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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Mice lacking the extracellular matrix protein MAGP1 display delayed thrombotic
occlusion following vessel injury
*Claudio C. Werneck,1 *Cristina P. Vicente,2 Justin S. Weinberg,3 Adrian Shifren,4 Richard A. Pierce,4
Thomas J. Broekelmann,3 Douglas M. Tollefsen,4 and Robert P. Mecham3
Departments of 1Biochemistry and 2Cell Biology, Institute of Biology, State University of Campinas-UNICAMP, Campinas, Brazil; and Departments of 3Cell
Biology and Physiology and 4Medicine, Washington University School of Medicine, St Louis, MO
Mice lacking the extracellular matrix protein microfibril-associated glycoprotein-1
(MAGP1) display delayed thrombotic occlusion of the carotid artery following
injury as well as prolonged bleeding from
a tail vein incision. Normal occlusion
times were restored when recombinant
MAGP1 was infused into deficient animals prior to vessel wounding. Blood
coagulation was normal in these animals
as assessed by activated partial thromboplastin time and prothrombin time. Platelet number was lower in MAGP1-deficient
mice, but the platelets showed normal
aggregation properties in response to
various agonists. MAGP1 was not found
in normal platelets or in the plasma of
wild-type mice. In ligand blot assays,
MAGP1 bound to fibronectin, fibrinogen,
and von Willebrand factor, but von Wille-
brand factor was the only protein of the 3
that bound to MAGP1 in surface plasmon
resonance studies. These findings show
that MAGP1, a component of microfibrils
and vascular elastic fibers, plays a role in
hemostasis and thrombosis. (Blood. 2008;
111:4137-4144)
© 2008 by The American Society of Hematology
Introduction
Upon injury to the vessel wall, platelets and protein components of
the coagulation pathway interact with the subendothelial extracellular matrix to arrest bleeding through formation of a hemostatic
plug. The extracellular matrix (ECM) component collagen has long
been known to be an important vessel wall protein that mediates
platelet tethering and promotes firm platelet adhesion. However,
collagen is not the exclusive subendothelial protein responsible for
von Willebrand factor (VWF) interaction.1,2 We now show that
another prominent vessel wall ECM component, microfibrilassociated glycoprotein-1 (MAGP1), also participates in hemostasis.
MAGP1 is a component of fibrillin-rich microfibrils. These 10to 15-nm structures serve multiple functions in the ECM, one of
which is to help structure elastic fibers.3-5 In large vessels,
microfibrils and elastic fibers produced by smooth muscle cells3 are
organized into elastic sheets, or lamellae, that are oriented circumferentially between the cell layers. MAGP1 is also produced by
endothelial cells, which are capable of synthesizing elastic fibers
under appropriate circumstances.6,7 The MAGP1-containing microfibrils remain at the surface of elastic lamellae where they can
easily interact with cells.8-10 In fact, microfibrils have been shown
to promote platelet adhesion and subsequent activation and
aggregation11,12 through an interaction mediated by von
Willebrand factor.13
The biologic function of MAGP1 is unknown. It is a relatively
small protein (⬃20 kDa) compared with fibrillin (⬃350 kDa), its
binding partner in the microfibril. The amino-terminal half of the
molecule contains trisaccharide and tetrasaccharide O-linked sugars, a site for tyrosine sulfation, and one or more glutamine residues
that act as amine acceptors for transglutaminase reactions.14 The
carboxyl-terminal half of the protein contains the molecule’s 13
cysteine residues and defines a fibrillin-binding domain.15
Other proteins that interact with MAGP1 include tropoelastin,16
collagen VI,17 decorin,18 biglycan,19 and Notch1.20 There is no
binding of MAGP1 to collagens I, II, and V.17 MAGP1 can also
influence the way that cells interact with ECM and, in this respect,
functionally resembles thrombospondin, tenascin, and other members of the matricellular family of ECM proteins. There is no
evidence that MAGP1 interacts with integrins, although interactions with other cell-surface matrix-binding proteins have not been
extensively studied.
To better understand the functional role of MAGP1, we used
gene targeting to inactivate the MAGP1 gene (Mfap2) in mice. The
targeting strategy was designed to delete exons 3 to 6, which
encode a portion of the signal peptide and nearly half of the coding
sequence of the molecule. Analysis of the founder lines confirmed
that the targeting strategy had successfully given rise to the
expected mutant allele. Progenies from founder lines were initially
propagated in a mixed background and then bred into the C57BL/6
and Black Swiss lines. Several traits with variable penetrance were
observed in the outbred Black Swiss mice, but most were absent in
the inbred C57 background (J.S.W., Thomas J. Brockelmann,
R.A.P., C.C.W., Fernando Segade, Russell H. Knutsen, and R.P.M.
Deficiency in microfibril-associated glycoprotein-1 leads to complex phenotypes in multiple organ systems. Submitted December
2007). One phenotype that persisted with complete penetrance in
both backgrounds was prolonged thrombosis and bleeding times
after vascular injury. In mice lacking MAGP1, thrombus
formation is delayed despite normal blood coagulation in vitro,
suggesting that MAGP-containing microfibrils play a role in
hemostasis and thrombosis.
Submitted July 20, 2007; accepted February 1, 2008. Prepublished online as
Blood First Edition paper, February 15, 2008; DOI 10.1182/blood-200707-101733.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
*C.C.W. and C.P.V. contributed equally to this work.
© 2008 by The American Society of Hematology
BLOOD, 15 APRIL 2008 䡠 VOLUME 111, NUMBER 8
4137
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4138
WERNECK et al
BLOOD, 15 APRIL 2008 䡠 VOLUME 111, NUMBER 8
Von Willebrand factor antigen
Methods
Reagents and antibodies
Tissue culture reagents were from the Washington University Tissue
Culture Support Center (St Louis, MO). Restriction endonucleases and
other enzymes were purchased from Promega (Madison, WI) or Roche
Applied Science (Indianapolis, IN). Human von Willebrand factor, fibrinogen, and fibronectin were purchased from Haematologic Technologies
(Essex Junction, VT). All other reagents were obtained from Sigma-Aldrich
(St Louis, MO), Fisher Scientific (Pittsburgh, PA), or Bio-Rad (Hercules,
CA) unless noted. Polyclonal MAGP-GST antibody was raised against the
amino-terminal half of bovine recombinant MAGP1.14 Monoclonal anti-V5
antibodies were purchased from Invitrogen (Carlsbad, CA).
Mice
MAGP1-deficient mice were generated by homologous recombination in
RW.4 129/SvJ-derived embryonic stem cells (J.S.W. et al, manuscript
submitted). Embryonic stem (ES) cells heterozygous for the knockout
construct were injected into C57BL/6 blastocysts, which were transferred
into the uteri of pseudopregnant females. The resulting chimeric animals
were mated to C57BL/6 females, and agouti offspring were screened by
Southern blotting of tail genomic DNA for germ-line transmission of the
targeted Mfap2 allele. Knockout-positive animals were initially bred to
Black Swiss females and later backcrossed for 10 generations into the
C57BL/6 strain, which was used for all studies in this report. The
Washington University Animal Studies Committee approved all experimental protocols and all mice were housed in a pathogen-free facility.
Photochemically induced carotid artery thrombosis in mice
The protocol of Eitzman et al21 was followed with slight modification.22
Male mice (10-12 weeks old) were anesthetized with pentobarbital, secured
in the supine position, and placed under a dissecting microscope. Following
a midline cervical incision, the right common carotid artery was isolated
and a Doppler flow probe (model 0.5 VB; Transonic Systems, Ithaca, NY)
was applied. Thrombosis was induced by injection of rose bengal dye
(Fisher Scientific) into the tail vein in a volume of 120 ␮L at a concentration
of 50 mg/kg using a 29-gauge needle. Just before injection, a 1.5 mW,
540 nm green light laser (Melles Griot, Carlsbad, CA) was applied to the
desired site of injury from a distance of 6 cm. The laser remained on until
thrombosis occurred. Flow in the vessel was monitored with the Doppler
probe for 150 minutes from the onset of injury, at which time the animal
was humanely killed. In some experiments, various doses of purified
recombinant MAGP1 or saline (control) were injected into the tail vein
5 minutes before induction of thrombosis.
Tail vein bleeding time
Tail vein bleeding time was determined as described by Broze et al.23
Briefly, mice were anesthetized (ketamine 75 mg/kg; medetomidine 1 mg/
kg intraperitoneally) and placed prone on a warming pad. A transverse
incision was made with a scalpel over a lateral vein at a position where the
diameter of the tail was 2.25 to 2.5 mm. The tail was immersed in normal
saline (37°C) in a hand-held test tube. The time from the incision to the
cessation of bleeding was recorded as the bleeding time.
Activated partial thromboplastin time
Citrate-anticoagulated mouse plasma (100 ␮L) was mixed with 100 ␮L
Alexin HS reagent (Sigma-Aldrich). After a 2-minute preincubation at
37°C, 100 ␮L of 0.25 M CaCl2 was added and the clotting time
was determined.
Prothrombin time
Citrate-anticoagulated mouse plasma (100 ␮L) was warmed to 37°C for
1 minute and then mixed with 200 ␮L Thrombomax HS with calcium
(Sigma-Aldrich) and the clotting time was determined.
An immunoturbidometric assay (STA Liatest VWF; Diagnostica Stago,
Parsippany, NJ) for von Willebrand factor antigen was performed on
citrate-anticoagulated mouse plasma according to the manufacturer’s
instructions. The assay was standardized with normal human plasma.
Analysis of blood cell counts and platelet aggregation
Peripheral blood was obtained by cardiac puncture from anesthetized
12-week-old MAGP1⫹/⫹ and MAGP1⫺/⫺ mice. Platelet number was
obtained using a Hemavet 850 automated hematologic analyzer (CDC
Technologies, Oxford, CT).
Platelet aggregation was monitored by measuring light transmission
through a suspension of stirred washed platelets (1-3 ⫻ 108/mL for mouse
and 2 ⫻ 108/mL for human) or platelet-rich plasma (PRP) using a PAP-4
aggregometer (Bio/Data, Horsham, PA). Aggregation reagents were obtained from Helena Laboratories (Beaumont, TX). Human and mouse
washed platelets and PRP were obtained as described by Cazenave et al.24
Ristocetin cofactor assay
Ristocetin cofactor assays were performed to determine the effect of
recombinant bovine MAGP1 on human von Willebrand factor activity.
The maximum slope of platelet agglutination was measured using a
PAP-4 aggregometer (Bio/Data) according to the manufacturer’s instructions. Reaction mixtures included 400 ␮L lyophilized human platelets
(Bio/Data), 50 ␮L ristocetin (AggRecetin; Bio/Data), and 50 ␮L of each
test sample. The test samples contained 1 part normal pooled plasma and
3 parts MAGP1 (0-100 ␮g/mL) diluted in Tris-buffered saline. Standard
curves were constructed with 12.5% to 100% plasma in the absence of
MAGP1. In experiments with added MAGP1, human plasma was used at
a fixed 50% dilution.
Recombinant bovine MAGP1 protein preparation
Full-length bovine MAGP1 cDNA was cloned into the pQE vector (Qiagen,
Valencia, CA) and M15 cells were transformed according to the manufacturer’s
instructions. Protein was produced and purified using the nickel-NTA agarose
system (Qiagen) by incubating bacterial lysates with nickel-NTA agarose and
washing according to the manufacturer’s instructions. Protein was eluted with
8 M urea buffer, pH 4.5, and analyzed for purity and integrity by Coomassie
stained gels. The eluted protein was then dialyzed against 2 changes, 4 L each, of
50 mM acetic acid. Protein concentration in dialyzed samples was quantified by
amino acid analysis prior to lyophilization.
Blood pressure, vascular distensibility, and cross-sectional
area
Mice were anaesthetized with 1.5% isoflurane inhaled through a nose cone
and kept warm by radiant heat. A catheter (Millar Instruments, Houston,
TX) was inserted into the right common carotid artery and the blood
pressure was monitored for 20 minutes. The isoflurane concentration was
reduced to 0.5% during this period for at least 5 minutes and the average
heart rate and systolic, diastolic, and mean pressure were recorded. In other
mice, the right common carotid artery was exposed and heparin (1000 units/
mL, approximately 50 units/mouse) was injected into the left ventricle to
prevent blood clots during dissection. The artery was removed, cannulated,
and mounted on a pressure and force arteriograph (Danish Myotechnology,
Copenhagen, Denmark). Pressure-diameter curves for the arterial segments
from wild-type and MAGP1⫺/⫺ animals were obtained as described.25,26
For vessel cross-sectional area measurements, wild-type and MAGP⫺/⫺
animals were anesthetized and the left heart was cannulated to allow
perfusion fixation with saline and then Histochoice (Amresco, Solon, OH)
fixative at 100 mmHg. The right carotid artery was then dissected free from
surrounding tissues and excised from the aortic arch to an area distal to the
carotid bifurcation. The vessels were imbedded in paraffin, bifurcation side
down, and cut on a microtome using 5-␮m sections. Triplicate sections
were taken at the bifurcation and every 100 ␮m from the bifurcation.
Verhoeff-van Gieson (VVG) staining of one section from each genotype at a
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BLOOD, 15 APRIL 2008 䡠 VOLUME 111, NUMBER 8
similar level was performed, and stained sections were imaged using
Axiovision software and a Zeiss Axioskop (Carl Zeiss Microimaging,
Thornwood, NY). Image J software (National Institutes of Health [NIH],
Bethesda, MD) was used to manually trace along the internal elastic lamina
(IEL) and the external elastic lamina (EEL) with medial area (expressed in
arbitrary units) defined as the area between the 2 lamellae.
Immunohistochemistry
Carotid arteries from thrombosis studies were harvested and frozen in
OCT (Sakura Finetek, Torrance, CA). Cross sections were obtained, and
the slides were air dried, treated with 4°C acetone, and incubated with
affinity-purified MAGP-GST antibody (1:1000 dilution). After incubation at room temperature for 1 hour, the slides were washed 3 times with
PBS and incubated with biotinylated antirabbit secondary antibody.
Slides were developed using Elite ABC and DAB kits (Vector Laboratories, Burlingame CA).
SDS-PAGE and Western blotting analysis
Proteins of interest were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) with or without 50 mM dithiothreitol.
After electrophoresis, the proteins were transferred from the gels to
nitrocellulose membranes for 1 hour at 70 V in 10 mM 3-[cyclohexylamino]1-propanesulfonic acid, 10% methanol (vol/vol). For Western blotting
analysis, the membranes were first blocked with 5% (wt/vol) nonfat dry
milk, 0.1% Tween 20 in phosphate-buffered saline (blocking buffer) for
1 hour at room temperature. The blots were then incubated with specific
primary antibodies (1:1000) for 1 hour, washed 3 times with blocking
buffer, and incubated with a 1:1000 dilution of a peroxidase-linked donkey
anti–rabbit IgG (GE Healthcare, Little Chalfont, United Kingdom) in
blocking buffer for 1 hour. After washing 3 times with PBS, the blots were
developed with the enhanced chemiluminescence (ECL) system (GE
Healthcare) according to the manufacturer’s instructions.
Immunoblot analysis was also used to assess circulating MAGP1
levels in mouse plasma. Mouse blood was collected from a transected
carotid artery directly into a tube containing ACD anticoagulant. Plasma
(2 ␮L) from either wild-type or MAGP1⫺/⫺ mice was separated by
SDS-PAGE using reducing conditions on a 12.5% polyacrylamide gel.
After transfer to nitrocellulose, the blot was developed using a
polyclonal rabbit antimouse MAGP1 antiserum (1:500 dilution). To
determine the limits of MAGP1 detection, 650 ng/mL, 25 ng/mL, or
1 ng/mL recombinant MAGP1 was added to MAGP1⫺/⫺ plasma and
analyzed by Western blot using identical conditions. Between 1 and
25 nm MAGP1 could be reliably detected using this technique.
Ligand blotting analysis
described.27
Ligand blotting was performed as previously
Briefly, proteins
were run on SDS-PAGE under either reducing or nonreducing conditions
and transferred to nitrocellulose as described in the preceeding paragraph.
The blots were blocked after transfer with 5% (wt/vol) nonfat dry milk and
0.1% Tween 20 in phosphate-buffered saline (blocking buffer) for 1 hour at
room temperature. Purified recombinant MAGP1 was added to the blocking
buffer to a final concentration of 1 ␮g/mL, and the blots were incubated for
90 minutes at 37°C under gentle shaking. Bound protein was detected with
MAGP-GST antibody (1 hour at 37°C) after 3 washes with blocking buffer
and then incubated with secondary antibody as described above. Ligand
blots in which MAGP1 was omitted from the blocking buffer or where
primary antibody was omitted were found to be negative.
Coimmunoprecipitation assay
Bovine MAGP1-V5–6His–tagged protein was expressed by SaOS2 cells
transfected with pcDNA3.1-MAGP1-V5–6His vector.15 After partial purification from the medium using Ni-NTA resin (Qiagen), samples were
dialyzed, lyophilized, and resuspended in 2 mL Tris-buffered saline (TBS).
Fibrinogen, von Willebrand factor, and fibronectin (100 ␮g each) were
iodinated using Iodogen (Pierce, Rockford, IL) and approximately 106 cpm of
each protein was incubated with 200 ␮L of the partially purified SaOS2
PROLONGED THROMBOSIS IN MAGP1-DEFICIENT MICE
4139
cell–derived MAGP1-V5–6His–tagged protein in 300 ␮L TBS, 2 mM CaCl2 for
2 hours at room temperature. The specific activity of each protein was as follows:
fibronectin ⫽ 937 000 cpm/␮g; fibrinogen ⫽ 410 000 cpm/␮g; VWF ⫽ 693 000
cpm/␮g. To reduce nonspecific binding, immobilized protein A gel (40 ␮L,
Immunopure; Pierce) was added and incubated for 1 hour. After centrifugation,
the supernatant was incubated with 40 ␮L immobilized protein A gel plus
anti-V5 antibody at 4°C overnight. The samples were centrifuged and the pellets
were washed 4 times with 2 mM CaCl2 in TBS buffer. SDS sample buffer
(50 ␮L) was added and the samples heated at 100°C for 5 minutes. An aliquot of
the mixture (20 ␮L) was subjected to SDS-PAGE on a 5% polyacrylamide gel
under reducing or nonreducing conditions followed by autoradiography. For
competition experiments, a 10-fold excess of unlabeled protein was added during
the overnight incubation.
Surface plasmon resonance assays
Interactions between proteins were studied by surface plasmon resonance
using the BIAcore X system, at 25°C (Uppsala, Sweden). Recombinant
bovine MAGP1 was covalently immobilized on the BIAcore CM-5 sensor
chip (carboxylated dextran matrix) according to the manufacturer’s instructions. The CM-5 chip was activated with a 1:1 mixture of 75 mg/mL
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 11.5 mg/mL Nhydroxysuccinimide for 7 minutes. MAGP1 (36 ␮g/mL in 10 mM sodium
acetate, pH 4.3) was injected over the CM-5 sensor chip for 7 minutes at a
flow rate of 10 ␮L/min (25°C). Remaining active groups on the matrix were
blocked with 1 M ethanolamine/HCl, pH 8.5. Immobilization of MAGP1
resulted in a surface concentration on the sensorchip of 3.9 ng/mm2.
Analytes were prepared in HBS-EP buffer (10 mM HEPES, pH 7.4,
150 mM NaCl, 3.4 mM EDTA and 0.005% [v/v[surfactant P20; BIAcore)
and injected at a flow rate of 20 ␮L/min. The nonlinear fitting of association
and dissociation curves according to a 1:1 model was used for the
calculation of kinetic constants (BIAevaluation software, version 3.2;
BIAcore). Individual experiments were performed 3 times.
Statistical analysis
Statistical significance was determined with the Student 2-tailed t test for
independent samples. P at less than .05 was considered significant.
Results
MAGP1-deficient mice have prolonged thrombosis and
bleeding times after vascular injury
Figure 1A compares the time to thrombus formation after photochemical injury of the common carotid artery of wild-type and
MAGP1-deficient mice. In MAGP1⫺/⫺ mice the occlusion time
was 99 plus or minus 16.3 minutes (mean ⫾ SD) compared with 57
plus or minus 6.7 minutes for wild-type animals (P ⬍ .005).
Interestingly, mice heterozygous for the MAGP1 deletion
(MAGP1⫹/⫺) showed intermediate values (73 ⫾ 17.8 minutes),
indicative of a gene dosage effect. Figure 1B is a representative
recording comparing carotid blood flow in wild-type and MAGP1⫺/⫺
mice. In addition to the prolonged time to cessation of flow, large
irregular spikes were frequently observed in the MAGP1⫺/⫺
animals. This is in contrast to what occurs in the wild-type vessel,
which shows a rapid, almost linear, cessation of flow once the clot
begins to form.
To examine whether the absence of MAGP1 also affects
hemostasis in the venous system, we measured the bleeding time in
the mouse tail vein.23 After incision, the tail was immersed in saline
kept at 37°C, and the bleeding time was taken as the time required
for the bleeding to stop. The bleeding times in the MAGP1deficient mice were almost double those of their MAGP1 wild-type
siblings (150 ⫾ 19 vs 81 ⫾ 15 seconds) showing that the absence
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4140
BLOOD, 15 APRIL 2008 䡠 VOLUME 111, NUMBER 8
WERNECK et al
+bMAGP
+bMAGP Ab
–bMAGP
+bMAGP Ab
A
MAGP-/-
MAGP+/+
B
Figure 2. Immunohistochemistry showing localization of infused, recombinant
bovine MAGP1 at the site of vascular injury. (A) Photomicrographs on the left are
cross sections of carotid arteries from MAGP1⫹/⫹ and MAGP1⫺/⫺ mice infused with
50 ␮g/kg recombinant bovine MAGP1 5 minutes prior to laser-induced injury. Vessels
were harvested after complete cessation of blood flow and frozen sections immunostained using a bovine MAGP1-specific antibody. Staining is evident in the thrombus
of both genotypes but is particularly prominent in the internal elastic lamina in the
MAGP1⫺/⫺ mouse (➚, and at higher power in panel B). Panels on the right show
staining with antibovine MAGP1 of injured vessels from animals not injected with
bovine MAGP1.
Figure 1. Effect of MAGP1 deficiency on thrombotic occlusion of the carotid
artery. (A) Blood flow in the common carotid artery was monitored continuously with
an ultrasonic flow probe. Local endothelial injury was induced by application of a
540-nm laser beam to the carotid artery followed by injection of rose bengal dye
(50 mg/kg) into the lateral tail vein. Shown is the time to occlusion of blood flow
following injury. Error bars indicate mean plus or minus standard deviation (n ⫽ 8 for
each group). **P ⬍ .005, *P ⬍ .05. (B) Representative blood flow recordings showing
the delayed occlusion time and stochastic flow pattern in MAGP1⫺/⫺ animals. Rose
bengal dye was injected at time ⫽ 0 minutes. (C) Infusion of recombinant MAGP1
re-establishes normal occlusion time in MAGP1⫺/⫺ mice. Recombinant bovine
MAGP1 was injected into the tail vein as a single bolus 5 minutes before rose bengal
injection. An equivalent bolus of saline served as the control. Error bars indicate mean
plus or minus SD (n ⫽ 6 for each group).
of MAGP1 affects hemostasis in both high (arterial) and low
(venous) blood pressure systems.
Normal occlusion time is restored in the MAGP1-deficient
mouse by injection of recombinant MAGP1
Injection of recombinant bovine MAGP1 into the tail vein
5 minutes before laser injury was able to reverse the extended
occlusion times documented in MAGP1-deficient mice. As seen
in Figure 1C, a MAGP1 dose of 50 ␮g/kg body weight was
sufficient to return the occlusion time values to those observed
in wild-type animals (⬃60 minutes). The effect was dose
dependent over the range of 0 to 100 ␮g/kg body weight. The
dose-dependent response is in agreement with results shown in
Figure 1A demonstrating that MAGP⫹/⫺ mice have occlusion
times intermediate between wild-type and MAGP⫺/⫺ animals.
The recombinant protein had no effect at 50 ␮g/kg on the
occlusion time of wild-type animals (not shown).
To insure that the injected amounts of MAGP1 were higher than
endogenous circulating MAGP1 levels, plasma from wild-type
animals was probed by immunoblot using antibodies to mouse
MAGP1. Plasma from MAGP1⫺/⫺ animals served as a negative
control. MAGP1⫺/⫺ plasma supplemented with known concentrations of recombinant MAGP1 served as a positive control and
defined the limits of detection for the assay. No MAGP1 was
detected in wild-type plasma using assay conditions that detected
as little as 25 ng MAGP1/mL plasma. Hence, even the lowest
level of MAGP1 infused in these experiments (25 ␮g/kg or
approximately 0.8 ␮g/mL plasma) is in excess over any endogenous protein that might exist at concentrations below those
detected in our assay.
Using an antibody that recognizes bovine but not mouse
MAGP1, immunohistochemistry of frozen sections of carotid
artery from MAGP1-injected mice documented recombinant protein in the thrombus and vessel wall of both wild-type and
MAGP1⫺/⫺ animals (Figure 2). Interestingly, intense reactivity was
associated with the internal elastic lamina of the MAGP1⫺/⫺
injured vessel with less detectable protein at the injury site in
wild-type animals. The presence of recombinant protein at the
thrombus-vessel wall interface is consistent with injected MAGP1
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BLOOD, 15 APRIL 2008 䡠 VOLUME 111, NUMBER 8
PROLONGED THROMBOSIS IN MAGP1-DEFICIENT MICE
MAGP1ⴚ/ⴚ (n)
P
aPTT, s
28.5 ⫾ 1.7 (4)
29.4 ⫾ 0.7 (3)
.57
PT, s
11.6 ⫾ 1.5 (3)
12.6 ⫾ 1.2 (4)
.33
VWF antigen, %*
42.3 ⫾ 6.4 (3)
44.5 ⫾ 17.1 (4)
.82
A
*Percentage of VWF concentration in normal human plasma.
P values are for comparison between genotypes.
binding to the subendothelial region after injury and facilitating
thrombus formation and anchorage to the vessel wall.
MAGP1 is not present in platelets
The possibility that platelets might contain MAGP1 was investigated by immunoblot of whole washed bovine platelet extracts
prepared as described by Cazenave et al.24 Bovine platelets were
40
30
20
10
B
Slope
Because abnormalities in coagulation cascade pathways can
interfere with hemostasis and thrombus formation, we determined the activated partial thromboplastin time and the prothrombin time to assess the functionality of the intrinsic and extrinsic
clotting pathways, respectively. Table 1 shows that the clotting
parameters are normal in the MAGP1-deficient mice, indicating
that the clotting pathways were unaffected by the absence of
MAGP1. Von Willebrand factor antigen levels were also similar
in wild-type and MAGP1-deficient mice (Table 1); in both
cases, the mean levels were approximately 40% that of human
pooled plasma.
Platelets play an important role during hemostatic plug
formation and many bleeding problems are related to abnormal
platelets. Therefore, we inspected several platelet parameters in
MAGP1 ⫺/⫺ mice. While MAGP1-deficient mice have a
significantly lower (P ⬍ .03) number of platelets
(770 ⫾ 202 ⫻109/L [770 000 ⫾ 202 000 platelets/␮L] blood,
n ⫽ 8) compared with wild-type animals (1020 ⫾ 159 ⫻ 109/L
[1 020 000 ⫾ 159 000 platelets/␮L] blood, n ⫽ 7), platelet function was essentially normal. Aggregation assays using plateletrich plasma (PRP) from MAGP1⫺/⫺ and wild-type mice with the
agonists collagen (Col, 10 ␮g/mL), adenosine 5⬘-diphosphate
(ADP, 20 ␮M), arachidonic acid (AA, 500 ␮g/mL), and epinephrine (Epi, 300 ␮M) showed no difference between the 2
genotypes. Figure 3A shows representative data for aggregation
of platelets from wild-type and MAGP1-deficient animals in
response to increasing concentrations of collagen. The results
indicated that platelets from both genotypes respond similarly.
We also tested whether MAGP1 had a direct effect on human
platelet aggregation by conducting aggregation studies with the
agonists mentioned above in the presence or absence of
recombinant MAGP1 (50 ␮g/mL). The results showed that, in
both cases, there were no differences in any of the aggregation
parameters, confirming that in vitro neither the absence nor
presence of MAGP1 alters platelet aggregation (Figure 3B).
Additional experiments were performed to determine the effect
of recombinant bovine MAGP1 on human von Willebrand factor
activity. As shown in Figure 3C, the ristocetin cofactor activity of
normal human plasma was unaffected by the presence of MAGP1
at concentrations of 1 to 100 ␮g/mL. These results suggest that
MAGP1 does not modulate the interaction between von Willebrand
factor and platelets.
Magp1+/+
50
0
5
10
20
50
ADP ADP+
Epi Epi+
AA AA+
Collagen (µg)
100
80
60
40
20
0
Col Col+
MAGP1
MAGP1
MAGP1
MAGP1
60
C
Ristocetin Cofactor %
MAGP1-deficient mice have normal coagulation and platelet
function in vitro
Magp1-/-
60
Final Aggregation %
MAGP1ⴙ/ⴙ (n)
Final Aggregation %
Table 1. Hemostatic parameters for MAGPⴙ/ⴙ and MAGPⴚ/ⴚ mice
4141
50
40
30
20
10
0
Control
1 µg/mL 10 µg/mL 100 µg/mL
[MAGP1]
Figure 3. Platelet function in MAGP1-deficient mice. (A) The percentage of
platelets that aggregate in response to different levels of collagen is the same for
both genotypes. (B) The presence of MAGP1 (50 ␮g/mL) has no effect on human
platelet aggregation induced by various agonists, including collagen (Col,
10 ␮g/mL), adenosine 5⬘-diphosphate (ADP, 20 ␮M), arachidonic acid (AA,
500 ␮g/mL), and epinephrine (Epi, 300 ␮M). Aggregation was monitored by
measuring light transmission through a suspension of stirred washed platelets
(1-3 ⫻ 108/mL for mouse and 2 ⫻ 108/mL for human) using an aggregometer.
Data are expressed as either the slope of the aggregation curve or as percentage
of cells that underwent aggregation. (C) Recombinant bovine MAGP1 has no
effect on the ristocetin cofactor activity of human plasma. All experiments
contained normal human plasma diluted 1:1 with Tris-buffered saline, yielding
50% ristocetin cofactor activity in the control sample. Error bars indicate mean
plus or minus standard deviation of 4 experiments. None of the values differed
significantly (P ⱖ .25).
used to obtain sufficient cells for rigorous biochemical analysis.
Figure 4A shows a Coomassie blue–stained gel indicating the
distribution and relative amounts of protein in the platelet extract
(lanes 1,2). Lane 3 contains partially purified recombinant MAGP1
expressed by transfected SaOS2 cells as a positive control.
Although not visible by Coomassie blue staining (lane 3), SaOS2
cell–derived MAGP1 protein was readily detected by immunoblot
with the MAGP1-specific antibody (Western blot, lane 3). There
was no immunoreactive MAGP1 detected in the platelet extracts
(Western blot, lanes 1,2).
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4142
BLOOD, 15 APRIL 2008 䡠 VOLUME 111, NUMBER 8
WERNECK et al
A
A
B
Std
1
2
3
1
2
3
– DTT
+DTT
– DTT
STD FN vWF Fb FN vWF Fb FN vWF
+DTT
Fb
FN
vWF
Fb
75 kDa
250 kD
37 kDa
MAGP1
25 kDa
150 kD
100 kD
Coomassie Blue
Western Blot
Coomassie Blue
Figure 4. Immunoblot of platelet extracts. Bovine washed platelets were boiled in
SDS sample buffer and subjected to SDS-PAGE under reducing and nonreducing
conditions. Protein bands were visualized by Coomassie blue staining or transferred
to nitrocellulose for immunodetection with an antibody to bovine MAGP1.
(A) Coomassie blue–stained gel. (B) Immunoblot analysis of proteins in panel A after
transfer to nitrocellulose. Lane 1: Platelet extract under nonreducing conditions (no
DTT). Lane 2: Platelet extract under reducing conditions (⫹ DTT). Lane 3: Semipurified bovine MAGP1 expressed by mammalian SaOS2 cells (⫹ DTT).
B
1 2
Ligand blot
3 4 5 6 7 8
9
250 kD
150 kD
MAGP1ⴚ/ⴚ mice have normal vessel structure and
blood pressure
100 kD
To determine whether the absence of MAGP1 alters vessel
structure and cardiovascular hemodynamics, which might impact
thrombus formation, blood pressure and vascular compliance were
compared between genotypes. Figure 5 shows that pressurediameter curves are identical for wild-type and MAGP1⫺/⫺ mice,
confirming that vessel diameter is the same for both animals at any
given pressure. There were also no significant differences in medial
cross-sectional area (287.2 ⫾ 91 units for wild-type vs 341 ⫾ 97
for MAGP1⫺/⫺) or in blood pressure (systolic/diastolic ⫽ 128/84
for wild-type vs 130/89 for MAGP⫺/⫺) between genotypes.
MAGP1 interacts with fibrinogen, fibronectin, and von
Willebrand factor
That MAGP1’s role in hemostatic plug formation does not appear
to be through a direct effect on platelet function suggests a possible
interaction with some other plasma or ECM protein. Candidate
plasma proteins were tested for binding to MAGP1 using 3
different assay techniques: ligand blots, coimmunoprecipitation,
and surface plasmon resonance binding. The results in Figures
6A,B document an interaction between MAGP1 and both von
Willebrand factor and fibrinogen. In the immunoprecipitation
Outer Diameter (microns)
900
800
700
600
Figure 6. Analysis of MAGP1 interaction with selected plasma proteins.
MAGP1’s ability to interact with plasma proteins was assayed by ligand blot (A) and
coimmunoprecipitation (B). (A) Lanes: FN indicates fibronectin; VWF, von Willebrand
factor; Fb, fibrinogen; and STD, molecular weight standards. The left side of the panel
is a Coomassie blue–stained gel (⫾ DTT) of the separated proteins. The right side
shows a ligand blot of the same proteins after transfer to nitrocellulose, incubation
with MAGP1, and bound MAGP1 detected with an antibody to MAGP1 after
extensive washing to remove unbound protein. (B) SDS-PAGE autoradiogram
showing coprecipitation of [125I]-labeled plasma proteins and V5-tagged MAGP1.
Lanes: 1, fibronectin with V5 antibody only (negative control); 2, fibronectin coprecipitated with MAGP1-V5 using V5 antibody; 3, fibronectin coprecipitated with MAGP1-V5
as in lane 2, but in the presence of 10-fold excess unlabeled fibronectin; 4, fibrinogen
with V5 antibody; 5, fibrinogen coprecipitated with MAGP1-V5 using V5 antibody; 6,
fibrinogen coprecipitated with MAGP1-V5 as in lane 5, but in the presence of 10-fold
excess unlabeled fibrinogen; 7, von Willebrand factor with V5 antibody; 8, von
Willebrand factor coprecipitated with MAGP1-V5 using V5 antibody; and 9, von
Willebrand factor coprecipitated with MAGP1-V5 as in lane 8, but in the presence of
10-fold excess von Willebrand factor. Vertical lines have been inserted to indicate
repositioned gel lanes.
experiments, the specificity of binding was confirmed by showing
that the addition of excess unlabeled fibrinogen or VWF to the
precipitation reaction blocked binding of labeled protein. No
interaction was observed between MAGP1 and fibronectin by
immunoblot, but an interaction with fibronectin was detected in the
coimmunoprecipitation experiments.
Further characterization of the protein interactions was obtained
using surface plasmon resonance with MAGP1 coupled to the sensor
chip and fibrinogen, fibronectin, or VWF injected as analyte. Under the
assay conditions tested, only von Willebrand factor bound to MAGP1,
with a calculated Kd of 2.05 ⫻ 10⫺7 M (Figure 7).
500
400
300
Discussion
0
25
50
75
100
125
150
175
Pressure (mm Hg)
Figure 5. Outer diameter versus pressure for the right carotid artery in
wild-type and MAGPⴚ/ⴚ mice. Pressure-diameter curve showing that the carotid
artery in wild-type (—) and MAGP⫺/⫺ (哹) animals has identical mechanical
properties, identical diameters, and equal pressures.
MAGP1 is an abundant protein found in elastic fibers in blood
vessels and in other elastic tissues. Its ability to bind multiple
proteins suggests that MAGP1 could be a bridging molecule to
facilitate the association and assembly of complex matrix
structures.16,18 As we show in this report, mice that lack MAGP1
display a bleeding abnormality characterized by prolonged tail
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BLOOD, 15 APRIL 2008 䡠 VOLUME 111, NUMBER 8
PROLONGED THROMBOSIS IN MAGP1-DEFICIENT MICE
700
600
500
RU
400
300
200
100
0
-100
-100
0
-200
100
200
300
400
500
600
700
Time (sec)
Figure 7. Characterization of MAGP1 and VWF interactions using surface
plasmon resonance. Different concentrations of von Willebrand factor were injected
over MAGP1 immobilized on a BIAcore CM-5 sensor chip. Sensorgram shows 6
different analyte concentrations (0.052 ␮M, 0.105 ␮M, 0.21 ␮M, 0.32 ␮M, 0.42 ␮M,
and 0.85 ␮M). One representative experiment is shown. The response difference
(the difference between experimental and control flow cells) is given in resonance
units (RU).
vein bleeding time and delayed thrombotic occlusion of the
carotid artery despite having normal blood coagulation parameters. Furthermore, platelet aggregation induced by various
agonists is similar between wild-type and knockout animals,
suggesting normal in vitro platelet function in the absence of
MAGP1. The absence of MAGP1 in platelets is consistent with
this conclusion. The bleeding and thrombotic abnormalities
have been observed in 2 genetic backgrounds (C57BL/6, this
study; and Black Swiss, not shown). MAGP1-deficient mice do
not manifest spontaneous hemostatic abnormalities in the absence of vascular injury.
The mechanism responsible for abnormal thrombotic occlusion is not immediately evident, but one possibility is that
MAGP1 deficiency diminishes the ability of the thrombus to
adhere to the injured blood vessel wall. Because MAGP1 does
not interact with integrins, it is unlikely that thrombus stabilization occurs directly through MAGP1-mediated integrin activation of platelets. Detection of MAGP1 in the thrombus and in the
vessel wall of MAGP1⫺/⫺ mice following infusion of MAGP1
protein suggests that it exerts its stabilizing effects by direct
interactions between components of the thrombus and components of the vascular matrix. Indeed, we have shown that
MAGP1 can interact with several proteins important to thrombus formation and platelet adhesion, including fibrinogen,
fibronectin, and VWF. This unusual property suggests that
MAGP1 functions to anchor protein components of the thrombus to the structural matrix of the vascular wall.
The location of MAGP1 in the vessel wall makes it ideal to
serve an anchoring function for the forming clot. MAGP1 is
produced by endothelial cells in culture where it colocalizes
with fibrillin to form a honeycomb network underneath the cell
layer.7 As a component of elastic fiber microfibrils, MAGP1 is
enriched in the elastic lamellae found in all elastic and muscular
arteries. In these vessels, the subendothelial matrix consists of
the endothelial basement membrane in close association with
the internal elastic lamina, such that upon endothelial injury or
denudation, the internal elastic lamina will be exposed and will
be a major surface for thrombus attachment. In smaller arteries
and veins, elastic fibers and microfibrils (and hence MAGP1)
are present in the vessel wall even though they do not form the
concentric lamellae seen in muscular and elastic arteries. Our
data are consistent with several studies showing that microfibrils
4143
promote platelet adhesion and aggregation11,12 through an
interaction mediated by VWF.13,28 While the microfibrillar
component that promotes VWF binding was not characterized in
these early studies, our findings identify this protein as MAGP1.
MAGP1 resembles the thrombospondins (TSPs) in its ability
to bind multiple proteins and in its propensity to modulate
cell-matrix interactions. Even the knockout animals share
similarities in that, like MAGP1-null mice, TSP-1– and TSP-2–
null mice have hemostatic defects.29 Mice that lack TSP-1, for
example, have enhanced thrombus embolization30 caused by
defective thrombus adherence to the injured blood vessel wall,
similar to what we have described for the MAGP1-deficient
mouse. TSP2 mice have a bleeding diathesis that manifests as a
prolonged bleeding time. In characterizing the TSP-1 phenotype, no indication was found for a role for TSP-1 in platelet
aggregation or in coagulation-mediated thrombosis, but evidence was presented for protection by TSP-1 of VWF cleavage
by ADAMTS13.30 Whether MAGP1 can serve a similar function
through its ability to bind VWF must await more detailed
mapping studies of MAGP1-binding sites within the VWF
molecule.
In conclusion, the results presented in this study implicate
MAGP1 and, hence, microfibrils and elastic fibers, in hemostasis
and thrombosis. Mice lacking MAGP1 have prolonged bleeding
after transection of the tail vein and prolonged thrombotic occlusion of the carotid artery after endothelial injury. Since in vitro
assays of blood coagulation and platelet function are normal in
these mice, the defect in MAGP1 deficiency probably involves
impaired interaction of the developing thrombus with components
of the vessel wall.
Acknowledgments
We thank Chris Ciliberto for excellent technical support, Yifang
Zhao for assistance with tissue processing and immunohistology,
and Russell Knutsen for the vascular compliance and blood
pressure studies. We also thank Dr Evan Sadler for providing the
von Willebrand factor antibody.
This work was supported by NIH grants HL71960, HL74138,
and HL53325 (R.P.M.), HL55520 (D.M.T.), and FAPESP no.
2006/06560-4 (C.C.W.). J.S.W. was supported by training grant
T32 HL007873 and by a National Science Foundation Graduate
Research Fellowship.
Authorship
Contribution: C.C.W. and T.J.B. performed in vitro assays and
platelet characterization studies; C.P.V. was responsible for the
carotid injury studies; J.S.W. prepared and purified recombinant
MAGP1 protein; A.S. contributed the immunohistology; R.A.P.
was responsible for the MAGP1 knockout mice; and D.M.T and
R.P.M. performed scientific oversight and data interpretation.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Robert P. Mecham, Department of Cell Biology and Physiology, Washington University School of Medicine,
Campus Box 8228, 660 South Euclid Ave, St Louis, MO 63110;
e-mail: [email protected].
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4144
BLOOD, 15 APRIL 2008 䡠 VOLUME 111, NUMBER 8
WERNECK et al
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2008 111: 4137-4144
doi:10.1182/blood-2007-07-101733 originally published
online February 15, 2008
Mice lacking the extracellular matrix protein MAGP1 display delayed
thrombotic occlusion following vessel injury
Claudio C. Werneck, Cristina P. Vicente, Justin S. Weinberg, Adrian Shifren, Richard A. Pierce,
Thomas J. Broekelmann, Douglas M. Tollefsen and Robert P. Mecham
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