PTP-1B is an essential positive regulator of platelet integrin signaling

ArticleinThe Journal of Cell Biology 170(5):837-45 · September 2005with28 Reads
Impact Factor: 9.83 · DOI: 10.1083/jcb.200503125 · Source: PubMed

Outside-in integrin alphaIIbbeta3 signaling is required for normal platelet thrombus formation and is triggered by c-Src activation through an unknown mechanism. In this study, we demonstrate an essential role for protein-tyrosine phosphatase (PTP)-1B in this process. In resting platelets, c-Src forms a complex with alphaIIbbeta3 and Csk, which phosphorylates c-Src tyrosine 529 to maintain c-Src autoinhibition. Fibrinogen binding to alphaIIbbeta3 triggers PTP-1B recruitment to the alphaIIbbeta3-c-Src-Csk complex in a manner that is dependent on c-Src and specific tyrosine (tyrosine 152 and 153) and proline (proline 309 and 310) residues in PTP-1B. Studies of PTP-1B-deficient mouse platelets indicate that PTP-1B is required for fibrinogen-dependent Csk dissociation from alphaIIbbeta3, dephosphorylation of c-Src tyrosine 529, and c-Src activation. Furthermore, PTP-1B-deficient platelets are defective in outside-in alphaIIbbeta3 signaling in vitro as manifested by poor spreading on fibrinogen and decreased clot retraction, and they exhibit ineffective Ca2+ signaling and thrombus formation in vivo. Thus, PTP-1B is an essential positive regulator of the initiation of outside-in alphaIIbbeta3 signaling in platelets.


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The Journal of Cell Biology, Vol. 170, No. 5, August 29, 2005 837–845
JCB 837
PTP-1B is an essential positive regulator of platelet
integrin signaling
Elena Garcia Arias-Salgado,
Fawaz Haj,
Christophe Dubois,
Barry Moran,
Ana Kasirer-Friede,
Barbara C. Furie,
Bruce Furie,
Benjamin G. Neel,
and Sanford J. Shattil
Department of Medicine, University of California, San Diego, La Jolla, CA 92093
Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215
utside-in integrin
3 signaling is required
for normal platelet thrombus formation and is
triggered by c-Src activation through an unknown
mechanism. In this study, we demonstrate an essential
role for protein–tyrosine phosphatase (PTP)–1B in this
process. In resting platelets, c-Src forms a complex with
3 and Csk, which phosphorylates c-Src tyrosine
529 to maintain c-Src autoinhibition. Fibrinogen binding
3 triggers PTP-1B recruitment to the
Csk complex in a manner that is dependent on c-Src and
specific tyrosine (tyrosine 152 and 153) and proline
(proline 309 and 310) residues in PTP-1B. Studies of
PTP-1B–deficient mouse platelets indicate that PTP-1B is
required for fibrinogen-dependent Csk dissociation from
3, dephosphorylation of c-Src tyrosine 529, and
c-Src activation. Furthermore, PTP-1B–deficient platelets
are defective in outside-in
3 signaling in vitro as
manifested by poor spreading on fibrinogen and de-
creased clot retraction, and they exhibit ineffective Ca
signaling and thrombus formation in vivo. Thus, PTP-1B is
an essential positive regulator of the initiation of outside-in
3 signaling in platelets.
Integrins mediate cell adhesion to extracellular matrix ligands.
In addition to localizing cells for proper biological function,
ligand binding to integrins initiates a process referred to as out-
side-in signaling (Hynes, 2002). Integrin signals collaborate
with signals from growth factor, cytokine, and G protein–coupled
receptors to regulate actin rearrangements and cell motility,
growth, differentiation, and survival (Juliano et al., 2004).
Because the cytoplasmic domains of integrin
are devoid of catalytic activity, integrins must associate with
intracellular enzymes to transduce signals. Associations between
integrins and specific receptor and nonreceptor protein kinases
have been demonstrated by biochemical, microscopic, and
biophysical techniques (Brunton et al., 2004; de Virgilio et al.,
2004). However, many of these associations take place rela-
tively late after adhesive ligand binding, suggesting that they
propagate rather than initiate outside-in signaling. One excep-
tion is in platelets, in which a constitutive association between
3 and c-Src is mediated by direct interaction of
3 cytoplasmic domain with the c-Src SH3 domain (Obergfell
et al., 2002; Arias-Salgado et al., 2003). A similar relationship
may pertain to c-Src and the related integrin,
3, in osteo-
clasts (Feng et al., 2001). Furthermore, in many cell types, a
close functional, if not physical, relationship exists between
Src family kinases and
1 or
2 integrins (Klinghoffer et al.,
1999; Suen et al., 1999; Brunton et al., 2004).
3 mediates fibrinogen-dependent platelet aggregation
and spreading on damaged vascular surfaces, whereas
promotes osteoclast adhesion to vitronectin or osteopontin
(Byzova et al., 1998; Shattil and Newman, 2004). Genetic
deficiency of
3 and
3 leads to defects in hemostasis
and bone remodeling, respectively (Hodivala-Dilke et al., 1999;
Feng et al., 2001). Adhesive ligand binding to
3 integrins
leads to c-Src activation and tyrosine phosphorylation of c-Src
substrates in platelets and osteoclasts (Feng et al., 2001; Obergfell
et al., 2002; Arias-Salgado et al., 2003). The close relationship
3 integrins and c-Src is underscored by defective
spreading of platelets that are deficient in multiple Src family
kinases (Obergfell et al., 2002) and by overlapping bone remod-
eling phenotypes in mice that are deficient in c-Src or
(Soriano et al., 1991; Hodivala-Dilke et al., 1999; McHugh et
al., 2000). Consequently, attention is now focused on how
integrins regulate c-Src to initiate outside-in signaling.
c-Src is maintained in an autoinhibited state by concerted
intramolecular interactions of the SH2 domain with a COOH-
terminal motif centered at phosphotyrosine 529 and of the SH3
Correspondence to Sanford J. Shattil:
Abbreviation used in this paper: PTP, protein–tyrosine phosphatase.
The online version of this article contains supplemental material.
Page 1
JCB • VOLUME 170 • NUMBER 5 • 2005838
domain with a polyproline sequence in the linker region be-
tween the SH2 and kinase domains (Sicheri and Kuriyan,
1997; Young et al., 2001; Harrison, 2003). As c-Src appears
to associate constitutively with
3 integrins via the c-Src
SH3 domain (Arias-Salgado et al., 2003), considerable reli-
ance may be placed on the SH2–phosphotyrosine 529 inter-
action to help maintain low c-Src activity in nonadherent
platelets. Thus, disruption of the SH2–phosphotyrosine 529
interaction by dephosphorylation of c-Src tyrosine 529
should facilitate c-Src activation during cell adhesion. Phos-
phorylation of c-Src tyrosine 529 is catalyzed by Csk, which
is associated with the
3–c-Src complex in resting plate-
lets (Okada et al., 1991; Obergfell et al., 2002; Arias-Salgado
et al., 2003). However, the identity of the protein–tyrosine
phosphatase (PTP) that dephosphorylates c-Src tyrosine 529
to promote initiation of
3 integrin signaling has remained
unknown. In this study, we used biochemical and genetic ap-
proaches to unambiguously identify PTP-1B, which is a ubiq-
uitous nonreceptor tyrosine phosphatase, as a phosphatase
that is required for dephosphorylation of c-Src tyrosine 529
and for c-Src activation downstream of
3. Moreover,
we demonstrate that PTP-1B is required for outside-in signal-
ing in platelets and for normal platelet thrombus formation in
living mice.
PTP-1B associates with
3 and is
required for integrin activation of c-Src
To explore how
3 regulates c-Src, we sought to identify
a PTP that localizes to the
3–c-Src complex in response
to fibrinogen binding to platelets. We reasoned that this might
reverse phosphorylation of c-Src tyrosine 529 by Csk and,
thereby, help to promote c-Src activation (Obergfell et al.,
2002; Arias-Salgado et al., 2003). A previous study has dem-
onstrated that PTP-1B is localized to internal membranes of
resting platelets and is cleaved by calpain in a platelet aggre-
gation–dependent manner (Frangioni et al., 1993). We found
that PTP-1B coimmunoprecipitated with
3 and c-Src
from detergent lysates of human and mouse platelets. How-
ever, unlike the associations of c-Src and Csk with
which are observed in resting platelets (Obergfell et al.,
2002), the association of PTP-1B with
3 and c-Src re-
quired fibrinogen binding to platelets. This was induced ei-
ther by MnCl
, which activates
3 directly (Fig. 1 a;
Litvinov et al., 2004), or by plating the cells on fibrinogen
(not depicted). PTP-1B recruitment to
3 in response to
and fibrinogen did not require PTP-1B cleavage by
calpain because platelet aggregation was avoided under these
unstirred conditions, and no such cleavage was observed. The
interaction of PTP-1B with
3 was specific and was ob-
served whether immunoprecipitation was performed with an-
tibodies to PTP-1B or
3 (Fig. 1 b). The interactions of
PTP-1B with
3 and c-Src were prevented by pretreat-
ment of platelets with 2
M SU6656 or 5
M PP2 to block
Src kinase activity (Fig. 1 a) or with 2 mM RGDS (Arg-Gly-
Asp-Ser) to inhibit fibrinogen binding.
Figure 1. Interactions between PTP-1B, IIb3, and c-Src in platelets.
(a) Washed human platelets were incubated for 15 min at RT with 250
g/ml fibrinogen in the presence or absence of 0.5 mM MnCl
. Some
samples were preincubated for 15 min with 2 M SU6656, 5 M PP2, or
5 M PP3; the latter is an inactive congener of PP2. Clarified lysates were
immunoprecipitated (IP) and probed on immunoblots as indicated. Vertical
lines in the blots indicate grouping of images from different parts of the
same gel. (b) Washed mouse platelets were incubated with MnCl
fibrinogen, and immunoblots of immunoprecipitates were probed as in a.
Control immunoprecipitations used normal rabbit serum (NRS) or rat IgG
(IgG). (c) Role of PTP-1B in platelet tyrosine phosphorylation. Fibrinogen
binding to PTP-1B
and PTP-1B
platelets was induced as in b. Lysates
were immunoblotted with antibodies to phosphotyrosine (pTyr) or c-Src
phosphotyrosine 418 and reprobed with antibodies to c-Src. (d) IIb3
surface expression in PTP-1B
(black bar) and PTP-1B
(hatched bar)
platelets was quantified by flow cytometry. Mean fluorescence intensities
are depicted in arbitrary units, and error bars represent means SEM of
three experiments. (e) PTP-1B is required for activation of integrin-associated
c-Src. Fibrinogen binding to PTP-1B
and PTP-1B
platelets was induced
as in b, and IIb3 immunoprecipitates were probed on immunoblots as
indicated. (f) PTP-1B is required for dissociation of Csk from the IIb3–
c-Src complex. Fibrinogen binding to PTP-1B
and PTP-1B
was induced as in b, and Csk immunoprecipitates were probed on immuno-
blots as indicated. Each immunoblot panel is representative of three to five
independent experiments.
Page 2
Dadke and Chernoff, 2002) but may also regulate the interac-
tion between Csk and the
3–c-Src complex.
Mechanism of PTP-1B–
To better understand the basis for interactions between PTP-1B,
IIb3, and c-Src during outside-in IIb3 signaling, mouse fi-
broblasts that were deficient in the ubiquitous Src family kinases
c-Src, c-Yes, and Fyn (SYF cells; Klinghoffer et al., 1999) were
stably transfected with IIb3. These IIb3-SYF cells express
PTP-1B endogenously, enabling examination of PTP-1B inter-
actions after transient transfection of c-Src. As observed with
platelets, IIb3-SYF cells expressing c-Src showed a fibrino-
gen-inducible association of PTP-1B with c-Src and IIb3
(Fig. 2 a). However, PTP-1B failed to associate with c-Src in
cells lacking IIb3 or with IIb3 in cells lacking c-Src. Thus,
the fibrinogen-dependent interaction of PTP-1B with IIb3 or
c-Src requires both integrin and tyrosine kinase.
To determine what portions of the c-Src molecule are re-
quired for these PTP-1B interactions, IIb3-SYF cells were
transfected with selected c-Src mutants. Coimmunoprecipitation
of PTP-1B with IIb3 did not occur with catalytically inactive
c-Src (K295R) or with c-Src lacking the SH3 domain (90–144).
Identical results were obtained with c-Src SH3 domain mutants
(W120F or 90–92) that were incapable of interacting with
polyproline type II motifs or the 3 cytoplasmic domain (unpub-
lished data). In contrast, c-Src tyrosine 529 (Y529F) and the
c-Src SH2 domain (150–246) were dispensable for the interac-
Fibrinogen-dependent PTP-1B recruitment to IIb3
and c-Src was also observed in response to platelet stimula-
tion with traditional agonists, such as ADP and thrombin (un-
published data). However, in the studies that follow, MnCl
or platelet adhesion were used to induce fibrinogen binding to
IIb3 to prevent or minimize generalized signaling via G
protein–coupled receptors and, thus, to facilitate direct as-
sessment of outside-in IIb3 signaling (Obergfell et al.,
2002; Arias-Salgado et al., 2003). Overall, these results indi-
cate that fibrinogen binding to IIb3 triggers recruitment of
PTP-1B to a plasma membrane complex of IIb3 and c-Src
in a manner that is dependent on Src kinase activity.
To establish whether PTP-1B is required for integrin acti-
vation of c-Src, platelets from knockout mice that were defi-
cient in PTP-1B (PTP-1B
) and wild-type (PTP-1B
) litter-
mates were studied (Klaman et al., 2000). Incubation of wild-
type platelets with MnCl
and fibrinogen caused an increase in
the tyrosine phosphorylation of numerous proteins. In contrast,
platelets showed markedly reduced fibrinogen-
dependent tyrosine phosphorylation (Fig. 1 c). Because several
of the phosphorylated proteins, including Syk (72 kD) and ad-
hesion and degranulation-promoting adaptor protein (130 kD),
are substrates of c-Src during outside-in IIb3 signaling, the
catalytic activity of c-Src in IIb3 immunoprecipitates was
assessed indirectly by monitoring the phosphorylation of acti-
vation loop tyrosine 418. Whereas fibrinogen binding to PTP-
platelets stimulated phosphorylation of c-Src tyrosine
418, this response was minimal or absent in PTP-1B
lets (Fig. 1, c and e). Platelets from heterozygous (PTP-1B
littermates responded normally (not depicted). The defective
responses of PTP-1B
platelets could not be explained by re-
duced surface expression of IIb3 receptors (Fig. 1 d). These
results suggest that PTP-1B
platelets have a fundamental
defect in IIb3 activation of c-Src.
To determine whether PTP-1B is required for fibrinogen-
dependent dephosphorylation of c-Src tyrosine 529, the phos-
phorylation state of tyrosine 529 was monitored with an anti-
body specific for nonphosphorylated tyrosine 529. Whereas
fibrinogen binding to wild-type platelets stimulated dephos-
phorylation of c-Src tyrosine 529 (as indicated by increased
immunoreactivity of the dephosphotyrosine 529 antibody), no
such dephosphorylation was observed in PTP-1B
In fact, the level of tyrosine 529 phosphorylation paradoxically
increased upon fibrinogen binding (Fig. 1 e). Thus, PTP-1B is
required for IIb3-dependent dephosphorylation of c-Src ty-
rosine 529, likely explaining the defective activation of c-Src in
The finding of relatively increased phosphorylation of
c-Src tyrosine 529 in fibrinogen-bound PTP-1B
suggested that PTP-1B may play some unexpected role in the
phosphorylation of tyrosine 529 by Csk. Csk is normally asso-
ciated with the IIb3–c-Src complex in resting platelets and
dissociates from it upon fibrinogen binding (Obergfell et al.,
2002). However, Csk failed to fully dissociate from IIb3 and
c-Src after fibrinogen binding to PTP-1B
platelets (Fig. 1 f).
Thus, PTP-1B may not only dephosphorylate c-Src tyrosine
529 upon fibrinogen binding to IIb3 (Arregui et al., 1998;
Figure 2. Structural features of c-Src that are required for interactions
ith PTP-1B and IIb3. (a) SYF cells or SYF cells stably expressing human
IIb3 (IIb3-SYF) were transiently transfected with wild-type c-Src or
empty vector. After 48 h, transfected cells were incubated at 37C for 15
min in the presence or absence of 1 mM MnCl
and 250 g/ml fibrino-
gen. Clarified lysates were immunoprecipitated with antibodies to c-Src or
3, and immunoprecipitates were probed on immunoblots as indicated.
Lysates were probed for PTP-1B as a loading control. (b and c) IIb3-SYF
cells were transfected with wild-type c-Src or an indicated c-Src mutant.
After 48 h, transfected cells were incubated in the presence or absence of
and fibrinogen as in a. Clarified lysates were immunoprecipitated
with antibodies to 3 (b) or c-Src (c) and with the precipitates probed on
immunoblots. Data are from a single experiment that was representative of
three that were performed.
Page 3
JCB • VOLUME 170 • NUMBER 5 • 2005840
tion of PTP-1B with IIb3 (Fig. 2 b). Similar results were ob-
tained for the interaction of PTP-1B with c-Src except that c-Src
tyrosine 529 was also required (Fig. 2 c). The requirement for the
c-Src SH3 domain might be explained by the direct binding of
SH3 to the 3 cytoplasmic domain (Arias-Salgado et al., 2003)
rather than binding of c-Src SH3 to PTP-1B. Together with the
platelet results (Fig. 1 a), these outcomes indicate that association
of PTP-1B with the IIb3–c-Src complex is regulated by c-Src
catalytic activity and by a process that requires tyrosine 529.
To establish what regions of PTP-1B are required for
these interactions, HA-tagged PTP-1B was cotransfected
with c-Src into IIb3-SYF cells. Wild-type PTP-1B and
two different phosphatase-inactive “substrate-trapping” mutants
(C215S and D181A) each interacted with IIb3 and c-Src
(Fig. 3 a). Interestingly, the interaction with c-Src was some-
what greater with the D181A PTP-1B mutant, which is known
to exhibit a higher affinity for binding to PTP-1B substrates
than the C215S mutant (Flint et al., 1997). These data are con-
sistent with a direct dephosphorylation of c-Src tyrosine 529 by
PTP-1B. In contrast to substrate-trapping mutants, the double
mutation of proline 309 and 310 to alanine prevented PTP-1B
interaction with IIb3 and c-Src, as did the double mutation
of tyrosine 152 and 153 to phenylalanine. These amino acid
residues may help to mediate interactions of PTP-1B with one
or more members of the integrin signaling complex during the
early phase of outside-in signaling (Dadke and Chernoff,
2002). In addition, they may enable the phosphorylation of
PTP-1B by c-Src because PTP-1B can phosphorylate c-Src in
vitro (Jung et al., 1998), and fibrinogen binding to IIb3-SYF
cells (Fig. 3 b) or platelets (Fig. 3 c) stimulated tyrosine phos-
phorylation of PTP-1B in a Src-dependent manner.
PTP-1B regulates platelet functions that
are dependent on outside-in
IIb3 signaling
Outside-in signaling via IIb3 facilitates platelet spreading
on fibrinogen and platelet thrombus formation under condi-
tions of flow (Phillips et al., 2001; Nesbitt et al., 2002; Shattil
and Newman, 2004). Therefore, these responses were com-
pared in PTP-1B
and PTP-1B
platelets. PTP-1B
lets attached but failed to spread on fibrinogen over 45 min,
whereas PTP-1B
platelets exhibited cytoskeletal reorganiza-
tion, filopodial and lamellipodial extensions, and varying de-
grees of spreading (Fig. 4 a, no agonist). When spreading was
assessed by computer analysis of mean platelet areas and the
percentage of platelets with filopodia or lamellipodia was
quantified, the differences between PTP-1B
and PTP-1B
platelets were statistically significant (P 0.001; Fig. 4 b).
Figure 3. Structural features of PTP-1B that are required for interactions
ith IIb3 and c-Src. (a) IIb3-SYF cells were transiently cotransfected
with c-Src and wild-type or mutant forms of HA-tagged human PTP-1B.
After 48 h, transfected cells were incubated with or without MnCl
fibrinogen as described in Fig. 2. Clarified lysates were immunoprecipi-
tated with antibodies to the HA tag, and precipitates were probed on
immunoblots as indicated. (b and c) PTP-1B is tyrosine phosphorylated in
response to fibrinogen binding. IIb3-SYF cells transfected with c-Src and
empty vector (b) or human platelets (c) were incubated with or without 0.5
mM MnCl
and 250 g/ml fibrinogen for 10 min. Some platelet samples
were preincubated for 15 min with c-Src inhibitors (5 M PP2 or 2 M
SU6656) or 5 M PP3 as a control. Clarified lysates were immunoprecip-
itated with an antibody to PTP-1B, and immunoprecipitates and lysates
were probed on immunoblots. Data are from a single experiment that was
representative of three that were performed. NRS, normal rabbit serum.
Figure 4. PTP-1B
platelets are defective in IIb3-dependent spreading
on fibrinogen. (a) Platelets from PTP-1B
and PTP-1B
mice were
plated on fibrinogen-coated coverslips for 40 min at RT in the presence or
absence of 100 M ADP. Adherent cells were fixed, permeabilized, and
stained with rhodamine-phalloidin (F-actin, red) and antiphosphotyrosine
antibodies (green). Images were acquired with a confocal fluorescence
microscope. Bar, 10 m. (b) Platelet surface areas from at least 25 images
were analyzed and depicted in the left panel as means SEM. The right
panel depicts the percentage of platelets containing one or more filopodia
and/or lamellipodia. At least 80 cells each were analyzed.
Page 4
The stimulation of platelets with a G protein–coupled re-
ceptor agonist such as ADP results in more rapid and uniform
platelet spreading on fibrinogen when compared with cells in-
cubated without agonist (Haimovich et al., 1993). Thus, in ad-
dition to IIb3 signaling, costimulatory pathways are in-
volved in full platelet spreading. In contrast to the spreading
defect of untreated PTP-1B
platelets, costimulation with
ADP resulted in uniform, full spreading (Fig. 4, a and b). PTP-
platelets adhered normally to fibrinogen (Fig. 5 a), and
they bound soluble fibrinogen normally in response to either
ADP, PAR4 receptor–activating peptide, or convulxin, which
is a glycoprotein VI agonist (Fig. 5 b). In addition, stirred PTP-
platelets that were incubated with 1–10 M ADP or 250
M PAR4 receptor–activating peptide exhibited an initial rate
and extent of aggregation that was equivalent to those of
platelets (unpublished data). On the other hand,
platelets mediated less fibrin clot retraction
than PTP-1B
platelets (P 0.05); this response is depen-
dent, in part, on IIb3-triggered changes in the actin cytoskel-
eton (Fig. 5 c; Phillips et al., 2001; Shattil and Newman, 2004).
Collectively, these results indicate that PTP-1B is required for
normal outside-in IIb3 signaling in platelets. However,
PTP-1B appears to be dispensable for agonist induction of sol-
uble fibrinogen binding to IIb3 and for ADP costimulation
of platelet spreading.
Thrombus formation can be studied in living mice by
real-time fluorescence and brightfield microscopy of cremaster
muscle arterioles that were subjected to laser injury (Falati et
al., 2002). Platelets from PTP-1B
and PTP-1B
mice were
labeled with the Ca
-sensitive fluorescent dye Fura 2 and
were reinfused into PTP-1B
and PTP-1B
mice, respec-
tively. Labeled donor platelets accounted for 20% of total
platelets in the recipients. This enabled quantification of fluo-
rescent platelet accumulation and mobilization of intracellular
in developing thrombi at sites of laser injury (Fig. 6 and
Videos 1 and 2, available at
full/jcb.200503125/DC1). As described previously for other
normal mouse platelets (Falati et al., 2002), PTP-1B
lets accumulated into a growing thrombus for 60–120 s, and
some platelets detached over the course of several minutes.
Platelet calcium mobilization increased over roughly the same
time course. In contrast, the quantity of PTP-1B
that incorporated into a growing thrombus was markedly re-
duced, and those platelets that did become incorporated tended
to detach rapidly and exhibited little calcium mobilization (Fig.
6 and Videos 1 and 2). Similar results were obtained when
labeled PTP-1B
platelets were reinfused into PTP-1B
mice, indicating that the defect was intrinsic to PTP-1B
platelets (17 thrombi were analyzed in three PTP-1B
not depicted). Thus, in this model of vascular injury, PTP-1B is
required for calcium mobilization and stable platelet accumula-
tion into growing thrombi.
mice did not exhibit spontaneous bleeding,
but their mean tail bleeding times were nominally longer than
those of controls (although this difference was not statistically
significant: PTP-1B
, 254 53 s; PTP-1B
, 198 19 s;
P 0.06, n 14 mice each). However, rebleeding from tail
wounds after initial bleeding had stopped occurred in 28% of
mice but in none of the controls. This pattern of re-
bleeding has also been observed in mice with a defect in out-
side-in signaling as a result of tyrosine-to-phenylalanine muta-
tions in the 3 cytoplasmic domain (Law et al., 1999a).
Src family kinases are key components of outside-in integrin
signaling to the actin cytoskeleton in hematopoietic and nonhe-
matopoietic cells (Klinghoffer et al., 1999; Obergfell et al.,
2002; Lowell, 2004). In particular, c-Src, the most abundant
Src family member that is expressed in platelets, can bind di-
rectly to the integrin 3 subunit, and fibrinogen binding to
IIb3 triggers c-Src activation (Obergfell et al., 2002; Arias-
Salgado et al., 2003). We sought to determine the mechanism
by which fibrinogen binding leads to c-Src activation and ex-
Figure 5. Role of PTP-1B in the interaction of platelets with fibrinogen
and fibrin. (a) Platelet adhesion. Washed platelets (1.5 10
in 50 l
incubation buffer) were incubated in fibrinogen-coated microtiter wells for
1 h at RT, and platelet adhesion was quantified. Platelets from at least
four mice were used to generate duplicate points at each fibrinogen con-
centration. (b) Soluble fibrinogen binding. Platelets were incubated at RT
for 20 min with 150 g/ml FITC-fibrinogen in the presence or absence of
ADP, convulxin, or PAR4 receptor–activating peptide (AYPGKF; Faruqi et
al., 2000). Fibrinogen binding was analyzed by flow cytometry. Data are
the means SEM of quadruplicate determinations from an experiment
that was representative of three that were performed. (c) Fibrin clot retraction
was assessed 2 h after the addition of thrombin and CaCl
to platelet-rich
plasma. Clot volumes, expressed as a percentage of the initial volume of
platelet-rich plasma, were significantly greater in PTP-1B
than in PTP
samples, indicating less clot retraction. Data represent means SEM of
seven experiments.
Page 5
JCB • VOLUME 170 • NUMBER 5 • 2005842
plore the physiological significance of this process. The results
establish that (1) fibrinogen binding to platelets leads to PTP-
1B recruitment to an IIb3-based signaling complex that in-
cludes c-Src and Csk; (2) recruitment of PTP-1B is required for
the dissociation of Csk from the complex, dephosphorylation
of c-Src tyrosine 529, and c-Src activation; (3) PTP-1B is re-
quired for IIb3-dependent platelet spreading on fibrinogen
and for normal fibrin clot retraction but not for the agonist-
induced activation of IIb3; and (4) deficiency of PTP-1B re-
sults in defective platelet thrombus formation in an in vivo
model of vascular injury.
Although PTPs frequently exert negative regulation of
signaling pathways, positive regulation has also been described
previously (Neel et al., 2003; Tonks, 2003). In fact, receptor ty-
rosine phosphatases such as RPTP- or nonreceptor phos-
phatases such as Shp2 promote outside-in integrin signaling in
fibroblasts, in some cases by dephosphorylating c-Src tyrosine
529 or the equivalent residue in another Src family kinase (Oh
et al., 1999; Su et al., 1999). Although PTP-1B has been impli-
cated in 1 integrin–dependent c-Src activation, this has been
observed only in immortalized fibroblasts and not in a primary
cell type (Cheng et al., 2001), raising the question as to its
physiological significance. Our data establish the in vivo rele-
vance of PTP-1B activation of c-Src downstream of a 3 inte-
grin. PTP-1B may also exert negative regulation of integrin
signaling by dephosphorylating c-Src substrates such as p130
Cas (Arregui et al., 1998; Liu et al., 1998; Cheng et al., 2001).
Multiple substrates for PTP-1B may exist in platelets, although
our results indicate that the dominant action of PTP-1B is the
positive regulation of IIb3 signaling through activation of
integrin-associated c-Src. In contrast to these results for PTP-
1B, platelets from motheaten viable mice that were deficient in
Shp1 catalytic function displayed normal IIb3-dependent
activation of c-Src (unpublished data) and a morphology upon
attachment to fibrinogen that is similar to wild-type platelets
(Lin et al., 2004; unpublished data).
The fibrinogen-dependent association of PTP-1B with
IIb3 was observed in human and mouse platelets and in a
fibroblast model system, enabling examination of its struc-
tural basis. PTP-1B recruitment to IIb3 required catalytic
competence and the SH3 domain of c-Src (Figs. 1 and 2).
Moreover, specific proline (proline 309 and 310) and tyrosine
(tyrosine 152 and 153) residues in PTP-1B were necessary
(Fig. 3), suggesting that a protein (or proteins) with SH3,
SH2, and/or phosphotyrosine-binding domains is involved in
mediating linkage of PTP-1B to the integrin complex. Al-
though the linker protein in platelets could be c-Src itself,
there is no evidence that the c-Src SH2 domain binds to PTP-
1B, and the c-Src SH3 domain may not be available to PTP-
1B when it engages the integrin 3 cytoplasmic domain
(Arias-Salgado et al., 2003). Thus, a model is proposed in
which PTP-1B is localized in resting platelets to internal
membranes (Frangioni et al., 1993). Then, fibrinogen binding
induces IIb3 oligomerization (Simmons et al., 1997; Buen-
suceso et al., 2003), triggering transautophosphorylation of in-
tegrin-associated c-Src. This event might not be sufficient for
full c-Src activation (Harrison, 2003), but low level activation
might enable c-Src to phosphorylate a protein that is capable
of recruiting PTP-1B to the IIb3 complex. After recruit-
ment, PTP-1B may become a substrate for c-Src (Fig. 3, b and c;
Jung et al., 1998) and induce further c-Src activation by pro-
moting Csk dissociation from the integrin complex and de-
phosphorylation of tyrosine 529 (Fig. 1 f).
Although additional studies will be required to determine
the mode of PTP-1B linkage to the IIb3 complex in plate-
lets, work in other cells has implicated scaffold or adaptor mol-
ecules, such as SHPS-1, PAG/Cbp, and Dok-1, in mediating in-
teractions between Src kinases, PTPs, and/or Csk in response
to growth factors or cell adhesion (Timms et al., 1999; Dube et
al., 2004; Zhang et al., 2004). However, SHPS-1 is poorly ex-
pressed in platelets, and PAG/Cbp does not interact with
IIb3 (Wonerow et al., 2002). Intriguingly, Dok-1 contains a
phosphotyrosine-binding domain and potential SH2-binding
sites and is a substrate for PTP-1B (Dube et al., 2004). Further-
more, Dok-1 binds directly to Csk (Shah and Shokat, 2002) and
may associate with integrin cytoplasmic domains (Calder-
wood et al., 2003). Dok-2, a homologue of Dok-1, is expressed
in platelets (Garcia et al., 2004). In preliminary studies, we
have found that Dok-2 coimmunoprecipitates with PTP-1B
from resting platelets. In addition, fibrinogen binding to plate-
Figure 6. Defective thrombus formation in PTP-1B
mice. PTP-1B
and PTP-1B
platelets were labeled ex vivo with Fura 2-AM and were
reinfused into PTP-1B
and PTP-1B
recipient mice, respectively. Then,
vessel walls of arterioles in recipient cremaster muscles were subjected to
laser injury, and the accumulation of fluorescent platelets into developing
thrombi was assessed. (a) Representative composite brightfield and fluo-
rescence images of Fura 2–labeled platelets up to 90 s after laser injury of
an arteriole. Green represents labeled platelets and yellow represents
cytoplasmic free calcium. Blood flow is from bottom to top. See Videos 1
and 2 (available at
DC1) for examples of thrombus formation in PTP-1B
and PTP-1B
mice, respectively. (b) Accumulation of fluorescent platelets into the devel-
oping thrombus. Fluorescent signal was detected at 510 nm after excitation
at 380 nm. FPlatelet is defined as the integrated fluorescence intensity
associated with platelets. (c) Calcium mobilization within fluorescent plate-
lets of the developing thrombus. Fluorescent signal was detected at 510
nm after excitation at 340 nm. FCalcium mobilization is defined as the
integrated fluorescence intensity associated with calcium mobilization.
Each curve in b and c is a composite of 18 independent thrombi gener-
ated in three mice (six thrombi per mouse). To analyze these data, all 18
curves were plotted versus time, and median values were determined at
each time point and depicted in the figure.
Page 6
lets stimulates tyrosine phosphorylation of Dok-2, dissociation
of Dok-2 from PTP-1B, and its association with Csk (un-
published data). However, a role for Dok-2 or any other Csk-
binding protein (Thomas et al., 1999) in regulating PTP-1B re-
cruitment to IIb3 and outside-in signaling remains to be
Altogether, these studies have established a new function
for PTP-1B by uncovering requirements for PTP-1B in inte-
grin-dependent c-Src activation, platelet spreading on fibrino-
gen, and clot retraction and platelet thrombus formation. De-
fects in both platelet spreading and clot retraction may be
adequately explained by the c-Src activation defect in PTP-
platelets because both responses require outside-in
IIb3 signaling (Phillips et al., 2001; Shattil and Newman,
2004). However, although thrombus formation under flow con-
ditions depends on signaling inputs from multiple platelet
receptors, including IIb3 (Ruggeri, 2002; Jackson et al.,
2003), it is legitimate to ask whether the impairment of c-Src
activation in PTP-1B
platelets is the cause of defects in
platelet calcium mobilization and thrombus formation, which
were observed in the microcirculation of PTP-1B
mice (Fig.
6). Although we cannot exclude the possibility of additional
unstudied signaling pathways that are affected by a deficiency
of PTP-1B, we found no defect in agonist-induced IIb3 acti-
vation, platelet aggregation, or agonist costimulation of platelet
spreading. Furthermore, the ligation of IIb3 is known to in-
duce calcium transients that are required for the formation of
stable platelet aggregates under conditions of flow and wall
shear stress, which are typical within arterioles (Mazzucato et
al., 2002; Nesbitt et al., 2002). In particular, IP
production and
calcium mobilization are triggered by Src-dependent activation
of phospholipase C during outside-in IIb3 signaling (Won-
erow et al., 2003). Thus, links between defective integrin acti-
vation of c-Src and reduced calcium mobilization provide a
plausible explanation for the reduced thrombus formation ob-
served in PTP-1B
In contrast to the reduced platelet thrombus formation in
cremasteric vessels of PTP-1B
mice, there was no spontane-
ous bleeding, although rebleeding from tail bleeding time
wounds was more frequent than in control mice. Thus, the de-
gree of any abnormality in hemostasis imposed by PTP-1B de-
ficiency may be dictated by the type, location, and extent of
vascular injury. The same might be true in the case of pharma-
cological inhibition of PTP-1B. Interestingly, one PTP-1B an-
tagonist of questionable specificity has been shown to reverse
platelet aggregation that is stimulated by cross-linking the
FcRIIa receptor (Ragab et al., 2003). Although the selectivity
of PTP-1B antagonists is still an issue, they are being evaluated
for the treatment of type 2 diabetes and obesity because PTP-
1B negatively regulates insulin and leptin receptor signaling in
nonhematopoietic tissues (Elchebly et al., 1999; Klaman et al.,
2000; Cheng et al., 2002; Zabolotny et al., 2002; Tonks, 2003;
Hooft van Huijsduijnen et al., 2004). Assuming that PTP-1B
antagonists with appropriate selectivity and toxicity profiles
can be developed, current studies indicate that these com-
pounds should be analyzed for their effects on platelet outside-
in IIb3 signaling.
Materials and methods
Reagents and antibodies
Mouse mAb to human PTP-1B and rabbit pAb to murine PTP-1B were ob-
tained from Calbiochem and Upstate Biotechnology, respectively. Anti-
bodies against c-Src (327 and 1671) and the integrin 3 subunit (SSA6
and 8053) were described previously (Arias-Salgado et al., 2003). Anti-
bodies to Csk (C-20) and the COOH terminus of c-Src (B-12) were ob-
tained from Santa Cruz Biotechnology, Inc. Phosphospecific antibody to
Src tyrosine 418 was obtained from Biosource International, and antibody
specific for the nonphosphorylated form of c-Src tyrosine 529 was ob-
tained from Cell Signaling Technology, Inc. Rat monoclonal anti–mouse
CD41 (integrin IIb subunit), FITC-conjugated hamster anti–mouse CD61
(integrin 3 subunit), and mouse mAb to Csk were from BD Biosciences.
Mouse mAbs 4G10 and PY20 to phosphotyrosine were obtained from
Upstate Biotechnology and BD Biosciences, respectively. Antibody HA.11
against the HA epitope tag was obtained from Covance. HRP-conjugated
secondary antibodies and HRP-conjugated protein A–Sepharose beads
were purchased from Bio-Rad Laboratories. HRP-conjugated anti–mouse
IgG TrueBlot (eBioscience) was used when necessary to eliminate interfer-
ence by the heavy chain of immunoprecipitating antibodies. FITC-conju-
gated anti–mouse IgG was obtained from Jackson ImmunoResearch Labo-
ratories. Purified human fibrinogen was purchased from Enzyme Research
Laboratories, Inc. Rhodamine-phalloidin was obtained from Molecular
Probes. Src kinase inhibitors PP2 and SU6656 and the control compound
PP3 were obtained from Calbiochem. Protein A– and protein G–Sepha-
rose beads were purchased from GE Healthcare. All other reagents were
obtained from Sigma-Aldrich.
Mouse strains
and PTP-1B
mice (SV129/C57BL6/J) were described previ-
ously (Klaman et al., 2000). Age- and sex-matched littermates were used
for each experiment. Mice were housed and handled in accordance with
institutional guidelines.
Cell lines, plasmids, and transfections
SYF cells (mouse embryonic fibroblasts deficient in c-Src, Fyn, and c-Yes)
were obtained from American Type Tissue Collection. Cells were main-
tained at 37C with 6% CO
in DME supplemented with 10% FBS, L-glutamine,
and antibiotics. The vector pCDM8/IIb has been described previously
(Hughes et al., 1995). Integrin 3 cDNA was subcloned into HindIII-XhoI
sites of pcDNA3.1/Zeo (Invitrogen). Expression vectors containing human
IIb and 3 subunits were cotransfected into SYF cells with LipofectAMINE
(Invitrogen). Stable transfectants (IIb3-SYF cells) were isolated by selec-
tive growth in medium containing 125 g/ml Zeocin (Invitrogen), and
clones expressing IIb3 were isolated by single cell sorting. A single
clone (A29) was used for the studies reported in this article, but similar re-
sults were obtained with three other independent clones.
Expression vectors for wild-type and mutant c-Src (K295R, Y529F,
90–144, and 150–246) and HA-tagged PTP-1B have been described
previously (Sells and Chernoff, 1995; Arias-Salgado et al., 2003). Mutant
PTP-1B constructs (C215S, D181A, P309/310A, and Y152/153F) were
generated using the Site-Directed Mutagenesis Kit (Stratagene), and muta-
tions were confirmed by direct DNA sequencing. Transient transfections of
SYF cells were performed with LipofectAMINE. After 24 h, cells were se-
rum starved in 0.5% FBS and were cultured for an additional 24 h before
further use.
Platelet isolation and functional assays
Human and mouse platelets were obtained from fresh anticoagulated
whole blood, washed, and resuspended to 3 10
cells/ml in a platelet
incubation buffer (Law et al., 1999b). A pool of platelets from at least four
mice was used for each experiment. FITC-fibrinogen binding to platelets
and platelet aggregation were measured as described previously (Law et
al., 1999b). Surface expression of IIb3 in mouse platelets was moni-
tored with a FITC-conjugated anti–mouse 3 antibody. Platelet spreading
was assessed by confocal microscopy after plating cells on immobilized fi-
brinogen (100 g/ml of coating concentration) for 40–90 min. Fluores-
cence images were acquired with a laser scanning confocal microscope
(model MRC 1024; Bio-Rad Laboratories) using a 60 oil immersion ob-
jective (Nikon). Platelet surface areas were measured using Image Pro Plus
software (Media Cybernetics, Inc.). Platelet adhesion was quantified by
an acid phosphatase assay after incubating 1.5 10
cells (50 l) for
1 h at RT in fibrinogen-coated microtiter wells (Law et al., 1999b). The
percentage of adherent platelets was determined by calculating the ratio
Page 7
JCB • VOLUME 170 • NUMBER 5 • 2005844
of bound/maximal signal at 405 nm, with maximal signal obtained from
wells with platelets not subjected to washing. Fibrin clot retraction was
studied by incubating 150 l of mouse platelet-rich plasma (2.2 10
platelets) in the presence of 1 U/ml thrombin and 3 mM CaCl
for 2 h at
RT in an aggregometer cuvette. A paper clip was added to facilitate clot
removal at the termination of the experiment. The volume of residual clot-
free plasma was determined, and clot volume was taken as 150 l minus
this value. Clot volume was expressed as a percentage of the original
150-l plasma volume.
Immunoprecipitation and immunoblotting
Cells were lysed in buffer containing 1% NP-40, 150 mM NaCl, 50 mM
Tris, pH 7.4, 1 mM sodium vanadate, 0.5 mM sodium fluoride, 1 mM leu-
peptin, and complete protease inhibitor cocktail (Roche Applied Science).
Lysates were clarified by centrifugation at 13,000 g for 10 min at 4C, and
100–500 g of protein from the soluble fraction was immunoprecipitated
using a relevant primary antibody and protein A– or protein G–Sepharose
beads. Immunoprecipitates were subjected to SDS-PAGE and immunoblot-
ting, and immunoreactive bands were detected by enhanced chemilumines-
cence (SuperSignal West Pico Substrate; Pierce Chemical Co.).
Platelet thrombus formation in vivo
Fluorescence and brightfield microscopy was used to capture real-time
digital images of Fura 2-AM–labeled platelets in developing thrombi of liv-
ing mice after a laser-induced injury to the arteriole wall in the cremaster
muscle (Falati et al., 2002). In brief, platelets from PTP-1B
and PTP-
mice were loaded with Fura 2-AM. Then, 250–300 10
platelets, corresponding to 20% of the total endogenous platelet num-
ber, were infused into the circulation of an anesthetized mouse. PTP-1B
and PTP-1B
donor platelets were infused into PTP-1B
or PTP-1B
recipient mice as indicated in each particular experiment. Vascular injury
was induced 10 min after platelet infusion. Real-time multichannel intravi-
tal microscopy was used to monitor two fluorescence channels and one
brightfield channel almost simultaneously. The accumulation of labeled
platelets within a developing thrombus was monitored at 510 nm after ex-
citation at 380 nm, and calcium mobilization within those platelets was
monitored at 510 nm after excitation at 340 nm. Mouse tail bleeding
times and the occurrence of rebleeding from tail wounds were assessed as
described previously (Law et al., 1999a).
Online supplemental material
Videos show thrombus formation in a cremasteric arteriole of a living PTP-
(Video 1) or PTP-1B
(Video 2) mouse at three frames/s for 3 min.
and PTP-1B
platelets were labeled with Fura 2, an arteriole in
a recipient cremaster muscle was subjected to laser injury, and the accumu-
lation of fluorescent platelets into the developing thrombus was assessed as
described above and in Fig. 6 a. Online supplemental material is available
We would like to thank Dr. Nick Prevost for outstanding technical assistance.
This work was supported by grants DK60838, HL56595, HL57900,
HL77645, and HL77817 from the National Institutes of Health.
Submitted: 23 March 2005
Accepted: 26 July 2005
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    • "Remarkably, we demonstrated a significant effect on collagen contraction. Higher contractile capacity of PTP1B-deficient cells may explain defects in clot retraction in platelets (Salgado et al., 2005), cell migration in fibroblasts (Hernández et al., 2006; Burdisso et al., 2013), axon elongation (Fuentes and Arrequi, 2009), and dendritic spine maturation (Fuentes et al., 2012). "
    [Show abstract] [Hide abstract] ABSTRACT: Cell contractility and migration by integrins depends on precise regulation of protein tyrosine kinase and Rho-family GTPase activities in specific spatiotemporal patterns. Here we show that protein tyrosine phosphatase PTP1B cooperates with β3 integrin to activate the Src/FAK signalling pathway which represses RhoA-myosin-dependent contractility. Using PTP1B null (KO) cells and PTP1B reconstituted (WT) cells, we determined that some early steps following cell adhesion to fibronectin and vitronectin occurred robustly in WT cells, including aggregation of β3 integrins and adaptor proteins, and activation of Src/FAK-dependent signalling at small puncta in a lamellipodium. However, these events were significantly impaired in KO cells. We established that cytoskeletal strain and cell contractility was highly enhanced at the periphery of KO cells compared to WT cells. Inhibition of the Src/FAK signalling pathway or expression of constitutive active RhoA in WT cells induced a KO cell phenotype. Conversely, expression of constitutive active Src or myosin inhibition in KO cells restored the WT phenotype. We propose that this novel function of PTP1B stimulates permissive conditions for adhesion and lamellipodium assembly at the protruding edge during cell spreading and migration.
    Full-text · Article · Dec 2015 · Biology Open
    • "While Src is involved in the downstream signaling of the GPIb–A1 interaction [23], it also constitutively associates with the cytoplasmic tails of β3 and is essential to αIIbβ3 outside-in signaling in platelets. Upon fibrinogen ligation, integrin clustering causes protein tyrosine phosphatase (PTP) 1B-dependent dissociation of c-terminal Src kinase (Csk) from cytoplasmic tails of β3 [98,99], which in turn activates Src. Activated Src is found to localize in filopodia and at the edges of spreading platelets. "
    [Show abstract] [Hide abstract] ABSTRACT: During clot formation, platelets are subjected to various different signals and cues as they dynamically interact with extracellular matrix proteins such as von Willebrand factor (vWF), fibrin(ogen) and collagen. While the downstream signaling of platelet-ligand interactions is well-characterized, biophysical cues, such as hydrodynamic forces and mechanical stiffness of the underlying substrate, also mediate these interactions and affect the binding kinetics of platelets to these proteins. Recent studies have observed that, similar to nucleated cells, platelets mechanosense their microenvironment and exhibit dynamic physiologic responses to biophysical cues. This review discusses how platelet mechanosensing is affected by the hydrodynamic forces that dictate vWF-platelet interactions and fibrin polymerization and network formation. The similarities and differences in mechanosensing between platelets and nucleated cells and integrin-mediated platelet mechanosensing on both fibrin(ogen) and collagen are then reviewed. Further studies investigating how platelets interact with the mechanical microenvironment will improve our overall understanding of the hemostatic process. Copyright © 2015. Published by Elsevier Ltd.
    Full-text · Article · May 2015 · Blood reviews
    • "* PI3 kinase LY-294002 CR ↓ [76] * PI3 kinase β Pik3cb-null, TGX-221 CR ↓ [75] Protein phosphatase 1 Pp1cg -/- CR ↓ [99] PTP1B Ptp1b -/- CR = / ↓ [100, 101] Cytoskeleton linked proteins Rac1 EHT-1864 CR ↓ [102] Rap1b Rap1b -/- CR ↓ [103] RhoA Rhoa -/- CR ↓ [79] Talin-1 Tln1-null CR ↓ [104] WASP Wasp -/- CR ↓ [81] Platelet signaling proteins (other) * Calpain Capn1 -/- CR ↓ [100] Cbl Cbl -/-, Cbl (LOF) CR ↓ [105] Gas6 Gas6 -/- TG = [14] Lnk Sh2b3 -/- CR ↓ [106] mTOR rapamycin CR ↓ [107] NF-κB BAY-117082 CR ↓ [108] PER2 Per2-null CR ↓ [109] Phosphodiesterase 3 milrinone TG ↓ [110] * Phospholipase Cγ2 Plcg -/- CR ↓ [111] SHIP1 (PIP 3 phosphatase) Inpp55-null CR ↓ [112] Plasma proteins * Factor XII F12 -/-, CTI TG ↓ [34] * Factor XI F11 -/- TG ↓ [34] * Factor VII FVIIai TG ↓ [34] * (Pro)thrombin melagatran TG ↓ [35] "
    [Show abstract] [Hide abstract] ABSTRACT: The coagulation process is activated by tight control mechanisms, in which platelets play prominent and unique roles. In thrombosis and hemostasis, activated platelets regulate the coagulation system in various ways: by exposing a phosphatidylserine surface for thrombin formation, by supporting fibrin formation, and by regulating the retraction of a fibrin clot. In this review we discuss the involvement of platelet receptors, other membrane proteins, downstream signaling proteins, cytoskeleton-linked proteins and plasma proteins in these procoagulant functions. Studies with both genetically modified mice and pharmacological inhibitors indicate that, for collagen-adhered platelets, in part common signaling pathways lead to phosphatidylserine exposure, generation of thrombin and fibrin, and retraction of the fibrin clot. However, prolonged Ca(2+) elevation leads to thrombin generation, whereas integrin-dependent signaling stimulates fibrin clot retraction. Contact-dependent signaling pathways, triggered by homotypic platelet-platelet interactions, act in particular via the integrin route. © 2014 Elsevier Ltd. All rights reserved.
    Full-text · Article · May 2014 · Thrombosis Research
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