天津医科大学-Tianjin Medical University€¦ · 22 1. Heemskerk JW, Vuist WM, Feijge MA,...

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Lactadherin Promotes Microvesicle Clearance to Prevent

Coagulopathy and Improves Survival of Severe TBI Mice†

Yuan Zhou,1,2 Wei Cai,3 Zilong Zhao,1 Tristan Hilton,2 Min Wang,3 Jason Yeon,2 Wei Liu,1

Fangyi Zhang,4 Fu-Dong Shi,1,5 Xiaoping Wu,2 Perumal Thiagarajan,6 Min Li,3* Jianning

Zhang,1* Jing-fei Dong2,7*

1 Tianjin Institute of Neurology; Departments of Neurosurgery and Neurology, Tianjin Medical

University General Hospital, Tianjin, China 2 Bloodworks Research Institute, Seattle, WA, USA 3 Institute of Pathology, Lanzhou University School of Basic Medical Sciences, Lanzhou, China 4 Department of Neurosurgery, University of Washington School of Medicine, Seattle, WA 5Department of Neurology, Barrow Neurological Institute, St. Joseph’s Hospital and Medical

Center, Phoenix, AZ, USA 6Departments of Pathology and Medicine, Baylor College of Medicine and Center for

Translational Research on Inflammatory Diseases, Michael E. DeBakey, VA Medical Center,

Houston, TX, USA 7 Division of Hematology, Department of Medicine, University of Washington, School of

Medicine, Seattle, WA, USA

*To whom correspondence should be addressed:

Jing-fei Dong, Bloodworks Research Institute, 1551 Eastlake Avenue East, Seattle, WA. Tel: 206

398 5914; Email: [email protected], Jianning Zhang, Department of Neurosurgery, Tianjin

Medical University General Hospital, Tianjin, China. Tel: 022-60817448; Email:

[email protected] or Min Li, Institute of Pathology, Lanzhou University School of

Basic Medical Sciences, Lanzhou, China. Tel: 0931 891 5021; Email: [email protected]

†This study is supported by NIH grants NS087296 and HL119391(JFD), Natural Science

Foundation of China State Key Program Grant 81330029 (JNZ) and Research Grants 81271361,

81271359 (JNZ) and 81672399 (ML), and the Biomedical Laboratory Research and

Development Award BX000502 from the Department of Veterans Affairs (PT).

WORD COUNTS

Abstract: 136

Text: 3292

Figure: 6

Supplemental Figure: 8

Blood First Edition Paper, prepublished online November 21, 2017; DOI 10.1182/blood-2017-08-801738

Copyright © 2017 American Society of Hematology

For personal use only.on November 21, 2017. by guest www.bloodjournal.orgFrom

http://www.bloodjournal.org/

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CONFLICT OF INTEREST: The authors claim no conflict of interest related to the study.

AUTHOR CONTRIBUTIONS:

Yuan Zhou: designed and performed experiments, analyzed data and wrote manuscript

Wei Cai: designed and performed experiments

Zilong Zhao: designed and performed experiments, and wrote manuscript

Tristan Hilton: designed and performed experiments, and wrote manuscript

Min Wang: designed and experiment experiments, and analyzed data

Jason Yeon: designed and performed experiments

Xiaoping Wu: designed and performed experiments, analyzed data, and wrote manuscript

Fangyi Zhang: Designed the study and wrote the manuscript

Fu-Dong Shi: Wrote manuscript

Perumal Thiagarajan: designed experiments, provided reagents, and wrote manuscript

Min Li: developed hypotheses, designed experiments, analyzed data, wrote manuscript

Jianning Zhang: developed hypotheses, designed experiments, analyzed data, and wrote

manuscript

Jing-fei Dong: developed hypotheses, designed experiments, analyzed data, and wrote

manuscript




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KEY POINT

1) Lactadherin promotes the clearance of circulating microvesicles through

phagocytosis.

2) Promoting microvesicle clearance prevents coagulopathy, reduces cerebral

edema, and improves neurological function in severe TBI mice.




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ABSTRACT

Coagulopathy is common in patients with traumatic brain injury (TBI) and predicts

poor clinical outcomes. We have shown that brain-derived extracellular microvesicles,

including extracellular mitochondria, play a key role in the development of TBI-

induced coagulopathy. Here, we further show in mouse models that the apoptotic cell-

scavenging factor lactadherin, given at a single dose of 400 μg/kg 30 min before

(preconditioning) or 30 min after cerebral fluid percussion injury, prevented

coagulopathy as defined by clotting time, fibrinolysis, intravascular fibrin deposition,

and microvascular bleeding of the lungs. Lactadherin also reduced cerebral edema,

improved neurological function, and increased survival. It achieved these protective

effects by enhancing the clearance of circulating microvesicles through

phosphatidylserine-mediated phagocytosis. Together, these results identify the

scavenging system for apoptotic cells as a potential therapeutic target to prevent TBI-

induced coagulopathy and improve the outcome of TBI.




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INTRODUCTION

In response to injury, cells shed membrane fragments and release intracellular granules,

through active microvesiculation1,2 or apoptosis.3,4 The former is initiated by

intracellular signals that activate the cysteine protease calpain, which then disrupts the

membrane-cytoskeleton by cleaving cytoskeletal proteins.5;6 Both microvesiculation and

apoptosis produce extracellular vesicles called microvesicles (MVs), which have

increasingly been recognized as a new class of mediators or delivery vehicles for

diverse biological processes and disease development.

We recently identified a causal role of brain-derived MVs (BDMVs) in the pathogenesis

of TBI-induced coagulopathy.7 Trauma-induced uncontrolled hemorrhage is a leading

cause of preventable deaths, accounting for 30% to 40% of all trauma fatalities.8,9 It is

caused by direct injury to the vasculature and secondary coagulopathy. Retrospective

and observational studies have consistently shown that coagulopathy is common in

patients with TBI,10-12 even though patients with isolated TBI lack the key causal factors

for coagulopathy found in patients with injuries to the body and limbs, i.e. substantial

blood loss and hemodilution due to fluid resuscitation.13,14 The underlying cause of TBI-

induced coagulopathy remains poorly understood. We have recently demonstrated that

TBI-induced coagulopathy develops from a hypercoagulable state induced by BDMVs

that are suddenly and substantially released from an injured brain into the




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circulation.7,15 Among these BDMVs, 55.2% are cell-free or membrane-embedded

mitochondria (mtMVs).15 Both BDMVs and mtMVs express abundant anionic

phospholipids on their surfaces: phosphatidylserine (PS) on membrane vesicles and

cardiolipin (CL) on mtMVs. These anionic phospholipid-bearing MVs induce a

hypercoagulable state that rapidly turns into consumptive coagulopathy. These

observations led us to hypothesize that enhancing the clearance of these procoagulant

MVs would prevent the development or reduce the severity of TBI-induced

coagulopathy.

Several molecules have been extensively demonstrated to participate in the clearance of

apoptotic cells. Among them is lactadherin (milk fat globule–epidermal growth factor 8

[MFGE-8]).16 This 41-46 kDa glycoprotein has an N-terminal epidermal growth factor

(EGF)-like domain(s) that contains an integrin-binding RGD sequence and two C-

terminal discoidin domains (C1 & C2), which bind PS with a high affinity.16-20 This

structure allows lactadherin to couple apoptotic cells with monocytes/macrophages to

facilitate phagocytosis.21-23 Because BDMVs and mtMVs share a key structural element

with apoptotic cells, i.e. the surface exposure of the anionic PS and CL, which are

recognized by annexin V in a similar fashion,7,15 we hypothesize that lactadherin

enhances the clearance of BDMVs and mtMVs to prevent coagulopathy and improve

outcomes of TBI. Here we report findings from a study designed to test this hypothesis.




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MATERIALS AND METHODS

This study was designed primarily to determine whether a high basal rate of

microvesicle clearance reduces or prevents TBI-induced coagulopathy. Most of the

experiments were therefore performed in mice preconditioned with lactadherin before

being exposed to TBI. However, lactadherin was also given to a subset of mice after TBI

to test its therapeutic potential.

Mouse model of TBI

Adult male C57BL/6J mice (12-16 wks and 22-25g, Jackson Laboratory, Bar Harbor, ME)

were subjected to fluid percussion injury (FPI).7,15 Briefly, saline from a Plexiglas

cylindrical reservoir was rapidly injected at a controlled pressure of 1.9±0.2 atm by a FPI

device (Custom Design & Fabrication, Richmond, VA) into an unstrained mouse

through a 3 mm-diameter cranial cavity with the dura matter intact (2.0-mm posterior

from the bregma and 2.0-mm lateral to the sagittal suture). Thirty minutes before FPI,

the mice received a single tail-vein infusion of either 400 μg/kg (~10 μg/mouse) of

purified lactadherin (Biomatik, Wilmington, DE) or an equal volume of the vehicle PBS.

A sham mouse received PBS and underwent the craniotomy but was not exposed to FPI.

Blood samples were collected 1 hr before and 3 hrs and 6 hrs after FPI to monitor acute

changes in microvesicle release and coagulation. In addition, the mice were recorded for

7-day survival, and the surviving mice were also evaluated for neurological function

using a modified Neurological Severity Score (mNSS) system24 on post-TBI days 1, 3,




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and 7. In addition to the full length lactadherin, a truncated variant (Q188-C312)

containing the PS-binding C1C2 domains, but not the integrin-binding EGF domain25

was tested similarly to distinguish between MV-scavenging and potential anticoagulant

activities of lactadherin. For data validation, lactadherin null mice and their wild-type

littermates were identically examined for FPI-induced release of BDMVs and

coagulopathy. Lactadherin-null mice (B6;129-Mfge8<tm1Osa>/OsaRbrc mice,

No.RBRC01726, supplemental method) were obtained from RIKEN BioSource Center

(Ibaraki 305-0074, Japan) with the consent of Dr. Shigekazu Nagata of Immunology

Frontier Research Center, Osaka University.21 The use of lactadherin-/- mice is approved

by the IACUC of the Bloodworks Research Institute.

In reciprocal experiments, non-injured mice were infused with 1.5 x 107 of purified

BDMVs/mouse followed immediately by 400 μg/kg of lactadherin or an equal volume

of PBS. They were evaluated similarly as those subjected to FPI. BDMVs were made by

freeze-thawing normal mouse brains. They have been found to be indistinguishable

from those purified from the plasma of TBI mice in morphology and procoagulant

activity.7 The number of BDMVs we infused was approximately 50% of what we had

previously detected in plasma samples from TBI mice.7 This number was used because

more than 85% of mice died within 30 min of an infusion of 3.0 x 107 MVs, as is found in

TBI mice, making the collection and analysis of blood and tissue samples very difficult.

This high mortality was determined to be caused by a rapid accumulation of MVs in the




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circulation through direct vascular infusion of BDMVs, as compared to the relatively

slow release of BDMVs from a traumatically injured brain.

Evans blue dye extravasation

To measure FPI-induced vascular leakage and cerebral edema, mice subjected to FPI

received 100 μl of 2% Evans blue (Sigma Aldrich, St. Louis, MO) through the tail vein.26

They were sacrificed 2 hrs after injection and perfused with PBS. The brains were

dissected, embedded on Tissue-Tek OCT medium (Sakura Fineteck, Torrance, CA),

sectioned (10 μm thickness), and fixed in ice-cold acetone for 10 min in the dark. The

brain sections were stained with DAPI for 30 min at room temperature and reviewed

under a fluorescence microscope (Olympus IX81, Waltham, MA). The brains were also

snap-frozen in liquid nitrogen, homogenized in formamide (1:20 w/v), and incubated at

60°C overnight. The brain homogenates were centrifuged at 16,000 x g for 30 min. Evans

blue in the supernatant was measured at OD620 nm in a SpectraMax M5 plate-reader

(Molecular Devices, Sunnyvale CA).

Flow cytometry

Neuronal MVs in the peripheral blood samples of TBI and control mice were identified

using flow cytometry, first by the particle size (< 1 μm determined using 0.5, 0.9, and 3

μm standard microbeads, Biocytex, Marseille, France) and then by their expression of

neuron-specific enolase (NSE) detected by a rabbit anti-mouse NSE antibody (Abcam,




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Cambridge, MA) followed by a PE-conjugated anti-rabbit IgG (eBioscience, San Diego,

CA) on an LSR II flow cytometer (Beckon Dickinson, San Jose, CA).7 An isotype-specific

IgG was used as the control. mtMVs were detected by the mitochondrial dye

MitoTracker® Green (Thermo Fisher Scientific).15 Sphero Accucount beads were used to

quantify MVs. Flow cytometry was also used to measure the binding of FITC-

conjugated lactadherin (Haematologic Technologies, Inc., Essex Junction, VT) to

purified BDMVs after 30 min of incubation at room temperature. All buffers used for

MV detection were filtered with a 0.1-μm filter (EDM Millipore, Billerica, MA) to reduce

non-cellular MV contamination. Annexin V, which is widely used to detect PS exposure

on microvesicles, was not used in most experiments except when lactadherin-/- mice

were studied, because annexin V binding to PS was competitively inhibited by

lactadherin.

Coagulation and fibrinolysis assays

To quantify a lactadherin-mediated reduction of MVs on plasma clotting time, MVs

from fixed volumes of plasma from TBI mice receiving lactadherin or PBS were mixed

with phospholipid-free normal plasma and the coagulation factor Xa (0.02 U/ml) to

induce clot formation as previously described.7,15 The clot formation was monitored at

37oC on a CoaScreener coagulation analyzer (American Labor Corp., Durham, NC).

Plasma levels of the fibrinolytic product D-dimer were measured with a commercial kit

according to the manufacturer’s instructions (Cloud-Clone Corp., Houston, TX).




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Microvesicle clearance and phagocytosis

For the MV clearance experiments, purified BDMVs were biotinylated using an EZ-

Link™ Sulfo-NHS-Biotin kit (ThermoFisher Scientific, Waltham, MA). The biotinylation

of the BDMVs was validated by their binding to FITC-conjugated streptavidin using

flow cytometry (supplemental Figure S1). After baseline blood sampling, 1.5 x 107 of

biotinylated BDMVs were infused into a mouse through the tail vein and blood samples

were collected 3 hrs and 6 hrs post-infusion. The livers were dissected, fixed in 4%

paraformaldehyde, and processed for histology to detect BDMV accumulation in the

liver by HRP-conjugated streptavidin (ZSGB-Bio SP-9000). For in vitro phagocytosis

testing, we used mtMVs as the surrogate to reduce the heterogeneity of BDMVs in their

compositions (membrane vesicles and intracellular granules) and sizes (0.1-1.0 μm).

Using mtMV as the surrogate for BDMV is valid because mtMVs account for 55.2% of

all annexin V+ MVs detected in FPI mice.15 mtMVs labeled with MitoTracker Green were

incubated with purified monocytes for 30 min at 37oC. After washing with PBS, the cells

were incubated with a V450-conjugated CD45 antibody for 30 min at 37oC. After

washing, ≥ 20,000 cells were analyzed using the Amnis ImageStreamX Mk II Imaging

Cytometer (Amnis, Seattle, WA) with a 60 x objective lens at a focal distance of 2.5 μm.

Purified PS, CL, and PC (200 μM each, Avanti Polar Lipids, Inc., Alabaster, AL) were

tested for blocking mtMV phagocytosis.

Tissue histology




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Mice were sacrificed 24 hrs after FPI to collect the lungs, which were fixed with 4%

formaldehyde overnight at room temperature, embedded and sectioned. The sections

were stained with hematoxylin and eosin (HE) to detect TBI-induced extravascular

erythrocyte accumulation and with phosphotungstic acid hematoxylin (PTAH) to detect

fibrin deposition to the vessel wall.7,15 The livers from mice injected with biotinylated

BDMVs were similarly processed and stained with HRP-streptavidin to measure

microvesicle clearance.

Statistical analysis

Categorical variables were expressed as percentages and continuous variables as the

mean ± SEM. The survival data were analyzed by Kaplan-Meier plot. All data were

analyzed using Sigma plot (V. 11.2) for paired t-test, one-way or repeated measures

ANOVA, as specified in each dataset. A p value of < 0.05 was considered to be

statistically significant.




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RESULTS

Lactadherin prevented FPI-induced coagulopathy, cerebral edema, and death.

Lactadherin preconditioning prevented an FPI-induced hypercoagulable state and

consumptive coagulopathy by reversing shortened clotting time (Figure 1A), reducing

the level of plasma D-dimer (Figure 1B), and preventing intravascular fibrin deposition

in the lungs (Figures 1C-1E). The lactadherin-preconditioned mice also had reduced

vascular leakage, as defined by the extravascular accumulation of erythrocytes (Figures

1F-1H) and an enlarged perivascular space of the lungs (Figure S2), resulting in

significantly reduced cerebral edema (Figures 2A-2E). The FPI-induced

thrombocytopenia was also corrected by lactadherin preconditioning (Figure 2F)

without significant changes to the total leukocyte and erythrocyte counts (Figure S3).

Lactadherin neither induced platelet aggregation nor altered the platelet aggregation

induced by collagen (Figure S4), suggesting that it did not affect platelet reactivity. By

preventing coagulopathy and cerebral edema, the 7-day mortality was reduced from

46.7% in FPI mice receiving PBS to 8.3% in those preconditioned with lactadherin

(Figure 2G). The surviving mice receiving lactadherin developed less severe

neurological dysfunction, measured by mNSS during the 7 day monitoring period

(Figure 2H).

To specifically examine the effect of lactadherin on BDMV-induced coagulopathy

without the confounding influence of FPI, we infused non-injured mice with purified




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BDMVs (1.5 x 107/mouse) followed immediately by lactadherin (400 μg/kg). In these

reciprocal experiments, lactadherin also prevented the development of a

hypercoagulable state, as measured by clotting time and plasma D-dimer (Figures S5B

& S5C). Thrombocytopenia similar to that found in FPI mice was also prevented by

lactadherin (Figure S5D), again without affecting leukocyte or erythrocyte counts

(Figure S6). Lactadherin reduced the 7-day mortality from 58.2%, in BDMV-infused

mice, to 10% (Figure 2I).

Lactadherin reduced circulating microvesicles

Lactadherin is known to remove apoptotic cells by coupling them to monocytes or

macrophages to facilitate phagocytosis.16 Because this scavenging activity is mediated

by the anionic phospholipid PS exposed on apoptotic cells, we hypothesized that

lactadherin could also promote the clearance of procoagulant BDMVs and mtMVs that

express PS and CL, respectively, to prevent FPI-induced coagulopathy (Figure 3A). We

found that FPI mice preconditioned with lactadherin had significantly lower levels of

neuronal microvesicles (NSE+) and mtMVs (MitoTracker Green+) than those receiving

PBS (Figure 3B). The total number of annexin V-binding MVs was also reduced, from

3.3x105 ± 5.8x103/μl in FPI mice, to 1.2x105±2.1x103/μl in those preconditioned with

lactadherin (the pre-injury baseline: 0.9x104±1.9x102/μl, Figure S5D). We focused on

annexin V-binding MVs because (1) our focus was on an anionic phospholipid-

mediated procoagulant activity of MVs and (2) lactadherin primarily removed anionic




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phospholipid-expressing MVs through the mechanism depicted in Figure 3A. The

reduction was similarly observed in non-injured mice infused with BDMVs followed by

lactadherin (Figure 3C). By contrast, higher levels of circulating NSE+ (Figure 3D) and

PS-expressing (Annexin V+) MVs (Figure 3E), a shortened clotting time (Figure 3F), and

a higher level of plasma D-dimer (Figure 3G) were observed in lactadherin-/- mice

subjected to FPI than in their wild-type littermates.

Lactadherin promoted microvesicle clearance

Several lines of experimental evidence support our hypothesis that lactadherin reduces

circulating MVs by promoting their clearance through phagocytosis. First, the plasma

level of biotinylated BDMVs injected into non-injured mice was significantly lower in

the mice receiving lactadherin than of those receiving PBS (Figure 4A). Second, more

biotinylated BDMVs were detected in sinusoids of the livers of mice receiving

lactadherin than those receiving PBS (Figure 4B). Third, mtMV phagocytosis by

monocytes was directly visualized using imaging flow cytometry (Figure 4C). The

phagocytosis was blocked by the anionic phospholipids CL or PS but not by the neutral

phosphatidylcholine (PC, Figure 4D), suggesting that anionic phospholipids were

involved in the process. This notion is further supported by the ability of the PS-binding

annexin V to block lactadherin binding to BDMVs (Figure 4E). Fourth, the

transmigration of BDMVs through a monolayer of cultured endothelial cells measured

in a transwell culture system (supplemental method) was prevented by lactadherin in




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the presence of monocytes and, to a lesser extent, in their absence (Figure 5A). Finally,

truncated lactadherin lacking the integrin-binding EGF domain (C1C2) was

significantly less active in reducing plasma NSE+ microvesicles (Figure 5B) and mtMVs

(Figure 5C). Mice receiving C1C2 developed an FPI-induced hypercoagulable state, as

demonstrated by a shortened clotting time (Figure 5D) and an elevated level of plasma

D-dimer (Figure 5E).

Dynamic changes of plasma lactadherin during acute TBI

We determined that lactadherin was present in the circulation of non-injured C57BL/6J

mice at 1.1±0.6 ng/ml. The ability of exogenous lactadherin to prevent FPI-induced

coagulopathy would, therefore, suggest that either the basal level of circulating

lactadherin is not sufficient to remove the excessive microvesicles acutely released from

an injured brain, or the lactadherin activity is suppressed by the injury. To distinguish

the two possibilities, we measured dynamic changes of plasma lactadherin during acute

FPI. We found that mouse plasma lactadherin increased steadily by 21.5±8.5% and

43.2±13.9% from the baseline at 3 hrs and 6 hrs post-TBI, respectively (Figure 6A).

Because the antigen level was closely correlated with the scavenging activity of

lactadherin (n = 21, R2 = 0.8689, p < 0.001), the finding suggests that the scavenging

activity was not suppressed during acute FPI. Furthermore, mice preconditioned with

lactadherin had twice the level of circulating lactadherin at 3 hrs but not at 6 hrs post-

FPI (Figure 6A), indicating rapid lactadherin consumption.




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Lactadherin as therapeutic

The rapid lactadherin consumption during acute FPI (Figure 6A) strongly suggests

therapeutic potential of lactadherin to prevent TBI-induced coagulopathy. We tested

this hypothesis by infusing lactadherin (400 μg/kg) into mice 30 min after FPI. This

single dose reduced circulating NSE+ MVs (Figure 6B) and mtMVs (Figure 6C) to levels

similar to those in the mice preconditioned with lactadherin before FPI (Figure 6D). It

also reversed the FPI-induced hypercoagulable state (Figures 6E & 6F) to a level similar

to that of mice preconditioned with lactadherin before FPI (Figure S7). For these

experiments, lactadherin was given 30 min after FPI because TBI-induced coagulopathy

develops within 60 min after injury in this mouse model.7,15




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DISCUSSION

We have recently demonstrated that brain-derived microvesicles, including

extracellular mitochondria, are released from traumatically injured brains into the

circulation and cause consumptive coagulopathy.7,15 Here, we further show that

lactadherin preconditioning prevented mice subjected to severe TBI from developing

coagulopathy and cerebral edema, resulting in significantly improved neurological

function and survival (Figures 1 & 2). Consistently with the findings from wild-type

mice, lactadherin-deficient mice had significantly higher levels of circulating neuronal

and mitochondrial microvesicles and developed more severe coagulopathy after TBI

(Figures 3D-3E). These lactadherin-deficient mice have previously been shown to have

significantly more platelet-derived microvesicles due to a lower rate of PS-mediated

phagocytosis, leading to enhanced thrombin generation and thrombosis.25

Several lines of evidence support our hypothesis that lactadherin exerted its protective

effects by enhancing phagocytosis-mediated microvesicle clearance. First, mice

preconditioned with lactadherin had lower levels of neuronal and mitochondrial

microvesicles (Figure 3B & 3C), faster clearance of biotinylated BDMVs from the

circulation (Figure 4A), and enhanced hepatic accumulation of BDMVs (Figure 4B). The

plasma levels of NSE+ microvesicles and mtMVs reduced by 72±29% and 29±8.8%

respectively in TBI mice preconditioned with lactadherin. Furthermore, while NSE+

microvesicles and mtMVs were measured in this study, those from platelets, endothelial




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cells, erythrocytes, and leukocytes are also likely released in response to TBI-induced

secondary ischemic and inflammation injures. Consistent with the notion, the total

number of annexin V-binding microvesicles that account for procoagulant microvesicles

in the circulation increased from 0.9x104±1.9x102/μl at the preinjury baseline to a post-

TBI level of 3.3x105 ± 5.8x103/μl. These blood cell-derived microvesicles have been

shown to express procoagulant anionic phospholipids6 and are removed by

lactadherin.22,25

Second, a truncated lactadherin lacking the integrin binding domain failed to promote

microvesicle clearance and TBI-induced coagulopathy (Figure 5). The finding that the

C1C2 domains of lactadherin are homologous to those of human coagulation factors V

and VIII17,18 and bind anionic phospholipids27 raises the possibility that lactadherin can

also reduce TBI-induced consumptive coagulopathy by acting as an anticoagulant.

However, this is unlikely because the truncated lactadherin, which bind PS with an

affinity similar to that of full-length lactadherin27, did not have the same protective

effect as the full-length lactadherin (Figures 5B-5E).

Third, the phagocytosis of mtMVs (as the surrogate of BDMVs) by monocytes was

directly visualized and found to be anionic phospholipid-dependent (Figures 4C-4E),

much like the lactadherin-mediated scavenging of apoptotic cells by monocytes and

macrophages.28,29 The phagocytosis reduced the rate of BDMV transmigration through




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the endothelium (Figure 5A), but we cannot exclude other actions of lactadherin. For

example, lactadherin may protect or restore the integrity of glycocalyx by interacting

with negatively charged sialic acids and phospholipids that are enriched in

glycocalyx.30-32 Trauma-induced disruption of glycocalyx has been reported to increases

the permeability of the blood-brain barrier.33,34 In addition, the RGD sequence in

lactadherin binds the integrins αvβ3 and αvβ5 to promote cell adhesion16,19,35,34,35 through

integrin-mediated intracellular signaling36 to repair injured endothelial barriers. This

lactadherin-integrin interaction may also promote phagocytosis of BDMVs by

endothelial cells, which have been shown to have a phagocytic activity,37 as lactadherin

also protects the endothelial barrier independent of monocytes (Figure 5A).

Finally, the rapid consumption of plasma lactadherin after TBI (Figure 6A) strongly

suggests that the intrinsic level of circulating lactadherin, though sufficient to maintain

a basal level of apoptotic cell-scavenging activity, is insufficient to remove a large

number of microvesicles released suddenly into the circulation. This insufficiency

develops despite the TBI-induced steady increase in circulating lactadherin (Figure 6A).

In summary, cellular microvesicles are heterogeneous in their morphologies, cargoes,

and activities. This heterogeneity leads them to have different effects on various tissues,

some detrimental and others beneficial. We have demonstrated in mouse models that

enhancing the clearance of procoagulant microvesicles that express the anionic




21

phospholipids PS and CL prevents TBI-induced coagulopathy and improves

neurological function in mice subjected to severe TBI. Our findings strongly suggest

that lactadherin, as an integral part of the intrinsic apoptotic cell-scavenging system, has

significant therapeutic potential for improving outcomes of TBI and other microvesicle-

driven pathologies.




22

REFERNCE 1. Heemskerk JW, Vuist WM, Feijge MA, Reutelingsperger CP, Lindhout T. Collagen but not fibrinogen surfaces induce bleb formation, exposure of phosphatidylserine, and procoagulant activity of adherent platelets: evidence for regulation by protein tyrosine kinase-dependent Ca2+ responses. Blood. 1997;90(7):2615-2625. 2. Siljander P, Farndale RW, Feijge MA, et al. Platelet adhesion enhances the glycoprotein VI-dependent procoagulant response: Involvement of p38 MAP kinase and calpain. Arterioscler Thromb Vasc Biol. 2001;21(4):618-627. 3. Shcherbina A, Remold-O'Donnell E. Role of caspase in a subset of human platelet activation responses. Blood. 1999;93(12):4222-4231. 4. Brown SB, Clarke MC, Magowan L, Sanderson H, Savill J. Constitutive death of platelets leading to scavenger receptor-mediated phagocytosis. A caspase-independent cell clearance program. J Biol Chem. 2000;275(8):5987-5996. 5. Fox JE, Austin CD, Reynolds CC, Steffen PK. Evidence that agonist-induced activation of calpain causes the shedding of procoagulant-containing microvesicles from the membrane of aggregating platelets. J Biol Chem. 1991;266(20):13289-13295. 6. Owens AP, 3rd, Mackman N. Microvesicles in hemostasis and thrombosis. Circ Res. 2011;108(10):1284-1297. 7. Tian Y, Salsbery B, Wang M, et al. Brain-derived microvesicles induce systemic coagulation in a murine model of traumatic brain injury. Blood. 2015;125(13):2151-2159. 8. Niles SE, McLaughlin DF, Perkins JG, et al. Increased mortality associated with the early coagulopathy of trauma in combat casualties. J Trauma. 2008;64(6):1459-1463; discussion 1463-1455. 9. Mitra B, Cameron PA, Mori A, Fitzgerald M. Acute coagulopathy and early deaths post major trauma. Injury. 2012;43(1):22-25. 10. Talving P, Benfield R, Hadjizacharia P, Inaba K, Chan LS, Demetriades D. Coagulopathy in severe traumatic brain injury: a prospective study. J Trauma. 2009;66(1):55-61; discussion 61-52. 11. Hulka F, Mullins RJ, Frank EH. Blunt brain injury activates the coagulation process. ArchSurg. 1996;131:923-927. 12. Stein SC, Smith DH. Coagulopathy in traumatic brain injury. Neurocrit Care. 2004;1(4):479-488. 13. Wafaisade A, Wutzler S, Lefering R, et al. Drivers of acute coagulopathy after severe trauma: a multivariate analysis of 1987 patients. Emerg Med J. 2010;27(12):934-939. 14. Maani CV, DeSocio PA, Holcomb JB. Coagulopathy in trauma patients: what are the main influence factors? Curr Opin Anaesthesiol. 2009;22(2):255-260. 15. Zhao Z, Wang M, Tian Y, et al. Cardiolipin-mediated procoagulant activity of mitochondria contributes to traumatic brain injury-associated coagulopathy in mice. Blood. 2016;127(22):2763-2772. 16. Hanayama R, Tanaka M, Miwa K, Shinohara A, Iwamatsu A, Nagata S. Identification of a factor that links apoptotic cells to phagocytes. Nature. 2002;417(6885):182-187. 17. Larocca D, Peterson JA, Urrea R, Kuniyoshi J, Bistrain AM, Ceriani RL. A Mr 46,000 human milk fat globule protein that is highly expressed in human breast tumors contains factor VIII-like domains. Cancer Res. 1991;51(18):4994-4998. 18. Couto JR, Taylor MR, Godwin SG, Ceriani RL, Peterson JA. Cloning and sequence analysis of human breast epithelial antigen BA46 reveals an RGD cell adhesion sequence presented on an epidermal growth factor-like domain. DNA Cell Biol. 1996;15(4):281-286. 19. Andersen MH, Berglund L, Rasmussen JT, Petersen TE. Bovine PAS-6/7 binds alpha v beta 5 integrins and anionic phospholipids through two domains. Biochemistry. 1997;36(18):5441-5446. 20. Shi J, Gilbert GE. Lactadherin inhibits enzyme complexes of blood coagulation by competing for phospholipid-binding sites. Blood. 2003;101(7):2628-2636. 21. Hanayama R, Tanaka M, Miyasaka K, et al. Autoimmune disease and impaired uptake of apoptotic cells in MFG-E8-deficient mice. Science. 2004;304(5674):1147-1150. 22. Dasgupta SK, Abdel-Monem H, Guchhait P, Nagata S, Thiagarajan P. Role of lactadherin in the clearance of phosphatidylserine-expressing red blood cells. Transfusion. 2008;48(11):2370-2376. 23. Dasgupta SK, Thiagarajan P. The role of lactadherin in the phagocytosis of phosphatidylserine-expressing sickle red blood cells by macrophages. Haematologica. 2005;90(9):1267-1268. 24. Chen X, Zhang KL, Yang SY, Dong JF, Zhang JN. Glucocorticoids Aggravate Retrograde Memory Deficiency Associated with Traumatic Brain Injury in Rats. JNeurotrauma. 2009;26(2):253-260.




23

25. Dasgupta SK, Abdel-Monem H, Niravath P, et al. Lactadherin and clearance of platelet-derived microvesicles. Blood. 2009;113(6):1332-1339. 26. Gao W, Zhao Z, Yu G, et al. VEGI attenuates the inflammatory injury and disruption of blood-brain barrier partly by suppressing the TLR4/NF-kappaB signaling pathway in experimental traumatic brain injury. Brain Res. 2015;1622:230-239. 27. Andersen MH, Graversen H, Fedosov SN, Petersen TE, Rasmussen JT. Functional analyses of two cellular binding domains of bovine lactadherin. Biochemistry. 2000;39(20):6200-6206. 28. Fadok VA, de Cathelineau A, Daleke DL, Henson PM, Bratton DL. Loss of phospholipid asymmetry and surface exposure of phosphatidylserine is required for phagocytosis of apoptotic cells by macrophages and fibroblasts. J Biol Chem. 2001;276(2):1071-1077. 29. Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL, Henson PM. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol. 1992;148(7):2207-2216. 30. Sahagun G, Moore SA, Hart MN. Permeability of neutral vs. anionic dextrans in cultured brain microvascular endothelium. Am J Physiol. 1990;259(1 Pt 2):H162-166. 31. Nag S. Cerebral endothelial surface charge in hypertension. Acta Neuropathol. 1984;63(4):276-281. 32. Simionescu N, Simionescu M, Palade GE. Differentiated microdomains on the luminal surface of the capillary endothelium. I. Preferential distribution of anionic sites. J Cell Biol. 1981;90(3):605-613. 33. Zhu J, Li X, Yin J, Hu Y, Gu Y, Pan S. Glycocalyx degradation leads to blood-brain barrier dysfunction and brain edema after asphyxia cardiac arrest in rats. J Cereb Blood Flow Metab. 2017:271678X17726062. 34. Wu F, Peng Z, Park PW, Kozar RA. Loss of Syndecan-1 Abrogates the Pulmonary Protective Phenotype Induced by Plasma After Hemorrhagic Shock. Shock. 2017;48(3):340-345. 35. Taylor MR, Couto JR, Scallan CD, Ceriani RL, Peterson JA. Lactadherin (formerly BA46), a membrane-associated glycoprotein expressed in human milk and breast carcinomas, promotes Arg-Gly-Asp (RGD)-dependent cell adhesion. DNA Cell Biol. 1997;16(7):861-869. 36. Ensslin MA, Shur BD. Identification of mouse sperm SED1, a bimotif EGF repeat and discoidin-domain protein involved in sperm-egg binding. Cell. 2003;114(4):405-417. 37. Dini L, Lentini A, Diez GD, et al. Phagocytosis of apoptotic bodies by liver endothelial cells. J Cell Sci. 1995;108 ( Pt 3):967-973.




24

FIGURES AND LEGENDS

Figure 1. Lactadherin reduced TBI-induced coagulopathy and vasculopathy: Clotting

time (A) and plasma D-dimer (B) of sham mice and FPI mice preconditioned with either

PBS or lactadherin (n = 16, one-way ANOVA). Representative images of PTAH-stained

lungs from a sham mouse (C) and FPI mice receiving PBS (D, red arrow indicates

extensive blue PTAH stain for intravascular fibrin deposition) or lactadherin (D, arrow

indicates significantly reduced intravascular PTAH stain, bar in C-E = 10 μm).

Representative images of H&E-stained lungs from a sham mouse (F) and FPI mice

receiving PBS (G) or lactadherin (H, bar in F-H = 20 μm). Perivascular space (*) is

enlarged with extravascular accumulation of erythrocytes (hemorrhage) in the lungs of

TBI mice receiving PBS. The C-H images are representatives of the 26 mice examined.

Figure 2. Lactadherin reduced cerebral edema and improved outcomes of FPI: (A)

Representative topical and cross-sectional views of brains from a sham mouse (left) and

from FPI mice preconditioned with PBS (middle) or lactadherin (right). (B-D)

Representative fluorescence images of brain cryosections from a sham mouse and mice

receiving lactadherin or PBS. (E) Levels (OD unite) of Evans blue in the supernatants of

tissue homogenates from FPI mice receiving different treatments (n = 9, one-way

ANOVA). (F) Platelet counts at the baseline (white bars) and 3 hrs after FPI (black bars)

of sham mice and TBI mice preconditioned with PBS or lactadherin (n = 16, one-way

ANOVA). A Kaplan-Meier survival analysis (G, n = 18, p < 0.005 vs. mice received PBS)

and mNSS (H, n =18, one-way ANOVA) of sham and FPI mice receiving PBS or

lactadherin. (I) A Kaplan-Meier survival analysis of BDMV-infused and control mice (p

< 0.005 vs. mice received PBS).




25

Figure 3: Lactadherin reduced MPs and its deficiency increased TBI-induced BDMV

release and coagulopathy: (A) A schematic illustration of lactadherin-mediated MV

clearance. (B) Levels of circulating NSE+ (scale on the left) and mtMVs (MitoTracker

Green+, scale on the right) measured 3 hrs after injury of sham mice and FPI mice

preconditioned with PBS or lactadherin (n = 15, one-way ANOVA). (C) Levels of

circulating NSE+ MVs (scale on the left) and mtMVs (scale on the right) in non-injured

mice infused with 1.5x107/mouse of BDMVs followed by lactadherin or PBS (n = 12, one-

way ANOVA). Plasma samples from lactadherin-/- mice and their wild-type littermates

were examined for dynamic changes in plasma levels of (D) NSE+ MVs, (E) annexin V-

binding MVs, (F) clotting time, and (G) plasma levels of D-dimer (n = 24, repeated-

measures ANOVA, * p < 0.001).

Figure 4. Lactadherin promoted BDMV clearance through phagocytosis: (A) Plasma

levels of biotinylated BDMVs infused into non-injured mice that also received

lactadherin or PBS (n = 12, paired t-test, * p < 0.01). (B) Representative images of liver

sections from mice infused with (1) PBS, (2) biotinylated BDMVs, and (3) biotinylated

BDMVs with lactadherin were stained with HRP-streptavidin (bar = 20 μm, biotinylated

BDMVs are stained in brown color); (4) integrated optical densities from scans from

multiple (n = 9, one-way ANOVA). (C) Bright field (left) and fluorescence images (right)

of CD45+ monocytes and MitoTracker Green+ mtMVs (right overlay) detected by

imaging flow cytometry. (D) mtMV binding to CD45+ monocytes in the absence and

presence of PS, CL, and PC (all 200 nM, n= 12, one-way ANOVA). (E) Blocking the

binding of FITC-lactadherin to BDMVs 100-fold excess unlabeled annexin V (n = 24,

one-way ANOVA)




26

Figure 5. BDMV clearance required the integrin-binding domain of lactadherin: (A)

The transmigration of PKH26-labeled BDMVs through activated endothelial cells in the

presence and absence of lactadherin and monocytes (n = 15, one-way ANOVA).

C57BL/6J mice (n = 16) were preconditioned with an equal molar concentration of either

lactadherin or its C1C2 domain (PBS as control) before being subjected to FPI. They

were then examined for plasma levels of (B) NSE+ MVs, (C) mtMVs, (D) clotting time,

and (E) plasma levels of D-dimer. Data presented in C-F were analyzed with repeated

measures ANOVA, * p< 0.01 and ** p < 0.001 vs. PBS-injected mice.

Figure 6: Plasma lactadherin and its therapeutic potential for TBI-induced

coagulopathy. (A) Plasma levels of lactadherin in sham mice and in FPI mice

preconditioned with lactadherin or PBS (n = 15, repeated measures ANOVA, * p < 0.001

vs. sham). Plasma levels of (B) NSE+ MVs and (C) mtMVs in C57Bl/6J mice subjected to

FPI and received 400 μg/kg of lactadherin or PBS 30 min after injury (sham mice as

control). (D) A comparison in plasma NSE+ (top) and mtMVs (bottom) between mice

preconditioned with lactadherin (a) and those receiving lactadherin after the injury (b, n

= 32, one-way ANOVA). (E) Clotting time and (F) plasma levels of D-dimer in mice

receiving lactadherin 30 min after TBI (n = 21, repeated measures ANOVA, *p< 0.01 and

#p < 0.05 vs. PBS-infused mice).




FPI

Plas

ma

D-d

imer

(ng/

ml)

Lactadherin-

--+

++ -

--+++

020040060080010001200140016001800

p < 0.001 p < 0.001

p < 0.001 p < 0.001B

LactadherinFPI -

-- ++ +

++

20

30

40

50

60

70

80

90

Clo

tting

tim

e (s

econ

d)

3 hr post 6 hr post

3 hr post 6 hr post

p < 0.001

p < 0.001 p < 0.001p < 0.001A

---+

FPI +

lact

adhe

rinFP

I + P

BSSh

am

PTAH stain HE stain

**

**

*C

D

E

F

G

H

Figure 1




FPI-PBS

FPI-PBS

Sham

Sham

FPI-lactadherin

FPI-lactadherin

A E

0

2

4

68

1012

1416

18

Evan

s bl

ue d

ensi

ty (O

DU

)

p < 0.001

p < 0.001

C DBFPI

Lactadherin-- -

+ ++ - -

-+++

0

200

400

600

800

1000

Plat

elet

cou

nt (x

103 /µl

) p = 0.049

p < 0.05

F

LactadherinFPI -

--+++

1.0

0.8

0.6

0.4

0.20 2 4 6

Days post FPI

Accu

mul

ativ

e su

rviv

al

Sham

FPI+PBS

FPI+lactadherin*

G1.0

0.8

0.6

0.4

0.20 2 4 6

Days post BDMP injectionAc

cum

ulat

ive

surv

ival

PBS

BDMP+PBS

BDMP+lactadherin*

IH p < 0.01p < 0.01

p < 0.05

p < 0.01p < 0.01

p < 0.05p < 0.05

0

2

4

6

8

10

12

14

mN

SS

LactadherinPost FPI

FPI ---

+++

---

+++

---

+++

day 1 day 3 day 7

Figure 2




0

2

4

6

8

10

12

% N

SE+

MV

% A

nnex

inV+

MV

Clo

tting

tim

e (s

ec.)

Perc

ent p

ositi

ve

5

0 0

10

15

20

25

30

5

10

15

20

25

30

Perc

ent p

ositi

ve

shamFPI+PBSFPI+lactadherin

PBSBDMV+PBSBDMV+lactadherin

p < 0.05

p < 0.01

0

2

4

6

8

10

12

14

NSE+ mtMV NSE+ mtMV

B C

p < 0.05

p < 0.01

p < 0.005

p < 0.001

p < 0.005p < 0.05

p < 0.01

p < 0.05AEGF EGF C1 C2N C

MV

monocyte/macrophage

PS or CLRGD

integrin

GFD E

D-d

imer

(ng/

mlx

100) **

*

**

*

*

0 3 6Post-FPI time (hr)




02468

101214161820

Lactadherin-/- Lactadherin+/+

Lactadherin-/-Lactadherin+/+



20

30

40

50

60

70

80

0

2

4

6

8

10

0

5

10

15

20

25

Figure 3




A

BL 1 3 6

0

2

4

6

8

% B

iotin

ylat

ed B

DM

V

post-injection time (hr)

+ lactadherin- lactadherin

**

Bright field Fluorescence

mtMVMonocytemtMV

endocytosedMonocyteC D

% m

tMV

inte

rnal

izat

ion

-- + --- +-

-- +-

CLPSPC

0

20

40

60

80

100p < 0.005

p < 0.01p < 0.01

p < 0.005

p < 0.05

p < 0.05

p < 0.01

BDM

V+la

ctad

herin

BDM

V+PB

SPB

S

B

02468

101214

Opt

ical

den

sity

BDMV+Lactadherin

BDMV+PBS

PBS

4

E

0246810121416

Mea

n flu

ores

cenc

e (A

U x

100

)

FITC-Lact - + + +- - + -- - +-Annexin V

Lactadherin

p < 0.005

p < 0.005

1

2

3Figure 4




A B C

D E

010

2030

4050

607080

MonocytesBDMV

BDM

V co

unt/µ

l (x

100)

NSE

+ m

icro

vesi

cle

(%)

mtM

V (%

)

-- - - +- - +

+ +

+

+

Lactadherin

p < 0.001

p < 0.001

p < 0.01

Clo

tting

tim

e (s

ec)

0200400600800

10001200140016001800

Plas

ma

D-d

imer

(ng/

ml)

60 3Post FPI time (hrs)



* *

FPI+PBSFPI+C1C2FPI+lactadherin

* **FPI+PBSFPI+C1C2FPI+lactadherin

*FPI+PBSFPI+C1C2FPI+lactadherin

20

100

30405060708090

LactadherinC1C2

FPI

- --

-

+

+ ++ ++ +

3 hr post 6 hr post

- - -

-

+-- - + - -- +

p < 0.01p < 0.01p < 0.01 p < 0.01

p < 0.01p < 0.01

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

0

2

4

6

8

10

12

Figure 5




0.80.6

1.01.21.41.61.82.02.22.42.6

Plas

ma

lact

adhe

rin (n

g/m

l)

***

* *#

FPI+PBSFPI+lactadherinSham

A B

E

C

20

30

40

50

60

70

80C

lotti

ng ti

me

(sec

.)N

SE+

MV

(% to

tal)

mtM

V (%

tota

l)

p < 0.05

p < 0.05

p < 0.05

p < 0.05

0

200

400

600

800

1000

1200

1400FD

D-d

imer

(ng/

ml)

0 3 6

Post-FPI time (hr)

0 3 6

Post-FPI time (hr)

0 3 6

Post-FPI time (hr)

0 3 6

Post-FPI time (hr)

* *

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5FPI+PBSFPI+lactadherinSham



0

2

4

6

8

10

12

LactadherinFPI

- --

+++ ++

3 hr post 6 hr post

- --

+0 2 4 6 8 10 12

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

Percent positive

mtMV

NSE+ MV

FPI

FPI

a

a

b

b

Lact

adhe

rinLa

ctad

herin

p <

0.00

1

p <

0.00

1

p <

0.00

1

p <

0.00

1

Figure 6




doi:10.1182/blood-2017-08-801738Prepublished online November 21, 2017;

Fu-Dong Shi, Xiaoping Wu, Perumal Thiagarajan, Min Li, Jianning Zhang and Jing-fei DongYuan Zhou, Wei Cai, Zilong Zhao, Tristan Hilton, Min Wang, Jason Yeon, Wei Liu, Fangyi Zhang, and Improves Survival of Severe TBI MiceLactadherin Promotes Microvesicle Clearance to Prevent Coagulopathy

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