(Circulation. 1997;96:2339-2347.)
© 1997 American Heart Association, Inc.
Articles |
Inhibition of Arterial Thrombosis by Recombinant Annexin V in a Rabbit Carotid Artery Injury Model
From the Divisions of Hematology and Cardiology, Department of Internal Medicine, University of Texas Health Science Center, Houston.
Correspondence to Claude R. Benedict, MD, DPhil, Department of Internal Medicine, Division of Cardiology, MSB 6.039, University of Texas Medical School, 6431 Fannin St, Houston, TX 77030.
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Abstract |
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 Top Abstract IntroductionMethodsResultsDiscussionReferences |
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Background The procoagulant effect of anionic phospholipid may play a major role in the development of arterial thrombosis.
Methods and Results Annexin V, a
calcium-dependent anionic-phospholipid–binding protein, was
expressed and isolated from Escherichia coli and its
antithrombotic effect examined in a rabbit carotid artery thrombosis
model. A partially occlusive thrombus was formed in the left carotid
artery by application of electric current to produce an 
50%
occlusion of the lumen. After the current was discontinued, flow ceased
completely within 42±12 minutes (n=6) because of continuing
platelet/fibrin thrombus formation. When annexin V was given
at doses of 2.8 to 16.6
µg · kg-1 · min-1
for a period of 180 minutes, starting at the time the current was
stopped, there was a dose-dependent inhibition of thrombus formation.
At a dose of 5.6
µg · kg-1 · min-1,
blood flow remained patent throughout the infusion and for an
additional 60 minutes after the infusion was stopped. In addition,
there was a decrease in thrombus weight (16±7.4 versus 2.0±1.0 g),
125I-fibrin deposition (45% reduction,
P<.001), and 111In-labeled platelet
accumulation (43% reduction, P<.001). Prior mixing of
annexin V with phosphatidylserine micelles
abolished the antithrombotic effect of annexin V, whereas mixing with
phosphatidylcholine micelles had no effect. The antithrombotic effect
of annexin V was not associated with bleeding tendency, as judged by
the amount of blood absorbed in a gauze pad placed in a surgical
incision extending to the muscle tissue and by the standard template
bleeding time.
Conclusions These observations support a potentially important role for anionic phospholipid exposure in platelets in arterial thrombosis, and inhibition of this activity could be a novel target for therapy in coronary thrombosis and stroke and after angioplasty.
Key Words: annexins • thrombosis • phospholipids • hemodynamics
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Introduction |
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Thrombosis of arteries plays a key role in the pathogenesis of a variety of ischemic syndromes, including unstable angina, myocardial infarction, and stroke. Arterial thrombus formation usually involves elements of vessel wall damage and platelet activation and aggregation. In addition to forming occlusive aggregates, activated platelets provide a highly efficient catalytic surface for the activation of prothrombin and factor X. The precise components of these catalytic surfaces are not fully elucidated, but anionic phospholipid constitutes a major component for binding sites for proteases and cofactors.1 2 In platelets and in most other cells examined, anionic phospholipids are present only in the inner leaflet of the lipid bilayer membrane.3 After the activation of platelets by certain agonists, anionic phospholipids move from the inner to the outer leaflet of the lipid bilayer; this rearrangement of membrane phospholipid is thought to be a major mechanism responsible for platelet procoagulant activity.4 5 6 In addition, cultured endothelial cells have also been shown to provide a phospholipid-dependent procoagulant surface in vitro.7 8 Furthermore, cells undergoing apoptosis have been shown to expose anionic phospholipid on their surfaces.9 Thus, the exposure at the sites of vascular injury of anionic phospholipids from activated platelets and other cells may play a major role in the initiation, growth, and extension of the thrombus and may provide a novel target for antithrombotic therapy.
The annexins are a family of calcium-dependent anionic-phospholipid–binding proteins.10 A member of this family, annexin V, was originally isolated from placenta, characterized as placental anticoagulant protein I, and sequenced.11 12 The same protein was also isolated and sequenced under different names.13 14 Annexin V binds anionic phospholipids with a very high affinity in a calcium-dependent manner.15 We have shown previously that annexin V binds to anionic phospholipid on platelets and blocks the binding of factors Xa and Va to platelets.16 17 In addition, annexin V has been shown to be an effective inhibitor of thrombus formation in a venous thrombus model and in vitro perfusion models.18 19 20 In the present study, we examined the potential role of annexin V as an inhibitor of arterial thrombosis in a carotid artery injury model developed in our laboratory. Our results indicate that intravenous infusion of annexin V can significantly inhibit thrombosis in this model without impairing the hemostatic response, even at doses that are three times greater than that required to inhibit thrombus formation.
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Methods |
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Preparation of Human Recombinant Annexin V
The polymerase chain reaction was used to amplify the cDNA from the initiator methionine to the stop codon with specific oligonucleotide primers from a human placental cDNA library (Clontec). The forward primer was 5'ACCTGAGTAGTCGCCATGGCACAGGTTCTC-3' and the reverse primer was 5'-CCCGAATTCACGTTAGTCATCTTCTCCACAGAGCAG-3'. The amplified 1.1-kb fragment was digested with Nco I and EcoRI and ligated into the prokaryotic expression vector pTRC 99A (Pharmacia Biotechnology Inc). The ligation product was used to transform competent Escherichia coli strain JM 105 and sequenced. The sequence of the amplified segment was identical to that published by Funakoshi et al.12
Recombinant annexin V was isolated from the bacterial lysates as described by Berger et al21 with some modification. An overnight culture of E. coli JM 105 transformed with pTRC 99A–annexin V was expanded 50-fold in fresh Luria-Bertani medium containing 100 mg/L ampicillin. After 2 hours, isopropyl ß-D-thiogalactopyranoside was added to a final concentration of 1 mmol/L. After 16 hours of induction, the bacteria were pelleted at 3500g for 15 minutes at 4°C. The bacterial pellet was suspended in TBS, pH 7.5, containing 1 mmol/L PMSF, 5 mmol/L EDTA, and 6 mol/L urea. The bacterial suspension was sonicated with an ultrasonic probe (model W185, Heat System-Ultrasonic, Inc) at a setting of 6 on ice for 3 minutes. The lysate was centrifuged at 10 000g for 15 minutes, and the supernatant was dialyzed twice against 50 vol TBS containing 1 mmol/L EDTA and once against 50 vol TBS.
Multilamellar liposomes were prepared according to the method of
Kinsky.22 PS (Sigma Chemical Co), lyophilized bovine brain
extract, cholesterol, and diacetylphosphate were dissolved
in chloroform in a molar ratio of 10:15:1 and dried in a stream of
nitrogen in a conical flask. TBS (5 mL) was added to the flask and
agitated vigorously in a vortex mixer for 1 minute. The liposomes were
washed by centrifugation at 3500g for 15
minutes, then incubated with the bacterial extract, and calcium
chloride was added to a final concentration of 5 mmol/L.
After 15 minutes of incubation at 37°C, the liposomes were sedimented
by centrifugation at 10 000g for 10
minutes, and the bound annexin V was eluted with 10 mmol/L
EDTA. The eluted annexin V was concentrated by Amicon ultrafiltration
and loaded onto a Sephacryl S 200 column (5x90 cm). The annexin V was
recovered in the included volume, whereas most of the liposomes were in
the void volume. Fractions containing annexin V were pooled and
dialyzed in 10 mmol/L Tris and 2 mmol/L EDTA,
pH 8.1, loaded onto an anion exchange column (Resource Q, Pharmacia
Biotechnology Inc), and eluted with a linear gradient of 0 to 200
mmol/L NaCl in the same buffer. The purified preparation showed
a single band in SDS-PAGE under reducing conditions (Fig 1
). For rabbit experiments, the annexin V
was dialyzed against HBS (10 mmol/L HEPES, 0.15
mol/L NaCl, pH 7.4) and sterile filtered with 0.2-µm
filters.
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Unilamellar micelles of PS were prepared as described
before.23 Purified phospholipids were obtained from Avanti
Polar Phospholipids, Inc. These micelles contained 20 mol%
diheptanoylphosphatidylcholine and 80%
dioleylphosphatidylserine (PS micelles) or
dipalmitoylphosphatidylcholine (PC micelles). Equal volumes of annexin
V (1 mg/mL) and phospholipid micelles (0.5 mg/mL) were
mixed immediately before administration to the animals. For
radiolabeled studies, annexin V was labeled with
[125I]NaI by the Iodo-Gen method to a specific activity
of 350 cpm/ng as described previously.24
Rabbit Model of Carotid Artery Thrombosis
Carotid artery thrombosis was induced with electric current as
described previously.25 26 27 28 Male New Zealand White rabbits
weighing 3.2 to 3.6 kg were anesthetized with ketamine
(15 mg/kg) and xylazine (15 mg/kg). The right femoral
artery was cannulated for recording of arterial
blood pressure with a microtransducer (Electromedics). The right
marginal ear vein was cannulated for administration of fluids and
drugs. The right femoral vein was cannulated for drawing blood samples.
The left common carotid artery was exposed by a median longitudinal
incision in the neck, and a 2.5-mm Doppler flow probe was placed on
the carotid artery without constricting the vessel. Proximal to the
Doppler flow probe, a 4-mm-long, 23-gauge stainless steel needle
electrode was inserted into the lumen of the carotid artery with
minimal trauma. This electrode was positioned within the lumen parallel
to the vessel wall. The bleeding was arrested by a piece of gel foam
(Upjohn), and the needle was stabilized by a "surround collar"
sutured around the vessel, which did not narrow the artery. After
instrumentation, a 30-minute control period was allowed. During this
time, blood pressure, heart rate, mean and phasic carotid artery blood
flow, and ECG were continuously monitored. After this control period,
thrombus formation was initiated by 150 µA of anodal current applied
to the needle electrode until a 50% increase in flow velocity was
recorded by the Doppler flow probe. This corresponds to an
50% decrease in cross-sectional area due to thrombus formation in
the lumen.25 To assess the degree and the variability of
carotid occlusion at this point, a separate group of 16 rabbits were
similarly instrumented, and current was applied until the carotid
artery flow velocity increased by 50%. At this time, current was
stopped and the carotid artery pressure was fixed at
arterial pressure by buffered
glutaraldehyde infused through a perfusion catheter
that was placed in the left common carotid artery proximal to the site
of thrombus formation. After fixation, the vessel was paraffin-embedded
and sectioned at 0.5-mm intervals. Twelve sections were made from each
vessel, starting at the site of needle insertion and moving distally.
After the sections were stained, planimetry was used to calculate the
mean percentage of vessel occlusion by thrombus from the section that
was most narrowed by thrombus in each vessel.
In initial studies, 4 rabbits were infused with annexin V (5.6
µg · kg-1 · min-1
IV) for 180 minutes without thrombus formation to determine whether
annexin V had any inherent hemodynamic effects. After
it was determined that annexin V did not any have direct effects,
thrombus formation studies were done. At the time of 50% increase in
flow velocity (50% occlusion of the vessel), the current was
discontinued and rabbits were randomly allocated into 1 of 11 different
treatment groups (see the Table) as
follows: group 1, excipient (HBS), n=6; group 2, annexin V 2.8
µg ·
kg-1 · min-1 for
180 minutes, n=6; group 3, annexin V 4.2
µg · kg-1 · min-1
for 180 minutes, n=6; group 4, annexin V 5.6
µg · kg-1 · min-1
for 180 minutes, n=6; group 5, a mixture of annexin V and PS micelles
(5.6
µg · kg-1 · min-1
of annexin V+2.8
µg · kg-1 · min-1
PS micelle) for 180 minutes, n=7; group 6, PS micelles 2.8
µg · kg-1 ·
min-1 for 180 minutes, n=7; group 7, a mixture
of annexin V and PC micelles (5.6
µg · kg-1 · min-1
of annexin V+2.8
µg · kg-1 · min-1
PC micelle) for 180 minutes, n=6; group 8, PC micelles 2.8
µg · kg-1 · min-1
for 180 minutes, n=4; group 9, heparin 35 U/kg as an IV bolus followed
by 0.5
U · kg-1 · min-1
for 180 minutes, n=6; group 10, heparin 35 U/kg as an IV bolus followed
by 0.25
U · kg-1 · min-1
for 180 minutes, n=5; and group 11, monoclonal antibody against rabbit
platelet IIb/IIIa receptor (0.5 mg/kg IV; AZ-1; gift from
Michael Ezekowitz, MD, Yale University Medical School, New Haven,
Conn), n=7. Where indicated, animals also received
125I-fibrinogen (3 µCi in 1 mL of saline IV) at the time
the current was stopped or 111In-labeled platelets as
described below. Fibrinogen was purified and radioiodinated
as described previously to a specific radioactivity of 130
µCi/mg.24 29
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111In-Labeling of Platelets
Before instrumentation of the animals, blood was collected to
label platelets with 111In-8-hydroxyquinoline (oxine)
as described previously.28 29 30 Blood (21 mL) was collected
into 4 mL of acid citrate dextrose and prostacyclin (10 µg/25
mL of blood), mixed, and centrifuged at 125g for 20
minutes at room temperature. The platelet-rich plasma was
centrifuged at 1100g for 5 minutes and the
platelet-poor plasma removed. The platelet pellet was suspended
in 300 µL of platelet-poor plasma for labeling.
[111In]InCl3 (Amersham Corp) was prepared in
0.3 mol/L acetate buffer, pH 5.3, to which 50 µL of oxine in
ethanol (1 mg/mL) was added. After 15 minutes, the reaction
mixture was extracted twice with 2 mL of methylene chloride and dried,
and the residue was dissolved in 30 µL of absolute ethanol.
Approximately 80% to 95% of the original radioactivity was recovered.
111In-oxine (250 to 300 µCi) was then added to
platelet suspensions for 30 minutes at 37°C, the mixture was
centrifuged at 1100g for 5 minutes to remove the
supernatant plasma, and the platelet pellet was resuspended in 1 mL
of autologous plasma. The labeling efficiency was in the range of 50%
to 80%. The in vivo viability of labeled platelets was determined
by calculating the percentage of administered radioactivity bound to
circulating platelets at different time intervals. At 2, 5, 10, 30,
60, and 120 minutes after the administration of
111In-labeled platelets, a 1-mL blood sample was
collected, platelets were isolated as described above, and
radioactivity was counted in both the platelet pellet and
platelet-poor plasma. The percentage recovery of the radiolabel,
calculated as described before,29 was found to be 80% to
90% in the platelet pellet.
Determination of the Accumulation of
125I-Fibrinogen/Fibrin and 111In-Labeled
Platelets
Deposition of 125I-fibrinogen/fibrin into carotid
vessel segments was quantified as previously
described.26 27 28 In animals infused with
125I-fibrinogen, either at the time of vessel occlusion or
60 minutes after the infusion was stopped, the left and the right
carotid arteries were carefully removed and freed of all the
surrounding fibrous tissue (carotid sheath). Then the left carotid
artery was weighed and divided into three 2-cm segments: just proximal
to the needle electrode insertion site into the vessel lumen; the site
of thrombus formation, which corresponded to the position of the
needle; and distal to the thrombus. Each segment was weighed,
radioactivity was determined, and the counts were normalized according
to the weight of the segment. Then the right carotid artery was weighed
and, if necessary, trimmed so that its weight was the same as that of
the left carotid artery, and radioactivity was determined. Accumulated
radioactivity in the left carotid artery segments was expressed as a
ratio of that measured from the uninstrumented right carotid
artery.
Radiolabeled platelets were reinjected into the rabbits when the current was discontinued. At the end of the study (60 minutes after total occlusion or when the vessel remained patent for 60 minutes after the infusion of drugs was stopped), the carotid artery was removed and the 111In-labeled platelet accumulation ratio was determined as described for the 125I-labeled fibrinogen/fibrin accumulation ratio.
Rabbit Bleeding Assays
We evaluated the homeostatic parameters by two
different methods. Template bleeding times were measured with the
Simplate device (Organon Teknika). Uniform incisions 10 mm long
and 1 mm deep were made on the ventral surface of the rabbit's
ear in such a way as to avoid the superficial veins. Blood was blotted
with filter paper (Whatman No. 4) every 30 seconds, avoiding the
incision. Bleeding time was defined as the interval between the time of
incision until blood did not stain the paper.
The incisional bleeding assay was a modification of previously published methods.19 20 A surgical incision 4 cm long and 1 cm deep was made in the anterior abdominal wall, which incised the first layer of the anterior abdominal wall muscles. A preweighed gauze pad was placed in the incision for 5 minutes, and the amount of blood absorbed into the gauze was weighed. Both of the bleeding assays were performed before administration of annexin V or heparin (baseline) and then 15, 60, 180, and 300 minutes after administration of the test samples.
Tissue and Plasma Concentrations of Annexin V
To examine the tissue distribution and in vivo clearance of
annexin V, 125I-labeled annexin V (1.5 µCi, specific
activity 350 cpm/ng) was given intravenously to rabbits
after the current was stopped, and 1-mL venous blood samples were
collected at various times (0 to 120 minutes) (n=3). At the end of the
study, the animals were killed by exsanguination under anesthesia, and
the amount of radioactivity was counted in liver, spleen, right kidney,
left kidney, brain, left ventricle (heart), aorta, left lung, right
lung, normal proximal uninvolved segment of the left common carotid
artery, the thrombosed segment of the same vessel, and the carotid
artery thrombus. The radioactivity was expressed as cpm/mg wet tissue
wt.
Assessment of Coagulation and Bleeding Parameters With
Annexin V Infusion
To determine the potential of annexin V to induce coagulation
and bleeding abnormalities, four incremental doses of annexin V (2.8,
4.2, 8.3, and 16.7
µg · kg-1 · min-1
IV) were given by continuous infusion for 60 minutes (n=4; each animal
received all four doses in ascending order over a 4-hour period), and
aPTT, bleeding time, and incisional bleeding were assessed at the end
of each dose before the next higher dose was begun.
Statistical Analysis
Data were analyzed by one-way ANOVA. In each figure, in
the text, and in the Table
, mean values±SD are shown.
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Results |
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Hemodynamic Effects of Annexin V
Intravenous infusion of annexin V into the instrumented rabbit model, without the presence of thrombosis, for 180 minutes (5.6 µg · kg-1 · min-1) (n=4) did not alter the blood pressure (pretreatment, 112±11 mm Hg versus 3 hours after infusion, 106±9 mm Hg), heart rate (pretreatment, 94±12 bpm versus 3 hours after infusion, 98±14 bpm), or carotid artery blood flow (pretreatment, 18.4±4.9 mL/min versus 3 hours after infusion, 19.2±3.8 mL/min). Thus, annexin V was devoid of any inherent hemodynamic effects detectable by these methods.
Time to Coronary Occlusion
Application of current to the left carotid artery for 47±21
minutes increased the flow velocity by 50%, as observed
previously.28 In the separate group of 16 animals, the
examination of the carotid artery at this time point showed a partially
occlusive thrombus that occupied 48.6±4% of the vessel lumen. The
scanning electron microscopic appearance of the partially occlusive
thrombus is shown in Fig 2A, which
demonstrates a predominantly platelet-covered surface for the
formed thrombus and underlying fibrin scaffolding.25 26
When the thrombus was removed, the underlying area of the carotid
artery closest to the electrode showed endothelial
removal and exposure of the subendothelial matrix (Fig 2B). When the current is discontinued at 50% occlusion, there is
progressive thrombus growth with complete occlusion of blood flow
within 42±12 minutes (Figs 3, top, and
4A and Table).
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When annexin V (5.6
µg · kg-1 · min-1)
was given, starting at the time when current was discontinued, blood
flow remained unchanged throughout the infusion and then for an
additional 60 minutes after the infusion was stopped (total duration of
maintenance of blood flow, >240 minutes) (Figs 3, bottom, and
4A and Table). Note that the phasic carotid artery blood flow has a
normal systolic and diastolic phasic blood flow
profile at the end of 240 minutes (Fig 3B). Because in the presence of
severe narrowing (high-grade stenosis) the phasic flow pattern
is gradually lost (flow is predominantly during systole), the
preservation of the normal carotid flow pattern by annexin V indicates
that there was no further narrowing of the vessel by ongoing thrombus
formation. This observation is further supported by the fact that
examination of the vessels from animals infused with annexin V revealed
only small, nonocclusive thrombi containing fibrin and platelets
compared with occlusive thrombi of the same apparent
histological composition in control animals infused
with HBS (Fig 4B). Hemodynamic variables
(arterial blood pressure, heart rate, and phasic and mean
carotid blood flow) were not affected by the infusion of annexin V.
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Annexin V inhibited thrombus formation in a dose-dependent manner,
being effective in all animals receiving 5.6
µg · kg-1 · min-1
but ineffective at a dose of 2.8
µg · kg-1 · min-1
(Fig 4A and Table). When animals were anticoagulated with heparin (35
U/kg as an IV bolus followed by 0.5
U · kg-1 · min-1),
the left carotid artery also remained patent (5 of 6 animals), although
lower levels of heparin (35 U/kg as an IV bolus followed by 0.25
U · kg-1 · min-1)
did not prevent carotid occlusion (Fig 4A and Table). Similarly, the
administration of monoclonal antibody to rabbit IIb/IIIa receptor (0.5
mg/kg IV) prevented carotid artery thrombosis (5 of 7 animals)
(Table). However, there was a significant reduction in platelet
counts after the administration of the antibody (before,
264±32x105/mm3 versus 4 hours after,
92±18x105/mm3, P<.001; 4 hours
after without antibody, 214±46x105/mm3,
P=NS). After the administration of monoclonal antibody to
IIb/IIIa, there was no significant increase in bleeding for the first 1
to 2 hours. After 3 to 4 hours, however, a marked increase in bleeding
at incisional sites, as well as significant blood loss by the
incisional method, was observed (data not shown). Lower doses of the
monoclonal antibody were ineffective in preventing carotid artery
thrombosis.
We investigated the effect of PS micelles on the antithrombotic
activity of annexin V. When annexin V at a dose equivalent to 5.6
µg · kg-1 · min-1
for 180 minutes was mixed with PS micelle at a dose equivalent to 2.8
µg · kg-1 · min-1
for 180 minutes and then infused, the effect of annexin V was markedly
diminished (Fig 4A and Table). Infusion of the same dose of PS or PC
micelles (2.8
µg · kg-1 · min-1
for 180 minutes) had no significant effect compared with animals
receiving HBS (Fig 4A and Table).
Radiolabeled Fibrinogen Accumulation in Thrombus
Because annexin V would be expected to interfere primarily with
the platelet procoagulant mechanism ultimately leading to the
generation of fibrin, it was important to assess its effect on the
deposition of radiolabeled fibrinogen/fibrin in the thrombosed carotid
segment (Fig 5A). Animals were infused
with 125I-fibrinogen at the time the current was stopped,
and 125I-fibrinogen/fibrin accumulation was measured. The
accumulated fibrinogen/fibrin was expressed as the ratio of
radioactivity in the segment from the instrumented artery to that of
the radioactivity in the corresponding segment of the contralateral
nonmanipulated artery. In the presence of annexin V,
125I-fibrinogen/fibrin accumulation ratios decreased in the
thrombosed segments by 47% (from 42±4.2 [untreated] to 22.1±4.7
[treated], P<.001), whereas there was no difference in
the segment proximal or distal to the lesion.
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Platelet Accumulation in Thrombus
Because platelets are an important component of the thrombus
in the electrically induced carotid thrombosis model and
thrombin-induced platelet aggregation is thought to contribute
significantly to this process, it was important to determine the effect
of annexin V on platelet contribution to intravascular clotting in
this setting (Fig 5B). To compare platelet deposition in the
thrombosed, proximal, and distal segments of the left carotid artery, a
platelet accumulation index using 111In-labeled
platelets was used. The counts in the left and right carotid
segments were compared as for labeled fibrinogen. In animals treated
with annexin V, the platelet deposition indices decreased by
42% (from 76.1±6.9 [untreated] to 44.0±9.7 [treated],
P<.001) in the thrombosed segment of the carotid artery.
There were no significant changes in the platelet deposition
indexes observed in the proximal or distal carotid artery segments.
These data suggest that annexin V also caused a significant decrease in
platelet deposition into the thrombus. To verify the viability of
labeled platelets, we determined the 111In counts in
blood as a function of time after injection of labeled platelets
into the rabbits. Two minutes after injection, 95% of the injected
counts were present; at 5 minutes, 78%; at 10 minutes, 64%; and
at 30 minutes, 62%. No significant decrease in 111In
activity was noted after 30 minutes (80% to 90% of this circulating
radioactivity was associated with the platelets). This strongly
suggests that the damaged platelets are rapidly removed by spleen
or other reticular-endothelial cells and that the
remaining circulating 111In was localized in normal
platelets.
Clearance and Tissue Distribution of Annexin V
Clearance of annexin V was rapid, with an 
-phase of 5
minutes, at the end of which 10% to 12% radioactivity remains in
circulation (Fig 6A). The majority of the
annexin V was cleared through the kidneys (data not shown). Among the
vascular structures, the highest ratio of blood to tissue was found in
the thrombus (Fig 6B). After the thrombus was removed, the thrombosed
left common carotid artery segment containing the thrombus did not show
a significantly higher count compared with other vascular structures,
such as right carotid artery or aorta. We also infused
125I-labeled annexin V starting at the time of initiation
of current (initiation of thrombus formation) (n=3). The objective was
to determine whether annexin V would bind to the carotid
arterial wall if there was no thrombus to cover the area of
injury. After 180 minutes of infusion, the left carotid artery was
carefully removed and counted, and it did not show a significantly
higher count compared with the uninstrumented right carotid artery or
the aorta. These results indicate that annexin V concentrates
preferentially in the thrombus and not in the vessel wall.
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Alterations in Hemostatic and Bleeding Parameters
Infusion of annexin V results in the prolongation of aPTT in
rabbit plasma (Fig 7). The control aPTT
was 26.9±4.6 seconds; with 2.8
µg · kg-1 · min-1
of annexin V infusion, it was 34.7±2.7 seconds; with 4.2
µg · kg-1 ·
min-1, 37.5±7.0 seconds; with 8.4
µg · kg-1 · min-1,
37.7±4.1 seconds; and with 16.8
µg · kg-1 · min-1,
36.0±3.3 seconds. Although the prolongation of aPTT was statistically
significant (P<.05), the increasing doses of annexin V
infusion did not cause a significant further prolongation in aPTT,
unlike heparin infusion (27.4±2.2 seconds with 35 U/kg bolus+0.25
U · kg-1 · min-1
IV and 55.0±8 seconds with 35 U/kg bolus+0.5
U · kg-1 · min-1
IV) (Fig 7). The effect of annexin V on hemostatic response to a
cutaneous abdominal wound was assessed after a standardized incision by
weighing the amount of blood absorbed by a sponge placed in the wound
for 5 minutes (Fig 8A). Animals infused
with either saline, annexin V, or a mixture of annexin V and PC or PS
micelles did not bleed excessively compared with untreated controls.
Consistent with this observation, when abdominal incision
bleeding was measured, no difference in accumulated radioactivity in
the gauze pad was noted between annexin V–treated and control animals
that had been infused with 125I-fibrinogen. In contrast,
animals receiving heparin at levels required to prevent occlusive
carotid thrombosis (0.5
U · kg-1 · min-1)
had markedly increased bleeding. The dose of heparin used was then
steadily decreased until a level was reached at which there was only a
moderate increase in bleeding from the abdominal wound (0.25
U · kg-1 · min-1)
(Fig 8A). At this concentration of heparin, however, occlusion of the
left carotid artery occurred at time intervals similar to those noted
in saline-treated control animals (Table). The contrast between the
increased bleeding tendency of animals receiving heparin (35 U/kg bolus
followed by 0.5
U · kg-1 · min-1,
the amount of heparin required to prevent formation of an occlusive
thrombus) and the apparent lack of bleeding in animals treated with
annexin V was also qualitatively evident throughout the experimental
manipulations with regard to blood loss at incisions in the chest wall
and catheter insertion sites. Neither annexin V and nor heparin
produced a significant change in template bleeding times (Fig 8B).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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After vessel wall injury, platelets rapidly adhere to the damaged vessel wall and to one another to form the primary hemostatic plug. In addition, after activation, platelets accelerate the generation of thrombin by providing an effective catalytic surface for the conversion of prothrombin by factor Xa (in the presence of factor VIIIa and Ca2+) and factor X by factor IXa (in the presence of factor VIIIa and Ca2+).1 2 3 Both factor Va and factor VIIIa were shown to bind with high affinity to a limited number of binding sites in platelets,
1000 sites for factor Va
and 400 sites for factor VIIIa.31 32 33 34 The occupancy of
Va receptor is essential for the subsequent binding of factor Xa,
whereas binding of factor VIIIa, though not essential for factor IXa
binding, enhances the binding affinity of IXa by
fivefold.33 Platelet-bound factor Xa catalyzes the
activation of prothrombin 300 000 times
(Kcat/Km) faster than in
solution, and platelet-bound factor IXa catalyzes the activation of
factor X 17 000 000 times faster than in
solution.1 2 31 32 33 34 The nature of the binding site has not
been fully elucidated, but anionic phospholipid is required for
formation of this binding site. The importance of the exposure of anionic phospholipid for hemostasis in vivo is demonstrated in Scott syndrome, a rare bleeding disorder associated with deficiency of platelet procoagulant activity.35 The platelets in this disorder have decreased exposure of anionic phospholipid after platelet activation and have reduced factor Va-Xa and factor IXa binding sites.36 37 Whether the exposure of anionic phospholipid is also important in the pathogenesis of thrombosis associated with vessel wall injury is not known. The expression of platelet anionic phospholipids in vitro requires a stronger stimulus than that for the induction of aggregation and secretion.4 6 16 17 Such strong stimuli may be provided in vivo in arterial injury by the exposure of subendothelial components to the adhering platelets. Furthermore, the high fluid shear stress typically seen at these sites may be an additional stimulus, resulting in a strong activation. The exposure of anionic phospholipid by these stimuli may play a significant role in the growth of platelet-rich thrombi in arteries as their growth is stabilized by a fibrin scaffold.
Our results show that in a model of arterial thrombosis, anionic phospholipids play a significant role in thrombus formation. The intravenous infusion of annexin V significantly inhibited thrombus formation in a dose-dependent manner, as judged by maintenance of blood flow with normal systolic and diastolic phasic profiles and inhibition of fibrin deposition and platelet accumulation at the site of arterial injury. Most significantly, the prior mixing of annexin V with PS micelles attenuated the antithrombotic effect of annexin V, whereas prior mixing with PC micelles had no effect. These results suggest that mechanistically, the anticoagulant effect of annexin V is mediated by its interaction with PS, presumably on activated platelets. Annexin V has been shown to be selectively taken up in the thrombi in vivo.38 It is also possible that annexin V may have similar effects on endothelial or other cell types at the site of vascular injury.
One intriguing finding to emerge from these studies was the lack of hemostatic compromise observed in rabbits treated with annexin V, even at three times the concentration required to induce an antithrombotic effect. In contrast, the dose of heparin required to maintain vascular patency induced significant bleeding at extravascular sites. Although one must be careful not to overinterpret these results, if this finding is verified in other models of arterial thrombosis, it may provide important clues to the inherent differences between the mechanisms involved in hemostasis and those involved in thrombosis. The two methods used to evaluate hemostasis, the template bleeding time and the abdominal incision method, both evaluate bleeding from capillary sources. Occlusion by an arterial thrombus requires the continued recruitment and incorporation of platelets into the developing thrombus after the first wave of platelets has recognized the site of injury. These platelets are laid down on a scaffold of fibrin, which requires activation of a soluble coagulation system. In small capillaries, on the other hand, thrombus growth is much less crucial to achieve a thrombus mass sufficient to occlude the vessel. Thus, the procoagulant activity associated with platelets may not be required. Another possible explanation for the disparity between the antithrombotic effect and the antihemostatic effect of fibrin is the observation that annexin V does not inhibit the activity of the tissue factor VIIa complex as effectively as it inhibits prothrombinase activity.39 In extravascular tissues, the tissue factor–initiated clotting mechanisms may be less amenable to inhibition. Finally, the differential effect can also be due to the most obvious differences between capillaries and arteries: the ratio of endothelial surface to blood volume. In capillaries, where this ratio is large, the altered endothelial surface may suffice to bring about the arrest of bleeding, whereas in large arteries, where this ratio is small, platelet procoagulant activity may have a predominant role. In any event, our data suggest that the platelet procoagulant activity is not as important for capillary hemostasis as for arterial thrombosis. Of interest, the Ms Scott for whom the Scott syndrome was named has a normal bleeding time.35
In conclusion, our experiments show that the inhibition of procoagulant activity of platelets by annexin V can attenuate experimentally induced arterial thrombosis in the absence of excessive bleeding. These observations lend support to the notion that anionic phospholipid exposure in platelets has an important role in arterial thrombosis. Inhibition of this activity may be a novel target for antithrombotic therapy in conditions associated with arterial injury, such as acute myocardial infarction and stroke, and after angioplasty.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by NIH grants HL-50653, HL-40860, and HL-50100 and by a Grant-in-Aid (93-14960) from the American Heart Association. We are grateful for the technical assistance of Gerald Todd and Anhquyen Le. Special thanks go to Dr Jose Lopez for the critical review of the manuscript and suggestions.
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Footnotes |
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Guest editor for this article was James H. Chesebro, MD, The Cardiovascular Institute, New York, NY.
Presented in part at the 68th Scientific Sessions of the American Heart Association, Anaheim, Calif, November 13-16, 1995, and published in abstract form (Circulation. 1995;92[suppl I]:I-805).
Received December 10, 1996; revision received April 14, 1997; accepted May 19, 1997.
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