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Regular Article
THROMBOSIS AND HEMOSTASIS
Macrophage receptor SR-AI is crucial to maintain normal plasma levels
of coagulation factor X
Vincent Muczynski,1,2 Amine Bazaa,1,2 Cécile Loubière,1,2 Amélie Harel,1,2 Ghislaine Cherel,1,2 Cécile V. Denis,1,2
Peter J. Lenting,1,2,* and Olivier D. Christophe1,2,*
1
Unité 1176 INSERM, Le Kremlin-Bicêtre, France; and 2Unité Mixte de Recherche S1176, Université Paris-Sud, Le Kremlin-Bicêtre, France
Beside its classical role in the coagulation cascade, coagulation factor X (FX) is involved
in several major biological processes including inflammation and enhancement of virus• SR-AI is the major receptor of induced immune responses. We recently reported that the long circulatory half-life of FX
FX at the macrophage surface. is linked to its interaction with liver-resident macrophages. Importantly, we now observed
that macrophages, but not undifferentiated monocytes, support this interaction. Using
• Macrophages use SR-AI to
cell biology approaches with primary and THP1-derived macrophages as well as transfected
control FX circulatory levels.
cells, we further identified the scavenger receptor type A member I (SR-AI) to be a
macrophage-specific receptor for FX. This result was confirmed using SR-AI–deficient mice, which exhibit reduced circulating
levels of FX in vivo and loss of FX-macrophage interactions in vitro. Binding studies using purified proteins revealed that FX binds
specifically (half-maximal binding, 3 mg/mL) to the extracellular domain of SR-AI. Altogether, we demonstrate that macrophages
regulate FX plasma levels in an SR-AI–dependent manner. (Blood. 2016;127(6):778-786)
Key Points
Introduction
Upon vessel damage, exposure of blood to collagen in the vessel
wall and release of material by vascular cells triggers the activation
of clotting factors. This provokes a series of events that eventually
produces fibrin and leads to the formation of a hemostatic plug.
During this process, called blood coagulation, zymogen factor X (FX)
is converted into the activated serine protease FXa via site-specific
proteolysis.1-5 The crucial role of FX in coagulation is demonstrated by the severe bleeding diathesis of patients with inherited
FX deficiencies.6 However, beyond its role in coagulation, FX is
also involved in nonhemostatic processes as first suggested by the
partial embryonic lethality observed in mice following deletion of
the F10 gene.7 Indeed, FX has been linked to fibroproliferative
diseases, including tissue remodeling, fibrosis, cancer, and inflammation
particularly after tissue injury in different pathologies.8,9 The mechanism
by which FX modulates these processes is incompletely understood but
seems to be related to the capacity of FXa to induce signaling pathways
via the proteinase-activated receptors 1 and 2 (PAR-1 and -2). Another
biological process involving FX concerns viral infections through its
binding to the surface of herpes simplex virus-1 or human species
C adenovirus. Whether such interactions promote the activation of
the innate immune response10,11 or, in contrast, facilitate adenovirus
and adenovirus-associated viruses infection12 is still a matter of debate.
In any case, in view of this multiplicity of FX biological functions,
further understanding of the regulatory mechanisms that control
FX circulatory levels is clearly required.
Interestingly, the circulatory half-life of FX (48 hours) is longer
than those of other structurally related coagulation factors such as
FVII (5 hours), FIX (18-24 hours), or protein C (4 hours).13-15 However,
the mechanisms underlying this particularly long half-life are still very
poorly described. In a previous study conducted in our laboratory, organ
biodistribution analysis identified the liver as a major target organ for
FX.16 At the cellular level, we demonstrated that FX binds to Kupffer
cells, the tissue-resident macrophages of the liver. Strikingly, we observed that macrophage inactivation in mice significantly reduced FX
plasma levels. This was surprising because plasma concentrations of
von Willebrand factor, a protein known to be degraded by macrophages, was increased under similar conditions.17 This strongly suggested that FX-macrophage interactions are crucial to the regulation of
its circulating levels. However, the cellular receptor(s) and mechanism(s)
involved in this interaction remained unknown.
In the present study, we aimed to explore the mechanism by which
macrophages preserve FX plasma levels. To this end, we investigated
the nature of the interaction between FX and macrophages and
attempted to identify the cellular receptor(s) involved. Using multiple
models both in vitro and in vivo, we identified the scavenger receptor
type A member I (SR-AI) as a previously unreported receptor for FX
and demonstrated its critical importance in maintaining normal FX
plasma levels.
Submitted May 19, 2015; accepted November 17, 2015. Prepublished online
as Blood First Edition paper, November 25, 2015; DOI 10.1182/blood-201505-647032.
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.
Methods
An extensive description of the experimental procedures can be found in the
supplemental Methods (see supplemental Data, available on the Blood Web site),
a brief summary of which is given below.
*P.J.L. and O.D.C. contributed equally to this manuscript.
The online version of this article contains a data supplement.
778
© 2016 by The American Society of Hematology
BLOOD, 11 FEBRUARY 2016 x VOLUME 127, NUMBER 6
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SR-AI IS CRUCIAL TO REGULATE PLASMA LEVELS OF FX
779
Proteins
Human plasma-derived FX was obtained from Cryopep, and was used
throughout the study unless indicated otherwise. The soluble extracellular
domains of human SR-AI (hSR-AI) and murine SR-AI (mSR-AI) were obtained
from R&D Systems. hSR-AI was used throughout the study, unless stated
otherwise.
Cell culture
Human macrophages were obtained both from THP1 (a monocytic leukemia
cell line) and purified CD141 cells. Murine macrophages were obtained
from CD1151 cells.18 CD1151 cells were isolated by positive selection
using the CD1151 sorting kit (Miltenyi Biotec). Differentiation into
macrophages for each of these cells is described in more detail in the
supplemental Methods. Complementary DNA encoding hSR-AI (Eurofins
Scientific) was cloned and transfected into HEK-293 cells (see supplemental Methods).
Microscopy analyses and immunofluorescence-based
quantification
Widefield-microscopy images were acquired on an AxioImager A1 (Carl Zeiss)
and further analyzed using ImageJ software for quantification. Confocal microscopy images were acquired on a LSM700 (Carl Zeiss) and cell stacks
were further deconvolved using Huygens software (Scientific Volume
Imaging). The thresholded Manders coefficient (tMC) used to verify
colocalization was calculated for each cell stack using the JACoP-plugin
in ImageJ software. All images were assembled using ImageJ software (see
supplemental Methods).
Flow cytometry
For flow cytometry analysis, THP1-derived macrophages or undifferentiated
THP1 cells were incubated with Alexa 488–labeled FX or donkey anti-goat
Fab92 as a control as described in supplemental Methods. Event collection
was performed using the Accuri C6 flow cytometer (BD Biosciences) and
events were analyzed with Kaluza software (Beckman Coulter).
Binding assay
Binding of FX (0-40 mg/mL) to the soluble extracellular domain of hSR-AI
(0.5 mg per well) in the presence or absence of potential competitors was
assessed in an immunosorbent assay as described in the supplemental Methods.
FIX and FX activity assays
Endogenous murine FX activity was measured through FX activation into
FXa by the Russel Viper Venom-X enzyme (RVV-X) and the subsequent
conversion of the chromogenic substrate S-2765. FIX activity was determined
using a 1-stage clotting assay.
Mice
Wild-type (wt) C57Bl/6 mice were purchased from Janvier Labs and SRAI–deficient C57Bl/6 mice (B6.Cg-Msr1tm1Csk/J) were from The Jackson
Laboratory. Blood samples were taken between 48 hours and 72 hours after
arrival of the mice. Experiments using gadolinium chloride (GdCl3)-6H2O
or polyclonal anti–mSR-AI antibodies were performed as described in the
supplemental Methods.
Statistical analyses
Statistical analyses were performed using GraphPad Prism software. For
flow cytometry data, a 2-way analysis of variance (ANOVA) followed
by Sidak posttest for multiple comparisons was performed. Statistical
significance of histograms was performed with Mann-Whitney nonparametric unpaired statistical tests. All graphs were built using GraphPad Prism
software.
Results
FX binds to differentiated macrophages but not to monocytes
Recently, FX was found to interact with macrophages of different
origin (Kupffer cells and THP1-derived macrophages).16 To explore
the specificity of FX binding to macrophages vs monocytic cells, we
compared binding of Alexa 488–labeled FX to undifferentiated
THP1 cells and THP1-derived macrophages. Following incubation
(1 hour at 37°C), flow cytometry analysis revealed a similar amount
of fluorescence for undifferentiated THP1 cells incubated either
with control Alexa 488–conjugated Fab92 or Alexa 488–labeled FX
(1.5 6 1.0 3 103 and 3.2 6 1.9 3 103 fluorescence units; P . .05),
suggesting that FX is unable to bind these cells (Figure 1A,C). In
contrast, THP1-derived macrophages incubated with Alexa 488–
conjugated FX displayed a significant 10-fold shift of fluorescence
compared with cells incubated with Alexa 488–conjugated Fab92
(25.9 6 2.9 3 103 and 3.0 6 0.9 3 103 fluorescence units, respectively, P , .0001), indicating efficient binding of FX to these cells
(Figure 1B-C). This result was confirmed using immunofluorescent staining (Figure 1D-E). No FX signal was visible using
undifferentiated THP1 cells, whereas a cluster-shaped FX staining
was observed for THP1-derived macrophages. Similar results
were obtained using primary monocytes and monocyte-derived
macrophages (data not shown). Binding of FX to THP1-derived
macrophages but not undifferentiated THP1 cells indicates that
candidate receptors for FX become available upon monocyte-tomacrophage differentiation.
FX colocalizes with SR-AI at the surface of
THP1-derived macrophages
Analysis of literature and proteome databases revealed several transmembrane receptors upregulated upon monocyte-to-macrophage
transition, 4 of which (mannose-macrophage receptor [MMR], dendritic
cell-specific–intercellular adhesion molecule-3 grabbing nonintegrin
[DC-SIGN], C-type lectin domain family 10 member A [CLEC10A],
and SR-AI), were selected arbitrarily for testing their capacity to act as
an FX receptor. We first performed Duolink–proximity ligation assay
(PLA) analysis, which generates red fluorescent spots if proteins (in
this case FX and 1 of the selected receptors) are located within a radius
of 40 nm. No fluorescent spots were formed when cells were incubated
with anti-FX and anti-receptor antibodies in the absence of FX
(Figure 2A-E), confirming the specificity of this approach. A similar
background signal was obtained for cells incubated with FX and
stained for FX with MMR, DC-SIGN, or CLEC10A (Figure 2A-C),
despite the presence of each of these receptors at the macrophage
surface (supplemental Figure 1). In contrast, distinct red spots were
detected for cells stained for FX and SR-AI (Figure 2D). This positive
signal between FX and SR-AI was also detected using primary human
CD141 monocyte-derived macrophages (Figure 2E). Subsequent
double immunofluorescent staining for FX and MMR or SR-AI was
performed using THP1-derived macrophages incubated with FX
and analyzed in confocal microscopy (Figure 2F-G). Little if any
colocalization was detected for FX and MMR (Figure 2F). Signals for
FX and SR-AI displayed similar staining patterns with clear areas of
colocalization (yellow indicated with white arrows). Reconstituted
orthoview (Figure 2G) shows colocalization between SR-AI and FX in
all 3 planes (XY, XZ, and YZ) that seems to be restricted to the surface
of macrophages. In order to verify this colocalization between FX and
SR-AI, cell stacks acquired in confocal microscopy were analyzed to
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FX and SR-AI had a tMC value of 0.53 6 0.23 (mean 6 standard
deviation [SD]; P 5 .0005; Figure 2I). This statistical difference in
tMC value confirms that the FX fluorescent signal truly overlaps
with that of SR-AI.
FX binds in vitro to SR-AI
Having identified colocalization of FX with SR-AI at the macrophage
surface, we then investigated binding of FX to the extracellular
fragment of SR-AI in an immunosorbent assay using purified
proteins. Little, if any, FX binding was detected to albumin-coated
control wells. In contrast, FX binding to SR-AI was dose-dependent
with a calculated half-maximal binding of 2.9 6 0.9 mg/mL (Figure 3A).
Subsequently, 3 potential competitors for SR-AI binding were tested.
First, acetylated low-density lipoprotein (Ac-LDL; a natural ligand)
failed to compete with FX (1 mg/mL) at concentrations up to 50 mg/mL,
suggesting that both proteins bind to distinct regions within SR-AI.
However, efficient dose-dependent inhibition was observed for
polyinosinic acid (a polyanionic compound known to interact with
SR-AI19) or polyclonal SR-AI–inhibiting antibodies (50% inhibitory
concentration 5 2.4 6 0.1 mg/mL and 2.9 6 0.2 mg/mL, respectively;
Figure 3B). Polyinosinic acid also inhibited this interaction dosedependently when tested at higher FX concentrations (5 mg/mL;
supplemental Figure 2A). Binding was further reduced significantly in
the presence of a soluble SR-AI fragment (supplemental Figure 2B).
Thus, FX binds specifically to human SR-AI in a system using purified
proteins.
Effect of FX on internalization of the SR-AI ligand Ac-LDL
Figure 1. FX binding to human monocytes and macrophages. (A-B) Undifferentiated THP1 (A) or THP1-derived macrophages (B) were incubated with Alexa
488–labeled FX or Alexa 488–labeled Fab92 as a control (10 mg/mL, 1 hour at 37°C)
and subsequently analyzed by flow cytometry for FX binding. Black curves represent
Fab92-incubated cells (control) whereas red curves represent Alexa 488-FX–incubated
cells. Representative plots of 3 different experiments are shown. (C) The mean FX
fluorescence was quantified and is expressed in arbitrary units. Each dot represents 1
experiment (N 5 5-6 in total) and bars represent the mean 6 SD. ***P , .001 in a
2-way ANOVA followed by the Sidak posttest for multiple comparison. (D-E)
Widefield microscopy images of immunofluorescent staining for FX (green) in
undifferentiated THP1 (D) or THP1-derived macrophages (E) incubated with 10
mg/mL FX (1 hour at 37°C). Nuclei and polymerized actin were counterstained
using DAPI (blue) and Alexa 647–labeled phalloidin (magenta), respectively.
Arrows indicate spots of FX staining. Bars represent 10 mm; objective, 363. DAPI,
4,6 diamidino-2-phenylindole; N.S., not significant.
calculate the tMC. This coefficient (ranging from 0 to 1) is a statistical
parameter allowing for the determination of whether 2 fluorescent
signals overlap truly or coincidentally. To make this calculation, we
compared cells stained for FX and MMR (negative control) or SR-AI.
FX staining intensity was similar for both experiments (Figure 2H),
ensuring that data analysis was unbiased by differences in FX
binding between experiments. Macrophages stained for FX and
MMR had a tMC value of 0.12 6 0.09, whereas cells stained for
We next compared binding and/or uptake of FX with the SR-AI ligand
Ac-LDL by THP1 macrophages using confocal immunofluorescent
microscopy. Alexa 488–conjugated Ac-LDL efficiently bound to
THP1 macrophages, whereas costaining of the cytoskeleton using
phalloidin revealed that Ac-LDL was located inside of the cell
(Figure 3C). Indeed, additional analysis indicated that Ac-LDL
colocalized with the early endosomal marker early endosome antigen-1
(EEA1) (Figure 3D). In contrast, costaining for FX and the cytoskeleton
revealed that FX remained at the cell surface (Figure 3E). Indeed, no
colocalization between FX and EEA1 could be detected (Figure 3F).
Finally, we tested whether co-incubation of Ac-LDL with FX (at its
plasma concentration of 10 mg/mL) would allow uptake of Ac-LDL.
Confocal microscopical analysis showed that Ac-LDL was endocytosed
by THP1 macrophages and delivered to the early endosomes in the
presence of FX, whereas simultaneously FX remained at the cell surface
(Figure 3G-H). Thus, despite the binding of FX to SR-AI, sufficient
SR-AI receptors remain to mediate binding and uptake of other ligands
for this receptor.
FX binds to SR-AI–transfected HEK-293 cells
To determine whether cellular SR-AI is able to act as a receptor for
FX, binding of FX to THP1 macrophages was assessed in the presence
of polyclonal anti–SR-AI antibodies. As depicted in Figure 4A-D,
these antibodies reduced FX binding to THP1 macrophages by .80%.
Additional binding experiments were performed using HEK-293
cells transfected or not with pcDNA6 encoding full-length hSR-AI
(pcDNA6/hSR-AI). Transfection with pcDNA6/hSR-AI induced
the abundant expression of SR-AI as assessed by immunofluorescent staining of nontransfected and transfected cells (Figure 4E-F).
Following incubation with FX (1 hour at 37°C), nontransfected HEK293 cells showed a weak background staining for FX (mean pixel
intensity per cell: 419 6 24 gray level units [GLU]; mean 6 standard
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Figure 2. Colocalization of FX in human macrophages. (A-E) Widefield microscopy images of Duolink-PLA assay between FX and MMR (A), DC-SIGN (B), CLEC10A (C),
or SR-AI (D) in THP1-derived macrophages, or between FX and SR-AI in CD141-derived macrophages (E) incubated with either PBS (top panels) or FX (bottom panels).
Nuclei were counterstained using DAPI. Bars represent 10 mm; objective, 340. Red spots indicate a distance ,40 nm between 2 antigens. Images are representative of
3 different experiments. (F-I) Confocal analysis of immunofluorescent staining for FX (red) and MMR (F) or SR-AI (G) in a THP1-derived macrophage incubated with FX. Cell
stack was reconstituted in orthoview to visualize colocalization of the 2 signals (right panels). Arrows indicate areas of colocalization. Z depth is 0.5 mm; bars represent 10 mm;
objective, 363. The mean FX fluorescence of the cells was quantified using Fiji software (H). tMC represents a statistical parameter verifying whether fluorescent signals truly overlap
and was calculated using JACoP plugin in Fiji for THP1-derived macrophages incubated with FX and immunostained for FX and MMR (negative control) or SR-AI (I). ***P , .001,
respectively, in the Mann-Whitney nonparametric unpaired statistical test. Dots represent each individual cell value and bars represent the mean 6 SD of 7 (FX/MMR) to 12 (FX/SR-AI)
cells from 3 independent experiments. PBS, phosphate-buffered saline.
error of the mean [SEM]; n 5 162 cells) (Figure 4G,I). In contrast, distinct bright spots of FX staining were observed with
HEK-293 pcDNA6/hSR-AI cells incubated with FX (Figure 4H).
Quantification revealed a significantly increased level of FX
fluorescence (mean pixel intensity per cell: 728 6 31 GLU;
n 5 218 cells; P , .0001) (Figure 4I). Moreover, HEK-293
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Figure 3. Differential binding of FX and Ac-LDL to SR-AI. (A-B) Increasing concentration of FX (0-40 mg/mL) (A) or increasing concentration of SR-AI inhibitor (Poly[I],
polyclonal anti–SR-AI antibody, or Ac-LDL; 1-50 mg/mL) along with 1 mg/mL FX (B) were incubated in microtiter wells coated with hSR-AI (0.5 mg per well). Bound FX was
probed using a peroxidase-labeled polyclonal anti-FX antibody and revealed by chromogenic conversion of tetramethylbenzidine. For the negative control, hSR-AI was
omitted during the coating (s in panel A). Data represent the mean 6 SD (n 5 3-7). (C-H) Confocal analysis of THP1-derived macrophages incubated (1 hour at 37°C)
with 150 nM Alexa 488–labeled Ac-LDL (C-D), 10 mg/mL Alexa 488–labeled FX (E-F), or preincubated with FX prior to the addition of Alexa 488–labeled Ac-LDL (G-H).
Polymerized actin was counterstained using Alexa 647–labeled phalloidin (C,E,G) or cells were immunostained for EEA-1 (D,F,H). Dotted lines define cell boundaries
based on phalloidin staining. Arrows indicate area of colocalization. Z depth is 0.5 mm; bars represent 10 mm; objective, 363. OD, optical density; PoAb, polyclonal
antibody; Poly[I], polyinosinic acid.
pcDNA6/hSR-AI cells incubated with FX showed a similar staining
pattern for SR-AI and FX with distinct areas of colocalization between
the 2 signals when analyzed using confocal microscopy (white
arrows in Figure 4J). Therefore, stable expression of SR-AI at
the cell surface confers to the cell the ability to bind FX.
SR-AI–deficient macrophages lack FX binding
To deepen the physiological context of our findings, we included
experiments using SR-AI–deficient C57BL/6 mice. First, CD1151
monocytes were purified from bone marrow of wt or SR-AI–deficient
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Figure 4. Binding of FX to cellular SR-AI. (A-D)
THP1-derived macrophages were preincubated with
PBS (A), nonspecific IgG (B), or a polyclonal anti–SR-AI
antibody (C) and further incubated with 10 mg/mL
Alexa 488–labeled FX (1 hour at 37°C). Images were
acquired in widefield microscopy and quantified for FX
fluorescence (D). (E-I) Immunofluorescent staining of
SR-AI (red) and FX (green) was performed in nontransfected HEK-293 cells (E and G, respectively) or
HEK-293 cells transfected with pcDNA6/hSR-AI (F and
H, respectively) incubated with 10 mg/mL FX (1 hour at
37°C). Images were acquired in widefield microscopy
and subsequently quantified for FX fluorescence (I).
Data are presented in mean pixel intensity per cell (D,I).
Boxes represent the median and 25th to 75th percentile,
and bars represent the 10th to 90th percentile (at least 5
different fields per experiment in 3 different experiments).
(J) Representative images of double immunostaining for
FX and SR-AI in HEK-293 pcDNA6/hSR-AI analyzed
using confocal microscopy. Nuclei and polymerized
actin were counterstained using DAPI (blue) and Alexa
647–labeled phalloidin (magenta), respectively. Objective, 363; bars represent 10 mm; Z depth is 0.4 mm (J)
and arrows indicate area of colocalization. ***P , .001,
respectively, in the Mann-Whitney nonparametric unpaired statistical test.
C57BL/6 mice and differentiated into macrophages. Immunostaining
for mSR-AI confirmed its lack of expression in cells isolated from
SR-AI–deficient mice (Figure 5A-B). After validating that human
FX interacts with mSR-AI (supplemental Figure 3B), cells were
incubated with human FX (10 mg/mL; 1 hour, 37°C). wt macrophages showed numerous areas of FX staining resembling those
observed in human THP1-derived macrophages (Figure 5A). Quantification of FX fluorescence gave a mean pixel intensity of 654 6 49
GLU (mean 6 SEM; n 5 97 cells; Figure 5C). However, FX staining
was absent for mSR-AI–deficient macrophages (Figure 5B) and
quantification evidenced a significant decrease in fluorescent signal
(mean pixel intensity 5 314 6 20 GLU; n 5 130 cells, P , .0001;
Figure 5C). The absence of FX binding to SR-AI–deficient macrophages points to SR-AI being critical for the accumulation of FX at the
macrophage surface.
Role of SR-AI in regulating FX plasma levels in vivo
We then measured endogenous murine FIX and FX activity in order
to investigate how the absence of mSR-AI affects FX plasma levels.
When compared with wt C57BL/6 mice, SR-AI–deficient mice
showed similar levels of mFIX (P . .05; Figure 6A). In contrast,
a marked significant decrease in mFX activity was observed:
34.5% 6 18.4% of the wt value (mean 6 SD; P , .0001; Figure 6A).
To further explore the role of mSR-AI in vivo, we injected wt mice
with polyclonal anti–mSR-AI or control antibodies (1.25 mg/kg) and
mFX levels were measured 6 hours and 24 hours after antibody
injection. Interestingly, endogenous mFX levels were reduced by
25% 6 9% and 40% 6 19%, respectively, upon anti–mSR-AI
antibody injection compared with control (P , .01; supplemental
Figure 3A). We then treated both wt- and SR-AI–deficient mice with
GdCl3 (50 mg/kg), which inactivates macrophages. Endogenous
mFX levels were decreased to 34% 6 6% 24 hours after GdCl3
application in wt mice (Figure 6A). In contrast, no further decrease
in endogenous mFX levels was seen in SR-AI–deficient mice
(35% 6 8% vs 34% 6 19% in GdCl3 and nontreated SR-AI–deficient
mice, respectively; P . .05; Figure 6A). Finally, we tested the effect of
inhibitory polyclonal anti–mSR-AI antibodies on the in vivo survival of
IV administered human FX over a 24-hour period. Compared with
control antibody-treated mice, the initial recovery of FX at 5 minutes
after injection was significantly reduced in anti–mSR-AI–treated mice
(59.2% 6 8.2% vs 44.9% 6 9.9%, for control and anti–SR-AI mice,
respectively; n 5 15-20 per group; P , .0001; Figure 6B). Furthermore, the mean residence time (MRT) was significantly shorter in
anti–SR-AI–treated mice compared with control antibody-treated mice
(MRT 5 5.4 hours [minimum-maximum: 5.3-5.7 hours] vs 3.9 hours
[minimum-maximum: 3.7-4.9 hours]; P , .05; Figure 6C). These data
point to the absence of functional SR-AI being associated with a reduced
circulatory half-life of FX, indicating that SR-AI is pertinent to sustain
normal levels of FX.
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Figure 5. FX binding to murine SR-AI–deficient macrophages. (A-C) CD1151-derived macrophages from wt (A) or SR-AI–deficient (B) C57BL/6 mice incubated with
human FX (10 mg/mL, 1 hour at 37°C) were double immunostained for murine SR-AI (red) and human FX (green). Images were acquired in widefield microscopy and
subsequently quantified for FX fluorescence (C). Data are presented in mean pixel intensity per cell. Boxes represent the median and 25th to 75th percentile, and bars
represent the 10th to 90th percentile (at least 5 different fields per experiment in 3 different experiments). Nuclei and polymerized actin were counterstained using DAPI (blue)
and Alexa 647–labeled phalloidin (magenta), respectively (A and B). Objective, 363; bars represent 10 mm; ***P , .001, respectively, in the Mann-Whitney nonparametric
unpaired statistical test.
Discussion
In this study, we have identified SR-AI as a receptor for FX on
macrophages and revealed that their interaction is of physiological
relevance to maintain normal FX plasma levels.
Previously, we have shown that FX is targeted to resident liver
macrophages, and that GdCl3-mediated macrophage inactivation
severely reduces FX plasma levels.16,20 Combined with the observation that FX remains at the surface of macrophages, rather than
being internalized and degraded,16,20 these data point to macrophages serving a protective role in maintaining FX plasma levels. To
better understand this protective role, we searched for receptors that
are involved in the binding of FX to the macrophage surface. To limit
the number of potential candidates, we first compared binding of FX
to resting monocytes and differentiated macrophages. Interestingly,
no binding of FX to resting monocytes was observed (Figure 1).
Previously, Mac-1 has been proposed as a potential FX receptor at
the surface of monocytes.21 However, these experiments were done
using adenosine 59-diphosphate– or ionomycin-stimulated monocytes, conditions that induce conformational changes within the
Mac-1 integrin.22 The notion that resting monocytes were used in our
experiments may explain the absence of FX binding to these cells
(Figure 1). It should further be noted that treatment of mice with a
Mac-1 inhibitor (ie, neutrophil-inhibitory factor) leaves FX levels
unchanged (data not shown), suggesting that Mac-1 is not involved
in the regulation of FX plasma levels.
A number of studies have investigated the changes in the transcriptome during monocyte-to-macrophage transition, revealing numerous receptors and other proteins that undergo important changes
in expression during this transition.23-25 Based on these studies, we
arbitrarily selected 4 candidates that could potentially act as receptor
for FX. Three of them (MMR, DC-SIGN, and CLEC10A) belong to
the C-type lectin receptors, characterized by their ability to bind
carbohydrate structures in a calcium-dependent manner. MMR and
DC-SIGN bind both mannose and fucose residues, whereas CLEC10A
specifically recognizes a- and b-linked N-acetylgalactosamine.26-28
Each of these carbohydrate structures is present in FX,29 providing a
rationale for a potential interaction between FX and these receptors.
However, despite their evident expression in THP1 macrophages
(supplemental Figure 1), no colocalization was found when assessed
via Duolink-PLA analysis (Figure 2). Also, classic double staining
did not reveal any colocalization between FX and any of these
receptors, as is illustrated for MMR in Figure 2.
The fourth candidate was SR-AI, also known as macrophagescavenger receptor or CD204. A first potential link between FX and
SR-AI became apparent via Duolink-PLA analysis. This experiment
revealed abundant colocalization of FX with SR-AI on both THP1and primary CD141-derived macrophages (Figure 2). Moreover, no
binding of deglycosylated FX to macrophage-expressed SR-AI
could be detected in this Duolink-PLA analysis (not shown), compatible with our previous finding that N-linked glycans on FX are needed
for binding to macrophages.16 To confirm the close proximity between
FX and SR-AI, classic double-staining experiments were performed
using THP1 macrophages as well as stably transfected cells expressing
SR-AI. In both cases, an apparent colocalization was observed when
cells were analyzed via confocal microscopy (Figures 2G and 4J).
Indeed, analysis of the fluorescent signals for FX and SR-AI on
THP1 macrophages revealed a statistically relevant overlap of both
signals (tMC 5 0.53 compared with tMC 5 0.12 for negative
control; P , .0005; Figure 2I). Importantly, FX binding was reduced
significantly to just above background levels, when analyzing
binding of FX to murine SR-AI–deficient CD1151 macrophages
(Figure 5). Moreover, binding of FX to THP1 macrophages
was inhibited by .80% in the presence of anti–SR-AI antibodies
(Figure 4A-D). These data point to SR-AI being the dominant receptor
for FX on macrophages, although our findings do not exclude the
presence of additional FX-binding receptors.
To further explore the interaction between FX and SR-AI, binding
experiments using the soluble extracellular portion of SR-AI were
performed. FX binding to immobilized SR-AI was saturable and
dose-dependent, and half-maximal binding was calculated to be
3 mg/mL (Figure 3), thus allowing receptor binding at physiological
plasma concentrations of FX (ie, 10 mg/mL). Polyclonal anti–SR-AI
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
BLOOD, 11 FEBRUARY 2016 x VOLUME 127, NUMBER 6
Figure 6. SR-AI and FX levels in vivo. (A) Citrated plasma was collected from wt or
SR-AI–deficient C57BL/6 mice prior or 24 hours after GdCl3 injection (50 mg/kg). FIX
and FX activity were measured (A) and results are expressed in percentage of the
normalized mean of wt values. (B-C) C57Bl/6 wt mice were injected in the caudal
vein with FX (10 mg per mouse) in the presence of control IgG or polyclonal
anti–mSR-AI antibodies (50 mg per mouse; 20 mouse per group). Plasma was
collected at various time points (5 minutes, 1 hour, 3 hours, 6 hours, and 24 hours;
4-5 mice per group; 2-3 collections per mouse) and residual FX antigen was measured. Data were fitted to a biexponential decay equation to calculate pharmacokinetic parameters. Shown are the recovery at 5 minutes (B) and MRT (C) for each
group. (A-B) Data represent the mean 6 SD. (C) Boxes represent mean 6 range of
the calculated MRT, with the range being obtained from curves plotted with the
minimum and maximum SD of residual FX levels. ***P , .001, respectively, in the
Mann-Whitney nonparametric unpaired statistical test.
antibodies and polyinosinic acid were able to compete for FX
binding in a dose-dependent manner in this system using purified
proteins (Figure 3), indicating that FX binding to SR-AI is specific.
Interestingly, binding was affected to a minor extent, if any, by the
SR-AI ligand Ac-LDL. This lack of inhibition enabled us to test
whether the presence of FX (which remains bound to SR-AI at the
cell surface; Figure 3E-F) still allows the uptake of other SR-AI
ligands, such as Ac-LDL. Confocal microscopy analysis clearly
showed that FX did not prevent the binding and uptake of Ac-LDL
by THP1 macrophages (Figure 3G-H), probably because of the large
excess of SR-AI receptors over FX molecules at the macrophages
surface.
SR-AI IS CRUCIAL TO REGULATE PLASMA LEVELS OF FX
785
It is intriguing to note that, to the best of our knowledge, FX is the
first nonmodified circulating protein identified to interact with SR-AI.
One could argue that purification of FX coincides with modifications
in the protein that induce binding to SR-AI. However, we did not
observe differences in SR-AI binding between plasma-derived FX or
recombinant FX (not shown), both of which are purified via 2 different methods. Furthermore, FX levels are reduced by 65% in
SR-AI–deficient mice. Should SR-AI binding require any FX
modification, then one would not expect that .60% of the FX population in plasma would be dependent on regulation by SR-AI. Previously identified ligands included modified LDL variants, glycated
albumin, glycated collagens, and b-amyloid fibrils.30 Such modified
proteins are undesirable in the circulation, which may explain why they
are efficiently eliminated from the circulation by SR-AI. For FX, a rather
distinct mechanism appears to be involved. Indeed, far from being
undesirable, appropriate plasma levels of FX are needed to sustain
normal hemostasis. This may explain why other than “classic” ligands
for SR-AI, FX is not endocytosed and degraded by macrophages even
upon prolonged incubation (up to 2 hours).16 Unexpectedly, chemical
depletion of macrophages using GdCl3 in wt mice results in FX levels
that are decreased to about 35% of normal (Figure 6A), demonstrating
that intact macrophages are needed to maintain FX plasma levels.16
Similarly, mice deficient for SR-AI are characterized by reduced FX
levels (34% of normal; Figure 6A). These levels are no further decreased
upon GdCl3 treatment in SR-AI–deficient mice (Figure 6A), suggesting
that removal of SR-AI appeared responsible for the reduction of FX
levels. Indeed, injection of anti–SR-AI antibodies significantly reduced
FX levels in wt mice (supplemental Figure 3A). Moreover, the presence
of SR-AI–blocking antibodies resulted in a significantly reduced
recovery and MRT of human FX that was coadministered with these
antibodies (Figure 6B-C). These data not only show that the interaction
between FX and SR-AI is of high physiological significance, but
also indicate that the FX/SR-AI interaction is fundamentally different
compared with the interaction between SR-AI and other ligands.
Apparently, SR-AI is critical in protecting FX in the circulation. This
may seem counterintuitive in view of the functions and normal mode
of action of macrophages in general and SR-AI in particular. However,
this observation is not without precedent. Previously, carcinoembryonic antigen has been described as a glycoprotein that is recycled by
macrophages rather than being degraded.31
Although our results show that SR-AI appears to play a prominent
role in regulating plasma FX levels, so far we can only speculate on
the reason why FX remains associated at the surface of macrophages
in an SR-AI–dependent manner. It is tempting to consider a proteinrecycling program resembling that of immunoglobulin G (IgG) and
albumin, which via the neonatal Fc receptor are rescued from cellular
catabolism.32,33 An essential difference is that the SR-AI/FX complex
likely forms an extracellular reservoir, whereas IgG and albumin are
recycled within the cells. Indeed, we have previously demonstrated
that macrophage-bound FX is released in a time-dependent fashion
from the macrophage surface as an intact, nonproteolyzed protein.16
The presence of such a reservoir would imply that circulating levels of
FX in plasma represent only a part of the total FX population present in
the vasculature. It should further be noted that our findings do not
exclude the presence of other FX-binding receptors, and it seems
likely that other partners at the macrophage surface contribute to
the physiological role of SR-AI to maintain plasma levels of FX.
Studies have been initiated to decipher this unusual mechanism that
protects FX.
Apart from its importance to maintain FX plasma levels, the FX/SRAI interaction at the macrophage surface may also be of relevance for
FX functioning beyond hemostasis, for instance in relation to the innate
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
786
BLOOD, 11 FEBRUARY 2016 x VOLUME 127, NUMBER 6
MUCZYNSKI et al
immune system. Indeed, both FX and SR-AI contribute to the innate
immune response via interactions with structural elements of viruses
such as herpes simplex virus-1 and adenoviruses.10,11,34,35 In view of
these observations, the possibility exists that the interaction between FX
and SR-AI participates in the complex process of pathogen recognition
and clearance, in which FX bridges SR-AI and the pathogen. Further
studies are needed to decipher the precise role of the potential
interaction between the FX/SR-AI complex and the innate immune
system.
Acknowledgments
The authors are grateful to Florence Fragnet and Anne-Lise Marville
for their everyday skillful administrative assistance. The authors
thank Pascal Roux and Dr Audrey Salles (Pasteur Institute, Paris,
France) for their help in accessing the confocal microscope facility
and interpretation of optical microscopy data, and Emilie Bouvier
and Alexandre Diet (Center for Breeding & Distribution of Transgenic Animals, Orléans, France) for their technical assistance.
This work was supported by research funding from Novo Nordisk
S/A. However, Novo Nordisk S/A had no role in the design of the
study or the analyses and interpretation of the data.
Authorship
Contribution: V.M., A.B., C.L., A.H., and G.C. performed the
experiments; V.M., C.V.D., P.J.L., and O.D.C. designed the study
and analyzed the data; V.M., P.J.L., and O.D.C. wrote the paper; and
all authors contributed to the editing of the final manuscript.
Conflict-of-interest disclosure: P.J.L. is a consultant for Novo
Nordisk S/A. The remaining authors declare no competing financial
interests.
Correspondence: Cécile V. Denis, INSERM U1176, 80 rue du
General Leclerc, 94276 Le Kremlin-Bicêtre cedex, France; e-mail:
[email protected].
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2016 127: 778-786
doi:10.1182/blood-2015-05-647032 originally published
online November 25, 2015
Macrophage receptor SR-AI is crucial to maintain normal plasma levels
of coagulation factor X
Vincent Muczynski, Amine Bazaa, Cécile Loubière, Amélie Harel, Ghislaine Cherel, Cécile V. Denis,
Peter J. Lenting and Olivier D. Christophe
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