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Mice Lacking the Extracellular Matrix Protein MAGP1 Display Delayed

Thrombotic Occlusion Following Vessel Injury

Claudio C. Werneck1*, Cristina P. Vicente2*, Justin S. Weinberg3, Adrian Shifren4,

Richard A. Pierce4, Thomas J. Broekelmann3, Douglas M. Tollefsen4, and Robert P. Mecham3

From the Departments of 1Biochemistry and 2Cell Biology, Institute of Biology, State

University of Campinas - UNICAMP, 13083-970, Campinas, SP, Brazil; 3Cell Biology

and Physiology and 4Medicine, Washington University School of Medicine,

St. Louis, Missouri, 63110

* These authors contributed equally to this work.

Short Title: Prolonged thrombosis in MAGP1-deficient mice

Corresponding author:

Dr. Robert P. Mecham

Washington University School of Medicine

Department of Cell Biology and Physiology

660 South Euclid Ave, Campus Box 8228

St. Louis, MO 63110

Email: [email protected]

Blood First Edition Paper, prepublished online February 15, 2008; DOI 10.1182/blood-2007-07-101733

Copyright © 2008 American Society of Hematology

For personal use only. at WASHINGTON UNIV SCH MEDICINE on March 12, 2008. www.bloodjournal.orgFrom

http://bloodjournal.hematologylibrary.org

http://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtl

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Abstract

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 Willebrand factor was the only protein of the three 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.




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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,

microfibril-associated glycoprotein-1 (MAGP1), also participates in hemostasis.

MAGP1 is a component of fibrillin-rich microfibrils. These 10-15 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 cells 3 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 aggregation 11,12 through an interaction mediated by von

Willebrand factor13.

The biological function of MAGP1 is unknown. It is a relatively small protein

(~20 kDa) compared to fibrillin (~350 kDa), its binding partner in the microfibril. The

amino-terminal half of the molecule contains tri- 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




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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 3-6, 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. Progeny 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

(Weinberg et al., manuscript submitted). 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.

Materials and 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 Inc. (Essex Junction, VT). All other reagents were obtained

from Sigma (St. Louis, MO), Fisher (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 (Weinberg et al., manuscript submitted). ES cells

heterozygous for the knockout construct were injected into C57BL/6 blastocysts, which




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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 germline 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 al. 21 was followed with slight modification 22. Male

mice (10 to 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, Pittsburgh, PA) into the tail vein in a

volume of 120 µl 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 five 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 IP) 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 2.25-2.5 mm. The tail was immersed in

normal saline (37oC) 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 (aPTT)

Citrate-anticoagulated mouse plasma (100 µL) was mixed with 100 µL Alexin HS




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reagent (Sigma, St. Louis, MO). After a 2-minute preincubation at 37°C, 100 µL of 0.25

M CaCl2 was added and the clotting time was determined.

Prothrombin Time (PT)

Citrate-anticoagulated mouse plasma (100 µl) was warmed to 37 °C for 1 min and

then mixed with 200 µl of Thrombomax HS with calcium (Sigma, St. Louis, MO) and the

clotting time was determined.

Von Willebrand Factor Antigen

An immuno-turbidometric 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 -/- mice. Platelet number was obtained using a Hemavet 850

automated hematological analyzer (CDC Technologies Inc., Oxford, CT).

Platelet aggregation was monitored by measuring light transmission through a

suspension of stirred washed platelets (1-3x108/mL for mouse and 2x108/mL for human)

or platelet rich plasma (PRP) using a PAP-4 aggregometer (Bio/Data Corporation,

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 µl of lyophilized human

platelets (Bio/Data), 50 µl of ristocetin (AggRecetin, Bio/Data), and 50 µl of each test

sample. The test samples contained 1 part normal pooled plasma and 3 parts MAGP1 (0-

100 µg/ml) diluted in Tris-buffered saline. Standard curves were constructed with 12.5-

100% plasma in the absence of MAGP1. In experiments with added MAGP1, human




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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,

Inc., 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 two changes, 4L 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 Inc., 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 five 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 unit/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 fixative at 100 mm Hg. 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 5µm sections. Triplicate sections were taken at the

bifurcation and every 100 µm from the bifurcation. Verhoeff-van Gieson (VVG) staining




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of one section from each genotype at a similar level was performed and stained sections

imaged using Axiovision software and a Zeiss Axioskop. Image J software (NIH) 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 two lamellae.

Immunohistochemistry

Carotid arteries from thrombosis studies were harvested and frozen in OCT

(Sakura Finetek, Torrance, CA). Cross-sections were obtained, the slides were air dried,

treated with 4˚C acetone, and incubated with affinity purified MAGP-GST antibody

(dilution 1:1000). After incubation at room temperature for 1 h, the slides were washed

three times with PBS and incubated with biotinylated anti-rabbit 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 SDS-PAGE with or without 50 mM

dithiothreitol. After electrophoresis, the proteins were transferred from the gels to

nitrocellulose membranes for 1 h at 70 V in 10 mM 3-[cyclohexylamino]- 1-

propanesulfonic acid, 10% methanol (v/v). For western blotting analysis, the membranes

were first blocked with 5% (w/v) nonfat dry milk, 0.1% Tween 20 in phosphate-buffered

saline (blocking buffer) for 1 h at room temperature. The blots were then incubated with

specific primary antibodies (1:1000) for 1 h, washed three times with blocking buffer and

incubated with a 1:1000 dilution of a peroxidase linked donkey anti-rabbit IgG

(Amersham Biosciences, Piscataway, NJ) in blocking buffer for 1 h. After washing three

times with PBS, the blots were developed with the ECL system (Amersham Biosciences,

Piscataway, NJ) 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. Two µl of plasma from either wild-type or MAGP1-/-

mice were 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 anti-mouse MAGP1 antiserum (1:500 dilution). To determine the limits




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of MAGP1 detection, 650 ng/ml, 25 ng/ml, or 1 ng/ml recombinant MAGP1 were 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

Ligand blotting was performed as previously described 27. Briefly, proteins were

run on SDS-PAGE under either reducing or non-reducing conditions and transferred to

nitrocellulose as described above. The blots were blocked after transfer with 5% (w/v)

nonfat dry milk and 0.1% Tween 20 in phosphate-buffered saline (blocking buffer) for 1

h at room temperature. Purified recombinant MAGP1 was added to the blocking buffer to

a final concentration of 1 µg/mL, and the blots were incubated for 90 min at 37 °C under

gentle shaking. Bound protein was detected with MAGP-GST antibody (1 h at 37 °C)

after three 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.

Co-Immunoprecipitation 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, Valencia, CA), samples were dialyzed,

lyophilized and re-suspended in 2 mL Tris-buffered saline (TBS).

Fibrinogen, von Willebrand factor, and fibronectin (100 g each) were iodinated

using Iodogen (Pierce, Rockford, IL) and ~ 106 cpm of each protein was incubated with

200 l of the partially purified SaOS2 cell-derived MAGP1-V5-6His tagged protein in

300 l of TBS, 2 mM CaCl2 for 2 h at room temperature. The specific activity of each

protein was: fibronectin=937,000 cpm/g; fibrinogen=410,000 cpm/g; vWF=693,000

cpm/g. To reduce nonspecific binding, immobilized protein A gel (40 µl)

(Immunopure, Pierce, Rockford, IL) was added and incubated for 1 h. After

centrifugation, the supernatant was incubated with 40 µl of immobilized protein A gel

plus anti V5 antibody at 4 0C overnight. The samples were centrifuged and the pellets

were washed 4 times with 2 mM CaCl2 in TBS buffer. 50 µl of SDS sample buffer was

added and the samples heated at 100 0C for 5 min. An aliquot of the mixture (20 l) was




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subjected to SDS-PAGE on a 5% polyacrylamide gel under reducing or non-reducing

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 N-hydroxysuccinimide for 7 min. MAGP1 (36 µg/mL in 10 mM sodium acetate,

pH 4.3) was injected over the CM-5 sensor chip for 7 min at a flow rate of 10 µl/min (25 oC). 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 were

injected at a flow rate of 20 µl/min. The non-linear 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). Individual experiments were performed three

times.

Statistical Analysis

Statistical significance was determined with the Student 2-tailed t test for

independent samples. P <0.05 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 ± 16.3 minutes (mean ± SD) compared to 57 ± 6.7

minutes for wild-type animals (P<.005). Interestingly, mice heterozygous for the

MAGP1 deletion (MAGP1+/-) showed intermediate values (73 ± 17.8 minutes),




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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 37º C and the bleeding time was taken as the time required

for the bleeding to stop. The bleeding times in the MAGP1-deficient mice were almost

double those of their MAGP1 wild-type siblings (150±19 vs. 81±15 seconds) showing

that the absence 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 µg/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 µg/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 µg/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 per ml of plasma. Hence, even the lowest level of




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MAGP1 infused in these experiments (25 µg/kg or approximately 0.8 µg/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 binding to the subendothelial

region after injury and facilitating thrombus formation and anchorage to the vessel wall.

MAGP1-deficient mice have normal coagulation and platelet function in vitro.

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 I 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 I); 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<0.03) number of platelets (770,000±202,000 platelets/µl of blood,

n=8) when compared with wild-type animals (1,020,000±159,000 platelets/µl of blood,

n=7), platelet function was essentially normal. Aggregation assays using platelet-rich

plasma (PRP) from MAGP1-/- and wild-type mice with the agonists collagen (Col, 10

µg/mL), adenosine 5’-diphosphate (ADP, 20 µM), arachidonic acid (AA, 500 µg/mL)

and epinephrine (Epi, 300 µM) showed no difference between the two genotypes. Figure

3A shows representative data for aggregation of platelets from wild-type and MAGP1-




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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 µg/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 1-100 µg/ml. These results suggest that MAGP1 does not

modulate the interaction between von Willebrand factor and platelets.

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 used in order to obtain sufficient cells for rigorous

biochemical analysis. Panel A of Figure 4 shows a Coomassie blue-stained gel

indicating the distribution and relative amounts of protein in the platelet extract (lanes 1

and 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 and 2).

MAGP1-/- mice have normal vessel structure and blood pressure

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

pressure-diameter 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




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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

three different assay techniques: ligand blots, co-immunoprecipitation, and surface

plasmon resonance binding. The results in Figures 6A and 6B document an interaction

between MAGP1 and both von Willebrand factor and fibrinogen. In the

immunoprecipitation 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 co-

immunoprecipitation 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 2.05x10-7 M (Figure 7).

Discussion

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 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.




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The absence of MAGP1 in platelets is consistent with this conclusion. The bleeding and

thrombotic abnormalities have been observed in two 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 co-localizes 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 promote

platelet adhesion and aggregation 11,12 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.




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MAGP1 resembles the thrombospondins (TSP) 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

embolization 30 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.




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Acknowledgement

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

Willibrand factor antibody. The research reported in this manuscript was supported by

National Institutes of Health HL71960, HL74138 and HL53325 to R.P.M., HL55520 to

D.M.T., and FAPESP #2006/06560-4 to 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 and J.S.W. prepared and

purified recombinant MAGP1 protein. A.S. contributed the immunohistology and R.A.P.

is responsible for the MAGP1 knockout mice. D.M.T and R.P.M. performed scientific

oversight and data interpretation. C.W. and C.P.V. contributed equally to this work.

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.,

Saint Louis, MO 63110; email: [email protected].




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Table I Hemostatic Parameters for MAGP+/+ and MAGP-/- Mice MAGP1+/+ MAGP1-/- aPTT (sec) 28.5 ± 1.7 (n=4) 29.4 ± 0.7 (n=3) P=0.57 PT (sec) 11.6 ± 1.5 (n=3) 12.6 ± 1.2 (n=4) P=0.33 vWF antigen (%)* 42.3 ± 6.4 (n=3) 44.5 ± 17.1 (n=4) P=0.82 * % of vWF concentration in normal human plasma. P values are for comparison between genotypes.




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Figure Legends 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 ± standard deviation (n=8 for each group). ** = P< 0.005, * = P<0.05.

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

min. C) Infusion of recombinant MAGP1 reestablishes 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 ± standard deviation (n=6 for each group).

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

µg/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 (arrow and at higher power in panel B). Panels on the right show staining with

anti-bovine MAGP1 of injured vessels from animals not injected with bovine MAGP1.

Figure 3- Platelet function in MAGP1-deficient mice. A) The percent of platelets that

aggregate in response to different levels of collagen is the same for both genotypes. B)

The presence of MAGP1 (50µg/ml) has no effect on human platelet aggregation induced

by various agonists, including collagen (Col, 10µg/ml), adenosine 5’-diphosphate (ADP,

20 µM), arachidonic acid (AA, 500 µg/ml) and epinephrine (Epi, 300 µM). Aggregation

was monitored by measuring light transmission through a suspension of stirred washed

platelets (1-3x108/mL for mouse and 2x108/mL for human) using an aggregometer. Data




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are expressed as either the slope of the aggregation curve or as percent 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 ± standard deviation of 4 experiments. None of

the values differed significantly (P ≥ 0.25).

Figure 4- Immunoblot of platelet extracts. Bovine washed platelets were boiled in SDS

sample buffer and subjected to SDS-PAGE under reducing and non-reducing conditions.

Protein bands were visualized by Coomassie blue staining or transferred to nitrocellulose

for immunodetection with an antibody to bovine MAGP1. Panel A- Coomassie blue

stained gel; Panel B- Immunoblot analysis of proteins in panel A after transfer to

nitrocellulose. Lane 1- Platelet extract under non-reducing conditions (no DTT); Lane 2-

Platelet extract under reducing conditions (+DTT); Lane 3- Semipurified bovine MAGP1

expressed by mammalian SaOS2 cells (+DTT).

Figure 5- Outer diameter vs. pressure for the right carotid artery in wild-type and

MAGP-/- mice. Pressure-diameter curve showing that the carotid artery in wild-type

(solid line) and MAGP-/- (hatched line) animals has identical mechanical properties,

identical diameters, and equal pressures.

Figure 6- Analysis of MAGP1 interaction with selected plasma proteins. MAGP1’s

ability to interact with plasma proteins was assayed by ligand blot (panel A) and co-

immunoprecipitation (panel B). Panel A: Lanes: FN- fibronectin; vWF- von Willebrand

factor; Fb- fibrinogen; 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. Panel B: SDS-PAGE autoradiogram showing co-precipitation

of [125I]-labeled plasma proteins and V5-tagged MAGP1. Lanes: 1- fibronectin with V5

antibody only (negative control); 2- fibronectin co-precipitated with MAGP1-V5 using




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V5 antibody; 3- fibronectin co-precipitated with MAGP1-V5 as in 2, but in the presence

of 10-fold excess unlabelled fibronectin; 4- fibrinogen with V5 antibody; 5- fibrinogen

co-precipitated with MAGP1-V5 using V5 antibody; 6- fibrinogen co-precipitated with

MAGP1-V5 as in 5, but in the presence of 10-fold excess unlabelled fibrinogen; 7- von

Willebrand factor with V5 antibody; 8- von Willebrand factor co-precipitated with

MAGP1-V5 using V5 antibody; 9- von Willebrand factor co-precipitated with MAGP1-

V5 as in 8, but in the presence of 10-fold excess von Willebrand factor. Vertical lines

have been inserted to indicate repositioned gel lanes.

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 six different

analyte concentrations (0.052 µM, 0.105 µM, 0.21 µM, 0.32 µM, 0.42 µM and 0.85 µM).

One representative experiment is shown. The response difference (the difference between

experimental and control flow cells) is given in resonance units (RU).




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