heparin (open circles) did not differ from that
obtained in the presence of heparin (closed circles). This
indicates that the presence of heparin did not influence the
k
306
in the partially active FVa intermediate. In a second
experiment, the effect of heparin on the direct cleavage at
Arg306 (k¢
306

) in FVa was determined from a time course of
Fig. 1. Effect of heparin on the APC-catalyzed inactivation of FVa.
Plasma purified human FVa was inactivated by recombinant wild-type
APC in the absence and presence of UFH as described in Materials
and Methods. (A) Time courses of inactivation of 1.5 n
M
FVa by
0.32 n
M
APC in the absence (s)orpresence(d)of25IUÆmL
)1
UFH.
(B) FVa (1.5 n
M
) was inactivated with 0.32 n
M
wild-type APC. After
5 min of incubation, indicated by the arrow, the reaction volume was
split into two equal volumes and transferred to two new tubes con-
taining either 25 IUÆmL
)1
UFH (final concentration, d) or an amount
of compensation buffer (s), and the monitoring of FVa activity in the
two reaction mixtures was continued. (C) Time courses of inactivation
of 0.70 n
M
FVa
Leiden
by 1.0 n
M

APC in the absence (s) or presence
(d)of25IUÆmL
)1
UFH. The mean of two experiments is given.
Inactivation time courses were very reproducible with variations per
time point < 7%. From time courses like these, apparent second-order
rate constants k
506
, k¢
306
and k
306
were calculated as described in
Materials and methods.
Ó FEBS 2004 Heparin and APC-catalyzed inactivation of FVa (Eur. J. Biochem. 271) 2727
FVa
Leiden
inactivation in the absence and presence of
heparin. From these data, an approximately 2–3-fold (2.48;
standard error: 0.47) stimulation of the rate of inactivation
(k¢
306
) by heparin was calculated (Fig. 1C) by fitting to a
single exponential. Table 1 summarizes all rate constants
obtained by fitting all time courses to the random-order
cleavage model as described in Materials and methods,
under the constraints that k
306
is minimally influenced by
heparin and using k¢

306
obtained from the FVa
Leiden
inactivation.
Concentration dependence of heparin effect on
k
506
To further characterize the influence of heparin on the APC-
catalyzed inactivation of FVa and to confirm the specificity
of the effects observed in Fig. 1, time courses of FVa
inactivation by wild-type APC were determined in the
presence of various concentrations of UFH (0.1–
55 IUÆmL
)1
), and rate constants for the inactivation were
calculated. The effect of heparin on k
506
was dose-dependent
and saturable, with 50% inhibition observed at
% 2IUÆmL
)1
UFH (Fig. 2).
The inhibitory effect was not specifically mediated by
unfractionated heparin because we found that 25 IUÆmL
)1
low molecular weight heparin (which is less than 18
saccharide residues in length) displayed similar inhibitory
activity to UFH. In contrast, equimolar amounts of
pentasaccharide did not influence the inactivation of FVa
by APC.

To relate the inactivation curves of FVa to specific
cleavages in FVa, Western blotting was performed (Fig. 3).
A monoclonal antibody (AHV 5146) against the FVa heavy
chain was used to visualize the inactivation fragments. In
the absence of heparin, the transient 75 kDa fragment
representing the heavy chain fragment 1–506, typical for the
partially active reaction intermediate (FVa
int
), can be seen
(pathway 2). The 30-kDa band represents the 307–506
fragment formed on cleavage at Arg306 in FVa
int
.Inthe
presence of UFH, the pattern of fragment generation was
considerably different from that obtained in its absence. The
amount of 75-kDa fragment was greatly reduced, and
instead a 60/62-kDa fragment accumulated, representing
the 307–709 fragment, which is the result of cleavage at
Arg306 in FVa. These data indicate that heparin strongly
influences cleavage at Arg506, and shifts the pathway
towards initial cleavage at Arg306 (pathway 1), after which
Arg506 can be cleaved, resulting in the formation of a
30 kDa fragment.
Kinetic analysis of cleavage of FVa at Arg506 by APC
To further characterize the inhibitory effect of heparin on
the Arg506 cleavage, we performed a kinetic analysis by
using nonlinear regression analysis of initial rates of FVa
inactivation by APC (initial rates representing almost
exclusively cleavage at Arg506) at FVa concentrations of
0.2–30 n

M
, in both the absence and presence of 25 IUÆmL
)1
heparin (Fig. 4). In the FVa assay, a lower concentration of
FXa (0.5 n
M
) was used than in the standard FVa assay, in
order to minimize the FXa cofactor activity of the reaction
intermediate, FVa
int
. This facilitated the determination of
loss of FVa cofactor activity during the initial stage of
inactivation and minimized the influence of the k
306
Table 1. Apparent second-order rate constants for the APC-catalyzed inactivation of FVa and FVaLeiden in the presence and absence of heparin. Rate
constants (
M
)1
Æs
)1
) for inactivation of normal FVa (0.50–1.5 n
M
)andFVa
Leiden
(0.50–1.5 n
M
) catalyzed by recombinant wild-type APC (0.037–
1.0 n
M
) obtained by fitting time courses of inactivation, such as presented in Fig. 1, to an integral time course equation as described in Materials and

Methods. Reactions were performed in 25 mm Hepes (pH 7.5), containing 150 m
M
NaCl, 3 m
M
CaCl
2
and 5 mgÆmL
)1
BSA in the presence of
25 l
M
phospholipid vesicles (10 : 90 DOPS/DOPC, mol/mol) at 37 °C in the absence or presence of 25 IUÆmL
)1
UFH. FVa activity was assayed as
described in Materials and Methods. Rate constants given are means from at least three experiments, with SEM being < 20%. Values for k¢
306
for
inactivation of normal FVa by APC were obtained from time courses of inactivation for FVa
Leiden
as described in Materials and methods. ND, Not
determined; k
506
and k
306
could not be determined in FVa
Leiden
because of the absence of the Arg506 cleavage site.
Rate constant
Normal FVa FVa
Leiden

– Heparin + Heparin – Heparin + Heparin
k¢
306
6.83 · 10
5
2.17 · 10
6
6.83 · 10
5
2.17 · 10
6
k
506
1.17 · 10
8
9.96 · 10
6
ND ND
k
306
1.55 · 10
6
1.49 · 10
6
ND ND
Fig. 2. Effect of varying heparin concentration on APC-mediated clea-
vage at Arg506 in FVa. Rate constants for cleavage at Arg506 in FVa
by wild-type APC were calculated for inactivations in the presence of
various concentrations of UFH. Inactivations were performed and
analyzed as described in Materials and methods. Data points represent

means ± SEM from three independent time courses of inactivation.
2728 G. A. F. Nicolaes et al.(Eur. J. Biochem. 271) Ó FEBS 2004
cleavage. The kinetic parameters obtained were K
m-app
¼
2.13 ± 0.49 n
M
(mean ± SEM) and k
cat-app
¼
0.68 ± 0.048 s
)1
in the absence of UFH, and K
m-app
¼
11.4 ± 1.49 n
M
and k
cat-app
¼ 0.26 ± 0.016 s
)1
in the
presence of UFH, which correspond to second-order rate
constants for cleavage at Arg506 (k
cat
/K
m
)of
3.2 · 10
8

M
)1
Æs
)1
and 2.3 · 10
7
M
)1
Æs
)1
, respectively. These
values are in reasonable agreement with the rate constants
obtained from fitting the time courses of FVa inactivation
(cf. Figure 1, Table 1). These data suggest that, under the
conditions tested, heparin increases the K
m
of APC for
cleavage of FVa at Arg506 5.5-fold and at the same time
decreases the k
cat
2.6-fold, thus acting as a mixed-type
inhibitor of APC. As this analysis was performed using
initial rates for inactivation of FVa that are almost
completely due to cleavage at Arg506, no individual kinetic
parameters can be deduced for the second cleavage at
Arg306.
Effects of heparin on FVa inactivation in the presence
of DEGR-FXa and protein S
To further verify that heparin inhibits cleavage at Arg506
and stimulates initial cleavage at Arg306, inactivation of

FVa by APC with and without heparin was performed
in the presence and absence of 20 n
M
DEGR-FXa,
which is known to completely and specifically block APC
cleavage at Arg506 [14]. The presence of DEGR-FXa
resulted in abrogation of the fast phase of the reaction,
as compared with inactivation in the absence of DEGR-
FXa. Addition of 25 IUÆmL
)1
UFH in the presence of
DEGR-FXa stimulated the APC-mediated inactivation
approximately twofold (data not shown), indicating that
the stimulatory effect of heparin on the cleavage at
Arg306 is also observed in the presence of FXa.
Theoretically, the addition of heparin may induce the
formation of a binary FXa–FXa complex, thus abolish-
ing the inhibitory effect of DEGR-FXa on the Arg506
cleavage. This possibility was excluded, however, because
time courses of FVa inactivation in the presence of
DEGR-FXa could be fitted, both with and without
heparin, to a mono-exponential equation, indicating
single cleavage at Arg306 only. The effect of UFH on
FVa inactivation by APC was also investigated in the
presence of 200 n
M
protein S. The resulting time course
of inactivation of FVa by APC became monophasic, an
effect known to be due to stimulation of the cleavage at
Arg306 [14]. Addition of 10 IUÆmL

)1
heparin resulted in
a somewhat slower inactivation rate (data not shown),
which presumably is due to the inhibitory effect of UFH
on the cleavage at Arg506.
Fig. 4. Kinetic analysis of the inactivation of FVa by APC in the pres-
ence and absence of heparin. Initial rates of FVa inactivation were
determined at various concentrations of FVa in the presence (d)or
absence (d)of25IUÆmL
)1
UFH, after incubation with 0.14 n
M
APC
(d)or0.04n
M
APC (s). The incubation was performed in 25 m
M
Hepes (pH 7.5), containing 150 m
M
NaCl, 3 m
M
CaCl
2
and
5mgÆmL
)1
BSA in the presence of 25 l
M
phospholipid vesicles
(10 : 90 DOPS/DOPC, mol/mol) at 37 °C. After different time inter-

vals the FVa activity was determined as described in Materials and
methods. Initial rates of FVa inactivation are expressed as n
M
FVa
inactivatedÆmin
)1
Æ(n
M
APC)
)1
. The solid lines represent a fit of the data
according to the Michaelis–Menten equation with K
m-app
¼ 11.4 n
M
and k
cat-app
¼ 0.26 s
)1
in the presence of UFH (d)andK
m-app
¼
2.1 n
M
and k
cat-app
¼ 0.68 s
)1
in the absence of UFH (s).
Fig. 3. Western blotting of FVa degradation. To identify fragment

generation during APC-catalyzed inactivation of FVa, plasma purified
human FVa (10 n
M
) was incubated with 25 l
M
phospholipid vesicles
(10 : 90 DOPS/DOPC, mol/mol) at 37 °Cin25m
M
Hepes (pH 7.5),
containing 150 m
M
NaCl, 3 m
M
CaCl
2
and 5 mgÆmL
)1
BSA. In the
absence of heparin (lanes 1–5), inactivation was started by the addition
of 0.33 n
M
wild-type APC. In the presence of 25 IUÆmL
)1
UFH (lanes
6–10), FVa inactivation was started by the addition of 3.9 n
M
wild-
type APC. Samples were withdrawn from the inactivation mixture and
subjected to SDS/PAGE. Subsequently, proteins were blotted on a
poly(vinylidene difluoride) membrane using a semidry blotting

apparatus. After transfer to poly(vinylidene difluoride) membranes,
heavy chain fragments were visualized using a monoclonal antibody
(AHV 5146) directed against the FVa heavy chain. Lanes 1–5 and 6–10
describe reaction samples of similar FVa activity with the percentages
of residual FVa cofactor activity being (no heparin, lanes 1–5) 100, 37,
20, 10 and 2 and (25 IUÆmL
)1
UFH, lanes 6–10) 100, 36, 17, 9 and 2,
respectively.
Ó FEBS 2004 Heparin and APC-catalyzed inactivation of FVa (Eur. J. Biochem. 271) 2729
Lack of heparin effect on FVa inactivation by
APC mutants deficient in heparin binding
To verify that the inhibition of APC-mediated FVa
inactivation by heparin is related to the ability of heparin
to bind APC, we used recombinant mutants of APC
harboring mutations in the heparin-binding site. The APC
variants chosen were: K37S/K38Q/K39Q with strongly
reduced affinity for heparin; K62N/K63D which has a
modest effect on the APC–heparin interaction; K37S/
K38Q/K39Q/K62N/K63D which shows no detectable
binding to heparin [21]. Table 2 shows the apparent
second-order rate constants obtained from time courses of
inactivation of FVa by the various APC variants. Results
obtained in the absence of heparin were consistent with
those on record [15]. APC variants carrying mutations in
the 37 loop had much lower rate constants for cleavage at
Arg506, whereas the rate constants for cleavage at Arg306
were similar to those obtained with wild-type APC. The
mutations in loop 60 did not affect k
506

and showed a
modestly increased k¢
306
(Table 2). In the presence of
heparin, the abilities of wild-type APC and the 60-loop
variant to cleave the Arg506 site in FVa were strongly
inhibited, with reduction of k
506
of 11.7-fold and 8.4-fold,
respectively, whereas only minor effects were seen on the
cleavage at Arg306. The addition of heparin during
inactivation of normal FVa by the loop-37 mutant
resulted in a further decrease in k
506
and a small
stimulation in the secondary cleavage at Arg306 (k
306
).
However, heparin did not influence Arg506 cleavage in
FVa by the APC variant that completely lacked the
heparin-binding capacity (37+60 loop), but a small
(inhibitory) effect on k¢
306
and k
306
was noted.
These observations suggest that, for heparin to exert its
inhibitory effect on the cleavage at Arg506, a normal
interaction with APC is required. It is likely that the
heparin-binding loop 37 of APC interacts directly with FVa

in an area adjacent to the Arg506 cleavage site and that this
interaction is severely hampered by heparin in the ternary
heparin–APC–FVa complex.
Electrostatic potentials of APC and the A domains of FVa
Loops 37, 60, 70 and 148 in APC form an electropositive
surface, while the catalytic triad is located in an
electronegative environment (Fig. 5). In FVa, both
Arg306 and Arg506 are in electropositive regions. How-
ever, moving towards the C-terminus, the prime side of
Arg306 is first positive then neutral and finally positive,
while the prime side of Arg506 is electronegative (Fig. 5).
At this site, two electronegative zones that possibly
interact with APC involve residues Asp513, Asp578 and
Asp577 (site 1) and Glu323, Glu374, Asp373, and Glu372
(site 2). A very negatively charged segment following
Cys656, which is the last residue in the homology model
based on ceruloplasmin, formed by Asp659-Asp660-
Asp661-Glu662-Asp663 may also play a role during the
docking of APC on to the FVa Arg506 site. In contrast,
the segment containing Arg306 protrudes outside the
surface of FVa, and, besides the residues directly fitting
into the catalytic groove, there are no obvious interacting
regions for APC.
Docking heparin-like molecule onto APC
As numerous problems have been noticed when trying to
dock heparin at the surface of a protein [41], we used three
different methods. To facilitate calculations, a negatively
charged peptide mimicking the overall charge distribution
and shape (see Materials and Methods) of a heparin
molecule was created and the validity of this approach was

tested. The validated protocol was next applied to dock the
heparin-like peptide on to APC. The lowest conformation
energies positioned the long axis of the peptide along a small
electropositive groove formed by loops 37 and 70 (Fig. 6).
These orientations are compatible with known experimental
data suggesting that APC loops 37, 60 and 70 are directly or
indirectly involved in heparin binding [21]. In our structural
model, loop 60 had no direct contact with the negatively
charged peptide, but the distance between Lys62 and Lys63
and negative groups on the peptide (6–8 A
˚
) was compatible
with electrostatic interactions and preorientation of heparin
on the APC surface during formation of an encounter
complex. However, it is important to note that direct
contact could occur between the heparin-like peptide and
loop 60 if flexibility had been allowed.
In the second docking approach, APC was maintained
rigid during the simulation but a real heparin molecule (10
sugar units, length 40 A
˚
) was used and flexibility was
tolerated (Fig. 6, inset). The top ranking conformation
Table 2. Apparent second-order rate constants for the inactivation of FVa and FVa
Leiden
catalyzed by several recombinant variants of APC in the
presence and absence heparin. Rate constants (
M
)1
Æs

)1
) for inactivation of normal FVa (0.80 n
M
)andFVa
Leiden
(0.80 n
M
) catalyzed by recombinant
wild-type APC (0.090–0.60 n
M
) obtained by fitting time courses of inactivation, such as presented in Fig. 1, to an integral time course equation as
described in Materials and Methods. Reactions were performed in 25 m
M
Hepes (pH 7.5), containing 150 m
M
NaCl, 3 m
M
CaCl
2
and 5 mgÆmL
)1
BSA in the presence of 25 l
M
phospholipid vesicles (10 : 90 DOPS/DOPC, mol/mol) at 37 °C in the absence or presence of 25 IUÆmL
)1
UFH. FVa
activity was assayed as described in Materials and Methods. Rate constants given are means from at least two experiments.
APC variant
In the absence of UFH In the presence of UFH
k¢

306
k
506
k
306
k¢
306
k
506
k
306
Wild-type 6.83 · 10
5
1.17 · 10
8
1.55 · 10
6
2.17 · 10
6
9.96 · 10
6
1.49 · 10
6
37-loop 4.09 · 10
5
3.24 · 10
6
5.93 · 10
5
ND

a
1.29 · 10
6
4.22 · 10
5
60-loop 2.60 · 10
6
1.65 · 10
8
3.86 · 10
6
3.38 · 10
6
1.97 · 10
7
2.48 · 10
6
37+60 loop 7.72 · 10
6
2.13 · 10
6
9.64 · 10
5
3.13 · 10
6
2.21 · 10
6
5.19 · 10
5
a

The value for k¢
306
in the presence of heparin in FVa
Leiden
, could not be reliably measured, implying it is < 4 · 10
5
M
)1
Æs
)1
.
2730 G. A. F. Nicolaes et al.(Eur. J. Biochem. 271) Ó FEBS 2004
positioned heparin against loops 37, 60 and 70. In this case,
heparin also had direct contact with positively charged
residues located on loop 60. The last approach followed a
protocol used for virtual ligand screening, and also
positioned the short sugar molecules in between loop 37
andloop70ofAPC.
Docking of APC on to FVa
To elucidate molecular interactions between APC and FVa
at cleavage sites Arg306 and Arg506, two theoretical
docking protocols were used [30,38]. Because the segment
containing Arg306 and Arg506 had to fit into the APC
Fig. 5. Molecular models of FVa and APC demonstrating positions of potentially important residues for the APC–FVa interaction. Top left: 3D
structure of Gla-domainless APC [27] shown with a view down the active site. The catalytic triad (from left to right), D102, H57 and S195, is colored
red. The light chain structure (in white) only includes epidermal growth factor (EGF)1 and EGF2 domains (residues 49–146). The SP domain
(yellow) runs from residues 16–244 (chymotrypsinogen nomenclature). Positively charged residues in loops 60, 37 and 70 play a key role in heparin
binding. Only the loops 37, 70 and 148 have been shown to form a binding exosite for FVa important for cleavage at Arg506 but not Arg306. Other
regions may be important for interactions with FVa but are not defined at present.Top right: molecular surface of APC color-coded according to its
electrostatic potentials (red, regions of negative potentials; blue, regions of positive potentials; white, neutral potential; a linear interpolation was

used to produce the color for surface potentials between )3 and +3 kTÆe
)1
). Bottom left: 3D model for the three A domains of FVa. The A1
domain is colored yellow, the A2 white, and the A3 green. The position of the C domains is not clearly defined at present, and a negatively charged
segment at the end of the A2 domain is missing. Two cleavage sites for APC (Arg306 and Arg506) are highlighted. Bottom right: molecular surface
of FVa domains A1, A2, and A3 color-coded according to electrostatic potentials. Preliminary docking of FVa Arg506 into the catalytic cleft of
APC suggests that the 37 loop may interact with the FVa D578 area (see text). Docking of FVa Arg306 into APC does not suggest significant
contact between the 37 and 148 loop exosites, and the 37 loop may point towards relatively neutral regions (see text).
Ó FEBS 2004 Heparin and APC-catalyzed inactivation of FVa (Eur. J. Biochem. 271) 2731
active site, and because we used essentially rigid-body
docking, several conformations of the loops displaying
Arg306 and Arg506 of FVa as well as the APC loop 148
were generated before docking computations. Test dockings
were performed, and a theoretical FVa model with struc-
tural changes at the level of loop 306 and 506 was selected
for further docking simulation (data not shown). Similarly,
a model of APC in which the loop 148 partially covers the
active site as compared with the structure present in the
PDB file 1aut was selected (Fig. 6).
When using the docking method of Norel et al.[38],we
noticed that, in the best-ranked complex, APC was
positioned very close to the Arg506 site in a conformation
basically suitable for cleavage at this site. Some interactive
reorientations of APC were needed in order to remove steric
clashes. This was performed using the orientation of a serpin
reactive loop crystallized into a serine protease as guideline
[40]. Interestingly, in none of the predicted complexes was
APC positioned next to the Arg306 site, suggesting that the
shape complementarity in this region is not optimal. With
the ICM method, models of the complex with APC docked

at FVa position 506 or 306 could be generated that are in
agreement with published experimental data. To develop
our two final models of the FVa–APC complex, we merged
the two sets of computations reported above and performed
limited interactive reorientations of the two proteins to
remove minor steric clashes (Fig. 7).
When APC is docked at position 506, the electropositive
loops 37, 70 and 148 of APC seem to have contact with the
electronegative area in FVa formed by residues Asp513,
Asp578 and Asp577. APC could also interact with FVa
residues Asp659-Asp660-Asp661-Glu662-Asp663, but these
residues are not present in the model as it was only possible
to predict the structure of the FVa A2 domain up to residue
656. FVa residues Glu323, Glu374, Asp373 and Glu372
could facilitate the docking process, but the role of these
residues is unknown. In the present model, APC loop 60
does not seem to have significant contact with FVa. When
superimposing the APC–heparin complex on to the APC–
FVa complex, we observed that heparin clashes against
FVa and/or could be very close to negatively charged
FVa residues Asp513, Asp578 and Asp577 and/or
Asp659-Asp660-Asp661-Glu662-Asp663 (Fig. 7A). Con-
tact between FVa and APC loop 148 only occurs when
the 148 loop is in a partially closed conformation. In the
PDB file 1aut, the 148 loop is open and with this
conformation, very limited direct contacts with FVa were
noted.
When APC is docked at position Arg306, only the
electropositive segment containing FVa Arg306 seems to
Fig. 6. APC–heparin docking and loop 148

predictions. APCisshownwithanorientation
similar to the one presented in Fig. 5. A neg-
atively charged peptide mimicking heparin
was docked on to the APC X-ray crystal
structure, and the lowest-energy conforma-
tions positioned the negatively charged ribbon
in direct contact with loops 37 and 70. This
positioning is compatible with known experi-
mental data. Loop 148 is not well defined in
the X-ray crystal structure, and different con-
formations were generated. The loops that
tend to be closed above the active site have
lower energies. The very open conformation
present in the APC PDB file represents only
one possible structure; the loop probably
oscillates between open and closed confor-
mations as observed for the equivalent loop of
thrombin.Inset:APCispresentedwiththe
peptide mimicking heparin (ribbon) docked
on to its surface and with one of the lowest
conformation energy obtained from the
docking of a flexible heparin molecule.
2732 G. A. F. Nicolaes et al.(Eur. J. Biochem. 271) Ó FEBS 2004
Fig. 7. Proposed models of APC docked at
Arg506 and Arg306. The 3D structure of APC
wasdockedontoFVaatpositionArg506(A)
or at position Arg306 (B). FVa is shown as a
solid surface with the different A domains
color coded as in Fig. 6 (bottom left). APC is
presented as a ribbon. Different loops are

colored and labeled for orientation. Some
residues expected to play a role in the inter-
action are mentioned (see text). The peptide–
heparin-like 3D structure was extracted from
our docking simulations and positioned on
top of APC. When APC is docked at Arg506,
heparin seems to disturb the interaction,
whereas when APC is at Arg306, heparin
has enough room and could bridge the two
molecules (see text).
Ó FEBS 2004 Heparin and APC-catalyzed inactivation of FVa (Eur. J. Biochem. 271) 2733
complement well the electronegative catalytic groove of
APC. The loops surrounding the active site of APC do not
seem to make strong contact with FVa besides the segment
to be cleaved (i.e. no major contacts besides the P5 to P5¢
segment were observed). When the APC–heparin complex is
superimposed on APC docked at position Arg306, enough
room appears available for heparin, and heparin could even
bridge FVa and APC. Some positively charged residues on
FVa such as Lys320, Arg321, Arg400 and Arg501 could
possibly contact heparin during this reaction.
Discussion
The recent demonstration of a heparin-binding site in the SP
domain of APC that overlaps with a secondary binding
exosite for FVa in APC [15,17,21] prompted us to reinves-
tigate the effect of heparin on the inactivation of FVa by
APC. We find that heparin has specific effects on the
inactivation of FVa by APC that have not been observed
before.
Thus, APC-mediated cleavage at Arg506 in FVa is

inhibited up to 12-fold by heparin, whereas heparin was
found to stimulate the slower cleavage at Arg306 severalfold
if the Arg506 site had not been cleaved first. Inactivation of
FV and FVa occurs via different mechanisms, with the
order of cleavage at Arg506 and Arg306 being reversed
during FV inactivation by APC [9], i.e. in FV, cleavage at
Arg306 precedes cleavage at Arg506. Given that Arg306-
cleaved FV is no longer capable of acquiring FVa cofactor
activity, subsequent inhibition of Arg506 cleavage by
heparin will not further affect the activity of FV. Therefore,
our data are compatible with the reported enhancement of
APC-catalyzed FV inactivation by heparin [22,23]. How-
ever, in contrast with Peta
¨
ja et al., we found heparin to
inhibit the APC-catalyzed inactivation of FVa, i.e. of
activated FV. This discrepancy may be due to the fact that
the concentrations of heparin used in our study were higher
and the assay conditions were different from those used by
Peta
¨
ja and coworkers.
The inhibitory effect of heparin was dependent on the
concentration of heparin, half-maximal inhibition being
observed at % 2IUUFHÆmL
)1
, which is higher than the
concentrations generally used in a clinical setting. Therefore,
our data should primarily be interpreted in a structure–
function context, heparin being considered as a probe for

investigation of the interaction between APC and FVa.
However, given the general abundance of heparin-like
structures on the surface of the vascular bed, and the lack of
good estimates of local concentrations of coagulation
proteins or reactants during hemostatic reactions, our data
may still have clinical ramifications.
There are two possible molecular explanations for the
inhibition of the Arg506 cleavage by heparin: (a) direct
binding of heparin to FVa close to the Arg506 site; (b)
binding of heparin to APC. Heparin binds to a cluster of
basic residues that are present on three conserved surface
loops in the SP domain of APC (loops 37, 60 and 70)
[17,21,26]. Loops 37 and 70 have been shown to contain
exosites that are important for the cleavage at Arg506 but
not for cleavage at Arg306 [15–17]. The observation that
heparin does not inhibit FVa inactivation by recombinant
APC variants that are unable to bind heparin supports the
mechanism in which heparin binding to the basic cluster in
APC is responsible for the observed inhibitory activity. A
direct effect of heparin on APC can also explain the
stimulation of the cleavage at Arg306. On the basis of
molecular modeling, we propose that, similar to the
mechanism by which heparin promotes PCI–APC complex
formation, heparin bridges APC to positively charged
protein patches in the neighborhood of Arg306, thereby
facilitating its cleavage by APC.
Because cleavage at Arg506 by the 37-loop APC mutants
is unaffected by heparin and as the activity of FVa in the
prothrombinase complex was not influenced by the presence
of heparin [42], we suggest no major direct influence of

heparin on the quality of FVa as a substrate for APC but we
do not exclude interactions of heparin with other sites in
FVa.
In FVa, the Arg506 cleavage site is predicted to be located
on a short loop, whereas Arg306 is positioned on a relatively
extended structure. As some loops surrounding the active
site of APC protrude relatively far into the solvent, it is
expected that contacts between APC and FVa are not
confined to those between the APC active site and the main
SandS¢ 506 subsites only. Exosites on both APC and FVa
should be found when docking Arg506 into the active site of
APC, whereas there might be fewer (if any) exosites
involved when Arg306 docks into APC. Two theoretical
molecular models for APC docking to the Arg506 and
Arg306 sites were developed and analyzed taking into
account our APC–heparin model and different conforma-
tions for APC loop 148 (Figs 6 and 7). When APC docks at
the Arg506 site, the electropositive loops 37 and 70 of APC
are in contact with the electronegative area in FVa formed
by residues Asp513, Asp578 and Asp577. The contacts
noted in our structure are also consistent with the model of
APC manually positioned at the FVa Arg506 site [43]. The
role of FVa residues Glu323, Glu374, Asp373 and Glu372
with regard to APC positioning at Arg506 remains to be
investigated. This also applies to a cluster of residues
(Asp659-Asp660-Asp661-Glu662-Asp663; not present in
the FV model) proposed by Pellequer et al. [43] to interact
with APC. Interestingly, when we positioned our APC–
heparin model on to FVa at position Arg506, heparin was
very close to the FVa electronegative regions 513–578–577

and possibly Asp659-Asp660-Asp661-Glu662-Asp663 and
could directly crash into FVa (i.e. fully compatible with the
observed decrease in k
506
).
Evaluation of the FVa Arg306 site gave a different
pattern from that observed for the Arg506 site. The size of
the Arg306 loop and the lack of negatively charged exosites
surrounding Arg306 does not allow clear contacts with APC
loops 37, 60, 70 and 148. In our model, these loops point
toward neutral regions of FVa, yet are relatively distant
from the molecular surface of FVa. When superimposing
our APC–heparin complex on the APC–FVa complex at
Arg306, we noticed that heparin could bridge APC and FVa
(i.e. consistent with increased k¢
306
). The exact region of FVa
involved in contacting heparin when APC is positioned at
Arg306 is not known, but we hypothesize that Lys320,
Arg321, Arg400 and Arg501 of FVa play a role.
In conclusion, our experimental findings, taken together
with the structural bioinformatics analyses, indicate that
binding of heparin to loop structures 37, 60 and 70 in APC
2734 G. A. F. Nicolaes et al.(Eur. J. Biochem. 271) Ó FEBS 2004
impairs the interaction of APC with FVa during the APC-
catalyzed cleavage at Arg506. Loops 37, 70, and 148 of APC
are, according to our structural models, which must be
regarded as speculative, proposed to contact FVa when the
cleavage site at Arg506 is approached and this contact is
directly perturbed by heparin. Negatively charged regions

are not present in the close vicinity of Arg306, and we found
no structural basis for a contact between exosites in APC
and FVa during approach of the cleavage site at Arg306
which probably contributes to the lower rate of cleavage at
Arg306 compared with that at Arg506.
Acknowledgements
This work was supported by grant no. 902-26-227 from the Dutch
Organization for Scientific Research (NWO; to G.N.), by grants from
the Swedish Research Council (07143), the So
¨
derberg Foundation, the
O
¨
sterlund’s Foundation, the Pa
˚
hlsson Trust, a Marie Curie training
grant QLK5-CT-2000–60007 (to K.W.S.) and research funds from the
University Hospital, Malmo
¨
. Support from INSERM and La Region
Ile-de-France are greatly appreciated. We would like to thank Dr Theo
Lindhout for helpful suggestions and for reading the manuscript. We
are grateful to Dr Fernandez-Recio and Molsoft scientists for
suggestions for running the ICM molecular modeling package, and
to Dr R. Norel for advice on running the docking package PPD.
We also thank OpenEye Scientific Software for providing the
FRED
package.
References
1. Nesheim, M.E., Taswell, J.B. & Mann, K.G. (1979) The con-

tribution of bovine Factor V and Factor Va to the activity of
prothrombinase. J. Biol. Chem. 254, 10952–10962.
2. Rosing, J., Tans, G., Govers-Riemslag, J.W.P., Zwaal, R.F.A. &
Hemker, H.C. (1980) The role of phospholipids and factor Va in
the prothrombinase complex. J. Biol. Chem. 255, 274–283.
3. Nicolaes, G.A.F. & Dahlba
¨
ck, B. (2002) Factor V and thrombotic
disease: description of a janus-faced protein. Arterioscler. Thromb.
Vasc. Biol. 22, 530–538.
4. Esmon, C.T. (2000) Regulation of blood coagulation. Biochim.
Biophys. Acta 1477, 349–360.
5. Dahlba
¨
ck, B. (2000) Blood coagulation. Lancet 355, 1627–1632.
6. Stenflo, J. (1984) Structure and function of protein C. Semin.
Thromb. Hemost. 10, 109–121.
7. Stenflo, J. (1991) Structure-function relationships of epidermal
growth factor modules in vitamin K-dependent clotting factors.
Blood 78, 1637–1651.
8. Esmon, C.T. (1989) The roles of protein C and thrombomodulin
in the regulation of blood coagulattion. J. Biol. Chem. 264, 4743–
4746.
9.Kalafatis,M.,Rand,M.,D.,Mann,K.&G.(1994)The
mechanism of inactivation of human factor V and human factor
Va by activated protein C. J. Biol. Chem. 269, 31869–31880.
10. Nicolaes, G.A.F., Tans, G., Thomassen, M.C.L.G.D., Hemker,
H.C.,Pabinger,I.,Varadi,K.,Schwarz,H.P.&Rosing,J.(1995)
Peptide bond cleavages and loss of functional activity during
inactivation of factor Va and factor VaR506Q by activated pro-

tein. J. Biol. Chem. 270, 21158–21166.
11. Nicolaes, G.A.F. & Dahlba
¨
ck, B. (2003) Congenital and acquired
activated protein C resistance. Sem. Vasc. Med. 3, 33–46.
12. Hockin, M.F., Cawthern, K.M., Kalafatis, M. & Mann, K.G.
(1999) A model describing the inactivation of factor Va by APC:
bond cleavage, fragment dissociation, and product inhibition.
Biochemistry 38, 6918–6934.
13. Gale, A.J., Xu, X., Pellequer, J.L., Getzoff, E.D. & Griffin, J.H.
(2002) Interdomain engineered disulfide bond permitting elucida-
tion of mechanisms of inactivation of coagulation factor Va by
activated protein C. Protein Sci. 11, 2091–2101.
14. Rosing, J., Hoekema, L., Nicolaes, G.A.F., Thomassen,
M.C.L.G.D., Hemker, H.C., Varadi, K., Schwarz, H.P. & Tans,
G. (1995) Effects of protein S and factor Xa on peptide bond
cleavages during inactivation of factor Va and factor Va
R506Q
by
activated protein C. J. Biol. Chem. 270, 27852–27858.
15. Friedrich, U., Nicolaes, G.A.F., Villoutreix, B.O. & Dahlba
¨
ck, B.
(2001) Secondary substrate-binding exosite in the serine-protease
domain of activated protein C important for cleavage at R506 but
not at R306 in factor Va. J. Biol. Chem. 276, 23105–23108.
16. Gale, A.J., Tsavaler, A. & Griffin, J.H. (2002) Molecular char-
acterization of an extended binding site for coagulation factor Va
in the positive exosite of activated protein C. J. Biol. Chem. 277,
28836–28840.

17. Yang, L., Manithody, C. & Rezaie, A.R. (2002) Contribution of
basic residues of the 70–80-loop to heparin binding and antico-
agulant function of activated protein C. Biochemistry 41, 6149–
6157.
18. Shen, L., Villoutreix, B.O. & Dahlba
¨
ck, B. (1999) Involvement of
Lys 62 (217) and Lys 63 (218) of human anticoagulant protein C in
heparin stimulation of inhibition by the protein C inhibitor.
Thromb. Haemost. 82, 72–79.
19. Gale, A.J., Heeb, M.J. & Griffin, J.H. (2000) The autolysis loop of
activated protein C interacts with factor Va and differentiates
between the Arg506 and Arg306 cleavage sites. Blood 96, 585–593.
20. Cooper, S.T., Whinna, H.C., Jackson, T.P., Boyd, J.M. & Church,
F.C. (1995) Intermolecular interactions between protein C inhi-
bitor and coagulation proteases. Biochemistry 34, 12991–12997.
21. Friedrich, U., Blom, A.M., Dahlba
¨
ck,B.&Villoutreix,B.O.
(2001) Structural and energetic characteristics of the heparin
binding site in antithrombotic protein C. J. Biol. Chem. 276,
24122–24128.
22. Peta
¨
ja, J., Fernandez, J.A., Gruber, A. & Griffin, J.H. (1997)
Anticoagulant synergism of heparin and activated protein C in
vitro. Role of a novel anticoagulant mechanism of heparin,
enhancement of inactivation of factor V by activated protein C.
J. Clin. Invest. 99, 2655–2663.
23. Fernandez, J.A., Peta

¨
ja, J. & Griffin, J.H. (1999) Dermatan sulfate
and LMW heparin enhance the anticoagulant action of activated
protein C. Thromb. Haemost. 82, 1462–1468.
24. Rosing, J., Bakker, H.M., Thomassen, M.C.L.G.D., Hemker,
H.C. & Tans, G. (1993) Characterization of two forms of human
factor Va with different cofactor activities. J. Biol. Chem. 268,
21130–21136.
25. Nicolaes, G.A.F., Villoutreix, B.O. & Dahlba
¨
ck, B. (1999) Partial
glycosylation of Asn2181 in human factor V as a cause of mole-
cular and functional heterogeneity. Modulation of glycosylation
efficiency by mutagenesis of the consensus sequence for N-linked
glycosylation. Biochemistry 38, 13584–13591.
26. Shen, L., Villoutreix, B.O. & Dahlba
¨
ck, B. (1999) Interspecies loop
grafting in the protease domain of human protein C yielding
enhanced catalytic and anticoagulant activity. Thromb. Haemost.
82, 1078–1087.
27. Mather, T., Oganessyan, V., Hof, P., Huber, R., Foundling, S.,
Esmon, C. & Bode, W. (1996) The 2.8 A
˚
crystal structure of
Gla-domainless activated protein C. EMBO J. 15, 6822–6831.
28. Villoutreix, B.O. & Dahlba
¨
ck, B. (1998) Structural investigation of
the A domains of human blood coagulation factor V by molecular

modeling. Protein Sci. 7, 1317–1325.
29. Honig, B. & Nicholls, A. (1995) Classical electrostatics in biology
and chemistry. Science 268, 1144–1149.
Ó FEBS 2004 Heparin and APC-catalyzed inactivation of FVa (Eur. J. Biochem. 271) 2735
30. Fernandez-Recio, J., Totrov, M. & Abagyan, R. (2002) Soft
protein–protein docking in internal coordinates. Protein Sci. 11,
280–291.
31. Berman, H.M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T.N.,
Weissig,H.,Shindyalov,I.N.&Bourne,P.E.(2000)TheProtein
Data Bank. Nucleic Acids Res. 28, 235–242.
32. Faham, S., Hileman, R.E., Fromm, J.R., Linhardt, R.J. & Rees,
D.C. (1996) Heparin structure and interactions with basic fibro-
blast growth factor. Science 271, 1116–1120.
33. Jin, L., Abrahams, J.P., Skinner, R., Petitou, M., Pike, R.N. &
Carrell, R.W. (1997) The anticoagulant activation of antithrombin
by heparin. Proc. Natl Acad. Sci. USA 94, 14683–14688.
34. Pellegrini, L., Burke, D.F., von Delft, F., Mulloy, B. & Blundell,
T.L. (2000) Crystal structure of fibroblast growth factor receptor
ectodomain bound to ligand and heparin. Nature (London) 407,
1029–1034.
35. Mulloy, B., Forster, M.J., Jones, C. & Davies, D.B. (1993) N.m.r.
& molecular-modelling studies of the solution conformation of
heparin. Biochem. J. 293, 849–858.
36. Langer, T. & Hoffmann, R.D. (2001) Virtual screening: an
effective tool for lead structure discovery? Curr. Pharm. Des. 7,
509–527.
37. McGann, M.R., Almond, H.R., Nicholls, A., Grant, J.A. &
Brown, F.K. (2003) Gaussian docking functions. Biopolymers 68,
76–90.
38. Norel, R., Sheinerman, F., Petrey, D. & Honig, B. (2001) Elec-

trostatic contributions to protein–protein interactions: fast ener-
getic filters for docking and their physical basis. Protein Sci. 10,
2147–2161.
39. Xiang, Z., Soto, C.S. & Honig, B. (2002) Evaluating conforma-
tional free energies: the colony energy and its application to the
problem of loop prediction. Proc. Natl Acad. Sci. USA 99, 7432–
7437.
40. YeS., Cech, A.L., Belmares, R., Bergstrom, R.C., Tong, Y.,
Corey, D.R., Kanost, M.R. & Goldsmith, E.J. (2001) The struc-
ture of a Michaelis serpin–protease complex. Nat. Struct. Biol. 8,
979–983.
41. Bitomsky, W. & Wade, R.C. (1999) Docking of glycosaminogly-
cans to heparin-binding proteins: validation for aFGF, bFGF,
andantithrombinandapplicationtoIL-8.J. Am. Chem. Soc. 121,
3004–3013.
42. Anderson, J.A., Fredenburgh, J.C., Stafford, A.R., Guo, Y.S.,
Hirsh, J., Ghazarossian, V. & Weitz, J.I. (2001) Hypersulfated low
molecular weight heparin with reduced affinity for antithrombin
acts as an anticoagulant by inhibiting intrinsic tenase and pro-
thrombinase. J. Biol. Chem. 276, 9755–9761.
43. Pellequer, J.L., Gale, A.J., Getzoff, E.D. & Griffin, J.H. (2000)
Three-dimensional model of coagulation factor Va bound to
activated protein C. Thromb. Haemost. 84, 849–857.
2736 G. A. F. Nicolaes et al.(Eur. J. Biochem. 271) Ó FEBS 2004