The integrin α6β1 modulation of PI3K and Cdc42 activities induces dynamic filopodium formation in human platelets

Article (PDF Available)inJournal of Biomedical Science 12(6):881-98 · January 2006with21 Reads
DOI: 10.1007/s11373-005-9021-2 · Source: PubMed
Abstract
Platelets are an ideal model for studying a rapid morphological change in response to various signal transduction systems. Morphological changes via the activation of integrin alphaIIbbeta3 in platelets have been investigated intensively. In contrast, activation via integrin alpha6beta1 is less well studied. Here, we provide the first biochemical evidence that integrins alpha6beta1 and alphaIIbbeta3 of platelets are associated with different membrane proteins. We also demonstrate that platelets activated by integrin alpha6beta1 show dynamic change by actively forming filopodia and never fully spreading over a period of more than an hour. In addition, platelets activated by integrin alpha6beta1 are different from those activated by integrin alphaIIbbeta3 in terms of cell-substrate contact and in their distribution pattern of actin, Arp2/3 and various phosphotyrosine proteins. The morphological appearance of platelets produced through integrin alpha6beta1 activation is highly dependent on PI3 kinase (PI3K) but less dependent on Src kinase. Suppression of PI3K activity in integrin alpha6beta1 activated platelets induces an increase in Cdc42 activity and more filopodium formation. However, both Cdc42 and PI3K activity are higher in platelets activated by integrin alpha6beta1 than in those activated by integrin alphaIIbbeta3. Taken together, this study demonstrates that the signals induced by integrin alpha6beta1 modulate at the level of PI3K and Cdc42 activity to allow platelets to actively form filopodia.
The integrin a6b1 modulation of PI3K and Cdc42 activities induces dynamic
filopodium formation in human platelets
Jui-Chin Chang
1
, Hsin-Hou Chang
2
, Chien-Ting Lin
1
& Szecheng J. Lo
1,3,
*
1
Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, 112, Taiwan;
2
Graduate
Institute of Molecular and Cellular Biology, Tzu-Chi University, Hualin, 970, Taiwan;
3
Department of Life
Science, Chang Gung University, Taoyuan, 333, Taiwan
Received 26 April 2005; accepted 3 August 2005
Ó 2005 National Science Council, Taipei
Key words: actin-cytoskeleton, Cdc42, filopodium, integrins, laminin receptor, morphological change,
rhodostomin, signal transduction
Summary
Platelets are an ideal model for studying a rapid morphological change in response to various signal
transduction systems. Morphological changes via the activation of integrin aIIbb3 in platelets have been
investigated intensively. In contrast, activation via integrin a6b1 is less well studied. Here, we provide the
first biochemical evidence that integrins a6b1 and aIIbb3 of platelets are associated with different mem-
brane proteins. We also demonstrate that platelets activated by integrin a6b1 show dynamic change by
actively forming filopodia and never fully spreadi ng over a period of more than an hour. In addition,
platelets activated by integrin a6b1 are different from those activated by integrin aIIbb3 in terms of cell
substrate contact and in their distribution pattern of actin, Arp2/3 and various phosphotyrosine proteins.
The morphological appearance of platelets produced through integrin a6b1 activation is highly dependent
on PI3 kinase (PI3K) but less dependent on Src kinase. Suppression of PI3K activity in integrin a6b1
activated platelets induces an increase in Cdc42 activity and more filopodium formation. However, both
Cdc42 and PI3K activity are higher in platelets activated by integrin a6b1 than in those activated by
integrin aIIbb3. Taken together, this study demonstrates that the signals induced by integ rin a6b1 mod-
ulate at the level of PI3K and Cdc42 activity to allow platelets to actively form filopodia.
Introduction
Platelets are derived from bone marrow megak-
aryocytes and appear as tiny (about 2 lmin
diameter) anucleated cell fragments that are discoid
in shape in the blood [1]. They play a fundamental
role in hemastasis and in the prevention of bleeding
from damaged blood vessels [2]. At the site where
vascular injury occurs, platelets adhere to exposed
subendothelial collagens and laminins and become
activated [3, 4]. Subsequently, a series of morpho-
logical changes occurs and von Willebrand factor
(vWf), fibronectin, fibrinogen, thrombospondin
and granula r components are secreted. This results
in the formation of a platelet adhesion plug by
platelet aggrega tion [2, 5–8]. Together with the
extracellular matrix (ECM), membrane adhesion
molecules on platelets, primarily the integrin recep-
tors, play a critical role in platelet clot formation.
Integrins, heterodimers of a and b subunits, are
a superfamily of adhesion receptors that media te
cell–cell and cell–ECM interaction and regulate
cell migration, morphogenesis, differentiation, sur-
vival and tumor metastasis [9–12]. Eight b and 18
a subunits can be assembled into 24 distinct
*To whom correspondence should be addressed. Fax: +886-
3-3283211; E-mail: losj@mail.cgu.edu.tw
Journal of Biomedical Science (2005) 12:881–898
881
DOI 10.1007/s11373-005-9021-2
integrins identified in mammals [13]. Integrin
aIIbb3 is the most abundant adhesion molecule
in platelets (about 80,000 molecules per cell) and is
only expressed in platelets. Four other integrins,
avb3, a2b1, a5b1, and a6b1 are also present in
platelets, but they are present in 80- to 300-fold
lesser amounts than aIIbb3 [14]. The ligands of
integrins aIIbb3, avb, and a5b1 are ECM that
contains the tripeptide of Arg-Gly-Asp (RGD),
such as fibrinogen, fibronectin, vWf, and vitronec-
tin. The ligands of integrin a2b1 and a6b1 are
collagens and laminins, respectively [13, 15, 16].
Laminins are abc heterotrimeric glycoproteins
present in at least 14 isoforms and they are
ubiquitously distributed in all basement mem-
branes including those of blood vessels [17]. The
corresponding receptors of laminins are found in
various types of cells and include integrin a6b1,
dystroglycan, and heparin sulfate proteoglycan
[17, 18]. Additionally, a 67 kDa non-integrin
protein on platelets acts as a laminin receptor
[19]. Since platelets contain, secrete, and adhere to
laminin-8 [20], activation of integrin a6b1on
platelets should play a physiological role.
A morphological change is the hallmark of the
progression during platelet activation. In the
resting state, a three-dimension mesh of cytoskel-
eton actin filaments is formed in the roun ded
platelet cytoplasm. In the activated state, the
filopodia of the platelets are extended and this
results in platelets undergoing full spreading,
adhesion, and retraction [21]. These dynamic
changes result from various kinetic process es and
signal transduction messages that are mediated by
complicated machinery, including the activation of
protein kinases, the elevation of the calcium
concentration, and the reorganization of the a ctin
filaments [5, 22]. The platelet morphological
changes that occur through activation by integrins
aIIbb3 and a2b1 are well characterized [3, 23–26],
but activation induced by integrin a6b1 is poorly
understood [4, 20, 27].
In this study, we tested by biochemical analyses
whether integrin aIIbb3 and a6b1 are located
within the same membrane raft and whet her they
share similar signal transduction molecules in-
volved in platelet morphological changes. W e also
employed time-lapse and interference reflection
light microscopy, scanning electron microscopy
and whole-mount transmission electron micros-
copy to investigate platelet morphological changes
and actin-cytoskeleton patterns after integrin a6b
1
is activated by eithe r laminin or anti-integrin a6
antibody. We observed the active formation of
filopodia and novel actin-filament pattern change
in such platelets, which are very different from the
morphological characteristics of platelets stimu-
lated by integrin aIIbb3. Results from PI3K and
Src inhibitors treatment and biochemical analyses
suggest a model when platelet integrin a6 b1is
activated where active filopodium formation oc-
curs via feedback loop regula tion and balancing of
the levels of PI3K and Cdc42.
Materials and met hods
Reagents and antibodies
The plasmid pGEX-GST-PAK for production of
GST-PAK was a gift from Dr. Y.-S. Chang
(Chang Gung University). The plasmid pGS T-
RHO for production of GST-rhodostomin was
constructed and described previously [28]. Gluta-
thione Sepharose 4B, protein A-Sepharose and
protein G-Sepharose beads were purchased from
Amersham Biosciences (Uppsla, Sweden). The Src
inhibitor PP1 was from BIOMOL Research Lab-
oratories (Plymouth Meeting, PA) and the PI3K
inhibitors, LY294002 and wortmannin, were
obtained from Calbiochem (La Jolla, CA). Sulfo-
NHS-Biotin for labeling platelet su rface proteins
was purchased from Pierce (Rockford, IL). Bovine
serum albumin (BSA), laminin and phosphatidyl-
inositol were bought from Sigma (St. Louis, MO).
Rhodamine-phalloidin was purchased from Molec-
ular Probe (Eugene, OR). [c)
32
P] ATP and ECL
kit were obtained from Perkin Elmer Life Sciences
(Boston, MA). Monoclonal antibody (mAb)
against integrin a6 (GoH3) for immunoprecipita-
tion and plate-coating was purchased from Immu-
notech (Marseille Cedex, France). Anti-integrin a6
mAb (BQ16) and anti-PI3 kinase p110 polyclonal
antibody (pAb) for imm unoblotting were bought
from Santa Cruze Biotechnology (Santa Cruze,
CA). Anti-integrin b1 mAb (JB1A) for immuno-
blotting was obtained from Chemicon (Temecula,
CA). Anti-Src mAb (GD11), anti-Syk mAb
(4D10.1), anti-PI3 kinase p85 pAb, anti-phosp-
hotyrosine mAb (4G10), anti-Rac mAb, and
anti-Cdc42 mAb for immunoprecipitation and
immunoblotting were purchased from Upstate
882
Biotechnology (Lake Placid, NY). Anti-Arp3 pAb
and anti-phosphotyrosine mAb (PY20) for immu-
nostaining were bought from Upstate Biotechnol-
ogy and Transduction Laboratories (Lexington,
KY), respectively.
Platelet preparation
Washed platelets were prepared as described
previously [29]. In brief, human blood was col-
lected in 1/6 volume acid-citrate-dextrose (ACD)
from healthy donors by venous puncture, an d was
centrifuged at 150 g for 30 min at room tem-
perature. The platelet rich plasma (PRP) superna-
tant was mixed with 5 mM EDTA and
re-centrifuged at 1000 g for 15 min. The pellet
was suspended in calcium-free Tyrode’s buffer
with apyrase (1 unit/ml) and incubated at 37 °C
for 20 min. After centri fugation, the washed pellet
was resuspended in 1 mM calcium-containing
Tyrode’s buffer.
Platelet adhesion assay
Acid-washed glass cover slips were coated with
substrates as previously reported for cell attach-
ment assay [30, 31]. In brief, 0.4 lM GST-rhodo-
stomin, 5 lg/ml laminin, or 2 lg/ml anti-a6
integrin antibody (GoH3) were coated on cover
slips at 37 °C for 1.5 h, and blocked with 5% BSA
in PBS, pH 7.4, at 37 °C for 1 h. Freshly prepared
and non-activated human platelets were incubated
on coated plates with substrate for 15–30 min at
37 °C. For drug treatment studies, non-activated
platelets were co-incubated with 20 lM PP1,
25 lM LY294002 or 100 nM wortmannin before
seeding onto protein-coated plates. After washed
with PBS, samples were then prepared for light
microscopy or electronic microscopy as described
below.
Time-lapse recording and interference reflection
microscopy (IRM)
Freshly prepared human platelets wer e seeded on
substrate-coated cover slips and observed imme-
diately using a LEICA DM IRBE inverted micro-
scope equipped with lenses for differential
interference contrast. Images were recorded using
Metamorph software as previously described [31].
Degrees of apposition between platelets and
substrates were analyzed using interference reflec-
tion microscopy [31]. Platelets adhered to sub-
strates were fixed with 4% glutaraldehyde and
then extracted with 0.5% Triton X-100. After
staining with rhodamine–phalloidin, simultaneous
epifluorescence and interference reflection micros-
copy was performed using a laser scanning confo-
cal microscope (LEICA, DMRE).
Scanning electron microscopy (SEM)
Platelets were attached on substrate-coated cover
slips and prepared for scanning electron micro-
scope [30, 31]. In brief, platelets were fixed with
glutaraldehyde, post-fixed with OsO
4
, and sub-
jected to alcohol (50–100%) dehydration, and
critical point drying. After gold coating, the
platelet-attached cover slips wer e examined under
a scanning electron microscope (Joel; JSM-5300)
at 15 kV.
Whole-mount transmission electron microscopy
(WMTEM)
Platelets were adhered to the formvar-supported
grids which were pre-coated with various sub-
strates. Platelets on the grids were fixed with
glutaraldehyde and post-fixed with 1% OsO
4
as
described by Lo et al. (1980) [32]. Platelets were
extracted with 0.5% Triton X-100 in PHEM buffer
containing 0.1% glutaraldehyde for 2 min. Plate-
lets were then fixed with 2.5% glutaraldehyde and
stained by 1.5% uranyl acetate. The platelets on
grids were visualized in a transmission electron
microscope (Joel; JEM-2000ExII).
Immunofluorescence staining
Platelets adhering to substrates were fixed with
fixation buffer (0.5% Triton X-100, 4% parafor-
maldehyde and 1.5 lM rhodamine–phalloidin in
PHEM buffer) for 20 min at room temperature.
To localize specific antigens, the fixed platelets
were blocked with 0.25% BSA in PBS, and treated
with 0.1% SDS for 1 min followed by incubation
with a primary antibody for at least 2 h. The
specimens were then incubated with the appropri-
ate secondary antibody and stained. Images were
obtained using a laser scanning confocal micro-
scope (LEICA, DMRE).
883
Immunoprecipitation and Western blotting
The immunoprecipitation assay was performed as
described previously [31]. Six-well culture plates
were coated with GST-rhodostomin or anti-a
6
integrin antibody as part of the cell adhesion
study; 500 ll washed platelets were ad ded and
incubated at 37 °C for 30 min. Five hundred ll
of ice-cold 2X lysis buffer (20 mM Tris, pH 7.5,
300 mM NaCl, 2 mM EDTA, 2 mM EGTA, 2%
NP-40, 2 mM PMSF, 2 mM Na
3
VO
4
, protease
inhibitor cocktail) was added to each well. The
debris was removed and the platelet lysate was
precleared and incubated with the appropriate
antibody and protein A- or protein G-Sepharose
at 4 °C overnight. After washing the Sepharose
three times, 2X Laemmi sample buffer was added
and to extract the proteins, which were separated
by SDS-PAGE. Protein phosphotyrosine was
probed by mAb 4G10 and blots were stripped
and reprobed with various antibodies as indi-
cated.
Small GTPase activity assay
The presence of activated cell ular Rac and Cdc42
were determined by precipitation with a GST
fusion protein GST-PAK1 that contains the Rac-
binding domain from the human PAK1 (amino
acids 70–132) as previously described [33]. Cul-
ture dishes (15-cm diame ter) were coated with the
various substrates and the platelets were added to
adhere for 30 min at 37 °C. After removing the
free platelets, the attached platelets were lysed
with lysis buffer (1% Triton X-100, 0.1% SDS,
50 mM Tris, pH7.4, 150 mM NaCl, 10 mM
MgCl
2
, 1 mM PMSF , 1 mM Na
3
VO
4
, protease
inhibitors). The lysates were scraped and cleared
by centrifugation at 13,000 g at 4 °C for 2 min.
The supernatants were incubated with GST-
PAK1 beads (20–50 lg) at 4 °C for 60 min. The
beads were then washed 3 times with lysis buffer,
eluted by 2X Laemmi sample buffer, and ana-
lyzed by Western blotting using specific antibod-
ies against Rac or Cdc42.
PI3 kinase activity assay
The measurement of PI3 kinase activity was per-
formed using immunoprecipitates of PI3K p85 as
described above. Immunoprecipitates were washed
three times with lysis buffer, three times with buffer
containing 0.1 M Tris, pH 7.4/ 5 mM LiCl/
0.1 mM Na
3
VO
4
, and twice with buffer containing
10 mM Tris, pH 7.4/ 150 mM NaCl/ 5 mM
EDTA/ 0.1 mM Na
3
VO
4
. Following by incuba-
tion with 1 0 ll (20 lg) of sonicated phosphatidyl-
inositol (resuspended in 20 mM MOPS, pH7.0,
1 mM EGTA) for 10 min on ice, the reaction was
carried out at 37 °C for additional 10 min by
adding 50 ll PI3K assay buffer (10 mM Tris,
pH 7.4, 100 mM NaCl, 5 mM EDTA, 10 mM
MgCl
2
,20lM ATP) containing 10 lCi [c)
32
P]
ATP. The reaction was terminated by adding 20 ll
6 N HCl. Phospholipids were extracted with
200 ll of a CHCl
3
/MeOH (1:1) mixture and
resolved by thin layer chromatography (Silica
Gel 60, Merck) with CHCl
3
/MeOH/H
2
O/
NH
4
OH (60:47:11.3:2) as the solvent. The plates
were air dried at the room temperature and the
radioactive products were analyzed by autoradi-
ography.
Results
In earlier studies, a recombinant protein of gluta-
thione S-transferase fused with rhodostomin
(GST-rhodostomin) was created [28]. This is an
Arg-Gly-Asp (RGD) tripeptide-containing snake
venom with a high potency to inhibit the preys’
platelet aggregation and was used to demonstrate
platelet morphological changes through activation
with integrin aIIb b3 [30, 31]. In this study, to
compare this with the morphological changes of
platelets that resulted from activation of integrin
a6b1, GST-rhodostomin, laminin and anti-a6
antibody (GoH3 mAb) were employed as sub-
strates. Platelet adhesion activities and cytoskele-
ton patterns were studied using various types of
light and electron microscopy.
Integrins a6b1 and aIIbb3 associate with different
membrane proteins of platelets
To assure anti-integrin a6 antibody (GoH3 mAb)
is a functional substrate for stimulating platelets to
induce adhesion and morphological changes, we
performed experiments involving precipitation and
immunoblotting [34]. Platelets were first labeled
884
with biotin then lysed with Triton X-100. The
lysates were precipitated with either GoH3 mAb or
GST-rhodostomin and were detect ed separately by
avidin-HRP, anti-a6 or anti-b1. As shown in
Figure 1, several surface-biotinylate d proteins
were precipitated by the GoH3 antibody with a
size ranging from 48 to 140 kDa and these have a
different pattern from those precipitated by GST-
rhodostomin (lane 1 vs. lane 2). The two major
protein bands (110 and 130 kDa in size) precipi-
tated by GST-rhodostomin are integrin aIIbb3as
has been previously demonstrated [34] and they
did not react with either anti-a6 or anti-b1 (lane 4
and 6). Conversely, the major protein bands (135
and 140 kDa) precipitated by GoH3 reacted with
anti-a6 and anti-b1, respectively (lane 3 and 5).
These results indicated that GoH3 could directly
precipitate the heterodimeric receptor of integrin
a6b1 and indirectly pull-down integrin a6b1 asso-
ciated membrane proteins, which form a complex
with the receptor through unknown chemical
forces. Judging by the various patterns of the
precipitated biotinylated proteins, seven extra
bands by GoH3 versus five extra bands by GST-
rhodostomin were present and we suggest that
integrin a6b1 and aIIbb3 are associated with these
membrane proteins and that the two groups of
proteins are located on different membrane rafts.
Platelets that adhered to GST-rhodostomin
were morphologically differen t from those that
adhered to GoH3 and laminin
To compare the morphological changes resulting
from activation of integrin a6b1 and aIIbb3,
freshly prepared human platelets were seeded on
glass cover slips coated with GST-rhodostomin,
GoH3, or laminin. After 15 min, platelets that
adhered to the cover slips were fixed and examined
using a differential interference contrast (DIC)
microscope. As shown in Figure 2, the platelets
activated by integrin aIIbb3 displayed a flat
morphology and were fully spread on GST-rho-
dostomin. In contrast, platelets with asteroid-
shaped morphology, thickening in the center and
numerous filopodia at the peripher y, appeared
when they attached to GoH3 and laminin. On
further examination the platelets attached to
GoH3 by SEM revealed filopodia per cell ranging
from 2 to 15 and their length ranged from 0.5 to
4 lm. This suggested that each platelet exhibited
various degree of adhesion activity. Nevertheless, a
Figure 1. Pull-down assay of integrin aIIbb3 and a6b1 and their associated proteins. Intact platelets were biotinylated first and the
lysates were precipitated by immobilized GoH3 mAb (lanes 1, 3, and 5) or GST-rhodostomin (lanes 2, 4 and 6). The precipitated
proteins were fractionated by SDS-PAGE and transferred onto PVDF membranes, then reacted with avidin-HRP (lanes 1 and 2),
anti-integrin a6 antibody (lanes 3 and 4), and anti-integrin b1 antibody (lanes 5 and 6) as indicated below the gels. Arrows indicate
integrin a6 (solid arrow) and b1 (empty arrow), respectively; arrowheads indicated integrin aIIb (solid arrowhead) and b3 (empty
arrowhead), respectively.
885
similar appearance for platelets adhering to lam-
inin and GoH3 (anti-integrin a6 antibody) sug-
gested that a non-fully spreading morphology of
the platelets must be the result of activation by
integrin a6b1. This supposition was supported by
observations that pre-incubating platelets with
laminin before seeding on GoH3-coated plates
reduced platelet adhesion and that the attached
platelets displayed a rounded shape similar to
those plated on BSA-coated cover slips (data not
shown).
Platelets form dynamic filopodia on GoH3- and
laminin-coated cover slips
To determine whether the time taken to fully
spread platelets on GoH3- and laminin-coated
plates requires is longer, DIC microscopy with
time-lapse recording was performed. Images of the
cells were photographed every 10 s and the entire
recording lasted at least for 1 h, or in some cases
for more than 2 h. The results showed that
platelets formed filopodia and lame llipodia within
15 min after attachment to substrate. However,
platelets continued ruffling for more than 1 h and
never appeared to fully spread when they adhered
to GoH3 and laminin (Figure 3a, the top first and
second row; supplement mo vies 1 and 2). When
platelets adhered to GST-rhodostomin, they also
displayed filopodia and lamellipodia and mem-
brane ruffling; nevertheless, they became fully
spread as a pancake shape all within 15 min
(Figure 3a, the third row; supplement movie 3).
Figure 3b shows the plot of membrane displace-
ment changes as a function of the time after
adhesion to substrate. The results clearly indicate
that platelets on GST-rhodostomin continuously
extended their membrane and became full y spread
and then remained steady in size, whereas those on
GoH3 were moving back and forth and never
became fully spread. From these results we con-
cluded that the dynamic formation of filopodia in
platelets activated by integrin a6b1 is not due to a
limitation in the time available to induce full
spreading; instead, it probably reflects signal
molecule fluctuation occurring inside the platelets.
The morphological appearance of platelets is
similar when they adhered to laminin and GoH3.
Figure 2. Images of platelets adhered to various substrates. Fresh platelets were plated on GST-rhodostomin, GoH3 mAb, and
laminin for 15 min and then fixed and photographed using a differential interference contrast (DIC) microscope or a scanning elec-
tron microscope (SEM) as indicated. The scale bar indicates 5lm.
886
To eliminate the possibility that the signals
induced by laminin may give rise to the morpho-
logical changes by binding to receptors other than
integrin a6b1, we employed GoH3, which only
recognizes integrin a6b1, as a substrate for the
following experiments.
Figure 3. Dynamic changes in platelets adhering to various substrates. (a) Time-lapse recording of platelet morphological changes
over a 10 s interval was performed and the entire recording time was 60 min. Fresh platelets were attached onto GoH3 mAb (the
top row), laminin (the second row), and GST-rhodostomin (the third row). The time sequence is indicated below each frame. The
scale bar indicates 5 lm. (b) The dynamic membrane extension of platelet was plotted as a function of time. Line 1 indicates a
platelet adhered to GST-rhodostomin while lines 2 to 4 represent three different platelets adhered to GoH3 mAb. The radius of
platelet membrane protrusion was measured and was represented as the percentage value compared with platelet fully spread on
GST-rhodostomin.
887
Actin-cytoskeleton patterns vary in platelets
adhering to GoH3 and GST-rhodostomin
Cell morphology is well known to highly correlate
with the arrangement of the cell’s internal cyto-
skeleton. We determined the cytoskeleton pattern
inside of platelets activated by integrin a6b1 and
aIIbb3 using whole mount transmission electron
microscopy (WMT EM). In order to produce clear
and sharp imag es of cytoskeleton, we removed the
membrane components with Triton X-100. The
filamentous structures of platelets are shown in
Figure 4a. Three differences in features were noted
between platelets adhering to GoH3 and to GST-
rhodostomin: (i) filaments radiated out from the
dense cell center to the filopodia of platelets
Figure 4. Actin-cytoskleton pattern of platelets adhering to GST-rhodostomin or GoH3 mAb. (a) Whole mount transmission elec-
tron microscopic (WMTEM) analyses of platelets adhering to GST-rhodostomin (the left panel) or to GoH3 mAb (the right pa-
nel). (b) Actin in platelets stained with rhodamine–phalloidin and observed under fluorescence microscopy (the left column). The
same field of cells was photographed with a DIC microscope (the right column). Arrow heads indicate the thick actin bundles pres-
ent in lamellipodia and arrows indicate actin bundles in filopodia. The scale bar in (a) is equal to 2 lm and 5 lm in (b).
888
adhering to GoH3, which contrasts wi th the lack
of a dense cell center appearance but the presence
of stress-fiber like filaments in platelets adhering to
GST-rhodostomin, (ii) there were short and heavy
bundles of filaments in the filopodia of platelets
adhering to GoH3 (Figure 4, arrows), and (iii)
there were heavy and wide bundles on the entire
peripheral rim of platelets adhering to GST-
rhodostomin (Figure 4, arrowheads). To verify
that the filamen tous structures appearing in
WMTEM are composed of actin, platelets were
stained with rhodamine–phalloidin and observed
under a fluorescence microscope. Fluorescence
images (Figure 4b) show that the actin pattern of
the platelets is highly correlated with the filamen-
tous structures in the images of WMTEM except
that there is an obvious actin ring in the cell center
of platelets adhering to GoH3 (the upper panels).
Consistent with the WMTEM results, the images
in the lower panels show that heavy actin staining
appears on the membrane periphery of the lamel-
lipodia and stre ss fibers appear across or radiating
out from the cell center zone of platelets that have
adhered to GST-rhodostomin. The actin-filament
distribution in platelets adhering to GoH3 was
different from that of platelets adhering to GST-
rhodostomin. There were neither actin bundles at
the cell periphery nor stress fibers in the cell center
in platelets adhering to GoH3 mAb. Based on the
morphology and the actin patterns of platelets
adhered to GoH3, three phases can be summarized
as follows: (i) platelets in the early attachment
stage appeared to have a high concentration of
actin at the cell center; (ii) platelets in the
intermediate attachment stage displayed ring bun-
dles at the center with several bundles radiated out
into filopodia and (iii) platelets in their most
spread form appeared to have only a weak actin
signal along the lamellipodia but a strong signal in
the short filopodia. Platelets repeated these three
phases and never appeared fully spread when
adhering to GoH3 as shown in the supplement
movie 1.
Characterization of cell-substrate contact
and distribution of actin related protein (Arp2/3)
complex and phosphotyrosine protein in platelets
adhered to GoH3 and GST-rhodostomin
The results of the different actin distribution
patterns for platelets adhering to GoH3 and
GST-rhodostomin led us to investigate whether
other actin-associated physiological differences
occurs inside platelets. IRM was used to study
the cell–substrate contact and immunofluorescence
microscopy was used to determine the Arp2/3
complex and phosphotyrosine protein distribu-
tion.
In a typical IRM image, dark areas show closer
contact between cell and substrate due to the
different refractive indexes of cell and glass sub-
strate, whereas light areas indicate wide distances.
Figure 5a shows that dark areas appeared for
almost all of the whole platelets except for the
lamellipodia region when platelets adhered to
GoH3 (upper panels). When we compared the
actin distribution, the dark areas did not com-
pletely match the actin distribution in the cell
center zone but were precisely arrayed in filopodia.
The dark areas of platelets adhered to GST-
rhodostomin were perfectly matched to actin
distribution including the peripheral region (Fig-
ure 5a, lower panels). A protected zone of adhe-
sion (PZA) described by Loike et al. (1993) [35]
was clearly seen in most platelets that adhered to
GST-rhodostomin but was absent in platelets that
adhered to GoH3. This indicated that platelets
adhering to GoH3 had a looser contact in the
peripheral region, which should be able to allow
filopodium extension and retraction.
Platelets adhered to GoH3 have more filopodia
while those adhered to GST-rhodostomin appear
to have prominent lamellipodia (Figure 2). Both
structures are heavily stained by rhodamine–phal-
loidin (Figure 4b). Presumably, actins appear in
un-branched bundles in filopodia and in branched
networks in lamellipodia and are regulated by
actin related protein 2/3 (Arp2/3) complex [36, 37].
To elucidate the distribution of Arp2/3 complex
and actin in platelets activated by integrin a6b1
and aIIbb3, immunostaining against Arp3 was
conducted. The results indicate that Arp2/3 was
lightly present scattered across the cytoplasm of
the platelets adhering to GoH3 but was abun-
dantly present in the lamellipodia of platelets
adhering to GST-rhodostomin (Figure 5b).
It is well known that a cascade of protein
phosphorylation occurs when integrins aIIbb and
a2b1 are activated [3, 26]. We investigated the
profile of tyrosine phosphorylated proteins in
platelets adhering to GoH3 and GST-rhodostomin
by immnoprecipitation followed by Western
889
Figure 5. Analyses of cell–substrate contact, Arp2/3 and phosphotyrosine protein distribution in platelets activated by GoH3 mAb
or GST-rhodostomin. (a) Platelets interacting with GoH3 mAb- or GST-rhodostomin-coated coverslips were fixed and stained with
rhodamine–phalloidin and then observed under fluorescence (the left column) or IRM (the right column) microscopy. Arrows indi-
cate the protected zone of adhesion (PZA), which was only observed in the platelets adhering to GST-rhodostomin. (b) Platelets
adhering to GoH3 mAb or GST-rhodostomin were probed with anti-Arp3 antibody labeled with FITC (the left column) and then
stained with rhodamine–phalloidin (the middle column). The merged pictures are shown in the right column. (c) Platelets adhering
to GoH3 mAb or GST-rhodostomin were probed with anti-phosphotyrosine antibody labeled with FITC (the left column) and
were stained with rhodamine–phalloidin (the middle column). The merged pictures are shown in the right column. Scale bar, 4 lm.
890
blotting and by immunofluorescence microscopy.
The Western blot results show that there were only
a few phosphorylated proteins that were different
when platelets were analyzed that had adhered to
GoH3, GST-rhodostomin and BSA (data not
shown). In contrast to these limited differences,
immunofluorescence microscopy shows distinct
patterns with the mAb 4G10 staining strongly in
filopodial tip of platelets adhering to GoH3 and
scattered in platelets adhering to GST-rhodosto-
min (Figure 5c).
Actin-cytoskeleton patterning of platelets adhered
to GoH3 is highly dependent on the PI3K signaling
pathway but is less dependent on the Src pathway
Experiments using inhibitors of Src kinase and
PI3K show a reduction in platelet adhesion and
spreading on fibrinogen and collagen and this has
been used to show that both kinases play impor-
tant roles in platelet activation induced by integrin
aIIbb3anda2b1 [3, 26]. We investigated using a
similar approach whether these two signal path-
ways also participate in platelet adhesion and
morphology changes when platelets are activated
by integrin a6b1. There was no significant change
in the number of platelets adhered to GoH3 in the
presence of 20 lM PP1 (Src kinase inhibitor),
100 nM wortmannin (PI3K inhibitor) and 25 lM
LY294002 (PI3K inhibitor) (Figure 6a), indicating
that two signal pathways have little or no effect on
platelet adhesion to GoH3. Moreover, the mor-
phology and acti n pattern of the platelets were
similar with or without PP1 (Figure 6b, upper
right panel). However, a dramatic change in actin
patterning and the formation of numerous filopo-
dia was observed in platelets adhered to GoH3
when they were treated with wortmannin and
LY294002 (Figure 6b, the lower panels). Both
actin patches and an increased number of filopodia
(2–10-fold) were observed.
To further examine the effect of the inhibitors
on the kinase activities inside of the platelets
adhering to GoH3, immunoprecipitation with
various antibodies (anti-Src, anti-Syk, and anti-
PI3K) and followed by probing with phosphoty-
rosine proteins were performed. Results indicate
that the phosphorylation level of Src and Syk was
3- and 2-fold higher, respectively, in platelets
adhering to GoH3 than in those adhering to
BSA (Figure 7a and b, lane 1 vs. lane 6), indicating
that the binding of integrin a6b1 activates the Src
kinase signaling pathway. The phosphorylation
level of Src and Syk was decreased about 50% in
platelets treated with PP1 (lane 2 of lower panel).
The phosphorylation level of Src was no signifi-
cant change in platelets treated with wortmannin
and LY294002 (Figure 7a, lanes 3 and 4 of lower
panel) while that of Syk increased 30–40%
(Figure 7b , lanes 3 and 4 of lower panel). Simi-
larly, the results for PI3K showed that p110, the
subunit of PI3K, had a 2-fold higher phosphory-
lation level in platelets adhering to GoH3 than
those adhering to BSA (Figure 7c, lane 1 vs. lane
6), indicating the engagement of integrin a6b1
activates the PI3K pathway as well. However, the
phosphorylation of p110 was red uced 4 fold when
platelets were treated PP1, wortmannin, and
LY294002 (Figure 7c, lanes 2 to 4 of lower panel).
Taken all these results together, we suggest that
there are two possible pathways to activate the
PI3K, one is through the Src pathway and the
other is undefined.
Inhibition of PI3K activity results in an elevation
of Rac and Cdc42 activity
Rho GTPase family is well known to be involved
in cell morphological changes by regulating the
actin-cytoskeleton reorganization. Lamellipodia
and filopodia formation is mediated by activation
of Rac and Cdc42, respectively [38, 39]. In order to
correlate the activities of Rac and Cdc42 to the
morphology of plate lets adhered to different sub-
strates and in the presence of various inhibitors;
we analyzed the activated Rac/Cdc42 proteins by
precipitation with GST-PAK1 and immunoblot-
ting with anti-Rac and anti-Cdc42 antibodies.
Since only the acti vated form of Rac/Cdc42 binds
to GST-PAK1, the amount of Rac/Cdc42 precip-
itated by GST-PAK1 represents the activation
level of Rac/Cdc42. Figure 8a shows that a similar
level of Rac activity (0.41 vs. 0.38) was present in
platelets that had adhered to either GoH3 or GST-
rhodostomin (upper panel). In contrast, more than
a two and half-fold higher activity for Cdc42 (0.85
vs. 0.34) was detected in platelets adhering to
GoH3 than those adhering to GST-rhodostomin
(lower panel). These results are consistent with the
results of Figure 2, which shows the morpholog-
ical differences, and indicate that Cdc42 plays a
more important role in filopodia formation.
891
Interestingly, in the presence of PP1, Rac and
Cdc42 activity increased 42% and 40%, respec-
tively, in platelets adhering to GoH3, while in the
presence of wortmannin, Rac and Cdc42 activity
increased 58% and 79%, respectively (Figure 8b).
These results clearly indicate that the inhibition of
Src leads to a lower increase in small GTPase
activity compared to inhibition of PI3K. This may
explain why the adherence of platelets to GoH3 in
the presence of PP1 did not result in an obvious
actin pattern change (Figur e 6b, the upper right
panel). On the other hand, the appearance of actin
patch formation and numerous filopodia might
result from the greater increase in Cdc42 activity in
the presence of the PI3K inhibitors (Figure 6b, the
lower panels).
Both PI3K and Cdc42 activities are higher
in platelet s adhering to GoH3 than in those adhering
to GST-rhodostomin
Based on the results from the studies with PI3K
inhibitors, one would predict that lower PI3K
activity would lead to higher Cdc42 activity. This
would suggest that PI3K activity is lower in
platelets adhered to GoH3 than in those adhering
to GST-rhodostomin. To verify this hypothesis,
PI3K was immunoprecipitated by anti-p85 anti-
body and probed with anti-ph osphotyrosine, anti-
p85 and anti-p110, separately. The results show a
band migrated at the 110 kDa position, presum-
ably p110, and this was phosphorylated in platelet s
adhering to both GoH3 and GST-rhodostomin
but not to BSA (Figure 9a lanes 1 and 2 vs. lane 3).
This is consistent with Figure 7c and other results
that show the PI3K pathway is activated when
there is engagement of integrin a6b1oraIIbb3.
However, a higher amount of phosphorylated
p110 as well as a higher production of PI(3)P
was found in platelets adhering to GoH3 than
those adhered to GST-rhodostomin (Figure 9a,
lane 2 vs. lane 1; Figure 9b, lane 1 vs. lane 2),
which suggest s that there is cross talk between the
PI3K and Cdc42 pathways. This might result in
the maintenance of a balance between PI3K and
Cdc42 levels in platelets adhered to GoH3, which
will lead to dynamic filopodium formation. Once
the balance of PI3K and Cdc42 breaks to a lower
Figure 6. The effect on platelet adhesion number and morphology after treatment with different kinase inhibitors. (a) Platelets were
pretreated with PP1, LY294002, wortmannin, or DMSO and plated on GoH3-coated coverslips for 15 min. After removal of any
unattached platelets by washing with buffer, the bound cells were fixed and scored from 10 to 15 different images from three inde-
pendent experiments. The graph showed the mean number (±SEM) of adhered platelets per 0.005 mm
2
. The number of adhering
cells showed no obvious difference with or without drug treatment. (b) Platelets with the same treatment as in (a) were fixed and
stained with rhodamine–phalloidin. The actin-cytoskeleton pattern was observed under fluorescence microscopy. No obvious pat-
tern changes were seen in cells treated with PP1 (the upper right panel) but there was an increase in the number of filopodia and
actin dots in cells treated with the PI3K inhibitors (the lower panels). Scale bar, 5 lm.
892
Figure 7. Biochemical analyses of Src, Syk, and PI3K activity in platelets adhered to GoH3 mAb in the presence of various inhibi-
tors. Washed platelets were pretreated with drugs or not as indicated and plated on the dishes coated with 5% BSA (control) or
GoH3 mAb for 30 min at 37 °C. Cell lysates were immunoprecipated by antibodies for Src (a), Syk (b), or PI3K p85 (c). The tyro-
sine-phosphorylation of the three kinases was determined by reacted with antibody 4G10 (the top rows). The same blots were then
stripped and reprobed with anti-kinase antibodies to indicate the total amount of kinases in the platelets (the second rows). The le-
vel of tyrosine-phosphorylation of the different kinases was quantified by the ratio of the density of the band detected by 4G10
over the density of total kinase and adjusted by the ratio that platelets adhered to BSA. Results are representative of three inde-
pendent experiments (the lower panels) (p<0.05).
893
ratio, the platelets, as in the situation of adhesion
to GST-rhodostomin, become fully spread within
minutes (Figure 3 and the supplement movie 3).
Discussion
Platelets contain five different integrin receptors
on their plasma membrane. However, whether
these integrins are present in the same membrane
domain and whether they share the same intra-
cellular signal molecules involved in platelet
morphological changes have never been explored.
In this paper, we demonstrate that a6b1 and
aIIbb3 are associated with different membrane
proteins (Figure 1) using a pull-down assay of
biotin-labeled proteins [34]. Coupling this assay
with a highly sensitive detection method using
avidin-horseradish peroxidase (HRP)-enhanced
chemiluminescence, we were able to identify 7
proteins co-existing with integrin aIIbb3 and 5
proteins co-existing with a6b1. Although the
features of these proteins will have to wait until
the results of MALDI-TOF mass spectrometric
analyses are available, based on the differences in
molecular weight, we have provided the first
biochemical evidence that the two integrins are
associated with different membrane proteins and
are thus located on different lipid raft microdo-
mains in platelets.
In addition to the difference in associated
proteins, integrin aIIbb3 and a6b1 activate differ-
ent signal pathways or different levels of various
signal molecules, although they share the same
molecules (Src, Syk, and PI3K), when they induce
platelet morphological changes. Platelets activated
by integrin a6b1 never appear to fully spread even
up to 2 h after attachment, whereas platelets
activated by integrin aIIbb3 become fully spread
within 15 min (Figures 2 and 3). We suggest that
there is no cross talk between activation by
integrin a6b1 and aIIbb3. Otherwise, platelets
adhered to GoH3 and laminin will release granules
and clotting factors, which would in turn indu ce
fully spreading. In endothelium cells, the integrin
a6b1 cooperates with a3b1 and can cross talk with
integrin avb3 when cells are exposed to laminin for
blood vessel developmen t [40, 41]. In oligoden-
drocytes, the signals through a6b1 activation
replace those activated by avb3 and there is a
switch from cell proliferation to cell survival and
differentiation [42] . These events are required for a
Figure 8. Biochemical analyses of Rac and Cdc42 activity in platelets adhered to different substrates for 30 minutes. (a) A compar-
ison of Rac and Cdc42 activity in platelets adhering to GST-rhodostomin (lane 1) and GoH3 mAb (lane 2). The presence of
GTPcS and GDP in this reaction was performed as positive (+) (lane 3) and negative ()) (lane 4) controls for Rac and Cdc42
activity, respectively. The relative activity of both GTPases was quantified and adjusted by the total amount of GTPase (the sec-
ond and fourth rows) as indicated. The ratio is indicated between gels. (b) A comparison of Rac and Cdc42 activity in platelets ad-
hered to GoH3 in the presence of PP1 (lane 2) and wortmannin (lane 3). The relative activity of the two GTPases was measured as
in (A) and adjusted by the activity in platelets without drug treatment (lane 1). Lane 4 is a positive control for the assay.
894
long-term, more than 24 h, interaction. On the
other hand, there is no need for cross-talk between
the integrins during platelet clot formation since it
happens only in a few minutes.
Unlike the integrin aIIbb3, which expresses
only in platelets, the integrin a6b1 expresses also in
eosinophils, neutrophils, and endothelium cells
[43–45]. The activation of integrin aIIbb3in
platelets is known to form blood clots through
morphological changes and aggregation while the
role of a6b1 in platelets is not yet fully understood,
but it may be similar to that in other cells, namely,
adhesion and/or migration. The present results,
which show that platelets adhering to laminin
actively forming filopodia, may represent the
physiological role in vivo. This supposition is
supported by knock-out experiments in mice,
which show that integrin b1 of platelets can
mediate shear-resistant adhesion independently of
aIIbb3 [4].
In this study, we also demonstrate for the first
time that platelets activated by integrin a6b1 are
different from those activated by integrin aIIbb3in
the following asp ects: (i) actin is present in a ring
Figure 9. Biochemical analyses of PI3K in platelets adhering to various substrates for 30 min. (a) The PI3K of platelets adhering
to GST-rhodostomin (lane 1), GoH3 mAb (lane 2), or BSA (lane 3) was precipitated by anti-p85 antibody and then probed with
4G10 to detect the phosphotyrosine form (the top gel), or with anti-p85 (the second row) or with anti-p110 (the third row) to de-
tect total PI3K. (b) The PI3K activity was measured by the production of phosphatidylinositol 3-phosphate (PI3P). The precipi-
tated PI3K was incubated in a solution containing phosphatidylinositol and [c
32
P] ATP and the production of PI3P was then
determined by thin layer chromatography and autoradiography. The position of PI3P is indicated. The total amount of PI3K from
various platelet conditions was determined by Western blotting using anti-p85 (the second row gel) and anti-p110 (the third row)
antibodies.
895
pattern in the center zone, (ii) actin in filopodia
concomitantly displays a high concentration of
phosphotyrosine proteins, and (iii) cell–substrate
contacts are present in the center and absent in the
periphery (Figures 4 and 5). These differences
result from either different signal pathways or/
and signals levels induced by the two integrins.
Figure 7 shows that both Src and PI3K are
activated when platelets adhere to GoH3 and
GST-rhodostomin. These results support the
hypothesis that the signal pathways induced by
various integrins in platelets are conserved [4]
because the cytoplasmic tails of the integrin a and
b chains are made up of highly conserved amino
acid sequence [46]. However, how integrin a6b1
and aIIbb3 induce different levels of PI3K and
Cdc42 activity as sho wn in Figures 8 and 9 is
not known. The difference may result from several
possibilities: (i) that there are different negative
regulators, such as SHIP1 (Src homology
2-domain containing 5-phosphatase) and various
SFKs, associated with a6b1 and aIIbb3 [42, 47],
(ii) that the inhibition and activation pa thways are
regulated in a temporal manner (see a review of
DeMali et al. 2003) [48], and (iii) that a feedback
loop occurs betw een PI3K and Cdc42 during
outside-in signaling [49, 50].
Two other lines of evidence showing that
activation of the different integrins, which creates
different levels of intracellular signal molecules,
may also be part of the explanation of the current
observations. Elevation of Rho GTPase activity
can occur through either b1orb3 integrin,
although in an opposite way to that previ-
ously reported [51, 52]. The frequency of calcium
oscillation in platelets varies when they adhere to
different substrates [31, 47, 53]. Previously, we also
observed that PI3K activity is coupled with
calcium oscillation [54]. Actin-cytoskeleton
changes depend on how PI 3K lipid products
coordinate the inter nal calcium stores and mem-
brane ion channels. Platelets appear with numer-
ous filopodia when integrin aIIbb3 is activated in
the presence of PI3K inhibitors or integ rin aIIbb3
is only partially activated [31, 54]. Therefore, the
level of PI3K may direct platelets to give rise to
filopodia or to be fully spread. Thus, the difference
between activation of integrin aIIbb3 and a6b1is
suggested to be such that the former can reach the
threshold level of PI3K while the latter can not
and that once plate lets become fully spread then
the PI3K activity drops and continues to fluctuate
at a lower level.
Based on the current study and previously
reports, we suggest a simple scheme regarding how
activation of integrin a6b1 causes active filopodi-
um formation in platelets. Upon platelets adhering
to laminin or GoH3, integrin a6b1 is activated and
both Src and PI3K are turned on. Src activation is
through the clustering of b1, which activates two
NPXY motifs at the C-terminus [46]. The acti-
vated Src then has a role as a positive and
upstream effector of PI3K [55, 56] since inhibition
of Src leads to decreased p110 activity (Figure 7c,
lane 2). PI3K can also be activated through a
lysine residue of integrin b1 transmembrane
domain (LLLIWKLLMII) [46]. However, the
former pathway to activate PI3K is less important
than the latter pathway because platelets treated
with PP1 show little or no change in actin
arrangement (Figure 6b, upper right panel). The
cross talk between PI3K and Cdc42 results in
oscillation of the calcium ion level and actin
regulator proteins, which, in turn, leads to accu-
mulation of phosphotyrosine proteins at the tip of
filopodia. The fluctuation of PI3K and Cdc42
drives filopodium extension and retraction and
this appears as dynamic filopodia formation. In
order to either confirm or disprove this control
model, a great deal of further research is required.
Since platelets are anucleated cell fragments, the
RNAi method is not applicable for studying the
effect of gene knock down. Platelets from various
gene knock out mice are the sources for verifying
the model.
Acknowledgements
We gratefully thank to Dr. Y.-S. Chang (Chang
Gung University) for providing us with the plas-
mid pGEX-GST-PAK and to Dr. Ralph Kirby
(National Yang-Ming University) for English
editing of this manuscript. Special thanks are
also due to the members of laboratory for their
long-term discussion and to Dr. Chi-Hung Lin
(National Yang-Ming University) and his labora-
tory members for helping image processing and
analysis. This work is supported by grants from
the National Science Council of Republic of Chi-
na (NSC90-2320-B-010-067 and NSC91-2320-B-
010-050).
896
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898
    • "Although platelets express at least five different integrins, most of our understanding on the contribution of PI3K is related to studies on the two major receptors: integrin a IIb b 3 and integrin a 2 b 1 . Indeed, to our knowledge the investigation of the possible involvement of PI3K in platelet activation induced by other integrin receptors is limited to the work of Chang and collaborators, who demonstrated the importance of PI3K for integrin a 6 b 1 -mediated filipodia formation (Chang et al., 2005). The affinity of integrins for their specific ligands is precisely modulated by a complex array of signals. "
    [Show abstract] [Hide abstract] ABSTRACT: Blood platelets are anucleated circulating cells that play a critical role in hemostasis and are also implicated in arterial thrombosis, a major cause of death worldwide. The biological function of platelets strongly relies in their reactiveness to a variety of extracellular agonists that regulate their adhesion to extracellular matrix at the site of vascular injury and their ability to form rapidly growing cell aggregates. Among the membrane receptors expressed on the cell surface, integrins are crucial for both platelet activation, adhesion and aggregation. Integrin affinity for specific ligands is regulated by intracellular signaling pathways activated in stimulated platelets, and, once engaged, integrins themselves generate and propagate signals inside the cells to reinforce and consolidate platelet response and thrombus formation. Phosphatidylinositol 3-Kinases (PI3Ks) have emerged as crucial players in platelet activation, and they are directly implicated in the regulation of integrin function. This review will discuss the contribution of PI3Ks in platelet integrin signaling, focusing on the role of specific members of class I PI3Ks and their downstream effector Akt on both integrin inside-out and outside-in signaling. The contribution of the PI3K/Akt pathways stimulated by integrin engagement and platelet activation in thrombus formation and stabilization will also be discussed in order to highlight the possibility to target these enzymes in effective anti-thrombotic therapeutic strategies. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Full-text · Article · Jun 2015
    • "Vero E6 cells (ATCC No. C1008) and HeLa cells (ATCC No. CCL- 2.2) were maintained in RPMI 1640 (BioWest, Miami, FL, USA), and NIH3T3 cells (ATCC No. CRL-1658) were maintained in Dulbecco's modified Eagle's medium (DMEM) (BioWest) containing 10% heatinactivated fetal bovine serum (Biological Industries, Beit Haemek, Israel), 2 mM l-glutamine (BioWest), 100 U/L of penicillin (BioWest ), 100 mg/mL of streptomycin (BioWest), and non-essential amino acid (BioWest). Cell adhesion and inhibition assays were performed according to our previously described methods (Chang et al., 1993Chang et al., , 1997a Chang and Lo, 1998; Chang et al., 1999 Chang et al., , 2001 Chang et al., , 2002 Chang et al., , 2003 Chang et al., 2005; Sun et al., 2005; Chang and Lo, 2007). Recombinant proteins (1.2 g/30 L/cover slip) were coated on cover slips at 37 @BULLET C for 1 h in 6-well microtiter plates, and then blocked with 5% bovine serum albumin (BSA) at 37 @BULLET C for a further hour. "
    [Show abstract] [Hide abstract] ABSTRACT: Emerging life threatening pathogens such as severe acute aspiratory syndrome-coronavirus (SARS-CoV), avian-origin influenzas H7N9, and the Middle East respiratory syndrome coronavirus (MERS-CoV) have caused a high case-fatality rate and psychological effects on society and the economy. Therefore, a simple, rapid, and safe method to investigate a therapeutic approach against these pathogens is required. In this study, a simple, quick, and safe cell adhesion inhibition assay was developed to determine the potential cellular binding site on the SARS-CoV spike protein. Various synthetic peptides covering the potential binding site helped to minimize further the binding motif to 10-25 residues. Following analyses, 2 peptides spanning the 436-445 and 437-461 amino acids of the spike protein were identified as peptide inhibitor or peptide vaccine candidates against SARS-CoV.
    Full-text · Article · Jun 2014
    • "Magnesium and manganese and cobaltact as cofactors for this interaction.6 α6β1 signaling to the platelets via phosphoinositide 3 kinase induces morphologic changes in the platelets.7 "
    [Show abstract] [Hide abstract] ABSTRACT: Platelets play an important role in hemostasis, inflammation, host defense, tumor growth and metastasis. Platelets receptors are instrumental in platelet-platelet aggregation and interaction of platelets with leukocytes, endothelial cells and coagulation factors. These receptors are also the targets for antiplatelet drugs. This review focuses on the role of platelet receptors in human physiology. Data were extracted from peer-reviewed journals using MEDLINE and EMBASE databases, and the following terms (platelets, platelet receptors, CD markers, integrins, tetraspanins, transmembrane receptors, prostaglandin receptors, immunoglobulin superfamily receptors) were used.
    Full-text · Article · Apr 2013
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