Modified Serpins for the Treatment of Bleeding Disorders
Field
This invention relates to modified serpin molecules with altered
specificity, in particular serpin molecules modified to have
increased specificity for anticoagulant proteases, such as activated
Protein C (APC) .
Background
Hemophilias are bleeding disorders, which are caused by a deficiency
in circulating plasma fV (hemophilia A , HA) or f x (hemophilia B ,
HB) (reviewed in Bolton-Maggs & Pasi, 2003) . This reduces the
activity o f the intrinsic tenase (Xase) and thereby the amount of
thrombin generated when tissue injury occurs. This leads to
uncontrolled bleeding after injury as well as spontaneous bleeding
into joints and soft tissue.
Hemophilia affects around 1 in 5,000 people. The 170,000 patients
identified in the World Federation of Hemophilia Global Survey is an
underestimate of the global health burden (World Federation of
Hemophilia, 2011) . The treatment costs are very high and treatment
is frequent and lifelong.
Standard treatments for hemophilia entail replacement of the
clotting factor affected, using either recombinant or plasma-derived
factors (reviewed in Mannucci, 2003; 2008) . However, a significant
proportion of patients treated in this manner will develop
inhibitory antibodies against the supplemented coagulation factor,
rendering the treatment ineffective (reviewed in Brettler, 1996) .
Inhibitors occur in 30% of treated patients with hemophilia
(reviewed in Teitel & Sholzberg 2013) but global estimates are low
due to high mortality in untreated inhibitor patients and a low
prevalence of inhibitors in many countries in which factor VIII
replacement therapy is not available. Another drawback of
conventional therapies is their expense, as well as the short halflife
of the injected clotting factor, necessitating frequent
treatments (reviewed in Lee et al, 2006) .
In the case where patients develop inhibitory antibodies, bypassing
agents are used for treatment of bleeding events (reviewed in
(Negrier et al, 2006) ). Bypassing agents reduce bleeding without
directly supplying the clotting factor affected; they yp ss' the
activity of the tenase complex. Examples of current bypassing agents
include recombinant fVI and FEIBA (Factor Eight_Bypassing
Activity), a prothrombin complex concentrate. These replacement
treatments are very expensive (Bohn et al, 2004; Di Minno e al,
2010; Gringeri et al, 2003; Escobar, 2010) and need to be given even
more frequently than the conventional therapies and in high doses
due to the short half-lives of both products (reviewed in Haya et
al, 2007). In addition, patient response has been shown to be
variable and unpredictable (reviewed in Berntorp, 2009) .
In addition, the short half-life of factor concentrates renders
standard replacement therapy of hemophilia suboptimal . This is
particularly evident in hemophilia A as factor VIII has a half-life
of less than 12 hours. Consequently, despite the availability of
treatment for both hemophilia A and B the bleeding rates are higher
in hemophilia A and chronic hemophilic arthropathy is more common.
This may be related to the short half-life of factor VIII and
consequently the difficulty in maintaining a hemostatic level of
factor VIII (Escobar and Sallah 2013) . In a national review of
treatment the annual frequency of bleeding in patients with severe
hemophilia A without inhibitors was 14 compared to 9 in patients
with hemophilia B (Nagel, et al 2011). The need for musculoskeletal
surgery was 3-times greater in patients with hemophilia A .
Tagariello et al found that patients with hemophilia A required
joint replacement three times more often than patients with
hemophilia B (Tagariello, et al 2009) . Lowe et al found that
hospitalization was required three times more frequently for
patients with hemophilia A compared to hemophilia B (Lowe and Ludlam
2008) .
Current treatments for bleeding disorders, such as hemophilia
therefore have a range of drawbacks .
Summary
The present inventors have recognised that the specificity of serpin
molecules can be engineered by modification of residues within the
reactive center loop (RCL) and have identified modified serpin
molecules with increased specificity for anticoagulant proteases.
These modified serpin molecules may be useful in therapy, for
example for the treatment of bleeding.
An aspect of the invention provides a modified serpin having
mutations at one or more of residues P4, P2, PI and PI' in the
reactive center loop (RCL) thereof.
Another aspect of the invention provides a modified serpin having
mutations at one or both of residues PI' and P2 and optionally
residues P4 and/or PI in the reactive center loop (RCL) thereof.
The mutations may increase the inhibition of activated Protein C
relative to the inhibition of thrombin.
The mutations may also increase the inhibition of activated Protein
C relative to the inhibition of other procoagulant proteases, such
as fVIIa, fIXa, fXa and fXIa.
Other aspects of the invention relate to the use of modified serpins
as described herein for the treatment of bleeding, for example
bleeding in patients with heritable bleeding disorders and acquired
bleeding, including trauma, surgery and in patients receiving
anticoagulant therapy.
Brief Description of Figures
Figure 1 shows the coagulation cascade and the regulatory role of
serpins in this cascade.
Figure 2 shows the results of a prothrombin time (PT) assay to
determine the effect of Protein C Inhibitor (PCI) with a 21 residue
N-terminal truncation (N-terminal residue is A22 of the wild-type
sequence, when counting residue 1 of the propeptide as residue 1 o f
the mature protein) and having K residues at the P2 and PI'
positions within the RCL (A22 P2KP1'K PCI) on coagulation through
the tissue factor pathway (extrinsic) . Pooled normal plasma from
three separate plasmas was incubated with either no PCI (black bar,
- ) or 5 A22 wild-type (WT) PCI (grey bar, WT) or 5 A22 P2KP1'K
PCI (white bar, P2KPl'K). Coagulation was initiated by the addition
of PT reagent, to initiate coagulation via the extrinsic pathway and
the time until clot formation measured. The assay was performed in
triplicate, error bars show the standard deviation. 2-fold diluted
plasma was used to increase the sensitivity of the assay for
inhibitors of coagulation. A22 P2KPl'K PCI has no effect on
coagulation in this prothrombin time (PT) assay. This result
indicates that there is no significant inhibition o f TF: fVIIa,
thrombin and other procoagulant proteases by A22 P2KP1'K PCI.
Figure 3 shows the results of an activated partial thromboplastin
time (aPTT) assay to determine the effect o f A22 P2KPl'K PCI on
coagulation through the contact activation pathway (intrinsic) . Fig
3A shows pooled normal plasma from three separate plasmas incubated
with either no PCI (black bar, - ) or 5 of A22 wild-type (WT) PCI
(grey bar, WT) or A22 P2KP1'K PCI (white bar, P2KP1'K). aPTT reagent
was added and the samples incubated for 5 min at 3 °C. Coagulation
was then initiated b y the addition of CaCl2, to initiate coagulation
via the intrinsic pathway and the time until clot formation
measured. Bars show averages o f at least three measurements, error
bars show the standard deviation. The assay was stopped at 300 s .
Samples shown at 300 s did not clot within the time of the
experiment and are marked with asterisks. Fig 3B shows the data from
A without A22 W T PCI samples to show a small effect on the clotting
time .
Figure 4 shows the results o f a prothrombin time assay (PT)
determining the effect of a full-length (FL) ai-antitrypsin
Pittsburgh (Pitts) mutant (M358R = PlR) that further comprises a
C232S mutation and P357 (P2) and S359K (Pl'K) mutations within the
RCL (FL o T Pitts C232S P2KP1'K) on coagulation through the tissue
factor pathway (extrinsic) . Pooled normal plasma from three separate
plasmas was incubated with either no (-, black bar) or 5 FL
AT Pitts C232S (Pitts, grey bar) or 5 FL oi Pitts C232S
P2KP1'K (P2KP1'K, white bar). Coagulation was initiated by the
addition of PT reagent and the time until clot formation measured.
Bars show averages of at least three measurements, error bars show
the standard deviation. 2-fold diluted plasma was used to increase
the sensitivity of the assay for inhibitors of coagulation. FL oii T
Pitts C232S P2KP1'K, unlike FL o i Pitts C232S does not prolong the
PT . This indicates there is no significant inhibitory effect towards
any of the procoagulant proteases, including TF-fVIIa, thrombin, or
fXa.
Figure 5 shows the results of an activated partial thromboplastin
time assay (aPTT) to determine the effect of FL Pitts C232S
P2KP1'K on coagulation through the contact activation pathway
(intrinsic) . Fig 5A shows pooled normal plasma from three separate
plasmas incubated with either no Oi (black bar) or increasing
concentrations of FL Pitts C232S (grey bars) or FL AT Pitts
C232S P2KP1'K (white bars). aPTT reagent was added and the samples
incubated for 5 in at 37 "C. Coagulation was then initiated by the
addition of CaCl2 and the time until clot formation measured. Bars
show averages of at least three measurements, error bars show the
standard deviation. The assay was stopped at 300 s . Samples shown at
300s did not clot within the time of the experiment and are marked
with asterisks. Fig 5B shows the data from A without FL OiiAT Pitts
C232S samples to show a small effect on the clotting time by FL 
Pitts C232S P2KP1'K. However, this effect does not increase dosedependently
.
Figure 6 shows that FL < Pitts C232S inhibits thrombin generation
in normal human plasma (NP) . Figs A-C show representative thrombin
generation curves for reactions containing increasing concentrations
of FL o lAT Pitts C232S in the presence of (A) no thrombomodulin (TM)
(B) 1.25 nM thrombomodulin (TM) (C) 10 nM thrombomodulin (TM) .
Curves show an average of duplicates. All assays were performed in
pooled normal human plasma (NP) from George King Biomedical.
Coagulation was initiated by the addition of CaCl2 and
TF/phospholipid (RB low TF and phospholipid reagent #5006210,
Technoclone GmbH) to activate coagulation through the extrinsic
pathway. Thrombin generation was measured through the cleavage of a
fluorogenic substrate (Z-Gly-Gly-Arg-AMC) . Fluorescence units were
converted to thrombin concentration by calibrating fluorescence
units against known concentrations of thrombin, using the
Technothrombin calibration kit (Technoclone) . Fig 6D shows mean
ETPs (endogenous thrombin potentials) , representing the total amount
of thrombin generated during the reactions. Bars show the mean of
two independent experiments performed in duplicate. Error bars
represent the standard deviation. The thrombomodulin (TM) used was
recombinantly produced from HEK-EBNA cells and consists of the
extracellular domain of TM.
Figure 7 shows that FL Pitts C232S P2KP1'K rescues the
anticoagulant effect of TM in normal human plasma (NP) . Fig 7A-C
show representative thrombin generation curves for reactions
containing increasing concentrations of FL o A T Pitts C232S P2KP1'K
in the presence of (A) no TM (B) 1.25 nM TM (C) 10 nM TM. Curves
show an average of duplicates. All assays were performed in pooled
normal human plasma (NP) from George King Biomedical. Coagulation
was initiated by the addition of CaCl2 and TF/phospholipid (RB low
TF and phospholipid reagent #5006210 Technoclone GmbH) to activate
coagulation through the extrinsic pathway. Thrombin generation was
measured through the cleavage of a fluorogenic substrate (Z-Gly-Gly-
Arg-AMC) . Fluorescence units were converted to thrombin
concentration by calibrating fluorescence units against known
concentrations of thrombin, using the Technothrombin calibration kit
(Technoclone GmbH) . Fig 7D shows mean ETPs (endogenous thrombin
potentials) , representing the total amount of thrombin generated
during the reactions. Bars show the mean of three independent
experiments performed in duplicate. Error bars represent the
standard deviation.
Figure 8 shows that FL i Pitts C232S abolishes thrombin
generation in human hemophilia A plasma (HA, fVHI-def icient) , and
human hemophilia B plasma (HB, fIX-def icient) . Graphs show the mean
ETPs from thrombin generation experiments spiked with increasing
concentrations of FL o iA T Pitts C232S in either (A) fVIII-def icient
plasma (less than 1% fVIII activity) or (B) fIX-def icient plasma
(less than 1% f x activity) with the indicated amounts o f added
thrombomodulin (TM) . All plasmas were from George King Biomedical.
Reactions were initiated by adding CaCl 2 and TF/phospholipid (RB low
TF and phospholipid reagent #5006210 Technoclone GmbH) with 1:4,000
final dilution o f Dade Innovin (Siemens) to activate coagulation
through the extrinsic pathway. Thrombin generation was measured
through the cleavage of a fluorogenic substrate (Z-Gly-Gly-Arg-AMC) .
Fluorescence units were converted to thrombin concentration by
calibrating fluorescence units against known concentrations of
thrombin, using the Technothrombin calibration kit (Technoclone) .
Mean ETPs are shown from at least two independent experiments
performed in duplicate. Error bars show standard deviations.
Figure 9 shows that FL i T Pitts C232S P2KPl'K rescues the effect
o f TM on human HA plasma (fVIII-def icient plasma) . Figs 9A-C show
representative thrombin generation curves for reactions containing
increasing concentrations o f FL iA T Pitts C232S P2KP1'K in the
presence of (A) no TM (B) 1.25 nM TM (C) 5 nM TM. Curves show an
average o f duplicates. All assays were performed in fVIII-def icient
plasma (less than 1% fVIII activity) from George King Biomedical.
Coagulation was initiated by the addition of CaCl2 and
TF/phospholipid {RB low TF and phospholipid reagent #5006210,
Technoclone GmbH) and 1:4,000 Dade Innovin (Siemens) to activate
coagulation through the extrinsic pathway. Thrombin generation was
measured through the cleavage of a fluorogenic substrate (Z-Gly-Gly-
Arg-AMC) . Fluorescence units were converted to thrombin
concentration b y calibrating fluorescence units against known
concentrations of thrombin, using the Technothrombin calibration kit
(Technoclone) . Fig 9D shows mean ETPs (endogenous thrombin
potentials) , representing the total amount of thrombin generated
during the reactions. Bars show the mean of two independent
experiments performed in duplicate. Error bars represent the
standard deviation.
Figure 10 shows that FL A T Pitts C232S P2KP1'K rescues the effect
of TM o human B plasma (fIX-def icient plasma) . (A-C)
Representative thrombin generation curves are shown for reactions
containing increasing concentrations o f FL Pitts C232S P2KPl'K
in the presence of (A) no TM (B) 1.25 nM TM (C) 5 nM TM. Curves show
an average of duplicates. All assays were performed in fIX-def icient
plasma (less than 1 % fix activity) from George King Biomedical.
Coagulation was initiated b y the addition o f CaCl and
TF/phospholipid (RB low TF and phospholipid reagent #5006210,
Technoclone GmbH) and 1:4,000 Dade Innovin (Siemens) to activate
coagulation through the extrinsic pathway. Thrombin generation was
measured through the cleavage of a fluorogenic substrate (Z-Gly-Gly-
Arg-AMC) . Fluorescence units were converted to thrombin
concentration by calibrating fluorescence units against known
concentrations of thrombin, using the Technothrombin calibration kit
(Technoclone) . Fig 10D shows mean ETPs (endogenous thrombin
potentials) , representing the total amount of thrombin generated
during the reactions. Bars show the mean of at least two independent
experiments performed in duplicate. Error bars represent the
standard deviation.
Figure 11 shows the effect o f FL o A T Pitts C232S and FL xiA T Pitts
C232S P2KP1'K in HB mouse plasma. Fig 11A-D show representative
thrombin generation curves for reactions containing increasing
concentrations of (A-B) FL cxiAT Pitts C232S or (C-D) FL T Pitts
C232S P2KP1'K in the presence of (A and C ) no TM (B and D ) 750 nM
soluble human TM. All assays were performed in HB mouse plasma
collected by tail bleed into citrate, centrifuged to remove red
cells and frozen at -80 °C . Coagulation was initiated b y the addition
o f CaCl2, TF/phospholipid (RB low TF and phospholipid reagent,
Technoclone) and 1:12,000 Dade Innovin (Siemens) to activate
coagulation through the extrinsic pathway. Thrombin generation was
measured through the cleavage of a fluorogenic substrate (Z-Gly-Gly-
Arg-AMC) . Fluorescence units were converted to thrombin
concentration b y calibrating fluorescence units against known
concentrations of thrombin, using the Technothrombin calibration kit
(Technoclone ). Fig E shows mean ETPs (endogenous thrombin
potentials) , representing the total amount of thrombin generated
during the reactions. Bars show the mean of four independent
experiments. Error bars represent the standard deviation. All assays
were performed at 33 °C.
Figure 12 shows that FL T Pitts C232S P2KP1'K reduces bleeding in
HB mice in a tail clip assay. Tail clip results show the total blood
loss over a 10 min collection period after tail transection at 3 mm
diameter for WT mice or HB mice. Mice were injected either with PBS,
FL oiiAT Pitts C232S or FL Pitts C232S P2KP1'K at the indicated
doses in a total injection volume o f 200 ΐ . Protein solutions were
made up to 200 ΐ using PBS. The volume blood loss was determined by
collection of blood from the tail into 14 ml saline. The collected
blood was spun down and the red cells lysed, followed by measurement
of the absorbance at 575 nm. A standard curve was constructed by
determining the absorbance at 575 nm after red cell lysis using
known volumes of collected blood. Blood loss in the experimental
samples was then calculated from this standard. Each point shown
indicates data from a single mouse and the horizontal lines show the
mean of all animals per group. P values were calculated using an
unpaired t-test . Circles indicate WT mice injected with PBS, squares
indicate HB mice injected with PBS, triangles indicate HB mice
injected with 7.5 mg/kg FL c T Pitts C232S, inverted triangles
indicate HB mice injected with 7.5 mg/kg FL OiiAT Pitts C232S P2KP1'K
and diamonds indicate HB mice injected with 15 mg/kg FL iAT Pitts
C232S P2KP1'K.
Figure 13 shows that FL oiiAT Pitts C232S P2KP1'K increases stable
clot formation in HB mice in a cremaster arteriole laser injury
model. HB mice were infused with a fluorescently tagged a-fibrin
antibody and a f luorescently tagged antibody for labeling platelets
through a jugular vein cannula. Controls indicate the baseline level
of clot formation after injury in mice infused with antibodies only.
m indicates the number of mice per condition, n indicates the number
of injuries performed for that condition. Light grey indicates no
clot, dark grey indicates a platelet only clot, and black represents
clots containing platelets and fibrin.
Figure 14 shows that FL iA Pitts C232S P2KP1'K increases stable
clot formation in B mice in a cremaster arteriole laser injury
model. Figure 14 shows representative raw data o f the results shown
in Figure 13. Fluorescence was quantified over time from all
injuries for each condition. Graphs display the median value from
all injuries in the indicated condition. Number of mice and total
number o f injuries were as follows: Control: 5 mice, 8 injuries; FL
o T Pitts C232S at 7.5 mg/kg: 1 mouse, 7 injuries; FL oi A T Pitts
C232S P2KP1'K at .5 mg/kg: 4 mice, 18 injuries; FL iA T Pitts C232S
P2KP1'K at 15 mg/kg: 3 mice, 20 injuries.
Figure 15 shows the inhibition o f thrombin and fXa b y mutants o f
oiiAT specific for APC over thrombin that were rescreened for fXa
inhibition. The resulting four mutants, which should have reduced
fXa inhibition were then tested for inhibition o f (A) thrombin and
(B) fXa, to determine if the screening had been successful in
producing APC-specific mutants which did not inhibit thrombin or
fXa. 1 serpin was incubated with 12.5 nM protease for different
amounts o f time. A t the indicated timepoints, the reaction was
stopped b y addition o f an excess of chromogenic substrate (S2238 for
thrombin, S2222 for fXa) . The residual enzyme activity was divided
b y the initial enzyme activity and the natural log o f this value
plotted against time. The slope o f these plots is the observed rate
constant, k . To obtain an estimate o f the second-order rate
constant k2, k b was divided b y the serpin concentration. Selected
values are shown in the figure, to illustrate the highest inhibition
o f thrombin and fXa, a s well a s the value for the only mutant that
did not substantially inhibit fXa (P2RPl'Q). The mutants shown all
have the Pitts (M358R, PlR) mutation.
Detailed Description
This invention relates to the modification o f serpins to increase
their specificity for APC relative to other coagulation proteases.
Modified serpin variants specific for APC may b e useful, for example
as procoagulants .
Since they are specific to APC, the modified serpins described
herein are expected to have few or no off-target effects and are
specific to pathways that are activated on injury. This allows the
modified serpins to be applied therapeutically and prophylactically,
as well as in emergency situations, i.e. trauma. The dosing of
modified serpins is predictable since they are suicide inhibitors,
meaning that one molecule of serpin cannot inhibit more than one
molecule of protease. In addition, because efficient natural
clearance mechanisms are specific for serpin: protease complexes
over serpins alone, the plasma half-lives of modified serpins are
likely to exceed those of current bypassing agents. For example, the
half-life of a modified serpin as described herein may be about 5
days .
Protein C (Gene ID 5624) is a vitamin K-dependent plasma
glycoprotein that is cleaved to its activated form (activated
Protein C , APC) b y the thrombin-thrombomodulin complex. Human
Protein C has the reference amino acid sequence of NP_000303.1 G I :
4506115 and may b e encoded by the reference nucleotide sequence of
NM_000312.3 G : 270483720. APC is an anticoagulant protease that
proteolytically cleaves fVa and fVIIIa (Figure 1 ) , thereby
attenuating the production of thrombin.
A modified serpin as described herein may have one or more mutations
in its reactive center loop (RCL) . For example, the modified serpin
may have one, two, three, four, or more than four mutations in its
RCL. The residues at one, two, three or all four of positions P4,
P2, PI and PI' may be mutated. For example, the residues at one or
both of positions Pi' and P2 and optionally Pl and/or P4 may be
mutated.
RCL residues are numbered herein according to the Schechter-Berger
nomenclature for substrates and inhibitors of serine proteases
(Schechter & Berger, 1967) . This standard nomenclature allows the
residue at specific positions in the RCL, such as positions PI' , PI,
P2 and/or P4, to be easily identified in any serpin sequence.
Preferably, the one or more mutations are the only mutations in the
RCL o f the modified serpin. For example, the RCL may consist o f the
sequence o f the RCL o f the wild-type serpin with mutations at one or
both of positions Pi' and P2 and optionally P I and/or P4.
The RCL of the modified serpin may have mutations at the PI' and P2
positions; mutations at the PI', P2 and Pi positions; mutations at
the Pi', P2 and P4 positions or mutations at the Pi', P2, P I and P4
positions; a mutation at the PI' position; mutations at the Pi' and
PI positions; mutations at the PI' and P4 positions or mutations at
the PI' , PI and P4 positions; a mutation at the P2 position;
mutations at the P2 and PI positions; mutations at the P2 and P4
positions, mutations at the P2, PI and P4 positions, a mutation at
the PI position, a mutation at the P4 position; or mutations at the
PI and P4 positions. The residues at other positions in the RCL may
b e unmutated wild-type residues.
Preferably, the residues at position PI'; positions PI' and P2;
positions Pi', PI and P2; positions P2 and PI; positions PI and PI';
positions PI', P2 and P4 or positions PI', Pi, P2 and P4 o f the RCL
are mutated. In some preferred embodiments, the residues at
positions Pi' , P and P2 are mutated.
The reactive center loop (RCL) o f a serpin is typically about 2 0
residues in length and contains the scissile Pl-Pl' bond that is
cleaved b y the target protease. The RCL extends from strand 5 o f
beta sheet A to strand 1 o f beta sheet C o f the serpin. Residues P17
Glu, P15 Gly and P14 Thr are conserved in serpins. For example, the
RCL of a serpin may comprise the consensus sequence P17 E , P16
E/K/R, P15 G , P14 T/S, P12-P9 (A/G/S) (Hopkins et al. 1993; Irving
et al. 2000). The RCL starts at residue P17 and usually ends at
residue P3' . RCLs may b e extended in some serpins, such a s PCI, b y
additional residues on the P ' side. For example, the RCL o f oi -
antitrypsin consists o f residues P17-P3' and the RCL o f PCI consists
o f residues P17-P6' . Examples of serpins with residues PI', Pi, P2
and P4 highlighted are shown in SEQ ID NOS : 1 to 11 below. The
residues that constitute the mature serpin sequences are also
indicated.
The residues in the other positions of the RCL in the serpin may be
unmodified i.e. they may be the native residues of the wild-type
serpin sequence. The modified serpin may therefore comprise an RCL
having a wild-type sequence with mutations at positions PI, PI', P2
and/or P4 as described above.
The one or more mutations in the reactive center loop (RCL) of the
modified serpin may comprise or consist of a mutation at the PI'
position. Preferably, the mutation is a substitution. The native Pi'
residue in the RCL of the wild-type serpin may be replaced with a
non-native residue in the modified serpin. For example, the native S
residue at the Pi' position in the wild-type sequence of or PCI
may be replaced with a residue other than S in the modified serpin.
The native PI' residue in the RCL of the wild-type serpin may be
replaced with a large polar residue, such as Q , N , Y ; a large
hydrophobic residue, such as I , M and V ; a positively charged
residue such as R , H or K ; or another residue such as C , A , 3 and E .
In some preferred embodiments, the Pi' residue may b e modified to a
large polar residue, such as Q , N , Y ; a large hydrophobic residue,
such as V ; or a positively charged residue such as R , H or K ; more
preferably, a positively charged or large polar residue, such as H ,
K , R , or Q ; most preferably K .
In other embodiments, the PI' residue may be unmodified in the
modified serpin. For example, the residue at the Pi' position in the
RCL of the modified serpin may be the residue that is present at the
Pi' residue in the wild-type serpin sequence.
The one or more mutations in the reactive center loop (RCL) of the
modified serpin may comprise or consist of a mutation at the P2
residue. Preferably, the mutation is a substitution. For example,
the native P2 residue in the RCL of the wild-type serpin may be
replaced with a non-native residue in the modified serpin.
The native P2 residue in the RCL of the wild-type serpin may be
replaced with a large polar residue, such as D , Q , N , Y , a large
hydrophobic residue such as , L , I , V and F , a positively charged
residue, such as R , H or K or another residue, such as C , A , T , S or
P .
In some embodiments the P2 residue in the modified serpin may be
other than P .
I n some preferred embodiments, the P2 residue may be modified to a
large polar residue, such as Q , N , Y , a large hydrophobic residue
such as W or a positively charged residue such as R , H or K , most
preferably a positive residue, such as H , K or R , preferably K .
In some embodiments, the P2 residue may be unmodified in the
modified serpin. For example, the residue at the P2 position in the
RCL of the modified serpin may be the residue that is present at the
P2 residue in the wild-type serpin sequence.
The one or more mutations in the RCL of the modified serpin may
comprise or consist of substitutions at the PI' and/or P2 residues
i.e. the residues located at the PI' and/or P2 positions in the RCL
of the wild-type serpin sequence may be replaced by other residues
in the modified serpin.
In preferred embodiments, the modified serpin has mutations at both
the P2 and the PI' positions of the RCL.
Suitable residues at the P2 and PI' positions of the modified serpin
are described above.
In some modified serpins described herein, at least one of the Pi'
and P2 residue may be a positively charged residue, such as R , H or
K or a large polar residue, such as D , Y , Q or N ; a large
hydrophobic residue, such as W , L , F , V , M or I ; or another residue,
such as L , C , A , E , T , S or P . Preferably, at least one of the PI'
and P2 residue is a positively charged residue, such as R , H or K a
large polar residue, such as Y , Q or N ; or a large hydrophobic
residue, such as W , L , F , V or .
Examples o f P2 and the PI' residues respectively in a modified
serpin as described herein include K , FK, RK, VK, LK, QK, CK, PK,
FR, HR, IR, SR, TR, VR, YR, AR, PR, RS, KS , QV, RV, RI, RH, KH, TH,
RC, RA, LY, QY, TY, DM, TM, WN, RN, HN, TN, KN, NN, PE, RQ, KQ and
TQ.
In some preferred embodiments, both the PI' and the P2 residue may
be modified to positively charged residues, such as K , H or R , most
preferably K .
In some embodiments, the P2 and the PI' residues respectively in a
modified serpin as described herein may b e other than PN, FS, QS,
AS, TS, HS, TA, PT, CC, PS, PT, P , PH, PA or PC. For example, the
P2 and PI' residues may b e other than PN, FS, QS, AS, TS, HS, TA,
PT, CC or PC in a PCI scaffold or other than PS, PT, PM, PH or PA in
an iiA T scaffold,
In some embodiments, the PI residue may b e unmodified in the
modified serpin. For example, the residue at the Pl position in the
RCL of the modified serpin may be the residue that is present at the
PI residue in the wild-type serpin sequence. For example, the Pl
residue in a modified PCI may b e an R residue.
In other embodiments, the Pl residue may b e mutated in the modified
serpin. For example, the one or more mutations in the reactive
center loop (RCL) o f the modified serpin further comprise a mutation
at the Pl residue. Preferably, the mutation is a substitution. The
native Pl residue in the RCL of the wild-type serpin may be replaced
with a non-native residue in the modified serpin.
In some embodiments, the PI residue may b e mutated or modified to a
positively charged residue such as H , K or R , preferably R .
Preferably, a native residue that is non-positively charged at the
P position of a wild-type serpin may be replaced by a positively
charged residue in the modified serpin. For example, M at the P
position of wild-type xiA T may b e replaced by a positively charged
residue, such as R , in a modified c A T . The Pittsburgh (Pitts)
variant of ¾ has a mutation at residue 358 which replaces the M
residue at the PI position with an R residue.
In some embodiments, the P residue may b e unmodified in the
modified serpin. For example, the residue at the P4 position in the
RCL of the modified serpin may be the residue that is present at the
P4 residue in the wild-type serpin sequence. For example, the P4
residue in a PCI scaffold may b e F and the P4 residue in an ii
scaffold may b e A .
In other embodiments, the P4 residue may be mutated in the modified
serpin. For example, the one or more mutations in the reactive
center loop (RCL) of the modified serpin further comprise a mutation
at the P4 residue.
Preferably, the mutation is a substitution. The residue at the P4
residue in the RCL of the wild-type serpin may be replaced with a
different residue in the modified serpin. For example, the P4
residue in a modified PCI may be mutated or modified to a residue
other F and the P4 residue in a modified OiiAT may b e mutated or
modified to a residue other than A .
Suitable residues in the P4 position o f the RCL o f the modified
serpin include S , R , V , C , , K , G , L , H , F , T , Q and A .
In examples of modified procoagulant serpins as described herein,
(1) the P4 residue is Q , the P2 residue is R , the PI residue is
and the PI' residue is N ;
(2) the P4 residue is K , the P2 residue is R , the I residue is R
and the PI' residue is H ;
(3) the P4 residue is s , the P2 residue is , the I residue is R
and the PI' residue is K ;
(4) the P4 residue is H , the P2 residue is R , the I residue is R
and the PI' residue is V ;
(5) the P4 residue is F , the P2 residue is K , the PI residue is R
and the PI' residue is K ;
(6) the P4 residue is F , the P2 residue is R , the I residue is R
and the PI' residue is K ;
(7) the P4 residue is F , the P2 residue is v , the PI residue is R
and the PI' residue is K ;
(8) the P4 residue is c , the P2 residue is L , the PI residue is R
and the PI' residue is K ;
(9) the P4 residue is the P2 residue is F , the I residue is R
and the PI' residue is ;
(10) the P4 residue is S , the P2 residue is H , the I residue is R
and the PI' residue is R ;
(11) the P4 residue is G r the P2 residue is I , the I residue is R
and the PI' residue is R ;
(12) the P4 residue is R , the P2 residue is Q , the I residue is R
and the PI' residue is V ;
(13) the P4 residue is T , the P2 residue is R , the PI residue is R
and the PI' residue is V
(14) the P4 residue is R , the P2 residue is R , the I residue is R
and the PI' residue is I ;
(15) the P4 residue is v , the P2 residue is R , the I residue is R
and the PI' residue is I ;
(16) the P4 residue is the P2 residue is R , the PI residue is R
and the PI' residue is I ;
(17) the P4 residue is T , the P2 residue is L , the I residue is R
and the PI' residue is Y ;
(18) the P4 residue is A , the P2 residue is Q , the I residue is R
and the PI' residue is Y ;
(19) the P4 residue is K , the P2 residue is D , the I residue is R
and the PI' residue is M ;
(20) the P4 residue is , the P2 residue is W , the I residue is R
and the PI' residue is N ;
(21) the P4 residue is A , the P2 residue is K , the PI residue is R ,
and the PI' residue is S ;
(22) the P4 residue is A , the P2 residue is , the I residue is
and the PI' residue is S ;
(23) the P4 residue is A , the P2 residue is P , the I residue is
and the PI' residue is E ;
(24) the P4 residue is A , the P2 residue is P , the I residue is R ,
a d the PI' residue is ;
(25) the P4 residue is A , the P2 residue is p . the residue is R ,
and the PI' residue is ;
(26) the P4 residue is A , the P2 residue is T , the I residue is R ,
and the PI' residue is M ;
(27) the P4 residue is A , the P2 residue is T , the I residue is R ,
and the PI' residue is H ;
(28) the P4 residue is A , the P2 residue is T , the I residue is R ,
and the PI' residue is Q ;
(29) the P4 residue is A , the P2 residue is T , the PI residue is R ,
and the PI' residue is N ;
(30) the P4 residue is A , the P2 residue is T , the I residue is R ,
a d the PI' residue is Y
(32) the P4 residue is A , the P2 residue is T , the I residue is
and the PI' residue is R ;
(33) the P4 residue is A , the P2 residue is R , the PI residue is R ,
and the PI' residue is A ;
(34) the P4 residue is A , the P2 residue is R , the I residue is
and the PI' residue is H ;
(35) the P4 residue is A , the P2 res idue is R , the I residue is R ,
and the PI' residue is C ;
(36) the P4 residue is A , the P2 residue is R the I residue is R ,
and the Pi' residue is N ;
(3 7 the P4 residue is A , the P2 residue is s , the I residue is R r
and the PI' residue is R ;
(38) the P4 residue is A , the P2 residue is , the residue is R r
and the PI' residue is ;
(39) the P4 residue is A , the P2 residue is K , the I residue is R,
and the PI' residue is H ;
(40) the P4 residue is A , the P2 residue is , the I residue is R ,
and the PI' residue is K ;
(41) the P4 residue is A , the P2 residue is v , the I residue is R ,
and the PI' residue is R ;
(42) the P4 residue is A , the P2 residue is Y , the I residue is R ,
and the PI' residue is R ;
(43) the P4 residue is A , the P2 residue is A , the PI residue is R ,
and the PI' residue is R ;
(44) the P4 residue is A , the P2 residue is c , the I residue is R ,
and the PI' residue is K ;
(45) the P4 residue is A , the P2 residue is w, the PI residue is R,
and the PI' residue is N ;
(46) the P4 residue is A , the P2 residue is H , the PI residue is
and the PI' residue is N ;
(47) the P4 residue is A , the P2 residue is Q , the I residue is R ,
and the PI' residue is K ; or,
(48) the P4 residue is A , the P2 residue is N, the PI residue is R,
and the PI' residue is N .
(49) the P4 residue is F , the P2 residue is F , the PI residue is R ,
and the PI' residue is K .
(50) the P4 residue is A , the P2 residue is , the PI residue is
and the PI' residue is Q ,
(51) the P4 residue is A , the P2 residue is R , the I residue is R ,
and the PI' residue is Q .
In some preferred modified serpins, the P4 residue is A , the P2
residue is K , the PI residue is R , and the PI' residue is K .
In some embodiments, the residues at the P4, P2 and the PI'
positions in a modified procoagulant serpin as described herein may
be other than HPN, DKN, HPE, FFS , LQS , HAS , Y S, AHS, ATA, LPT , ACC,
APT, APA, APM, APH, APS and VPC, respectively. For example, a
modified PCI may have residues other than HPN, DKN, HPE, FFS, LQS,
HAS, YTS, AHS, ATA, LPT, ACC and VPC at the P4, P2 and the PI'
positions and a modified iAT may have residues other than APT, APA,
PM or APH at the P4, P2 and the PI' positions. In some embodiments,
the combination of residues at the P4, P2 and the PI' positions in a
modified procoagulant serpin as described herein may b e nonnaturally
occurring i.e. the combination of residues at the P4, P2
and the PI' positions is not found in the parent wild-type (i.e.
unmodified) serpin or in other wild-type serpins .
A modified serpin a s described herein may comprise the sequence of a
wild-type (i.e. unmodified) serpin, preferably a mature wild-type
serpin, with one or more mutations in the RCL thereof as described
above, and optionally one or more additional mutations outside the
RCL .
The sequences o f wild-type serpins are well-known in the art, and
may include SEQ ID NOS: 1 to 11 a s set out herein. The sequences o f
wild-type serpins may include the sequence o f mature wild-type
proteins .
The mature protein C inhibitor (PCI) sequence including its
propeptide corresponds to residues 2 0 to 406 o f SEQ ID NO: 1 . The
mature oii-antichymotrypsin corresponds to residues 2 6 to 423 o f SEQ
ID NO: 2 . The mature Cl-esterase inhibitor sequence corresponds to
residues 23-500 o f SEQ ID NO: 3 . The mature 2-antiplasmin sequence
corresponds to residues 28-491 o f SEQ ID NO: 4 . The mature
antithrombin (ATIII) sequence corresponds to residues 33-464 o f SEQ
ID NO: 5 . The mature heparin cofactor II sequence corresponds to
residues 20-499 o f SEQ ID NO: 6 . The mature ai-antitrypsin (c A T )
sequence corresponds to residues 25-418 o f SEQ ID NO: 7 . The mature
kallistatin sequence corresponds to residues 21-427 o f SEQ ID NO: 8 .
The mature plasminogen activator inhibitor sequence corresponds to
residues 24-402 o f SEQ ID NO: 9 . The mature protein Z dependent
inhibitor sequence corresponds to residues 22-444 o f SEQ ID NO: 10.
The mature protease nexin 1 isoform a sequence corresponds to
residues 20-398 o f SEQ ID NO: 11.
Other than mutations o f residues in the RCL as described above, a
modified serpin may have 50 or fewer amino acid residues altered
relative to a wild-type serpin amino acid sequence (for example the
mature serpin sequence of one of SEQ ID NOS 1 to 11, preferably SEQ
ID NO: 1 or 7 ) , preferably 45 or fewer, 40 or fewer, 30 or fewer, 20
or fewer, 15 or fewer, 10 or fewer, 5 or fewer or 3 or fewer. For
example, a modified serpin may comprise the sequence of a wild-type
serpin with 50 or fewer, 45 or fewer, 40 or fewer, 30 or fewer, 20
or fewer, 15 or fewer, 10 or fewer, 5 or fewer or 3 or fewer amino
acid residues mutated or altered, in addition to the one, two, three
or four amino acid residues in the RCL of the serpin that are
mutated or altered as described above (i.e. the residues at
positions PI' and/or P2 and optionally PI and/or P4).
An amino acid residue in the wild-type amino acid sequence may be
altered or mutated by insertion, deletion or substitution,
preferably substitution for a different amino acid residue. Such
alterations may be caused by one or more of addition, insertion,
deletion or substitution of one or more nucleotides in the encoding
nucleic acid.
For example, a modified serpin may comprise the amino acid sequence
of residues 25-418 of SEQ ID NO: 12 having 50 or fewer mutations,
wherein said mutations are at positions other than P4, P2, PI and
Pl' i.e. the P4 residue at in the RCL of the modified serpin is A ,
the P2 residue is K , Pl residue is R and the PI' residue is K .
The P4 residue in the modified serpin of SEQ ID NO: 12 is located at
position 379 (355 of the mature protein) , the P2 residue is located
at position 381 (357 of the mature protein) , the P residue is
located at position 382 (358 of the mature protein) , and the Pl'
residue is located at position 383 (359 of the mature protein).
The modified serpin may share at least 50% sequence identity with
the wild-type amino acid sequence of a wild-type serpin, for example
the mature serpin sequence of any one of SEQ ID NOS: 1 to 11,
preferably SEQ ID NO: 1 or 1 , at least 55%, at least 60%, at least
65%, at least 70%, at least about 80%, at least 90%, at least 95%,
at least 98% or at least 99% sequence identity.
For example, a modified serpin may comprise an amino acid sequence
having at least 50% sequence identity to residues 25-418 of SEQ ID
NO: 12, wherein the P4 residue in the RCL of the modified serpin is
A , the P2 residue is K , PI residue is R and the PI' residue is K .
Sequence identity is commonly defined with reference to the
algorithm GAP (Wisconsin GCG package, Accelerys Inc, San Diego USA) .
GAP uses the Needleman and Wunsch algorithm to align two complete
sequences that maximizes the number of matches and minimizes the
number of gaps. Generally, default parameters are used, with a gap
creation penalty = 12 and gap extension penalty = 4 . Use of GAP
may be preferred but other algorithms may be used, e.g. BLAST (which
uses the method of Altschul et al. (1990) J . Mol. Biol. 215: 405-
410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS
USA 85: 2444-2448), or the Smith-Waterman algorithm (Smith and
Waterman (1981) J . Mol Biol. 147: 195-197), or the TBLASTN program,
of Altschul et al. (1990) supra, generally employing default
parameters. In particular, the psi-Blast algorithm may b e used
(Nucl. Acids Res. (1997) 25 3389-3402). Sequence identity and
similarity may also be determined using Genomequest ¾ software (Gene-
T , Worcester MA USA) .
Sequence comparisons are preferably made over the full-length of the
relevant sequence described herein.
Preferably, a modified procoagulant serpin as described herein
comprises the RCL consensus P17 E , P16 S/E/K/R, P15 G , P14 T/S, P12-
P9 (A/G/S) .
A modified serpin may further comprise one or more residues that are
conserved in wild-type serpin sequences. For example, a modified
serpin may comprise some or all of the following residues (numbered
according to their position in lAT ): 33F, 49N, 53S, 54P, 56S, 61L,
67G, 72T, 80L, 130F, 147F, 1571, 158N, 161V, 165T, 167G, 1691, 180T,
184L, 186N, 190F, 191K, 192G, 194W, 198F, 203T, 208F, 218V, 220M,
221 , 277Y, 254L, 255P, 289P, 290K, 299L, 303L, 307G, 312F, 316A,
327L, 334H, 342E, 344G, 347A, 369P, 370F, 383L, 384F, 386G, and 391P
(Irving et al 2008). The corresponding conserved residues in other
serpin sequences may be readily determined using routine sequence
analysis .
Mutations or variations outside the RCL of the modified serpin may
include replacement of o e or more Cys residues in the modified
serpin, such as the C232 (numbering according to the mature
sequence) residue of i T , to abolish disulfide bridge formation or
other modifications; deletion or substitution of residues at the N
terminus of the wild-type sequence, for example to facilitate
expression; or mutation or modification of residues in the heparin
binding sites of the modified serpins (i.e. helix D or helix H ) in
order to alter the heparin binding activity of the modified serpin.
In some embodiments, the modified serpin may have an N terminal
truncation relative to the wild-type serpin. For example, the
modified serpin may have a 10 to 30 residue truncation at the N
terminus, preferably about 20 residues.
One or more residues in the modified serpin may be non-natural amino
acids, modified amino acids or D-amino acids. The use of such amino
acids is well-known to those of skill in the art
A modified serpin as described herein may display the secondary
structure of the wild-type serpin, for example a modified serpin
may display a structure comprising 3 beta sheets, 8-9 alpha helices
and a flexible RCL of about 20 residues.
In some preferred embodiments, the modified serpin may consist of
the amino acid sequence of a wild-type serpin with one or more
mutations in the reactive center loop (RCL) thereof as described
herein .
Preferably, the modified serpin is non-immunogenic in a human. For
example, the wild-type serpin may be a human serpin, preferably a
human plasma serpin.
The wild-type serpin may be C i -antichymotrypsin (SERPINA3) , 2-
antiplasmin (SERPINF2) antithrombin (ATIII) (SERPINC1) , heparin
cofactor II (HCII) (SERPINDl) , protein C inhibitor (PCI) (SERPINA5),
ai-antitrypsin () (SERPINAl) , Kallistatin (SERPINA4),
Plasminogen activator inhibitor-1 (SERPINE1), Cl-esterase inhibitor
(SERPING1), protease nexin 1 (SERPINE2) or Protein Z-dependent
inhibitor (SERPINA10) (Whisstock et al JBC. 285 32 24307-24312, Rau
et al Journal of Thrombosis and Hemostasis, 5 (Suppl. 1): 102-115,
Huntington, Journal of Thrombosis and Hemostasis, 9 (Suppl. 1): 26-
34) .
Preferably, the wild-type serpin is ATIII, HCII, PCI or A T , most
preferably PCI or (Huntington e t al Cell. Mol. Life Sci. 66
(2009) 113-121; Li et al JBC. 283 51 36039-36045; and Li et al PNAS
2008 105 4661-4666) .
xi -antichymotrypsin (SERPINA3; Gene ID 12) may have the reference
amino acid sequence of NP_001076.2 GI: 50659080 (SEQ ID NO: 2 ) and
may be encoded by the reference nucleotide sequence of NM_001085.4
GI:73858562.
Cl-esterase inhibitor (SERPING1; Gene ID 710) may have the reference
amino acid sequence of NP_000053.2 G : 73858568 (SEQ ID NO: 3 ) and
may be encoded by the reference nucleotide sequence of NM_000062.2
GI:73858567.
Oi2 -antiplasmin (SERPINF2 Gene ID 5345) may have the reference amino
acid sequence of NP_000925.2 GI :115583663 (SEQ ID NO: 4 )and may be
encoded by the reference nucleotide sequence of NM_001165920 .
GI:260064047
Antithrombin (ATIII) (SERPINC1 Gene ID 462) may have the reference
amino acid sequence of NP_000479.1 G I : 4502261 (SEQ ID NO: 5 ) and
may be encoded by the reference nucleotide sequence of NM_000488.3
GI:254588059
Heparin cofactor II (HCII) (SERPIND1 Gene ID3053) may have the
reference amino acid sequence o f NP_000176.2 G : 73858566 (SEQ ID
NO: 6 ) and may b e encoded b y the reference nucleotide sequence o f
NM_000185.3 GI:73858565
Protein C inhibitor (PCI) (SERPINA5 Gene ID 5104) may have the
reference amino acid sequence o f NP_000615.3 G I : 194018472 and may
b e encoded b y the reference nucleotide sequence o f NM_000624.5
G I :401782581 . In some preferred embodiments, the protein C inhibitor
(PCI) may b e an allelic variant resulting from a substitution at SNP
rs6115, V7AR___0 308 1 (the S45N variant, numbering according to mature
protein including the propeptide) and having the sequence shown in
SEQ ID NO: 1 . Residues 1-19 o f SEQ ID NO: 1 correspond to the signal
sequence. In plasma, PCI may exist in a full-length form which
includes the propeptide o f residues 20-25 o f SEQ ID NO: 1 (i.e.
residues 20-406 o f SEQ ID NO: 1 ) o r N terminally cleaved form which
lacks the propeptide (i.e. residues 26-406 o f SEQ ID NO: 1 ) .
cxi-antitrypsin (o T ) (SERPINAl Gene ID 5265) may have the reference
amino acid sequence o f NP_000286.3 G :50363217 (SEQ ID NO: 7 ) and
may b e encoded b y the reference nucleotide sequence o f NM_000295.4
G :189163524 .
Kallistatin (SERPINA4 Gene ID 5267) may have the reference amino
acid sequence o f NP__006206.2 G I : 21361302 (SEQ ID NO: 8 ) and may b e
encoded b y the reference nucleotide sequence o f NM__006215.2 G :
21361301.
Plasminogen activator inhibitor-1 (SERPINE1 Gene ID 5054) may have
the reference amino acid sequence o f NP_000593.1 G : 10835159 (SEQ ID
NO: 9 ) and may b e encoded b y the reference nucleotide sequence o f
NM_000602.4 GI: 383286745.
Protein Z-dependent inhibitor (PZI) (SerpinAlO; Gene ID 51156) may
have the reference amino acid sequence o f NP_057270.1 G I : 7705879
(SEQ ID NO: 10) and may b e encoded b y the reference nucleotide
sequence o f NM_016186.2 G I : 154759289.
Protease nexin 1 (PN1) (SerpinE2; Gene ID 5270) may have the
reference amino acid sequence of NP_001130000 .1 G : 24307907,
NP_001130002.1 GI: 211904152 or NP_006207.1 GI : 211904156 (SEQ ID
NO: 11) and may be encoded by the reference nucleotide sequence of
NM_001136528.1 GI : 211904151, NM_001136530 .1 GI : 211904155 or
NM_006216.3 GI: 211904150.
The PI', PI, P2 and P4 residues that may be mutated as described
above are highlighted in bold in SEQ ID NOS : 1 to 11.
The one or more mutations in the RCL alter the specificity of the
modified serpin relative to the unmodified wild-type serpin. The
modified serpin displays increased selectively for anticoagulant
proteases over procoagulant proteases compared to the wild-type
serpin .
Preferably, the one or more mutations within the RCL increase the
inhibition of APC by the modified serpin relative to the inhibition
of other coagulation proteases, in particular one or more
procoagulant proteases out of thrombin, fXa, fVIIa, fIXa and fXIa.
For example, the one or more mutations in the RCL of the modified
serpin may increase the inhibition of APC by the modified serpin
relative to the inhibition of thrombin. The selective inhibition of
APC relative to thrombin may be increased in the presence or absence
of heparin.
In addition, the one or more mutations in the RCL of the modified
serpin may increase the inhibition of APC by the modified serpin
relative to the inhibition of 1 , 2 , 3 or all 4 of the procoagulant
proteases fXa, fVIIa, fIXa and fXIa.
A serpin modified as described herein displays greater inhibition of
APC relative to thrombin and other procoagulant proteases than the
unmodified wild-type serpin.
The modified serpin may show greater inhibition of APC than
inhibition of thrombin. For example, inhibition of APC by the
modified serpin may be at least 5 fold more, at least 10 fold more
at least 100 or at least 1000 fold more than inhibition of thrombin
by the modified serpin. In some embodiments, the modified serpin may
inhibit APC with a second-order rate constant k ) that is at least
5 fold more, at least 10 fold more at least 100 or at least 1000
fold more than the second-order rate constant for the inhibition of
thrombin. Preferably the stoichiometry of inhibition of the modified
serpin for APC is 1 .
Preferably, a modified serpin as described herein may bind and
inhibit APC but display no binding or inhibition or substantially no
binding or inhibition of thrombin.
The one or more mutations in the RCL may also increase the
inhibition of APC relative to the inhibition of 1 , 2 , 3 , or all 4 of
fVIIa, fIXa, fXa and fXIa. Inhibition of APC relative to fVIIa,
fIXa, fXa and/or fXIa may be increased in the presence or absence of
heparin.
For example, the modified serpin may display greater inhibition of
APC relative to 1 , 2 , 3 , or all 4 of fVIIa, fIXa, fXa and fXIa than
the wild-type serpin.
The modified serpin may inhibit APC more than it inhibits fVIIa. For
example, inhibition of APC by the modified serpin may be at least 2
fold more, at least 10 fold more, at least 100 more, or at least
1000 fold more than the inhibition of f a by the modified serpin.
The modified serpin inhibits APC with a second-order rate constant
( 2) that is at least 2 fold more, at least 10 fold more, at least
100 more, or at least 1000 fold more than the second-order rate
constant for the inhibition of fVIIa.
The modified serpin may inhibit APC more than it inhibits fIXa. For
example, inhibition of APC by the modified serpin may be at least 2
fold more, at least 10 fold more, at least 100 more or at least 1000
fold more than the inhibition of fIXa by the modified serpin. The
modified serpin inhibits APC with a second-order rate constant (k
that is at least 2 fold more, at least 10 fold more, at least 100
more, or at least 1000 fold more than the second-order rate constant
for the inhibition of fIXa.
The modified serpin may inhibit APC more than it inhibits f a . For
example, inhibition of APC by the modified serpin may be at least
1.5 fold more, at least 2 fold more, at least 10 fold more at least
100 or at least 1000 fold more than the inhibition of fXa by the
modified serpin. The modified serpin inhibits APC with a secondorder
rate constant ( ¾ ) that is at least 1.5 fold more, at least 2
fold more, at least 10 fold more, at least 100 more, or at least
1000 fold more than the second-order rate constant for the
inhibition of fXa.
The modified serpin may inhibit APC more than it inhibits fXIa. For
example, inhibition of APC by the modified serpin may be at least 2
fold more, at least 10 fold more at least 100 or at least 1000 fold
more than the inhibition of fXIa by the modified serpin. The
modified serpin inhibits APC with a second-order rate constant (k 2)
that is at least 2 fold more, at least 10 fold more, at least 100
more, or at least 1000 fold more than the second-order rate constant
for the inhibition of fXIa.
A modified serpin as described herein may be part of a fusion
protein which contains one or more heterologous amino acid sequences
additional to the modified serpin sequence. For example, the fusion
protein comprising the modified serpin may further comprise one or
more additional domains which improve the stability,
pharmacokinetic, targeting, affinity, purification and production
properties of the modified serpin.
Suitable additional domains include immunoglobulin Fc domains.
Immunoglobulin Fc domains are well-known in the art and include the
human IgGl Fc domain. A human immunoglobulin Fc domain may be
located at the N-terminal or C- terminal end of the modified serpin.
Modified serpins as described herein may be provided using synthetic
or recombinant techniques which are standard in the art.
In some embodiments, the modified serpin may be produced as a fusion
protein further comprising an affinity tag, which may, for example,
be useful for purification. An affinity tag is a heterologous
peptide sequence which forms one member of a specific binding pair.
Polypeptides containing the tag may be purified by the binding of
the other member of the specific binding pair to the polypeptide,
for example in an affinity column. For example, the tag sequence may
form an epitope which is bound by an antibody molecule.
Suitable affinity tags include for example, glutathione-Stransferase,
(GST), maltose binding domain (MBD) , MRGS (H) , DYKDDDDK
(FLAG™), T7-, S- (KETAAAKFERQHMDS) , poly-Arg (R -e) , poly-His (H - i ) ,
poly-Cys (C ) poly-Phe (F ) poly-Asp (D5-i ), SUMO tag (Invitrogen
Champion pET SUMO expression system) , Strept-tag II (WSHPQFEK) , cmyc
(EQKLISEEDL) , Influenza-HA tag (Murray, P . J . et al (1995) Anal
Biochem 229, 170-9), Glu-Glu-Phe tag (Stammers, D . K . et al (1991)
FEBS Lett 283, 298-302), Tag. 100 (Qiagen; 12 aa tag derived from
mammalian MAP kinase 2 ) , Cruz tag 09™ (MKAEFRRQESDR, Santa Cruz
Biotechnology Inc.) and Cruz tag 22™ (MRDALDRLDRLA, Santa Cruz
Biotechnology Inc.). Known tag sequences are reviewed in Terpe
(2003) Appl. Microbiol. Biotechnol. 60 523-533. In preferred
embodiments, a poly-His tag such as (H) 6, His-SUMO tag (Invitrogen
Champion pET SUMO expression system), or MRGS (H) may be used.
The affinity tag sequence may b e separated from the modified serpin
after purification, for example, using site-specific proteases.
In some embodiments, the modified serpin may be coupled to an
appropriate signal leader peptide to direct secretion of the fusion
polypeptide from cell into the culture medium. A range of suitable
signal leader peptides are known in the art. The signal leader
peptide may b e a serpin signal sequence or may be heterologous to
the modified serpin i.e. it may be a non-serpin signal sequence. For
example, an a-factor secretion signal or BiP signal sequence may be
employed. Preferably, the signal peptide is removed by posttranslational
processing after expression of the polypeptide.
Modified serpins as described herein may be isolated, in the sense
of being free from contaminants, such as unmodified serpins and
other polypeptides and/or serum components.
Modified serpins as described herein may inhibit one or more
activities of activated protein C (APC) . For example, modified
serpins as described herein may inhibit the proteolytic cleavage of
one or more APC substrates, such as fVa or fVIIIa. For example,
binding of the modified serpin to APC may result in an at least 5-
fold, at least 10-fold, or at least 15-fold decrease in the
proteolytic cleavage of fVa, fVIIIa and/or another APC substrate. In
some embodiments, binding of APC by the modified serpin may result
in no detectable cleavage of fVa or fVIIIa substrate by APC.
Techniques for measuring APC activity, for example by measuring the
proteolytic cleavage of APC substrates in vitro are standard in the
art and are described herein. Suitable assays for use in determining
APC activity include standard kinetic assays, for example to measure
rate constants, and coagulation assays, including thrombin
generation assays (TGA) .
Techniques for measuring the activity of procoagulant proteases, for
example by measuring the proteolytic cleavage of chromogenic
substrates in vitro are standard in the art and are described
herein. Suitable assays for use in determining protease activity
include standard kinetic assays, for example to measure rate
constants, and coagulation assays, including thrombin generation
assays (TGA) , prothrombin time assays (PT) and activated partial
thromboplastin time assays (aPTT) .
In some embodiments, modified serpins as described herein may be
further modified by chemical modification, for example by
PEGylation, or by incorporation in a liposome, to improve their
pharmaceutical properties, for example by increasing in vivo halflife.
A modified serpin as described herein may b e attached to one or more
polyethylene glycol (PEG) or other moieties to increase the in vivo
half-life of the modified serpin (Cantin et al . 2002, Am. J . Respir.
Cell Mol. Biol. 27; 659-665) . For example, a modified serpin may be
mono-pegylated or poly-pegylated (for example, with 2-6 PEG
moieties). Suitable pegylation methods are well known in the art.
The effect o f a modified serpin on coagulation and bleeding may b e
determined. Suitable techniques are standard in the art. For
example, the effect of a modified serpin on thrombin generation may
be determined using a thrombin generation assay (TGA) or an
activated partial thromboplastin time assay or a prothrombin time
assay described herein. Suitable i vivo models include cremaster
arteriole laser injury models, F e C l; carotid artery models and tail
clip assays as described herein. Other suitable coagulation models
are well known in the art.
Other aspects o f the invention provide a nucleic acid encoding a
modified serpin as described above and a vector comprising such a
nucleic acid.
Suitable vectors can be chosen or constructed, containing
appropriate regulatory sequences, including promoter sequences,
terminator fragments, polyadenylation sequences, enhancer sequences,
marker genes and other sequences as appropriate. Preferably, the
vector contains appropriate regulatory sequences to drive the
expression of the nucleic acid in mammalian cells. A vector may also
comprise sequences, such as origins of replication, promoter regions
and selectable markers, which allow for its selection, expression
and replication in bacterial hosts such as E . coli.
Vectors may be plasmids, viral e.g. phage, or phagemid, as
appropriate. For further details see, for example, Molecular
Cloning: a Laboratory Manual: 3rd edition, Russell et al ., 2001,
Cold Spring Harbor Laboratory Press. Many known techniques and
protocols for manipulation of nucleic acid, for example in
preparation of nucleic acid constructs, mutagenesis, sequencing,
introduction of DNA into cells and gene expression, are described in
detail in Current Protocols in Molecular Biology, Ausubel et al .
eds . John Wiley & Sons, 1992.
A nucleic acid or vector as described herein may be introduced into
a host cell .
Another aspect of the invention provides a recombinant cell
comprising a nucleic acid or vector that expresses a polypeptide
comprising or consisting of a modified serpin as described above.
A range of host cells suitable for the production of recombinant
modified serpins are known in the art. Suitable host cells may
include prokaryotic cells, in particular bacteria such as
Escherichia coli and Lactococcus lactis and eukaryotic cells,
including mammalian cells such as CHO and CHO-derived cell lines
(Lee cells), HeLa, COS, HEK293 and HEK-EBNA cells, amphibian cells
such as Xenopus oocytes, insect cells such as Trichoplusia ni, Sf9
and Sf21 and yeast cells, such as Pichia pastoris .
Techniques for the introduction of nucleic acid into cells are well
established in the art and any suitable technique may be employed,
in accordance with the particular circumstances. For eukaryotic
cells, suitable techniques may include calcium phosphate
transf ection, DEAE-Dextran, electroporation, liposome-mediated
transfection and transduction using retrovirus or other virus, e.g.
adenovirus, AAV, lentivirus or vaccinia. For bacterial cells,
suitable techniques may include calcium chloride transformation,
electroporation and transfection using bacteriophage.
Marker genes such as antibiotic resistance or sensitivity genes may
be used in identifying clones containing nucleic acid of interest,
as is well-known in the art.
The introduced nucleic acid may be on an extra-chromosomal vector
within the cell or the nucleic acid may be integrated into the
genome of the host cell. Integration may be promoted by inclusion of
sequences within the nucleic acid or vector which promote
recombination with the genome, in accordance with standard
techniques .
In some embodiment, nucleic acid encoding a modified serpin as
described herein may be contained in a vector suitable for
administration to an individual e.g. for gene therapy applications.
Suitable vectors include retroviral vectors, lentiviral vectors,
adenoviral vectors and A T vectors.
The introduction may be followed by expression of the nucleic acid
to produce the encoded modified serpin. In some embodiments, host
cells (which may include cells actually transformed although more
likely the cells will be descendants of the transformed cells) may
be cultured in vitro under conditions for expression of the nucleic
acid, so that the encoded serpin polypeptide is produced. When an
inducible promoter is used, expression may require the activation of
the inducible promoter.
The expressed polypeptide comprising or consisting of the modified
serpin may be isolated and/or purified, after production. This may
be achieved using any convenient method known in the art. Techniques
for the purification of recombinant polypeptides are well known in
the art and include, for example HPLC, FPLC or affinity
chromatography. In some embodiments, purification may be performed
using an affinity tag on the polypeptide as described above.
Another aspect of the invention provides a method of producing a
modified serpin comprising expressing a nucleic acid encoding a
modified serpin as described above in a host cell and optionally
isolating and/or purifying the modified serpin thus produced.
Polypeptides comprising or consisting of a modified serpin produced
as described may be investigated further, for example the
pharmacological properties and/or activity may be determined.
Methods and means of protein analysis are well-known in the art.
A modified serpin as described herein, nucleic acid encoding a
modified serpin or a recombinant cell expressing a modified serpin,
may be useful in therapy. For example, a modified serpin as
described herein, nucleic acid encoding a modified serpin or a
recombinant cell expressing a modified serpin may be administered to
an individual for the treatment of bleeding.
Whilst a modified serpin may be administered alone, modified serpins
will usually be administered in the form of a pharmaceutical
composition, which may comprise at least one component in addition
to the modified serpin e.g. a nucleic acid encoding the modified
serpin or recombinant cell expressing the modified serpin. Thus
pharmaceutical compositions may comprise, in addition to the
modified serpin, nucleic acid or cell, a pharmaceutically acceptable
excipient, carrier, buffer, stabilizer or other materials well known
to those skilled in the art. The term "pharmaceutically acceptable"
as used herein pertains to compounds, materials, compositions,
and/or dosage forms which are, within the scope of sound medical
judgement, suitable for use in contact with the tissues of a subject
(e.g., human) without excessive toxicity, irritation, allergic
response, or other problem or complication, commensurate with a
reasonable benefit/risk ratio. Each carrier, excipient, etc. must
also be "acceptable" in the sense of being compatible with the other
ingredients of the formulation. The precise nature of the carrier or
other material will depend on the route of administration, which may
be by bolus, infusion, injection or any other suitable route, as
discussed below.
In some embodiments, modified serpins, nucleic acids or cells may b e
provided in a lyophilised form for reconstitution prior to
administration. For example, lyophilised serpins may be reconstituted
in sterile water and mixed with saline prior to
administration to an individual.
For parenteral, for example sub-cutaneous or intra-venous
administration, e.g. by injection, the pharmaceutical composition
comprising the modified serpin, nucleic acid or cell may be in the
form of a parenterally acceptable aqueous solution which is pyrogenfree
and has suitable H , isotonicity and stability. Those of
relevant skill in the art are well able to prepare suitable
solutions using, for example, isotonic vehicles, such as Sodium
Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
Preservatives, stabilizers, buffers, antioxidants and/or other
additives may be employed as required including buffers such as
phosphate, citrate and other organic acids; antioxidants, such as
ascorbic acid and methionine; preservatives (such as
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride; benzethonium chloride; phenol, butyl or
benzyl alcohol; alkyl parabens, such as methyl or propyl paraben;
catechol; resorcinol; cyclohexanol ; 3'-pentanol; and m-cresol); low
molecular weight polypeptides; proteins, such as serum albumin,
gelatin or immunoglobulins; hydrophilic polymers, such as
polyvinylpyrrolidone; amino acids, such as glycine, glutamine,
asparagines, histidine, arginine, or lysine; monosaccharides,
disaccharides and other carbohydrates including glucose, mannose or
dextrins; chelating agents, such as EDTA; sugars, such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions, such as
sodium; metal complexes (e.g. Zn-protein complexes); and/or nonionic
surfactants, such as TWEEN™, PLURONICS™ or polyethylene
glycol (PEG). Suitable carriers, excipients, etc. can be found in
standard pharmaceutical texts, for example, Remington's
Pharmaceutical Sciences, 18th edition, Mack Publishing Company,
Easton, Pa., 1990.
Pharmaceutical compositions and formulations may conveniently be
presented in unit dosage form and may be prepared by any methods
well known in the art of pharmacy. Such methods include the step of
bringing into association the modified serpin with the carrier which
constitutes one or more accessory ingredients. In general, the
compositions are prepared by uniformly and intimately bringing into
association the active compound with liquid carriers or finely
divided solid carriers or both, and then if necessary shaping the
product .
Preferably, modified serpins, nucleic acids or cells as described
herein are formulated in a pharmaceutical composition for intra
venous or sub-cutaneous administration.
A pharmaceutical composition comprising a modified serpin, nucleic
acid or cell may be administered alone or in combination with other
treatments, either simultaneously or sequentially dependent upon t v
condition to be treated.
A modified serpin, nucleic acid or cell as described herein may be
used in a method of treatment of the human or animal body, including
therapeutic and prophylactic or preventative treatment (e.g.
treatment before the onset of a condition in an individual to reduce
the risk of the condition occurring in the individual; delay its
onset; or reduce its severity after onset) . The method of treatment
may comprise administering a modified serpin to an individual in
need thereof.
An individual suitable for treatment as described above may be a
mammal, such as a rodent (e.g. a guinea pig, a hamster, a rat, a
mouse), murine (e.g. a mouse), canine (e.g. a dog), feline (e.g. a
cat), equine (e.g. a horse), a primate, simian (e.g. a monkey or
ape), a monkey (e.g. marmoset, baboon), an ape (e.g. gorilla,
chimpanzee, orang-utan, gibbon), or a human.
In some preferred embodiments, the individual is a human. In other
preferred embodiments, non-human mammals, especially mammals that
are conventionally used as models for demonstrating therapeutic
efficacy in humans (e.g. murine, primate, porcine, canine, or rabbit
animals) may be employed. The inhibition of human and murine APC
by modified serpins without the inhibition of human or murine
thrombin is shown below.
Administration is normally in a "therapeutically effective amount"
or "prophylactically effective amount", this being sufficient to
show benefit to a patient. Such benefit may be at least amelioration
of at least one symptom. The actual amount administered, and rate
and time-course of administration, will depend on the nature and
severity of what is being treated, the particular mammal being
treated, the clinical condition of the individual patient, the cause
of the disorder, the site of delivery of the composition, the method
of administration, the scheduling of administration and other
factors known to medical practitioners.
A composition may be administered alone or in combination with other
treatments, either simultaneously or sequentially dependent upon the
circumstances of the individual to be treated.
Prescription of treatment, e.g. decisions on dosage etc, is within
the responsibility of general practitioners and other medical
doctors and may depend on the severity of the symptoms and/or
progression of a disease being treated. Appropriate doses of
therapeutic polypeptides are well known in the art (Ledermann J .A .
et al. (1991) Int. J . Cancer 47: 659-664; Bagshawe K.D. et al.
(1991) Antibody, Immunoconjugates and Radiopharmaceuticals 4 : 915-
922) . Specific dosages may be indicated herein or in the Physician's
Desk Reference (2003) as appropriate for the type of medicament
being administered may be used. A therapeutically effective amount
or suitable dose of a modified serpin may be determined by comparing
its in vitro activity and in vivo activity in an animal model.
Methods for extrapolation of effective dosages in mice and other
test animals to humans are known. The precise dose will depend upon
a number of factors, including whether the modified serpin is for
prevention or for treatment, the size and location of the area to be
treated, the precise nature of the modified serpin and the nature of
any detectable label or other molecule attached to the modified
serpin .
A typical modified serpin dose will be in the range of 0.1 mg/kg to
lOOmg/kg, for example 1 to 80mg/kg. For example, a dose in the range
100 ]ig to 1 g may be used for systemic applications, and 1 pg to 1
g for topical applications. An initial higher loading dose,
followed by one or more lower doses, may be administered. This is a
dose for a single treatment of an adult patient, which may be
proportionally adjusted for children and infants, and also adjusted
for other modified serpin formats in proportion to molecular weight.
Treatments may be repeated at daily, twice-weekly, weekly or monthly
intervals, at the discretion of the physician. The treatment
schedule for an individual may be dependent on the pharmoco kinetic
and pharmacodynamic properties of the modified serpin composition,
the route of administration and the nature of the condition being
treated.
Treatment may be periodic, and the period between administrations
may be about one week or more, e.g. about two weeks or more, about
three weeks or more, about four weeks or more, about once a month or
more, about five weeks or more, or about six weeks or more. For
example, treatment may be every two to four weeks or every four to
eight weeks. Treatment may be given before, and/or after surgery,
and/or may be administered or applied directly at the anatomical
site of trauma, surgical treatment or invasive procedure. Suitable
formulations and routes of administration are described above.
In some embodiments, modified serpins as described herein may be
administered as sub-cutaneous injections. Sub-cutaneous injections
may be administered using an auto-injector, for example for long
term prophylaxis/treatment.
In some preferred embodiments, the therapeutic effect of the
modified serpin may persist for several half-lives, depending on the
dose .
Modified serpins described herein inhibit APC without inhibiting or
substantially inhibiting procoagulant factors, such as thrombin, and
may be useful in the treatment of bleeding and bleeding disorders;
in particular disorders caused by reduced thrombin generation or
increased APC anticoagulant pathway activity.
Hemostasis is the normal coagulation response to injury i.e. the
prevention of bleeding or hemorrhage, for example from a damaged
blood vessel. Hemostasis arrests bleeding and hemorrhage from blood
vessels in the body. Modified serpins may promote hemostasis i.e.
they may promote or increase the arrest of bleeding and hemorrhage
from blood vessels in the body, for example in individuals with
bleeding disorders or trauma.
Aspects of the invention provide; a modified serpin as described
herein for use in a method of treatment of the human or animal body;
a modified serpin as described herein for use in a method of
treatment of bleeding or the promotion of hemostasis; the use of a
modified serpin as described herein in the manufacture of a
medicament for the treatment of bleeding or the promotion of
hemostasis; and a method of treatment of bleeding or the promotion
of hemostasis comprising administering a modified serpin as
described herein to an individual in need thereof.
Bleeding may include bleeding or hemorrhage from blood vessels in
the body.
An individual suitable for treatment with a modified serpin as
described herein may have a bleeding disorder.
Bleeding disorders may be caused or associated with reduced thrombin
generation or increased activity of the APC anticoagulant pathway.
Bleeding disorders may include any heritable or acquired bleeding
disorder in which there is reduced thrombin generation, reduced
fibrin clot formation or reduced clot stability. For example,
bleeding disorders may include congenital deficiencies of factors
VII, VIII, X , IX, X I and XIII; combined V and VIII deficiency;
prothrombin deficiency; fibrin deficiency; and rare deficiencies of
other clotting factors; hemophilia A , B and C ; increased bleeding
associated with hyperf ibrinolysis ; increased bleeding due to reduced
platelet count or reduced platelet function; and von Willebrand
Disease .
Acquired bleeding disorders may include dilutional coagulopathy
associated with major blood loss, bleeding resulting from trauma and
surgery and the effects of anticoagulant therapy.
In some embodiments, the individual may be resistant to exogenous
coagulation factors, such as exogenous fVIII or fix. For example,
exogenous fV or f x may elicit an immune response in the
individual, for example the production of inhibitory alloantibodies .
An individual suitable for treatment with a modified serpin as
described herein may have an acquired bleeding disorder, such as
bleeding related to trauma, surgery or anti-coagulant therapy.
For example, an individual suitable for treatment with a modified
serpin as described herein may have suffered a trauma; or may have
undergone or be undergoing surgery or anti-coagulant therapy.
Suitable individuals may be bleeding or at risk of bleeding from one
or more blood vessels in the body.
In some embodiments, a modified serpin as described herein may be
useful in the prevention or treatment of i ) bleeding in patients
with clotting factor alloantibodies; ii) bleeding in patients at
high risk of inhibitor development, for example to avoid development
of alloantibodies; iii) bleeding in patients with factor VIII
deficiency in the absence of inhibitors; iv) bleeding in patients
with congenital bleeding disorders, for example a congenital
bleeding disorder for which there is no current recombinant optimal
replacement therapy, such as severe factor VII deficiency, factor X I
deficiency, combined VIII & V deficiency, factor X deficiency and
factor V deficiency; v ) bleeding in patients with hemophilia, for
example patients for whom replacement therapy is inappropriate or
unavailable; or vi) acquired bleeding, including bleeding related to
trauma, surgery, and anticoagulant therapy.
Other aspects of the invention provide the use of a modified serpin
as described herein as a procoagulant and the use of a modified
serpin to inhibit APC in the treatment of bleeding.
Various further aspects and embodiments of the present invention
will be apparent to those skilled in the art in view of the present
disclosure .
Other aspects and embodiments of the invention provide the aspects
and embodiments described above with the term "comprising" replaced
by the term "consisting of" and the aspects and embodiments
described above with the term "comprising" replaced by the term
"consisting essentially of".
It is to be understood that the application discloses all
combinations of any of the above aspects and embodiments described
above with each other, unless the context demands otherwise.
Similarly, the application discloses all combinations of the
preferred and/or optional features either singly or together with
any of the other aspects, unless the context demands otherwise.
Modifications of the above embodiments, further embodiments and
modifications thereof will be apparent to the skilled person on
reading this disclosure, and as such these are within the scope of
the present invention.
All documents and sequence database entries mentioned in this
specification are incorporated herein by reference in their entirety
for all purposes.
"and/or" where used herein is to be taken as specific disclosure of
each of the two specified features or components with or without the
other. For example "A and/or B" is to be taken as specific
disclosure of each of (i) A , (ii) B and (iii) A and B , just as if
each is set out individually herein.
Certain aspects and embodiments of the invention will now b e
illustrated by way of example and with reference to the figures
described above and the tables described below.
Table 1 shows second-order rate constants for the inhibition of
thrombin and APC by A22 PCI (PCI with an N-terminal truncation,
starting at Ala22, numbering uses the mature protein sequence,
including the propeptide), FL o i T Pitts (full length ii with the
P1R Pittsburgh (Pitts) mutation) and their variants. Standard errors
are shown. * The rate constant of inhibition for thrombin by the
P2 P1' variant of PCI is an estimate as after initial inhibition,
reactions do not appear to approach complete inhibition, potentially
due to serpin being shuttled down the substrate pathway or
dissociation o f the covalent serpin :protease inhibitory complex
Table 2 shows second-order rate constants for the inhibition of
thrombin and APC by PCI and variants in the presence o f heparin.
Standard errors are shown. * the rate constant o f inhibition for
thrombin by the P2KP1'K variant of PCI is an estimate from the
initial slope of the plot of residual thrombin activity versus time.
Complete inhibition is not achieved, potentially because of serpin
being shuttled down the substrate pathway or dissociation of the
covalent serpin :protease inhibitory complex.
Table 3 shows second-order rate constants for the inhibition o f fXa
b y oi T Pitts and PCI and their variants. Standard errors are shown.
Table 4 shows second-order rate constants for the inhibition o f fXIa
b y PCI and the PCI P2KP1'K variant. APC inhibition is shown for
comparison (from Table 1 ) . Standard errors are shown.
Table 5 shows second-order rate constants for the inhibition o f
thrombin and APC by PCI variants generated b y targeted random
mutagenesis. Standard errors are shown. Constants for T and P2KP1'
PCI are shown for comparison. * The rate constant of inhibition for
thrombin by the P2KP1' variant of PCI is an estimate as after
initial inhibition, reactions do not appear to approach complete
inhibition, potentially due to serpin being shuttled down the
substrate pathway or dissociation of the covalent serpin:protease
inhibitory complex.
Table 6 shows second-order rate constants for the inhibition of fXIa
by FL Pitts C232S and its P2 P1 'K variant. APC inhibition is
shown for comparison (from Table 1 ) . Standard errors are shown.
Table 7 shows a fraction of the RCL sequences o f the PCI variants
determined by targeted random mutagenesis to be specific for APC
inhibition over thrombin inhibition. Sequences shown are from an
initial experiment in which 88 mutants were assessed. The P4, P2 and
Pi' residues that were varied in this experiment are shown in bold.
T PCI and 2PCI sequences are shown for comparison.
Table 8 shows a fraction of the RCL sequences o f the PCI variants
determined by targeted random mutagenesis to be specific for APC
inhibition over thrombin inhibition. Sequences shown are from a
larger experiment in which 460 mutants were assessed. The P4, P2 and
PI' residues that were varied in this experiment are shown in bold.
WT PCI and 2PCI sequences are shown for comparison.
Table 9 shows a fraction o f the RCL sequences o f the variants
determined by targeted random mutagenesis to be specific for APC
inhibition over thrombin inhibition. Sequences are shown compared to
both W T and « Pitts. The P2 and PI' residues are shown in
bold, the PI residues are underlined. Prefixes show the library of
origin for the particular mutant with mutants denoted P2.nr coming
from the P2 variant library Pi' .nr. coming from the PI' variant
library and mutants labelled 1-5. nr. coming from plates 1-5 of the
P2P1' variant library.
Table 10 shows second-order rate constants for the inhibition of
thrombin and APC b y a subset o f variants of ii T determined b y
targeted random mutagenesis to be more specific for APC than for
thrombin. Standard errors are given. The second-order rate constants
for thrombin and APC inhibition by FL AT Pitts C232S P2KP1'K and
FL i Pitts C232S are given for comparison.
Table 11 shows the results of PT and aPTT assays to investigate
inhibition of procoagulant proteases by hits from random mutagenesis
on the FL A T Pitts C232S background. PT assays were performed
using 1/4 diluted plasma to increase the sensitivity o f the assay
and performed in triplicate, except for the reactions shown for FL
T Pitts C232S P2RP1'C, which were performed in duplicate. The
error shown is the standard deviation. aPTT assays were single
experiments, no error is shown. For both PT and aPTT a buffer only
control, where instead o f protein, buffer (TBS) was added to the
plasma was used as a control. Increases in PT or aPTT with respect
to the control are an indication of inhibition of procoagulant
proteases. For both PT and aPTT assays, the serpin mutants were used
at a 5 concentration. For comparison, the P2K and Pl'K mutants
are shown as an average of triplicates. A s shown from inhibition
rate constants in Tables 1 and 3 , these mutants show high
specificity for APC over thrombin, but inhibit fXa significantly.
They are therefore good comparator for an inhibition of procoagulant
proteases other than thrombin. ND indicates not determined.
Table 12 shows second-order rate constants for the inhibition of fXa
by iA T variants from targeted random mutagenesis . The mutants
evaluated here showed specificity for APC over thrombin, substantial
APC inhibition and showed no prolongation o f the PT . Most also
showed only minor aPTT prolongation. Because o f these features, they
were selected for further analysis. For comparison, fXa inhibition
b y FL oi-AT Pitts C232S and FL iA T Pitts C232S P2KPl'K is also shown
(from Table 3 ) .
Table 13 shows a summary of the characterization of two more mutants
of found b y combining information from rational and random
mutagenesis. FL A T Pitts C232S and FL i T Pitts C232S P2KP1'K
C232S are shown for comparison (data from Tables 1 and 3 and Figure
5 ) . aPTTs for P2RP1'Q and P2KP1'Q are the average o f four separate
measurements, the error shown is the standard deviation. The value
obtained for plasma with buffer is shown in brackets, again with the
standard deviation shown as the error. The aPTTs shown for Pitts and
P2KPl'K are the result of at least three separate measurements, the
error shown is the standard deviation. The value shown in brackets
is the value obtained for plasma with buffer, shown with the
standard deviation as the error. All aPTTs were obtained using a
final concentration of 5 serpin. FL o AT Pitts C232S at this
concentration rendered the plasma unclottable. The value of 300 s
shown is the cut-off for the assay. Second-order rate constants for
the inhibition of thrombin, APC and fXa by the variants are shown
with the standard error.
Experiments
The coagulation cascade and the regulatory role of serpins in this
cascade are shown in Figure 1 . Two pathways lead to activation of
the coagulation cascade, the extrinsic cascade (or tissue factor
pathway) and the intrinsic pathway (contact activation pathway) . The
main physiological pathway of activation is believed to be the
extrinsic pathway. In this pathway, tissue factor (TF) is exposed on
the surface of the damage blood vessel. TF can then bind fVIIa and
fVII. TF:fVIIa activates fVII, as well as TF:fVII spontaneously
activating to TF:fVIIa. TF:fVIIa activates f to fXa and this
activates prothrombin to thrombin ( f Ila) ; the central protease of
the coagulation cascade. Thrombin activates platelets by cleavage of
protease activated receptors (PARs) and cleaves fibrinogen to
fibrin. Fibrin is crosslinked by fXIIIa, which is itself activated
by thrombin, to form the stable fibrin clot. Thrombin in addition
activates a positive feedback mechanism to potentiate its own
formation. It activates fVIII to fVIIIa and fV to fV . fVIIIa binds
to fIXa to form the intrinsic tenase (Xase) complex. The intrinsic
Xase activates more fX. This fXa can bind to fVa to form
prothrombinase . Prothrombinase activates prothrombin to thrombin and
is responsible for most of the thrombin generated after initiation
of coagulation. In addition to thrombin's positive feedback
mechanism, thrombin can also shut down its own activation via a
negative feedback mechanism. When it binds its cofactor
thrombomodulin (TM) , the thrombin:TM complex can activate protein C
(PC) to activated protein C (APC) . APC cleaves and inactivates both
fVIIIa and fVa, effectively shutting down thrombin generation.
Serpins are important inhibitors o f the coagulation cascade. The
inhibitory actions of the serpins protein C inhibitor (PCI) ,
antithrombin (AT ), heparin cofactor II (HCII) and cxi-antitrypsin
(o A T ) are shown in Figure 1 .
Below w e describe the conversion of serpins into specific inhibitors
of APC for use as procoagulant agents (treatment, prophylaxis,
bypassing or synergistic) in the treatment of bleeding disorders
such as hemophilia. The modifications described here for both PCI
and show a s a proof o f principle that small changes in these
proteins can b e used to create specific APC inhibitors.
PCI was first described as the physiological inhibitor o f APC and
therefore served as a starting point for these investigations
(Suzuki e t al, 1983; 1984) . However, PCI is promiscuous and also
inhibits thrombin, thrombin:TM, fXIa, fXa and TF:fVIIa (Elisen e t
al, 1998; Mosnier et al, 2001; Suzuki et al, 1984; Fortenberry e t
al, 2011; Meijers e t al, 1988). A s a consequence, PCI can function
a s both a pro- and an anticoagulant. Its activity is regulated b y
binding to glycosaminoglycans , such as heparin and heparan sulfates
(Pratt & Church, 1992; Pratt e t al, 1992; L i & Huntington, 2008) .
For an overview o f the role of PCI in the coagulation cascade, see
Figure 1 .
oilA T is the natural inhibitor o f neutrophil elastase (Kalsheker,
1989) . Unlike other serpins in the coagulation cascade that have
either Arg o r Leu at PI, <¾ has a Met instead. This makes it a
very poor inhibitor of coagulation proteases. Nevertheless, because
o f the high concentration o f o i T in plasma, it is believed to
inhibit APC to a physiologically significant degree (Heeb & Griffin,
1988) . Mutagenesis o f the P i residue has shown that the use o f an
Arg or Leu at P drastically improves inhibition of coagulation
proteases b y i T (Heeb e t al, 1990) . This is exemplified b y the
Pittsburgh (M358R (P1R) ; Pitts) variant o f iA T that causes a severe
bleeding disorder (Owen e t al, 1983).
To develop serpins specific for inhibition o f APC over procoagulant
proteases, PCI and A T Pittsburgh (PlR, i Pitts) were used as
template serpin scaffolds. All proteins used in this study were
expressed from Escherichia coli cultures (Rosetta2 (DE3) LysS ,
Novagen) using the pETSUMO expression vector (Invitrogen) and
purified using a combination of Ni-chromatography, anion-exchange
and in the case of PCI, heparin affinity chromatography. The SUMOtag
was subsequently removed by SUMO protease cleavage and the tag
removed by tandem Ni-anion exchange chromatography for A T
Pittsburgh (Pitts, PlR) and tandem anion exchange-heparin
chromatography for PCI. The PCI construct is N-terminally truncated,
starting at Ala22 (A22, numbering according to the mature protein
sequence, starting with the propeptide) . The Pitts (PlR)
construct is full-length (FL) and has an additional C232S mutation
to abolish intermolecular disulfide bond formation and other
modifications during expression and purification (Yamasaki e al,
2011) . Due to the expression vector used, the OiiAT Pitts construct
has a Ser (S) as its first residue instead o f a Glu (E) . The C232S
and ElS mutations are not expected to alter the activity of aiAT.
Lys residues were introduced at different positions into the RCL of
PCI and Pittsburgh and the resulting mutants tested for
inhibition of thrombin and APC. In the initial stages of this study,
inhibitors were not screened or tested for their inhibition of other
coagulation proteases on the premise that once thrombin inhibition
was abolished in favor of APC inhibition, the inhibitor could
potentially b e additionally modified if it had significant residual
inhibitory activity towards other coagulation proteases. The RCL
residues are numbered according to the Schechter-Berger nomenclature
for serpins (Schechter & Berger, 1967) .
Rate constants of thrombin and APC inhibition were measured under
pseudo-first-order conditions using an excess of serpin over
protease (Table 1 ) . Serpin and protease were incubated together for
varying lengths o f time and the residual activity was determined by
adding an excess of chromogenic substrate for the protease (S2238
for thrombin and S2366 for APC) , Residual protease activity was then
measured by following the absorbance at 405 nm. Plots of residual
protease activity over time gave the observed rate constant o s . The
second-order rate constant, k , is the slope of the plot of 0
versus serpin concentration (fitted using a linear regression
model) . Standard errors of the slope are shown.
The lysine mutations introduced at P2 and PI' were highly effective
at increasing the specificity of PCI and for APC over thrombin
for all variants shown in Table 1 . Generally, thrombin inhibition
was greatly reduced in all cases. APC inhibition was also reduced
for all mutants but not nearly to the same degree. Both serpins
initially inhibited thrombin better than APC. This was reversed for
all mutants tested.
PCI, unlike iA T , binds heparin and this binding considerably
increases its inhibition of thrombin and APC (Pratt & Church, 1992).
We therefore tested the inhibition of thrombin and APC by the Pl'K
and P2KP1'K PCI mutants in the presence of heparin to see if the
swap in specificity seen in Table 1 would persist. Rate constants
were measured under pseudo-first-order conditions using an excess of
PCI over protease. PCI was preincubated with an equimolar
concentration of unf ractionated heparin for 30 min prior to the
experiment. PCI: heparin and protease were incubated together for
varying lengths of time and the residual activity after certain
timepoints determined by adding an excess of chromogenic substrate
for the protease mixed with polybrene to bind the heparin. Plots of
residual protease activity over time gave the observed rate constant
s . The second-order rate constant, k , is the slope of the plot of
k s versus serpin concentration (fitted using a linear regression
model) . Standard errors of the slope are shown. The value calculated
for the inhibition of thrombin by P2KPl'K PCI was an estimate from
the initial slope of the plot of residual thrombin activity versus
time, as the graph suggests that complete inhibition is not
achieved. This might be due to the substrate pathway or complex
dissociation. The second-order rate constants are shown in Table 2 .
A s for the inhibition in the absence of heparin, the Pl'K and
P2KP1'K mutants of PCI, unlike the WT protein were specific for APC
over thrombin (Table 2 ) .
These experiments showed that introducing only one or two
modifications in the serpin RCL was sufficient to abolish or greatly
reduce the inhibition o f thrombin both in the presence and absence
of cof actors. The inhibition of APC was reduced but still
considerable, especially for the variants of i and the PCI
variants in the presence o f heparin. However, the specificity of PCI
and iA T Pitts is not limited to thrombin and APC. Both these
serpins also inhibit fXa, another procoagulant protease. In order
not to inhibit coagulation, our variants also need to b e specific
for APC over fXa. We therefore also determined the rate constants of
inhibition o f PCI and i and their variants for fXa (Table 3 ) .
Rate constants were measured under pseudo-first-order conditions
using an excess of serpin over protease. Serpin and protease were
incubated together for varying lengths of time and the residual
activity determined b y adding an excess o f chromogenic substrate
(S2222) for the protease. Plots of residual protease activity over
time gave the observed rate constant k0 bs. The second-order rate
constant, k2, is the slope of the plot o f k0 b versus serpin
concentration (fitted using a linear regression model) . Standard
errors of the slope are shown (Table 3 ) .
A s seen before for thrombin, WT PCI inhibited fXa better than APC.
iA T Pitts inhibited APC better than fXa, but the inhibition of fXa
was still considerable. The inhibition of fXa was still significant
for Pl'K PC , P2K i T Pitts and Pl'K i Pitts (Table 3). The
P2KP1'K variants of oiiAT Pitts and PCI were both highly specific for
APC over fXa, with absent or negligible inhibition of fXa and were
therefore considered lead candidates. The Pl'K variant of PCI is
also of interest as its inhibition o f fXa is very slow in the
absence o f heparin. The presence of heparin accelerates the rate of
APC inhibition significantly, which could potentially skew the
specificity ratio in favor o f APC.
The PCI lead compounds will be discussed first, followed by the i
lead compound.
To investigate the properties of A22 P2KPl'K PCI in a more complex
plasma system and to rule out any negative effects on the
procoagulant proteases, a prothrombin time assay (PT) was performed.
This assay measures the time until clot formation in plasma after
coagulation is initiated via the extrinsic pathway. A22 T PCI
showed a small increase in the clotting time, whereas P2KP1'K showed
a smaller increase, consistent with reduced inhibitory activity
towards procoagulant proteases (Figure 2).
In addition, we wanted to rule out any effect of the PCI mutant on
the contact activation pathway of coagulation. To do so, rate
constants of inhibition were measured for the inhibition of fXIa and
an aPTT assay was done. This assay is similar to the PT assay except
that it measures coagulation initiated via the intrinsic pathway.
Second-order rate constants of inhibition for inhibition of fXIa by
PCI and the P2 Pl 'K variant were measured under pseudo-first-order
conditions using an excess of serpin over protease. Serpin and
protease were incubated together for varying lengths of time and the
residual activity determined by adding an excess of chromogenic
substrate for the protease (S2366) . Plots of residual protease
activity over time gave the observed rate constant bs - The secondorder
rate constant, k2, is the slope of the plot of 0 s versus
serpin concentration (fitted using a linear regression model) .
Standard errors of the slope are given. fXIa inhibition by A22
P2KPl'K PCI did not go to completion over the course of the
experiment, potentially due to serpin cleavage by the protease.
Compared to WT, the P2KP1'K mutant showed a much reduced inhibition
towards fXIa and greater specificity for APC than for fXIa (Table
4 ) -
The aPTT assay showed that WT PCI was a potent inhibitor of the
contact activation pathway, potentially due to inhibition of fXIa or
fX a (Figure 3 ) . The P2KP1 ' mutant showed a small increase in the
aPTT. However, since the contact activation pathway primarily
activates coagulation through fIXa activation, inhibition of contact
activation to a small extent is likely to be insignificant in
hemophiliacs as they are deficient in either the main target of
contact activation ( fix ) or its cofactor ( fVI I ) .
The results shown here so far therefore show that both A22 Pl'K PCI
and A22 P2KP1'K PCI are promising, APC-specific lead compounds for
development into bypassing agents for the treatment of hemophilia.
To generate additional PCI mutants with specificity for APC over
thrombin, a targeted random mutagenesis strategy was employed on the
PCI scaffold. The residues targeted were P4, P2 and PI'. The random
approach is based on a selection for APC inhibition and against
thrombin inhibition by testing inhibitory activity of bacterial
lysates, after PCI expression in a 96-well format.
The assay was calibrated using the most specific PCI mutant
generated from the rational mutagenesis combined with the testing
for specificity outlined above; A22 P2KP1'K PCI. WT PCI was used as
an additional control. The negative selection against inhibition of
thrombin was achieved by incubating bacterial lysates for a time
period such that A22 WT PCI showed complete inhibition and the
incubation period extended from that timepoint on to also select
against minor inhibition. Positive selection for APC inhibition was
calibrated such that both WT and P2KP1'K PCI fell into the
intermediate range of APC inhibitory activity so we would be able to
determine both increases as well as decreases in inhibition.
In an initial assay, 88 variants were screened for thrombin and APC
inhibitory activity. Cultures were grown, induced and protein
expressed in 96-well plates. Cells were lysed by the addition of a
lysis buffer and the lysates assayed for inhibitory activity against
thrombin and APC by incubating the lysate with the protease for 1 h
for thrombin and 30 min for APC. Residual protease activity was then
read by addition of a chromogenic substrate to the protease. A22 WT
PCI and A22 P2KPl'K PCI were used as controls. Any lysate with
higher or equivalent residual thrombin activity and lower or
equivalent residual APC activity compared to P2KP1'K PCI was
considered to be a promising APC-specific candidate. These sequences
are shown in Table 7 . P4, P2 and P ' positions are shown in bold.
The mutant with the greatest APC inhibitory activity from this set
of experiments (D8; P4QP2RPl'N) was characterized in a preliminary
fashion and was shown to be more specific for the inhibition of APC
than thrombin, whilst utilizing different mutations than P2KP1'K
(Table 5 ) . This indicated that it would be possible to make
additional mutations in the serpin RCL, which would have equivalent
effects to the mutations already described.
To generate a larger dataset, a further 460 mutants were screened in
both the positive and negative selection assays. Cultures were
grown, induced and protein expressed in five 96-well plates. Cells
were lysed by the addition of a lysis buffer and the lysates assayed
for inhibitory activity against thrombin and APC by incubating the
lysate with the protease for 1 h for thrombin and 30 min for APC.
Residual protease activity was then read by addition of a
chromogenic substrate to the protease (S2238 for thrombin, S2366 for
APC). A22 T PCI and A22 P2KP1'K PCI were used as controls. Any
lysate with higher or equivalent residual thrombin activity and
lower or equivalent residual APC activity compared to P2KPl'K PCI
was considered to be a promising APC-specific candidate. From this
dataset, colonies were picked and sequenced for serpins that showed
better inhibition of APC and worse or equivalent inhibition of
thrombin than P2KP1'K and these were retested in triplicate in the
same assay to verify that these were indeed true hits and not false
positives. From this retest a set of 15 out of the 17 mutants found
in the initial screen was determined to be better or equivalent at
inhibiting APC than P2KP1'K and worse or equivalent at inhibiting
thrombin (Table 8). Sequences of variants that were positive after
retesting are shown in Table 8 . Interestingly, the random
mutagenesis confirmed the beneficial effects of positively charged
residues in the P2 and P ' positions. However alternative RCL
compositions were also found.
Preliminary second-order rate constants for the inhibition o f
thrombin and APC b y random mutagenesis PCI variants were measured
under pseudo first-order conditions using an excess of serpin over
protease. Serpin and protease were incubated together for varying
lengths of time and the residual activity determined by adding an
excess of chromogenic substrate for the protease. Plots o f residual
protease activity over time gave the observed rate constant 0 bs- The
second-order rate constant, k2, is the slope o f the plot of k0bs
versus serpin concentration (fitted using a linear regression
model) . WT and P2KPl'K are shown for comparison. Rate constants are
shown in Table 5 . The error shown is the standard error o f the
slope .
Preliminary characterization of some of the selected mutants from
the random mutagenesis assay showed that the selected mutants were
at least functionally equivalent to P2KPl PCI, and some had a
slightly improved rate of APC inhibition (Table 5 ) . These
experiments strongly suggest that P2KP1'K is not the only
composition which could be utilized to generate APC-specific
serpins.
The main lead compound based on Pitts from the rate constants
shown in Tables 1 and 3 was FL ¾ Pitts C232S P2KPl'K. This mutant
was shown not to inhibit thrombin and only slowly inhibited fXa, but
retained APC inhibition (Tables 1 and 3 ) .
To investigate the properties o f this mutant in a more complex
plasma system and to rule out any negative effects on the
procoagulant proteases, a prothrombin time assay (PT) was performed.
This assay measures the time until clot formation after coagulation
is initiated via the extrinsic pathway. A s expected, the
anticoagulant i Pitts showed an increase in clotting time, due to
its inhibition of thrombin and fXa (Figure 4 ) . In contrast, the
P2KPl'K mutant of o T Pitts showed no increase in clotting time and
therefore did not interfere with normal coagulation in this assay.
The data so far indicated that the P2KP1'K mutant o f ai T Pitts did
not interfere with procoagulant proteases in either the extrinsic
(tissue factor) pathway or the common pathway of coagulation. In
addition we wanted to determine whether FL iA T Pitts C232S P2KPl'K
affected the intrinsic (contact activation) pathway. Second-order
rate constants of inhibition of fXIa b y FL i Pitts C232S and the
P2KPl'K variant were measured under pseudo first-order conditions
using an excess of serpin over protease. Serpin and protease were
incubated together for varying lengths o f time and the residual
activity determined by adding an excess o f chromogenic substrate for
the protease (S2366) . Plots of residual protease activity over time
gave the observed rate constant k • The second-order rate constant,
k2, is the slope o f the plot of k0b versus serpin concentration
(fitted using a linear regression model) . Standard errors of the
slope are given.
fXI is activated during the contact activation pathway and it feeds
into the common pathway of coagulation by activating fix. In
addition, fXI is activated once coagulation is initiated by
thrombin. fXIa was inhibited to a significant degree by FL o A T
Pitts C232S, however this inhibition was greatly reduced for FL oiiAT
Pitts C232S P2KP1'K (Table 6).
Because a small degree o f inhibition of fXIa b y the P2KP1'K mutant
of i T Pitts was detected and to determine any potential negative
effect on fXIIa, we additionally performed an aPTT assay. This assay
is similar to the PT assay except that it measures coagulation
initiated via the intrinsic pathway of coagulation. The aPTT could
therefore be used to detect any negative effect on fXIa and the
contact activation pathway of coagulation. In this assay, plasma
incubated with FL T Pitts C232S did not clot within the time o f
the assay except for one reaction with 0.67 serpin (Figure 5A) .
FL aiAT Pitts C232S P2KPl'K showed a small increase in clotting
time, but there was no dose-dependent increase (Figure 5B) . This
indicates that the fXIa inhibitory activity of FL c A T Pitts C232S
P2KPl'K is likely too slow to significantly affect the contact
activation pathway. In addition, the contact activation pathway
activates the coagulation cascade via activation of fIXa. Since
hemophiliacs lack either fix or its essential cofactor fV , the
role of a small degree of inhibition of the contact activation
pathway in hemophiliacs is likely to be minimal.
To investigate whether the P2KPl'K mutant of o Pitts was able to
inhibit APC in a plasma system, we used a modified thrombin
generation assay (TGA) . Thrombin generation was measured in human
pooled normal plasma (NP) in the presence and absence of recombinant
soluble thrombomodulin (TM) . This TM was expressed and purified from
a HEK-EBNA expression system and comprises the soluble extracellular
domain. TM is not normally present in the TGA because
physiologically it is a transmembrane protein, present on the
endothelial membrane and largely absent from plasma. Therefore,
there is no activation of the PC pathway in a normal TGA or other
coagulation assays utilizing plasma, such as the PT and aPTT assays.
Adding TM to the assay allows PC activation and thereby might give a
more realistic picture of thrombin generation in vivo. Assays shown
in Figures 6 and 7 were performed in pooled normal human plasma (NP)
from George King Biomedical. Coagulation was initiated by the
addition of CaCl 2 and TF/phospholipid (RB low TF and phospholipid
reagent, Technoclone) to activate coagulation through the extrinsic
pathway. Thrombin generation was measured through the cleavage of a
fluorogenic substrate (Z-Gly-Gly-Arg-AMC) . Fluorescence units were
converted to thrombin concentration by calibrating fluorescence
units against known concentrations of thrombin, using the
Technothrombin calibration kit (Technoclone) .
Addition of TM to pooled normal plasma reduced thrombin generation
in a concentration-dependent manner. From this experiment we chose
two concentrations of TM to knock down thrombin generation to either
intermediate levels (1.25 nM TM final assay concentration) or to low
levels (10 nM TM final assay concentration) . These concentrations
were used in subsequent assays to evaluate the ability of FL iA
Pitts C232S P2KP1'K to inhibit APC in plasma.
Addition of FL A Pitts C232S to normal human plasma (NP) reduced
thrombin generation at all concentrations used, likely due to the
inhibition of thrombin as well as fXa (Figure 6 ) . In contrast, FL
ii T Pitts C232S P2KPl'K had no effect on NP in the absence of TM
(Figure 7A) . However in the presence of TM, FL c iA T Pitts C232S
P2KP1'K dose-dependently rescued thrombin generation (Figure 7B-D) .
This effect is the result o f specific inhibition of APC b y FL 
Pitts C232S P2KP1'K.
In order to perform the same experiments in fVIII- or fIX-def icient
plasma, it was necessary to increase the amount of tissue factor
(TF) used to initiate the assay because at the baseline conditions
(RB trigger only) , there was no detectable thrombin generation in
factor deficient plasma. To demonstrate the effect of an increase in
TF on thrombin generation, the reactions were spiked with different
dilutions of T F reagent (Dade Innovin, Siemens) in addition to the
RB reagent used to trigger the assay at baseline conditions. The
concentration of TF in the Innovin reagent is not disclosed by the
manufacturer, however previous measurements have shown it to b e
around 7.36 nM (Duckers e t al, 2010) . Increasing the T F trigger
shortened lag-time and increased both peak thrombin and endogenous
thrombin potential (ETP) in human NP, fVIII-def icient (HA) and flXdef
icient (HB) plasma. From these experiments we chose an Innovin
dilution of 1:4,000 in the final reaction to initiate thrombin
generation. RB reagent, which contains both phospholipids and TF was
added because of the need to add phospholipids to the assay.
Because the use of factor-deficient plasma required modification of
the assay parameters, we repeated the TM titration experiment for
human normal pooled plasma in human HA plasma with the addition of
1:4,000 Innovin. The assay was performed in human fVIII-def icient
plasma (less than 1% fVIII activity) from George King Biomedical.
Coagulation was initiated by the addition of C Cl and/or
TF/phospholipid (RB low TF and phospholipid reagent, Technoclone)
with 1:4,000 final dilution of Dade Innovin (Siemens) to activate
coagulation through the extrinsic pathway. Thrombin generation was
measured through the cleavage of a fluorogenic substrate (Z-Gly-Gly-
Arg-AMC) . Fluorescence units were converted to thrombin
concentration by calibrating fluorescence units against known
concentrations of thrombin, using the Technothrombin calibration kit
(Technoclone ). Thrombomodulin (TM) was found to reduce thrombin
generation in the thrombin generation assay (TGA) in fVIII-def icient
plasma (HA) .
From this experiment, 1.25 nM and 5 nM TM were selected for
subsequent experiments. The high TM concentration used was lower
than for NP, mainly because the total thrombin generation in HA
plasma in the assay conditions used was lower.
The effects o f both FL Pitts C232S and FL iA T Pitts C232S
P2KP1'K on HA and HB plasma were comparable to the results from
pooled NP. FL i Pitts inhibited thrombin generation in the
presence and absence of TM in both HA (fVIII-def icient) and HB
plasma (fIX-def icient) (Figure 8). FL i T Pitts C232S P2KP1'K could
rescue the effect of TM on thrombin generation in both fV - and
fIX-deficient plasma and had no effect in the absence of TM (Figures
9 and 10) . This indicates that FL T Pitts C232S P2KP1'K can
inhibit APC and have a procoagulant effect in factor-deficient
plasma. This means it could potentially promote clot formation and
reduce bleeding in hemophilia patients. The magnitude of this
procoagulant effect will b e determined by the relative contribution
of the protein C system to the reduction in thrombin generation in
vivo. The in vitro experiments shown here cannot be used to predict
the likely efficacy of this mutant in vivo, however they do show
that in complex plasma systems FL oiiAT Pitts C232S P2KPl'K can
inhibit APC and does not interfere with the procoagulant pathways,
and that these effects are independent of the presence or absence o f
f x and fVIII.
In order to verify our in vitro data we wanted to use in vivo mouse
models of hemophilia. However, to verify that the effect of human
aiAT on mouse plasma would be comparable to the effect seen in human
plasma w e first performed a TGA in mouse plasma. This was done using
a modified TGA protocol (Bunce et al, 2011; Ivanciu et al, 2011) .
These modifications were necessary because of the increased
concentrations of inhibitory proteins in mouse plasma that preclude
TGA assays under standard conditions (Tchaikovski e t al, 2007; Bunce
et al, 2011; Ivanciu et al, 2011) . Comparable to the human system,
there was no thrombin generation in HB mouse plasma under the
baseline conditions o f the assay. Therefore, w e performed a
titration spiking in different concentrations of Innovin. A
concentration of 1:12,000 Innovin was chosen for subsequent assays.
Because no murine TM was available, we used soluble human TM as used
in the human plasma TGAs to promote APC formation in the mouse TGA
assay. The concentration required to see any effect of human TM in
HB mouse plasma was ~ 100-fold higher than seen in human plasma.
This could b e explained b y the observation that human TM knock-in
mice show reduced ability to activate murine PC (Raife e t al, 2011) .
This indicated that human TM is less potent at promoting murine PC
activation than murine TM. A concentration of 750nM human TM was
used in subsequent experiments.
Different concentrations of both FL cxi T Pitts C232S and FL 
Pitts C232S P2KP1'K were then added to the determined conditions in
mouse HB plasma both in the absence and presence of TM to compare
the effect of these mutants in mouse plasma to the previous TGA
results in human plasma.
FL T Pitts C232S reduced thrombin generation in mouse HB plasma
in the absence of TM as seen in human plasma (Figure 11A) . However
in the presence o f TM, at the lowest concentration, there was a
partial rescue of thrombin generation (Figure 11B) . This may
potentially b e due to a difference in the relative rates of
inhibition for murine thrombin and murine APC by FL ii T Pitts
compared to the rates seen in humans, such that the generated APC is
inhibited prior to thrombin inhibition. When the concentration o f FL
Pitts C232S is increased to such levels that all APC has been
inhibited, thrombin is also inhibited. This could explain the
results seen in Figure 11B, but was not further investigated. FL
iA T Pitts C232S P2KPl'K rescued thrombin generation knocked-down by
TM addition in HB mouse plasma as it did in human plasma (Figure
11D) . However, when FL Pitts C232S P2KP1'K was added to HB
mouse plasma in the absence of TM, an increase in thrombin
generation was also observed (Figure 11C) . It is possible that this
effect relates to the method of blood collection employed for these
experiments. To collect plasma, the tail was transected and blood
collected into citrate. This was then spun down and the plasma
removed and frozen. The injury inflicted for blood collection leads
to activation of the coagulation system and may cause generation of
APC in the plasma prior to the experiment. Additionally, mice do not
have PCI in their plasma (Zechmeister-Machhart et al, 1996) , which
may increase the circulating half-life of APC, such that it is not
inactivated prior to the TGA assay. An alternative explanation,
which cannot be ruled out at present would be an off-target
procoagulant effect in mouse plasma. However, since this effect is
not seen in human plasma, this would involve the inhibition of a
mouse-specific anticoagulant protease.
To investigate a potential in vivo effect of FL Pitts C232S
P2KPl'K and to determine if it could potentially be useful as either
a prophylactic agent or a treatment for hemophilia we used two in
vivo mouse assays; tail clip and a cremaster arteriole laser injury
model. The mice used were male, fix knockout mice on the BALB/c
background (Lin et al, 1997; Ivanciu et al, 2011) .
In the tail clip assay, protein or buffer was injected through the
tail vein and after a 5 min incubation period, the tail was
transected at a diameter of 3 mm and placed in 14 ml 37 °C saline
solution in a 37 °C water bath. Blood was collected for 10 min and
the resulting blood loss was quantified by measuring total
hemoglobin after red cell lysis by measuring absorbance at 575 n
(Ivanciu et al, 2011) . The volume blood loss was calculated by
making a standard curve where known volumes of blood collected by
tail transection were processed in a similar manner to the tail clip
samples. After red cell lysis, the absorbance at 575 nm was
determined and plotted against the volume blood loss to generate the
standard curve. Tail clip assays showed a potent procoagulant effect
of FL c iA T Pitts C232S P2KP1'K (Figure 12) . At the dose of 15 mg/kg,
blood loss of the HB mice was restored to the level of T mice
injected with PBS (Figure 12) . Lower dose FL A T Pitts C232S
P2 Pl 'K also showed a trend to reduction of bleeding with respect to
HB mice although this was not statistically significant. FL 
Pitts C232S showed no significant effect on blood loss at 7.5 mg/kg.
Another vivo model used for evaluating procoagulant agents is the
intravital cremaster arteriole laser-induced injury model (Falati e t
al, 2002) . In this system, a cannula is inserted into the mouse
jugular vein to allow infusion o f the therapeutic protein as well as
fluorescently-tagged antibodies to fibrin and platelets. The
cremaster muscle is then spread out for imaging. Clot formation
after laser-induced injury to the arterioles is imaged and
quantified using fluorescence microscopy.
For an overall qualitative assessment, injuries were sorted into
three categories: no clot (no fluorescence detected) , platelet clot
(only platelets visible, these clots were generally unstable and
dissolved over the course o f the imaging) and platelets + fibrin
(both platelet and fibrin fluorescence visible and clot remained
stable over the course of the imaging) . This showed that there was a
dose-dependent increase in stable platelet and fibrin clot formation
with increasing concentration of FL Pitts C232S P2KP1'K (Figure
13) . All images were quantified for platelet and fibrin
fluorescence. The median value for each timepoint was calculated and
the results plotted in Figure 14. These data included the
quantification from all images, regardless of their category
assigned for Figure 13. The plots o f the median show that control or
FL x A T Pitts C232S infused mice exhibit no clot formation, whereas
both high and low dose FL Pitts C232S P2KP1'K showed platelet
aggregation and fibrin deposition at the site of the injury. No
difference could be detected between the two doses in terms of
platelet aggregation. For fibrin, there was a dose-dependent
increase in fibrin deposition for FL iA Pitts C232S P2 P1 'K and no
fibrin for either control or FL T Pitts C232S infused mice
(Figure 14) .
Taken together, these results show that FL aiAT Pitts C232S P2KP1'K
has a procoagulant effect in both in vitro assays and in vivo models
of hemophilia. The in vivo experiments were all done in mouse models
of hemophilia B , however TGA results in human plasma (Figures 9 and
10) and the proposed mechanism of action of FL i Pitts C232S
P2KP1'K indicate that its procoagulant effect should be independent
of fix or fV I deficiency and could therefore be used in both
hemophilia A and B . The procoagulant effect seen n vivo was
sufficient to reduce bleeding to the same levels as seen for T mice
(Figure 12) indicating that serpins that inhibit APC can be used for
treatment of bleeding disorders and not only as a prophylactic or an
adjuvant to existing treatments.
A targeted random mutagenesis strategy was also employed on the T
scaffold in order to explore potential additional APC specific
mutants on the i scaffold.
Three different i T variant libraries were generated on the FL i T
Pitts C232S background: one randomised at P2, one randomised at PI'
and a third library randomised at both P2 and P '. The resulting
plasmid libraries were transformed into the Rosetta2 (DE3 )pLysS
expression strain and protein expressed in 96-well plates. Bacteria
were lysed and the lysates assayed for thrombin and APC inhibition.
For the single variant libraries, 88 colonies were assayed per
library. For the double (P2P1') variant library, 460 colonies were
assayed. FL ίPitts and FL i Pitts P2KP1'K (the lead APC
specific variants) were expressed on all assay plates as a
reference. Variants with higher or equal APC inhibitory activity
(lower or equal residual APC activity) and lower or equal thrombin
inhibitory activity (higher or equal residual thrombin activity)
compared to P2KP1'K A T were deemed candidates for variants
specific for APC over thrombin. A subset of candidate variants was
picked and re-assayed in the same setup to verify the results from
the first screen. Mutants showing similar properties in both assays
were then sequenced. The resulting sequences are shown in Table 9 .
To verify the ability of this assay to pick out variants, which were
specific for APC over thrombin, nine variants identified in Table 9
were expressed on a larger scale in E . coli and purified. The
second-order rate constant of inhibition with respect to thrombin
and APC was then determined for each mutant. The results are shown
in Table 10. These results confirmed that all variants tested had a
higher specificity for APC than for thrombin unlike the FL i T
Pitts C232S variant.
Certain types of residues were favored in the oiiAT Pitts scaffold at
both P2 and Pi' positions (Table 9 ) . Specificity was primarily
conferred by the presence o f bulky polar (Q, N , Y ) bulky hydrophobic
( ) or positively charged residues (R, H , K ) at P2 and Pi'. Other
residues seen at these positions included C , A , T , S and V . These
medium to small residues were accompanied in the double variant
library b y a complementing large positively charged residue (R, K )
or large polar residue (Y, N , Q ) at the other position, which likely
has a larger influence on the specificity swap. However, there may
b e a cooperative effect of these mutations as well, especially where
the PI' is R . Pl'R showed variable results in the single variant
library screen and may have some residual thrombin inhibitory
activity. The P2 P is known to be important for thrombin cleavage o f
substrates (Gallwitz et al, 2012). Simple removal of this residue
coupled to a specificity swapping mutation at PI ' may be sufficient
to generate an APC specific inhibitor with little residual thrombin
inhibitory activity. Especially T at P2 might have some effect on
its own as, out of its partnering residues at Pi' (Q, N , Y , R ) , only
R was identified in the single residue PI' variant library as being
sufficient to cause a specificity swap by itself.
Interestingly, non-specific mutants that clustered around the AT
Pitts control were shown to be largely c A T Pitts. All retained the
P2 P , showing its importance in maintaining thrombin inhibitory
activity. The PI' was more variable, consistent with the
distribution o f mutants in the Pi' variant library. Comparing the
spread of variants in the P2 and Pi' libraries, thrombin seemed to
b e more tolerant to Pi' than to P2 mutations. However, the
appearance o f favorable residues in the double variant libraries
that were not present in the single variant libraries indicate that
the effects of these residues on specificity may be cooperative and
double mutations may be more effective than single mutations at
increasing specificity.
The random mutagenesis results presented above showed that it was
possible to generate mutants that showed specificity for APC over
thrombin other than the lysine mutants already identified. So far,
the random mutagenesis strategy was used, assuming that once
specificity over thrombin was obtained, these mutants would also
show some degree of specificity for APC over other procoagulant
proteases. To test this, the PT and aPTTs of the random mutants in
Table 10 were tested. These results are displayed in Table 11. None
of the mutants had a significant effect on the PT unlike previously
shown for FL oilAT Pitts C232S (Figure 4 ) . This provides indication,
that the mutants have largely lost their inhibitory activity towards
procoagulant proteases. However, aPTTs are more sensitive to the
presence of inhibitors. Therefore, the measure of the aPTT might
give a more accurate representation of smaller residual inhibition.
Previous experiments (Figure 5 ) showed that FL lAT Pitts C232S
rendered plasma unclottable in the aPTT assay. In contrast, only one
of the mutants evaluated here showed this effect (P2TPl'N). Some
mutants, such as 2 1 and P2KPl'N showed only a relatively small
prolongation of the aPTT and were therefore potentially interesting.
From these results, we selected four mutants, P2R, P2QP1'K, P2KP1
and P2KP1'N (all on the Pitts, PlR background). These showed either
a high inhibition of APC over thrombin (Table 10) and for some also
a low prolongation of the aPTT (21 and P2KP1'N). Because they
did not prolong the PT, it is unlikely that they inhibit TFrfVIIa.
The most likely candidates for prolongation of the aPTT would be
inhibition of fXIa or fXa. Of these, fXa inhibition would most
inhibit the initial stages of coagulation and was therefore
considered to be a more significant barrier for a successful
inhibitor. Inhibition constants for the inhibition of fXa by these
four mutants were therefore determined as described above (Table
12) . P2R showed significant inhibition of fXa, which may be the
reason for its prolongation of the aPTT. The other three mutants
showed lower fXa inhibition, but none of the mutants were as
specific as the previously identified P2KP1'K mutant. Nevertheless,
P2KP1'N, P2QP1'K and P2 P1 represent additional promising
candidates for further development as they show specificity for APC
over thrombin and fXa. In addition, their inhibition of APC is
roughly 2-fold increased with respect to the P2KPl'K mutant
described earlier. This faster inhibition might in part compensate
for the slightly reduced specificity.
However, these results indicated that selecting for specificity for
APC over thrombin is not completely sufficient to design an
inhibitor which also shows specificity over fXa and other
procoagulant proteases. Therefore, the random mutagenesis strategy
was extended further. Mutants, which previously had been selected
for specificity for APC over thrombin, were rescreened against fXa
and mutants selected, which had reduced fXa inhibition, while
maintaining low thrombin inhibition and APC inhibition.
Four additional mutants were identified from this additional screen.
These all had the P1R mutation and in addition had either P2RP1 ,
P2RP1'Q, P2WP1'I or P2WP1'H. To verify the specificity of these
mutants, an initial experiment was performed, using only one
concentration of serpin and testing its inhibition of thrombin and
fXa. APC inhibition was not considered at this stage, because
mutants were selected based on their low inhibition of thrombin and
fXa. Serpin and protease were incubated for different times and at
the indicated timepoints, the reaction was stopped by addition of an
excess of chromogenic substrate. Residual protease activity was
divided by the initial protease activity and the natural log of this
value plotted against time (Figure 15) . The slope of this line
divided by the serpin concentration gives an estimate of the secondorder
rate constant of inhibition. These assays showed that while
all mutants hardly inhibited thrombin, with the fastest inhibitor,
P2WP1'H having a second-order rate constant of -50.3 M .s (compare
to the inhibition constant of FL Pitts C232S, 2.928 x 105 M^.s
). However, P2RP1 , P2WP1 'I and P2WP1'H showed significant
inhibition of fXa. The second-order rate constant was only ~10-fold
reduced for these mutants compared to FL AT Pitts C232S (4,070.1 M
'-.s for P2RP1'A compared to 4.13 x 104 M s- 1 for FL Pitts
C232S) . P2RP1'A, P2WP1 'I and P2WP1'H showed similar fXa inhibition
to each other.
Only one mutant showed significant selectivity against both thrombin
and fXa. This mutant had a P2RPl'Q in addition to the Pitts (P1R)
mutation. Because of its selectivity, it was interesting for more
thorough investigation. Previous results from both the random and
rational mutagenesis studies indicated that R and K residues perform
reasonably similarly. W e therefore also generated a P2KPl'Q mutant
on the PlR background as it was expected from the results shown here
to have similar properties. The results from measurements of
inhibition constants and aPTTs (experiments performed as described
before) for both mutants are shown in Table 13. The P2KP1'K mutant
is shown for comparison. Both P2KP1'Q and P2RP1'Q showed very low
inhibition of thrombin and f a . In addition, there was also hardly
any effect on the aPTT . APC inhibition was significant, being only
slightly reduced in comparison to P2KP1'K. Therefore, these two
mutants would be expected to perform similarly to P2KPl'K and may
represent other potentially promising alternative molecules for
further development.
We evaluated inhibition of murine thrombin and APC by FL «iAT Pitts
C232S P2KPl'K. Thrombin and APC were obtained from recombinant
sources. The proteases used are truncated with respect to the plasma
version, including only the EGF2-protease domains for APC (Gladomainless
APC) and the protease domain for thrombin. Therefore, we
also tested the human versions of these proteases to ensure that any
differences were due to a species difference, rather than a
construct difference. Human and murine thrombin showed none or very
little reactivity with FL Pitts C232S P2KPl'K by SDS-PAGE,
indicating that in this respect, results from model systems would be
relevant to the human system. Second-order rate constants of
inhibition were (8.14 ± 0.58) x 103 M_ .s_ , for human Gla-domainless
APC, (3.80 ± 0.37) x 103 M^.s for murine Gla-domainless APC,
compared to (14.88 ± 1.87) x 103 M^.s for human plasma APC. These
results provided indication that while the reactivity of the mutant
in mouse models would likely be lower than in humans, i.e. the
relative dose for the same effect might need to be higher, the
effect in terms of protease inhibition is likely to be similar.
The data presented show as a proof-of-principle that the serpin
scaffold can be used to generate specific APC inhibitors, using only
very few mutations, that these inhibitors can have procoagulant
activities both in vitro and in vivo and as such show promise as
procoagulant agents for treatment and prophylaxis of bleeding
disorders such as hemophilia.
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Second-order rate constants of inhibition s )
Fold
Variant Thrombin APC inhibition
APC/thrombin
Ά22 T PCI 28.21 + 1.51 0.68 ± 0.032 0.02
A22 PI 'K PCI 0.022 + 0.0024 0.88 ± 0.074 40
A22 P2KP1 ' PCI ~ 0 .03* 0.28 + 0.013 9.3
FL T Pitts C232S 292 .76 ± 17 .60 108.16 + 7.086 0 .4
FL i T Pitts C232S P2 0.051 + 0.0028 64.82 ± 7.14 1,271
FL A T Pitts C232S Pl'K 0.17 ± 0.017 95 .66 + 1 .70 563
FL oiiAT Pitts C232S no inhibition no thrombin
15.14 + 1.68
P2KP1 'K after 4 h inhibition
Table 1
Table 2
Table 3
Second-order rate constants of inhibition ( .s-1)
Inhibition of
Variant fXIa APC
APC/fXIa
A22 T PCI 8.59 + 0.43 0.68 ± 0.032 0.08
A22 P2 P1 ' PCI 0.023 + 0.0052 0.28 ± 0 .013 12 .2
Table 4
Table 5
Table 6
Table 7
10
20
25
Table 8
P6 P5 P4 P3 P2 PI I ' P2 ' P3 ' P4 '
WT L E A I P M s I P P
Pitts L E A I P R s I P P
P2 .Gil L E A I K R s I P P
P2 .F10 L E A I R R s I P P
P2.D8 L E A I K R s I P P
P2.G8 L E A I R R s I P P
P2 .E7 L E A I R R s I P P
P2 .D10 L E A I R R s I P P
P2 .G4 L E A I R R s I P P
P2 .F4 L E A I R R s I P P
PI' .H8 L E A I P R I P P
PI' .All L E A I P R R I P P
PI' .F10 L E A I P R E I P P
PI' .F9 L E A I P R K I P P
PI' .F4 L E A I P R E I P P
4 .G9 L E A I T R N I P P
4 .G4 L E A I R K I P P
3.E5 L E A I R R A I P P
3.B6 L E A I S R R I P P
3.B2 L E A I K R N I P P
3.A10 L E A I T R Y I P P
2 .HI L E A I R R H I P P
2 .C6 L E A I T R R I P P
1 .H10 L E A I V R R I P P
1 .Bll L E A I R R C I P P
1 .A12 L E A I K R H I P P
2 ,E5 L E A I T R R I P P
3 .G9 L E A I Y R R I P P
3 .F4 L E A I A R R I P P
3 .C9 L E A I C R K I P P
2 .H5 L E A I K R N I P P
2.E7 L E A I R N I P P
1 .B2 L E A I S R R I P P
5.C12 L E A I H R N I P P
5.A6 L E A I R R I P P
4 .El L E A I P R K I P P
4 .C12 L E A I N R N I P P
3 .F8 L E A I T R M I P P
3 .CIO L E A I T R H I P P
2 .E8 L E A I K R S I P P
1 .H9 L E A I R I P P
Table 9
Second-order rate constants of inhibition .s- )
Inhibition of
Variant Thrombin APC
APC/thrombin
FL AT Pitts
292.76 + 17.60 108.16 ± 7.086 0.4
C232S
FL Pitts no inhibition No thrombin
15.14 + 1.68
C232S P2KP1'K after 4 h inhibition
FL AT Pitts
0.042 ± 0.0024 61.12 ± 6.26 1455.2
C232S P2R
FL cxiAT Pitts 131.57 ± 13.32
0.68 ± 0.068 193.5
C232S Pl'R
FL a AT Pitts 0.15 ± 0.015 2.99 ± 0.29 19.9
C232S Pl'E
FL aiAT Pitts
0.27 + 0.047 62.37 ± 2.46 231.0
C232S P2TP1'N
FL aiAT Pitts
C232S 21 Ύ 0.023 + 0.0014 5.70 ± 0.83 247 .8
FL i Pitts
0.0038 ± 0.0013 33.41 + 6.36 8792.1
C232S P2QP1'K
FL aiAT Pitts no inhibition No thrombin
28.84 ± 3.05
C232S 21 Ή after 2 h inhibition
FL aiAT Pitts 37.80 + 2.48
0.015 + 0.0026 2520.0
C232S P2KP1'N
FL aiAT Pitts
C232S P2RP1'C 0.034 + 0.0094 24.55 + 2.15 722 .1
Table 10
Variant PT (s) aPTT (s)
Plasma 27.2 + 0.8 60.3
FL OiiAT Pitts C232S P2K 27.0 ± 0.5 107 .2
FL OiiAT Pitts C232S Pl'K 27.1 ± 0.4 228 .1
FL OiiAT Pitts C232S P2 27.8 ± 0.4 111 .6
FL ciiAT Pitts C232S Pl'R 28.1 ± 0.5 287
FL Pitts C232S Pl'E 27.2 + 0.4 84
FL o iAT Pitts C232S P2TP1'N 29.5 ± 0.9 >300
FL Pitts C232S 21 28.3 ± 0.8 185.4
FL Pitts C232S P2QPl'K 27.9 ± 0.3 111.5
FL AT Pitts C232S 21 Ή 27.4 ± 0.9 77 .8
FL aiAT Pitts C232S P2KP1'N 27.8 ± 0.3 81.9
FL aiAT Pitts C232S P2RPl'C 28.5 ± 0.6 ND
Table 11
Table 12
Table 13
Sequences
1 mqlflllclv llspqgaslh rhhpremkkr vedlhvgatv apssrrdftf dlyralasaa
61 psqniffspv sismslamls lgagsstkmq ileglglnlq kssekelhrg fqqllqelnq
121 prdgfqlslg nalftdlvvd Iqdtfvsamk tlyladtfpt nfrdsagamk qindyvakqt
181 kgkivdllkn ldsnavvimv nyiffkakwe tsfnhkgtqe qdfyvtsetv vrvpmmsred
241 qyhylldrnl scrvvgvpyq gnatalfilp segkmqqven glsektlrkw lkmfkkrqle
301 lylpkfsieg syqlekvlps lgisnvftsh adlsgisnhs niqvsemvhk avvevdesgt
361 raaaatgtif tfrs arlnsq rlvfnrpflm fivdnnilfl gkvnrp
SEQ ID NO: 1 Protein C inhibitor (PCI)
Mature protein, including the propeptide, corresponds to residues
to 40 6 . Signal sequence corresponds to residues 1-19. Propeptide
corresponds to residues 20-25. Residues P4, P2, PI and PI' of the
RCL bold and underlined.
1 mermlpllal gllaagfcpa vlchpnspld eenltqenqd rgthvdlgla sanvdf afsi
61 ykqlvlkapd knvifsplsi stalaflslg ahnttlteil kglkfnltet seaeihqsf
121 hllrtlnqss delqlsmgna mfvkeqlsll drftedakrl ygseafatdf qdsaaakkli
181 ndyvkngtrg kitdlikdld sqtmmvlvny iffkakwemp fdpqdthqsr ylskkkwvm
241 vpmmslhhlt ipyfrdeels ctvvelkytg nasalf ilpd qdkmeeveam llpetlkrwr
301 dslefreige lylpkfsisr dynlndillq lgieeaftsk adlsgitgar nlavsqvvhk
361 avldvfeegt easaatavki tlls alvetr tivrfnrpf 1 miivptdtqn iffmskvtnp
421 kqa
SEQ ID NO: 2 Alpha-l-antichymotrypsin
Mature protein corresponds to residues 26 to 423. Residues P4, P2,
Pi and PI' of the RCL bold and underlined
1 masrltlltl lllllagdra ssnpnatsss sqdpeslqdr gegkvattvi skmlfvepil
61 evsslpttns ttnsatkita nttdepttqp ttepttqpti qptqpttqlp tdsptqpttg
121 sfcpgpvtlc sdleshstea vlgdalvdfs Iklyhafsam kkvetnmafs pfsiaslltq
181 vllgagentk tnlesilsyp kdftcvhqal kgfttkgvts vsqifhspdl airdtfvnas
241 rtlysssprv Isnnsdanle Ixntwvaknt nnkisrllds Ipsdtrlvll naiylsakwk
301 ttfdpkktrm epfhfknsvi kvpmmnskky pvahfidqtl kakvgqlqls hnlslvilvp
361 qnlkhrledm eqalspsvfk aimeklemsk fqptlltlpr ikvttsqdml simekleffd
421 fsydlnlcgl tedpdlqvsa mqhqtvlelt etgveaaaas aisv art llv fevqqpflfv
481 Iwdqqhkfpv fmgrvydpra
SEQ ID NO: 3 Cl-esterase inhibitor
Mature protein corresponds to residues 23-500. Residues P4, P2, Pi
and PI' of the RCL bold and underlined
1 mallwgllvl swsclqgpcs vfspvsamep lgrqltsgpn qeqvspltll klgnqepggq
61 talksppgvc srdptpeqth rlarammaf t adlfslvaqt stcpnlilsp lsvalalshl
121 algaqnhtlq rlqqvlhags gpclphllsr lcqdlgpgaf rlaarmylqk gfpikedf e
181 qseqlfgakp vsltgkqedd laninqwvke ategkiqefl sglpedtvll llnai gf
241 wrnkfdpslt qrdsf hldeq vpve qa rtyplrwfll eqpeiqvahf p f nn sf v
301 Ivpthfewnv sqvlanlswd tlhpplvwer ptkvrlpkly lkhqmdlvat lsqlglqelf
361 qapdlrgise qslvvsgvqh qstlelsevg veaaaatsiamsrmslssf s vnrpf Iffif
421 edttglplfv gsvrnpnpsa prelkeqqds pgnkdflqsl kgfprgdklf gpdlklvppm
481 eedypqfgsp k
SEQ ID NO: 4 2-Antiplasmin
Mature protein corresponds to residues 28-491. Residues P4, P2, P
and PI' of the RCL for inhibition of chymotrypsin in bold, residues
for the inhibition of plasmin underlined.
1 mysnvigtvt sgkrkvylls llligfwdcv tchgspvdic takprdipmn pmciyrspek
61 katedegseq kipeatnrrv welskansrf attf yqhlad skndndnif 1 splsistaf
121 mtklgacndt lqqlmevf f dtisektsdq ihf faklnc rlyr ankss klvsanrlf g
181 dksltfnety qdiselvyga klqpldf en aeqsraaink wvsnktegri tdvipseain
241 eltvlvlvnt iyf kglwksk fspentrkel fykadgescs asmmyqegkf ryrrvaegtq
301 vlelpfkgdd itmvlilpkp ekslakveke Itpevlqewl deleemmlvv hmprf riedg
361 fslkeqlqdm glvdlf spek sklpgivaeg rddlyvsdaf hkaf levnee gseaaastav
421 viagrs lnpn rvtf kanrpf Ivfirevpln tiifmgrvan pcvk
SEQ ID NO: 5 Antithrombin (ATIII)
Mature protein corresponds to residues 33-464. Residues P4, P2, P and
PI' of the RCL bold and underlined
1 mkhslnalli fliitsawgg skgpldqlek ggetaqsadp qweqlnnknl smpllpadf h
61 kentvtndwi pegeedddyl dlekif sedd dyidivdsls vsptdsdvsa gnilqlf hgk
121 sriqrlniln akf afnlyrv lkdqvntf dn ifiapvgist amgmislglk getheqvhsi
181 lhfkdfvnas skyeittihn Ifrklthrlf rrnf gytlrs vndlyiqkqf pilldf ktkv
241 reyyfaeaqi adfsdpafis ktnnhimklt kglikdalen idpatqmmil nciyfkgswv
301 nkfpvemthn hnf lnerev vkvsmmqtkg n laandqel dcdilqleyv ggismlivvp
361 hkmsgmktle aqltprvver qks tnrtr evllpkf e knynlveslk Imgirmlfdk
421 ngnmagisdq riaidlfkhq gtitvneegt qattvttvgf pls tqv t vdrpflfliy
481 ehrtscllfm grvanpsrs
SEQ ID NO: 6 Heparin cofactor II
Mature protein corresponds to residues 20-499. Residues P4, P2, Pi
and PI' of the RCL bold and underlined
1 mpssvswgil llaglcclvp vslaedpqgd aaqktdtshh dqdhptfnki tpnlaef fs
61 lyrqlahqsn stnif fspvs iataf amlsl gtkadthdei leglnf nlte ipeaqihegf
121 qellrtlnqp dsqlqlttgn glfIseglkl vdkf ledvkk lyhseaftvn fgdteeakkq
181 indyvekgtq gkivdlvkel drdtvf alvn yif fkgkwer p evkdteee dfhvdqvttv
241 kvpmmkrlgm fniqhckkls swvllmkylg nataif flpd egklqhlene lthdiitkfl
301 enedrrsasl hlpklsitgt ydlksvlgql gitkvf snga dlsgvteeap lklskavhka
361 vltidekgte aagamf leai pms ippevkf nkpf fImie qntksplfmg k vnptqk
SEQ ID NO: 7 ai-antitrypsin ()
Mature protein corresponds to residues 25-418. Residues P4, P2, P
and Pi' o f the RCL bold and underlined
1 mhlidyllll Ivgllalshg qlhvehdges csnsshqqil etgegspslk iapanadfaf
61 rfyyliaset pgkniffspl sisaayamls lgacshsrsq ileglgfnlt elsesdvhrg
121 fqhllhtlnl pghgletrvg salflshnlk flakflndtiti avyeaklfht nfydtvgtiq
181 lindhvkket rgkivdlvse lkkdvlmvlv nyiyfkalwe kpfissrttp kdfyvdentt
241 vrvpmitilqdq ehhwylhdry Ipcsvlrmdy kgdatvffil pnqgkmreie evltpemlmr
301 wnnllrkrnf ykklelhlpk fsisgsyvld qilprlgftd Ifskwadlsg itkqqkleas
361 ksfhkatldv deagteaaaa tsfaik ffs a qtnrhilrfn rpflvvifst stqsvlflgk
421 vvdptkp
SEQ ID NO: 8 Kallistatin
Mature protein corresponds to residues 21-427. Residues P4, P2, Pi
and Pi' o f the RCL bold and underlined
1 mqmspaltcl vlglalvfge gsavhhppsy vahlasdfgv rvfqqvaqas kdrnvvfspy
61 gvasvlamlq lttggetqqq iqaamgfkid dkgmapalrh lykelmgpwn kdeisttdai
121 fvqrdlklvq gfmphffrlf rstvkqvdfs everarfiin dwvkthtkgm isnllgkgav
181 dqltrlvlvn alyfngqwkt pfpdssthrr lfhksdgstv svpmmaqtnk fnytefttpd
241 ghyydilelp yhgdtlsmfi aapyekevpl saltnilsaq lishwkgnmt rlprllvlpk
301 fsletevdlr kplenlgmtd mfrqfqadft slsdqeplhv aqalqkvkie vnesgtvass
361 stavivsarm apeeiimdrp flfvvrhnpt gtvlf gqv ep
SEQ ID NO: 9 Plasminogen activator inhibitor
Mature protein corresponds to residues 24-402. Residues P4, P2, Pi
and Pi' o f the RCL bold and underlined
1 k vpsllls vllaqvwlvp glapspqspe tpapqnqtsr vvqapkeeee deqeaseeka
61 seeekawlma srqqlakets nfgfsllrki s rhdgn vf spfgmslamt glmlgatgpt
121 etqikrglhl qalkptkpgl Ipslfkglre tlsrnlelgl tqgsfafihk dfdvkerffn
181 lskryfdtec vpmnfrnasq akrlmnhyin ketrgkipkl fdeinpetkl ilvdyilfkg
241 kwltpfdpvf tevdtfhldk yktikvpmmy gagkfastfd knfrchvlkl pyqgnatmlv
301 vlmekmgdhl aledylttdl vetwlrnmkt. rnmevffpkf kldqkyemhe llrqmgirri
361 fspfadlsel satgrnlqvs rvlqrtviev dergteavag ilseit aysm ppvikvdrpf
421 hfmiyeetsg mllflgrvvn ptll
SEQ ID NO: 10 Protein Z dependent inhibitor
Mature protein corresponds to residues 22-444. Residues P4, P2, Pi
and Pi' of the RCL bold and underlined
1 mnwhlplfll asvtlpsics hfnplsleel gsntgiqvfn qivksrphdn ivisphgias
61 vlgrtilqlgad grtkkqlamv mrygvngvgk ilkkinkaiv skknkdivtv anavfvknas
121 eievpfvtrn kdvfqcevrn vnfedpasac dsinawvkne trdmidnlls pdlidgvltr
181 lvlvnavyfk glwksrfqpe ntkkrtfvaa dgksyqvpml aqlsvfrcgs tsapndlwyn
241 fielpyhges ismlialpte sstplsaiip histktidsw msimvpkrvq vilpkftava
301 qtdlkeplkv Igitdmfdss kanfakittg senlhvshil qkakievsed gtkasaatta
361 iliars sppw fivdrpflff irhnptgavl fmgqinkp
SEQ ID NO: 11 Protease nexin 1
Mature protein corresponds to residues residues 20-398 - isoform a .
Residues P4, P2, PI and PI' of the RCL bold and underlined
1 mpssvswgil llaglcclvp vslaedpqgd aaqktdtshh dqdhptfnki tpnlaefafs
61 lyrqiahqsn stniffspvs iatafamlsl gtkadthdei leglnfnlte ipeaqlhegf
121 qellrtlnqp dsqlqlttgn glflseglkl vdkfledvkk lyhseaftvn fgdteeakkq
181 indyvekgtq gkivdlvkel drdtvfalvn yiffkgkwer pfevkdteee dfhvdqvttv
241 kvpmmkrlgm fniqhckkls swvllmkylg nataifflpd egklqhlene Ithdiitkfl
301 enedrrsasl hlpklsitgt ydlksvlgql gitkvfsnga dlsgvteeap Iklskavhka
361 vltidekgte aagamfleai krkippevkf nkpfvflmie qntksplfmg kvvnptqk
SEQ ID NO: 12 Modified Serpin in scaffold
Mature protein corresponds to residues 25-418. Residues P4, P2, PI
and PI' of the RCL bold and underlined
Claims
1 . A modified serpin having mutations at one or both of residues
Pi' and P2 and optionally Pi and/or P4 in the reactive center loop
(RCL) thereof.
2 . A modified serpin according to claim 1 wherein said mutations
increase the inhibition of one or more of anticoagulant proteases
selected from activated Protein C and thrombin :thrombomodulin
complex relative to the inhibition of one or more procoagulant
proteases selected from thrombin, fVIIa, fXa, fIXa and fXIa.
3 . A modified serpin according to claim 1 or claim 2 wherein said
mutations increase the inhibition of activated Protein C relative to
the inhibition of thrombin.
4 . A modified serpin according to any one of the preceding claims
wherein the RCL of the modified serpin consists of the amino acid
sequence of the RCL of a wild-type serpin with said mutations
therein .
5 . A modified serpin according to any one of the preceding claims
having a mutation at the PI' position.
6 . A modified serpin according to claim 5 wherein the PI' residue
is mutated to Q , N , Y , , R , H , K , C , A , I , T , S , , P , E or V .
7 . A modified serpin according to claim 6 wherein the Pi' residue
is mutated to Q , N , Y , R , H , K , C , A , I , S , M , E or V .
8 . A modified serpin according to claim 7 wherein the Pi' residue
is mutated to Q , H , K or R .
9 . A modified serpin according to claim 8 wherein the PI' residue
is mutated to K .
10. A modified serpin according to claim 8 wherein the PI' residue
is mutated to Q .
11. A modified serpin according to any one of the preceding claims
having a mutation at the P2 position.
12. A modified serpin according to claim 11 wherein the P2 residue
is mutated to D , Q , N , Y , W , F , L , C , A , I , T , S , M , H , K , R , P or
V .
13. A modified serpin according to claim 12 wherein the P2 residue
is mutated to D , Q , N , Y , W , F , L , C , A , I , T , S , H , K , R , P or V .
14. A modified serpin according to claim 13 wherein the P2 residue
is mutated to H , K or R
15. A modified serpin according to claim 14 wherein the P2 residue
is mutated to K .
16. A modified serpin according to any one of the preceding claims
having mutations at the PI' and P2 positions.
17. A modified serpin according to claim 15 wherein the P2 and the
PI' residues respectively in the modified serpin are KK, FK, RK, VK,
LK, QK, CK, P , FR, HR, IR, SR, TR, VR, YR, AR, PR, RS , KS, QV, RV,
RI, RH, KH, TH, RC, RA, LY, QY, TY, DM, TM, WN, RN, HN, TN, KN, NN,
PE, RQ, KQ or T .
18. A modified serpin according to claim 17 wherein the P2 and the
PI' residues respectively in the modified serpin are K
19. A modified serpin according to claim 17 wherein the P2 and the
PI' residues respectively in the modified serpin are R and Q .
20. A modified serpin according to claim 17 wherein the P2 and the
PI' residues respectively in the modified serpin are K and Q .
21. A modified serpin according to any one of the preceding claims
wherein the modified serpin comprises a mutation at P4 .
22. A modified serpin according to claim 21 wherein the P4 residue
is mutated to F , S , R , V , C , W , K , G , L , H , T , Q or A .
23. A modified serpin according to any one of the preceding claims
wherein the modified serpin comprises a mutation at PI.
24. A modified serpin according to claim 23 wherein the Pl residue
is mutated to R .
25. A modified serpin according to any one of claims 1 to 2 4
wherein the modified serpin has an R residue at the PI position.
26. A modified serpin according to any one of claims 1 to 25
wherein said mutations in the RCL consist of a mutation at Pl' .
27. A modified serpin according to any one of claims 1 to 25
wherein the mutations in the RCL of the modified serpin consist of
mutations at positions Pl' and P2 .
28. A modified serpin according to any one of claims 1 to 25
wherein the mutations in the RCL of the modified serpin consist of
mutations at positions Pl' , P2 and Pl .
29. A modified serpin according to any one of claims 1 to 25
wherein the mutations in the RCL of the modified serpin consist of
mutations at positions Pl', P2 and P4.
30. A modified serpin according to any one of claims 1 to 25
wherein the mutations in the RCL of the modified serpin consist of
mutations at positions Pl , P2, P4 and Pl.
31. A modified serpin according to any one of claims 1 to 25
wherein the mutations in the RCL of the modified serpin consist of a
mutation at position P2 .
32. A modified serpin according to any one of claims 1 to 25
wherein the mutations in the RCL of the modified serpin consist of
mutations at positions P2 and P .
33. A modified serpin according to any one of claims 1 to 25
wherein the mutations in the RCL of the modified serpin consist of
mutations at positions P2 and PI .
34. A modified serpin according to any one of claims 1 to 25
wherein the mutations in the RCL of the modified serpin consist of
mutations at positions PI, P2 and P .
35. A modified serpin according to any one of claims 1 to 25
wherein the mutations in the RCL of the modified serpin consist of
mutations at position P .
36. A modified serpin according to any one of claims 1 to 25
wherein the mutations in the RCL of the modified serpin consist of
mutations at positions P4 and PI.
37. A modified serpin according to any one of claims 1 to 25
wherein the mutations in the RCL of the modified serpin consist of
mutations at positions P4r Pi and PI'.
38. A modified serpin according to any one of claims 1 to 25
wherein the mutations in the RCL of the modified serpin consist of
mutations at positions Pi' and PI.
39. A modified serpin according to any one of claims 1 to 25
wherein the mutations in the RCL of the modified serpin consist of
mutations at positions Pi' and P .
40. A modified serpin according to any one of claims 1 to 25
wherein the mutations in the RCL of the modified serpin consist of a
mutation at position PI.
41. A modified serpin according to any one of the claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is Q , the
P2 residue is R and the PI' residue is N .
42. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is K , the
P2 residue is R and the PI' residue is H .
43. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is S , the
P2 residue is L and the Pi' residue is K .
44. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is H , the
P2 residue is R and the PI' residue is V .
45. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is F , the
P2 residue is K and the PI' residue is K .
46. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is F , the
P2 residue is R and the Pi' residue is K .
47. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is F , the
P2 residue is V and the PI' residue is K .
48. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is C , the
P2 residue is L and the PI' residue is K .
49. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is F , the
P2 residue is F and the PI' residue is R .
50. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is S , the
P2 residue is H and the PI' residue is R .
51. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is G , the
P2 residue s I and the PI' residue is R .
52. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is R , the
P2 residue is Q and the Pi' residue is V .
53. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is T , the
P2 residue is R and the PI' residue is V .
54. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is R , the
P2 residue is R and the PI' residue is I .
55. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is V , the
P2 residue is R and the PI' residue is I .
56. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is L , the
P2 residue is R and the PI' residue is I .
57. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is T , the
P2 residue is L and the Pi' residue is Y .
58. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is A , the
P2 residue is Q and the PI' residue is Y .
59. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue is W , the P2 residue is W and the PI' residue
is N ; or the P4 residue is K , the P2 residue is D and the PI'
residue is M .
60. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is A , the
P2 residue is K , and the Pi' residue is K .
61. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is A , the
P2 residue is K , and the Pi' residue is S .
62. A modified serpin according to any one of claims 1 to 40 wherein
the P4 residue in the RCL of the modified serpin is A , the P2
residue is R , and the PI' residue is S .
63. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is A , the
P2 residue is P , and the PI' residue is E .
64. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is A , the
P2 residue is P , and the Pi' residue is R .
65. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is A , the
P2 residue is P , and the PI' residue is K .
66. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is A , the
P2 residue is T , and the Pi' residue is M .
67. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is A , the
P2 residue is T , and the PI' residue is H .
68. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is A , the
P2 residue is T , and the Pi' residue is Q .
69. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is A , the
70. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is A , the
P2 residue is T , and the Pi' residue is Y .
71. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is A , the
P2 residue is T , and the PI' residue is R .
72. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is A , the
P2 residue is R , and the PI' residue is A .
73. A modified serpin according to any one of claims 1 to 40
wherein the P 4 residue in the RCL of the modified serpin is A , the
P2 residue is R , and the PI' residue is H .
74. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is A , the
P2 residue is R , and the PI' residue is C .
75. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is A , the
P2 residue is R , and the PI' residue is N .
76. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is A , the
P2 residue is S , and the Pi' residue is R .
77. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is A , the
P2 residue is K , and the PI' residue is N .
78. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is A , the
P2 residue is K , and the Pi' residue is H .
79. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is A , the
P2 residue is V , and the PI' residue is R .
80. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is A , the
P2 residue is Y , and the PI' residue is R .
81. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is A , the
P2 residue is A , and the Pi' residue is R .
82. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is A , the
P2 residue is C , and the PI' residue is K .
83. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is A , the
P2 residue is W , and the Pi' residue is N .
84. A modified serpin according to any one of claims 1 to 40
wherein the P 4 residue in the RCL of the modified serpin is A , the
P2 residue is H , and the Pi' residue is N .
85. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is A , the
P2 residue is Q , and the Pi' residue is K .
86. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is A , the
P2 residue is N , and the Pi' residue is N .
87. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is F , the
88. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is A , the
P2 residue is R , and the Pi' residue is Q .
89. A modified serpin according to any one of claims 1 to 40
wherein the P4 residue in the RCL of the modified serpin is A , the
P2 residue is K , and the Pi' residue is Q .
90. A modified serpin according to any one of the preceding claims
wherein the PI residue in the RCL of the modified serpin is R .
91. A modified serpin according to claim 90 wherein the P4 residue
in the RCL of the modified serpin is A , the P2 residue is K , the P
residue is R and the P ' residue is K .
92. A modified serpin according to claim 90 wherein the P4 residue
in the RCL of the modified serpin is A , the P2 residue is K , the PI
residue is R and the PI' residue is Q .
93. A modified serpin according to claim 90 wherein the P4 residue
in the RCL of the modified serpin is A , the P2 residue is R , the PI
residue is R and the Pi' residue is Q .
94. A modified serpin according to any one of the preceding claims
that comprises the sequence of a wild-type serpin with 50 or fewer
amino acid residues mutated.
95. A modified serpin according to any one of the preceding claims
that comprises an amino acid sequence that has at least 70% sequence
identity to the sequence of a wild-type serpin.
96. A modified serpin according to any one of the preceding claims
that comprises the amino acid sequence of a wild-type serpin with
said mutations in the reactive center loop (RCL) thereof.
97. A modified serpin according to any one of claims 94 to 96
wherein the wild type serpin is selected from the group consisting
of oci-antichymotrypsin (SERPINA3) , Cl-esterase inhibitor, -
antiplasmin (SERPINF2) antithrombin (ATIII) (SERPINC1) , heparin
cofactor II (HCII) (SERPIND1), protein C inhibitor (PCI) (SERPINA5)
or ai-antitrypsin (o^AT) (SERPINA1), Kallistatin (SERPINA4),
Plasminogen activator inhibitor (SERPINE1), Protease nexin 1
(SERPINE2) and Protein Z-dependent protease inhibitor (SERPINA10) .
98. A modified serpin according to any one of claims 94 to 97
wherein the wild type serpin sequence is selected from the group
consisting of the mature serpin sequences of 3EQ ID NOS 1 to 11.
99. A modified serpin according to any one of claims 94 to 98
wherein the wild type serpin is protein C inhibitor (PCI)
(SERPINA5) .
100. A modified serpin according to claim 99 wherein the wild type
serpin sequence is residues 20 to 406 of SEQ ID NO: 1 .
101. A modified serpin according to any one of claims 94 to 98
wherein the wild type serpin is ai-antitrypsin () (SERPINA1) .
102. A modified serpin according to claim 101 wherein the wild type
serpin sequence is residues 25-418 of SEQ ID NO: 7 .
103. A modified serpin according to any one of claims 94 to 98, 101
or 102 comprising an amino acid sequence having at least 70%
sequence identity to residues 25-418 of SEQ ID NO: 12, wherein the
P4 residue in the RCL of the modified serpin is A , the P2 residue is
K , PI residue is R and the PI' residue is K .
104. A modified serpin according to any one of claims 94 to 98 or
101 to 103 comprising the amino acid sequence of residues 25-418 of
SEQ ID NO: 12 with 50 or fewer mutations, wherein the P4 residue in
Lhe RCL of the modified serpin is A , the P2 residue is , Pi residue
is R and the Pi' residue is K .
105. A modified serpin according to any one of claims 94 to 98 or
101 to 104 comprising the amino acid sequence of residues 25-418 of
SEQ ID NO: 12.
106. A nucleic acid encoding a modified serpin according to any one
of claims 1 to 105.
107. A vector comprising a nucleic acid according to claim 106,
108. A recombinant cell comprising a vector which expresses a
modified serpin according to any one of claims 1 to 105.
109. A recombinant cell according to claim 108 comprising a vector
according to claim 107.
110. A pharmaceutical composition comprising a modified serpin
according to any one of claims 1 to 105, a nucleic acid according to
claim 106, a vector according to claim 107 or a recombinant cell
according to claim 108 or 109 and a pharmaceutically acceptable
excipient .
111. A method of producing a pharmaceutical composition comprising
admixing a modified serpin according to any one of claims 1 to 105,
a nucleic acid according to claim 106, a vector according to claim
107 or a recombinant cell according to claim 108 or 109 with a
pharmaceutically acceptable excipient.
112. A method of treatment of bleeding or promotion or hemostasis
comprising administering a modified serpin according to any one of
claims 1 to 105, a nucleic acid according to claim 106, a vector
according to claim 107 or a recombinant cell according to claim 108
or 109 to an individual in need thereof.
113. A modified serpin according to any one of claims 1 to 105, a
nucleic acid according to claim 106, a vector according to claim 107
or a recombinant cell according to claim 108 or 109 for use in the
treatment of the human or animal body.
114. A modified serpin according to any one of claims 1 to 105, a
nucleic acid according to claim 106, a vector according to claim 107
or a recombinant cell according to claim 108 or 109 use in the
treatment or prevention of bleeding or the promotion of hemostasis
in an individual.
115. Use of a modified serpin according to any one of claims 1 to
105, a nucleic acid according to claim 106, a vector according to
claim 107 or a recombinant cell according to claim 108 or 109 in the
manufacture of a medicament for the treatment or prevention of
bleeding or the promotion of hemostasis in an individual.
116. A method according to claim 112, a modified serpin, nucleic
acid, vector or cell for use according to claim 114 or a use of
claim 115, wherein the individual has a bleeding disorder.
117. A method or a modified serpin, nucleic acid, vector or cell
for use or use according to claim 116, wherein the bleeding disorder
is hemophilia.
118. A method according to claim 112 or a modified serpin, nucleic
acid, vector or cell for use according to claim 114 or a use
according to claim 115 wherein the individual is a trauma patient.
119. Use of a modified serpin according to any one of claims 1 to
105, a nucleic acid according to claim 106, a vector according to
claim 107 r a recombinant cell according to claim 108 or 109 as an
in vitro or in vivo procoagulant.