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

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

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

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),

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

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

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

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

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

) 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

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.

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

– 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

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

)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-

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

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

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

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

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

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

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

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

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

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