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THROMBOSIS AND HEMOSTASIS
A role for factor XIIa–mediated factor XI activation in thrombus formation in vivo
Qiufang Cheng,1 Erik I. Tucker,2 Meghann S. Pine,3 India Sisler,3 Anton Matafonov,1 Mao-fu Sun,1 Tara C. White-Adams,2
Stephanie A. Smith,4 Stephen R. Hanson,2 Owen J. T. McCarty,2 Thomas Renné,5 András Gruber,2 and David Gailani1,6
1Department
of Pathology, Vanderbilt University, Nashville, TN; 2Division of Biomedical Engineering and Hematology/Oncology, Oregon Health and Sciences
University, Portland, OR; 3Department of Pediatrics, Vanderbilt University, Nashville, TN; 4Department of Internal Medicine, College of Medicine, University of
Illinois at Urbana-Champaign, Urbana, IL; 5Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden; and 6Department of
Medicine, Vanderbilt University, Nashville, TN
Mice lacking factor XII (fXII) or factor XI
(fXI) are resistant to experimentally–
induced thrombosis, suggesting fXIIa activation of fXI contributes to thrombus
formation in vivo. It is not clear whether
this reaction has relevance for thrombosis in primates. In 2 carotid artery injury
models (FeCl3 and Rose Bengal/laser),
fXII-deficient mice are more resistant to
thrombosis than fXI- or factor IX (fIX)–
deficient mice, raising the possibility that
fXII and fXI function in distinct pathways.
Antibody 14E11 binds fXI from a variety of
mammals and interferes with fXI activation by fXIIa in vitro. In mice, 14E11 prevented arterial occlusion induced by FeCl3
to a similar degree to total fXI deficiency.
14E11 also had a modest beneficial effect
in a tissue factor–induced pulmonary embolism model, indicating fXI and fXII contribute to thrombus formation even when
factor VIIa/tissue factor initiates thrombosis. In baboons, 14E11 reduced plateletrich thrombus growth in collagen-coated
grafts inserted into an arteriovenous
shunt. These data support the hypothesis
that fXIIa-mediated fXI activation contributes to thrombus formation in rodents
and primates. Since fXII deficiency does
not impair hemostasis, targeted inhibition of fXI activation by fXIIa may be a
useful antithrombotic strategy associated with a low risk of bleeding complications. (Blood. 2010;116(19):3981-3989)
Introduction
Initiation of fibrin formation by contact activation requires proteolytic conversion of plasma factor XII (fXII) to the protease factor
XIIa (fXIIa) on a surface.1-3 FXIIa activates the next zymogen in
the coagulation cascade, factor XI (fXI), to factor XIa (fXIa),
which in turn converts factor IX (fIX) to factor IXa␤ (fIXa␤). This
series of reactions, referred to as the intrinsic pathway of coagulation, drives thrombin generation and fibrin formation in the
activated partial thromboplastin time (aPTT) assay used by clinical
laboratories. A role for fIX in hemostasis is not in question, as its
deficiency causes the severe bleeding disorder hemophilia B.
However, the importance of the intrinsic pathway, as a whole, to
clot formation and stability at a site of injury is probably limited, as
fXII deficiency is not associated with abnormal bleeding,1,2 and
fXI-deficient patients have a variable hemorrhagic disorder with
milder symptoms than hemophiliacs.2,4 Current models of thrombin generation address these phenotypic differences by incorporating additional mechanisms for protease activation. Thus, fIX is
activated by the factor VIIa/tissue factor complex in addition to
fXIa,3,5 while fXI can be activated by thrombin.3,6
Mice lacking fXII, like their human counterparts, do not have a
demonstrable bleeding abnormality,7 supporting the premise that
fXIIa activation of fXI is not required for hemostasis.8 Given this, it
was surprising to observe that mice lacking fXII9 or fXI10 were
resistant to arterial thrombotic occlusion. While this suggested
contact activation might play an important role in pathologic
coagulation, if not hemostasis, it was not clear that fXIIa was
mediating its prothrombotic effect through fXI. We developed an
antibody against mouse fXI (14E11) that prolongs time to clot
formation in plasma by interfering with fXI activation by fXIIa.
Based on the phenotypes of fXII- and fXI-deficient mice, we
postulated that 14E11 would inhibit thrombus formation in vivo,
despite selectively interfering with a reaction not required for
hemostasis. Here we show that 14E11 affects fXIIa-dependent
coagulation in plasmas from multiple species and report on its
effects in mouse and primate thrombosis models.
Submitted February 21, 2010; accepted June 28, 2010. Prepublished online as
Blood First Edition paper, July 15, 2010; DOI 10.1182/blood-2010-02-270918.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
© 2010 by The American Society of Hematology
BLOOD, 11 NOVEMBER 2010 䡠 VOLUME 116, NUMBER 19
Methods
Reagents
Pooled normal and fXII-deficient plasmas were from George King BioMedical. fIX, fXI, and fXIa were from Haematologic Technologies. fXIIa,
high-molecular-weight kininogen (HK), and corn trypsin inhibitor (CTI)
were from Enzyme Research Laboratories. Z-Gly-Gly-Arg-AMC was from
Bachem. Dioleoylphosphatidylcholine:dioleoylphosphatidylserine (7:3 w/w)
was from Avanti Polar Lipids. S-2366 was from Diapharma. Bovine serum
albumin (BSA), rabbit brain cephalin (RBC), kaolin, and O-phenylenediamine (OPD) were from Sigma-Aldrich.
Monoclonal antibodies
FXI-deficient Balb-C mice were immunized with 25 ␮g of recombinant
mouse fXI11 diluted 1:1 with Freund adjuvant (200 ␮L total) by intraperitoneal injection. Two 25-␮g booster doses in incomplete Freund adjuvant
were given 3 and 7 weeks later, and hybridomas were generated by standard
protocols. Media were tested for capacity to recognize mouse fXI by
enzyme-linked immunosorbent assay and to prolong the aPTT of mouse
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3982
CHENG et al
and human plasmas. Clones of interest were subcloned twice by limiting
dilution. Clone 14E11 was expanded in a CL1000 bioreactor (Integra
Biosciences), and immunoglobulin G (IgG) was purified by cation exchange and thiophilic agarose chromatography. 14E11 was biotinylated
using an EZ-link sulfo-NHS-biotinylation kit (Thermo Scientific). Generation and characterization of monoclonal IgG O1A6, which binds to the A3
domain of human fXI and interferes with activation of factor IX by fXIa,
has been described.6,12
14E11 binding to fXI and fXIa
FXI or fXIa (2 ␮g/mL, 100 ␮L/well) in 50mM Na2CO3 pH 9.6 was
incubated overnight at 4°C in Immulon 2HB microtiter plates (Thermo
Scientific). Wells were blocked with 150 ␮L of phosphate-buffered saline
(PBS) with 2% BSA for 1 hour at room temperature (RT). One hundred
microliters of biotinylated 14E11 (100 to 10⫺7␮M) in 90mM HEPES
(N-2-hydroxyethylpiperazine-N⬘-2-ethanesulfonic acid) pH 7.2, 100mM
NaCl, 0.1% BSA, 0.1% Tween-20 (HBS) were added, with incubation for
90 minutes at RT. After washing with PBS-0.1% Tween-20 (PBST), 100 ␮L of
strepavidin-horseradish peroxidase (HRP; Thermo Scientific, 1:8000 dilution in
HBS) was added, with incubation at RT for 90 minutes. After washing, 100 ␮L of
substrate solution (12 mL of 30mM citric acid, 100mM Na2HPO4 pH 5.0,
one tablet OPD, 12 ␮L of 30% H2O2) was added. Reactions were stopped
after 10 minutes with 50 ␮L of 2.5M H2SO4. Absorbance at 495 nm was
measured on a SpectroMax 340 microplate reader (Molecular Devices).
BLOOD, 11 NOVEMBER 2010 䡠 VOLUME 116, NUMBER 19
Solution was added. Reactions were stopped after 10 minutes with 50 ␮L of
2.5M H2SO4, and absorbance at 495 nm measured.
Chromogenic assay for fXI activation
FXI (170nM) in PBS with 1 mg/mL BSA was incubated at 37°C for
15 minutes with or without 600nM 14E11. At time zero, fXIIa, HK, and
kaolin, all in PBS with 1 mg/mL BSA, were added so that final concentrations were fXI (85nM), fXIIa (17nM), HK (70nM), and kaolin (0.5 mg/
mL). At various time points, 50-␮L of aliquots were supplemented with CTI
(600nM final concentration) and then mixed with an equal volume of
1.0mM S-2366. Changes in absorbance at 405 nm were measured.
Thrombin generation assay
Thrombin generation was measured by following cleavage of Z-Gly-GlyArg-AMC (415␮M) in fXII-deficient plasma on a Thrombinoscope (Thrombinoscope BV),6 in the presence of 14E11 (300nM) or vehicle. Plasma
(80 ␮L) was mixed with 20 ␮L of Tyrode buffer pH 7.4 containing PC:PS
vesicles (30␮M) and either tissue factor (2.3pM) or fXIIa (10 or 100nM).
CTI was added to a final concentration of 4␮M for reactions with tissue
factor (TF). Final concentrations: 5␮M PC:PS vesicles, 0.23pM TF or
1-10nM fXIIa. Clotting was started with 20 ␮L of 20mM HEPES pH 7.4,
100mM CaCl2, 6% BSA, and fluorescence was monitored (emission ␭
460 nm). Assays were run twice in triplicate, and endogenous thrombin
potential (ETP) was determined with Thrombinoscope Analysis software,
version 3.0.
14E11 binding site on fXI
Biotinylated 14E11 was the primary antibody in Western blots of recombinant fXI with individual apple domains replaced with corresponding
domains from human prekallikrein (PK),13,14 or individual fXI apple
domains attached to tissue plasminogen activator (tPA).15 Detection was
with strepavidin-HRP and chemiluminescence.
Effect of 14E11 on plasma coagulation
aPTT assays. Plasma (50 ␮L) anticoagulated with 0.38% sodium citrate
was mixed with 50 ␮L of PBS (with or without 14E11 [0 to 320nM]) and
50 ␮L of aPTT reagent (aPTT-ES; Helena Laboratories) at 37°C for
3 minutes. Fifty microliters of CaCl2 (20mM) was added and time to clot
formation determined on an Amelung KC4 Analyzer (Sigma-Aldrich).
Modified fXIa PTT. FXIa (30nM) in 25mM Tris-HCl pH 7.4, 100mM
NaCl (TBS) with 0.1% BSA (TBSA) was incubated at RT for 30 minutes,
with or without 14E11 (300nM). FXIa (65 ␮L) was mixed with 65 ␮L of
fXI-deficient plasma and 65 ␮L of RBC at 37°C. After 30 seconds, 25mM
CaCl2 (65 ␮L) was added and time to clot formation measured.
Modified fXIIa PTT. 65 ␮L of fXIIa (1-100nM) in TBSA was mixed
with 65 ␮L of normal plasma (with or without 14E11 [300nM]), and 65 ␮L
of RBC at 37°C. After 30 seconds, 25mM CaCl2 (65 ␮L) was added and
time to clot formation determined.
FIX activation by fXIa
FIX (100nM) was incubated with fXIa (1nM) in TBS with 5mM CaCl2 at
RT with or without 200nM 14E11 or O1A6. At intervals, 20-␮L samples
were placed in sodium dodecyl sulfate (SDS) sample buffer. Samples were
size-fractionated on 10% nonreducing SDS-polyacrylamide gels and transferred to polyvinylidene fluoride membranes. Chemiluminescent Western
blotting was performed using goat anti-human fIX IgG (Affinity Biologicals).
HK-fXI binding assay
Immulon 2HB microtiter plates were coated with HK (30nM) in 50mM
carbonate buffer pH 9.6 (100 ␮L per well) overnight at 4°C. Wells were
blocked with 150 ␮L of PBS-2% BSA for 1 hour at RT. Biotinylated fXI
(10nM) and unlabeled 14E11 (25pM to 1␮M) were added to the wells (total
volume 100 ␮L) and incubated at RT for 90 minutes. After washing with
PBST, 100 ␮L of strepavidin-HRP (1:8000 in HBS) was added and
incubated at RT for 90 minutes. After washing, 100 ␮L of Substrate
Platelet adhesion assay
Glass coverslips were incubated with fXI, fXIa, or fibrinogen (50 ␮g/mL)
for 1 hour at RT.16 Surfaces were blocked with denatured fatty acid–free
BSA (5 mg/mL) for 1 hour, then incubated with vehicle, 14E11 or
nonspecific IgG (20 ␮g/mL) for 20 minutes. Purified human platelets
(2 ⫻ 107/mL) were incubated on protein-coated coverslips for 45 minutes
at 37°C and imaged using Köhler illuminated Nomarski differential
interference contrast optics with a Zeiss 63⫻ oil immersion 1.40 NA
plan-apochromat lens on a Zeiss Axiovert 200M microscope (Carl Zeiss).
Numbers of adherent platelets was computed using ImageJ software
(National Institutes of Health).16
Immunoprecipitation of plasma fXI
Plasma anticoagulated with sodium citrate or EDTA was obtained from the
following species: African elephant, (Loxodonta africana), Amur tiger
(Panthera tigris altaica), baboon (Papio anubis), cattle (Bos taurus),
chicken (Gallus gallus domesticus), dog (Canis lupus familiaris), domestic
cat (Felis silvestris catus), giant anteater (Myrmecophaga tridactyla), horse
(Equus ferus caballus), llama (Lama glama), pig (Sus scrofa domestica),
rabbit (Oryctolagus cuniculus), and raccoon (Procyon lotor). Fifty microliters of Affigel-10 beads (BioRad) bound with 14E11 (3 mg/mL) were
rocked at RT for 2 hour with 500 ␮L of plasma and 500 ␮L of TBS. Beads
were washed with 1 mL of TBS and then eluted with 50 ␮L of SDSnonreducing sample buffer. Eluates were size fractionated on 7.5%
SDS-polyacrylamide gels, followed by Western blotting using biotinylated
14E11 antibody.
Mouse carotid artery thrombosis models
Procedures for mice were approved by the Institutional Animal Care and
Use Committee of Vanderbilt University. C57Bl/6 mice deficient in fIX,17
fXI,18 or fXII7 were described. Mice with combined fXI and fXII
deficiencies were generated from these lines. Anesthesia was with 50 mg/kg
intraperitoneal pentobarbital. The right common carotid artery was fitted
with a Doppler flow probe (Model 0.5 VB; Transonic System). 14E11
(1.0 mg/kg) was infused into the internal jugular vein 15 minutes before
injury. In the ferric chloride model,10 thrombus formation was induced by
applying two 1 ⫻ 1.5-mm filter papers (GB003; Schleicher & Schuell)
saturated with FeCl3 (2.5% to 15% solution) to opposite sides of the artery
for 3 minutes, followed by rinsing with normal saline. Flow was monitored
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BLOOD, 11 NOVEMBER 2010 䡠 VOLUME 116, NUMBER 19
FACTOR XIIa ACTIVATION OF FACTOR XI
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Figure 1. Binding properties of 14E11. (A) Coomassie blue–stained 10% polyacrylamide gel of human (H) and mouse (M) recombinant fXI. (B-C) Western blots of
nonreducing 10% polyacrylamide gels of mouse (B) and human (C) normal (N) and fXI-deficient (XI⫺/⫺) plasmas, using biotinylated-14E11 for detection. rXI in panel B indicates
recombinant mouse fXI control. (D) Binding of biotinylated-14E11 to immobilized mouse fXI (E), human fXI (F), or human fXIa (䡺), as described in “Methods.” (E) Western blot
of nonreducing 10% polyacrylamide gel of human fXI (hXI), human prekallikrein (PK), and human fXI in which the A1, A2, A3, or A4 domain has been replaced with the
corresponding domain from PK. Position for fXI dimer is indicated to the right by “D,” and for monomeric PK by “M.” Note fXI with the PK A4 domain is a monomer because A4
mediates fXI dimer formation. (F) Western blot (left panel) of a nonreducing 10% polyacrylamide gel of human fXI (hXI) and individual human fXI apple domains linked to tPA.
The right panel is a stained gel showing the recombinant apple domain-tPA chimeras. Note the A4 chimera forms a dimer. For panels A-C and E, positions of molecular mass
standards in kDa are at the left of the figures, and for panel F at the right.
for 30 minutes. For the laser injury model,19 Rose Bengal (75 mg/kg) was
infused through the internal jugular vein, and the carotid artery was
illuminated with a 1.5-mW 540-nm laser (Melles Griot) positioned 6 cm
from the vessel. Flow was monitored for 120 minutes.
Mouse pulmonary embolism model
Mice anesthetized with 50 mg/kg intraperitoneal pentobarbital were fitted
with an MP100 transducer (AD Instruments) to monitor respiration, and
14E11 (1.0 mg/kg) or vehicle was infused into the internal jugular vein.
After 15 minutes, 100 ␮L of a 1:4 dilution of STA-Neoplastin CI Plus
(⬃ 1nM TF; Diagnostica Stago) was infused into the inferior vena cava
over 30 seconds.20 Time to cessation of breathing was determined.
Baboon thrombosis model
Nonterminal thrombosis studies were performed on a male baboon (Papio
anubis) with an exteriorized femoral arteriovenous shunt.11,21 Protocols
were approved by the Institutional Animal Care and Use Committee of the
Oregon Health and Sciences University. Briefly, thrombus formation was
initiated by introducing a 20-mm long by 4-mm diameter vascular graft
(polytetrafluorethylene [Gore-Tex]; Gore & Co.) coated with collagen into
the shunt. Flow rate through the shunt was restricted to 100 mL/min (mean
shear rate 265/s). Thrombus formation was assessed by quantitative
imaging of 111In-labeled platelet accumulation within, and downstream of
the graft (10 cm), using a GE-400T gamma scintillation camera interfaced
with a NuQuest InteCam computer system. Fibrin deposition in the graft
was determined by direct measurement of 125I-labeled fibrinogen as
described.21
Results
Antibody 14E11
Antibodies were raised against mouse fXI (Figure 1A) in fXIdeficient mice and screened for ability to prolong the aPTT of
mouse plasma. The IgG 14E11 was selected for further study
because it also prolonged the aPTT of human plasma. On Western
blots, 14E11 recognizes a single band of appropriate size
(⬃ 160 kDa) in normal mouse (Figure 1B) and human (Figure 1C)
plasma, but not in fXI-deficient plasma. 14E11 has a high affinity
for mouse and human fXI, and human fXIa in a solid phase binding
assay (apparent Kd ⬃ 2-3pM; Figure 1D). fXI is a dimer of 80-kDa
subunits, each containing 4 apple domains (A1-A4) and a protease
domain. PK, a monomeric homolog of fXI, has an identical
structure. Previously, we used human fXI/PK chimeras to show that
the A2 domain is required for 14E11 binding to fXI (Figure 1E).6
Western blots using individual fXI apple domains indicate the
14E11 binding site is likely located entirely within A2 (Figure 1F).
Anticoagulant properties of 14E11
14E11 prolonged the aPTT of mouse and human plasma (Figure 2)
with maximum inhibition at 25-50nM. This is similar to the plasma
fXI concentration in humans (20-45nM),2 and probably in mice.12,18
The prolongation of the clotting times were similar to those of
fXI-deficient plasmas. The aPTT of fXI-deficient mouse plasma, or
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3984
CHENG et al
Figure 2. Effect of 14E11 on the aPTT assay. Mouse (E) or human (F) plasma
supplemented with 14E11 (10⫺5 to 100␮M) were tested in an aPTT assay, as
described in “Methods.”
plasma from a wild-type mouse treated with 14E11 is 48-70 seconds.
This modest prolongation, relative to human fXI-deficient plasma,
is a consistent finding. 14E11 did not affect the prothrombin time of
BLOOD, 11 NOVEMBER 2010 䡠 VOLUME 116, NUMBER 19
human plasma. In an assay using fXIa to trigger clotting, 14E11 did
not affect clotting times, indicating it does not interfere with fXIa
activation of fIX. This was confirmed by Western blot analysis in a
purified protein system (Figure 3A). In contrast, fIX activation is
reduced by antibody O1A6 (Figure 3A), which binds to the A3
domain6 near an area required for fIX activation.13 Cumulatively,
these data indicate 14E11 interferes with fXI activation, but not
fXIa activity. This was supported by studies in which clotting was
triggered by addition of fXIIa. Here, 14E11 prolonged clotting
times to an extent consistent with 90% inhibition of fXI activation
by fXIIa.
We previously reported that 14E11 effects fXI activation by
fXIIa in a purified protein system (rate reduced ⬃ 50%),6 but does
not completely explain its potent effect in the aPTT assay. fXI
forms a complex in plasma with HK.14,22,23 In the aPTT, HK
enhances fXI activation by fXIIa by facilitating fXI binding to the
contact surface.1,2 In a solid phase competition assay, 14E11
inhibited binding of fXI to immobilized HK (apparent Ki of
⬃ 10-20nM; Figure 3B). A modification of the method of Baglia et
al24 was used to measure activation of fXI by fXIIa in the presence
of HK on a kaolin (aluminum silicate) surface. 14E11 significantly
reduced the rate of fXI activation in this HK-dependent system
(Figure 3C).
Figure 3. Activities of antibody 14E11. (A) Nonreducing Western blots showing fIX (100nM) activation by fXIa
(1nM) in the presence of control vehicle (top), 200nM
14E11 (middle), or 200nM O1A6 (bottom). Blots were
developed with a polyclonal anti-human fIX IgG. Arrows
to the right of each blot indicate zymogen fIX (IX) and
fIXa␤ (IXa). Time of incubation in minutes is shown at the
bottom, and positions of molecular mass standards are at
the left of the figure. (B) Competition binding assay in
which biotinylated fXI is allowed to bind to immobilized
HK in the presence of 14E11 (10⫺5 to 100␮M). Bound fXI
was detected with strepavidin-HRP, as described in
“Methods.” Each symbol is the average of results for
duplicate experiments. (C) Factor XI (85nM) was activated by fXIIa (17nM) in the presence of HK (70nM and
kaolin (0.5 mg/mL) in the presence (F) or absence (E) of
300nM 14E11. At various time points, samples were
removed from the reaction into CTI, and fXIa generated
was determined with a chromogenic substrate, as described in “Methods.” Each symbol is the average of
results for duplicate experiments. (D) Thrombin generation in fXII deficient plasma initiated by addition of Ca2⫹
and fXIIa (10nM curves 1 and 3, or 1nM curves 2 and 4) in
the absence (curves 1 and 2) or presence (curves 3 and
4) of 300nM 14E11. (E) Thrombin generation in fXII
deficient plasma initiated by addition of Ca2⫹ in the
absence (curve 1) or presence (curvess 2, 3, and 4) of
tissue factor (0.23pM). Reaction for curves 2, 3, and 4
included control vehicle, 300nM 14E11, or 300nM O1A6,
respectively.
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BLOOD, 11 NOVEMBER 2010 䡠 VOLUME 116, NUMBER 19
FACTOR XIIa ACTIVATION OF FACTOR XI
3985
fXI is a noncovalently associated dimer, this is not apparent under
the denaturing conditions of SDS-gel electrophoresis. Horse fXI
also has a substantial fraction of protein without the interchain
bond. As the horse cDNA codes for Cys321, formation of the
interchain bond is likely incomplete. The pattern for the African
elephant is unexplained, but was noted in 3 unrelated members of
the species. FXI is a relatively recent addition to the coagulation
mechanism, making its appearance during mammalian evolution.26
The negative result with chicken plasma is expected, as birds do not
have a fXI gene.26
Mouse arterial thrombosis models
Figure 4. 14E11 immunoprecipitates of fXI from mammalian plasmas. Western
blots of nonreducing 7.5% polyacrylamide gels of fXI from mammal plasmas
immunoprecipitated with 14E11 linked to agarose beads. The primary detection
antibody was biotinylated-14E11. Positions of molecular mass standards are shown
on the left.
14E11 significantly reduced thrombin generation in a plasma
thrombin generation assay when coagulation is initiated by adding
fXIIa in the absence of surface (Figure 3D). Previous work with
this system showed that fXI can be activated in the absence of
fXIIa, most likely by thrombin.6 This pathway, which is thought to
serve a role in hemostasis,6,8 is demonstrated in Figure 3E. Here, a
low concentration of TF (0.23pM) produced a burst of thrombin
generation in the presence of fXI (ETP 1092nM) that was not
affected by 14E11 (ETP 1118nM). This is consistent with the
selective effect of 14E11 on fXIIa-mediated fXI activation. In
contrast, O1A6 reduced thrombin generation approximately 50%
(ETP 547nM), as expected with inhibition of fIX activation by
fXIa. Taken as a whole, the data indicate 14E11 exerts an inhibitory
effect on fXI activation, but not fXIa activity, in plasma in the
absence or presence of a surface. In the absence of a surface, this
may be due to direct inhibition of fXIIa activation of fXI. In the
presence of a surface (contact activation), inhibition of the fXI-HK
interaction likely also contributes.
14E11 recognizes fXI from multiple species
Give the unexpectedly high affinity of 14E11 for human fXI, we
tested the antibody in plasmas from other species used in vascular
research. 14E11 had a potent effect on the aPTT assay in pig, rabbit,
and baboon plasma (supplemental Figure 1, available on the Blood
Web site; see the Supplemental Materials link at the top of the
online article), and in rhesus macaque and rat plasma (not shown).
14E11 immunoprecipitated fXI from plasmas of a variety of
placental mammals (Figure 4). Rabbit fXI migrates at a relatively
low molecular mass because it lacks Cys321, which forms the
interchain bond connecting the fXI dimer subunits.25 While rabbit
Application of FeCl3 to the exterior of blood vessels causes severe
endothelial damage and occlusion by platelet-rich thrombi.27 fXIIand fXI-deficient mice are resistant to mesenteric artery occlusion
induced by a 20% FeCl3 solution.9 We studied carotid artery
occlusion in fIX-, fXI-, or fXII-deficient mice using graduated
FeCl3 concentrations (Figure 5A).10 In our hands, the artery
typically occludes in 5-15 minutes or not at all. Because our mouse
lines were maintained for many years, they were rebackcrossed
through 6 generations to C57/Bl6 mice from a single vendor to
compensate for genetic drift. Vessel occlusion occurs consistently
in wild-type mice treated with 3.5% FeCl3, but not 2.5% FeCl3.
Mice lacking fIX, fXI, or fXII were resistant to occlusion at 5%
FeCl3, with some mice occluding at 7.5% FeCl3. fIX and fXI null
mice all occluded at 10% FeCl3, but half of fXII-null mice tested at
10% FeCl3, and one at 12.5% FeCl3 did not. fXII- and fXI-deficient
mice were tested in a model where carotid artery thrombosis is
induced with a laser after infusion of Rose Bengal (Figure 5B).19 In
this model, there is probably less endothelial damage and collagen
exposure than with FeCl3.27 Mean time to vessel occlusion in wild
type mice (40 ⫾ 13 minutes, ⫾ 1 standard deviation, n ⫽ 16) was
significantly shorter than for fXI-deficient mice (59 ⫾ 13 minutes,
n ⫽ 12,P ⫽ .0012) or fXII-deficient mice (79 ⫾ 17 minutes, n ⫽ 15
P ⫽ .0001). Time to occlusion was also significantly longer for
fXII-deficient mice than for fXI-deficient mice (P ⫽ .0031)
The results are consistent with several possible mechanisms.
fXIIa may contribute to thrombus formation through fXI activation. However, given the different degrees of response of fXI- and
fXII-deficient mice to vessel injury, the proteins could contribute
through distinct pathways. Alternatively, fXIIa may work through
both fXI-dependent and -independent processes. We tested mice
with combined fXI- and fXII-deficiency in the FeCl3 model to look
for an additive effect indicating the proteins operate in distinct
pathways. Results for these animals were similar to those for
fXII-deficient mice (Figure 4A), consistent with fXIIa activating
fXI. As 14E11 selectively interferes with fXIIa-mediated fXI
activation in vitro, we tested it in the FeCl3 model. Intravenous
infusion of 1.0 mg/kg 14E11 into wild-type mice prolonged the
aPTT to a similar degree as adding 14E11 directly to plasma. The
same effect was obtained 5 hours after intraperitoneal administration. Western blots of plasma obtained at intervals after 14E11
infusion indicate the antibody does not clear fXI protein from
plasma (supplemental Figure 2). In the FeCl3 model, 1.0 mg/kg
14E11 protected mice from carotid occlusion induced by 7.5%
FeCl3, but not 10% FeCl3 (Figure 5C), with the antithrombotic
effect lasting approximately 48 hours. Smaller doses of 14E11 were
less effective at preventing thrombosis (supplemental Figure 3).
Cumulatively, the results are consistent with the premise that fXI is
activated by fXIIa in the thrombosis models. However, it seems
likely that fXIIa is influencing thrombus formation partly by a
fXI-independent mechanisms.
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3986
CHENG et al
BLOOD, 11 NOVEMBER 2010 䡠 VOLUME 116, NUMBER 19
Figure 5. Mouse arterial thrombosis models. (A) C57Bl/6
mice of the genotypes indicated at the right of the panel
were tested in a carotid artery thrombosis model at FeCl3
concentrations from 2.5% to 15% (10 animals per concentration), as described in “Methods.” Bar heights indicate
percent of mice in each group with patent arteries
30 minutes after applying FeCl3. (B) Time to carotid
arterial occlusion in wild-type (n ⫽ 16), fXI-deficient
(n ⫽ 12), or fXII-deficient (n ⫽ 15) mice in a Rose Bengallaser injury model, as described in “Methods.” Error bars
indicate 1 standard deviation. Time to occlusion is significantly longer for fXI⫺/⫺ than wild-type mice (*P ⫽ .0012),
for fXII⫺/⫺ than wild-type mice (**P ⫽ .0001) and for
fXII⫺/⫺ than fXI⫺/⫺ mice (**P ⫽ .0031). (C) Results from
the FeCl3 carotid artery thrombosis model (panel A),
comparing the effect of treatment with an intravenous
infusion of 14E11 (1.0 mg/kg) into wild-type mice (2) to
results for 7.5% and 10% FeCl3 from panel A.
Pulmonary embolism model
We predicted that thrombosis triggered by a high concentration of
TF would be relatively unaffected by fXI or fXII deficiency and,
therefore, unresponsive to 14E11. C57Bl/6 mice were studied in a
model of lethal pulmonary embolism. Wild-type mice typically
developed respiratory arrest in 100-300 seconds (mean 195 ⫾ 97
seconds) after infusion of rabbit TF into the inferior vena cava
(Figure 6).20,28 As expected, most fXI-deficient mice succumbed to
TF infusion; however, time to respiratory arrest (392 ⫾ 273
seconds) was significantly longer than for wild-type mice
(P ⫽ .0065), and a similar trend was seen in fXII-null mice
(462 ⫾ 318 seconds, P ⫽ .0024). Infusion of 14E11 before TF
infusion also significantly prolonged survival (430 ⫾ 285 seconds,
P ⫽ .0027) compared with untreated wild-type animals. Comparable results were obtained with human TF (data not shown).
14E11 in a primate thrombosis model
Previously, we described the effects of anti-fXI antibodies on
thrombus formation in collagen-coated Gore-Tex grafts inserted
into arteriovenous shunts in baboons.12,21 Antibody O1A6, which
blocks fIX activation by fXIa, had a limited effect on platelet
binding to the graft, but caused a major defect in growth of
platelet-rich thrombi.12 14E11 was tested in this model in a male
baboon. Twenty-four hours after a subcutaneous injection of 14E11
(1 mg/kg), the aPTT increased from 31.4 seconds to 71.9 seconds
and remained above baseline for 4 days (Figure 7A). When
collagen-coated grafts were introduced into the shunt before 14E11
treatment, 111In-labeled platelets bound to the grafts (Figure 7B),
with robust downstream formation of platelet-rich thrombi (Figure
Figure 6. Mouse pulmonary embolism model. C57Bl/6 mice were given a single
100-␮L bolus of rabbit brain tissue factor (⬃ 1nM solution), and time to cessation of
respiration was measured. The mice tested are wild-type (WT: F), fXI-deficient
(fXI⫺/⫺: f), fXII-deficient (fXII⫺/⫺: Œ) and WT treated with 1.0 mg/kg 14E11 (). Each
symbol represents one animal, and bars within the column of symbols indicate the
mean occlusion time. The box plots indicate the 25th, 50th, and 75th percentile for
each group, with the whiskers showing the 10th and 90th percentile.
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BLOOD, 11 NOVEMBER 2010 䡠 VOLUME 116, NUMBER 19
Figure 7. Baboon AV shunt thrombosis model. (A) Shown are aPTT clotting times
(see “Methods”) of plasma from a male baboon treated with a single subcutaneous
injection of 14E11 (1 mg/kg) at time zero. (B and C) Platelet deposition over time for
4 separate collagen coated grafts, 2 inserted into the shunt before treatment with
14E11 (E, 䡺) and 2 inserted 24 hours after a 1-mg/kg subcutaneous dose of 14E11
(F, f). Deposition of 111I-labeled platelets was measured with a gamma scintillation
camera, as described in “Methods.” Panel B shows accumulation of platelets within
the graft, while panel C shows the bulk of platelet accumulation in a thrombus forming
in the 10 cm immediately downstream of the graft.
7C). 14E11 did not affect platelet deposition on the graft surface
(Figure 7B), similar to prior results with heparin or aspirin.21
However, there was almost complete absence of downstream
thrombus growth (Figure 7C). Fibrin deposition within the grafts
was reduced approximately 40% after 14E11 treatment compared
with pretreatment controls (average 1.15 mg and 1.74 mg,
respectively).
Discussion
Patients with severe fXI deficiency are predisposed to excessive
bleeding after trauma to sites such as the oropharynx or urinary
FACTOR XIIa ACTIVATION OF FACTOR XI
3987
tract.2,4 FXII deficiency does not compromise hemostasis.1,2 Taking
these established observations into account, current models of
thrombin generation often omit fXII, with fXI activation mediated
by another protease such as thrombin.3,6,29 These hypotheses are
being reconsidered, at least as they pertain to pathologic coagulation, in light of the finding that fXII-deficient mice are resistant to
thrombosis.9 fXI-deficient mice are also resistant to thrombosis,9,10
consistent with the idea that thrombin generation driven by fXIIa
activation of fXI supports occlusive thrombus growth. However, it
has not been established that fXI is the major (or sole) target of
fXIIa, nor is it clear that the mouse data are relevant to thrombus
formation in humans. The antibody 14E11 provides us with a tool
to begin to address these questions, which have major implications
for development of therapeutic inhibitors that target the intrinsic
pathway.
fIX-, fXI-, and fXII-deficient mice have not previously been
compared in a single study. In our hands, fXII-deficient mice are
relatively more resistant to arterial occlusion than fIX- or fXIdeficient animals, suggesting at the very least that fXII also makes a
fXI-independent contribution to thrombosis. It is even conceivable
that fXII and fXI operate in distinct pathways. As 14E11 selectively
targets the fXIIa-mediated fXI activation in vitro, we tested it in the
FeCl3 model. In wild-type mice, 14E11 had a comparable effect to
fXI deficiency, without clearing fXI from the plasma, supporting
the hypothesis that thrombus formation in this model requires fXI
activation by fXIIa.
14E11 has a partial inhibitory effect on fXI activation by fXIIa
in a purified protein system.6 Inhibition of this reaction is more
robust in plasma, particularly when coagulation is triggered
through contact activation by addition of a surface. The proposed
binding site on fXI for fXIIa is on the A4 domain,30 at a
considerable distance from the 14E11 binding site on A2. FXI
forms a complex with HK in plasma, and 14E11 inhibits this
interaction. The 4 fXI apple domains form a disk-like structure,31
and HK is predicted to bind along a channel on the surface of A2
that extends between A1 and A4.14,31,32 HK-deficient mice have
delayed time to thrombus formation in the Rose Bengal-laser injury
model,33 consistent with the premise that interfering with the
fXI-HK interaction could affect thrombus formation in vivo.
Considering the available data, 14E11 appears to have a complex
effect on contact activation-initiated coagulation, directly interfering with fXIIa activation of fXI, and the fXI-HK interaction
required for binding fXI to the contact surface. It must be
considered that 14E11 could interfere with a fXI function in vivo
not required in the aPTT or thrombin generation assay, such as fXI
binding to platelets.34 FXI binds to 2 platelet receptors: glycoprotein Ib (GPIb)35 and ApoER2.16 Previously, we showed that
blocking ApoER2 prevents platelet binding to fXI;16 however,
14E11 does not interfere with this interaction in vitro (supplemental Figure 4).
While the data for 14E11 in mice support the premise that fXIIa
activates fXI, the resistance of fXII-deficient mice to thrombosis is
not completely explained by one pathway. The consistent observation that the aPTT of fXII-deficient plasma is longer than that of
fXI-deficient plasma suggests there are prothrombotic targets for
fXIIa other than fXI, although they have not been defined in vivo.
For example, fXIIa can activate factor VII, priming the extrinsic
pathway of coagulation.36,37 Platelets have been shown to support
fXII activation38,39; however, recent work raises the intriguing
possibility that fXIIa activates platelets. Four protease-activated
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3988
CHENG et al
receptors (PARs) have been described that are cleaved by thrombin
or other proteases. Mouse platelets primarily express PAR3 and
PAR4. Thrombin cleaved PAR3 functions as a cofactor, supporting
PAR4 cleavage and subsequent platelet activation.40 Mao et al41
recently reported that fXIIa cleaves PAR3, activating PAR4-null
murine platelets. As PAR3-null mice are resistant to FeCl3- or
TF-induced thrombosis,20 fXIIa cleavage of PAR3 may explain the
differences we observed between fXII- and fXI-deficient mice in
arterial thrombosis models. If this is the case, it is possible that fXII
makes a greater contribution to thrombosis in mouse models than in
humans, as human platelets primarily express PARs 1 and 4, with
PAR3 serving a lesser role.40
Indeed, in contrast to fXI, a direct relationship between fXII and
thrombosis in humans is not established. High plasma fXI levels
are associated with increased risks for arterial42 and venous
thrombosis43, and incidences of stroke44 and deep venous thrombosis45 are reduced in severe fXI deficiency. Inhibition of fIX
activation by fXIa produces an antithrombotic effect in several
species,46 including primates,12,21 supporting an important role for
fXIa in thrombus formation in humans. Paradoxically, a link has
been suggested between fXII deficiency and thrombosis dating
back to the death of the first described fXII-deficient patient from a
pulmonary embolism.47 Recently, Doggen et al42 described an
inverse relationship between fXII levels and risk of myocardial
infarction, bringing the issue back to the fore. A large study by
Endler et al48 also showed mortality from cardiovascular disease
generally increases as fXII levels decrease. Bradford et al49 noted
that fXII inhibits thrombin-induced platelet aggregation by interfering with thrombin binding to GP1b. These data do not make a
strong case for fXII contributing to thrombosis, and raise the
possibility that therapeutic intervention directed at fXII/XIIa could
actually increase thrombotic risk. The affinity of 14E11 for baboon
fXI provided us with an opportunity to test the hypothesis that
fXIIa-mediated activation of fXI contributes to thrombus formation
in primates.
The pan-specific nature of 14E11 is surely a consequence of
raising antibodies in fXI-deficient mice, producing clones that
recognize conserved epitopes that are not antigenic in wild-type
animals. 14E11 should prove useful in thrombosis models in a
variety of mammalian species. Previously, we showed that blocking fIX activation by fXIa, a reaction required for hemostasis in
humans, reduced platelet accumulation in a baboon thrombosis
model.12 14E11 had a similar impressive effect in this model,
indicating a role for fXIIa in thrombosis in primates. While these
data support targeting fXI activation as an antithrombotic strategy,
they do not directly address the concerns raised regarding the safety
of directly inhibiting fXII/fXIIa as a means to this end. The study
by Endler et al48 contained a subset of patients with severe fXII
deficiency ( ⬍ 10% of normal plasma level). Surprisingly, the risk
of cardiovascular death in this group was similar to that for the
population mean, and not a continuation of the trend of increased
mortality with lower fXII levels observed within the normal fXII
range. This suggests severe fXII deficiency (and perhaps the state
produced by a potent fXII/XIIa inhibitor) is distinctly different
from a low fXII level within the normal range in humans.
Development of specific fXIIa inhibitors will be important for
definitively establishing the contributions of fXII and fXI to
thrombosis in nonhuman primates (and ultimately in humans) and
the relative safety of fXIIa inhibition.
Finally, we tested 14E11 in a TF-induced pulmonary embolism
model, with the expectation that it would have a little effect. We
BLOOD, 11 NOVEMBER 2010 䡠 VOLUME 116, NUMBER 19
reasoned that a bolus of TF large enough to produce respiratory
arrest in under 5 minutes would induce substantial fIX activation
through factor VIIa/TF, overwhelming contributions from fXIa.
Interestingly, fXI and fXII deficiency, as well as 14E11, had a
modest beneficial effect. Previously, we showed that XI inhibition
reduces propagation of thrombi initiated by TF-coated surfaces in
baboons.21 More recently, Dow et al28 described a locus on
chromosome 11 in Cast/EiJ mice that confers resistance to the
lethal effects of TF infusion. Congenic C57Bl/6 mice carrying
Cast/EiJ chromosome 11 were significantly more resistant to TF
infusion than C57Bl/6 controls. The single plasma abnormality
identified in the congenic strain was reduced fXI activity.28 These
results raise the possibility that feedback activation of fXI and fXII
after initiation of coagulation by factor VIIa/TF, or contact
activation on the clot surface itself, contribute to thrombus growth
even in situations where TF is a primary driver of thrombosis.
Acknowledgments
We thank Dr. Joost Meijers (Academic University, Amsterdam,
The Netherlands) for fXI apple domain-tPA fusion constructs, Dr.
Darrell Stafford (University of North Carolina, Chapel Hill, NC)
for fIX-deficient mice, and Dr. Sally Nofs (Nashville Zoo at
Grassmere, Nashville, TN) for mammal plasma samples. We are
also grateful to Drs. Douglas Tollefsen and Li He (Washington
University, St. Louis, MO) for consultation on the Rose Bengallaser injury thrombosis model.
This work was supported by awards HL81326 and HL58837
from the National Heart, Lung, and Blood Institute (D.G.), and
09GRNT2150003 and 0910025G from the American Heart Association (O.J.T.M.).
Authorship
Contribution: Q.C. conducted FeCl3, TF-pulmonary embolism,
Rose Bengal-laser injury studies, and analyzed data; E.I.T. developed 14E11 and tested it in baboons and coagulation assays; M.S.P.
performed FeCl3 studies, fXI immunoprecipitation, and Western
blot analyses; I.S. performed FeCl3 and TF pulmonary embolism
studies; A.M. performed thrombin generation studies; M-f.S.
prepared mouse fXI and performed HK binding and fIX activation
studies; T.C.W.-A. performed platelet adhesion assays; S.A.S.
provided veterinary consultation, designed experiments, and prepared mammal plasma; S.R.H. assisted and consulted on 14E11
studies in baboons; O.J.T.M. characterized 14E11 properties in
platelet-based systems; T.R. developed fXII-deficient mice, analyzed data, and assisted in manuscript writing; A.G. developed the
14E11 antibody, performed baboon studies, analyzed data, and
assisted in manuscript writing; and D.G. analyzed data and wrote
the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: David Gailani, M.D., Division of Hematology/
Oncology, Vanderbilt University, 777 Preston Research Bldg, 2220
Pierce Ave, Nashville, TN 37232-6305; e-mail: dave.
[email protected].
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BLOOD, 11 NOVEMBER 2010 䡠 VOLUME 116, NUMBER 19
FACTOR XIIa ACTIVATION OF FACTOR XI
3989
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2010 116: 3981-3989
doi:10.1182/blood-2010-02-270918 originally published
online July 15, 2010
A role for factor XIIa−mediated factor XI activation in thrombus
formation in vivo
Qiufang Cheng, Erik I. Tucker, Meghann S. Pine, India Sisler, Anton Matafonov, Mao-fu Sun, Tara C.
White-Adams, Stephanie A. Smith, Stephen R. Hanson, Owen J. T. McCarty, Thomas Renné, András
Gruber and David Gailani
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