Tài liệu Báo cáo khoa học: Mouse recombinant protein C variants with enhanced membrane affinity and hyper-anticoagulant activity in mouse plasma pptx

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

VIIIa on anionic phospholipid membranes, thereby
Keywords
anticoagulation; Gla domain; mouse protein C;
mouse plasma; protein–membrane
interactions
Correspondence
B. Dahlba
¨
ck, Department of Laboratory
Medicine, Division of Clinical Chemistry,
Wallenberg Laboratory, Entrance 46, Floor
6, Lund University, University Hospital,
S-20502 Malmo
¨
, Sweden
Fax: +46 40 337044
Tel: +46 40 331501
E-mail:
(Received 1 July 2009, revised 4 September
2009, accepted 9 September 2009)
doi:10.1111/j.1742-4658.2009.07371.x
Mouse anticoagulant protein C (461 residues) shares 69% sequence identity
with its human ortholog. Interspecies experiments suggest that there is an
incompatibility between mouse and human protein C, such that human
protein C does not function efficiently in mouse plasma, nor does mouse
protein C function efficiently in human plasma. Previously, we described a
series of human activated protein C (APC) Gla domain mutants (e.g.
QGNSEDY-APC), with enhanced membrane affinity that also served as
superior anticoagulants. To characterize these Gla mutants further in
mouse models of diseases, the analogous mutations were now made in

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

, maximal concentration of thrombin; ETP, endogenous thrombin potential; Gla protein, c-carboxyglutamic
acid-containing protein; DOPS, 1,2-dioleoyl-sn-glycero-3-[phospho-
L-serine]; FU, fluorescence units; PE, phosphatidylethanolamine; POPC,
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPE, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; PS, phosphatidylserine;
R
max
, maximum surface coverage; RU, response units; SPR, surface plasmon resonance; T
max
, time required to reach maximum thrombin
generation.
6586 FEBS Journal 276 (2009) 6586–6602 ª 2009 The Authors Journal compilation ª 2009 FEBS
efficiently turning off the major driving force of coag-
ulation. Although historically known for its role in
anticoagulation, APC was recently revealed to have
cytoprotective, anti-inflammatory and anti-apoptotic
functions. These new functions of APC are related
to the ability of APC to bind endothelial protein C
receptor and activate protease activated receptor 1,
triggering intracellular signaling [2–4]. Moreover,
recombinant human APC was recently shown to inhi-
bit integrin-mediated neutrophil migration by a direct
interaction with leukocyte b1 and b3 integrin receptors
[5]. APC appears to play a central role in the patho-
genesis of sepsis and associated organ dysfunction. In
patients with sepsis, the APC system malfunctions at
almost all levels. First, plasma levels of the zymogen
protein C are low or very low because of impaired syn-
thesis, consumption and degradation by proteolytic
enzymes such as neutrophil elastase [6]. Furthermore,
significant down-regulation of thrombomodulin caused

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 relation to their Gla
ligands is almost identical in the two proteins [12]. The
conformational transition induced by the cooperative
binding of Ca
2+
turns the N-terminal part of the Gla
domain inside out, exposing the hydrophobic x-loop
to solvent and burying the majority of the Gla resi-
dues. Of the seven Ca
2+
, the majority are buried and
are integral to maintain the membrane-binding confor-
mation [9,13]. A few of the Gla-bound Ca
2+
are acces-
sible to solvent and may play a role in membrane
binding. A membrane-bound structure of a Gla pro-
tein does not exist, hampering our understanding of
how the Gla domain engages and reversibly binds to a
membrane surface. Thus, it is also unclear how the
Gla domains of the previously engineered mutants
(e.g. human QGNSEDY-APC; see below) have been
selectively altered to enhance membrane binding. How-
ever, it does appear that electrostatic, hydrophobic and
specific lipid headgroup interactions are all involved in
mediating the interaction. The nature of the phospho-
lipid membrane also contributes to binding efficiency,
with phosphatidylserine (PS) and, to a lesser extent,
phosphatidylethanolamine (PE) being generally
accepted as most important membrane phospholipids

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

treatment of different diseases (e.g. thromboembolism
and sepsis) [13]. The low-affinity binding of APC to
negatively-charged phospholipid membranes may be
adequate under normal healthy conditions because
protein S serves as a specific cofactor to increase the
membrane binding of APC at certain locations. The
situation may be different under pathologic conditions
such as sepsis, where a higher membrane-binding abil-
ity of APC could potentially be beneficial, in particular
because protein S and FV may be consumed under
these conditions. High affinity mouse APC variants
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 6587
will allow the in vivo elucidation of the biologic conse-
quences of the enhanced membrane-binding ability of
protein C and may open a path for the development
of APC variants with improved therapeutic potential
in sepsis, as well as other thromboembolic disorders.
Although human protein C variants with enhanced
affinity and function can be created, interspecies
incompatibility in functional assays using human pro-
tein C in animal model systems, prompted us to char-
acterize several mouse protein C variants in the
present study. Individual amino acids residues within
the Gla domain contribute to the membrane affinity
differences reported for the Gla proteins. In the 44-res-
idue Gla domain of human and mouse protein C,
there are eight amino acid differences. Thus, wild-type
mouse protein C as well as seven variants mutated at
three regions (positions 10, 12, 23, 32 and 33) of 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

c-carboxylation of glutamic acid residues is either known to occur or may occur are indicated by X. The numbering at the top refers to the
mouse protein C sequence. Highlighted residues are different with respect to wild-type mouse protein C. Sequences used for comparison
were obtained from NCBI with accession numbers: protein C for mouse (NP_032960.2), human (NP_000303.1), rat (NP_036935.1), bovine
(XP_585990.3) and human prothrombin (NP_000497.1).
Fig. 2. SDS-PAGE analysis of recombinant mouse protein C and
APC. Purified proteins (8 lg) were incubated with human thrombin-
thrombomodulin for 0 h (odd lanes) or 24 h (even lanes). Thrombin
catalysis was stopped with excess hirudin and subjected to 12%
SDS-PAGE under reducing conditions. Approximately 0.1 lg of pro-
tein C (odd numbered lanes) or APC (even numbered lanes) was
applied to each lane and visualized by silver staining. Protein C vari-
ants and molecular weight markers (MWM) ran in each lane are
indicated. The location of heavy chain (HC), light chain (LC) and
thrombin (IIa) is also indicated.
Anticoagulant mouse protein C variants M. J. Krisinger et al.
6588 FEBS Journal 276 (2009) 6586–6602 ª 2009 The Authors Journal compilation ª 2009 FEBS
heavy chains migrated to similar positions. All proteins
were fully activated by the human thrombin–TM com-
plex, as demonstrated by the shift of the heavy chains
to slightly lower molecular weight positions. The amid-
olytic activities of activated protein C mutants were
comparable with that of wild-type protein C (data not
shown). The proteins bound Ca
2+
similar to their
human counterparts, as judged by the shift in mobili-
ties in native agarose gel electrophoresis in the pres-
ence of Ca
2+
compared to EDTA (data not shown).

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

D
 8 lm), with
a value lower than that of the human ortholog
(K
D
= 2.1 lm) [15] and comparable with the bovine
ortholog (K
D
= 9.2 lm) [14], as assessed previously
under similar experimental conditions. Using 0-20-80
or 20-10-70 membranes, mouse protein C variants that
show a considerable improvement in membrane affin-
ity over wild-type are mutant II (12-fold K
D
decrease),
mutant III (six-fold K
D
decrease) and mutants V and
VII (three- to four-fold K
D
decrease). Equilibrium
binding dissociation constants, using 0-10-90 mem-
branes, could only be determined for the high affinity
proteins. A further improvement in membrane binding
of the variants is shown in terms of membrane bind-
ing occupancy at the saturating protein concentration,
a parameter experimentally determined as R
max
. For
0-20-80 membranes, the respective binding R

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

= 3005 RU,
whereas binding to 0-20-80 is improved with
K
D
= 0.66 lm ⁄ R
max
= 3569 RU. PE has been shown
to enhance the assembly and function of several clot-
ting factor complexes [22,23]. We also show that POPE
influences the binding of mouse protein C. Comparing
the binding data of 20-10-70 and 0-10-90 membranes
(Fig. 3E,F and Table 1), we observe the effect that
POPE has on membrane binding when holding DOPS
at a fixed concentration. Substantial improvements in
both the number of binding sites and average affinity
are observed with the POPE containing membrane.
Importance of Ca
2+
on membrane binding
Because the Gla protein–membrane interaction is
highly dependent on Ca
2+
, we also investigated how
the introduced mutations affect membrane binding as
a function of Ca
2+
concentration. Figure 4A presents
a representative sensorgram showing the effect of Ca
2+
on the interaction of mutant II, at fixed concentration,

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

membrane-binding Gla protein.
Fig. 3. Protein–membrane interaction of wild-type and variant mouse protein C to liposomes of varying phospholipid composition. Protein C
variants (wild-type, I–VII at 0.1 l
M) or running buffer was injected for 8 min (association), over either the (A) 0-20-80 or (B) 0-10-90 or (C)
20-10-70 POPE-DOPS-POPC membrane bilayer surface, to determine their relative binding efficiencies. Dissociation under running buffer
conditions was followed for an additional 8 min. Ca
2+
concentrations used throughout were 5 mM. The SPR response curves are shown
after background correction using a blank 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). Similar amounts of 0-20-80 (5539 RU), 0-10-90
(5738 RU) and 20-10-70 (5997 RU) liposomes were immobilized allowing comparisons. Protein labeling is shown. Insets show the same data
on a smaller scale highlighting the low affinity binders. Note y-axis scale differences in (B). Steady-state binding of mouse protein C wild-type
(
), mutant II ( ), III (.) and VII ( ), over either the (D) 0-20-80 or (E) 0-10-90 or (F) 20-10-70 POPE-DOPS-POPC membrane bilayer surface,
was measured using the indicated protein concentrations. Responses obtained at equilibrium were used to generate a binding isotherm
fitted to a one-site binding hyperbola using nonlinear least squares analysis. Binding isotherms were used to determine K
D
reported in
Table 1 and R
max
. Additional details are provide in the Experimental procedures.
Anticoagulant mouse protein C variants M. J. Krisinger et al.
6590 FEBS Journal 276 (2009) 6586–6602 ª 2009 The Authors Journal compilation ª 2009 FEBS
Wild-type protein C and mutant II have different
maximum surface coverage (R
max
) and therefore any
attempt to draw conclusions based on absolute
response values is erroneous. Furthermore, because

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

19% of its potential binding. This indicates that the
mutations introduced in mutants II and III lower the
Ca
2+
concentration requirement for effective binding,
thereby improving membrane affinity. A similar trend
in fractional binding site occupancy is observed for the
0-20-80 and 0-10-90 membranes (data not shown).
Mouse APC variants with hyper-anticoagulant
activity in mouse plasma
The generation of thrombin is severely diminished in
mouse plasma when an APC variant with high affinity
for membranes is included in the reaction (Fig. 6A and
Table 1). Compared at equivalent concentrations
(0.5 nm), mutant III completely abolished thrombin
generation, whereas mutants I, II, V and VII and, to a
lesser extent, mutant IV caused a down-regulation of
thrombin generation compared to wild-type APC,
which did not have an anticoagulant effect at this con-
centration. Although wild-type recombinant APC can
function as an effective anticoagulant (Fig. 6B), as also
shown by Tchaikovski et al. [26], strikingly lower con-
centrations of APC mutants II and III (Fig. 6C,D)
were required to achieve an identical anticoagulant
result. For example, to observe a similar measurable
down-regulation difference of thrombin generation rel-
ative to thrombin generation in the absence of added
APC [as assessed by either maximal concentration of
thrombin (C
max

) 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

(lM)
a
C
max
(FUÆmin
)1
)
e
ETP (FU)
e,f
0-20-80
b
0-10-90 20-10-70 1 nM
g
1nM
g
Buffer control – – – 276 ± 32 5677 ± 171
Wild-type 7.26 ± 0.50
c
NA
d
8.77 ± 0.75 241 ± 20 5380 ± 50
Mutant I 5.55 ± 0.26 NA 4.96 ± 0.16 105 ± 28 2813 ± 682
Mutant II 0.66 ± 0.04 2.45 ± 0.13 0.67 ± 0.05 4 ± 7 737 ± 118
Mutant III 1.32 ± 0.16 8.70 ± 0.98 1.44 ± 0.18 0 ± 0 658 ± 88
Mutant IV 7.10 ± 0.71 NA 6.79 ± 0.45 165 ± 25 4094 ± 674
Mutant V 2.05 ± 0.20 8.40 ± 1.15 2.28 ± 0.17 15 ± 18 927 ± 401
Mutant VI 7.47 ± 0.46 NA 5.99 ± 0.45 247 ± 18 5467 ± 67
Mutant VII 2.55 ± 0.25 8.77 ± 0.75 2.67 ± 0.23 24 ± 26 1175 ± 531
a

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

reduced protein C levels suffering from severe sepsis
(PROWNESS study) [8]. The results of this and several
other proceeding clinical trials [27] lead us to develop
an APC molecule with enhanced anticoagulant activity
with the purpose of investigating the effect of APCs
with increased anticoagulant activity in vivo on differ-
ent thromboembolic diseases. APC has species specific-
ity in its anticoagulant function that may, to a certain
extent, be a result of its interaction with protein S
[21,28,29]. Therefore, we were unable to continue work
in mouse models of sepsis or disseminated intravascu-
lar coagulation with our previously developed human
APC variants (e.g. QGNSEDY-APC). In the present
study, we created several mouse APC variants with
improved membrane binding characteristics and hyper-
anticoagulant activity. Binding ability was established
using a SPR membrane binding assay and anticoagu-
lant activity was assessed by a thrombin generation
assay. The most active variant mutant III (P10Q⁄
S12N ⁄ D23S ⁄ Q32E ⁄ N33D or QNSED), which is the
A
B
C
D
Fig. 4. Effect of Ca
2+
on the mouse protein C–membrane inter-
action. (A) Protein C mutant II (1 l
M) was injected for 4 min (associ-
ation) over a 0-10-90 POPE-DOPS-POPC membrane bilayer surface

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-

rium were used to generate a binding isotherm fitted to a one-site
binding hyperbola using nonlinear least squares analysis. Note that
the 50 m
M Ca
2+
data points were excluded from fitting because
high Ca
2+
concentrations appear to inhibit the membrane inter-
action. Binding isotherms were used to determine half-maximal
Ca
2+
concentration or [Ca
2+
]
½ max
reported in Table 2. Data are
representative of three experiments. Additional details are provide
in the Experimental procedures.
Anticoagulant mouse protein C variants M. J. Krisinger et al.
6592 FEBS Journal 276 (2009) 6586–6602 ª 2009 The Authors Journal compilation ª 2009 FEBS
mouse equivalent of the human QGNSEDY-APC vari-
ant, are discussed in more detail below.
In the ETP assay using mouse plasma, mutant III
served as a far superior anticoagulant compared to
wild-type APC. Mutant III had a 50-fold higher activ-
ity, thereby efficiently down-regulating thrombin gener-
ation, such that a mere 0.02 nm concentration was
required to reduce either C
max

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+

concen-
tration, an optimal phospholipid composition and,
lastly, structures intrinsic to the protein (e.g. optimal
arrangement of residues in the Gla domain). For any
given Gla protein, a good membrane can compensate
for binding at sub-optimal Ca
2+
concentrations.
Conversely, an optimal Ca
2+
concentration can com-
pensate for binding at sub-optimal membrane compo-
sitions. Thus, the results obtained in the present study
indicate that any one of these three factors can com-
pensate for two of the others if they are presented in a
sub-optimal manner.
The protein C mutations act as excellent reporters
of how these residues influence the membrane interac-
tion. Single and double amino acid substitutions at
three separate regions of the protein C Gla domain
were introduced at positions 10 ⁄ 12, 23 and 32 ⁄ 33.
Mutagenesis at position 10 ⁄ 12, as in mutant I (QN),
caused a small (1.5-fold) gain in affinity compared to
wild-type. The major affinity improvement came from
combined mutagenesis at position 32 ⁄ 33 with mutant
V (ED) having a 3.5-fold higher affinity than wild-
type. Similarly, this double mutagenesis caused an
eight-fold higher affinity in mutant II (QNED) relative
to mutant I (QN). A Glu residue (converted to Gla
residue upon post-translational modification) intro-

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+

).
Table 2. Half-maximal binding [Ca
2+
] as functions of various mem-
brane compositions were determined by SPR for mouse protein C.
Ca
2+
titration determined at fixed (1 lM) protein concentration.
Membrane (POPE-DOPS-POPC)
a
[Ca
2+
]
1 ⁄ 2 max
(mM)
b
0-20-80 0-10-90 20-10-70
mPC wild type 15.9 ± 7.0
c
15.2 ± 11.1 10.8 ± 3.43
mPC mutant II 3.7 ± 1.6 7.2 ± 3.4 3.7 ± 1.5
mPC mutant III 3.6 ± 1.6 6.7 ± 3.5 3.7 ± 1.6
mPC mutant V 9.0 ± 5.0 16.7 ± 12.0 8.0 ± 3.7
Human prothrombin 1.4 ± 0.6 2.9 ± 0.9 1.8 ± 0.6
a
Membranes used were 100 nm extruded liposomes with
synthetic phospholipids: POPE-DOPS-POPC (mol % indicated).
b
Half-maximum binding [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

[16]. Mutagenesis at less sites in human protein C, as
in variants GNED (approximately one-fold ⁄ 1.5-fold),
QGN (1.1-fold ⁄ one-fold) and SEDY (1.6-fold ⁄ one-
fold), had negligible or minor improvements in affinity
and anticoagulant activity, respectively [16]. Although
a single amino acid in some Gla proteins can signifi-
cantly influence affinity upon mutagenesis, there
appears to be other additional mechanisms that can
control the affinity of Gla proteins, as illustrated with
Pro10 of the human, bovine and mouse protein C
orthologs. A Pro at position 10, occurring naturally or
introduced by site-directed mutagenesis was shown to
significantly lower the membrane affinity of Gla pro-
teins. In bovine protein C, P10H mutagenesis results in
a ten-fold affinity increase, whereas, with human pro-
tein C, H10P mutagenesis results in a five-fold affinity
A
B
C
D
Fig. 6. Inhibition of thrombin generation in mouse plasma by
mouse APC and variants. (A) Mouse plasma was incubated with
either 0.5 n
M extrinsically added wild-type mouse APC, or variant
(I–VII), or in the absence of added protein (as indicated on curves).
Thrombin generation was initiated with 0.25 p
M tissue factor,
10 l
M phospholipid liposomes and 16.7 mM CaCl
2

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-

ulant activity that is not dependent on membrane
binding. In human proteins, protein S binds to the
protein C Gla domain. Although this interaction has
been mapped to Gla regions C-terminal to the 23 site
[31,32], it may still influence the interaction to the
cofactor and thus influence the anticoagulant activity
of APC. An influence of position 23 on the interaction
to APC substrates FVa and FVIIIa also cannot be
ruled out. The same reasoning can be applied when
comparing membrane binding and anticoagulant activ-
ity data of mutant V and mutant VII, which also only
vary at position 23. Thus, 23S appears to be better for
membrane binding, but 23D appears to instill a better
anticoagulant property in mouse APC.
Membrane binding capacity is a measure of the Gla
protein packing density to sites provided by the mem-
brane at saturating protein concentrations. Of rele-
vance for this discussion is the variation of membrane
binding site occupancy amongst the different mouse
protein C variants (Fig. 3D–F). For example, steady-
state R
max
for wild-type and mutant II differ by
approximately five-fold to a 0-20-80 membrane sur-
face. In the classical view of a simple bimolecular
(1 : 1) interaction, a receptor site can be saturated with
different affinity analytes (of equal molecular weight),
which, by definition, will all have an equivalent satura-
tion response (i.e. occurring when all receptor sites are
occupied) that will be approached by a specific analyte

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

engage with these membrane binding sites with an
overall higher averaged affinity then wild-type mouse
protein C. These two processes allow substantially
more protein to bind membrane when eqimolar con-
centrations of these proteins are compared.
Membrane binding on- and off-rates are probably
important specifications for the functions of protein
C ⁄ APC as well as the other Gla proteins. Association
with the membrane is likely a dynamic process involv-
ing several bound intermediates. An initial membrane
engagement step (termed electrostatic docking) [33],
followed by the association of an unknown number of
PS headgroups, hydrophobic x-loop insertion into the
Fig. 7. Concentration-dependent inhibition of thrombin generation
in mouse plasma by mouse APC. Mouse plasma was incubated in
the absence of protein or with the indicated concentration of extrin-
sically added wild-type mouse APC or variant (I–VII). Thrombin gen-
eration was initiated as described in Fig. 6. % C
max
is expressed as
the maximum concentration of thrombin generated in the presence
of APC divided by the maximum concentration of thrombin gener-
ated in the absence of APC. Mean values from duplicate determi-
nations from a single experiment (data for 0.1, 5 and 10 n
M) and
the mean ± SD from three independent experiments each with
duplicate determinations (data for 0.5 and 1 n
M) are shown (log
2
scale).

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

identical patch would exist on a 10% and 20%
PS-containing membrane, with the only difference
being that the surface area of the PS patch would be
two-fold greater in the 20% PS liposomes. Consistent
with the binding to a few PS molecules, it has been
estimated previously, in experiments using phospho-
lipid containing nanodiscs [34] or liposomes [33,35],
that approximately up to five PS molecules can be uti-
lized to bind a Gla domain to a membrane surface.
These derived values are in line with the physical
dimensions of a Gla domain having a membrane foot-
print that occupies an area of approximately 3.9 nm
2
[36] or the equivalent of six phospholipid headgroups
(one phospholipid headgroup has a surface area of
 0.7 nm
2
) [37]. In addition, bovine Gla proteins, pro-
tein Z and factor X, were shown to cluster only a
small number (approximately four) of fluorescent-
labeled anionic phospholipids [38]. Thus, the simple
model membranes used in the present study, composed
of even two types of synthetic phospholipids (DOPS
and POPC), likely provide several compositionally dif-
ferent binding sites that Gla proteins can engage with.
Because binding to 100% POPC liposomes is insignifi-
cant, the different binding sites utilized by the different
variants must be a function of DOPS content. Thus,
an enticing hypothesis is that the high affinity variants
can utilize membranes sites with lower PS content that

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

2+
concentrations, approximately 65–90% of
protein C is expected to be in a membrane binding
conformation. An ordered Gla domain is a prerequi-
site enabling a subsequent membrane interaction. Gla
domain destabilizing mutations thus also reduce mem-
brane affinity. Furthermore, externally bound calcium
ions are considered to form a direct link between pro-
tein and membrane, thereby providing an additional
mode of interaction [40]. Some putative conclusions
can be made on the mutations made in the present
study and their involvement in Ca
2+
-dependent mem-
brane binding. Because wild-type mouse protein C and
mutant V (ED) have very similar Ca
2+
-dependent
membrane binding properties, the QN to ED muta-
tions at the 32 ⁄ 33 site are not suspected to significantly
stabilize the Gla domain in a Ca
2+
limiting environ-
ment or help in providing additional Ca
2+
bridging
anchoring points to the membrane. However, further
mutagenesis at the 10 ⁄ 12 site (PS to QN; e.g. as in
mutants II and III) improved the Ca
2+

-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

2+
limiting concentrations and ⁄ or pro-
vide additional direct membrane anchoring points via
Ca
2+
. Interestingly, human prothrombin, which also
lacks a Pro at position 10 and has an Asn at position
12, also has excellent membrane binding properties at
low Ca
2+
concentrations.
In conclusion, several high affinity, high binding
capacity, Gla domain mutated mouse protein C vari-
ants were established, which, upon activation, served
as superior anticoagulants during tissue factor-depen-
dent coagulation. These activated protein C variants
may therefore be beneficial for the treatment of severe
sepsis and should be studied further in appropriate
mouse models.
Experimental procedures
Reagents
A full-length mouse protein C cDNA clone was the gener-
ous gifts from E. M. Conway (University of Leuven,
Leuven, Belgium). The TM cDNA clone was purchased
from ATCC (Rockville, MD, USA) and soluble TM was
produced as described previously [42]. Human prothrombin
was purchased from Enzyme Research Laboratories,
Kordia (Leiden, The Netherlands). Human prethrombin-1
was prepared as described previously [43]. Human thrombin
was purchased from Haematological Technologies (Essex

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-

strate I-1140 (Z-Gly-Gly-Arg-7-amino-4-methylcouma-
rinÆHCl, 300 lm) was obtained from Bachem (Bubendorf,
Switzerland) and stored as 2.5 mm stock in 2.5% dimethyl-
sulfoxide at )80 °C. Citrated platelet-free plasma pooled
from 48 CD-1 mice was from Sera Laboratories Interna-
tional Ltd (Haywards Heath, UK). Mouse plasma was cen-
trifuged for 2 min at 17 000 g to remove any precipitate,
stored in aliquots at )70 °C and thawed once at 37 °C just
prior to use. One out of every two samples from the sup-
plier was discarded because it did not contain any clotting
activity. Inter-lot variability of functional mouse plasma
was less than 5% by peak height and almost indistinguish-
able by lag-phase and time to peak parameters (results not
shown). Hepes buffer and BSA were obtained from Sigma
(St Louis, MO, USA). All SPR reagents including L1 sen-
sor chips were purchased from GE Healthcare (Uppsala,
Sweden). All other reagents and buffer components were
commercially available and of highest the purity.
Mutagenesis, construction and stable expression
of recombinant mouse protein C and variants
Mutagenesis was performed using QuikChange (Stratagene,
La Jolla, CA, USA) in accordance with the manufacturer’s
instructions (primer sequences and mutagenesis strategy are
described in the Supporting information) [14]. In total, the
following seven mutants were created: mutant I, P10Q ⁄
S12N (QN); mutant II P10Q ⁄ S12N ⁄ Q32E ⁄ N33D (QNED);
mutant III, P10Q ⁄ S12N ⁄ D23S ⁄ Q32E⁄ N33D (QNSED); mu-
tant IV, P10Q ⁄ S12N ⁄ D23S (QNS); mutant V, Q32E ⁄ N33D
(ED); mutant VI, D23S (S); and mutant VII, D23S ⁄ Q32E ⁄
N33D (SED). All mutations were confirmed by DNA

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

the Gla domain [15].
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 6597
Mouse protein C purification
The purification of mouse recombinant protein C and vari-
ants was based on the method described previously for
human and bovine recombinant protein C [14] with some
modifications. Serum-free conditioned medium (5 L) from
stably transfected cells containing each recombinant mouse
protein C was supplemented with 5 mm benzamidine and
4mm EDTA, centrifuged at 6000 g at 4 °C for 10 min and
batch absorbed with 40 mL of Q Sepharose Fast Flow
matrix (Amersham Biosciences, Uppsala, Sweden) by over-
night incubation at 4 °C. The gel slurry was packed in a col-
umn, extensively washed with 20 mm Tris-HCl, 150 mm
NaCl (pH 7.4) and eluted with buffer plus 10 mm CaCl
2
.
Protein C containing fractions were collected and CaCl
2
was
removed by overnight dialysis against 20 mm Tris, 50 mm
NaCl (pH 7.4) using Dialys Spectra ⁄ Por 4 dialysis mem-
branes with MW cut-off: 12–14 000 (Spectrum Laboratories
Inc., Rancho Dominguez, CA, USA) and the proteins reab-
sorbed to a second HiprepÔ 16 ⁄ 10 Q FF column (Amer-
sham Biosciences) after which it was eluted with a NaCl
gradient (starting solution: 20 mm Tris, 50 mm NaCl, pH
7.4, limiting solution: 20 mm Tris, 1 m NaCl, pH 7.4). Pro-
teins were concentrated using Amicon Ultra centrifugal filter

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

hirudin was added and the
APC reaction products were assessed by SDS-PAGE fol-
lowed by silver staining. Either the preparation was used as
is, or the thrombin-hirudin-TM was removed by chroma-
tography on a HiprepÔ 16 ⁄ 10 Q FF column as described
for protein C purification. Enzymatically active APC con-
centrations were determined by active site titration using
the APC chromogenic substrate S-2366 essentially as
described previously [14]. The activity of mouse APC was
consistent for all preparations of wild-type and variants of
APC (variation: < 5%)
Phospholipid liposome preparation
Liposomes were prepared as described previously [46] with
minor revisions. Briefly, lyophilized lipids were suspended
in chloroform to approximately 30–100 mm and concentra-
tions were established by inorganic phosphate determina-
tion, as described previously [47]. The appropriate molar
ratios of phospholipid in chloroform were dried, first under
a stream of nitrogen and then under vacuum, for at least
3 h. The resultant lipid residue was resolubilized in HBS
(10 mm Hepes, 150 mm NaCl, pH 7.4) to a final concentra-
tion of approximately 10 mm. The resultant multilamellar
vesicle suspension was subjected to a rapid freeze-thaw
technique five times by cycling in liquid nitrogen and warm
water. Large unilamellar vesicles were generated by extru-
sion of multilamellar vesicles under pressure through two
stacked Nucleopore polycarbonate filters (Avestin Inc.,
Ottawa, Canada) with a 100 nm pore size (19 passes) using
a LiposoFast microextruder (Avestin Inc.). Membrane com-
position is stated as mol % POPE-DOPS-POPC. The mean

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

2
) and
flow cells were equilibrated until the baseline stabilized to
less than 0.05 RUÆmin
)1
. Association and dissociation times
each were typically in the range 4–12 min. Equilibrium
response data were collected for each protein at several
concentrations typically spanning ten-fold below and, if
possible, ten-fold above the K
D
of the interaction, with
10 lm being the highest concentration tested. Prethrombin-
1, a Gla domain-less fragment of prothrombin known not
to interact with membranes, was used as a negative control.
Either a blank or 100% POPC liposome control flow cell
was used to subtract RU as a result of the refractive index
Anticoagulant mouse protein C variants M. J. Krisinger et al.
6598 FEBS Journal 276 (2009) 6586–6602 ª 2009 The Authors Journal compilation ª 2009 FEBS
of the protein solution and any instrument noise. No bind-
ing was detected in either control flow cells for any of the
proteins tested. The immobilized liposome surface could be
regenerated by removing the Ca
2+
-dependent membrane-
bound protein (prothrombin, protein C wild-type and vari-
ants) with an injection of 10 mm EDTA (pH 8.0), which
returned the baseline to the value prior to introducing pro-
tein. Equilibrium data (R
eq

) 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.

To determine the effect of Ca
2+
, the immobilized phos-
pholipid liposomes were equilibrated with buffer containing
a variable Ca
2+
concentrations (0, 0.5, 1, 2, 5, 10, 20 and
50 mm). Proteins were diluted such that HBC and Ca
2+
concentration were exactly matched to running buffer and
allowed to equilibrate for at least 30 min prior to injection.
After protein injections and sensorgrams were completed
for a single Ca
2+
concentration, running buffer was
exchanged and primed three times with the subsequent
HBC. Buffers were randomized. No binding was detected
in buffer lacking Ca
2+
for any of the proteins tested.
Protein binding equilibrium data (R
eq
) were fitted to a one-
site binding hyperbola according to the relationship R
eq
=
R
max
ÆC ⁄ ([Ca
2+

]
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

tissue factor (a validation of thrombin generation in mouse
plasma and the effect of exogenously added tissue factor is
provided in Fig. S2), 10 lm phospholipid liposomes (20-20-
60 POPE-DOPS-POPC) and 16.7 mm CaCl
2
in buffer
(25 mm Hepes, 175 mm NaCl, pH 7.4, containing 0.5%
BSA) and continuously followed with the fluorogenic sub-
strate I-1140 (Z-Gly-Gly-Arg-AMC; 300 lm)at37°C. All
concentrations are final. Prior to the start of the reaction,
reagents were incubated at 37 °C: tissue factor, phospho-
lipid liposomes and CaCl
2
were incubated together for 1 h,
the fluorogenic substrate was incubated for 30 min and the
mouse plasma was thawed for 2 min. Reagents were added
and mixed in a 96-well plate in the order (added vol-
ume ⁄ well, incubation time): six-fold buffer diluted mouse
plasma (60 lL, 3 min), APC sample (20 lL, 5 min), fluoro-
genic substrate (20 lL, 3 min) and tissue factor ⁄ lipo-
some ⁄ CaCl
2
(20 lL), after which readings were taken
immediately at 1 min intervals for approximately 60 min.
Each experiment was preformed in duplicate. The first
derivative of the fluorescence versus time plot (not shown)
was taken using GraphPad Prism, version 4 (graphpad
Software Inc., San Diego, CA, USA) to obtain the thromb-
ogram (or fluorescence ⁄ time versus time plot). Fluorescence
data were not corrected for the inner filter effect or sub-

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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Supporting information
The following supplementary material is available:
Fig. S1. Gla-specific antibody M3B recognizes mouse
protein C variants.
Fig. S2. Thrombin generation in mouse plasma and
the effect of tissue factor concentration.
Doc. S1. Primer sequences and mutagenesis strategy
for recombinant mouse protein C variants.
This supplementary material can be found in the
online version of this article.
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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