Mouse recombinant protein C variants with enhanced
membrane affinity and hyper-anticoagulant activity in
mouse plasma
Michael J. Krisinger
1
, Li Jun Guo
1
, Gian Luca Salvagno
2
, Gian Cesare Guidi
2
, Giuseppe Lippi
2
and Bjo
¨
rn Dahlba
¨
ck
1
1 Department of Laboratory Medicine, Division of Clinical Chemistry, Lund University, University Hospital, Malmo
¨
, Sweden
2 Clinical Chemistry Section, Department of Morphological-Biomedical Sciences, University Hospital of Verona, Italy
Introduction
Protein C is a vitamin K-dependent c-carboxyglutamic
acid-containing protein (Gla protein) found in human
and mouse plasma at a concentration of approximately
70 nm [1]. This zymogen is efficiently converted by the
thrombin–thrombomodulin complex to the multifunc-
tional serine protease activated protein C (APC). With
its cofactor, protein S, APC degrades factors Va and

mouse protein C. In total, seven mutants (mutated at one or more of
positions P
10
S
12
D
23
Q
32
N
33
) and wild-type protein C were expressed and
purified to homogeneity. In a surface plasmon resonance-based membrane-
binding assay, several high affinity protein C mutants were identified. In
Ca
2+
titration experiments, the high affinity variants had a significantly
reduced (four-fold) Ca
2+
requirement for half-maximum binding. In a
tissue factor-initiated thrombin generation assay using mouse plasma, all
mouse APC variants, including wild-type, could completely inhibit throm-
bin generation; however, one of the variants denoted mutant III (P10Q ⁄
S12N ⁄ D23S ⁄ Q32E ⁄ N33D) was found to be a 30- to 50-fold better anti-
coagulant compared to the wild-type protein. This mouse APC variant will
be attractive to use in mouse models aiming to elucidate the in vivo effects
of APC variants with enhanced anticoagulant activity.
Abbreviations
APC, activated protein C; C
max

by pro-inflammatory cytokines such as tumor necrosis
factor-a and interleukin-1 has been demonstrated,
resulting in diminished protein C activation [7]. The
protective effects of APC supplementation in patients
with severe sepsis complicated with disseminated intra-
vascular coagulation [8] remain to be fully elucidated
and are likely the result of its ability to modulate
multiple biochemical pathways [7].
A prerequisite for Gla protein-membrane binding is
the saturation of seven Ca
2+
sites in the N-terminal
Gla domain, which changes its tertiary structure from
an unfolded and nonfunctional conformation to a
tightly folded membrane-binding domain [9,10]. This
Ca
2+
binding requires the presence of Gla residues.
The Gla domains within the protein C family comprise
44 amino acids and contain between nine and 11 Gla
residues, which mediate the Ca
2+
interaction. In
human protein C, a detailed analysis of the function of
each of these Gla residues has been evaluated [11]. Of
the Gla residues, nine are strictly conserved through-
out the Gla proteins. From crystal structures of the
Gla domain of prothrombin and factor VIIa, the
placement of the seven Ca
2+

in promoting efficient binding, complex assembly and
enzyme catalysis in vivo.
Membrane affinity of a Gla protein often correlates
with its membrane localized activity. Strategies used to
increase the affinity of the Gla protein–membrane
interaction involve Gla-domain mutation (for human
protein C) [14–16], Gla domain substitution [17] and
covalent dimerization of the Gla protein [18]. We have
previously created several Gla-domain mutated human
protein C variants with enhanced anticoagulant activ-
ity. One of these variants with several Gla domain
mutations, QGNSEDY-human APC (H10Q ⁄ S11G ⁄
S12N ⁄ D23S ⁄ Q32E ⁄ N33D ⁄ H44Y), bound phospholipid
membranes with increased (approximately seven-fold)
affinity compared to wild-type [16]. QGNSEDY-
human APC was shown to be potent in both a human
plasma-based clotting assay (20-fold better) [16] and a
FVa-degradation assay, cleaving R306 (18-fold) and
R506 (four-fold) more efficiently [19]. However, the
variant had no antithrombotic effect when used in a
rat model of arterial thrombosis [20,21]. The lack of
effect was possibly a result of species–species differ-
ences between human protein C and the rat hemostatic
system. The reason for the poor anticoagulant effect of
human APC in rat plasma remains unknown but may
be a result of rat FVa ⁄ FVIIIa being poor substrates
for human APC [21].
APC variants with enhanced anticoagulant activity
resulting from improved membrane-binding ability
may prove more efficient than wild-type APC in the

Gla domain were purified and characterized. The
results obtained indicate that the functional improve-
ments were closely related to enhanced membrane
affinity. The mutant with highest function, mutant III
(P10Q ⁄ S12N ⁄ D23S ⁄ Q32E ⁄ N33D), showed reduced
Ca
2+
dependence for membrane binding and a 30-50-
fold inhibition improvement over wild-type in tissue-
factor-dependent thrombin generation in mouse
plasma. Overall, the proteins described in the present
study provide insight into the Gla protein–membrane
interaction and identify new reagents with varying
degrees of anticoagulant potency that may be of use
for testing in murine models of sepsis and thrombo-
embolic disorders.
Results
Expression and characterization of mouse protein
C variants
To determine whether the mutations previously made
in human protein C result in a similar enhancement of
both membrane affinity and anticoagulant activity in a
mouse system, the analogous mutations were made in
mouse protein C. Wild-type and seven variants of
mouse protein C (Fig. 1) were expressed and purified.
SDS-PAGE analysis of the purified proteins (Fig. 2)
demonstrated slightly different mobilities of the light
chains, an effect caused by the mutations, whereas the
Fig. 1. Gla domain sequence alignment from different species and mouse protein C variants used in the present study. N-terminal Gla
sequence (1–44) is shown and defined between the propeptidase and chymotrypsin cleavage sites. Positions in the sequence at which

The proteins were found to be c-carboxylated, as
judged by western blotting using a Gla-specific anti-
body (Fig. S1).
Membrane binding ability of wild-type and
variants of mouse protein C
To determine the functional significance of the substi-
tuted Gla domain residues, we measured membrane
binding properties by surface plasmon resonance (SPR).
Chips were coated with 0-20-80, 0-10-90 and 20-10-70
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine ⁄
1,2-dioleoyl-sn-glycero-3-[phospho-l-serine] ⁄ 1-palmitoyl-
2-oleoyl-sn-glycero-3-phosphocholine (POPE-DOPS-POPC)
liposomes, whereas a control surface was either left
blank or coated with 100% POPC. We first measured
the binding of each protein at equi-molar concentration
(100 nm) to estimate their relative membrane binding
abilities (Fig. 3A–C). Noticeably, mutants II and III
stand out from the other proteins analyzed, obtaining
the highest responses for all membrane types. Mutants
V and VII also show a significant binding-response
enhancement, whereas mutations introduced into
mutants IV and VI had little effect relative to the
wild-type protein (Fig. 3A–C, insets). Figure 3D–F
shows the equilibrium binding analysis of the protein–
membrane interactions, and the K
D
values determined
from the curve fitting are summarized in Table 1. The
affinities of wild-type mouse protein C for 0-20-80 and
20-10-70 liposomes are comparable (K

max
deter-
mined for wild-type [722 response units (RU)], and
mutants II (3569 RU), III (4380 RU) and VII
(2060 RU), is clearly different, as is also evident from
an inspection of Fig. 3D (or the other membranes in
Fig. 3E,F). All variants were tested using the same
immobilized membrane preparation. Thus, different
variants are able to utilize a different number of bind-
ing sites on the membrane surface. For example,
mutant II can utilize approximately five times as many
binding sites on a 0-20-80 membrane as wild-type
protein C.
Importance of the liposome phospholipid
composition on membrane binding
Simple model membranes composed of one, two or
three synthetically-derived phospholipids were used to
assess membrane binding. Membranes composed
entirely of POPC were inert to binding, whereas DOPS
or DOPS with POPE-containing liposomes were neces-
sary to obtain a binding response. By varying the
DOPS composition, we were able to show binding
specificity in terms of DOPS content. Doubling the
DOPS content from 10 to 20 mol % resulted in
increased binding sites with enhanced affinity
(Fig. 3D,E and Table 1). For example, mutant II binds
to 0-10-90 with K
D
= 2.45 lm ⁄ R
max

with 0-10-90 membranes. Wild-type protein C at
20 mm Ca
2+
is included as a standard for comparison.
As expected, the Gla protein–membrane interaction
M. J. Krisinger et al. Anticoagulant mouse protein C variants
FEBS Journal 276 (2009) 6586–6602 ª 2009 The Authors Journal compilation ª 2009 FEBS 6589
is highly dependent on Ca
2+
, with maximum binding
occurring at approximately 10 mm Ca
2+
. Figure 4B–
D shows the equilibrium binding analysis of the
protein–membrane interactions at different Ca
2+
con-
centrations, and [Ca
2+
]
1 ⁄ 2 max
determined from the
curve fitting are summarized in Table 2. Employing a
20-10-70 membrane, half-maximum Ca
2+
concentra-
tions required for mutant II (3.7 mm) and III
(3.7 mm) are much improved compared to wild-type
( 11 mm) and approach that of plasma-derived
human prothrombin (1.8 mm), comprising an efficient

human prothrombin (72 kDa) has a different molecu-
lar mass than that of mouse protein C (56 kDa), abso-
lute response values cannot be directly compared.
However, calculating the fraction of binding relative to
R
max
(equilibrium response at indicated Ca
2+
divided
by R
max
at saturating Ca
2+
, i.e.  20 mm Ca
2+
), as
shown in Fig. 5, reveals the Ca
2+
-dependent mem-
brane binding differences amongst the proteins.
Mutant II and III display a much improved fractional
membrane occupancy at physiologically relevant Ca
2+
concentrations (1–5 mm) [24,25] compared to wild-
type. For example, at 2 mm Ca
2+
, prothrombin
already obtains over 70% of its potential binding and
protein C mutants II and III each have approximately
50%, whereas wild-type protein C has obtained a mere

) or endogenous thrombin potential
(ETP)], the concentrations required for wild-type,
mutant II and mutant III were 1, 0.08 and 0.02 nm,
respectively. Similarly, thrombin generation was com-
pletely inhibited at the concentrations tested for wild-
type (16 nm), mutant II (1.28 nm) and mutant III
(0.5 nm). Figure 7 reflects these findings and summa-
rizes how each of the recombinant APC variants at
several concentrations influences the generation of
thrombin in mouse plasma. Interestingly, thrombin
generation parameters of lag-phase and time required
to reach maximum thrombin generation (T
max
) are not
significantly altered by the addition of the tested APC
molecules. Furthermore, a three-fold higher concentra-
tion of mutant III was required to obtain a similar
Table 1. Effect of Gla-domain mutations on mouse protein C ⁄ APC. Membrane dissociation constants (K
D
) at various membrane composi-
tions were determined by SPR for mouse protein C. C
max
and ETP generated in mouse plasma were determined using mouse APC. Further
details on methodology and experimental conditions are provided in Fig. 3 (membrane binding) and Fig. 6 (thrombin generation).
Protein C
Membrane affinity (POPE-DOPS-POPC)
Activated protein C
Thrombin generation in mouse plasma
K
D

K
D
is a representative determination from three experiments.
b
Membranes used were 100 nm extruded liposomes with synthetic
phospholipids: POPE-DOPS-POPC (mol %).
c
SE from one-site binding hyperbola fitting.
d
NA, not available. Concentrations tested did not
allow determination of K
D
.
e
SD from three independent experiments.
f
ETP determined after 60 min.
g
Concentration of APC added to
assay.
M. J. Krisinger et al. Anticoagulant mouse protein C variants
FEBS Journal 276 (2009) 6586–6602 ª 2009 The Authors Journal compilation ª 2009 FEBS 6591
anticoagulant activity (assessed by either C
max
or ETP)
in human plasma compared to mouse plasma (data
not shown), further highlighting the importance of
using proteins from the same species.
Discussion
Recombinant APC has been used to treat patients with

at variable Ca
2+
concentration to determine binding efficiency as a
function of Ca
2+
concentration for this high affinity binder. Dissoci-
ation under running buffer conditions was followed for an additional
6 min. Ca
2+
concentration (mM) is indicated near the appropriate
curve. The SPR response curves are shown after background cor-
rection using a 100% POPC control flow cell. Binding to the control
surface was not apparent and no evidence of nonspecific binding
was evident from an injection of Gla-less, prethrombin-1 (10 l
M,
not shown). Wild-type protein C (1 l
M)at20mM Ca
2+
was also
included for comparison (indicated with an arrow: 20 wild-type).
(B–D) Steady-state binding of mouse protein C wild-type (
),
mutant II (
), III (.), V (+) and human prothrombin (h), each at
1 l
M, over either the (B) 0-20-80 or (C) 0-10-90 or (D) 20-10-70
POPE-DOPS-POPC membrane bilayer surface was measured using
the indicated Ca
2+
concentrations. Responses obtained at equilib-

or ETP. Complete inhi-
bition of thrombin generation was obtained at a
mutant III concentration of 0.5 nm, which is 30-fold
lower than the concentration of wild-type APC
required to give similar inhibition. Previous work
based on a tissue factor-dependent clot-based assay in
normal human plasma showed that human APC vari-
ant QGNSEDY had a 20-fold higher anticoagulant
potential than human wild-type APC [16]. Although
this was a simple end point assay, the anticoagulant
potency of human APC variant QGNSEDY in human
plasma parallels that of mouse APC mutant III in
mouse plasma. Mutant III, although only containing
five mutations, is equivalent to the human QGNSEDY
variant because the wild-type mouse Gla domain
already has G at position 11 and Y at position 44.
Thus, the seven Gla domain residues introduced in
human APC (Q10, G11, N12, S23, E32, D33 and Y44)
are all present in mouse APC mutant III.
The high sensitivity of SPR detection allowed us to
accurately analyze even low affinity proteins, such as
wild-type mouse protein C, to membrane binding site
saturation. The results obtained are thus based on the
combined analysis of binding affinity, R
max
and quali-
tative kinetics. Efficient binding of protein C, as well
as other Gla proteins, to membrane is dependent on
three complimenting factors: an optimal Ca
2+

duced at position 32 in factor VII has been suggested
to bind an additional Ca
2+
during membrane binding
[30], although it does not appear to serve the same role
Fig. 5. Fractional membrane binding site occupancy by the various
protein C variants at different Ca
2+
concentrations. Equilibrium
binding responses for mouse protein C wild-type (
), mutant II ( ),
III (.), V (
+
) and human prothrombin (h), using a 20-10-70 POPE-
DOPS-POPC membrane, were obtained at the indicated Ca
2+
con-
centration as described in Fig. 4D. Equilibrium binding responses
were normalized for fractional occupancy for each individual
protein. % R
max
is expressed as the equilibrium response at the
indicated Ca
2+
concentration divided by R
max
at saturating Ca
2+
concentration (20 mM Ca
2+

] determined from one-site binding
hyperbola fitting. Representative determination from three experi-
ments.
c
SE determined from one-site binding hyperbola fitting.
M. J. Krisinger et al. Anticoagulant mouse protein C variants
FEBS Journal 276 (2009) 6586–6602 ª 2009 The Authors Journal compilation ª 2009 FEBS 6593
in mouse protein C (see discussion on Ca
2+
below).
An acidic residue at position 23, such as Asp in pro-
tein C, has been speculated to instill low affinity to
Gla proteins. However, the D23S mutation had little
effect on binding affinity; for example, when compar-
ing mutant VI (S) and wild-type. The D23S mutation
appears even inhibitory if mutant II (QNED) and III
(QNSED) are compared. Similarly, the gain of func-
tion observed with mutant I (QN) was reversed by
having the D23S mutation present, as in mutant IV
(QNS). It is noteworthy that the multi-site mutations
introduced often resulted in synergistic affinity effects
and were not simply the additive sum of individual
mutations. As such, there appears to be an intra-
molecular synergism between the 10 ⁄ 12 (QN) and
32 ⁄ 33 (ED) sites in mouse protein C. Our previous
work with human protein C showed that variant
QGNSEDY had increased membrane affinity (3.5- to
seven-fold) and was more potent as an anticoagulant
in a TF-dependent clotting (PT) assay (approximately
five-fold longer prolonged clot time) than wild-type

and followed
continuously with the fluorogenic substrate I-1140 (Z-Gly-Gly-Arg-7-
amino-4-methylcoumarinÆHCl, 300 l
M)in25mM Hepes, 175 mM
NaCl (pH 7.4) containing 0.5% BSA at 37 °C. All indicated concen-
trations are final concentrations. Mouse plasma (10 lL) was used
in a final reaction volume of 120 lL. The first derivative of a typical
experiment (n = 3) is shown. (B–D) Concentration-dependent inhibi-
tion of thrombin generation in mouse plasma by mouse APC is
shown. Mouse plasma was incubated with the indicated concentra-
tion (n
M) of extrinsically added mouse APC (B) wild-type, (C) mutant
II or (D) mutant III using the conditions described above.
Anticoagulant mouse protein C variants M. J. Krisinger et al.
6594 FEBS Journal 276 (2009) 6586–6602 ª 2009 The Authors Journal compilation ª 2009 FEBS
decrease [14]. Mouse protein C contains the ‘low affin-
ity’ Pro10, but did not gain a significant increase in
affinity by mutagenesis to P10Q because mutant I
(QN) had only a modest 1.5-fold affinity increase com-
pared to wild-type protein. Thus, work on human,
bovine and mouse protein C mutagenesis illustrates the
intricacy of improving the Gla protein–membrane
interaction.
Mutant II has a modest (two-fold), but significant,
affinity enhancement compared to mutant III,
although, unexpectedly, mutant III has better antico-
agulant activity. The only differences between mutant
II and mutant III is the amino acid at position 23 (D
in mutant II and S in mutant III). It is conceivable
that the amino acid at position 23 may affect anticoag-

concentration specified by the K
D
of the receptor–ana-
lyte interaction. This was clearly not observed when
the saturation binding levels of the protein C variants
were compared, for any of the phospholipid mem-
branes tested, implying a different mode of binding
between the proteins. This indicates that the mem-
brane, even in simple model membranes, provides a
number of binding sites that are not isolated and
homogenous in nature, but rather heterogeneous. For
example, it can be envisioned that a Gla protein can
engage with a membrane site containing a variable
number of PS molecules each displaying a different
affinity. The results obtained in the present study indi-
cate that the high affinity mutants (e.g. II and III) can
utilize ‘poor’ binding sites that low affinity mutants
(e.g. wild-type) cannot engage with, and, thus, the high
affinity mutants have access to more total binding sites
resulting in a higher R
max
. The membrane binding
capacity difference observed amongst the variants
argues for the existence of several binding sites com-
posed of a variable number of PS molecules. Thus, we
conclude that two processes make the binding of
mouse protein C mutants more efficient to membrane
than wild-type. First, variants such as mutants II and
III are able to utilize a higher number of membrane
binding sites. Second, these variants are also able to

M. J. Krisinger et al. Anticoagulant mouse protein C variants
FEBS Journal 276 (2009) 6586–6602 ª 2009 The Authors Journal compilation ª 2009 FEBS 6595
membrane core and, finally, the chelation of additional
calcium ions between membrane and protein, all pro-
vide independent free-energy contributions to bound
intermediates. It is not surprising that the kinetic bind-
ing data describing the sum of these kinetic events
could not be adequately simulated by simple binding
models commonly used in the kinetic fitting of pro-
tein–protein interaction data. On a qualitative basis,
the kinetic shapes of the interaction profiles (Fig. 3A–
C) were not drastically different amongst the protein C
variants analyzed. Thus, from the data obtained in the
present study, we conclude that, in addition to utilizing
a higher number of binding sites, mutants II and III
gained their modest (approximately ten-fold) mem-
brane binding affinity enhancement by minor increases
in the overall on-rate and ⁄ or minor decreases in the
overall off-rate.
The results obtained in the present study support
that the binding of a Gla protein is to a few PS mole-
cules rather than a PS microdomain (a membrane PS-
rich regions containing over 20 grouped PS molecules).
We have shown that doubling the PS concentration in
the membrane results in an increase in the number of
binding sites (compare Fig. 3D and 3E) and also in an
average site with increased affinity. If Gla proteins
were to bind exclusively to a PS microdomain, then
the affinity is expected to remain conserved for lipo-
somes with varying PS concentration. That is, the

low affinity variants cannot engage with.
PE improved the binding of the mouse protein C
variants when the DOPS concentration was held con-
stant (Fig. 3E,F). We do not know at present whether
the affinity and binding site enhancement is mediated
directly by POPE (e.g. POPE binding sites) or whether
POPE causes indirect effects on DOPS (e.g. membrane
rearrangement).
The free Ca
2+
concentration in plasma is 1–1.5 mm
[24,25]. Protein C ⁄ APC binds to PS in membranes
released by activated platelets in the platelet plug,
within which the Ca
2+
concentration increases to
3–5 mm [24]. These variations in Ca
2+
concentration,
in vivo, may serve a regulatory function in hemostasis
[12]. The Gla domain structure is heavily dependent on
seven Ca
2+
coordinated by several Gla residues at the
core of the domain. The Ca
2+
concentration required
to cause 50% transition of protein C molecules is
approximately 0.5 mm [39] and, thus, under in vivo
Ca

-dependent
membrane binding by up to four-fold. According to
the X-ray structure of bovine prothrombin fragment 1
in complex with Ca
2+
[41], the 10 ⁄ 12 site resides near
the x-loop and the linear array of bound calcium ions
of the Gla domain. Indeed, the importance of the
10 ⁄ 12 site is also apparent when comparing the Ca
2+
-
dependent membrane binding data of mutants V and
II (Fig. 5). Differing only at the 10 ⁄ 12 site, mutant II
(Q10 and N12) has a [Ca
2+
]
1 ⁄ 2 max
that is two-fold
lower than mutant V (P10 and S12). Similar findings
for the 10 ⁄ 12 site were observed when comparing wild-
type and mutant I (data not shown). A recent model
Anticoagulant mouse protein C variants M. J. Krisinger et al.
6596 FEBS Journal 276 (2009) 6586–6602 ª 2009 The Authors Journal compilation ª 2009 FEBS
of the membrane bound Gla domain of FVIIa showed
that the inner calcium ions are required for optimal
folding, allowing membrane insertion, and the outer
calcium ions provide direct anchoring points to the
membrane [40]. The residues at positions 10 and 12
are expected to either influence the stability of the Gla
domain at Ca

Junction, VT, USA). All proteins were judged to be greater
than 98% pure from an overloaded Coomassie blue stained
SDS-PAGE gel. Protein concentrations were determined by
absorbance using the extinction coefficients (E
280
1%,
1 cm) and molecular weights given by the supplier: human
prothrombin: 13.8, 72 000; human prethrombin-1: 17.8,
49 900; mouse protein C: 14.5 (inferred from human pro-
tein C), 56 000 (estimate). The Gla-specific monoclonal
antibody M3B was a kind gift from J. Stenflo (Lund
University, Malmo
¨
, Sweden) and was described previously
[44]. Hirudin was purchased from Pentapharm (Basel,
Switzerland). Chromogenic APC substrate (S-2366) was
purchased from DiaPharma (Mo
¨
lndal, Sweden). Synthetic
phospholipids; POPE, DOPS and POPC were purchased
from Avanti Polar Lipids, Inc. (Alabaster, AL, USA).
Recombinant tissue factor (thromboplastin) was from Dade
InnovinÒ (Marburg, Germany) and dissolved according to
the manufacturer’s instructions and stored at 4 °C. The tis-
sue factor concentration in the thromboplastin stock is
400 ngÆmL
)1
(8.5 nm) according to T. M. Hackeng (Cardio-
vascular Research Institute, Maastricht, The Netherlands,
personal communication). The thrombin fluorogenic sub-

sequencing before transfection. The cDNAs corresponding
to wild-type protein C and protein C variants (QN, QNED,
QNSED, QNS, ED, S and SED) were all inserted into the
eukaryotic expression vector the pcDNA3, transfected into
HEK 293 cells (CRL-1573 ATCC), high-expressing colonies
selected, and recombinant protein C purified, as previously
described [45]. Briefly, the transfected cells were selected
in DMEM ⁄ F-12 (Invitrogen, Carlsbad, CA, USA) supp-
lemented with 10% fetal bovine serum containing
0.25 mgÆmL
)1
Geneticin G418 (Invitrogen) for 3–5 weeks.
G418 resistant colonies were picked and grown in serum free
medium containing 10 lgÆmL
)1
vitamin K
1
(Sigma). Colo-
nies expressing protein C at high levels, assessed by dot blot
assay using polyclonal sheep anti-(mouse protein C) serum
(Haematological Technologies), were picked for further
expression, as described previously [14,45]. High-producing
clones were isolated and grown until confluence in the pres-
ence of 10 lgÆmL
)1
vitamin K
1
. HEK 293 cells expressing
human protein C were previously shown to faithfully
perform c-carboxylation on all glutamic acid residues within

devices with a 10 000 MW cut-off (Bedford, MA, USA) and
then stored at )80 °C. The concentrations of proteins were
established by measuring A
280
. After purification, the puri-
ties of all mouse recombinant protein C preparations were
determined by SDS-PAGE followed by silver staining.
Preparation of mouse activated protein C
Incubation of mouse protein C with human or bovine
thrombin using conditions known to work well for human
protein C [14] were found to result in extra inappropriate
cleavages in the light chain. Many conditions were tried
and finally we determined that the thrombin–TM complex
was required to obtain reliable activation of mouse protein
C. Thus, to generate mouse APC with appropriate cleavage
pattern and full activation, recombinant mouse protein C
mutants (100 lgÆmL
)1
) were incubated with human throm-
bin (2 l gÆmL
)1
) in the presence of human soluble thrombo-
modulin (1 lgÆmL
)1
) and incubated for 24 h in 50 mm
Tris-HCl, 150 mm NaCl, 5 mm CaCl
2
(pH 7.4) at 37 °C.
After incubation, 100 UÆmL
)1

diameter of large unilamellar vesicles by this method is
approximately 110 ± 25 nm. Liposomes were stored at
4 °C and were used within 1 week of production.
Measurement of protein–membrane interactions
by SPR
Binding of mouse protein C variants and of prothrombin
to phospholipid liposomes was quantified by SPR using a
Biacore 2000 instrument (Uppsala, Sweden) at 24 °C. Prior
to lipid immobilization, the lipophilic LI sensor chip was
washed with 40 mm octyl glucoside (1 min at 20 lLÆmin
)1
).
Synthetically-derived phospholipid liposomes (500 lm) were
injected for 17 min at a 3 lLÆmin
)1
flow rate in HBS run-
ning buffer. Liposomes were immobilized to a response of
5000–7000 RU. The chips were washed five times with
10 mm EDTA (pH 8.0) injections (2 min at 20 lLÆmin
)1
),
after which the membrane surface was stable, as indicated
by an insignificant loss in the SPR signal for the subsequent
12 h (data not shown). Liposomes derived from naturally-
derived phospholipids slowly dissociated from the L1 chip
and were thus unsuitable for interaction experiments. For
protein binding experiments, running buffer was changed to
HBC (HBS with 10 mgÆmL
)1
BSA and 5 mm CaCl

) was fitted to a one-site binding
hyperbola according to the relationship R
eq
= R
max-
ÆC ⁄ (K
D
+ C), where R
max
is the binding at saturation or
maximum surface coverage, C corresponds to the injected
analyte concentration and K
D
is the equilibrium dissocia-
tion constant. We are aware of the fact that an analysis of
the overall binding curve is a relatively severe simplification
and that a complex set of reactions is involved in the mem-
brane binding process of any Gla protein [33,41,48–50].
However, this simplification appears to be justified because
the binding curves showed no signs of cooperativity when
analyzed by scatchard plots (M. J. Krisinger, E. L. Pryzdial
and R. T. MacGillivray, unpublished results). BSA (0.1%)
was included to block any nonspecific protein–lipid and
protein–protein interactions [51]. Experiments were carried
out with replicate analyte concentrations, which were essen-
tially identical. Experiments were also measured in random
order with respect to analyte concentrations. All buffers
were filtered through a 0.22 lm filter and degassed before
use. All stock solutions were briefly centrifuged before use
to remove any potential precipitate.

]
1 ⁄ 2 max
+ C), where R
max
is the binding
at saturation (maximum surface coverage), C corresponds
to the Ca
2+
concentration and [Ca
2+
]
1 ⁄ 2 max
is the Ca
2+
concentration required to reach the binding midpoint of the
titration.
Measurement of thrombin generation
Fluorescence measurements over time were taken in a
96-well plate Tecan infinite 200 fluorometer equipped with
a 360 nm excitation ⁄ 460 nm emission filter set (Mo
¨
lndal,
Sweden) and magellan software (Gro
¨
edig, Austria). Black
flat bottom 96-well plates (Nalge Nunc International,
Rochester, NY, USA) were used. Each well of a pre-
warmed plate contained 10 lL of normal pooled mouse
plasma in a total reaction volume of 120 lL. For a typical
experiment, thrombin generation was initiated with 0.25 pm

strate consumption; therefore, thrombin concentration was
expressed as the rate of development of fluorescence inten-
sity (fluorescence units or FU), calculated for every reading
(FUÆmin
)1
). The a2 macroglobulin–thrombin complex,
which forms during thrombin generation in plasma, was
not corrected for because we merely report relative throm-
bin generation rates using an identical plasma sample.
Thrombin generation parameters assessed were: lag-time
(min), maximal concentration of thrombin (C
max
or peak
height in FUÆ min
)1
), the time required to reach maximum
thrombin generation (T
max
in min) and ETP (area under
the curve in total FU determined after 60 min).
Acknowledgements
This work was supported by grants from the Swedish
Research Council (#71430), the Swedish Heart-Lung
Foundation and research funds from the University
Hospital in Malmo
¨
(to B.D.) and an Anna-Greta Crafo-
ords Foundation Research Scholarship (to M.J.K.).
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Anticoagulant mouse protein C variants M. J. Krisinger et al.
6602 FEBS Journal 276 (2009) 6586–6602 ª 2009 The Authors Journal compilation ª 2009 FEBS