From the Departments of Internal Medicine (P.M.F., W.P.F.) and Surgery
(C.K.O.), University of Michigan Medical School, Ann Arbor; the Ann Arbor
Veterans Affairs Hospital (W.P.F.); and the Center for Molecular and Vascular
Biology (P.C.), University of Leuven (Belgium).
Methods and Results—We developed a murine arterial
injury model and applied it to wild-type (PAI-1 +/+) and
PAI-1–deficient (PAI-1 -/-) animals.
FeCl3 was used to induce carotid artery thrombosis. Thrombi
consisted predominantly of dense platelet aggregates,
consistent with the histology of thrombi in large-animal
arterial injury models and human acute coronary
syndromes. To examine the role of PAI-1 in regulating
endogenous clearance of platelet-rich
arterial thrombi, thrombi were induced in 22
PAI-1 +/+ mice 14 PAI-1 -/- mice.
Twenty-four hours later, the amount of residual thrombus was determined
by histological analysis of multiple transverse
sections of each artery. Residual thrombus was detected in 55 of 85
sections (64.7%) obtained from PAI-1 +/+ mice compared
with 19 of 56 sections (33.9%) from PAI-1 -/- mice
(P=.009). Computer-assisted planimetry analysis
revealed that mean thrombus cross-sectional area was 0.033±0.027
mm2 in PAI-1 +/+ mice versus
0.016±0.015 mm2 in PAI-1 -/- mice
(P=.048).
Conclusions—PAI-1 is an important determinant of
thrombolysis at sites of arterial injury.
Application of this model to other genetically altered mice should
prove useful for studying the molecular determinants of
arterial thrombosis and thrombolysis.
Thrombosis Protocol
Histological and Morphometric Analyses
Platelet Aggregation Studies
Statistical Analyses
Application of Carotid Injury Model to PAI-1–Deficient
Mice
The significant difference in residual thrombus observed between
wild-type and PAI-1–deficient mice 24 hours after injury could be
explained by reduced thrombus formation and/or by enhanced
thrombolysis in PAI-1 -/- mice. To address
this issue, we studied thrombus generation after vascular injury in
PAI-1 +/+ (n=8) and PAI-1 -/- (n=4) mice. Mice
were subjected to arterial injury with 10%
FeCl3 for 3 minutes. Thereafter, a miniature
Doppler flow probe (model 0.5VB, Transonic Systems) that interfaced
with a Transonic model T106 flowmeter was applied to the carotid
artery, and blood flow was recorded with a computerized data
acquisition program (WinDaq, DATAQ Instruments). All 4 vessels of
PAI-1 -/- mice occluded within 30 minutes of initiation of
injury, whereas 7 of 8 vessels from PAI-1 +/+ mice occluded
within 30 minutes. These results suggested that PAI-1 -/-
mice did not exhibit a major defect in thrombus formation after injury
compared with PAI-1 +/+ mice. Because several minutes were
necessary to properly position the flow probe on the artery, precise
determination of the time of arterial occlusion was not
possible in 7 of 12 mice studied, because the vessel had already
thrombosed by the time the flow probe was properly positioned.
Recorded times to occlusion after initiation of injury were <7.5,
<9.5, 15.5, and 21 minutes in PAI-1 -/- mice versus
<6.0, <6.5, <8.5, <15, <15, 17, and 16 minutes in PAI-1
+/+ mice (blood flow was monitored for a total of 45 minutes after
injury in the 1 PAI-1 +/+ animal that did not develop
occlusive carotid artery thrombosis). To determine whether PAI-1
deficiency was associated with a primary defect in platelet
aggregation, pooled platelet-rich plasma was prepared from
PAI-1 +/+ mice (n=2) and PAI-1 -/- mice (n=2),
and in vitro platelet aggregation studies were performed (Fig 5
Modification of Injury Model for Study of Thrombus
Formation
Detection of less residual thrombus after injury in PAI-1
-/- mice does not necessarily imply enhanced
thrombolysis compared with PAI-1 +/+ mice,
because it was not possible in our study to confirm equal amounts of
thrombus deposition in each group. However, studies in which a flow
probe was used to confirm thrombus formation did not suggest a
diminished tendency to form thrombi in PAI-1 -/- mice. In
addition, in vitro studies revealed no significant difference in
platelet aggregation between experimental groups. Similarly,
histological analysis of injured carotid
segments did not reveal differences in thrombus composition or the
extent of injury between groups. Carmeliet et
al30 demonstrated that PAI-1 -/-
mice exhibit normal tail bleeding times and normal blood loss after
amputation of the caecum compared with PAI-1 +/+ mice.
Together, these observations suggest that there is no significant
difference in clot formation after injury between PAI-1 +/+
and PAI-1 -/- mice and that the reduction in residual
thrombus that we observed 24 hours after injury in PAI-1
-/- mice resulted from enhanced thrombolysis.
However, because thrombosis and thrombolysis can occur
concurrently in vivo,7 it is possible that
diminished thrombus formation also contributed to our results.
Regardless of mechanism, our studies demonstrate the important role of
PAI-1 in determining the net amount of platelet-rich thrombus
present in arteries after injury.
Characterization of FeCl3-Induced Thrombosis and
Comparison With Other Models
In summary, we have developed a murine model of carotid artery injury
that we have used to demonstrate the important role of PAI-1 in
regulating the acute clearance of arterial,
platelet-rich thrombi. The model is easy to perform and does not
require the use of specialized surgical techniques or equipment to
induce vascular injury. Because many other components of the murine
blood coagulation and fibrinolytic systems have been modified by gene
targeting approaches,49 50 51 52 53 54 55 this model should
prove useful for studying the function of other blood coagulation and
fibrinolysis factors within the distinct environment of
the acutely injured artery.
Received July 17, 1997;
revision received October 2, 1997;
accepted October 21, 1997.
© 1998 American Heart Association, Inc.
Basic Science Reports
Regulation of Arterial Thrombolysis by Plasminogen Activator Inhibitor-1 in Mice
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Background—Platelet-rich
arterial thrombi are resistant to lysis by
plasminogen activators. However, the mechanisms
underlying thrombolysis resistance are poorly defined.
Plasminogen activator inhibitor-1
(PAI-1), which is present in plasma, platelets, and vascular
endothelium, may be an important determinant of the
resistance of arterial thrombi to lysis. However, in vitro
studies examining the regulation of platelet-rich clot lysis by
PAI-1 have yielded inconsistent results.
Key Words: thrombosis • thrombolysis • carotid arteries • plasminogen activators
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
Vascular injury
and subsequent thrombus formation are key events in the pathogenesis of
several arterial diseases, including unstable angina
pectoris, myocardial infarction, and stroke.1 The
blood fibrinolytic system, which functions to degrade intravascular
fibrin, constitutes a critical response mechanism to
arterial injury and thrombosis. Endogenously
mediated fibrinolysis is initiated by t-PA and u-PA,
enzymes that convert plasminogen to the fibrinolytic
protease plasmin.2 However, the rate and extent
of thrombolysis after arterial injury can
vary considerably in humans. In some individuals, arterial
thrombi lyse spontaneously within several hours, whereas in others,
thrombi fail to lyse, resulting in permanent vascular
occlusion.3 This phenomenon is of considerable
clinical importance in the setting of acute myocardial infarction,
because early restoration of normal coronary artery blood flow
is strongly associated with improved survival, and even delayed
thrombolysis appears to exert beneficial effects on
cardiac function.4 5 However, the molecular
determinants of the lysis of arterial thrombi, which are
characterized by a high platelet content, are not well understood.
Although plasminogen activators trigger
activation of the fibrinolytic pathway, multiple cofactors,
inhibitors, and proteases contribute to the regulation of
vascular fibrinolysis.2 6 Several
studies suggest that PAI-1, which inhibits fibrinolysis
by binding irreversibly to the active site of t-PA and u-PA, is a major
determinant of the resistance of platelet-rich clots to lysis by
t-PA.7 8 9 10 11 However, other studies suggest that
PAI-1 plays only a minor role in regulating the lysis of
platelet-rich clots.12 13 These contrasting
results probably resulted from the variable concentrations of
platelets, t-PA, PAI-1, and other factors that were used in
different in vitro experiments. However, the concentrations of
fibrinolytic components at sites of arterial injury are not
well defined.14 Therefore, in vivo experiments
are necessary to examine the true function of PAI-1 in regulating
thrombolysis at sites of arterial injury.
The capacity to manipulate the mouse genome by gene targeting
approaches has provided a powerful tool for studying protein function
in vivo.15 16 Mice lacking PAI-1 have been
genetically engineered,17 and these animals
exhibit a phenotype consistent with that observed in
humans with PAI-1 deficiency.18 19 To address the
role of PAI-1 in the regulation of arterial
thrombolysis, we developed a murine model of
arterial injury and thrombosis and used it to study both
wild-type and PAI-1–deficient mice.
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
Animals
C57BL/6J mice were purchased from Jackson Laboratory, Bar
Harbor, Me. PAI-1–deficient mice were generated by homologous
recombination in embryonic stem cells, as described
previously.17 To eliminate potential effects of
strain differences on experimental results, consecutive generations of
mice carrying the null PAI-1 allele were backcrossed repeatedly to
C57BL/6J mice. Only mice that were the product of five or more
backcrosses to C57BL/6J were used in experiments comparing
PAI-1–deficient (PAI-1 -/-) mice with wild-type C57BL/6J
(PAI-1 +/+) mice. Genotyping of mice was performed by
polymerase chain reaction analysis of tail DNA as described
previously.20 All animal care and experimental
procedures complied with The Guide for Care and Use of Laboratory
Animals, Department of Health, Education, and Welfare Publication
No. NIH 78-23, and were approved by the University of Michigan
Committee on Use and Care of Animals.
Mice (6 to 8 weeks old; weight, 
25 g) were
anesthetized by inhalation of 1% isoflurane and secured in the
supine position under a dissecting microscope (Zeiss). A midline
cervical incision was made, and the left common carotid artery was
isolated by blunt dissection. FeCl3 (Mallinckrodt
Chemical) was used to induce vascular injury.21 A
1x2-mm strip of filter paper saturated with 10%
FeCl3 solution was applied to the adventitial
surface of the surgically exposed carotid artery for 3.0 minutes, then
removed. The surgical incision was sutured with 4–0 monofilament, and
the animal was returned to its cage. Operators were blinded to PAI-1
genotype while performing the thrombosis protocol. Twenty-four
hours later, mice were anesthetized, and the injured carotid
segments were isolated and perfusion-fixed by manual injection of 4%
formaldehyde (1.0 mL) into the heart via a 25-gauge needle. The injured
carotid artery segment and 1 mm of proximal and 1 mm of
distal uninjured artery were excised (total length of excised
arterial segment, 4 mm), placed in formaldehyde
fixative, and later embedded in paraffin for
histological analyses.
To quantify the amount of thrombus present in carotid
arteries 24 hours after injury, four evenly spaced transverse sections
(distance between sections, 1 mm) were prepared from each
paraffin-embedded carotid artery and subjected to hematoxylin-eosin
staining. Each transverse section was scored for the presence or
absence of intraluminal thrombus. In addition, a computer-assisted
planimetry program (Image-Pro Plus, Media Cybernetics) was used to
measure the cross-sectional area of thrombus for each transverse
section, and the mean of these values was calculated for each artery.
Examiners were blinded to the genotype (ie, PAI-1
+/+ or -/-) of specimens during all histological
analyses. To determine the composition of thrombi, some carotid
artery specimens were analyzed by transmission electron
microscopy as described previously.22
Blood was collected into citrate anticoagulant from
anesthetized mice by inferior vena cava puncture
with a 25-gauge needle. PRP was prepared by centrifuging blood
(120g for 6 minutes) at room temperature in a swing-out
rotor. The platelet count of PRP was adjusted with citrated
platelet-poor plasma to 2x108
platelets/mL. Samples (200 µL) of PRP were placed in 7.5x55-mm
siliconized flat-bottom tubes, and standard aggregometry was performed
with a model PAP-4 aggregometer (Bio/Data Corp).
Data are presented as mean±SD. The generalized
estimating equations method was used to determine whether a significant
difference existed between experimental groups in the prevalence of
intraluminal thrombus.23 The Wilcoxon
rank-sum test was used to determine whether a significant difference
existed in mean thrombus cross-sectional areas between experimental
groups. Student's unpaired t test was used to compare in
vitro aggregation of wild-type and PAI-1–deficient platelets.
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
Murine Model of Acute Arterial Injury
We performed a series of studies to determine the feasibility of
using mice to study arterial thrombosis and
thrombolysis. The common carotid artery was selected
for analysis because it was easily approached via a midline
cervical incision. After performing the procedure on 3 to 5 mice,
operators could routinely isolate the carotid artery in <10 minutes.
We used topical application of FeCl3 to the
exposed carotid artery to induce vascular injury and
thrombosis.21 Completely occlusive carotid artery
thrombosis was well tolerated by mice, presumably because of collateral
cerebral blood flow provided by the contralateral carotid artery. After
recovery from anesthesia, mice appeared healthy,
demonstrating normal ambulation, feeding, and grooming. The operative
mortality rate of carotid artery surgery was <5%. Fig 1
demonstrates the use of Whatman 1
filter paper to apply FeCl3 to the
arterial surface. Control experiments performed in >50
wild-type mice revealed that 10% FeCl3 induced
completely occlusive thrombosis within 30 minutes (determined by visual
inspection of the artery under the dissecting microscope) in >90% of
mice, whereas application of filter paper alone (ie, no
FeCl3) to carotid arteries did not induce
thrombosis. To determine the composition of thrombi induced by
FeCl3, injured carotid artery segments were
perfusion-fixed immediately after induction of thrombosis, excised, and
examined histologically. Light microscopic
analysis of hematoxylin-eosin–stained specimens from 10
consecutive mice revealed that thrombus composition was uniform from
animal to animal (Fig 2A). Transmission
electron microscopy confirmed that thrombi consisted predominantly of
dense platelet aggregates (Fig 2B), consistent with the
composition of arterial thrombi observed in humans and
large-animal thrombosis models.
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Figure 1. Induction of vascular injury in murine carotid
artery. A 1x2-mm strip of filter paper saturated with 10%
FeCl3 was applied to surface of surgically exposed left
carotid artery for 3 minutes, as described in "Methods." Forceps
elevate carotid artery, which is viewed through a dissecting microscope
(magnification x16).
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Figure 2. Histological analysis of
acute carotid artery thrombi. A, Light microscopy. Platelet-rich,
occlusive thrombus is observed (hematoxylin-eosin stain, magnification
x20). B, Transmission electron microscopy. Thrombus within lumen of
artery is composed predominantly of platelets. Note serpiginous
internal elastic lamina, covered by single layer of
endothelial cells (lead citrate–uranyl acetate stain,
magnification x3400).
To examine the role of PAI-1 in regulating
endogenously mediated arterial
thrombolysis, we applied the
FeCl3 model to wild-type (PAI-1 +/+)
and PAI-1–deficient (PAI-1 -/-) mice. Twenty-two
PAI-1 +/+ and 14 age- and sex-matched PAI-1 -/-
mice were studied. Each group was subjected to identical carotid artery
injury with 10% FeCl3 as described in
"Methods." There was no difference in operative bleeding between
the two experimental groups. Twenty-four hours later, the injured
carotid segments were perfusion-fixed and excised. Examiners were
blinded to PAI-1 genotype during all evaluations of tissue
samples. To determine whether the amount of residual intraluminal
thrombus differed between experimental groups, each segment of injured
carotid artery was cross-sectioned at four evenly spaced intervals as
described in "Methods," and transverse sections were prepared for
histological analysis (Fig 3). Residual thrombus (either completely
or partially occlusive) was detected in 55 of 85 sections (64.7%)
obtained from PAI-1 +/+ mice, compared with 19 of 56
sections (33.9%) from PAI-1 -/- mice (P=.009,
Fig 4A). The cross-sectional area of
thrombus was measured for each transverse section by computer-assisted
planimetry, and the mean thrombus area for each artery was calculated
(Fig 4B). Mean residual thrombus area was 0.033±0.027
mm2 (95% CI, 0.022 to 0.044
mm2) in PAI-1 +/+ mice versus
0.016±0.015 mm2 (95% CI, 0.008 to
0.024 mm2) in PAI-1 -/- mice
(P=.048). Subgroup analysis of patent and occluded
vessels revealed an equal proportion of patent arteries in both groups
(9 of 22 PAI-1 +/+ mice versus 6 of 14 PAI-1
-/- mice). However, significantly less residual mural thrombus was
observed in patent vessels from PAI-1 -/- mice than in
those from PAI-1 +/+ mice (0.001±0.002 versus
0.011±0.015 mm2, respectively,
P<.05). Similarly, increased thrombus was present in
occluded vessels from PAI-1 +/+ mice compared with
PAI-1 -/- mice (0.052±0.021 versus 0.027±0.010
mm2, respectively, P<.05).
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Figure 3. Transverse sections of carotid arteries 24 hours
after FeCl3 injury. A, PAI-1 +/+ mouse. B,
PAI-1 -/- mouse. Note noncircumferential,
full-thickness arterial injury induced by FeCl3
(hematoxylin-eosin stain, magnification x20).
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Figure 4. Effect of PAI-1 on arterial
thrombolysis. Carotid arteries of PAI-1
+/+ (n=22) and PAI-1 -/- (n=14) mice were harvested 24
hours after 10% FeCl3 injury. Four evenly spaced
transverse sections were prepared from each artery and analyzed
histologically. A, Prevalence of transverse sections
containing intraluminal thrombus. B, Mean thrombus cross-sectional area
within all, occluded, and patent arteries. Occluded vessels were
defined as those in which 
1 transverse section demonstrated complete
filling of lumen with thrombus. Error bars represent 95% CIs.
*P<.05, **P <.01 vs
PAI-1 +/+ mice.). There was no significant difference
in ADP-induced aggregation between PAI-1 +/+ and
PAI-1 -/- platelets (64.5±4.9% in PAI-1
+/+ mice versus 58±4.2% in PAI-1 -/- mice,
P=.29). We also compared thrombus composition in
PAI-1 +/+ and PAI-1 -/- mice, because this
parameter is an important determinant of the rate of clot
lysis.24 No gross differences were observed—ie,
all thrombi appeared to be platelet-rich. Because leukocytes
migrate into thrombi within 24 hours after initial formation and
leukocyte proteases, such as elastase, degrade
fibrin,3 25 we compared thrombus leukocyte
density between experimental groups. No significant differences were
observed (1.4±0.4 leukocytes per high-power field in PAI-1
+/+ mice versus 1.4±0.4 leukocytes per high-power field in
PAI-1 -/- mice). Similarly, no differences in the
histological appearance of injured arterial
walls was observed between PAI-1 +/+ and PAI-1
-/- mice. Topical application of FeCl3 produced
full-thickness, noncircumferential vascular injury (Fig 3
). Medial
cross-sectional area, an index of the severity of vessel injury, did
not differ between groups (0.015±0.0026 mm2
in PAI-1 +/+ mice versus 0.015±0.0041
mm2 in PAI-1 -/- mice).
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Figure 5. Aggregation of PAI-1 +/+ and
PAI-1 -/- platelets. Pooled PRP prepared from
PAI-1 +/+ (n=2) and PAI-1 -/- (n=2)
mice was aggregated by addition of ADP (20 µmol/L).
Our experiments with the vascular flow probe suggested that the
carotid artery model would prove useful for quantitatively studying
blood flow and thrombus formation in mice. Such a system would be
useful in the analysis of mice with genetic modifications in
factors that regulate carotid artery blood flow (eg, adrenergic
receptors) or platelet deposition after arterial injury
(eg, platelet or endothelial cell integrins).
Carotid artery blood flow in anesthetized mice (n=14) was
1.1±0.7 mL/min. Mice could be maintained under general
anesthesia for at least 1 hour with stable heart and
respiratory rates and stable carotid artery blood flows. As shown in
Fig 6A, high-quality flow tracings could
be obtained from the mouse carotid artery. To better study thrombosis
after injury, we modified our protocol to allow induction of vascular
injury while the flow probe was positioned on the artery. This was done
with a smaller strip of filter paper (0.5x1.0 mm) saturated with
higher concentrations of FeCl3 (25% to 50%)
applied proximal to the flow probe. Mean time to occlusion was
13.6±5.1 minutes after 25% FeCl3 injury (n=4)
and 5.6±4.9 minutes after 50% FeCl3 injury
(n=5). Cyclic flow reductions, which result from repetitive formation
and embolization of thrombi at sites of vascular
injury,26 were observed (Fig 6B). These
observations indicate that the carotid artery model demonstrates
several features observed in large-animal models and therefore should
prove useful in murine studies of blood flow regulation and thrombosis
after vascular injury.
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Figure 6. Monitoring of carotid artery blood flow. Miniature
Doppler flow probe (7.2 MHz) was used to monitor carotid artery
blood flow in mice. A, Phasic carotid artery flow before and after
FeCl3 injury. B, Mean carotid artery blood flow in
different mouse subjected to FeCl3 injury (50%) for 1.5
minutes. Artifactual reduction in flow during induction of injury is
due to removal of saline from surgical site to allow application of
FeCl3. Note that mean blood flow initially declines
gradually and that repetitive, transient increases in flow are
observed. Thereafter, flow declines rapidly, with complete loss of flow
occurring 14 minutes after injury. After loss of flow in left carotid
artery was documented, flow probe was placed on right carotid artery.
Flow rate of 1.6 mL/min was observed (data not shown).
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
Platelet-rich thrombi are more resistant to lysis by
t-PA than platelet-poor thrombi.24 However,
the mechanisms underlying this phenomenon are not well defined. In this
report, we studied the role of PAI-1 in regulating the
endogenous clearance of platelet-rich
arterial thrombi in mice. PAI-1 is abundant in
platelets, and its secretion from vascular
endothelial cells is stimulated by factors released
from activated platelets.27 Several
in vitro studies suggest that PAI-1 is the dominant factor underlying
platelet-mediated clot lysis
resistance.7 8 10 11 However, other studies have
concluded that PAI-1 plays only a minor role in inhibiting the lysis of
platelet-rich clots.12 13 These discordant
results are probably explained by the different concentrations of t-PA
and platelets that were used in different in vitro studies, which
do not necessarily reflect those attained in vivo at sites of
arterial injury. Furthermore, in vitro experiments do not
account for the role of the vascular wall in the regulation of clot
lysis. Earlier studies examining the role of PAI-1 in regulating clot
lysis in vivo involved the injection of preformed clots into veins or
the injection of whole blood/thrombin mixtures into isolated
arterial segments.7 28 29 30 31 Therefore,
the role of PAI-1 in regulating the clearance of platelet-rich
thrombi that form in response to arterial injury has not
been adequately addressed. Our study has demonstrated that compared
with wild-type mice, mice lacking PAI-1 exhibit a significant reduction
in intraluminal thrombus 24 hours after arterial injury.
These results suggest that PAI-1 inhibits the lysis of
platelet-rich thrombi that form in vivo in response to
arterial injury and are consistent with our earlier
studies demonstrating that in vitro clots containing human
PAI-1–deficient platelets lyse more rapidly than clots with normal
PAI-1 content.32 In addition to delaying
restoration of blood flow in occluded arteries, inhibition of
arterial thrombolysis by PAI-1 would be
expected to retard clearance of nonocclusive mural thrombi. Mural
arterial thrombi appear to contribute to the development of
atherosclerosis, because they may be incorporated into
developing lesions or serve as a source of platelet-derived growth
factors that stimulate smooth muscle cell
proliferation.33 Therefore, our results are
consistent with the recent report that PAI-1 deficiency
protects apolipoprotein E–deficient mice from the development of
atherosclerosis.34 Although the
results of murine studies must be extended with caution to human
diseases, our studies relate to the variable
thrombolysis that is observed in humans with acute
coronary artery occlusion. It is not well understood why
spontaneous thrombolysis is observed in some
individuals but not in others. Plasma PAI-1 concentrations vary widely
in the normal population,35 36 which may account,
at least partially, for the variable degree of
endogenous thrombolysis that is observed
after arterial thrombosis in humans.
In addition to studying the biological function of PAI-1, a goal
of our study was to develop a reliable model of arterial
injury and thrombosis in mice. Kurz et al21 first
described the use of FeCl3 to induce
arterial thrombosis in rats weighing 375 to 450 g. Our
studies demonstrate that this method can be applied effectively to
animals weighing <25 g and that FeCl3 produces
platelet-rich thrombi. The FeCl3 model
exhibits several features that suggest that it will prove useful for
studying the molecular determinants of arterial thrombosis
and thrombolysis in transgenic mice. It allows for
uniform injury from animal to animal, because the concentration of
FeCl3, the size of the arterial
segment being injured, and the duration of injury can be precisely
controlled. Because FeCl3 does not produce
circumferential vessel injury, it allows induction of thrombosis
without complete destruction of vascular cells that mediate subsequent
thrombolysis. Carotid artery blood flow can be
monitored accurately in mice, and complete vascular occlusion is well
tolerated because of collateral blood flow. In addition, the relevance
of the murine model to large-animal thrombosis models is supported by
the fact that cyclic flow variations are observed in the mouse carotid
artery after FeCl3
injury.26 A variety of methods have been used to
study thrombosis in rodents. These include application of electrical,
mechanical, and other forms of energy to different vascular sites,
intravenous injection of clots formed in vitro, and
injection of blood/thrombin mixtures into isolated vascular
segments.31 37 38 39 40 41 42 Only a few of these models
have been applied to mice. Carmeliet et al30 and
Palabrica et al43 modified the hamster
pulmonary embolism model of Stassen et
al37 to study the regulation of
thrombolysis in mice, whereas
Pierangeli44 used "pinch" injury to induce
femoral vein thrombosis. In contrast to these experimental systems, our
model includes the key components of arterial injury and
high-velocity laminar flow that are necessary to generate
platelet-rich thrombi. The mechanism by which
FeCl3 induces thrombosis is not well defined.
Iron induces formation of highly reactive oxidant species and
potentiates the sensitivity of endothelial cells to
oxidant damage.45 Recent studies indicate that
FeCl3 induces thrombosis by triggering expression
of tissue factor, a major determinant of thrombosis at sites of
atherosclerotic plaque rupture.46 47 Carmeliet et
al48 used perivascular electric injury to study
vascular wound healing and neointima formation in mouse
femoral arteries. Induction of thrombi by electrical injury may depend
on the generation of reactive iron species, because only
iron-containing electrodes efficiently cause thrombus
formation.21 Therefore, it is possible that
topical FeCl3 and electrical current delivered by
iron-containing electrodes induce vascular injury by similar
mechanisms.
Selected Abbreviations and Acronyms
PAI-1
=
plasminogen activator inhibitor-1
PRP
=
platelet-rich plasma
t-PA
=
tissue plasminogen activator
u-PA
=
urokinase plasminogen activator
Acknowledgments
This work was supported in part by National Institutes of Health
grants HL-02728 (Dr Fay) and HL-07853–01 (Dr Farrehi) and by American
Heart Association, Michigan Affiliate, Fellowship Award 26F967 (Dr
Farrehi). We are grateful to Drs Tom Wakefield and Benedict Lucchesi
for sharing laboratory equipment, Randy Westrick and Andrew Parker for
technical assistance, and Jennifer Sievers for statistical assistance.
We also thank Dr David Gordon, Chris Edwards, and Lingling Xu for
assistance with tissue preparation and histological
analyses. Finally, we thank Dr David Ginsburg for critically
reviewing the manuscript.
Footnotes
Reprint requests to William P. Fay, MD, University of Michigan Medical Center, MSRB III Room 7301, 1150 W Medical Center Dr, Ann Arbor, MI 48109-0644.
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
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J. Liu, M. E. Fitzgerald, M. C. Berndt, C. W. Jackson, and T. K. Gartner Bruton tyrosine kinase is essential for botrocetin/VWF-induced signaling and GPIb-dependent thrombus formation in vivo Blood, October 15, 2006; 108(8): 2596 - 2603. [Abstract] [Full Text] [PDF]
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M. Rucker, T. Schafer, C. Scheuer, Y. Harder, B. Vollmar, and M. D. Menger Local Heat Shock Priming Promotes Recanalization of Thromboembolized Microvasculature by Upregulation of Plasminogen Activators Arterioscler. Thromb. Vasc. Biol., July 1, 2006; 26(7): 1632 - 1639. [Abstract] [Full Text] [PDF]
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