Antithrombotic Efficacy of a Novel Murine Antihuman
Factor IX Antibody in Rats
Giora Z. Feuerstein, Arun Patel, John R. Toomey, Peter Bugelski, Andrew J. Nichols,
William R. Church, Richard Valocik, Paul Koster, Audrey Baker, Michael N. Blackburn
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Abstract—A murine antihuman factor IX monoclonal antibody (BC2) has been generated and evaluated for its capacity to
prolong the activated partial thromboplastin time (aPTT) in vitro and ex vivo and to prevent arterial thrombosis in a rat
model in vivo. BC2 extended aPTT to a maximum of 60 to 80 seconds at 100 to 1000 nmol/L in vitro (rat and human
plasma, respectively) and ex vivo (rat) after dosing of rats up to 6 mg/kg in vivo. BC2, administered as bolus (1 to 6
mg/kg) followed by infusion (0.3 to 2 mg z kg21 z h21), dose-dependently prevented thrombosis of an injured rat carotid
artery (FeCl3-patch model), increased time to artery occlusion, and reduced incidence of vessel occlusion. BC2 efficacy
in preventing arterial thrombosis exceeded that of heparin (bolus 15 to 120 U/kg followed by infusion 0.5 to 4.0 U z
kg21 z min21), whereas the latter rendered the blood incoagulable (aPTT.1000 seconds). BC2 demonstrated complete
antithrombotic efficacy also as a single bolus given either as prevessel or postvessel injury as evidenced by reduction
of thrombus mass (from 4.1860.49 to 1.8060.3 mg, P,0.001), increasing vessel patency time (from 14.960.9 minutes
to 58.361.7 minutes, P,0.001) and decreasing incidence of vessel occlusion from 100% to 0% in vehicle- versus
BC2-treated rats, respectively. BC2 (3 mg/kg, IV) administered in a single bolus resulted in 50% reduction in thrombus
mass (P,0.01), extended vessel patency time (P,0.001), extended aPTT only 4-fold, and had no effect on blood loss
via a tail surgical wound; heparin, at doses that reduced thrombus mass to a similar extent, extended aPTT beyond 1000
seconds (over 500-fold) and increased blood loss from 1.860.7 to 3.360.6 mL (P,0.001). These data suggest that BC2
may provide enhanced therapeutic efficacy in humans at lesser interference with blood hemostasis than heparin.
(Arterioscler Thromb Vasc Biol. 1999;19:2554-2562.)
Key Words: thrombosis n coagulation n factor IX n heparin n aspirin n monoclonal antibodies
D
for careful patient monitoring.7 Thus the state-of-the-art in
treatment of acute thrombotic disorders comprises a combination of antithrombotic agents including aspirin, heparin,
fibrinolytics (when appropriate), and the GPIIb/IIIa receptor
antagonists. This arsenal of agents, however, carries significant liabilities of which bleeding, especially cerebral hemorrhages, remains a major medical problem.7 The persistent
untoward consequences of contemporary antithrombotic regimens provide an impetus for exploration for superior antithrombotic agents that extend desired therapeutic efficacy,
reduce liabilities, and overall, provide a significant broadening of the therapeutic index. In the past few years, research
interest expanded toward additional anticoagulant targets
such as specific thrombin, factor Xa, and tissue factor/factor
VIIa inhibitors as antithrombotic agents.10,11 We have explored other sites in the coagulation cascade to identify
alternative ways to combat the thrombotic process.
Factor IX (FIX) is a key coagulation factor essential for
amplification of coagulation that results in thrombus formation.12–14 Severe deficiency in FIX leads to bleeding (Hemo-
espite substantial efforts to prevent and treat thrombotic
events, arterial thrombosis continues to be the major
cause of death in adult populations of developed countries.
Although contemporary medicine offers several medical
strategies to combat thrombosis, the unmet medical need is
significant as none of the available therapeutics provides the
desired efficacy and safety profile. Aspirin (ASA) remains
the primary antiplatelet agent of proven efficacy for chronic,
secondary prevention of arterial thrombosis,1,2 whereas ticlopidine and clopidogrel (ADP inhibitors) add marginal benefits3,4 over ASA. Heparin and coumadin are proven anticoagulants for acute and chronic thrombotic disorders,
respectively.5–7 The most recent novel antithrombotic agent
introduced for adjunctive treatment of thrombotic events
associated with vascular interventions is abciximab (ReoPro™), an antibody that blocks platelet integrin GPIIb/IIIa
(the fibrinogen receptor) and hence, platelet aggregation.8,9
However, these anticoagulants and the GPIIb/IIIa antagonists
may have restricted therapeutic capacity due to significant
adverse effects such as bleeding, thrombocytopenia, and need
Received April 28, 1998; revision accepted February 22, 1999.
From SmithKline Beecham Pharmaceuticals, Departments of Cardiovascular Pharmacology (G.Z.F., J.R.T., A.J.N., R.V., P.K.), Protein Biochemistry
(A.P.), Experimental Pathology (P.B.), and Structural Biology (A.B., M.N.B.), King of Prussia, Pa; and Green Mountain Antibodies (W.R.C.)
Burlington, Vt
Correspondence to Michael N Blackburn, PhD, Department of Structural Biology, UE-0447, SmithKline Beecham Pharmaceuticals, 709 Swedeland
Road, Box 1539, King of Prussia, PA 19406-0939. E-mail [email protected]
© 1999 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
2554
Feuerstein et al
Monoclonal Antibodies to FIX/FIXa Prevent Thrombosis
2555
Figure 1. Experimental design of the rat
arterial thrombosis model. Rx indicates
time of drug administration; BS, time of
blood sample withdrawn; TM, time of
thrombus extraction from the carotid
vessel; and aPTT, activated partial
thromboplastin time. The period of injury
represents the time of exposure of the
vessel to the patch soaked with 50%
FeCl3.
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philia B, Christmas disease).15 Lollar and Fass16 showed that
active-site blocked factor IXa (FIXai) inhibits clot formation
in vitro. Benedict et al demonstrated that administration of
FIXai prevents thrombosis in experimental animal models,17
indicating that modulation of FIX function can be achieved to
gain therapeutic efficacy in thrombosis. Bajaj and coworkers18 have described antibody mediated inhibition of
FIX coagulant activity but did not explore in vivo application
of the antibody as an antithrombotic agent. We hereby report
a novel murine antihuman FIX/IXa antibody (BC2) that
possesses high efficacy in the prevention of thrombosis in a
rat arterial thrombosis model. In addition, comparison of BC2
with aspirin (ASA) and heparin or a combination of BC2 with
ASA resulted in superior antithrombotic efficacy with limited
extension of aPTT and blood loss.
Medical) was placed on the artery to measure blood flow. A cannula
(Tygon, 0.02x0.04 in, Norton Performance Plastics) was inserted
into the jugular vein for drug administration. The left femoral artery
was then isolated and cannulated for measurement of blood pressure
and collection of blood samples.
Procedure of Carotid Artery Lesion
To initiate thrombosis in the carotid artery, a 6.5-mm diameter
circular patch of glass micro-filter paper saturated with FeCl3
solution (50%) was placed on the carotid artery downstream from the
flow probe and kept for 10 or 15 minutes as described previously.22
In this well characterized model, thrombus formation is usually
completed within 15 to 20 minutes.
Measurement of aPTT and Prothrombin Time
One mL of arterial blood was drawn from the femoral artery into
3.8% citrate solution and centrifuged; aPTT and prothrombin time
Materials and Methods
BC2 Production and Assay
Monoclonal antibodies were generated against purified human FIX
using female Balb/C mice as described by Jenny et al.19 Hybridoma
medium was assayed in an ELISA format using FIX immobilized
onto microtiter plates, and wells containing anti-FIX antibodies were
subcloned by limiting dilution. Anti-FIX antibody from the hybridoma, BC2, was produced from ascites tumors in mice and purified by
conventional means20 and stored in buffer containing 0.02 mol/L tris,
0.15 mol/L NaCl, pH 7.40. An MLA Electra 800 Automatic
Coagulation Timer (Medical Laboratory Automation, Inc) was used
to measure aPTT. Purified human factor IX and IXa were purchased
from Hematologic Technologies (Essex Junction, VT). Molecular
weight fractionated heparin with an average molecular weight of
15 40021 was used for all in vitro experiments. Unfractionated
heparin was obtained from A.H. Robbins (Elkin-Sinns, Inc, Cherry
Hill, NJ) and was dissolved in normal saline.
Assay of Factor IXa Activity
The effect of BC2 on factor IXa activity was determined by
measuring the prolongation of clotting times when clotting was
initiated by the addition of preactivated factor IXa to factor IX
deficient plasma. The factor IXa concentration was adjusted to 30
nmol/L, which gave a clot time of approximately 30 seconds.
Animal Preparation
Male Sprague-Dawley rats (Charles River, Raleigh, NC) weighing
300 to 490 g were anesthetized with sodium pentobarbital (55 mg/kg,
IP). The rats were placed dorsal on a heated (37°C) surgical board
and an incision was made in the neck; the trachea was isolated and
cannulated with PE-240, Intramedic tube (Clay Adams, Parsippany,
NJ). The left carotid artery and jugular vein were then isolated. A
Parafilm M sheet (4 mm2, American National Can) was placed under
the carotid artery, and an electromagnetic blood flow probe (Carolina
Figure 2. Effect of BC2 on in vitro clotting time. A, Rat citrated
plasma (Œ) or human citrated reference plasma (F) (100 uL)
were incubated with 100 uL of Dade Actin Activated Cephaloplastin Reagent plus BC2 at the indicated concentrations for 3
minutes at 37°C. Samples of human plasma were also incubated with heparin (f). Coagulation was initiated by addition of
100 uL of 0.02 mol/L Ca21, and the time to clot formation was
measured with an MLA Automatic Coagulation Electra 800
Timer. B, Increasing concentrations of BC2 were added to samples of Factor IX deficient human plasma. Clotting was initiated
by addition of 30 nmol/L Factor IXa and 0.005 mol/L Ca21, and
the prolongation of the clotting time was measured.
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Figure 3. Scanning electron microscopy
of the thrombus. A, Shows a massive
thrombus in the carotid artery of a
vehicle-treated rat in which the carotid
artery was injured by FeCl3 patch. The
thrombus is rich in platelets and fibrin. B,
Internal side of the injury zone of BC2treated rats. Insert depicts an intact endothelium of sham lesion carotid arteries
with endothelial cell lining. Injured zone
of BC2-treated rats shows no intact endothelial cells, few adherent platelets,
and some minor aggregates.
(PT) were monitored by a fibrometer (BB1L, Baxter Dade) with
standard procedures. aPTT and PT values are represented in seconds.
opened lengthwise, placed on scanning electron microscopy (SEM)
stubs, and sputter-coated with gold.
Monitoring Blood Loss From Surgical Wound
Experimental Design for Assessment of
Therapeutic Interventions
To monitor blood loss, a tail surgical cut model was used as
described previously.23 Briefly, the rat tail was cut at 30% of its
length from its end using a new 21 surgical blade (Bard-Parker). The
proximal end of the tail was placed into a tube and blood was
permitted to drip freely into a reservoir of 3.8% citrate solution (1
mL) for 15 minutes. This procedure followed the 60-minute period
of the experiment proper. The animals were then overdosed with
sodium pentobarbital.
Scanning Electron Microscopy of Rat
Thrombosis Model
Segments of rat carotid artery were collected from sham, FeCl3
injury, or FeCl316 mg/kg BC2 injected rats 15 minutes before
injury. The arteries were perfusion-fixed with formaldehyde and
ligated above and below the lesioned area. Fixed arteries were
dehydrated with graded ethanol (30% to 100%), incubated in
hexamethyldisilazane, and dried in a desiccator. Dried arteries were
Pretreatment Experiment
Figure 1 depicts the experimental design of studies aimed to explore
the effect of BC2 administered as bolus1infusion before the onset of
injury. This protocol was also used for comparison of the BC2 effect
with that obtained with heparin, ASA, and combination of BC2 or
heparin with ASA. In all studies, ASA was administered at 5 mg/kg,
IV bolus 15 minutes before vessel injury, whereas heparin or BC2
were administered as bolus followed by infusion as depicted in
Figure 1. Heparin or BC2 pretreatments started 15 minutes before
placement of the FeCl3 patch on the carotid artery. All drug infusions
continued to the end of the experimental period, 60 minutes from
start of vessel injury. Blood samples were collected for aPTT and PT
assay at 60 minutes (end of study). Carotid artery blood flow was
continuously monitored. At the end of the experiment, the thrombus
was extracted from the carotid artery and weighed.
Feuerstein et al
Monoclonal Antibodies to FIX/FIXa Prevent Thrombosis
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Postinjury Protocol
The same experimental procedures were used as in the pretreatment
procedure except that heparin and BC2 administration commenced at
the end of the injury period. ASA, when administered, was given 15
minutes before injury. Only bolus administration of BC2 was used in
the postinjury treatment experiments, whereas heparin was always
administered as bolus followed by infusion. In this series of
experiments, the injury period (FeCl3 patch placement) was confined
to 10 minutes only.
Data Analysis
All data in the text and figures are mean group values6standard error
of mean for the indicated number of rats in each group. ANOVA and
Bonferoni tests for multiple comparisons were used for between
group analyses and a value P,0.05 accepted as significant.
Results
Effect of BC2 on aPTT, PT, and Activated Factor
IX In Vitro
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Figure 2 demonstrates a steep dose-dependent effect of
heparin on aPTT in vitro. In contrast to heparin, which
prolonged clotting times of normal human plasma to .350
seconds at 200 nmol/L, the anti-FIX mAb, BC2, extended the
aPTT from 18 seconds to a plateau of 60 seconds in rat
plasma and from 28 seconds to 85 to 90 seconds in human
plasma at .200 nmol/L antibody. In parallel experiments,
anti-FIX antibody was found to cause no change in the PT.
ELISA experiments show that BC2 reacts with FIX and with
activated factor IX (FIXa) but not with factor X, factor XI, or
prothrombin, demonstrating the specificity of BC2.
To identify potential animal species for use in the in vivo
analysis of BC2, plasma from rabbit, dog, rat, guinea pig, pig,
baboon, and cynomolgus monkey were screened using the
aPTT assay. Of these species, only plasma from rat (Figure 2)
and the nonhuman primates were inhibited by the antibody,
indicating that FIX from rat and nonhuman primates crossreacts with BC2.
To determine whether BC2 can block the activity of FIXa,
BC2 was mixed with 30 nmol/L FIXa and added to FIX
deficient plasma. As shown in Figure 2B, increasing concentrations of BC2 prolonged the clotting time from 30 seconds
(no BC2) to 65 seconds at 800 nmol/L antibody demonstrating that BC2 binding blocks factor X activation by FIXa.
SEM of Carotid Thrombus
SEM of sham arteries revealed an essentially normal endothelium with rare scattered platelets (Figure 3B, inset). Few
breaks in the endothelium are noted, probably the result of
mechanical damage during surgery. No evidence of thrombus
formation was observed in the sham rats.
SEM of the arteries treated with FeCl3 revealed mural
thrombi that occupied a large portion of the lumen of the
vessel (Figure 3A). The thrombi were composed of aggregated platelets, red blood cells, and amorphous and fibrillar
proteinaceous material. The proteinaceous material is consistent with fibrin. The endothelium of the arteries was mostly
obscured by the large thrombi (Figure 3). Where visible, the
endothelium overlying the region treated with FeCl3 was
covered by numerous adherent platelets and amorphous
proteinaceous material.
SEM of the arteries treated with FeCl3 from rats treated
with BC2 revealed the lumen of the vessels to be largely free
of thrombus (Figure 3B). The endothelium overlying the
Figure 4. Time (A) and dose (B) dependence of BC2 induced
aPTT prolongation. Rats (n56) were treated with BC2 at 0.3,
1.0, 3.0, or 6.0 mg/kg, IV bolus. Blood samples for measurement of aPTT were taken pretreatment and at 15, 30, or 60 minutes after BC2 treatment. Asterisks denote statistical significance. *P,0.01, **P,0.001, and ***P,0.0001.
region treated with FeCl3 showed extensive damage. Some
areas were covered by adherent platelets and some platelet
aggregates, but there was little or no proteinaceous material.
Selection of BC2 Dose Based on aPTT
Time/Dose Relationships
Figure 4 depicts the effect of various IV bolus doses of BC2
on aPTT throughout the duration of the experimental protocol. The study demonstrates that only a dose of 3 mg/kg and
above resulted in extension of aPTT and that 6 mg/kg
maintained aPTT extension of 3.5- to 4-fold over basal levels
throughout the designated experimental protocol.
Effect of Pretreatment With BC2, Heparin, ASA,
or BC21ASA on Thrombus Weight, aPTT, PT,
and Vessel Occlusion in FeCl3-Induced
Arterial Thrombosis
Figure 5 illustrates the effects of heparin (dose-response, 15
to 120 U/kg, bolus10.5 to 4 U z kg21 z min21, infusion),
aspirin (5 mg/kg), BC2 (1 to 6 mg/kg), bolus10.3 to 2 mg z
kg21 z h21 (infusion), and combination of BC2 or heparin plus
ASA on thrombus mass. Heparin dose-dependently suppressed thrombus formation in the carotid artery as reflected
by thrombus weight at the end of the experimental period.
However, the residual thrombus found in the vessels of rats
treated with the highest dose of heparin was still approximately 20% of that in the vehicle-treated rats. ASA per se did
not reduce the thrombus mass significantly, whereas the
combination of heparin (30 U/kg11 U z kg21 z min21) and
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Arterioscler Thromb Vasc Biol.
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Figure 5. Effect of heparin, ASA, or BC2 on thrombus mass in
FeCl3 injured rat carotid arteries with pretreatment protocol. V
represents saline vehicle-treated rats; H15 to H120, heparintreated rats at 15, 30, 60, or 120 U/kg bolus followed by 0.5, 1,
2, or 4 U z kg21 z min21 infusion over the duration of the study.
ASA indicates aspirin, 5 mg/kg, IV bolus 15 minutes before
injury; BC2 Veh, Tris-buffered saline treated rats. BC2-1 to
BC2-6 denotes BC2 treatment at 1, 3, or 6 mg/kg followed by
infusion of 0.3, 1 or 2 mg z kg21 z h21 over the duration of the
study. Number of rats56 to 14. Asterisks denote statistical significance from vehicle-treated rats. *P,0.01.
ASA (5 mg/kg) resulted in a residual thrombus that equaled
that found in the heparin group alone. BC2 at 3 or 6 mg/kg
bolus followed by 1 or 2 mg z kg21 z h21 effectively reduced
thrombus mass as compared with its vehicle control; in fact,
the residual thrombus mass at the highest BC2 dose was
negligible. Interestingly, the lowest dose of BC2, 1 mg/kg
bolus (a dose that had no significant effect when given alone),
when combined with ASA (5 mg/kg, a dose of no effect per
se) resulted in complete abolishment of thrombus formation.
Figure 6 demonstrates the incidence of occlusion (%), as
determined by the absence of measurable blood flow, of the
Figure 7. Effect of heparin, ASA, or BC2 on aPTT and PT in the
pretreatment study. V represents saline vehicle-treated rats; H15
to H120, heparin-treated rats at 15, 30, 60, or 120 U/kg bolus
followed by 0.5, 1, 2, or 4 U z kg21 z min21 infusion over the
duration of the study. ASA indicates aspirin, 5 mg/kg, IV bolus
15 minutes before injury; BC2 Veh, Tris-buffered saline treated
rats. BC2-1 to BC2-6 denotes BC2 treatment at 1, 3, or 6
mg/kg followed by infusion of 0.3, 1, or 2 mg z kg21 z h21 over
the duration of the study. Number of rats56 to 14. Asterisks
denote statistical significance from vehicle-treated rats.
*P,0.01. A, Measurement of ex vivo aPTT. The dotted line
depicts a ratio of aPTT extension of 3.5-fold over baseline. B,
Measurement of ex vivo PT.
Figure 6. Effect of heparin, ASA, or BC2 on incidence of vessel
occlusion. V represents saline vehicle-treated rats; H15 to H120,
heparin-treated rats at 15, 30, 60, or 120 U/kg bolus followed
by 0.5, 1, 2, or 4 U z kg21 z min21 infusion over the duration of
the study. ASA indicates aspirin, 5 mg/kg, IV bolus 15 minutes
before injury; BC2 Veh, Tris-buffered saline treated rats. BC2-1
to BC2-6 denotes BC2 treatment at 1, 3, or 6 mg/kg followed
by infusion of 0.3, 1, or 2 mg z kg21 z h21 over the duration of
the study. Numbers on dashed line above respective bars represent percent occlusion of the vessels at the end of the study.
Number of rats56 to 14. Asterisks denote statistical significance
from vehicle-treated rats. *P,0.01.
carotid arteries in the various treatment groups. Heparin
treatment resulted in reduction of the incidence of vessel
occlusion from 100% in the vehicle group to no occlusion at
the highest dose. ASA alone had a tendency to reduce the
incidence of occlusion (not significant), whereas ASA and
heparin (30 U/kg11 U z kg21 z min21), which had no significant effect when administered alone, significantly reduced
the incidence of vessel occlusion. BC2 at the medium and
higher doses, as well as the combination of the lowest dose
and ASA, completely prevented vessel occlusion.
Figure 7A shows the effect of the various pretreatments on
the aPTT, which was performed with a limitation of 1000
seconds. A dashed line denoting a 3.5-fold increase over
baseline aPTT is depicted. Heparin dose-dependently increased aPTT; the 2 highest doses rendered the plasma
incoagulable as the aPTT reached 1000 seconds in every
animal. The medium dose (H30) that failed to maintain vessel
patency in the majority of animals extended aPTT 10-fold
over baseline. ASA had no effect on aPTT but potentiated the
extension of the aPTT by the medium dose of heparin (H30).
BC2 at 3 or 6 mg/kg doses extended aPTT but even at the
highest doses did not exceed a 3.5-fold prolongation of aPTT;
interestingly, in contrast to the synergy ASA had on heparin
Feuerstein et al
Monoclonal Antibodies to FIX/FIXa Prevent Thrombosis
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Figure 8. Effect of single bolus injection of BC2 versus heparin
infusion administered before carotid injury on thrombus mass,
carotid artery patency time, incidence of occlusion, and aPTT.
BC2 was administered as a single 3 mg/kg bolus IV Heparin
was administered as 60 U/kg bolus followed by infusion at 2
U z kg21 z min21. A, Thrombus mass; B, carotid patency time
and incidence of occlusion (depicted as % over the respective
bar); and C, aPTT (dashed bar denotes 3.5-fold ratio over baseline). Number of rats56 to 14. Asterisks denote statistical significance compared with vehicle control. *P,0.01 and **P,0.001.
extension of aPTT, no such augmentation was observed when
BC2 was administered with ASA.
Figure 7B depicts the effect of the various treatments on
PT. Neither ASA nor BC2 treatment regimens extended PT.
All doses of heparin, and especially the 2 highest doses,
significantly prolonged PT.
Effect of BC2 Administered as a Single Bolus
Before Carotid Lesion on Thrombus Weight,
Incidence of Vessel Occlusion, and Patency Time
and aPTT
Comparison between a single bolus injection of BC2 with
heparin infusion at a dose that produced maximal extension
of aPTT (Figure 8) demonstrates that both treatments resulted
in similar efficacy in reducing thrombus mass and extension
of vessel perfusion time; BC2 was somewhat more effective
in preventing vessel occlusion (10% compared with 27% in
the heparin group) (Figure 8B). A marked difference was
clearly noted in the effect of BC2 versus heparin on aPTT
(Figure 8C); BC2 increased aPTT by ,3.5-fold, whereas
heparin rendered the plasma incoagulable (aPTT.1000
seconds).
Effect of Murine IgG Administration on aPTT,
PT, and Blood Loss of Anesthetized Rats
To control for possible effects of murine immunoglobulin on
hemostatic parameters of rats, a bolus dose of murine IgG at
6 mg/kg was administered IV as per the protocol described
for pretreatment studies. Figure 9 demonstrates that a 6
2559
Figure 9. Effect of murine immunoglobulin on hemostatic parameters. Murine IgG was administered to rats (n56) as a 6
mg/kg bolus before FeCl3 injury of the carotid artery. At the end
of the 60-minute protocol blood samples were taken for measurement of aPTT and PT and thrombus mass was determined.
A, Thrombus weight; B, time to occlusion; C, aPTT (left panel)
and PT (right panel).
mg/kg, IV bolus dose of a murine IgG, corresponding to the
highest dose of BC2, does not result in alteration of thrombus
weight, time to occlusion, aPTT, or PT.
Effect of BC2 or Heparin Administered After
Completion of Carotid Injury on Thrombus Mass,
Vessel Patency Time, Incidence of Carotid Artery
Occlusion, aPTT and PT
Figures 10A and 10B compare the effect of BC2 or heparin
administered after the completion of the FeCl3 patch injury,
on thrombus mass, incidence of vessel occlusion, and carotid
artery patency time. In vehicle-treated control rats, complete
vessel occlusion occurs approximately 5 minutes after the end
of the injury period. BC2 administered as a single bolus (3
mg/kg) significantly inhibited thrombus formation in the
vessel and reduced the occlusion rate by 67%. Furthermore,
vessel patency was extended to almost the complete duration
of the experiment (60 minutes). A dose-response study of
heparin administered after the completion of the injury
demonstrated that heparin treatment also reduced thrombus
mass and increased vessel patency time and occlusion frequency, as demonstrated in the pretreatment experiments.
Whereas BC2 prolonged the aPTT to 64 seconds, heparin (at
all doses) dramatically extended the aPTT (600 to 1000
seconds) (Figure 11A). Furthermore, at the 2 highest doses of
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Figure 10. Effect of BC2 single bolus or heparin infusion on
thrombus mass, incidence of occlusions, and carotid patency
time in post-injury treatment study. Rats were treated with BC2
(3 mg/kg single IV bolus) or heparin (30, 60, 120, or 240 U/kg
bolus plus 1, 2, 4, or 8 U z kg21 z min21 infusion) at the end of
the 10 minute FeCl3 patch injury period. A, Thrombus mass at
the end of the 60-minute protocol. Values above the dashed
horizontal line represents incidence of vessel occlusion. B,
Carotid patency time. Number of rats56 to 14. Asterisks denote
statistical significance compared with vehicle control. *P,0.01,
**P,0.001.
heparin, a significant extension of PT was also observed
(Figure 11B). In marked contrast, BC2 (3 mg/kg) had no
effect on PT.
Effect of BC2 or Heparin on Blood Loss from
Surgical Cut
The effect of BC2 (3 mg/kg, IV bolus) or heparin (doseresponse) on blood loss is illustrated in Figure 12. Blood
volume was monitored over a 15-minute period by free flow
after the completion of experimental protocol, ie, 60 minutes
post injury. BC2 treatment did not result in an increase in
blood loss as compared with vehicle control. In contrast,
heparin treatments (60 to 240 U/kg bolus12 to 8 U z kg21 z
min21) resulted in a significant increase in blood loss over the
prespecified experimental period. The dose of heparin that
did not result in increased blood loss also failed to reduce
thrombus mass or diminish the incidence of vessel occlusion
(see Figure 10A).
To confirm that BC2 administration as a bolus followed by
infusion does not result in excessive blood loss, we have
repeated the experiment vide supra using a bolus treatment (2
mg/kg) followed by infusion of 1 mg z kg21 z h21 throughout
the experimental period. As depicted in Figure 13, the
extended infusion paradigm has not resulted in blood loss
over that in the vehicle-treated rats, although aPTT was
significantly extended in accord with previous data with
bolus dose of BC2 (see Figures 8 and 11).
Figure 11. Effect of BC2, single bolus, or heparin infusion on
aPTT and PT in the post-injury treatment protocol. Rats were
treated with BC2 (3 mg/kg single IV bolus) or heparin (30, 60,
120, or 240 U/kg bolus plus 1, 2, 4, or 8 U z kg21 z min21 infusion) at the end of the 10-minute FeCl3 patch injury period. A,
aPTT determined at the end of the 60-minute protocol; B, PT.
Number of rats56 to 14. Asterisks denote statistical significance
compared with vehicle control. *P,0.01, **P,0.001.
Effect of BC2 on FIX Activity
Because aPTT may not linearly relate to FIX activity, we
have explored the effect of BC2 on FIX activity versus aPTT
extension in the same experiment. BC2 was dosed in vivo and
FIX activity and aPTT monitored ex vivo. Figure 14 indicates
that significant reduction in Factor IX activity is associated
with minor change in aPTT and almost complete reduction in
FIX (.90%) extends aPTT by approximately 3.5- to 4-fold.
In the context of the efficacy studies, significant blockade of
thrombus formation and 90% reduction of vessel occlusion
Figure 12. Effect of BC2, single bolus, or heparin infusion on
blood loss from surgical wound in the post-injury protocol.
Blood loss after a surgical cut to the rat tail was performed as
described under Methods. Rats were treated with BC2 (3 mg/kg
single IV bolus) or heparin (30, 60, 120, or 240 U/kg bolus plus
1, 2, 4, or 8 U z kg21 z min21 infusion) at the end of the
10-minute FeCl3 patch injury period. Number of rats56 to 14.
Asterisks denote statistical significance compared with vehicle
control. *P,0.01, **P,0.001.
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Monoclonal Antibodies to FIX/FIXa Prevent Thrombosis
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Figure 14. Effect of BC2 on factor IX activity. Fifteen minutes
after rats (n56) were treated with BC2 at the indicated concentrations, blood samples were taken for measurement of FIX
activity (A) and aPTT (B). Activity measurements used factor IX
deficient human plasma and are expressed relative to a standard curve obtained with human reference plasma. Asterisks
denote statistical significance compared with vehicle control.
*P,0.05, **P,0.001.
Figure 13. Effect of BC2 bolus plus infusion on blood loss,
aPTT, and PT. Rats (n56) were treated with BC2 (2 mg/kg bolus
plus infusion of 1 mg z kg21 z h21). After a 1-hour treatment
period blood loss was measured with the surgical tail cut
method described under Methods. A, Blood loss; B, aPTT; and
C, PT. Asterisks denote statistical significance compared with
vehicle control. *P,0.01.
incidence can be achieved with 3 mg/kg doses of BC2, which
decrease FIX activity levels to approximately 50% of the
normal levels.
Effect of Human FIX on BC2-Induced aPTT
Extension In Vivo
To assess the capacity of human FIX (hFIX) to serve as an
antidote to BC2, rats were administered BC2 and aPTT
monitored 30 minutes later. At that time, rats were injected
with human FIX (3 or 5 mg/kg, IV bolus) or vehicle and
aPTT assayed 30 minutes later. Figure 15 depicts extension of
aPTT from 17.660.3 to 45.262.3 seconds and 44.861.6
seconds (P,0.001) at 30 and 60 minutes post BC2, respectively. Human FIX dose-dependently reduced aPTT toward
baseline, although incompletely (aPTT at 60 minutes was
24.760.5 seconds after injection of 5 mg/kg hFIX).
mapping studies will be required to define the specific
sequence recognized by BC2. The observation that BC2
inhibits rat FIX enabled the development of an extensive
portfolio of in vivo animal pharmacology.
In vivo efficacy of BC2 was demonstrated in a standard
arterial thrombosis model in rats where severe vessel wall
(and endothelial cells) injury is rapidly induced by Fe13mediated oxygen radical formation.22 This has been clearly
confirmed in our study by performing SEM and rapid and
complete occlusion of the vessel as directly measured arterial
blood flow in the carotid artery. BC2 was most efficacious in
blocking thrombus formation when administered either before or shortly after the injury to the vessel. The effects of
Discussion
The present experiments describe the in vitro and in vivo
anticoagulant and antithrombotic properties of BC2, a novel
antihuman FIX monoclonal antibody that blocks the coagulant activity of FIX and FIXa. Initial studies indicate that BC2
binds to the Gla domain of human FIX, and the observation
that BC2 cross-reacts with rat FIX suggests that the epitope is
conserved between human and rat FIX. More detailed epitope
Figure 15. Effect of human FIX on BC2-induced increase in
aPTT. Anesthetized rats (n56) were administered 3 mg/kg BC2,
bolus IV. After 30 minutes the rats were injected with vehicle
(hFIX V) or human FIX (3 or 5 mg/kg). After an additional 30 minutes, blood was sampled for measurement of aPTT. Asterisks
denote significance difference from vehicle-treated, BC2 injected
rats. **P,0.001.
2562
Arterioscler Thromb Vasc Biol.
October 1999
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BC2 were dose-dependent and showed potential synergy with
ASA (the latter had little effect in blocking thrombosis when
administered on its own in this model). The antithrombotic
efficacy of BC2 was reflected by thrombus mass reduction,
increased perfusion time, and reduced incidence of vessel
occlusion. In both preinjury or postinjury protocols, BC2
efficacy equaled or exceeded that of heparin, even when the
latter was provided at very high doses. However, although
heparin extended aPTT and PT and increased (doubled) the
volume of blood loss after a surgical cut, BC2 had no effect
on PT nor did BC2 treatment result in increased blood loss
from the surgical wound. Indeed, complete protection against
thrombosis was only achieved at heparin doses that rendered
the plasma incoagulable (aPTT.1000 seconds). Most interesting, BC2 extension of aPTT ex vivo was limited to 60 to
70 seconds, which was similar to its plateau effect in vitro.
Finally, the specificity of BC2 in blocking FIX and the
reversibility of anti-FIX antibody therapy was proven by the
administration of its antidote, FIX. Indeed, an almost 3-fold
extension of the aPTT induced by BC2 infusion in rats was
rapidly and dose-dependently reversed by human FIX administered at the peak of BC2 effect. The reason for the
incomplete reversal of BC2-induced aPTT extension may be
the result of species specificity of human FIX interacting with
the rat proteins that comprise the factor X activating complex.
Despite the central location of FIX in the coagulation
cascade, where it is activated by tissue factor/factor VIIa
complex and by factor XIa24 and is the primary activator of
factor X on activated platelets,13 factor IX/IXa has received
little attention as a potential target for therapeutic intervention
in thrombotic disorders.25,26 Our studies with this novel
anti-FIX antibody clearly demonstrate the capacity of specific
inhibition of FIX to prevent acute arterial thrombosis. These
studies thus extend previous studies reporting antithrombotic
efficacy of FIXai in canine models of arterial thrombosis and
cardiopulmonary bypass surgery.17,27 Preliminary studies
conducted in our laboratory also demonstrate that treatment
with BC2 results in extended patency of the blood vessel;
thus a single bolus injection of BC2 not only increased the
incidence of vessel patency 60 minutes after BC2 administration but also 24 hours later at a time when aPTT was within
normal range. This phenomenon may reflect the capacity of
the vessel wall to initiate repair when coagulation is interrupted at FIX/IXa limiting the generation of factor Xa and
thrombin. Taken together, our studies with BC2, as well as
those with FIXai, demonstrate that efficacy in blocking
thrombosis can be achieved with limited prolongation of the
aPTT and with less resultant increase in bleeding compared
with heparin.
Acknowledgment
The authors wish to acknowledge the excellent technical contribution
of J. Vasko-Moser and S. Tirri for preparation of the manuscript.
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Antithrombotic Efficacy of a Novel Murine Antihuman Factor IX Antibody in Rats
Giora Z. Feuerstein, Arun Patel, John R. Toomey, Peter Bugelski, Andrew J. Nichols, William
R. Church, Richard Valocik, Paul Koster, Audrey Baker and Michael N. Blackburn
Arterioscler Thromb Vasc Biol. 1999;19:2554-2562
doi: 10.1161/01.ATV.19.10.2554
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