A Rapid Pro-Hemostatic Approach To Overcome Direct Oral Anticoagulants

articles
A rapid pro-hemostatic approach to overcome direct oral

anticoagulants
2016 Nature America, Inc. All rights reserved.
Nabil K Thalji13, Lacramioara Ivanciu13, Robert Davidson1,2, Phyllis A Gimotty4, Sriram Krishnaswamy1,3 &
Rodney M Camire13
Direct inhibitors of coagulation factor Xa (FXa) or thrombin are promising oral anticoagulants that are becoming widely
adopted. The ability to reverse their anticoagulant effects is important when serious bleeding occurs or urgent medical
procedures are needed. Here, using experimental mouse models of hemostasis, we show that a variant coagulation factor,
FXaI16L, rapidly restores hemostasis in the presence of the anticoagulant effects of these inhibitors. The ability of FXa I16L to
reverse the anticoagulant effects of FXa inhibitor depends, at least in part, on the ability of the active site inhibitor to hinder
antithrombin-dependent FXa inactivation, paradoxically allowing uninhibited FXa to persist in plasma. Because of its inherent
catalytic activity, FXaI16L is more potent (by >50-fold) in the hemostasis models tested than a noncatalytic antidote that is
currently in clinical development. FXaI16L also reduces the anticoagulant-associated bleeding in vivo that is induced by the
thrombin inhibitor dabigatran. FXaI16L may be able to fill an important unmet clinical need for a rapid, pro-hemostatic agent to
reverse the effects of several new anticoagulants.
Clot formation is a homeostatic mechanism that prevents blood loss
following vascular injury. Its dysregulation can lead to bleeding or
pathological activation of coagulation and thrombosis. In clinical use
for over six decades, the pharmacologic oral anticoagulant warfarin
is a mainstay of care for thrombosis1; however, limitations of warfarin therapy prompted the development of new or direct oral anticoagulants (NOACs or DOACs, respectively)1,2. These small molecules
reversibly bind the active site of FXa35 or thrombin6 to inhibit enzyme
function. Clinically, DOACs are as or more effective than warfarin and
are approved in Europe and the United States for stroke prevention
in patients with atrial fibrillation, thromboprophylaxis following
orthopedic surgery, and initial and extended treatment of venous
thromboembolism712. However, as with warfarin, DOAC treatment
is associated with a clinically significant risk of bleeding13,14. Bleeding
complications with warfarin are managed by anticoagulant reversal
with vitamin K, fresh-frozen plasma or prothrombin complex concentrates (PCCs)1. In contrast, the FXa inhibitors lack clinically approved
countermeasures1518, and an antidote19,20 for the thrombin inhibitor
dabigatran was only recently approved by the US Food and Drug
Administration (FDA) (in November 2015).
Reversal strategies for direct FXa or thrombin inhibitors fall
into two categories antidotes that bind and sequester the inhibitor19,21,22, or bypassing approaches that enhance thrombin formation
and hemostasis in the presence of the inhibitor2327. Examples of the
former include idarucizumab, an antibody that binds dabigatran19,20,
and andexanet alfa, a catalytically inactive recombinant FXa variant
lacking the membrane-binding 4-carboxyglutamic acid domain

(GD-FXaS195A; Portola Pharmaceuticals) that binds FXa inhibitors21. Because their mechanism of action involves stoichiometric
binding to the inhibitor, hundreds of milligrams of these proteins are
needed for a therapeutic effect20,28,29. Both antidotes are efficacious
as reversal agents in preclinical models and in clinical trials15,19,22.
Unlike idarucizumab that targets only dabigatran, GD-FXaS195A binds
all FXa inhibitors through active site interactions, making it an attractive antidote. However, because GD-FXaS195A has a conformationally
intact active site and functional exosites, it can still interact with coagulation proteins, including tissue factor pathway inhibitor (TFPI), an
important negative regulator of coagulation30,31. Although many of
these binding interactions would be blunted in the presence of the
FXa inhibitor, they could still be relevant, as GD-FXaS195A needs to
be administered at high concentrations29.
As an alternative approach, a procoagulant hemostatic agent that
is effective at catalytic concentrations could be a viable strategy to
rapidly stop bleeding induced by DOACs. However, existing bypassing agents that have been developed for hemophilia treatment (for
example, recombinant factor VIIa (rFVIIa)) show mixed results in
reversing the effects of FXa or thrombin inhibitors in pre-clinical
model systems23,26,27. Here we tested a bypassing strategy with a FXa
variant that was previously developed in our group3234. Substitution
of isoleucine at position 16 (numbered after chymotrypsinogen35) with
leucine yields a FXa variant (FXaI16L) with an impaired conformational
transition from zymogen to protease (a zymogen-like FXa species).
1Division
of Hematology, Department of Pediatrics, Childrens Hospital of Philadelphia (CHOP), Philadelphia, Pennsylvania, USA. 2Raymond G. Perelman Center
for Cellular and Molecular Therapeutics, Childrens Hospital of Philadelphia, Philadelphia, Pennsylvania, USA. 3Division of Hematology, Department of Pediatrics,
University of Pennsylvania, Perelman School of Medicine, Philadelphia, Pennsylvania, USA. 4Department of Biostatistics and Epidemiology, University of
Pennsylvania, Perelman School of Medicine, Philadelphia, Pennsylvania, USA. Correspondence should be addressed to R.M.C. (rcamire@mail.med.upenn.edu).
Received 23 December 2015; accepted 16 June 2016; published online 25 July 2016; doi:10.1038/nm.4149
nature medicine advance online publication
Zymogen-like variants, such as FXaI16L, have impaired active site function, are resistant to active site inhibitors and have longer plasma halflives than wild-type (WT) FXa3234. Notably, their impaired activity is
rescued after binding the cofactor FVa on damaged cellular surfaces to
form the prothrombinase complex, resulting in robust thrombin generation at the injury site. These properties allow zymogen-like FXa variants to effectively bypass the clotting defect in hemophilic mice without
excessive activation of coagulation at therapeutic doses34. These properties might also enable these variants to avoid inhibition and restore
hemostasis in the presence of direct FXa or thrombin inhibitors. Here
we demonstrate that FXaI16L is an effective bypassing agent that counteracts DOACs in vitro and in vivo, using established mouse hemostasis
models. Reversal of the effects of FXa inhibitors is achieved, at least
in part, through an unexpected mechanism that involves competition
between the pharmacologic inhibitor (rivaroxaban) and the endogenous plasma proteinase inhibitors (such as antithrombin (AT)) for
binding to FXa. Our findings also have broader implications by providing new insight into the mechanisms by which the seemingly simple
DOACs function. Furthermore, this work highlights the efficacy of
zymogen-like FXa in mitigating the anticoagulant effects of the direct
oral FXa and thrombin inhibitors in multiple models of injury.
RESULTS
FXaI16L overcomes FXa inhibitors in vitro
Because clinical coagulation assays are not particularly sensitive to
the effects of direct FXa inhibitors36,37, we used thrombin-generation
100
20
40
60
Time (min)
80
100
125
100
75
50
25
0
0.03
0.10
0.30
1.00
I16L
Concentration of FXa
3.00 10.00
(nM)
b 125
Normalized peak thrombin (% of NHP)
200
assays (TGA) to determine whether FXaI16L could mitigate the effects

of rivaroxaban in normal human plasma (NHP). Within the therapeutically useful range (170830 nM)38, rivaroxaban inhibited thrombin
generation in NHP in a dose-dependent manner, reducing peak
thrombin levels (Fig. 1a,b). At a fixed concentration of rivaroxaban
(500 nM), addition of FXaI16L improved peak thrombin generation;
full normalization was achieved at 3 nM FXaI16L (Fig. 1c). Even in
the presence of 2.5 M rivaroxaban, which simulates excessive anticoagulation, addition of FXaI16L increased peak thrombin generation to
near-normal levels (Fig. 1d). Similar results were obtained with apixaban, another direct FXa inhibitor (data not shown). We also studied
the effects of rivaroxaban and FXaI16L in whole blood from normal
human donors by using rotational thromboelastography (ROTEM)
to evaluate whether additional blood components influence the
results. Addition of rivaroxaban at both therapeutic (Supplementary
Fig. 1a,b) and supratherapeutic (Supplementary Fig. 1c,d) concentrations prolonged the time to initial clot formation (ROTEM CT).
The addition of FXaI16L (0.3 or 3.0 nM) improved the ROTEM profile, as measured by the ROTEM CT in rivaroxaban-treated blood.
These results illustrate the effectiveness of FXaI16L in counteracting direct FXa inhibitors, regardless of whether it was assessed in
plasma or whole blood.
To compare the relative in vitro efficacy and potency of FXaI16L
to other anticoagulant reversal strategies, we expressed and purified
GD-FXaS195A. Our preparation of GD-FXaS195A was comparable to
that used in a previous report, as assessed by a modified coagulation
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25
0
0.2
0.4
0.8
1.6
3.2
Concentration of rivaroxaban (M)
6.4
125
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75
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0
0.03
0.10
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3.00 10.00
S195A
Concentration of GD-FXa
(M)
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50
25
0
0.03
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0.30
1.00
3.00 10.00
Concentration of FXaI16L (nM)
300
Normal human plasma

200 nM rivaroxaban
400 nM rivaroxaban
800 nM rivaroxaban
400
Concentration of thrombin (nM)
Articles
125
FXaI16L
GD-FXaS195A
100
75
50
25
0
102
101
100
101
102
103
104
Concentration of FXa (nM)
Figure 1 Effects of FXaI16L and GD-FXaS195A on thrombin generation in rivaroxaban-treated plasma. (a) Representative thrombin-generation tracings
(of four experiments) in platelet-poor NHP for different rivaroxaban concentrations. (b) Peak thrombin values generated at different rivaroxaban
concentrations in NHP, relative to that in NHP without rivaroxaban. (c,d) Normalized peak thrombin values generated after the indicated concentrations
of FXaI16L were titrated into NHP supplemented with 500 nM (c) or 2.5 M (d) rivaroxaban. (e) Peak thrombin values generated following titration of
the indicated concentrations of GD-FXaS195A into NHP containing 500 nM rivaroxaban. (f) Peak thrombin values generated in the presence of 500 nM
rivaroxaban and the indicated concentrations of FXa I16L (blue) or GD-FXaS195A (red), as plotted on a semi-logarithmic scale. In bf, all experiments were
performed in quadruplicate, and all measurements are shown as mean s.d.
advance online publication nature medicine
articles
addition of mFXaI16L (1 mg/kg; Fig. 2a). In this assay, GD-FXaS195A
was largely ineffective at a dose of 5 mg/kg, whereas a dose of 25 mg/kg
normalized the ROTEM CT.
To extend these findings in vivo, we used the FeCl3 carotid arterial thrombosis model34,40 (Fig. 2bf and Supplementary Fig. 4).
Rivaroxaban (0.5 mg/kg) prolonged the time to occlusion, and higher
doses of rivaroxaban (1 mg/kg) prevented carotid artery occlusion
(Fig. 2b). Administration of mFXaI16L (0.25 mg/kg or 1 mg/kg) 30 min
after injury (Fig. 2c) rapidly restored occlusion (Fig. 2d). This experimental design was advantageous because both the anticoagulant
effects of rivaroxaban and the pro-hemostatic effects of mFXa I16L
could be observed in the same animal. The mean time to occlusion
after mFXaI16L treatment was faster than that in control mice that
were not treated with rivaroxaban or mFXaI16L, consistent with the
concept that mFXaI16L generates thrombin by bypassing the intrinsic
and extrinsic coagulation pathways. Administration of GD-FXaS195A
(25 mg/kg) did not restore clot formation (Fig. 2d), suggesting that
it might not be as effective when administered after an injury has
occurred. As a control, 30 min after administration of GD-FXaS195A,
we infused mFXaI16L (1 mg/kg) and observed occlusion at the site of
injury (data not shown), confirming that the injury was sufficient to
produce carotid thrombosis.
DOACs are taken for many days to maintain a therapeutic steady
state. The oral FXa inhibitors are highly protein bound, and these
drugs are also distributed to extravascular tissues41. To determine
whether these factors could alter the effectiveness of FXaI16L in the FeCl3 model, mice were
given daily infusions of rivaroxaban. The time
to vessel occlusion was evaluated after the last
dose on day four. As expected, mice that were
given rivaroxaban did not form an occlusive
thrombus during the 30-min observation
period. Despite the multi-day infusion with
FXaI16L restores hemostasis in mice treated with rivaroxaban

For our studies, we expressed and purified mouse FXaI16L (mFXaI16L)
to avoid the possibility of inter-species incompatibility34 and used
the ex vivo ROTEM assay to measure the ROTEM CT and thereby
determine an effective dose. We found that rivaroxaban administered to WT mice at 1 mg per kg body weight (mg/kg) substantially increased the ROTEM CT, as compared to that in control mice
(Fig. 2a). Although the dose of rivaroxaban we used is higher than
its therapeutic range in humans (~5-fold higher), this dose is in line
with what others have used in preclinical studies21. The rivaroxabaninduced prolongation of the ROTEM CT was normalized after the
P = 0.0005
Time to complete occlusion (min)
n.s.
25
15
10
5
0
20
10
.0
25
5.
0
1.
5
0.
a
iv
cl
hi
Ve
Surgery
(30 min)
0.5
1.0
e
Rivaroxaban
infusion
Measured
Measured
blood
blood
flow (30 min) flow (30 min)
2.5
5.0
f
P < 0.004
30
20
10
7.5%
FeCl3
injury
(2 min)
Protein
infusion
(1 min)
Surgery
(30 min)
P = 0.0032
n.s.
40
Vehicle
Rivaroxaban (mg/kg)
mFXaI16L GD-FXaS195A
(mg/kg)
(mg/kg)
+ riva
+ riva
7.5%
FeCl3
injury
Protein
(2 min)
infusion
Rivaroxaban
infusion
Measured
blood
flow (30 min)
n.s.
40
n.s.
P < 0.004
P = 0.0024
30
20
10
I16L
mFXa
(mg/kg)
+ riva
I16L
mFXa
(mg/kg)
+ riva
00
00
25
.
5.
50
50
2.
25
0.
Ve
hi
S195A
GD-FXa
(mg/kg)
+ riva
0.
e
cl
0
.0
25
1.
00
25
0.
10
0.
a
iv
R
cl
0
iv
30
40
Clot time (min)
P < 0.0001
20
Ve
hi
assay21 (Supplementary Fig. 2). Titration of GD-FXaS195A into NHP

that was anticoagulated with either 500 nM rivaroxaban (Fig. 1e)
or 2.5 M rivaroxaban (Supplementary Fig. 3) confirmed that
GD-FXaS195A restores thrombin generation when present at concentrations equal to or higher than that of the inhibitor. However,
compared to FXaI16L, GD-FXaS195A was approximately 300-fold less
potent (Fig. 1f), reflecting mechanistic differences between these
proteins. We also evaluated factor eight inhibitor bypassing activity
(FEIBA), an activated PCC (aPCC) that is used to bypass the intrinsic
pathway defect in hemophilia with inhibitors39. In TGA experiments,
the aPCC did not substantially restore thrombin generation, as
measured by peak thrombin levels, in NHP supplemented with rivaroxaban (Supplementary Fig. 3).
S195A
GD-FXa
(mg/kg)
+ riva
Figure 2 In vivo effects of FXaI16L and

GD-FXaS195A in mice anticoagulated with
rivaroxaban. (a) ROTEM clot times after
administration of vehicle or administration of
1 mg/kg rivaroxaban (riva) and the indicated
concentrations of mFXaI16L or GD-FXaS195A to
hemostatically normal C57BL/6 mice. (b) Time
to complete occlusion of the mouse carotid
artery following FeCl3 injury in mice treated
with vehicle or the indicated concentrations
of rivaroxaban. Blood flow was monitored for
30 min after the injury. (c,d) Schematic of the
FeCl3-injury model in which FXa is delivered
after injury (c) and quantification of time to
carotid occlusion (measured for up to 30 min)
for rivaroxaban-treated (1 mg/kg) mice that were
injected with the indicated concentrations of
mFXaI16L or GD-FXaS195A 30 min after FeCl3
injury (d). (e,f) Schematic of FeCl3-injury model
in which FXa is delivered before injury (e) and
quantification of time to carotid artery occlusion
(measured for up to 30 min) in rivaroxabantreated (1 mg/kg) mice that were infused with
the indicated concentrations of mFXaI16L or
GD-FXaS195A before FeCl3 injury (f). In a,b,d,f,
each point represents a single mouse, and
horizontal black lines represent the mean for
each group. Adjusted P values were obtained
by using an analysis of variance (ANOVA) test;
n.s., not significant.
Articles
Figure 3 Blood loss following tail-clipping. Vehicle or rivaroxaban

(1 mg/kg) was administered to mice 1 min before tail-clipping. 5 min
following tail-clipping, mice (n = 5 per group) received PBS (black bars)
or the indicated concentrations of mFXaI16L (blue bars) through the
jugular vein. Blood was collected for 10 min, and this blood loss was
measured. Data are presented as mean s.e.m. Adjusted P values were
obtained by an ANOVA test; n.s., not significant.
Assessment of the procoagulant potential of FXaI16L

A concern with any pro-hemostatic agent is its potential for causing
thrombotic events. In hemophilic mice given a therapeutic dose of
FXaI16L (0.45 mg/kg), this does not appear to be an issue, as markers
of coagulation activation are not meaningfully altered34. Furthermore,
in WT mice, multi-day infusions (0.45 mg/kg; one or two times daily
for 3 d) of FXaI16L are also well tolerated34. To further characterize
the procoagulant potential of FXaI16L, we evaluated its effects both
in vitro and in vivo in WT mice. As expected, addition of FXaI16L to
P = 0.0024
P = 0.0037
P = 0.0101
250
200
Blood loss (l)
rivaroxaban, mFXaI16L (0.25 mg/kg) that was given after the FeCl3
injury was still effective (Supplementary Fig. 5), and its effectiveness
in this setting was comparable to that observed for mice that received
mFXaI16L (0.25 mg/kg) after a single dose of rivaroxaban (Fig. 2d).
These data show that FXaI16L treatment can restore clot formation
in the presence of steady-state concentrations of rivaroxaban under
conditions that mimic long-term anticoagulant therapy.
Reversal of anticoagulation is sometimes necessary before invasive
procedures. To test the effects of mFXaI16L in such a scenario, rivaroxaban-treated mice were injected with mFXaI16L 1 min before FeCl3
injury (Fig. 2e); administration of mFXaI16L (0.5 mg/kg) resulted in
complete occlusion of the carotid artery (Fig. 2f), with occlusion
times comparable to those of control mice. GD-FXaS195A also restored
vascular occlusion in this model at the dosage (25 mg/kg) predicted by
ex vivo experiments, whereas a lower dose (5 mg/kg) was ineffective.
Taken together, these results indicate that FXaI16L can overcome the
effects of rivaroxaban when given before or after an injury.
To assess the efficacy of FXaI16L in reducing rivaroxaban-associated
bleeding in mice, we performed a tail-transection assay. Prior work
with GD-FXaS195A showed that this agent is efficacious when given
before injury in similar types of models in both rats and mice 21.
Because FXaI16L would most likely be used during an active bleeding episode, we examined whether it would reduce blood loss
when infused after an injury. As compared to vehicle-treated mice,
rivaroxaban-treated (1 mg/kg) mice showed a substantial increase
in total blood loss following tail transection (Fig. 3). Treatment with
mFXaI16L reduced total blood loss in a dose-dependent manner when
administered after injury. These data highlight the procoagulant
potential and effectiveness of FXaI16L in reducing blood loss following a severe hemostatic challenge.
To further evaluate FXaI16L in vivo, we used a microcirculatory
model of hemostasis following laser injury to mouse cremasteric arterioles34,40. As compared to vehicle-treated mice, infusion of rivaroxaban (1 mg/kg) before vessel injury prevented fibrin deposition
and considerably decreased platelet accumulation at the site of laser
injury (Fig. 4). Infusion of 25 or 50 mg/kg GD-FXaS195A into rivaroxaban-treated mice resulted in a small but dose-dependent increase
in platelet and fibrin accumulation (Fig. 4ac). In contrast, infusion
of a much lower dose of mFXaI16L (1 mg/kg) into rivaroxaban-treated
mice produced a greater increase in both platelet and fibrin accumulation. These data, together with the data from the other hemostasis
models tested, indicate that FXaI16L can restore thrombin generation
in vivo in diverse vascular beds.
n.s.
150
100
50
Vehicle
Riva
0.5
1.0
mFXaI16L (mg/kg)
+ riva
normal human plasma promotes thrombin generation, increasing

peak thrombin levels <2-fold (Supplementary Fig. 6a). Notably,
when increasing amounts of FXaI16L were added, the increase in peak
thrombin generation seemed to level off at ~2 nM FXaI16L. We speculate that this result is related to the amount of FVa that is generated
in situ during the assay, as FXaI16L had no effect on thrombin generation in the absence of FV (data not shown). Alternatively, this result
could represent a limitation of the TGA, if the maximal amount of
thrombin achievable in the assay under these conditions was reached.
Furthermore, using the FeCl3 model, we found that administration of FXaI16L at 1 mg/kg shortened the time to occlusion in WT
mice from 6.5 to 5 min, again suggesting its potential for causing
procoagulant effects (Supplementary Fig. 6b). This procoagulant
potential, however, was not associated with a substantial alteration
of markers of coagulation. Infusion of FXaI16L at 1 mg/kg, in the presence or absence of rivaroxaban, had no effect on the levels of platelets
and fibrinogen, and it only modestly affected thrombinantithrombin
levels (Supplementary Fig. 6c). In addition, the levels of D-dimers,
a degradation product of polymerized fibrin and a systemic marker
of coagulation, were below the limit of detection (<15 ng/ml) in all
control and experimental groups (data not shown). These data show
that, at the doses examined, FXaI16L has potent pro-hemostatic effects
in the context of an injury, but it does not seem to systemically activate coagulation. We speculate that this lack of activation reflects
the poor catalytic function of FXaI16L in the zymogen-like state, which
is fully rescued only when FVa is available32,33.
Inhibition of FXaI16L by rivaroxaban
A possible mechanism by which FXaI16L can overcome the anticoagulant effects of rivaroxaban relates to the lower affinity of FXaI16L,
as compared to that of WT FXa (FXaWT), for active site inhibitors,
such that FXaI16L activity would be less sensitive than FXaWT to rivaroxaban inhibition. Indeed, we observed a ~38-fold difference in the
inhibitory constant (Ki) of rivaroxaban for FXaWT as compared to
the Ki for FXaI16L (Supplementary Fig. 7a). Despite this difference,
however, FXaWT and FXaI16L were similarly effective at reversing the
effects of rivaroxaban in TGA experiments (Supplementary Fig. 7b).
This result indicates that the ability of FXaI16L to overcome the effects
of rivaroxaban cannot be fully explained by differences in the affinity
of the free enzyme for the inhibitor. Furthermore, and consistent with
our previous observations that the activity of FXaI16L is rescued after
articles
a
b
90 s
Vehicle + HBS
0s
30 s
90 s
15
150 s
12
9
6
3
0
90 s
150 s
c
5
30 s
Fibrin (MFI 10 )
Rivaroxaban (1 mg/kg) + HBS

0s
Rivaroxaban (1 mg/kg) + GD-FXaS195A (50 mg/kg)
0s
30 s
90 s
150 s
Rivaroxaban (1 mg/kg) + mFXa
(1 mg/kg)
60
120
180
Time (s)
2.0
Vehicle + HBS
Rivaroxaban + HBS
l16L
Rivaroxaban + 1 mg/kg mFXa
S195A
Rivaroxaban + 25 mg/kg GD-FXa
S195A
Rivaroxaban + 50 mg/kg GD-FXa
1.5
1.0
0.5
0
I16L
Vehicle + HBS
Rivaroxaban + HBS
Rivaroxaban + 1 mg/kg mFXal16L
Rivaroxaban + 25 mg/kg GD-FXaS195A
Rivaroxaban + 50 mg/kg GD-FXaS195A
150 s
5
30 s
Platelets (MFI 10 )
0s
60
120
180
Time (s)
Figure 4 Reversal of rivaroxaban by FXaI16L and GD-FXaS195A in a laser-injury model. (a) Digital composite fluorescence and bright-field images of
representative thrombi in WT mice treated with vehicle and HEPES-buffered saline (HBS), rivaroxaban (1 mg/kg) and HBS, rivaroxaban (1 mg/kg) and
GD-FXaS195A (50 mg/kg), or rivaroxaban (1 mg/kg) and mFXaI16L (1 mg/kg) before (0 s) and 30, 90 or 150 s after laser-induced injury of the cremasteric
blood vessel wall. Platelets (red) were detected by an Alexa-Fluor-555-labeled rat anti-CD41 F(ab) 2, and fibrin (green) was detected with Alexa-Fluor488-labeled anti-fibrin; areas of overlap are in yellow. Scale bars, 10 m. (b,c) Platelet (b) and fibrin (c) accumulation over time following laser injury
in mice treated with vehicle + HBS (n = 3 mice; 22 injuries), rivaroxaban + HBS (n = 3 mice; 15 injuries), rivaroxaban + 25 mg/kg GD-FXa S195A
(n = 3 mice; 14 injuries), rivaroxaban + 50 mg/kg GD-FXa S195A (n = 1 mouse; 5 injuries) and rivaroxaban + 1 mg/kg mFXa I16L (n = 3 mice; 15 injuries).
Median fluorescence intensity (MFI) for platelet (b) and fibrin (c) fluorescence is plotted versus time.
assembly in the prothrombinase complex32, the Ki values of rivaroxaban for FXaWT and for FXaI16L in prothrombinase, using a purified
system, were the same (Supplementary Fig. 7a). Although this result
points to the equivalence of FXaWT and FXaI16 inhibition by rivaroxaban, it fails to explain how either of these proteins can generate
thrombin in the presence of rivaroxaban, considering that the inhibitor concentration is more than tenfold greater than its Ki for FXaWT or
FXaI16L (either as free enzymes or in prothrombinase). Collectively,
these data suggest that other aspects of the regulation of active FXa in
plasma must contribute to the bypassing effects of FXaI16L.
FXa activity paradoxically persists in rivaroxaban-treated plasma
Normally, FXa that is introduced into plasma is irreversibly inhibited
by AT and other plasma inhibitors, resulting in rapid first-order decay
of its activity, which has a half-life of 23 min42,43. A hallmark of
FXaI16L is its resistance to these plasma inhibitors33,34. As a measure
of this, we assessed the kinetics of FXaAT complex formation by
ELISA after addition of FXa to plasma in the presence of rivaroxaban. Increasing concentrations of rivaroxaban inhibited FXaWTAT
complex formation (Fig. 5a). As expected, FXaI16L was resistant to AT,
but rivaroxaban further decreased the rate of AT inhibition of FXaI16L
(Fig. 5b). Notably, the rates of AT inhibition of FXaWT and FXaI16L,
which differed by ~20-fold in the absence of rivaroxaban, were nearly
the same in the presence of 1 M rivaroxaban (Supplementary Table 1).
To further study the decrease in FXaAT complex formation in the
presence of rivaroxaban, we used saturating amounts of biotinylated
Glu-Gly-Arg-chloromethylketone (B-EGRCK) to covalently trap and
label all FXa species (free and rivaroxaban-bound) that were not irreversibly inhibited by AT. B-EGRCK labeling of FXaWT and FXaI16L
was increased in rivaroxaban-containing samples as compared to
that in rivaroxaban-free samples (Fig. 5c,d and Supplementary

Table 2). Together, the levels of FXaAT and B-EGRCK-labeled FXa
account for most of the FXa added to the system. We also measured
FXaAT formation and B-EGRCK labeling kinetics in a purified system with physiologic concentrations of purified AT44 and obtained
comparable results to those in plasma (Supplementary Fig. 8
and Supplementary Table 3).
The ability of rivaroxaban to diminish FXaAT complex formation
over time suggests a mechanism by which both FXaWT and FXaI16L
persist and function in the presence of rivaroxaban. Specifically, these
data are consistent with equilibration between a pool of FXa that
is irreversibly inhibited by AT and a pool of FXa that is reversibly
inhibited by rivaroxaban. Because rivaroxaban and AT compete for
binding to FXa, the two pools (FXaAT and FXarivaroxaban) must
be separated by free, uninhibited FXa (Fig. 5e). This steady-state level
of free FXa is probably responsible for the thrombin that is generated
in the presence of rivaroxaban. Direct measurement of this free FXa,
however, is complicated by the fact that any probe would perturb the
equilibrium between FXa and rivaroxaban. As an alternative, we used
the known rate constants for FXa inhibition by AT45 and the on and
off rates for rivaroxaban binding to FXa46 to calculate the amount of
free FXa present at steady state. In the absence or presence of rivaroxaban, the calculated free FXa levels drop rapidly after initial mixing
(Fig. 5f,g, blue). In the absence of rivaroxaban, this decrease in free
FXa is due to inhibition by AT (Fig. 5f, orange). However, in the presence of rivaroxaban, the majority of FXa reversibly forms a complex
with the FXa inhibitor (Fig. 5g, green), with an associated decrease in
FXaAT complex formation (orange). Without rivaroxaban, the free
FXa concentration decays exponentially, such that with 25 nM added
FXa, less than 10 pM free enzyme remains after 10 min (Fig. 5h,
Rivaroxaban
100 nM rivaroxaban
1 M rivaroxaban
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WT
AT
1.7 107 M1s1
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40
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25
FXaWTRiva
20
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15
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i
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FXaWT(pM)
5 nM rivaroxaban
50 nM rivaroxaban
0.1
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40
60
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FXaWTRiva
30
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60
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species (nM)
species (nM)
10
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100 120
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100 120
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80
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100 nM rivaroxaban
1 M rivaroxaban
20
Time (min)
60
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Concentration of
hFXal16LBEGRCK (nM)
FXa
15
Time (min)
WT
black). Notably, in the presence of rivaroxaban, following a rapid initial decrease, free FXa levels reach a nonzero steady state (Fig. 5h,
magenta and cyan), which causes a paradoxical and persistent increase in
free FXa at pharmacologic concentrations of free rivaroxaban (Fig. 5i).
Because we have previously shown that 30100 pM free FXa is sufficient for normal thrombin generation in plasma from individuals
with hemophilia33, these free-FXa levels probably account for the
restoration of hemostasis in the presence of rivaroxaban.
To determine whether the in silico results (Fig. 5ei) that were
obtained for FXaWT apply to FXaI16L, we performed similar simulations for the zymogen-like variant. Unlike FXaWT, the kon and koff
rates for rivaroxaban binding to FXaI16L are not known. This limitation was accommodated by using two separate kinetic models to
calculate the amount of free FXaI16L in the presence of rivaroxaban
(Supplementary Fig. 9). The first model (Supplementary Fig. 9a)
assumes that FXaI16L is a single species that has a 15-fold slower kon
rate for rivaroxaban (with an identical koff rate) and a 15-fold lower
rate of inhibition by AT as compared to that for FXaWT. Simulation
using this model yielded much higher concentrations of free FXaI16L
(Supplementary Fig. 9b) than FXaWT (Fig. 5h and Supplementary
Fig. 9e), which is consistent with our experimental results (Fig. 5d).
However, after correction for the 15-fold lower proteolytic activity
of FXaI16L, our simulation results were nearly identical to those for
FXaWT (Fig. 5h and Supplementary Fig. 9c). In the second model
(Supplementary Fig. 9d), we assumed that FXaI16L exists in two conformations, a zymogen-like conformation, FXaI16L (z), and a proteaselike conformation, FXaI16L (p), that are in reversible equilibrium, with
a Keq = 15 favoring the zymogen-like state. Kinetic calculations were
performed by assuming that FXaI16L (p) had identical kinetic properties to FXaWT, and that FXaI16L (z) does not react with inhibitors or
substrates. In the absence of rivaroxaban, the calculated concentration
of FXaI16L (p) (Supplementary Fig. 9f) was much higher over time
as compared to that of FXaWT (Fig. 5h and Supplementary Fig. 9e).
However, in the presence of 50 nM rivaroxaban, the concentrations
of FXaWT and FXaI16L (p) were comparable (Supplementary Fig. 9f).
Together, these two models suggest that competition between rivaroxaban and AT probably has a role in the mechanism by which FXaI16L
can overcome the effects of direct FXa inhibitors.
20
100 120
80
Rivaroxaban
100 nM rivaroxaban
1 M rivaroxaban
25
Concentration of
hFXal16LAT (nM)
Concentration of
hFXaWTAT (nM)
Concentration of
hFXaWTBEGRCK (nM)
Figure 5 Distribution of FXa in plasma in the presence or absence of

rivaroxaban. (a,b) Kinetics of FXaAT formation after addition of 25 nM
hFXaWT (a) or hFXaI16L (b) to FX-deficient plasma containing no (),
100 nM or 1 M rivaroxaban. Solid lines represent fit of the points to a
single exponential rise. (c,d) B-EGRCK labeling of 25 nM hFXaWT (c) or
hFXaI16L (d) following incubation in FX-deficient plasma in the absence
() or presence of 100 nM or 1 M rivaroxaban, as quantified by ELISA.
Solid lines in c represent fitting to single-exponential decay, except for
the 1 M line, which represents a smoothed connection of the points. The
solid lines in d also represent a smoothed connection of the points. Data
points in ad indicate the mean of three separate experiments s.e.m.
(e) Scheme depicts AT-inhibited FXaWT and rivaroxaban-inhibited FXaWT,
separated by free FXaWT. Known rate constants for each kinetic step are
indicated. (f,g) Concentrations of different FXaWT species in the absence
(f) or presence (g) of 50 nM rivaroxaban were calculated using known
rate constants and an initial (t = 0) concentration of 25 nM free FXaWT.
FXaWTAT complex levels, free FXaWT levels and FXaWTrivaroxaban
complex levels are shown. (h) Free FXaWT levels (calculated as in f,g) at
different rivaroxaban concentrations (0 nM, 5 nM or 50 nM) are plotted
versus time on a semi-logarithmic scale. (i) Free FXaWT levels, calculated
from h at 10 or 30 min, are plotted versus rivaroxaban concentration.
The therapeutic range of rivaroxaban concentrations that are free in
plasma (non-protein bound) is indicated.
Concentration of free
FXaWT (nM)
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30
35
600
Therapeutic range
10 min
400
200
30 min
50
100
Concentration of rivaroxaban (nM)
On the basis of our model (Fig. 5e), direct FXa inhibitors establish a new equilibrium that not only diminishes the rate of FXaAT
complex formation, but also establishes a small but functionally relevant pool of free FXa when FXa is added to the system. To further
probe this model, we experimentally tested the effect of disrupting
this equilibrium. The addition of fondaparinux, a heparin derivative
that accelerates AT inhibition of FXa, markedly enhanced the kinetics of FXaWTAT complex formation (Supplementary Fig. 10a and
Supplementary Table 4), and B-EGRCK labeling was correspondingly reduced (Supplementary Fig. 10b and Supplementary Table 2).
The addition of increasing amounts of rivaroxaban blunted the effect
of fondaparinux and reduced FXaAT complex formation. Similar
results were obtained with FXaI16L (Supplementary Fig. 10c,d). This
redistribution of FXa away from AT, even in the presence of fondaparinux, is not specific to rivaroxaban, as p-aminobenzamidine, a
FXa active site inhibitor with fast dissociation kinetics47, also inhibited FXaAT complex formation (data not shown). Taken together,
these data show that any active sitedirected FXa inhibitor can disrupt
FXaAT complex formation and establish a small pool of free FXa.
articles
The size of the pool will depend on the rate of generation of endogenous FXa or on how much exogenous FXa is administered.
Procoagulant function of FXaI16L in the presence of heparins
The greatly accelerated inhibition of FXaI16L by ATfondaparinux
(Supplementary Fig. 10c; squares), as compared to that by AT alone
(Fig. 5b; squares), suggests that FXaI16L would probably be ineffective
in mitigating the anticoagulant effects of fondaparinux or enoxaparin.
For example, although the procoagulant activity of FXaI16L persisted
for >30 min in FX-deficient plasma, its activity was completely eliminated by fondaparinux or enoxaparin following a 30-min incubation
period, as assessed by TGA (Supplementary Fig. 10e). However,
after a much shorter incubation period (1 min) with either of the
anticoagulants, appreciable FXaI16L procoagulant activity persisted.
Similarly, thrombin generation in NHP that was anticoagulated with
fondaparinux (750 nM) or enoxaparin (1 IU/ml) could be restored
to near-normal values 1 min after the addition of FXaI16L (1 nM)
(Supplementary Fig. 10e). If either of the anticoagulants and FXaI16L
were pre-incubated together for 15 min in plasma before the start of
the assay, however, FXaI16L was ineffective in enhancing thrombin
formation (Supplementary Fig. 10e). These observations with plasma
suggest that FXaI16L could have at least some procoagulant effects in
the presence of fondaparinux or enoxaparin, but this might be limited
by FXaI16L half-life and other factors.
FXaI16L overcomes the effects of dabigatran
AT also inhibits other coagulation enzymes, most notably thrombin,
in an active sitedependent manner. On the basis of our findings
with FXa, we predicted that small-molecule active site inhibitors of
thrombin should compete with AT for thrombin inhibition. This
hypothesis is supported by a prior study that showed that benzamidine, a nonspecific serine protease active site inhibitor, decreased
the rate of thrombin inhibition by AT48. To study this further, we
quantified the kinetics of thrombin inhibition by AT in the presence of
dabigatran, a direct thrombin inhibitor, using an ELISA that specifically detects the thrombinAT complex (TAT). Dabigatran markedly
inhibited TAT formation at concentrations within the therapeutic
range of the anticoagulant (Fig. 6a), suggesting that competition
between reversible active site inhibitors and plasma inhibitors is not
unique to FXa, but rather a common property of serine proteases.
The observation that dabigatran competes with AT for binding to
thrombin probably means that a persistent steady-state amount of free
thrombin is formed in the presence of dabigatran. A notable implication of this concept is that, just as with FXa and rivaroxaban, the addition or generation of more thrombin should increase this steady-state
level of free enzyme and thereby overcome the anticoagulant effect of
dabigatran. However, direct addition of thrombin is not practical as a
Dabigatran
100 nM dabigatran
1 M dabigatran
n.s.
P = 0.0121 P = 0.0387
250
40
Blood loss (l)
30
20
10
200
150
100
50
tra
n
m
da F
bi Xa l1
ga
6
tra L
n
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50
ig
a
20 30 40
Time (min)
ab
10
Ve
hi
cl
e
0
0
Concentration of TAT (ng/ml)
Figure 6 Influence of dabigatran on TAT levels and effects on blood

loss following tail-clipping. (a) Kinetics of TAT complex formation were
quantified by ELISA after mixing of 0.5 nM thrombin in re-calcified
prothrombin-deficient plasma in the absence () or presence of 100 nM
or 1 M dabigatran as detailed in the Online Methods. The solid line in
the dabigatran data represent fitting to a single-exponential rise with
offset, whereas the 100 nM and 1 M data could not be fit well, and
thus the lines represent a smoothed connection of the points. Error bars
indicate the s.e.m. (b) Vehicle or dabigatran (1 mg/kg) was administered
to mice 1 min before tail-clipping. 5 min after tail-clipping, mice (n = 5
per group) received PBS (black bars) or mFXaI16L (1 mg/kg; blue bar)
through the jugular vein. Blood was collected for 10 min, and this blood
loss was measured. Data are presented as mean s.e.m. Adjusted P
values were obtained by using an ANOVA test; n.s., not significant.
therapeutic reversal strategy because, unlike FXa, thrombin does not

bind membranes directly and thus would not be localized to the site of
vascular injury. It has also been suggested that thrombin added in low
concentrations can function as an anticoagulant through its interactions with thrombomodulin49. However, we hypothesized that FXaI16L
administration in the setting of dabigatran anticoagulation would
result in localized generation of thrombin, thereby increasing the levels of free thrombin and restoring hemostasis. Indeed, in TGA studies
in vitro, FXaI16L was able to alter the thrombin-generation profile in
the presence of dabigatran, as measured by changes in the lag time
(Supplementary Fig. 11). To determine whether FXaI16L can restore
hemostasis in vivo in the presence of dabigatran, we measured blood
loss following tail transection in mice. Dabigatran-treated (1 mg/kg)
mice experienced a more than threefold increase in blood loss as compared to vehicle-treated (control) mice (Fig. 6b). Infusion of mFXaI16L
after the injury resulted in a significant (P = 0.0387) decrease in blood
loss, indicating that mFXaI16L can reverse the effects of dabigatran in
vivo. Together with the results of using the FXa inhibitor, these data
suggest that FXaI16L could be used as a rapid-onset pro-hemostatic
to treat bleeding induced by DOACs. Although other mechanisms
could contribute to its pro-hemostatic effects, the generation of local
thrombin by FXaI16L and the ability of dabigatran to protect thrombin
from AT inhibition probably have a role in these effects.
DISCUSSION
Development of an effective and rapid reversal strategy for DOACs is
imperative as their use becomes more widespread. Here we tested a
rapid bypassing approach for reversal of oral FXa and thrombin anticoagulants with a zymogen-like variant of FXa, FXaI16L. Administration
of FXaI16L reduced blood loss and normalized hemostasis following
tail transection in mice that were treated with dabigatran. Moreover,
FXaI16L reversed the effects of rivaroxaban in vivo in mouse models of
large-vessel thrombosis and microcirculatory injury, and in a mouse
tail-bleeding assay. Notably, in vitro and in the large-vessel thrombosis
model, FXaI16L was >50-fold more potent than GD-FXaS195A, an
antidote that sequesters rivaroxaban by molecular engagement. The
higher potency of FXaI16L as compared to that of GD-FXaS195A derives
from their distinct mechanisms of action.
Initially, we postulated that the zymogen-like character of FXaI16L,
which renders it resistant to active site inhibitors, would allow for
thrombin formation in the presence of rivaroxaban. However, FXaWT
and FXaI16L were both highly effective at restoring thrombin generation in the presence of the FXa inhibitor. As a possible explanation for
these findings, we discovered that rivaroxaban shifts the distribution
of FXa from an irreversible complex with AT to a reversible complex
with rivaroxaban. Because the pathway for conversion of the rivaroxaban-inhibited enzyme to the complex with AT requires the formation
Articles
of free uninhibited FXa, a steady-state amount of free FXa is formed
that is not seen in the absence of rivaroxaban. Thus, pharmacologically
relevant concentrations of rivaroxaban produce a paradoxical increase
in free FXa. It is notable that the increase in free FXa would be most
relevant, or only relevant, when FXa is administered exogenously.
Normally, the overall result of rivaroxaban therapy is a net decrease
in endogenous FXa activity; although a pool of free FXa will be generated, its magnitude will most probably be insignificant.
Our findings reveal that rivaroxaban-mediated inhibition of FXaAT
complex formation is not a result of the kinetics of rivaroxaban binding, but is, instead, a consequence of the kinetics of AT inactivation
of FXa. Specifically, rivaroxaban is able to prevent FXaAT complex
formation only because the initial binding of AT to FXa is normally
extremely weak45. This is why fondaparinux, which enhances the binding of AT to FXa45, eliminates the effect of rivaroxaban on the formation of the FXaAT complex. A notable implication of these findings
is that it would not be possible to engineer an active site antagonist of
FXa that does not substantially disrupt the FXaAT interaction, such
that the approach of inhibiting the FXa active site may have more complex biological implications than anticipated. However, the ability of
fondaparinux to rapidly eliminate the FXarivaroxaban pool suggests
a specific mechanistic countermeasure to this problem.
The mechanism of dabigatran reversal by FXaI16L is almost certainly different from that of rivaroxaban reversal. In the setting of
rivaroxaban reversal, administration of FXaI16L directly increases the
free concentration of inhibited enzyme. In contrast, in the setting
of dabigatran reversal, we suspect that FXa I16L results in the in situ
generation of thrombin after FVa becomes available. Kinetic studies confirmed that dabigatran prevents thrombin inhibition by AT,
meaning that dabigatran likely prolongs the half-life of thrombin,
allowing for a steady-state amount of free enzyme to persist.
Thus, any thrombin generated by FXaI16L necessarily increases the
steady-state level of free thrombin at the site of vascular injury.
Another possible contribution to the mechanism of dabigatran
reversal is that FXaI16L may result in some degree of FV or even
FVIII activation. It has been previously shown that addition of FVa
to plasma can reverse the effects of both direct FXa inhibitors and
direct thrombin inhibitors50, and thus any FVa that is generated by
FXaI16L might enhance its pro-hemostatic effects.
FXaI16L has numerous features that make it an attractive agent
for alleviating the anticoagulant effects of rivaroxaban and dabigatran.
In vivo characterization of FXaI16L revealed that it is substantially
more potent and possibly more effective than antidote approaches,
especially when given after an injury. Its high potency is advantageous,
because low quantities of the protein can be used. From a safety perspective, catalytic amounts of FXaI16L are unlikely to interact appreciably with plasma proteins, including endogenous protease inhibitors.
Furthermore, even though both FXaI16L and GD-FXaS195A are variants
of FXa and immunogenic risk cannot absolutely be ruled out, the
ability to use FXaI16L at lower doses may decrease this risk. Finally,
the observation that FXaWT may also reverse the effects of rivaroxaban raises the question of whether FXaWT can be used as a therapeutic
reversal agent. In principle, this could be possible; however, FXaWT
has inherent prothrombotic risks that may be minimized by using
FXaI16L, as it is zymogen-like and has a dependence on FVa for rescue
of its full protease activity3234. Moreover, FXaWT typically has an
extremely short half-life in plasma. In the presence of rivaroxaban,
its half-life is prolonged, but this would not be the case in the setting
of dabigatran reversal. Thus, only FXaI16L could act as a reversal agent
for both direct FXa and thrombin inhibitors.
In conclusion, we demonstrate the efficacy of FXaI16L as a potential rapid-onset, universal pro-hemostatic bypassing agent for both
major classes of DOACs. Through comparisons of FXaI16L with a noncatalytic antidote, we have shown that, although both are efficacious,
pro-hemostatic agents such as FXaI16L are substantially more potent.
We also characterized a previously unreported competition between
direct FXa or thrombin inhibitors and intrinsic protease regulatory
proteins that should be taken into account in the engineering of new
anticoagulants. Our findings show that a bypassing approach, such
as the use of FXaI16L, can be efficacious for the reversal of DOACs.
Further work is needed to determine whether an antidote or bypassing approach is most beneficial to control bleeding.
Methods
Methods and any associated references are available in the online
version of the paper.
Note: Any Supplementary Information and Source Data files are available in the
online version of the paper.
Acknowledgments
This work was supported in part by the US National Institutes of Health (NIH)
grants F30 HL-120487 (N.K.T.), T32 HL-007439 (N.K.T.), T32 GM-007170
(N.K.T.) and P01 HL-74124 (R.M.C. and S.K.), the PennCHOP Blood Center
for Patient Care and Discovery (P.A.G.) and research funding from Pfizer (R.M.C.).
We are grateful to V. Arruda (Childrens Hospital of Philadelphia and University
of Pennsylvania) and L. Brass (University of Pennsylvania) for useful suggestions
and critical review of the manuscript.
AUTHOR CONTRIBUTIONS
N.K.T. designed, coordinated and performed experiments; L.I. and R.D. designed
and performed experiments; N.K.T., S.K. and R.M.C. designed experiments and
analyzed data; P.A.G. analyzed data; and N.K.T., L.I., S.K. and R.M.C. wrote the
paper. All authors discussed the results and commented on the manuscript.
COMPETING FINANCIAL INTERESTS
The authors declare competing financial interests: details are available in the online
version of the paper.
Reprints and permissions information is available online at http://www.nature.com/
reprints/index.html.
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and pharmacodynamic profile of rivaroxaban. Clin. Pharmacokinet. 53, 116
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Haemophilia 10 (suppl. 2), 39 (2004).
40. Whinna, H.C. Overview of murine thrombosis models. Thromb. Res. 122
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41. Scaglione, F. New oral anticoagulants: comparative pharmacology with vitamin K
antagonists. Clin. Pharmacokinet. 52, 6982 (2013).
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68906895 (1984).
43. Olson, S.T. et al. Role of the antithrombin-binding pentasaccharide in heparin
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(1983).
45. Izaguirre, G. et al. Conformational activation of antithrombin by heparin involves
an altered exosite interaction with protease. J. Biol. Chem. 289, 3404934064
(2014).
46. Perzborn, E. et al. Rivaroxaban: a new oral factor Xa inhibitor. Arterioscler. Thromb.
Vasc. Biol. 30, 376381 (2010).
47. Craig, P.A., Olson, S.T. & Shore, J.D. Transient kinetics of heparin-catalyzed protease
inactivation by antithrombin III. Characterization of assembly, product formation
and heparin dissociation steps in the factor Xa reaction. J. Biol. Chem. 264,
54525461 (1989).
48. Olson, S.T. & Shore, J.D. Transient kinetics of heparin-catalyzed protease inactivation
by antithrombin III. The reaction step limiting heparin turnover in thrombin
neutralization. J. Biol. Chem. 261, 1315113159 (1986).
49. Esmon, C.T. & Owen, W.G. The discovery of thrombomodulin. J. Thromb. Haemost.
2, 209213 (2004).
50. Smith, S.A. & Morrissey, J.H. Polyphosphate as a general procoagulant agent.
J. Thromb. Haemost. 6, 17501756 (2008).
ONLINE METHODS
Reagents. Z-Gly-Gly-Arg-AMC was from Bachem Bioscience Inc.

Technothrombin thrombin calibrator and reagent RB were from Diapharma
Group, Inc. Pooled normal human plasma (NHP) and human factor X
(FX)-deficient plasma were obtained from George King Biomedical, Inc.
Rivaroxaban and dabigatran were from Selleck Chemicals. All tissue culture
reagents were from Invitrogen, except for insulintransferrinsodium selenite,
which was from Roche. Ferric chloride (FeCl3), o-phenylenediamine dihyrdochloride (OPD), Gly-Pro-Arg-Pro-amide (GPRP) and hexadimethrine
bromide (polybrene) were from Sigma-Aldrich. Corn trypsin inhibitor (CTI),
biotinylated Glu-Gly-Arg-chloromethylketone (B-EGRCK) and biotinylated
Phe-Pro-Arg-chloromethylketone (B-FPRCK) were from Haematologic
Technologies. Activated prothrombin complex concentrates (aPCCs, FEIBA
nanofiltered) were purchased from Baxter International, Inc. Spectrozyme
FXa (SpecXa) was from American Diagnostica, Inc. Fondaparinux sodium
was from Apotex Corp. Innovin was from Dade Behring. Rat antimouse
CD41 antibody, prepared as a F(ab)2 fragment, was from BD Bioscience.
Mouse antihuman fibrin monoclonal antibody (clone 59D8), which crossreacts with mouse fibrin, has been previously described51,52. These antibodies were conjugated with Alexa Fluor 555 or Alexa Fluor 488, using the
Alexa Fluor Protein Labeling Kit according to the manufacturers instructions (Molecular Probes/Invitrogen). Affinity-purified goat antihuman
FX polyclonal IgG (catalog no. GAFX-AP) and peroxidase-conjugated
affinity-purified sheep antihuman antithrombin polyclonal IgG (catalog
no. SAAT-APHRP) were obtained from Enzyme Research Laboratories.
Horseradish-peroxidase-conjugated streptavidin was purchased from Life
Technologies. Enzygnost TAT micro-ELISA kit was purchased from Siemens.
The D-dimer ELISA kit, Asserachrom D-Di, was purchased from Diagnostica
Stago. The Fibrinogen ELISA kit was from Innovative Research.
Proteins. The FX activator from Russells viper venom, RVVX-CP was purified
as previously described53. Recombinant hFXI16L and mFXI16L were expressed
in human embryonic kidney 293 (HEK293) cells, purified from medium and
activated by using RVVX-CP as previously described54,55. hFXS195A was expressed
in HEK293 cells and purified from the medium by using a protocol identical to
that used for hFXaI16L. hFXS195A was then activated to hFXaS195A with RVVX-CP
and purified in the same way as the other recombinant FXa molecules. To generate GD-FXaS195A, hFXaS195A was digested with chymotrypsin as previously
described56 and purified using a Poros HQ/20 column and eluting with a NaCl
gradient in 20 mM Tris, pH 8.3, then dialyzing against 20 mM HEPES, 150 mM
NaCl, pH 7.4. Recombinant hFVa was prepared as previously described57.
Human AT was purified from plasma as previously described58.
aPCCs). The reaction was initiated immediately by adding Z-Gly-Gly-Arg-AMC

in 15 mM CaCl2 (50 l; 0.5 mM final). Fluorescence (ex = 360 nm, em = 460 nm)
was measured at 1-min intervals for 90 min at 37 C, using a Spectramax M2e
(Molecular Devices) plate reader. The Technothrombin calibrator kit was used to
convert raw fluorescence intensity to thrombin concentration. Thrombograms
(nM thrombin versus time) were made to determine the lag time, peak height
and endogenous thrombin potential (ETP).
Rotational thromboelastography (ROTEM). The institutional review board of
the Childrens Hospital of Philadelphia Research Institute approved the human
phlebotomy and blood use performed in this study. Informed consent was
obtained from all subjects. For human studies, whole blood from five healthy
donors was collected into CTI (final concentration 25 g/ml) and one-tenth
volume of 3.2% sodium citrate. 300 l of blood was added to a pre-warmed
(37 C) ROTEM cup along with rivaroxaban and hFXaI16L at the indicated concentrations. The reaction was initiated with 11.8 mM CaCl2 and a 1:17,300
dilution of innovin. Data was exported using the manufacturers export tool
and plotted as time versus elasticity. The clot time (CT) was determined as the
time to reach an amplitude of 2 mm of elasticity.
For mouse ex vivo ROTEM studies, rivaroxaban was formulated for intravenous injection by using a mixture of polyethylene glycol 400, water and glycerol
(968 g, 590 g and 58 g, respectively), a modification of a previously reported
protocol59. The DMSO stock of rivaroxaban was diluted in the injection solution
to a concentration of 0.25 mg/ml, such that the DMSO concentration in the final
solution was 0.5%. Rivaroxaban was infused via the lateral tail vein before anesthetizing the mouse (pentobarbital, intraperitoneal injection). The right jugular
vein and infrahepatic inferior vena cava (IVC) were exposed. Proteins (mFXaI16L
or GD-FXaS195A) diluted in HBSPEG were infused directly into the jugular
vein using a needle crossing the pectoral muscle and getting into the jugular
vein. One minute after protein infusion, blood (400 l final volume, including
anticoagulants) was withdrawn from the infrahepatic IVC using a 22G needle
preloaded with one-tenth volume 3.2% sodium citrate and CTI (100 g/ml final).
The blood samples were analyzed by ROTEM as described above.
Clotting assays. 1 M rivaroxaban was incubated in NHP along with various concentrations of GD-FXaS195A for 30 min at room temperature (25 C)21.
After incubation, 50 l of each plasma sample was incubated at 37 C for
60 s. Coagulation was then initiated by addition of 100 l of innovin, and
time to clot formation was measured with a Start4 coagulation machine
(Diagnostica Stago).
Ferric chloride carotid artery injury model. FeCl3-induced carotid artery

injury was performed with minor modifications to previously established protocols34. Rivaroxaban or vehicle was infused via lateral tail vein injection, using the
same protocol used in the ex vivo studies described above. After intraperitoneal
administration of anesthesia (pentobarbital), the right jugular vein was exposed
to allow for direct injection of proteins. The right common carotid artery from
the sternal origin of the sternocleidomastoid muscle to the carotid bifurcation
was exposed. Baseline carotid blood flow was recorded with a miniature Doppler
flow probe (Model 0.5PSB; Transonic Systems) positioned around the carotid
artery. A 2 mm 1 mm piece of Whatman no. 1 filter paper soaked in 7.5% FeCl3
(0.46 M) was applied to the adventitial surface of the artery for 2 min. The paper
was then removed, the area was flushed with PBS, and blood flow was monitored
continuously for 30 min. Time to complete occlusion was defined as the time
from the end of the injury period to the time when blood flow had decreased
by >90% of the baseline level for at least five continuous minutes. After the
30-min monitoring period, reversal of rivaroxaban was attempted by infusing
protein (mFXaI16L or GD-FXaS195A) or vehicle via direct jugular vein injection.
Following protein injection, blood flow was monitored for an additional 30 min,
and time to complete occlusion was recorded as the time between protein infusion and the beginning of a 5-min sustained occlusive event as defined above.
In some experiments, protein was injected via direct jugular vein injection
1 min before FeCl3 injury. In all experiments, complete occlusion was verified
at the site of the injury by visually observing an opaque clot.
Thrombin-generation assays (TGAs). TGAs in NHP were performed as previously described33, with a slight modification to accommodate the addition
of rivaroxaban and reversal agents as appropriate. 40 l NHP was added to a
microtiter plate (Nunc; F16 black Maxisorp) along with 10 l Technothrombin
RB (2 pM TF, 4.0 M phospholipid). 3 l rivaroxaban or dabigatran, dissolved in
20 mM HEPES, 150 mM NaCl, 0.1% PEG-8,000, pH 7.4 (HBSPEG), was added
to NHP along with 2 l of reversal agent (hFXaWT, hFXaI16L, GD-FXaS195A or
Reversal of rivaroxaban following multiple doses. Hemostatically normal

mice were injected with rivaroxaban (1 mg/kg) every 24 h via lateral tail vein
injection for 4 d. 10 min following the final injection, the right common carotid
artery was injured with FeCl3 as described above. Blood flow was monitored
for 30 min, and then mFXaI16L was infused via direct jugular vein injection.
Blood flow was monitored for an additional 30 min and time to carotid artery
occlusion was determined.
Mice. Wild-type (WT) male C57BL/6J mice purchased from Jackson Laboratory
were used in all experiments; no exclusion criteria were used. For ex vivo ROTEM
studies and all ferric chloride (FeCl3) carotid artery injury experiments, blood
was collected from 8- to 10-week-old male mice weighing 2030 g. For intravital
imaging and tail-clip studies, mice were 11- to 12-week-old males that weighed
2535 g. Experimental approval was obtained from the Childrens Hospital of
Philadelphia Institutional Animal Care and Use Committee.
nature medicine
doi:10.1038/nm.4149
Tail clip assay. The tail clip assay was performed with minor modifications
to previously established protocols34. Following intraperitoneal administration
of anesthesia (pentobarbital), mice were injected with rivaroxaban, dabigatran
or vehicle via direct jugular vein injection as described in the ex vivo studies
mentioned above. One minute after anticoagulant injection, the distal portion
of the tail was then transected at a diameter of 3 mm and placed in a conical
tube containing 14 ml of saline at 37 C. The tail was allowed to bleed into a
tube of saline for 5 min. Protein or PBS was then infused by direct jugular vein
infusion. The injured tail was moved to a fresh tube of saline, and blood was collected for 10 min. Quantitative assessment of blood loss in the second tube was
determined by measuring total hemoglobin by absorbance at 575 nm following
red cell lysis as described36. Quantitative assessment of hemoglobin content was
converted to total blood loss (l) by using appropriately established standard
curves generated by each method.
Intravital imaging of thrombus formation. Evaluation of hemostasis following
laser injury to mouse cremasteric arterioles has been previously described60,
and our specific experimental system has been detailed34,6163. Rivaroxaban
and proteins were formulated as described for ex vivo ROTEM experiments and
FeCl3 injury experiments, but were injected via jugular vein cannulus instead
of the tail vein. After allowing rivaroxaban to distribute for 5 min, Alexa-Fluor555-labeled rat antiD41 F(ab)2 and Alexa-Fluor-488-labeled anti-fibrin antibodies to detect platelets and fibrin, respectively, were infused and laser injury
was performed to the vessel wall of the cremasteric arterioles. In some experiments, rivaroxaban-treated mice were infused with the reversal agent (either
mFXaI16L or GD-FXaS195A, dissolved to the appropriate concentration in 20 mM
HEPES, 150 mM NaCl, pH 7.4). Cremasteric arterioles of 3050 m in diameter
were injured using a pulse-nitrogen dye laser applied through the microscope
objective. Bright-field and fluorescence images were collected over 34 min at
4 frames/s, and the data were analyzed with Slidebook 6 software (Intelligent
Imaging Innovations). The kinetics of clot formation were analyzed by determining median fluorescence intensity over time in ~1422 thrombi from three
animals per group. For the 50 mg/kg GD-FXaS195A group, one animal was used,
and five injuries were made.
Assessment of the pro-thrombotic potential of FXaI16L. Hemostatically normal mice were injected with rivaroxaban (1 mg/kg) or vehicle, and 1 min later
they were infused with either PBS or mFXaI16L (1 mg/kg). 30 min after infusion,
blood (500 l final volume, including anticoagulants) was collected from the
infrahepatic IVC using a 22G needle preloaded with one-tenth volume 3.2%
sodium citrate. Levels of platelets were determined from whole blood using a
veterinary automated hemocytometer (Hemavet, Drew Scientific). The remaining samples were centrifuged (190g for 15 min, then 1,100g for 15 min) to isolate
platelet-poor plasma. Fibrinogen, TAT and D-dimer levels were measured using
the ELISA kits described above, following the manufacturers instructions.
Kinetic characterization of FXa variant inhibition by rivaroxaban. Increasing
concentrations of rivaroxaban were added to the chromogenic substrate SpecXa
(100300 M depending on the enzyme used) in HBSPEG buffer containing
2 mM CaCl2. The reaction was initiated with addition of hFXaWT or hFXaI16L and
A405nm was monitored over time to measure FXa amidolytic activity (depending
on the experiment and the enzyme, 26 nM FXa was used to ensure sufficient
signal). Initial velocities were plotted against inhibitor concentration and fit to
the quadratic velocity equation for tight-binding competitive inhibition to determine Ki values. To determine inhibition kinetics of FXa in the prothrombinase
complex, experiments were repeated in the presence of 30 nM FVa and 50 M
phospholipid vesicles (80% phosphatidylcholine, 20% phosphatidylserine).
Quantification of FXaAT complex formation in human plasma. 0, 100 nM
or 1 M rivaroxaban was added to recalcified (5 mM CaCl2 final) human FXdeficient plasma at room temperature along with 1.33 mM GPRP and 1 M
dabigatran to prevent clotting. 25 nM hFXaWT or hFXaI16L was added to aliquots
of the plasma mixture at different time points in a reverse time course, and all
samples were quenched simultaneously with 50 M B-EGRCK. Samples were
allowed to incubate with B-EGRCK for 10 min and were then diluted tenfold
with ELISA blocking buffer (PBS, 0.1% Tween-20, 6% BSA, pH 7.4). In some
doi:10.1038/nm.4149
experiments, 750 nM fondaparinux was added to the plasma mixture before

addition of FXa, and in these studies the reaction was quenched with 50 g/ml
polybrene (to neutralize the fondaparinux64) in addition to 50 M B-EGRCK.
FXaAT standards were prepared by incubating 500 nM FXaWT or FXaI16L with
5 M hAT, 6 M fondaparinux and 5 mM CaCl2 in HBSPEG for 30 min.
The standard was then serially diluted in FX-deficient plasma to make FXaAT
standards ranging from 030 nM. For experiments with fondaparinux, standards also contained 750 nM fondaparinux and 50 g/ml polybrene to account
for matrix effects. All FXaAT standards were diluted tenfold with ELISA
blocking buffer before use. FXaAT levels in the samples were measured using
a sandwich ELISA. 96-well immunoassay plates were incubated overnight at
4 C with 100 l 10 g/ml affinity-purified goat antihuman FX polyclonal IgG
diluted in 50 mM sodium carbonate, pH 9.6. Plates were washed with PBS + 0.1%
Tween-20, pH 7.4, and then blocked with blocking buffer at room temperature
for 90 min. Plates were then washed and incubated with 100 l of the tenfolddiluted sample described above for 1 h at 37 C. After washing again, plates were
incubated with 100 l of 2 g/ml peroxidase-conjugated affinity-purified sheep
antihuman antithrombin III polyclonal IgG at 37 C for 1 h. Following a final
wash step, 100 l of freshly prepared OPD solution (1 mg/ml OPD in 10 mM
sodium citrate, pH 4.5 and 0.006% hydrogen peroxide) was added to each well
and allowed to incubate for 2 min. The reaction was stopped with addition of
50 l 3M H2SO4. Plates were incubated at room temperature for 30 min before
reading A490nm in a SpectraMax 190 microplate reader (Molecular Devices).
FXaAT concentrations were determined using the generated standard curve.
Control experiments indicate that this ELISA does not recognize FXa complexed
with 2-macroglobulin.
Quantification of FXaAT complex formation in a purified system. 0, 5 nM
or 50 nM rivaroxaban was added to 2.6 M hAT in HBSPEG and 5 mM CaCl2.
25 nM hFXaWT or hFXaI16L was then added and incubated as described above.
Reactions were stopped with addition of 50 M B-EGRCK and allowed to incubate with B-EGRCK for 10 min before tenfold dilution with ELISA blocking
buffer. FXaAT complex formation was quantified using the ELISA described
above. Standards were also prepared as above, but diluted into HBSPEG instead
of FX-deficient plasma.
Quantification of FXaB-EGRCK complex formation. FXaB-EGRCK
standards were prepared by incubating 500 nM FXaWT or FXaI16L with 50 M
B-EGRCK in HBSPEG for 30 min and then serially diluting it in FX-deficient
plasma to make FXaB-EGRCK standards ranging from 030 nM. For experiments with fondaparinux, standards also contained 750 nM fondaparinux and
50 g/ml polybrene to account for matrix effects. FXaBEGRCK labeling was
measured using a novel ELISA. The ELISA was nearly identical to the FXaAT
ELISA described above, but with the following modifications: samples and
standards were diluted 150-fold instead of tenfold. Instead of the ATIII-detection
antibody, 100 l 0.25 g/ml horseradish-peroxidase-conjugated streptavidin,
diluted in ELISA dilution buffer, was added. All other steps were identical.
Determination of the kinetics of TAT complex formation. 0, 100 nM or 1 M
dabigatran was added to recalcified (5 mM CaCl2 final) human prothrombindeficient plasma at room temperature along with 1.33 mM GPRP to prevent
clotting. 500 pM plasma-derived human thrombin was added to aliquots of
the plasma mixture at different time points in a reverse time course, and all
samples were quenched simultaneously with 50 M B-FGRCK. Samples were
then analyzed for TAT content with the Enzygnost TAT micro kit according to
the manufacturers instructions.
Determination of FXa distribution using known rate constants. We used
KinTeK Explorer (KinTeK Corp.) to calculate the concentration of the FXaAT
complex, the FXarivaroxaban complex and free FXa over time. 25 nM free FXa,
3.4 M AT and different concentrations of rivaroxaban were used as starting
conditions, and the FXa distribution was determined over time, subject to the
following expressions:
E + AT E AT
E+ R ER
nature medicine
where E represents free FXa, R represents free rivaroxaban, AT represents

antithrombin, ER represents rivaroxaban-bound FXa, and EAT represents
AT-bound FXa. Rivaroxaban association (1.7 107 M1s1) and dissociation
(5 103 s1) rate constants were used for the first expression46, and the secondorder rate constant for AT inhibition (4 103 M1s1) of FXa was used for the
second expression45.
Statistical analysis. A test for equality of variance among the groups was done
using Levenes test. If this test was significant, then Welch ANOVA (which
assumes equal variances among the groups) was used instead of the standard
ANOVA. TukeyKramers procedure was used following a significant standard
ANOVA to obtain adjusted P values for pairwise contrasts. Since TukeyKramers
procedure assumes equal variances, Bonferronis method was used to obtain
adjusted P values when the Welch ANOVA was used. For intravital experiments, data were considered nonparametric and were presented as medians. In
these experiments, the Wilcoxon rank-sum test was used for statistical analysis.
P < 0.05 was considered statistically significant.
For all animal experiments, the determination of sample sizes is based on
power calculations from similar, prior experiments from our group. Based on
these kinds of analyses, we had confidence that the sample sizes chosen were
appropriate and sufficiently powered. Furthermore, the group sizes for the injury
models presented were comparable to those previously used in the literature.
The investigator was not blinded to the mice in the animal experiments, and
the mice were not randomized.
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doi:10.1038/nm.4149

A Rapid Pro-Hemostatic Approach To Overcome Direct Oral Anticoagulants

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A rapid pro-hemostatic approach to overcome direct oral

2016 Nature America, Inc. All rights reserved.

lacking the membrane-binding 4-carboxyglutamic acid domain

nature medicine advance online publication

Normalized peak thrombin (% of NHP)

assays (TGA) to determine whether FXaI16L could mitigate the effects

Concentration of FXaI16L (nM)

Normalized peak thrombin (% of NHP)

Normal human plasma

Normalized peak thrombin (% of NHP)

Normalized peak thrombin (% of NHP)

Concentration of thrombin (nM)

Normalized peak thrombin (% of NHP)

2016 Nature America, Inc. All rights reserved.

Concentration of FXa (nM)

advance online publication nature medicine

FXaI16L restores hemostasis in mice treated with rivaroxaban

Time to complete occlusion (min)

nature medicine advance online publication

Time to complete occlusion (min)

Clot time (min)

2016 Nature America, Inc. All rights reserved.

assay21 (Supplementary Fig. 2). Titration of GD-FXaS195A into NHP

Figure 2 In vivo effects of FXaI16L and

2016 Nature America, Inc. All rights reserved.

Figure 3 Blood loss following tail-clipping. Vehicle or rivaroxaban

Assessment of the procoagulant potential of FXaI16L

Blood loss (l)

normal human plasma promotes thrombin generation, increasing

advance online publication nature medicine

Rivaroxaban (1 mg/kg) + HBS

Rivaroxaban (1 mg/kg) + GD-FXaS195A (50 mg/kg)

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Rivaroxaban (1 mg/kg) + mFXa

nature medicine advance online publication

that in rivaroxaban-free samples (Fig. 5c,d and Supplementary

1.7 107 M1s1

4.3 103 M1s1

Figure 5 Distribution of FXa in plasma in the presence or absence of

2016 Nature America, Inc. All rights reserved.

advance online publication nature medicine

2016 Nature America, Inc. All rights reserved.

nature medicine advance online publication

Concentration of TAT (ng/ml)

Figure 6 Influence of dabigatran on TAT levels and effects on blood

therapeutic reversal strategy because, unlike FXa, thrombin does not

2016 Nature America, Inc. All rights reserved.

advance online publication nature medicine

2016 Nature America, Inc. All rights reserved.

nature medicine advance online publication

2016 Nature America, Inc. All rights reserved.

Reagents. Z-Gly-Gly-Arg-AMC was from Bachem Bioscience Inc.

aPCCs). The reaction was initiated immediately by adding Z-Gly-Gly-Arg-AMC

Ferric chloride carotid artery injury model. FeCl3-induced carotid artery

Reversal of rivaroxaban following multiple doses. Hemostatically normal

2016 Nature America, Inc. All rights reserved.

experiments, 750 nM fondaparinux was added to the plasma mixture before

2016 Nature America, Inc. All rights reserved.

where E represents free FXa, R represents free rivaroxaban, AT represents

51. Weiler-Guettler, H. et al. A targeted point mutation in thrombomodulin generates

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