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PLATELETS AND THROMBOPOIESIS
Transforming growth factor-␤–induced protein (TGFBIp/␤ ig-h3) activates
platelets and promotes thrombogenesis
Ha-Jeong Kim,1 Pan-Kyung Kim,1 Sang Mun Bae,1 Hye-Nam Son,1 Debraj Singh Thoudam,1 Jung-Eun Kim,2
Byung-Heon Lee,1 Rang-Woon Park,1 and In-San Kim1
Departments of 1Biochemistry and Cell Biology and 2Molecular Medicine, Cell and Matrix Research Institute, School of Medicine, Kyungpook National University
School of Medicine, Daegu, Republic of Korea
Transforming growth factor-␤–induced
protein (TGFBIp)/␤ig-h3 is a 68-kDa extracellular matrix protein that is functionally
associated with the adhesion, migration,
proliferation, and differentiation of various cells. The presence of TGFBIp in
platelets led us to study the role of this
protein in the regulation of platelet functions. Upon activation, platelet TGFBIp
was released and associated with the
platelets. TGFBIp mediates not only the
adhesion and spread of platelets but also
activates them, resulting in phosphatidylserine exposure, ␣-granule secretion, and
increased integrin affinity. The fasciclin 1
domains of TGFBIp are mainly responsible for the activation of platelets. TGFBIp promotes thrombus formation on type
I fibrillar collagen under flow conditions
in vitro and induces pulmonary embolism
in mice. Moreover, transgenic mice, which
have approximately a 1.7-fold greater
blood TGFBIp concentration, are significantly more susceptible to collagen- and
epinephrine-induced pulmonary embolism than wild-type mice. These results
suggest that TGFBIp, a human platelet
protein, plays important roles in platelet
activation and thrombus formation. Our
findings will increase our understanding
of the novel mechanism of platelet activation, contributing to a better understanding of thrombotic pathways and the development of new antithrombotic therapies.
(Blood. 2009;114:5206-5215)
Introduction
Platelet adhesion and activation are critical initial events in the process
of hemostasis and thrombosis.1 Platelet activation is notified by a few
characteristic molecular changes such as an increase in the affinity of the
fibrinogen receptor, integrin ␣IIb␤3, changing to procoagulant surface,
and ␣-granule secretion.2-5 These events initially are triggered by
subendothelial collagens exposed at sites of blood vessel injury.
However, collagen is not exclusively a thrombogenic factor. Initial
platelet-collagen interaction is rapidly reversible and insufficient for
stable adhesion. Stable adhesion requires additional interaction with
extracellular matrix (ECM) proteins such as fibrinogen and von
Willebrand factor (VWF). However, injured vessels of mice lacking
both fibrinogen and VWF also are occluded by thrombi.6 Various
soluble stimuli released from platelets and also existing in plasma,
including fibrinogen and fibronectin, strengthen platelet adhesion and
recruit more platelets into the growing thrombus.7,8 Interestingly, in the
case of fibronectin, families with a congenital deficiency of fibronectin
may show abnormal wound healing but no bleeding.9 In conditional
knockout mice with reduced plasma fibronectin levels, normal initial
platelet adhesion is observed, but thrombus formation is delayed.10
These results led to the suspicion that another adhesive protein may
serve as a substitute.
Transforming growth factor-␤–induced protein (TGFBIp) has
been identified and cloned as a major TGF-␤–responsive gene in
the lung adenocarcinoma cell line A549: TGF-␤–induced gene
human clone 3, abbreviated to ␤ig-h3.11,12 It is secreted from
several cell lines, including epithelial cells, endothelial cells,
keratinocytes, fibroblasts, and monocytes and exists in the ECM. It
is a 68-kDa protein that consists of 4 fasciclin 1 domains (FAS1
domain) and a carboxy-terminal arginyl-glycyl-aspartic acid (RGD)
sequence, both of which potentially bind integrins as a cell
attachment site. It plays a crucial role in cell adhesion, migration,
proliferation, and differentiation as a ligand of several integrins,
such as ␣3␤1, ␣v␤5, and ␣v␤3.11-17 As one of the ECM components,
TGFBIp is associated with other ECM molecules, including
collagen, fibronectin, laminin, and glycosaminoglycan.12 In addition, it also exists in blood and accumulates at inflammatory sites
such as fibrosis, rheumatoid arthritis, atherosclerosis, diabetic
kidney, diabetic angiopathy, and wound healing.18-21 Considering
that the expression of TGFBIp in several cell types has been known
to be highly induced by TGF-␤ and TGF-␤ that is abundantly
present in platelets, TGFBIp could possibly be produced in
platelets and play an important role in mediating platelet functions.
Until now, however, nothing has been reported on the presence of
TGFBIp in platelets and its role in platelet functions.
In this report, we established the existence of TGFBIp in human
platelets for the first time and discovered that it is released from activated
platelets. It can bind to the surface of platelets and can induce platelet
activation, resulting in the promotion of thrombogenesis.
Submitted March 24, 2009; accepted August 11, 2009. Prepublished online as Blood
First Edition paper, September 8, 2009; DOI 10.1182/blood-2009-03-212415.
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.
An Inside Blood analysis of this article appears at the front of this issue.
The online version of this article contains a data supplement.
5206
Methods
Mice and reagents
Eight-week-old male C57BL/6 mice were used. Alb-TGFBIp mice were
generated as described previously.22 These mice were maintained under
© 2009 by The American Society of Hematology
BLOOD, 10 DECEMBER 2009 䡠 VOLUME 114, NUMBER 25
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BLOOD, 10 DECEMBER 2009 䡠 VOLUME 114, NUMBER 25
specific pathogen–free conditions, and the use committee established at
Medical College of Kyungpook National University and Kyungpook
National University Hospital approved the protocol.
Prostaglandin E1 and fibrinogen were purchased from Sigma-Aldrich.
The following mouse monoclonal antibodies were obtained from Chemicon
International Inc: anti-␣5␤1 (MAB1969, for blocking assay), anti-␣5␤1
(MAB1999, for FACS), and anti-␣IIb␤3 (MAB1207). Anti–P-selectin
antibody and annexin V–fluorescein isothiocyanate (FITC) were obtained
from BD Pharmingen. Polyclonal rabbit anti-TGFBIp antibody is obtained
and purified as described previously.15,16,23 Fibrinogen-FITC was obtained
from Invitrogen. Recombinant TGFBIp proteins and each of the domains
(Do1, Do2, Do3, Do4, Do4-RGD, and Do1-4) were induced and purified as
described previously.15,16 Triton X-114 (Sigma-Aldrich) was used to
remove endotoxin from bacterial cell lysate.24 The proteins were kept at
⫺20°C for up to 2 months until use.
Platelet isolation and detection of TGFBIp expression
Approval was obtained from the Kyungpook National University Hospital
Institutional Review Board for these studies. Informed consent was
provided according to the Declaration of Helsinki. Blood from healthy
nonsmoking and aspirin-free donors was collected into polypropylene
syringes containing anticoagulant and was used within 3 hours. The final
concentration of anticoagulant was 0.38% (wt/vol) sodium citrate. Prostaglandin E1 was added to a final concentration of 1 ␮mol/L. Platelet-rich
plasma was prepared by centrifuging the blood at room temperature for
13 minutes at 230g. Platelets were then pelleted by centrifuging the
platelet-rich-plasma at room temperature for 10 minutes at 1000g. The
platelet pellets were then washed twice in resuspension buffer (HEPESbuffered Tyrode solution, pH 6.5; containing 5 mmol/L HEPES [N-2hydroxyethylpiperazine-N⬘-2-ethanesulfonic acid], 4 mmol/L NaH2PO4,
137 mmol/L NaCl, 2.6 mmol/L KCl, 1 mmol/L MgCl2, 5 mmol/L glucose,
and 1 ␮g/mL bovine serum albumin [BSA]) containing 5.0 U/mL apyrase.
After centrifugation at room temperature for 10 minutes at 1000g, platelets
were resuspended to a final concentration of 109 platelets/mL in platelet
buffer (5.0 mmol/L HEPES, pH 7.3; containing 140 mmol/L NaCl and
1 mg/mL BSA).3 To identify TGFBIp in platelets, washed platelets were
allowed to attach to fibronectin. Adherent platelets were immunostained
with anti-CD41 and anti-TGFBIp antibodies. For the immunoblotting
assay, platelets were activated with thrombin with or without stirring, and
the pellet and supernatant were then collected. After sodium dodecyl sulfate
polyacrylamide gel electrophoresis, we performed an immunoblotting
assay with polyclonal rabbit anti-TGFBIp antibody. Surface binding
TGFBIp was measured by flow cytometry with an anti-TGFBIp antibody
and FITC-conjugated anti–rabbit immunoglobulin (IgG) antibody. For
reverse transcriptase polymerase chain reaction (RT-PCR), total RNA was
extracted from isolated platelets by the use of a commercial kit (Fast Track
2.0; Invitrogen). RT-PCR was performed with primers as follows: primer
pair for TGFBIp, upstream primer 5⬘-AGATCGAGGACACCTTTGAG-3⬘
and downstream primer 5⬘-TTGTTCAGCAGGTCTCTCAG-3⬘ (size of the
expected products: 184 bp); primer pair for VWF, upstream primer
5⬘-TCTGTGGATTCAGTGGATGCA-3⬘ and downstream primer 5⬘CGTAGCGATCTCCAATTCCAA-3⬘ (size of the expected products: 84 bp);
primer pair for glyceraldehyde 3-phosphate dehydrogenase (GAPDH),
upstream primer 5⬘-CGAGAATGGGAAGCTTGTCA-3⬘ and downstream
primer 5⬘-GGAAGGCCATGCCAGTGA-3⬘ (size of the expected products:
508 bp). The PCRs were conducted as follows: 30 seconds at 94°C,
30 seconds at 56°C, and 30 seconds at 72°C for 40 cycles.
Platelet adhesion assay
The number of adherent platelets was essentially determined as previously
described.25 In brief, 100 ␮L of 5 ⫻ 108 platelets/mL were added to
TGFBIp-coated microtiter plates (for adhesion assay on immobilized
TGFBIp) or noncoated microtiter plates (for adhesion assay with soluble
TGFBIp) with soluble-phase TGFBIp (1 ␮g/mL or 5 ␮g/mL) or fibrinogen
(5 ␮g/mL) at 37°C for the indicated time. For immobilization of TGFBIp,
flat-bottomed 96-well plates were incubated with the indicated concentration of TGFBIp at 4°C overnight. After platelets were allowed to adhere,
THROMBOGENIC EFFECT OF TGFBIp
5207
nonadherent platelets were removed by aspiration, and wells were washed
3 times with 200 ␮L of Tyrode buffer. The acid phosphatase assay was used
to quantitate the number of adherent platelets. In brief, after the adhesion
and washing procedure had been performed as described previously, the
substrate solution (0.1 mol/L citrate buffer pH 5.4, containing 5 mmol/L
p-nitrophenyl phosphate and 0.1% Triton X-100; 150 ␮L per well) was
added and incubated for 1 hour at room temperature. After the reaction was
stopped, the color was developed by the addition of 100 mL of 2N NaOH,
and absorbance was finally measured at 405 nm by the use of a microplate
reader (Bio-Rad model 550 microplate reader). For the inhibition assay, washed platelets were incubated with the indicated functionblocking antibodies (1 ␮g/mL) for 30 minutes at 37°C and were then
allowed to adhere.
Spreading assay
A platelet suspension (100 ␮L containing 5 ⫻ 107 platelets) was added to
TGFBIp-coated microtiter (for adhesion assay on immobilized TGFBIp) or
noncoated microtiter plates (for adhesion assay with soluble TGFBIp) with
soluble-phase TGFBIp (1 ␮g/mL or 5 ␮g/mL) or fibrinogen (5 ␮g/mL),
platelets were allowed to adhere (1 hour or 15 minutes at 37°C), and wells
were washed with Tyrode buffer to remove nonadherent platelets. Adherent
platelets were fixed with 3.7% paraformaldehyde and permeabilized with
0.2% Triton X-100 in phosphate-buffered saline (PBS; 140 mmol/L NaCl,
3 mmol/L KCl, 10 mmol/L Na2HPO4, and 2 mmol/L KH2PO4; pH 7.4).
After washing twice with PBS, the fixed platelets were stained for F-actin
with FITC-conjugated phalloidin for 20 minutes and washed with PBS at
least 3 times before analysis. Fluorescence was visualized by the use of a
Zeiss Axiovert S100 microscope with a monochromatic light source and a
charge-coupled device camera. Metamorph software was used for capturing
images and subsequent analysis.
In vitro perfusion experiment
Platelet thrombi are formed by the perfusion of whole human blood over
fibrillar type I collagen. Glass coverslips immobilized with thrombogenic substrates (collagen type I) were assembled in a parallel plate
rectangular flow chamber. Whole blood with or without TGFBIp (at final
concentrations of 0.25, 0.5, 1, 2, 4, and 8 ␮g/mL) was perfused through
the chamber by aspiration with a syringe pump (Harvard Apparatus) at
an estimated shear stress of 0.2 dyn/cm2 (flow rate of 0.22 mL/min) for
5 minutes. The perfusion chamber was mounted on the stage of an
inverted microscope for real-time visualization of platelet interactions
with the immobilized substrates and platelet-platelet aggregation formation.26 Videos were recorded by the use of StreamPix digital video
recording software (Norpix). The frame capture rate was set to
10 frames per second. The area of thrombi formation was analyzed by
the use of Metamorph software. Thrombus sizes were measured for at
least 5 different fields per experiment.
Flow cytometric analysis
Isolated washed platelets were stained for flow cytometric analysis by
the use of a standard indirect procedure. In brief, platelets were
incubated with soluble-phase TGFBIp (1 ␮g/mL or 5 ␮g/mL) or fibrinogen (5 ␮g/mL) for 15 minutes at room temperature. The samples were
then washed and fixed with 1% formaldehyde. Platelets were incubated
with annexin V–FITC, fibrinogen-FITC, anti–P-selectin–FITC antibody,
or control isotype-matched mouse IgG-FITC for 30 minutes at room
temperature. After incubation, platelets were washed and analyzed in a
BD FACSAria system.
Thrombosis assays
Pulmonary thromboembolism experiments were performed as described
previously.27 In brief, 8-week-old C57BL/6 male mice under anesthesia
were injected with 100 ␮L of saline containing the indicated concentration
of collagen or TGFBIp and 100 ␮g/kg epinephrine via tail vein injection.
After 10 minutes, lungs were removed, fixed overnight with 10% neutral
buffered formalin, and embedded in paraffin. Sections were stained with
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5208
KIM et al
BLOOD, 10 DECEMBER 2009 䡠 VOLUME 114, NUMBER 25
Figure 1. Secretion of TGFBIp from activated human platelets and TGFBIp binding on activated platelet surfaces. (A) TGFBIp exists in platelets. Washed platelets were
allowed to adhere on fibronectin and were stained with anti-CD41 (red) and anti-TGFBIp (green) antibodies. Attached platelets and TGFBIp expression were observed under a
fluorescence microscope. (Top) Low magnification (⫻400; white bar represents 10 ␮m). (Middle and bottom) High magnification (⫻1000; white bar represents 5 ␮m).
(B) TGFBIp on resting (left) and thrombin-activated (right) platelet surfaces was detected by FACS analysis. Black shading denotes immunoglobulin G control, and the gray line
denotes TGFBIp. Presence of TGFBIp in resting and activated platelets (thrombin activation) was analyzed by immunoblotting. Washed platelets were incubated with (C) or
without stirring (D), during which they were activated by thrombin. Cell (cell lysate) and supernatant (sup) were separated by centrifugation at 18 000g and were immunoblotted
by anti-TGFBIp antibody. ␤-actin was loaded as a control. (E) TGFBIp mRNA exists in platelets. Total RNA was isolated and subject to RT-PCR for the analysis of TGFBIp,
VWF, and GAPDH mRNA expression. (F) Measurement of the amount of TGFBIp in platelets. The amount of TGFBIp in platelets was semiquantitatively measured by Western
blot analysis.
hematoxylin and eosin, and images were captured by the use of a Leica DM
LS microscope (4⫻/0.1) and a Spot color digital camera (National
Diagnosis).
Data analysis
Results are expressed as the mean plus or minus SD of at least
3 independent experiments. The statistical significance of differences
between test groups was used for statistical comparison (Sigma Stat; SPSS
Science). Statistical relevance was determined by the use of analysis of
variance (ANOVA). P values less than .05 were considered significant.
Results
TGFBIp is present in platelets, secreted upon activation, and
then associated with platelets
To investigate the expression of TGFBIp in human platelets,
isolated platelets from healthy volunteers were allowed to attach to
immobilized fibronectin. After washing, adherent platelets were
immunostained with anti-CD41 (as a specific platelet marker) and
anti-TGFBIp antibodies. As shown in Figure 1A (top), the TGFBIp
exists in human platelets. Both TGFBIp and CD41 are located in
cytoplasm of platelets in resting condition (Figure 1A middle);
however, when they are activated by thrombin, both proteins also
are located at the membrane (Figure 1A bottom). The presence of
TGFBIp on the surface of activated platelets was further confirmed
by fluorescence-activated cell-sorting (FACS) analysis. As shown
in Figure 1B, TGFBIp expression was shifted to platelet surface
after activation.
To further study the presence of TGFBIp in resting and
activated platelets, we separated platelet-released supernatant
and cell lysate by centrifugation. In resting platelets, TGFBIp
was found in cell lysate, but after platelets had been activated by
thrombin, TGFBIp was located in platelet-released supernatant
(Figure 1C). Interestingly, when supernatant was collected
without stirring (in this condition, secreted protein from activated platelets can bind to the platelet surface), TGFBIp was
found in cell lysate but not in the supernatant (Figure 1D). These
results suggest that TGFBIp secreted by activated platelet exists
on the platelet surface in association with activated platelet
membrane.
To confirm that TGFBIp is produced in platelets, we measured
TGFBIp mRNA along with 2 control messages for VWF and
GAPDH. As shown in Figure 1E, all 3 mRNAs were detected in
platelets, suggesting that TGFBIp is produced in platelets. To
quantify the amount of TGFBIp in platelets, we performed a
semiquantitative immunoblot assay estimating that 3.4 ng of TGFBIp is present in 108 platelets (Figure 1F).
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BLOOD, 10 DECEMBER 2009 䡠 VOLUME 114, NUMBER 25
THROMBOGENIC EFFECT OF TGFBIp
E
0.6
0.6
0.5
0.5
Resting
Resting
Activation
Activation
∗
∗
∗
∗
0.4
0.4
0.3
0.3
∗
∗
0.2
0.2
∗
∗
∗
∗
FN열 1
계
TGFBIp
계
열2
2
∗
80
80
60
60
∗
40
40
20
20
0.1
0.1
0.0
0.0
120
120
100
100
Adhesion %
Absorbance (405nm)
Absorbance
(405nm)
A
BSA
BSA
0.2
0.2
0.5
0.5
22
55
10
10
00
20
20
IgG
β1
α5β
β3
αIIbβ
µg/mL)
TGFBIp (µ
B
100
F 100
FN
계열1
TGFBIp
계열2
BSA
Fibronectin
Pixel/cell
80
80
TGFBIp
D
C
∗
0.4
0.4
∗
0.3
0.3
0.2
0.2
0.1
0.1
0.0
0.0
60
60
∗
40
40
∗
20
20
∗
9090
Pixel/cell
0.5
0.5
A b s o rb a n c e ( 4 0 5 n m )
Absorbance (405nm)
5209
00
IgG
α5β
β1
αIIbβ
β3
6060
3030
00
BSA
FN
TGFBIp
1
BSA
2
FN
3
TGFBIp
Figure 2. Adhesion and spreading of human platelets on immobilized TGFBIp. (A) Washed platelets were allowed to attach to immobilized TGFBIp for 1 hour with or
without thrombin. Adhered platelets were detected by the acid phosphatase assay as described in “Platelet adhesion assay.” (B) Washed platelets were attached to BSA,
fibronectin (FN), and TGFBIp (each 10 ␮g/mL) for 1 hour, and attached platelets were stained for anti-CD41 antibody and observed under a fluorescence microscope. The
white bar represents 10 ␮m. The amounts of (C) platelet adhesion and (D) platelet spreading (pixels/cells) in the presence of TGFBIp, BSA, or fibrinogen were determined.
Platelet adhesion was detected by the acid phosphatase assay, and spreading was detected by use of the MetaMorph program. (E) Integrin-dependent platelet adhesion on
TGFBIp. After washed platelets were incubated with integrins ␣5␤1 and ␣IIb␤3, or immunoglobulin G as a control (each 1 ␮g/mL) for 30 minutes, they were allowed to mix with
immobilized TGFBIp (10 ␮g/mL) or fibronectin (FN; 10 ␮g/mL) for 1 hour. Platelet adhesion was detected by the acid phosphatase assay. (F) Integrin-dependent platelet
spreading on TGFBIp. Platelet spreading (pixels/cell) on TGFBIp or fibronection (FN) was detected by use of the MetaMorph program. All results are shown as the
means ⫾ SD of 3 different experiments and ANOVA. *P ⬍ .05 compared with BSA or immunoglobulin G.
Immobilized TGFBIp supports adhesion and spread of human
platelets
As an ECM protein and a ligand of integrin, TGFBIp mediates the
adhesion of various cells. To investigate the role of TGFBIp in
platelet adhesion, washed platelets were allowed to attach on
immobilized TGFBIp for 1 hour with or without thrombin. Both
resting and activated platelet adhesion was found to increase in a
dose-dependent manner (Figure 2A). We confirmed that TGFBIp
was properly immobilized in a dose-dependent manner, reaching
the plateau at 10 ␮g/mL (supplemental Figure 1, available on the
Blood website; see the Supplemental Materials link at the top of the
online article). Next, we tested whether TGFBIp can also mediate
the spread of platelets. As shown in Figure 2B-D, TGFBIp
efficiently increased platelet spreading. Interestingly, although the
adhesion activity of TGFBIp is less than that of fibronectin (Figure
2C), spreading activity was observed in TGFBIp but not in
fibronectin (Figure 2D). Platelets adhesion was not induced by
boiled TGFBIp (supplemental Figure 2A) and adhesion to TGFBIp
was specifically blocked by anti-TGFBIp antibody (supplemental
Figure 2B).
To test which integrins are involved in TGFBIp-mediated adhesion,
we used monoclonal antibodies that block the function of integrin
subunits. The adhesion of platelets to the immobilized TGFBIp was
inhibited only by an antibody against integrin ␣5␤1, by 50% (Figure 2E).
We also tested other integrin function–blocking antibodies such as
␣IIb␤3, ␣V␤3, ␣2␤1, and ␣6␤1, but none of them showed any profound
effect on the adhesion of platelets to TGFBIp (data not shown). In
contrast, immobilized TGFBIp-induced spreading was inhibited not
only by antibodies against integrin ␣5␤1 but also by antibodies against
integrin ␣IIb␤3 (Figure 2F).
The TGFBIp protein is composed of 4 homologous internal repeat
domains, referred to as fasciclin 1 (FAS1), that are known to mediate
cell adhesion with the comparable activity to wild-type TGFBIp.16,17,28
To assess whether these FAS1 domains of TGFBIp are involved in
platelet adhesion and spreading, washed platelets were incubated with
each domain (as shown in Figure 3A) of TGFBIp. The adhesion of
platelets to each immobilized FAS1 domain of TGFBIp also was dose
dependent (Figure 3B). Each FAS1 domain (Do1, Do2, Do3, and Do4)
and Do1-4 is almost equally efficient in mediating platelet adhesion
compared with the wild-type TGFBIp (Figure 3C). There was no
profound difference between Do4 and Do4-RGD, which suggests that
the role of the RGD motif in TGFBIp is dispensable in mediating
platelet adhesion. Interestingly, in contrast to adhesion, the spreading of
platelets occurred only with wild-type TGFBIp and Do4-RGD (Figure
3D), which suggests the importance of the RGD motif rather than the
FAS1 domain of TGFBIp in platelet spreading. Taken together, these
results suggest that the FAS1 domain is responsible for the adhesion
through integrin ␣5␤1, whereas the RGD motif is responsible for
spreading through integrin ␣5␤1 and ␣IIb␤3.
Soluble-phase TGFBIp also induces platelet adhesion and
spreading but through different kinetics
We assessed the effect of soluble TGFBIp on platelet adhesion and
spreading by transferring the washed platelets to noncoated plates
with soluble TGFBIp. Soluble-phase TGFBIp induced adhesion of
washed human platelets in a dose-dependent manner (Figure
4A-B), with adhesion activity comparable with that of soluble
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5210
BLOOD, 10 DECEMBER 2009 䡠 VOLUME 114, NUMBER 25
KIM et al
A
I
II
III
Soluble TGFBIp induces platelet activation
IV RGD His-taq
TGFBIp
Do1
Do2
Do3
Do4
Do4-RGD
Do1-4
B
∗
Absorbance (405nm)
0.7
0.7
0.6
0.6
0.5
0.5
0.4
0.4
0.3
0.3
0.2
0.2
0.1
0.1
11
13
15
17
19
21
Do1-4
9
Do4-RGD
7
Do4
5
Do3
3
Do2
Do1
BSA
1
TGFBIp
0
0
23
25
C
Adhesion %
120
120
100
100
8080
6060
4040
2020
00
1
2
TGFBIp
Do1
3
Do2
4
Do3
5 Do4-RGD
6
7
Do4
Do1-4
D
To address the role of TGFBIp in activating platelets, we analyzed
phosphatidylserine (PS) exposure, P-selectin exposure, and integrin ␣IIb␤3 binding affinity after the stimulation of platelets with
TGFBIp. After washed platelets were incubated with soluble
TGFBIp for 15 minutes, PS and P-selectin exposure were detected
by annexin V-FITC and anti–P-selectin–FITC antibody, respectively. Likewise, integrin ␣IIb␤3 binding affinity was examined by
fibrinogen binding activity. As shown in Figure 5A, all 3 platelet
activation markers were increased by TGFBIp treatment in a
dose-dependent manner, showing the clear role of soluble TGFBIp
in platelet activation.
Further, we attempted to determine whether the FAS1 domains
of TGFBIp also can mediate platelet activation by measuring PS
exposure upon treatment of each FAS1 domain. Similar to
TGFBIp, PS exposure was increased by each FAS1 domain (Do1,
Do2, Do3, and Do4), Do4-RGD, and Do1-4 (Figure 5B). We
measured another activation marker, phosphorylated focal adhesion kinase, which was found to be markedly increased not only by
TGFBIp but also by each FAS1 domain (data not shown). As
expected, each FAS1 domain mediated washed platelet adhesion
(Figure 5C) and spreading (Figure 5D), but their individual
activities were somewhat lower than that of TGFBIp. Interestingly,
RGD does not seem to play a role in the activation of platelets by
TGFBIp because there was no significant difference between Do4
and Do4-RGD in mediating adhesion and spread. These results
indicate that FAS1 domains of TGFBIp are responsible for the
activation of platelets.
Pixel/cell
120
120
90
90
∗
∗
Soluble-phase TGFBIp promotes thrombus formation in vitro
60
60
30
30
00
1
2
3
BSA
TGFBIp
Do1
4
Do2
5
6 Do4-RGD
7
8
Do3
Do4
Do1-4
Figure 3. Adhesion and spreading of human platelets on the FAS1 domain of
TGFBIp. (A) Schematic representation of TGFBIp, which consists of 4 homologous
internal repeat domains called Fasciclin 1 (FAS1), and various deletion constructs of
TGFBIp for each FAS1 and other modifications. (B) Dose-dependent adhesion of the
immobilized FAS1 domain of TGFBIp is shown. Washed platelets were allowed to
adhere to the indicated FAS1 domain and are presented as platelet adhesion to each
immobilized FAS1 domain of TGFBIp (1, 2, 5, and 10 ␮g/mL). Washed platelet
adhesion (C) and spreading (D) on TGFBIp and each of the indicated FAS1 domains
were detected. Platelet adhesion was detected by the acid phosphatase assay,
spreading was detected by the use of the MetaMorph program. Results are
expressed as a percent of TGFBIp adhesion. All results are shown as the
means ⫾ SD of 3 different experiments and ANOVA. *P ⬍ .05 compared with BSA.
fibrinogen. Soluble-phase TGFBIp also induced platelet spreading,
whereas fibrinogen did not (Figure 4C).
Next, we incubated washed platelets with soluble-phase TGFBIp or boiled TGFBIp protein on noncoated plates for various
indicated time intervals. As shown in Figure 4D, we found that
soluble TGFBIp-induced platelet adhesion was initiated from
10 minutes after incubation and nearly reached the peak within
1 hour, followed by a marginal increase, whereas the adhesion of
platelets to immobilized TGFBIp was initiated more slowly after
incubation. Platelet adhesion was not observed until 30 minutes of
incubation and reached the peak at 3 hours of incubation. Therefore, platelet adhesion induced by soluble-phase TGFBIp occurred
faster than that induced by immobilized TGFBIp, and soluble
TGFBIp is more likely to activate platelets before directly mediating adhesion and spreading by itself.
To explore the role of TGFBIp in thrombus formation, we first
tested the effect of TGFBIp on platelet deposition to type I collagen
under flow conditions. Washed platelet perfusions (5 minutes at
shear rate 500⫺1) over type I collagen were performed in the
presence or absence of soluble TGFBIp, and platelet deposition
was monitored. In the presence of 2 ␮g/mL TGFBIp, the deposition
and spread of platelets were significantly increased, but interestingly a greater concentration of TGFBIp (5 ␮g/mL) did not
further enhance the rates of platelet adhesion and spread
(Figure 6A-B).
To test the effects of TGFBIp on thrombus formation, a
thrombus-formation assay was performed in vitro. Whole blood
from aspirin-free donors was perfused over type I collagen in the
presence or absence of soluble TGFBIp for 5 minutes, and platelet
deposition was monitored. As shown in Figure 6C and D, when
soluble TGFBIp was added to whole blood, the surface coverage of
generated thrombi was greater than that of whole blood without
soluble TGFBIp, and thrombus size was dependent on the concentration of soluble TGFBIp. The surface coverage reached a peak at
2 ␮g/mL TGFBIp and decreased at greater concentrations. Each
FAS1 domain also induced thrombus formation on collagen type I
(data not shown). These results suggest that soluble TGFBIp
induces thrombus formation in vitro, which is dependent on
FAS1 domains.
Increased peripheral-blood TGFBIp promotes pulmonary
embolism
To determine whether circulating TGFBIp affects thrombus formation in vivo, a collagen/epinephrine mouse model of thrombosis
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BLOOD, 10 DECEMBER 2009 䡠 VOLUME 114, NUMBER 25
THROMBOGENIC EFFECT OF TGFBIp
A
D
absorbance (405nm)
B
Fibrinogen
5µg/mL
µ
1.01
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
00
TGFBIp
1µg/mL
µ
∗
∗
∗
1.5
1.5
1.2
1.2
0.9
0.9
0.6
0.6
0.3
0.3
∗
00
∗
BSA
1
Fg
2
5
3
0.5
TGFBIp
4
5
1
2
C
Pixel/cell
TGFBIp
5µg/mL
µ
absorbance (405 nm)
BSA
70
70
60
60
50
50
40
40
30
30
20
20
10
10
00
∗
∗
Immobilized TGFBIp
계열4
Immobilized BSA
계열5
Immobilized fibronectin
계열6
Soluble TGFBIp
계열1
Soluble bolied TGFBIp
계열2
Soluble fibrinogen
계열3
1.8
1.8
5211
00
1
60
2
120
3
180
4
240
5 (hour)
300
6
5 (µg/mL)
∗
∗
1
BSA
2
Fg
5
3
0.5
4
5
TGFBIp
1
2
6
5 (µ
µg/mL)
Figure 4. Effect of soluble TGFBIp on platelet. A platelet suspension (100 ␮L containing 5 ⫻ 107 platelets) was added to a noncoated chamber slide with soluble-phase
TGFBIp (1 ␮g/mL or 5 ␮g/mL) or fibrinogen (5 ␮g/mL). Platelets were allowed to adhere (15 minutes at 37°C), and wells were washed with Tyrode buffer. (A) Fixed platelets
were stained for F-actin with FITC-conjugated phalloidin and were observed under a fluorescence microscope. The white bar represents 10 ␮m. Representative of platelet
adhesion (B) and platelet spreading (pixels/cells) (C) in the presence of soluble-phase TGFBIp (0.5, 1, 2, and 5 ␮g/mL) or fibrinogen (Fg; 5 ␮g/mL). Platelet adhesion was
detected by the acid phosphatase assay, spreading was detected using the MetaMorph program. (D) Time course adhesion of human washed platelets by soluble and
immobilized TGFBIp. Platelet suspensions containing the indicated protein were dispensed in triplicate wells of microplates. After unbounded platelets were removed, adherent
platelets were detected by the acid phosphatase assay as described in “Platelet adhesion assay.” All results are shown as the means ⫾ SD of 3 different experiments and
ANOVA. *P ⬍ .05 compared with BSA.
80
80
∗
Thrombin
Do1-4
Do2
Do3
∗
∗
7
8
Do1-4
Do1
∗
6
∗
Do1
40
40
20
20
00
Do2
1
2
3
Do3
4
5
6
7
Do4
Do1-4
Fg
Do4-RGD
∗
∗
TGFBIp
5
∗
Do4-RGD
Do4
Do2
4
∗
Do4
Do3
Do1
3
Do3
TGFBIp
∗
Do2
con
60
60
BSA
∗
Do4-RGD
D
Annexin-V FITC
∗
Do4
Fibrinogen-FITC
BSA
count
α-P-selectin-FITC
2
∗
Do1
Annexin-V-FITC
B
1
TGFBIp 5µ
µg/mL
TGFBIp
count
TGFBIp 1µg/mL
TGFBIp
Resting
∗
0.7
0.7
0.6
0.6
0.5
0.5
0.4
0.4
0.3
0.3
0.2
0.2
0.1
0.1
00
BSA
IgG-FITC
absorbance (405nm)
C
Pixel/cell
count
A
8
Figure 5. Platelet activation by soluble TGFBIp. (A) Washed platelets were incubated with soluble TGFBIp (1 ␮g/mL) for 15 minutes. Phosphatidylserine and P-selectin
exposure and binding affinity of fibrinogen were detected by annexin V–FITC, anti–P-selectin-FITC antibody, and fibrinogen-FITC, respectively, by the use of FACS analysis.
(B) Washed platelets were incubated with the soluble FAS1 domain (Do1, Do2, Do3, Do4, Do4-RGD, and Do1-4; each 1 ␮g/mL), fibrinogen (Fg; 5 mg/mL, as negative control),
and thrombin (1 U/mL, as positive control) for 15 minutes, and PS exposure (annexin V–FITC) was analyzed by FACS. Washed platelets were allowed to adhere on noncoated
slides for 15 minutes, and platelet adhesion (C) and platelet spreading (D) were assessed (pixels/cells) in the presence of soluble each indicated protein (1 ␮g/mL). Platelet
adhesion was detected by the acid phosphatase assay, and spreading was detected by the use of the MetaMorph program. All results are shown as the means ⫾ SD of
3 different experiments and ANOVA. *P ⬍ .05 compared with BSA.
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BLOOD, 10 DECEMBER 2009 䡠 VOLUME 114, NUMBER 25
KIM et al
Adherent cell count/HPF
A
C
∗
250
250
∗
200
200
con
Thrombin
150
150
100
100
50
50
00
01
12
23
TGFBIp (µg/mL)
54
D
B
25000
50
Pixel/cell
TGFBIp
∗
∗
20000
40
∗
30
15000
20
10000
10
5000
00
01
2
3
1
2
TGFBIp (µg/mL)
54
Surface coverage (%)
5212
Donor 1
계열1
Donor 2
계열2
Donor 3
계열3
Donor 4
계열4
Donor 5
계열5
80
80
60
60
40
40
20
20
00
0
0
2
2
4
4
6
6
µg/mL)
TGFBIp concentration (µ
8
8
10
10
Figure 6. Thrombus formation in vitro by soluble-phase TGFBIp. Washed platelet perfusions (5 minutes at shear rate 500⫺1) over type I collagen were performed in the
absence or presence of soluble TGFBIp (1, 2, and 5 ␮g/mL). Platelet adhesion (A) and platelet spreading (B) under flow conditions were determined. Aspirin-free donors were
perfused over type I collagen in the presence or absence of soluble TGFBIp for 5 minutes, and platelet deposition (thrombus formation) was monitored in vitro. (C) Microscopic
picture (⫻200 magnification) showing thrombus formation in the presence or absence of soluble TGFBIp. The scale bar represents 10 ␮m. (D) Representative histograms
showing the size of thrombus formation at different concentrations (2-8 ␮g/mL) of TGFBIp. All results are shown as the means ⫾ SD of 3 different experiments and ANOVA.
*P ⬍ .05 compared with 0 ␮g/mL TGFBIp.
was used.29,30 C57BL/6 mice were given either TGFBIp/epinephrine or boiled TGFBIp/epinephrine via tail vein injection. Microscopic and histologic analysis revealed extensive pulmonary thromboembolism in TGFBIp-injected mice (Figure 7A). As shown in
Figure 7B, TGFBIp induced pulmonary embolism in a dosedependent manner, but boiled protein did not. TGFBIp can induce
pulmonary embolism more efficiently than collagen because 0.4 mg/
kg TGFBIp induced more than 6-fold greater numbers of thrombi
than did 0.5 mg/kg collagen (Figure 7B-C). The thrombogenic
activities of TGFBIp seem to act synergistically with collagen
because we demonstrated that suboptimal doses of both TGFBIp
and collagen-induced dramatic pulmonary embolism, as shown in
Figure 7C. This result suggests that TGFBIp can act as a platelet
activating factor in vivo, like collagen.
To test whether endogenously produced TGFBIp can induce
pulmonary embolism, we used Alb-hTGFBIp transgenic mice that
have approximately 1.7-fold increased serum TGFBIp levels.22
These Alb-hTGFBIp transgenic mice (TGFBIp TG) exhibited no
gross developmental abnormalities, except anterior segment dysgenesis of the eye in some cases,22 and had normal platelet and white
blood cell counts (Table 1). Thus, these transgenic mice are suitable
for examination of the effect of TGFBIp on platelet function. The
transgenic mice did not develop pulmonary emboli, but when they
were treated with either epinephrine alone or together with a
suboptimal dose of collagen (1 mg/kg), they showed significant
development of pulmonary emboli compared with control mice
(Figure 7D).
Discussion
Mediators from activated platelets act in an autocrine/paracrine
fashion and activate or prime approaching platelets. Here, we show
that TGFBIp is secreted from activated platelets and is then
associated with the surface of activated platelets in serum factorfree conditions, which suggests that TGFBIp could function as an
autocrine and/or paracrine factor. In fact, we showed that exogenously added soluble TGFBIp induced the activation of platelets,
resulting in a change in morphology (spreading), PS exposure,
␣-granule secretion, and increased integrin ␣IIb␤3 affinity.
TGFBIp mediates the adhesion of several cells through the
interaction of the FAS1 domain and integrins.12 Platelets express
several integrins, including ␣IIb␤3, ␣V␤3, ␣2␤1, ␣5␤1, and ␣6␤1.
Among these, integrin ␣V␤3 is known to interact with TGFBIp in
human umbilical vein endothelial cells,11,31 but it does not appear to
be responsible for the adhesion of platelets to TGFBIp. Instead,
integrin ␣5␤1 is partially responsible for mediating platelet adhesion to TGFBIp because approximately only 50% of platelet
adhesion was inhibited by integrin ␣5␤1 function-blocking antibody, which suggests that other integrins or other molecules also
are involved in mediating adhesion to TGFBIp. However, it is
unlikely that other integrins are involved because we found that
several function-blocking antibodies against integrins that have
been known to be expressed in platelets did not affect platelet
adhesion. TGFBIp is also known to bind proteoglycans such as
small leucine-rich biglycan and decorins.12,32 The presence of
proteoglycans on human platelets33 suggests that proteoglycans on
platelets could be the receptor of TGFBIp.
Integrin ␣IIb␤3 is the most abundant integrin on platelets and
megakaryocytes and is known to play important roles in hemostasis. In resting platelets, ␣IIb␤3 exists in an inactive conformation.
The inactive form of ␣IIb␤3 does not bind most physiologic ligands,
including immobilized fibrinogen and fibronectin. Once integrin
␣IIb␤3 is activated, it binds several soluble proteins in plasma,
including fibrinogen, VWF, and fibronectin. Thus, activation of
␣IIb␤3 is one of the key processes in thrombogenesis. Collagen,
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BLOOD, 10 DECEMBER 2009 䡠 VOLUME 114, NUMBER 25
A
Collagen
Emboli count/HPF
B
10
10
66
∗
∗
∗
44
boiled TGFBIp
TGFBIp
TGFBIp
계열1
Boiled TGFBIp
계열2
88
THROMBOGENIC EFFECT OF TGFBIp
∗
22
00
10
0.05
2
0.1
3
0.2
4
0.4
5
TGFBIp (mg/kg)
Emboli count/HPF
C
col 0
col 0.5
col 1.0
col 1.5
14
14.0
12
12.0
10
10.0
8
8.0
(mg/kg)
6
6.0
4
4.0
2
2.0
0
0.0
10
0.05
2
0.1
3
TGFBIp (mg/kg)
Emboli count/HPF
D
88
66
C57BL/6 wt mice
계열1
TGFBIp TG mice
계열2
44
∗
∗
∗
22
00
1
PBS
2
epinephrine
3
collagen
Figure 7. Increased peripheral blood TGFBIp promotes pulmonary embolism.
C57BL/6 mice were given with either TGFBIp/epinephrine or a similar volume of boiled
protein/epinephrine as a control via tail vein injection. (A) Hematoxylin and eosin staining of
the lungs of mice (n ⫽ 3 per group) given collagen (2 mg/kg), TGFBIp (0.4 mg/kg), and
boiled TGFBIp (0.4 mg/kg) is shown. Extensive pulmonary thromboemboli (➔) are shown
in collagen- and TGFBIp-injected mice compared with boiled TGFBIp-injected mice.
Representative sections (⫻40 magnification) are shown. The scale bar represents
400 ␮m. (B) TGFBIp-induced pulmonary emboli occurred in a dose-dependent manner.
(C) Representative of the suboptimal doses of both TGFBIp- and collagen-induced
pulmonary embolism. (D) Pulmonary embolism formation in Alb-hTGFBIp transgenic mice.
Mice were intravenously injected with 0.5 mg/kg collagen and 100 ␮g/kg epinephrine, and
the formation of pulmonary emboli was subsequently determined. The pulmonary embolism count was detected in at least 5 different fields. All results are shown as the
means ⫾ SD of 3 different experiments and ANOVA. * P ⬍ .05 compared with 0 mg/kg of
the TGFBIp-injected group.
thrombin, and adenosine diphosphate, which are released at sites of
vascular injury, have been known to initiate the activation of ␣IIb␤3.
In this report, we show that TGFBIp and its FAS1 domain also
trigger ␣IIb␤3 activation. This finding suggests that TGFBIp plays
an important role in thrombogenesis, similar to collagen or
thrombin.
5213
The binding of ligands to ␣IIb␤3 mostly occurs via the RGD
sequence, even though its initial adhesion is not mediated by RGD.
In the case of fibrinogen, initial cell attachment is mediated by the
␥400-411 sequence of fibrinogen, whereas secondary spreading is
mediated by the RGD sequence. In addition, other sequences
outside of this RGD region are sometimes required for full
adhesive activity.34 TGFBIp also contains the RGD motif that
could also be involved in platelet spreading. Indeed, TGFBIp
mediates the spread of platelets only via its RGD sequence but not
via FAS1 domains. Accordingly, the spread of platelets on TGFBIp
was inhibited by the ␣IIb␤3 integrin function-blocking antibody.
Our findings suggest that initial adhesion of platelets to immobilized TGFBIp is mediated by FAS1 domains through ␣5␤1, in part,
and that their spread is then mediated by the RGD motif via ␣IIb␤3
integrin.
The adhesion of platelets on immobilized TGFBIp occurred
30 minutes after incubation, whereas soluble-phase TGFBIp initiated adhesion as early as 10 minutes after incubation. Consequently, it seems that soluble TGFBIp activates platelets first and
then mediates their adhesion and spread. Upon the binding of
ligand, ␣IIb␤3 elicits a series of outside-in intracellular events that
includes activation of kinases and phosphatases, changes in
cytoskeletal reorganization, and regulation of protein synthesis.
Outside-in signaling through ␣IIb␤3 amplified events initiated by
thrombin and other agonists is necessary for full platelet spreading,
platelet aggregation, granule secretion, and the formation of a
stable platelet thrombus.35,36 Although it is not clear that soluble
TGFBIp binds integrin ␣IIb␤3, we have shown that soluble TGFBIp
and FAS1 domains induce an increase of integrin ␣IIb␤3 affinity, the
phosphorylation of FAK, and a change in cytoskeletal reorganization. Taken together, these findings suggest that soluble TGFBIp is
more likely to activate platelets initially, and once it is immobilized
in association with other matrix proteins also may mediate the
adhesion and spread of platelets.
The first step in the homeostatic cascade is platelet interaction
with the exposed ECM at the sites of injury.37 Among the
macromolecular constituents of the ECM, collagen is considered to
play a major role in this process, because in vitro it not only
supports platelet adhesion through direct and indirect pathways, but
it also directly activates the cells initiating aggregation and
coagulant activity.38 Rapid conversion to stable adhesion requires
additional contacts between the platelets and the ECM. TGFBIp is
an ECM protein that binds to multiple ligands, including integrins;
type I, II, and IV collagen; laminin; fibronectin; and glycosaminoglycan.39,40 At the site of blood vessel injury, TGFBIp may bind to
exposed collagen and other matrix molecules. Indeed, we found
that when TGFBIp was immobilized with collagen type I or
fibrinogen (data not shown), washed human platelet adhesion and
spreading was increased. These results indicate that collagenbound TGFBIp may mediate platelet adhesion and spreading as a
cofactor of collagen.
Considering the platelet activation activity of TGFBIp, we
addressed the effect of TGFBIp on thrombus formation. We
Table 1. Complete blood count of wild-type C57BL/6 mice and Alb-hTGFBIp transgenic mice
RBC,
ⴛ106/␮L
HGB, g/dL
HCT, %
PLT, ⴛ103/␮L
WBC,
ⴛ103/␮L
TGFBIp concentration,
ng/␮L
C57BL/6 (n ⫽ 10)
7.58 ⫾ 1.60
11.78 ⫾ 2.61
32.86 ⫾ 7.16
606.47 ⫾ 127.62
6.12 ⫾ 3.13
207.17 ⫾ 35.20
TGFBIp TG (n ⫽ 10)
6.66 ⫾ 1.93
10.33 ⫾ 3.09
29.44 ⫾ 8.46
561.33 ⫾ 143.64
6.19 ⫾ 2.16
341.84* ⫾ 60.44
HCT indicates hematocrit; HGB, hemoglobin; PLT, platelets; RBC, red blood cell count; TGFBIp, transforming growth factor-␤–induced protein; and WBC, white blood cell
count.
*P ⬍ .05 (analysis of variance) compared with wild-type mice.
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5214
BLOOD, 10 DECEMBER 2009 䡠 VOLUME 114, NUMBER 25
KIM et al
demonstrate that under physiologic flow conditions, TGFBIp
promotes thrombus formation on collagen in vitro. The thrombogenic activity of TGFBIp was further confirmed in vivo. Because
collagen and epinephrine are initial activating factors of platelets,
they can induce pulmonary embolism by intravenous coinjection.
Similarly, pulmonary embolism was induced when TGFBIp was
administered with epinephrine. Even TGFBIp induced pulmonary
emboli at a concentration of 0.05 mg/kg, which is not enough for
collagen to induce thrombosis. This finding suggests that TGFBIp
is much more potent than type I collagen in inducing thrombus
formation. Indeed, 0.4 mg/kg TGFBIp strongly induced pulmonary
thrombosis in mice, whereas 0.5 mg/kg collagen barely induced
pulmonary thrombosis. Assuming that the average weight of a
mouse is 20 g and the average blood volume is 2 mL, the injected
TGFBIp concentration, 0.05 mg/kg, is approximately 500 ng/mL
in peripheral blood. This concentration is 2.5 times greater than the
physiologic concentration of 200 ng/mL. Although Alb-hTGFBIp
transgenic mice have a blood TGFBIp concentration of approximately 340 ng/mL, they are significantly susceptible to the production of pulmonary thrombosis by collagen and epinephrine, which
suggests that even a small increase of blood TGFBIp concentration
can cause thrombus formation in certain pathologic conditions.
Taking all in vitro and in vivo data into consideration, it is strongly
suggested that TGFBIp can act as a thrombogenic factor in platelets
and blood.
After adhering to vascular lesions, platelets can rapidly recruit
additional platelets to the site of injury, which is necessary to
achieve hemostasis and can also recruit different types of leukocytes, which set off host defense responses. Activated platelets
release inflammatory and mitogenic mediators into the local
microenvironment, thereby altering the chemotactic and adhesive
properties of monocytes and endothelial cells.41 Activated platelets
or platelet microparticles also release chemokines that can trigger
the recruitment of monocytes or promote their differentiation into
macrophages.42-44 Therefore, TGFBIp, which is capable of activating platelets, is supposed to be actively involved in forming the
local microenvironment. In addition, in our previous report, we
showed that TGFBIp itself acts as a chemotactic molecule for
monocytes and mediates the adhesion of monocytes,17 thus suggesting that a local high concentration of TGFBIp released from or
associated with the platelets can also promote monocyte recruitment and mediate heterotypic monocyte-platelet aggregation.
In conclusion, we first demonstrated that TGFBIp is present in
the platelets and is released upon activation, and we also present
evidence that TGFBIp can activate the platelets, leading to
thrombus formation. Our finding will increase understanding of the
novel mechanism of platelet activation, contributing to a better
understanding of thrombotic pathways, and may subsequently
inform the development of new antithrombotic therapies.
Acknowledgments
This study was supported by a grant from the National R&D
Program for Cancer Control, Ministry for Health, Welfare &
Family Affairs, Republic of Korea (0720550-2); by the Korea
Science and Engineering Foundation (KOSEF) grant funded by the
Korean government (MEST; No. R11-2008-044-03 001-0); by a
grant of convergence technology for PET radiopharmaceuticals
from the National Research Foundation of Korea; and by the Brain
Korea 21 Project in 2009.
Authorship
Contribution: H.-J.K., P.-K.K., and S.M.B. designed and performed
experiments; H.-N.S. and J.-E.K. supplied Alb-hTGFBIp transgenic
mice; D.S.T., B.-H.L., and R.-W.P. supervised; I.-S.K. supervised
experiments; H.-J.K. and I.-S.K. wrote the manuscript; and D.S.T.,
B.-H.L., R.-W.P., and I.-S.K. edited the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: In-San Kim, Department of Biochemistry and
Cell Biology, Cell and Matrix Research Institute, Kyungpook
National University School of Medicine, 101 Dongin 2-ga, Junggu, Daegu 700-422, Republic of Korea; e-mail: [email protected].
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2009 114: 5206-5215
doi:10.1182/blood-2009-03-212415 originally published
online September 8, 2009
Transforming growth factor-β−induced protein (TGFBIp/β ig-h3)
activates platelets and promotes thrombogenesis
Ha-Jeong Kim, Pan-Kyung Kim, Sang Mun Bae, Hye-Nam Son, Debraj Singh Thoudam, Jung-Eun
Kim, Byung-Heon Lee, Rang-Woon Park and In-San Kim
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