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Blood, Vol. 93 No. 6 (March 15), 1999:
pp. 1825-1830
RAPID COMMUNICATION
Vitronectin Inhibits the Thrombotic Response to Arterial Injury in Mice
By
William P. Fay,
Andrew C. Parker,
Maria N. Ansari,
Xianxian Zheng, and
David Ginsburg
From the Departments of Internal Medicine and Human Genetics and the
Howard Hughes Medical Institute, University of Michigan, Ann Arbor, MI.
 |
ABSTRACT |
Vitronectin (VN) binds to plasminogen activator inhibitor-1 (PAI-1)
and integrins and may play an important role in the vascular response
to injury by regulating fibrinolysis and cell migration. However, the
role of VN in the earliest response to vascular injury, thrombosis, is
not well characterized. The purpose of this study was to test the
hypothesis that variation in vitronectin expression alters the
thrombotic response to arterial injury in mice. Ferric chloride
(FeCl3) injury was used to induce platelet-rich thrombi in
mouse carotid arteries. Wild-type (VN +/+, n = 14) and
VN-deficient (VN  /, n = 15) mice, matched for age and
gender, were studied. Time to occlusion after FeCl3 injury
was determined by application of a Doppler flowprobe to the carotid
artery. Occlusion times of VN / mice were significantly
shorter than those of VN +/+ mice (6.0 ± 1.2 minutes
v 17.8 ± 2.3 minutes, respectively, P < .001).
Histologic analysis of injured arterial segments showed that thrombi
from VN +/+ and VN / mice consisted of dense
platelet aggregates. In vitro studies of murine VN +/+ and
VN / platelets showed no significant differences in
ADP-induced aggregation, but a trend towards increased thrombin-induced
aggregation in VN / platelets. Purified, denatured VN
inhibited thrombin-induced platelet aggregation, whereas native VN did
not. Thrombin times of plasma from VN / mice (20.5 ± 2.1 seconds, n = 4) were significantly shorter than those of
VN +/+ mice (34.2 ± 6.7 seconds, n = 4, P < .01), and the addition of purified VN to VN / plasma
prolonged the thrombin time into the normal range, suggesting that VN
inhibits thrombin-fibrinogen interactions. PAI-1-deficient mice (n = 6) did not demonstrate significantly enhanced arterial thrombosis compared with wild-type mice (n = 6), excluding a potential indirect antithrombin function of VN mediated by interactions with PAI-1 as an
explanation for the accelerated thrombosis observed in VN / mice. These results suggest that vitronectin plays a previously unappreciated antithrombotic role at sites of arterial injury and that
this activity may be mediated, at least in part, by inhibiting platelet-platelet interactions and/or thrombin procoagulant activity.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
VITRONECTIN (VN) IS A major plasma
protein that is also found in platelets and the extracellular matrix of
many tissues.1,2 VN binds multiple ligands, including
integrins,3 plasminogen activator inhibitor-1
(PAI-1),4 the urokinase receptor (uPAR),5 collagen,6 complement C5b-7,7 and
heparin.8 These interactions suggest that VN plays an
important role in regulating several biologic processes, such as cell
adhesion and migration, hemostasis, and immune defense.9 VN
is a single-chain 78-kD glycoprotein that consists of an N-terminal
somatomedin B domain and two hemopexin-type domains. The somatomedin B
domain contains an Arg-Gly-Asp (RGD) sequence that binds integrins and
serves as a cell attachment site.3 Plasma VN exists in a
native conformation that does not bind integrins.10 Binding
to certain ligands, such as PAI-1 or thrombin-antithrombin III, induces
conformational changes in VN that expose binding sites for integrins,
heparin, and other molecules.11,12 In addition, VN exists
in both monomeric and multimeric forms that may serve distinct biologic
functions.13
VN appears to play an important role in the response of the blood
vessel to injury. VN may control the clearance of vascular thrombi by
binding and stabilizing PAI-1, a key regulator of
fibrinolysis.4 VN may regulate neointima formation after
injury through interactions with  V 3 and
uPAR, receptors expressed on the surface of migrating vascular smooth
muscle cells.14,15 However, the role of VN in thrombosis,
the earliest response of the blood vessel to injury, is not well
defined. VN binds to platelet glycoproteins IIb/IIIa (II 3) and
V3 and may mediate platelet adhesion and
aggregation at sites of vascular injury.16 In vitro studies
have yielded conflicting results regarding the role of VN in platelet
function. Asch and Podack17 showed that anti-VN antibodies
inhibit platelet aggregation in vitro, suggesting that VN contributes
to platelet accumulation at sites of vascular injury. However, Mohri
and Ohkubo18 demonstrated that VN inhibited platelet
aggregation and competed with fibrinogen and von Willebrand factor for
binding to glycoprotein IIb/IIIa, suggesting that VN may prevent
platelet-dependent thrombosis. In addition to its platelet
interactions, VN may control the thrombotic response to vascular injury
by regulating thrombin function. The capacity of PAI-1 to inhibit
thrombin is accelerated greater than 200-fold by VN.19,20
In addition, thrombin-antithrombin III complexes bind VN, suggesting
that VN controls clearance of thrombin from the
circulation.21 Recently, VN-deficient mice were generated by a gene-targeting strategy.22 Mice lacking VN develop
normally and do not exhibit any discernible phenotypic abnormalities.
In this study, we have subjected wild-type (VN +/+) mice and
VN-deficient (VN /) mice to carotid artery injury
to test the hypothesis that VN is an important determinant of the acute
thrombotic response to vascular injury. Our results suggest that VN
plays an important antithrombotic role in vivo.
| |
MATERIALS AND METHODS |
Mice.
C57BL/6J mice were purchased from Jackson Labs (Bar Harbor, ME). The
generation of VN-deficient mice by homologous recombination in
embryonic stem cells has been reported previously.22
PAI-1-deficient (PAI-1 /) mice were a gift from
Dr P. Carmeliet (University of Leuven, Leuven, Belgium).23
To eliminate potential
effects of genetic differences among mouse strains on experimental
results, consecutive generations of mice carrying the null VN allele
were backcrossed to C57BL/6J mice. Only mice that were the product of
 8 backcrosses were used in experiments comparing VN
/ mice with VN +/+ mice. PAI-1
/ mice used in experiments were the product of greater
than 10 backcrosses to the C57BL/6J genetic background. Genotyping of
mice was performed by polymerase chain reaction (PCR) analysis of tail
DNA as described previously.22,24 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.
Reagents.
Native human VN was purchased from Molecular Innovations Inc (Royal
Oak, MI). Human -thrombin was from CalBiochem (La Jolla, CA).
Thrombin substrate (Spectrozyme TH) was from American Diagnostica (Greenwich, CT). Ferric chloride (FeCl3) was from
Mallinckrodt Chemical (Paris, KY). Human VN purified by
heparin-affinity chromatography, reptilase (Atroxin), and human
fibrinogen (plasminogen-free) were from Sigma (St Louis, MO).
Thrombosis protocol.
A previously described carotid artery thrombosis protocol was
used.25,26 Adult mice (6 to 8 weeks old; weight, ~25 g)
were anesthetized by intraperitoneal injection of pentobarbital (120 mg/kg). The left common carotid artery was surgically exposed and a
miniature Doppler flowprobe (Model 0.5VB; Transonic Systems, Ithaca,
NY) was placed on the surface of the artery. Sodium chloride solution
(0.9%) was placed in the surgical wound to allow Doppler monitoring,
and baseline blood flow was recorded using a Transonic Model T106
flowmeter. Thereafter, sodium chloride solution was removed from the
wound and filter paper (0.5 × 1.0 mm) saturated with 10%
FeCl3 was applied to the adventitial surface of the carotid artery, immediately proximal to the flow probe. After 3.0 minutes, the
filter paper was removed, saline solution was again placed in the
wound, and carotid blood flow was monitored (ie, it was not possible to
monitor carotid artery blood flow during the application of
FeCl3). Time to thrombotic occlusion after initiation of
arterial injury was defined as the time required for blood flow to
decline to 0 mL/min. If the carotid artery was observed to be
thrombosed at the earliest time point that flow could be monitored
after initiation of injury (ie, 3.0 minutes), time to occlusion was recorded as  3.0 minutes. The operator was blinded to mouse genotype while performing all experiments.
Histologic analyses.
For some animals, the arterial vasculature was perfusion fixed
immediately after completing the thrombosis protocol, as described previously.27 Injured arterial segments were excised,
embedded in paraffin, sectioned, and subjected to hematoxylin and eosin staining.
Bleeding assay.
Mice (6 to 8 weeks old) were anesthetized by intraperitoneal injection
of phenobarbital (100 mg/kg) and placed in a restraining chamber from
which the tail protruded. The distal 1 mm of the tail was amputated and
the tail was immersed for 10 minutes in 1 mL of 0.9% NaCl warmed to
37°C. Blood loss was determined by measuring the absorbance of
saline at 560 nm and comparing the result to a standard curve
constructed from known volumes of mouse blood.
In vitro platelet aggregation.
Platelet aggregation was studied using a previously described
microtiter plate assay.28 Blood was collected into citrate anticoagulant from anesthetized mice by inferior vena cava puncture with a 25-gauge needle. Platelet-rich plasma (PRP) was prepared by
centrifuging blood (120 g for 6 minutes) in 0.5 mL polypropylene tubes
at room temperature in a swing-out rotor. After adjustment to a
platelet count of 2.5 × 108/mL by the addition of
citrated platelet-poor plasma, 95 µL of count-adjusted PRP was placed
in 96-sample microtiter plate wells and incubated at 37°C in a
SpectraMax 340 microtiter plate reader (Molecular Devices, Sunnyvale,
CA). ADP (12.5 µmol/L) was added, and the absorbance of wells at 595 nm was monitored at 20- to 30-second intervals. Plates were shaken
automatically for 15 seconds between each reading. The percentage of
aggregation was calculated as described.28 Gel-filtered
platelets suspended in Tyrode's buffer,29 prepared by
Sepharose 2B chromatography (Sigma), were used to study
thrombin-induced aggregation as described above.
Coagulation and hematologic assays.
Platelet-poor plasma was prepared by centrifuging citrated mouse blood
for 8 minutes at 16,000g. Thrombin times, activated partial
thromboplastin times (APTTs), and reptilase times were performed using
a KC4A Micro apparatus (Amelung GmbH, Lemgo, Germany) according to the
manufacturer's instructions. Thrombin amidolytic activity was measured
by incubating thrombin and Spectrozyme TH at room temperature in 0.01 mol/L Tris-HCl, 0.14 mol/L NaCl, pH 7.5, and monitoring the absorbance
of reaction mixtures at 405 nm in a microtiter plate reader. Platelet
counts and hematocrits of citrated whole blood were measured using a
Model H-10 blood cell counter (Texas Instruments Laboratories, Houston,
TX). Fibrinogen/fibrin degradation products (FDP) were measured with a
Staphylococcal clumping factor assay (Catalog 850-ST; Sigma) according
to the manufacturer's instructions. Plasma fibrinogen concentrations were determined by the fibrin clot opacity method, as
described.30,31 In this assay, the limit optical density of
dilute plasma during prolonged incubation with thrombin is directly
proportional to plasma fibrinogen concentration. Briefly, 20 µL of
citrated plasma were added to a spectrophotometer cuvette containing
400 µL of 0.05 mol/L Tris-HCl (pH 7.5), 0.15 mol/L NaCl.
Thrombin/calcium solution (20 µL) was added to yield final
concentrations of 0.45 U/mL and 0.9 mmol/L, respectively. After 10 minutes of incubation at room temperature, absorbance at 340 nm was
measured. Fibrinogen concentration in pooled plasma prepared from 4 VN +/+ mice was defined as 100%. Fibrinogen levels in pooled
(n = 4 mice) VN / plasma were determined by
comparison to a standard curve constructed from dilutions of pooled
VN +/+ plasma. Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and Western blotting of diluted human plasma
was performed with the PhastSystem (Pharmacia, Uppsala, Sweden).
Primary antibody was goat antimouse fibrinogen (Accurate Inc, Westbury,
NY). Secondary antibody was peroxidase-conjugated rabbit antigoat IgG
(Zymed Labs, South San Francisco, CA). Blots were developed by the
chemiluminescence method (ECL reagent kit; Amersham, Little Chalfont, UK).
Statistical analyses.
Data are presented as mean ± 1 standard error of the mean (SEM),
unless otherwise indicated. The two-sample t-test or
Mann-Whitney Rank Sum test were used to determine if significant
differences existed between experimental groups.
| |
RESULTS |
Arterial thrombosis studies.
Experimental groups consisted of 14 VN +/+ mice (5 male and 9 female) and 15 VN / mice (6 male and 9 female).
Representative carotid artery blood flow tracings before and after
vascular injury are shown in
Fig 1.
Thrombotic occlusion occurred 3.0 minutes after initiation of
vascular injury in 8 of 15 VN / mice, but only in
1 of 14 VN +/+ mice (Table 1).
Median occlusion times were 16.4 minutes for VN +/+ mice and
3.0 minutes for VN / mice. Mean occlusion times
(calculated by using values of 3.0 minutes as occlusion times for mice
whose arteries were already occluded when flow monitoring was resumed
after vascular injury) were significantly shorter in VN
/ mice (6.0 ± 1.2 minutes) than in VN +/+
mice (17.8 ± 2.3 minutes, P < .001). Blood platelet counts and hematocrits did not differ significantly between VN +/+ and VN / mice
(Table 2). APTTs, reptilase times, and
bleeding in response to tail tip amputation were similar between
groups. Plasma fibrinogen measured by the fibrin clot opacity method
did not differ between groups (100% ± 10.8% and 93.8% ± 7.2% in VN +/+ mice and VN / mice,
respectively; P > .7). Similarly, Western blot analysis of
diluted plasma samples showed no apparent differences in plasma
fibrinogen between VN +/+ mice and VN /
mice (data not shown). Fibrinogen/fibrin degradation products were not
detectable in pooled serum obtained from VN +/+ mice (n = 4) or
VN / mice (n = 4). Control experiments showed
that the FDP assay readily detected murine fibrin degradation products
(data not shown). Mean carotid artery blood flow prior to injury did
not differ between VN +/+ mice and VN /
mice (0.78 ± 0.2 mL/min and 0.85 ± 0.2 mL/min, respectively,
P > .2). No significant differences in gross or microscopic
appearance of uninjured arteries were noted between VN +/+ and
VN / mice (n = 3 each group, data not shown).
Histologic analysis of thrombi (n = 4) recovered immediately after
carotid injury showed that they consisted predominantly of platelets,
with no discernible differences between genotypes (Fig
2).
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| Fig 1.
Carotid artery blood flow tracings obtained from a
wild-type mouse and a VN-deficient mouse. Period of ferric chloride
injury is indicated by hatched bar. Artifactual reduction in flow
during injury is due to removal of saline from the surgical site to
allow application of FeCl3. Thrombotic occlusion occurs
18.7 minutes and 3.0 minutes after initiation of injury in the
VN +/+ mouse and theVN / mouse, respectively.
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| Fig 2.
Transverse sections of carotid arteries excised
immediately after ferric chloride-induced thrombosis. (A) VN
+/+ mouse. (B) VN / mouse. Thrombi consist of
dense platelet aggregates (hematoxylin and eosin staining, original
magnification × 200). The diameter of the mouse carotid artery is
approximately 0.5 mm.
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Characterization of in vitro platelet function.
Given the platelet-rich composition of carotid artery thrombi generated
in this model, platelet aggregation studies were performed to compare
platelet function of VN +/+ and VN /
mice. Pooled samples of platelet-rich plasma were prepared and in vitro
aggregation was induced with ADP (12.5 µmol/L). As shown in
Fig 3A, no significant differences were
observed between VN +/+ and VN / mice.
There was a trend towards enhanced thrombin-induced aggregation of
washed VN / platelets compared with VN
+/+ platelets (Fig 3B). Consistent with this observation, addition of
heparin-affinity-purified (ie, denatured-renatured) human VN (200 µg/mL) to washed VN / platelets significantly
inhibited thrombin-induced aggregation (P < .005; Fig 4). However, native human VN (350 µg/mL) had no detectable effect on thrombin-induced aggregation (data
not shown).
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| Fig 3.
In vitro function of mouse platelets. (A) ADP-induced
platelet aggregation. Citrated PRP was prepared from VN +/+
mice (n = 2) and VN / mice (n = 2). After adjusting
platelet counts to 2.5 × 108/mL, ADP (12.5 µmol/L)-induced platelet aggregation was studied in 96-well
microtiter plates that were warmed to 37°C and automatically
shaken. (B) Thrombin-induced platelet aggregation. Washed platelets
were prepared from VN +/+ mice (n = 3) and VN
/ mice (n = 3) and suspended in Tyrode's buffer at a
concentration of 2.5 × 108/mL. Thrombin (1 U/mL)-induced
platelet aggregation was studied as described for ADP. Data points
represent the mean of triplicate experiments ± 1 SD.
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| Fig 4.
Effect of purified VN on in vitro platelet aggregation.
Gel-filtered VN / platelets (2.0 × 108/mL)
were incubated with thrombin (1 U/mL) in the absence ( ) or presence
( ) of denatured human VN (200 µg/mL), and platelet aggregation was
studied as described in Fig 3. Data points represent the mean of
triplicate experiments ± 1 SEM.
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Effects of VN on thrombin function.
Because thrombin is an important determinant of platelet-dependent
arterial thrombosis,32 we performed experiments to test the
hypothesis that VN produces its anticoagulant effect by inhibiting thrombin procoagulant activity. Thrombin times were performed by adding
human -thrombin (4.4 U/mL) to pooled samples (n = 4) of citrated
plasma. Thrombin times of plasma from VN / mice (20.5 ± 2.1 seconds) were significantly shorter than those of VN +/+ mice (34.2 ± 6.7 seconds, P < .01).
Furthermore, addition of native human VN (350 µg/mL) to VN
/ mouse plasma prolonged the thrombin time into the
normal range, whereas the addition of an equal volume of buffer
containing bovine serum albumin (BSA; 350 µg/mL) had no effect
(Table 3). VN also inhibited clotting of
purified human fibrinogen (thrombin times, 32.9 ± 4.9 seconds and
42.9 ± 2.4 seconds in the absence and presence of VN [350 µg/mL], respectively; P < .05), but had no effect on
thrombin amidolytic activity, measured by thrombin (25 nmol/L)
hydrolysis of low molecular weight substrate (Spectrozyme TH; 150 µmol/L; data not shown).
Effects of VN on PAI-1 function.
VN accelerates the capacity of PAI-1 to inhibit thrombin by greater
than 200-fold.19,20 If the antithrombotic effect of VN
observed in our model were mediated by enhancement of the antithrombin function of PAI-1, then PAI-1 deficiency would be expected to mimic VN
deficiency, resulting in accelerated thrombus formation. To test this
hypothesis, we measured the time to occlusive thrombus formation after
FeCl3 carotid artery injury in PAI-1 +/+ mice (n = 6) and PAI-1 / mice (n = 6).
However, mean occlusion times did not differ significantly between
groups (15.5 ± 1.9 minutes and 13.8 ± 2.4 minutes for
PAI-1 +/+ mice and PAI-1 / mice, respectively; P > .5).
| |
DISCUSSION |
In this study, we observed a significantly enhanced rate of thrombus
formation after arterial injury in VN / mice
compared with VN +/+ mice. We used ferric chloride injury to
trigger thrombosis in our experiments. This method has been used in a
variety of species and vascular sites to trigger platelet-dependent
thrombosis.26,33,34 Iron ions enhance conversion of
O2 and H2O2 to
oxidizing species, such as hydroxyl radical, that injure endothelial
cells and markedly increase tissue factor expression in vitro and in
vivo.35-38 The markedly enhanced rate of thrombosis in
VN / mice compared with VN +/+ mice
suggests that VN plays a previously unsuspected role in protecting the
injured arterial wall against thrombus formation.
Our in vitro studies suggest that the antithrombotic effect of VN may
be mediated by inhibition of platelet-platelet and thrombin-substrate interactions. VN binds platelet glycoprotein IIb/IIIa,16,18 providing a mechanism by which VN may modulate platelet function. Because VN contains a single RGD and mutagenesis of this sequence blocks platelet binding,3 monomeric VN would not be
expected to support platelet aggregation. In fact, prior in vitro
studies demonstrated that VN inhibits platelet aggregation and competes with fibrinogen and von Willebrand factor for binding to platelets, leading to the hypothesis that VN inhibits platelet-dependent thrombosis.18 Our experiments provide the first in vivo
data to support this hypothesis. We observed that VN purified under denaturing conditions, which expose its cell attachment
site,10 inhibited thrombin-induced platelet aggregation.
However, native VN, which does not bind integrins,10 did
not. A hypothesis that is consistent with our in vivo and in vitro data
is that plasma VN undergoes a conformational change at sites of
vascular injury, thereby exposing its integrin-binding site and
inhibiting platelet-platelet interactions by competing with fibrinogen,
von Willebrand factor, or other factors for binding to glycoprotein
IIb/IIIa on activated platelets. Such a negative feedback mechanism
could serve to prevent excessive platelet accumulation and vascular
occlusion after injury to the blood vessel wall. The proportion of VN
capable of binding heparin is less than 2% in plasma, but increases
over threefold upon formation of serum,39 suggesting that
activation of the coagulation cascade triggers conversion of native VN
to a conformationally altered form capable of binding platelets.
Several factors generated or released at sites of arterial injury bind
VN and expose its RGD site, including thrombin-antithrombin complex,
PAI-1, and C5b-7.7,11,12 This functional activation of VN
by ligand binding also may explain the inhibition of platelet
aggregation by anti-VN antibodies in experiments by Asch and
Podack.17 It is possible that the antibodies used in these
experiments induced a conformational change in VN that exposed its RGD
site, thereby inhibiting platelet aggregation by enabling VN to compete
with fibrinogen for binding to platelet glycoprotein IIb/IIIa. In
addition to the plasma, VN is present in platelets and the blood vessel wall.40,41 Although platelet VN and blood vessel wall VN
exist to a significant extent as multimeric forms capable of supporting platelet aggregation,13 we observed accelerated platelet
thrombus formation in VN /mice. These results
suggest that, in this model, the platelet or vessel wall pools of VN
are not required for platelet accumulation at sites of arterial injury
and that the antithrombotic properties of VN are dominant over its
potential procoagulant function.
An additional mechanism by which VN may inhibit thrombosis is by
downregulating thrombin function. We demonstrated that thrombin induces
clot formation more rapidly in VN-deficient plasma than in normal
plasma and that addition of purified VN to VN-deficient plasma prolongs
the thrombin time. Similar VN effects were observed on thrombin
clotting of purified fibrinogen. VN deficiency had no effect on
reptilase clotting times, suggesting that VN does not affect
polymerization of fibrin monomer. VN did not inhibit thrombin
amidolytic activity. Together, these results suggest that VN inhibits
thrombin-fibrinogen interactions by binding to thrombin at a site
distinct from its active-site, as suggested previously by Naski et
al.20 A direct antithrombin effect of VN could contribute
to the enhanced thrombotic response observed in VN
/ mice. In addition to its direct effects, it has been proposed that VN may inhibit thrombin function indirectly by
accelerating PAI-1-mediated thrombin inhibition greater than
200-fold.19,20 However, we did not observe a significantly
accelerated thrombotic response in PAI-1 / mice,
which appears to exclude VN-dependent thrombin inhibition by PAI-1 as a
mechanism responsible for the longer thrombosis times observed in
VN +/+ mice.
In summary, we have shown that VN-deficient mice form occlusive
arterial thrombi at an accelerated rate compared with wild-type mice.
We hypothesize that the antithrombotic properties of VN are mediated by
its interaction with platelet glycoprotein IIb/IIIa and by its capacity
to inhibit thrombin-fibrinogen interactions, although we cannot exclude
that other mechanisms may be operative as well. These findings
represent the first phenotypic abnormality observed in VN
/ mice and suggest an important role for VN in inhibiting
platelet-dependent thrombosis at sites of arterial injury, a previously
unrecognized function of this adhesive glycoprotein.
| |
ACKNOWLEDGMENT |
The authors thank Randal Westrick for assistance with mouse breeding
and genotyping, Mary Ellen Wechter for assistance with phlebotomy, and
Drs Alvin Schmaier and Benedict Lucchesi for sharing laboratory equipment.
| |
FOOTNOTES |
Submitted July 30, 1998; accepted December 16, 1998.
Supported by National Institutes of Health Grants No. HL-57346 and
HL-49184. D.G. is a Howard Hughes Medical Institute investigator.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to William P. Fay, MD, University of Michigan
Medical Center, 7301 MSRB III, 1150 W Medical Center Dr, Ann Arbor, MI
48109-0644; e-mail: wfay{at}umich.edu.
| |
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ABSTRACT
INTRODUCTION