The Journal of Experimental Medicine
JEM © The Rockefeller University Press $30.00
Vol. 204, No. 13, December 24, 2007 3103-3111 www.jem.org/cgi/doi/10.1084/jem.20071800
BRIEF DEFINITIVE REPORT
Thrombosis and hemostasis depend on platelet
function. Upon disruption of vascular integrity,
platelets adhere to sites of injury and aggregate,
thereby preventing excessive bleeding ( 1 ).
Stable platelet adhesion to the injured blood
vessel and subsequent aggregation in turn de-
pend on integrin adhesion receptors. This point
is well illustrated in patients with genetic defects
in integrin subunits ? IIb or ? 3 that cause the
bleeding disorder Glanzmann thrombasthenia due
primarily to defective platelet aggregation or in
animals lacking all ( ? 2 ? 1, ? 5 ? 1, and ? 6 ? 1) ( 2 )
or certain ( ? 2 ? 1) ( 3 ) platelet ? 1 integrins that
manifest milder bleeding defects due to reduced
platelet adhesion to vascular surfaces ( 3, 4 ).
The ability of platelets to increase integrin af-
fi nity (operationally defi ned as integrin activation)
is critical for normal platelet function. Circulating
platelets are usually in a resting state. Upon
stimulation through agonist receptors, such as
those for ADP, collagen, or thrombin, signaling
events within the platelet lead to complex bio-
logical eff ects including activation of ? 1 and ? 3
integrins ( 5 ). Activated ? IIb ? 3 then binds plasma
proteins such as fi brinogen, leading to platelet
aggregation, whereas activation of ? 1 integrins
leads to adhesion of platelets to vessel wall com-
ponents such as collagen ( 1, 5 ). The molecular
events that link agonist receptors to integrin
activation are incompletely understood; however,
experiments in cultured cells have indicated that
this signaling results in increased association of
the cytoplasmic protein talin with the integrin ?
subunit cytoplasmic domain, inducing an increase
in integrin affi nity via a long-range allosteric
change in the integrin ’ s conformation ( 6 ). The
requirement of talin for integrin activation has
been examined in vitro; however, its role in vivo
remains to be determined.
Talin is a 270-kD protein composed of a
50-kD head domain and a 220-kD rod domain.
It was identifi ed in platelets where it comprises
Mark H. Ginsberg:
The online version of this article contains supplemental material.
Talin is required for integrin-mediated
platelet function in hemostasis
Brian G. Petrich , 1 Patrizia Marchese , 2 Zaverio M. Ruggeri , 2 Saskia Spiess , 1
Rachel A.M. Weichert , 1 Feng Ye , 1 Ralph Tiedt , 3 Radek C. Skoda , 3
Susan J. Monkley , 4 David R. Critchley , 4 and Mark H. Ginsberg 1
1 Department of Medicine, University of California, San Diego, La Jolla, CA 92093
2 Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037
3 Department of Experimental Hematology, University Hospital Basel, 4031 Basel, Switzerland
4 Department of Biochemistry, Henry Wellcome Building, University of Leicester, Leicester LE1 9HN, UK
Integrins are critical for hemostasis and thrombosis because they mediate both platelet
adhesion and aggregation. Talin is an integrin-binding cytoplasmic adaptor that is a central
organizer of focal adhesions, and loss of talin phenocopies integrin deletion in Drosophila .
Here, we have examined the role of talin in mammalian integrin function in vivo by selec-
tively disrupting the talin1 gene in mouse platelet precursor megakaryocytes. Talin null
megakaryocytes produced circulating platelets that exhibited normal morphology yet
manifested profoundly impaired hemostatic function. Specifi cally, platelet-specifi c deletion
of talin1 led to spontaneous hemorrhage and pathological bleeding. Ex vivo and in vitro
studies revealed that loss of talin1 resulted in dramatically impaired integrin ? IIb ? 3-
mediated platelet aggregation and ? 1 integrin – mediated platelet adhesion. Furthermore,
loss of talin1 strongly inhibited the activation of platelet ? 1 and ? 3 integrins in response
to platelet agonists. These data establish that platelet talin plays a crucial role in hemosta-
sis and provide the fi rst proof that talin is required for the activation and function of
mammalian ? 2 ? 1 and ? IIb ? 3 integrins in vivo.
TALIN IS REQUIRED FOR PLATELET INTEGRIN FUNCTION | Petrich et al.
embryonic days 8.5 and 9.5 due to defects in cell migration
before and during gastrulation ( 14 ). Thus, based on studies in
vitro and in invertebrates, talin is essential for the function of
certain integrins and for integrin activation. To examine the
role of talin in integrin function in vivo , we selectively de-
leted talin1 in platelets and megakaryocytes in mice and found
that platelet talin1 is essential for hemostasis because it is re-
quired for the function and activation of platelet ? 2 ? 1 and
? IIb ? 3 integrins.
RESULTS AND DISCUSSION
Platelet-specifi c deletion of talin1
Global genetic deletion of talin1 in mice is lethal by embryonic
day 9 ( 14 ). To circumvent this early embryonic lethality, we
deleted talin1 specifi cally in platelet precursor megakaryocytes
3 – 8% of total platelet protein ( 7 ). The head domain contains
binding sites for ? 1A, ? 1D, ? 2, ? 3, ? 5, and ? 7 ( 8 ) integrin
subunits and for another membrane protein, layilin ( 9 ). The
rod domain contains binding sites for vinculin and F-actin.
Thus, talin serves as a critical link between integrins and the
actin cytoskeleton ( 10 ). Furthermore, in invertebrates, talin is
necessary for formation of the integrin-associated cytoplas-
mic protein complex that includes proteins such as paxillin,
vinculin, integrin-linked kinase, PINCH, and parvin ( 11 ).
Lack of talin phenocopies lack of integrins in Drosophila ,
probably due to disrupted linkage to the actin cytoskeleton
( 11, 12 ). There are two mammalian talin isoforms encoded by
distinct genes: talin1 is expressed ubiquitously, and talin2 is
highly expressed in brain and striated muscle ( 13 ). In mice, global
deletion of talin1 results in embryonic lethality between
Figure 1. Deletion of talin1 in platelets and megakaryocytes. (A) Scheme of the targeting strategy. Homologous recombination of the Tln1 conditional
targeting vector into the Tln1 gene of embryonic stem cells introduced a loxP site (triangle) downstream of exon 4 and a fl oxed Neo cassette upstream of exon
1 to generate the targeted Neo allele (fl N). Partial Cre-mediated recombination in vivo was used to delete only the fl oxed Neo cassette leaving fl oxed exons 1 – 4
(conditional allele, fl ). In cells expressing both the Tln1 conditional allele and PF4-Cre, Cre recombinase – mediated recombination will result in deletion of cod-
ing exons 1 – 4, generating the Tln1 -deleted allele (2). E, EcoRI; probe, external probe for Southern analysis. Primers used for genotyping mice are indicated.
(B) Demonstration of homologous recombination in embryonic stem cells by Southern blotting. Genomic DNA from embryonic stem clones was digested with
EcoRI and probed with an external probe shown in A. The wild-type allele gives rise to a 13.8-kb band (wt/wt), whereas the targeted allele gives rise to a 7.6-kb
band due to introduction of an internal EcoRI site (fl N/wt). (C) PCR genotyping of mice possessing the conditional allele. Genomic DNA isolated from ear bi-
opsies of Tln1 fl /fl , Tln1 fl /+ , and Tln1 fl /+ mice was analyzed by PCR using the primer pair shown in A. (D) Coomassie blue – stained SDS-PAGE gel of platelet lysates
from Tln1 fl /fl Cre + and Tln1 fl /fl Cre 2 mice shows reduction of talin expression in Tln1 fl /fl Cre + samples, whereas other protein bands are expressed at similar levels.
(E) Immunostaining of freshly isolated bone marrow samples showing reduced talin expression in CD41 + megakaryocytes from Tln1 fl /fl Cre + mice. Bar, 20 mm.
JEM VOL. 204, December 24, 2007
BRIEF DEFINITIVE REPORT
hemorrhage, most often localized to the abdominal cavity,
was observed in 8% of 1 – 2-d-old Tln1 fl /fl Cre + mice ( Fig. 2 B ).
By 3 wk of age, 45% fewer Tln1 fl /fl Cre + than Tln1 fl /fl Cre ?
mice were alive. In addition, 12 out of 76 Tln1 fl /fl Cre + mice
were found dead between 3 and 9 wk of age compared with
3 out of 139 Tln1 fl /fl Cre ? mice ( Fig. 2 B ). Tln1 fl /fl Cre + mice
that survived to 8 wk of age had a 95% incidence of gastroin-
testinal bleeding compared with 7% of Tln1 fl /fl Cre ? litter-
mates as judged by an assay for fecal blood ( Fig. 2 C ).
Gastrointestinal bleeding in Tln1 fl /fl Cre + mice was associated
with profound anemia as manifested by signifi cantly reduced
by crossing mice harboring a fl oxed talin1 allele ( Tln1 ) ( Fig.
1 A ) with platelet factor 4 – Cre (PF4-Cre) mice that express Cre
recombinase selectively in platelets and megakaryocytes ( 15 ).
Mice homozygous for the fl oxed Tln1 allele and positive for
the PF4-Cre transgene ( Tln1 fl /fl Cre + ) had slightly reduced
platelet counts compared with Tln1 fl/fl Cre ? littermates
(Table S1, available at http://www.jem.org/cgi/content/full/
jem.20071800/DC1) that were still in the normal range ( 16 ).
SDS-PAGE analysis of platelet lysates revealed a selective loss
in the band corresponding to talin in Tln1 fl /fl Cre + platelets
( Fig. 1 D ). In addition, loss of talin was observed by immuno-
fl uorescence in CD41 + megakaryocytes from the bone mar-
row of Tln1 fl /fl Cre + mice ( Fig. 1 E ). Deletion of talin in Tln1 fl /fl
Cre + mice was only detectable in platelets and megakaryocytes
as CD41 ? bone marrow cells from Tln1 fl /fl Cre + mice showed
similar low levels of talin immunofl uorescence as that from
Tln1 fl /fl Cre ? mice (not depicted), consistent with PF4-Cre –
mediated recombination being selective for the megakaryocytic
lineage as reported previously ( 15 ).
These results show that terminal megakaryocyte develop-
ment and platelet formation do not require talin. Large mega-
karyocytes from Tln1 fl /fl Cre + mice were devoid of talin as
judged by staining of bone marrow cells with the 8d4 mono-
clonal antibody. Importantly, we targeted the talin1 allele and not
talin2 , an isoform that is very similar to talin1 ( 13 ). Nevertheless,
hematopoietic cells express little talin2 ( 13 ). Furthermore, the
8d4 antibody reacts with both talin isoforms, and thus the ab-
sence of 8d4 staining of megakaryocytes from Tln1 fl /fl Cre +
mice confi rms the elimination of talin expression in mature
megakaryocytes. Previous work establishes that the PF4-cre
mice we used express Cre specifi cally in megakaryocytes, in-
cluding large megakaryocytes ( 15 ), and we observed that talin
was still present in other cells of the hematopoietic lineage in
the Tln1 fl /fl Cre + mice. In spite of elimination of most of the
talin, we observed abundant megakaryocytes in the bone mar-
row of the platelet talin1 – defi cient mice. Talin-defi cient platelets
were present at normal abundance, indicating that talin is not
required for the formation of platelets from megakaryocytes.
It is noteworthy that there was a slight reduction in platelet
count in Tln1 fl /fl Cre + mice relative to Tln1 fl /fl Cre ? littermates.
This reduction was not due to the presence of the PF4-Cre
transgene in the absence of the fl oxed Tln1 allele because
Tln1 fl /+ Cre + mice had platelet counts similar to that of Tln1 fl /fl
Cre ? mice (755 ± 24 vs. 777 ± 74, × 10 9 platelets/ml ± SEM,
Tln1 fl /fl Cre ? vs. Tln1 fl /+ Cre + ). Hence, it will be of interest in
future work to examine the response of these animals to chal-
lenges to megakaryocytopoiesis.
Bleeding diathesis in platelet talin – defi cient mice
Despite normal platelet counts in adult Tln1 fl /fl Cre + mice
(Table S1), these mice showed dramatically impaired hemo-
stasis. In a tail bleeding assay, Tln1 fl /fl Cre + mice bled contin-
uously for the 10-min duration of the assay, whereas Tln1 fl /fl
Cre ? mice stopped bleeding an average of 4.6 min after tail
resection ( Fig. 2 A ). Platelet talin defi ciency was also associ-
ated with spontaneous bleeding. Lethal spontaneous internal
Figure 2. Reduced survival and perinatal hemorrhage in Tln1 fl /fl
Cre + mice. (A) Tln1 fl /fl Cre + mice have prolonged bleeding times in a tail
bleeding assay. Time to cessation of bleeding after tail resection was re-
corded for up to 10 min, at which time bleeding was stopped by cauter-
ization. (B) Example of 1-d-old Tln1 fl /fl Cre + pup found dead with visible
internal hemorrhage (arrows). Table showing reduced survival of Tln1 fl /fl
Cre + mice. The number of animals obtained from Tln1 fl /fl Cre 2 × Tln1 fl /fl
Cre + breeding at 3 wk of age is shown. (C) Incidence of fecal blood in
Tln1 fl /fl Cre + and Tln1 fl /fl Cre 2 mice at 8 – 10 wk of age was determined by a
guaiac-based hemoccult assay. (D) Peripheral red blood cell counts from
10-wk-old Tln1 fl /fl Cre + and Tln1 fl /fl Cre 2 littermates.
TALIN IS REQUIRED FOR PLATELET INTEGRIN FUNCTION | Petrich et al.
the common carotid artery. Complete occlusion of the carotid
arteries of Tln1 fl /fl Cre ? mice occurred 7.0 ± 0.9 min after
injury, whereas none of the Tln1 fl /fl Cre + mice tested showed
reduced fl ow during the 20-min assay ( Fig. 3 ). Histological
examination of the carotid arteries of these animals after the
thrombosis experiment indicated a similar extent of ferric
chloride – induced vessel injury in Tln1 fl /fl Cre + and Tln1 fl /fl Cre ?
mice (Fig. S2, available at http://www.jem.org/cgi/content/
full/jem.20071800/DC1). Collectively, our results show that
deletion of talin1 in platelets leads to markedly impaired
hemostasis and thrombus formation in vivo.
? 2 ? 1 integrin – mediated adhesion of platelets to exposed
subendothelial collagen after vascular trauma is thought to be
a key step in hemostasis. To examine the ability of Tln1 fl /fl Cre +
platelets to adhere to collagen under physiological conditions,
we measured platelet adhesion and thrombus formation to
red blood cell counts and hemoglobin concentration ( Fig. 2 D
and Table S1).
The hemostatic defects observed in Tln1 fl /fl Cre + mice are
at least as severe as those observed in ? 3 integrin null mice
( 17 ). In our hands, 22.9% fewer ? 3 ? / ? than ? 3 +/+ off spring
from ? 3 +/ ? by ? 3 +/ ? matings survived to 3 wk of age, a
smaller reduction in survival compared with Tln1 fl /fl Cre +
mice (45% fewer Tln1 fl /fl Cre + than Tln1 fl /fl Cre ? ). However,
because the Tln1 fl /fl and ? 3 null mice were both on mixed
genetic backgrounds (C57BL/6-Sv129), it is not possible to
make defi nitive statements regarding the relative hemostatic
impairment in these mice.
Platelet talin is required for thrombus formation
Thrombus formation in mice with talin-defi cient platelets
was examined in vivo by ferric chloride – induced injury of
Figure 3. Impaired thrombus formation in Tln1 fl /fl Cre + mice in vivo and ex vivo. (A) Time to occlusion of the carotid artery was determined with a
Doppler fl ow probe after a 3-min application of 10% ferric chloride. The experiment was stopped 20 min after injury in all animals. (B) Adhesion of Tln1 fl /fl
Cre + , Tln1 fl /fl Cre 2 , and b3(L476A) platelets to collagen and subsequent thrombus formation in fl owing blood were analyzed by epifl uorescence and confo-
cal microscopy at a shear rate of 1,500 s 21 . Heparinized whole blood containing 10 mM mepacrine to render platelets fl uorescent was perfused over glass
coated with fi brillar type I collagen for 2 min. Tln1 fl /fl Cre 2 platelets adhere to the collagen-coated surface and form thrombi (larger aggregates of bright
fl uorescence). In contrast, Tln1 fl /fl Cre + platelets form only transient contacts resulting in sparse coverage of platelets on the surface. b3(L746A) platelets
form a monolayer that cover much of the surface but do not form thrombi, as seen by the lack of highly fl uorescent aggregates that form with Tln1 fl /fl
Cre 2 platelets. Images shown are single frames from a real-time recording (Video S1). Bar, 20 mm. (C) Percent of the collagen-coated surface covered with
platelets was calculated as the number of fl uorescent pixels (due to adhesion of fl uorescently labeled platelets) divided by the total number of pixels (rep-
resenting the total surface). *, P < 0.0005; NS, not signifi cant. (D) Quantifi cation of the volume of the thrombi formed on the collagen-coated surface
after perfusion for 2 min with blood from Tln1 fl /fl Cre + , Tln1 fl /fl Cre 2 , and b3(L746A) mice. Confocal serial Z-section reconstructions of the platelet thrombi
were used to calculate the thrombi volume as described previously (reference 32 ). *, P < 0.0005.
JEM VOL. 204, December 24, 2007
BRIEF DEFINITIVE REPORT
these data fi rmly establish talin as a critical regulator of ? IIb ? 3
integrin activation in vivo.
The impaired adhesion of Tln1 fl /fl Cre + platelets to colla-
gen noted above suggests that ? 2 ? 1 integrin activation may
also be impaired in Tln1 fl /fl Cre + platelets. To examine the
activation of ? 1 integrins in Tln1 fl /fl Cre + platelets, we mea-
sured the binding of the conformation-sensitive ? 1 integrin
antibody 9EG7 to agonist-stimulated platelets. Tln1 fl /fl Cre ?
platelets bound signifi cantly more 9EG7 upon stimulation.
This response was largely ablated in platelets from Tln1 fl /fl
Cre + mice ( Fig. 4 C ). These results show that talin expression
is required for agonist-induced activation of both ? IIb ? 3 and
? 2 ? 1 integrins in platelets. In addition, these data suggest
that impaired activation of ? 2 ? 1 integrins contributes to the
spontaneous bleeding observed in Tln1 fl /fl Cre + mice.
collagen in fl owing blood. Platelets from Tln1 fl /fl Cre ? mice
stably adhered to the collagen-coated surface and subse-
quently formed platelet-rich thrombi visible as highly fl uo-
rescent aggregates ( Fig. 3 B, middle ). In contrast, Tln1 fl /fl Cre +
platelets formed only transient contacts with the collagen-
coated surface and did not form thrombi ( Fig. 3 and Video S1,
which is available at http://www.jem.org/cgi/content/
full/jem.20071800/DC1). Interestingly, platelets from
? 3(L746A) mice, in which ? 3 integrin – talin interactions are
selectively disrupted ( 18 ), formed stable adhesions to collagen
indicated by the platelet monolayer shown in Fig. 3 B and
quantifi ed in Fig. 3 C . Nevertheless, the ? 3(L746A) plate-
lets failed to undergo the integrin ? IIb ? 3 – mediated platelet –
platelet interactions required for thrombus formation ( Fig. 3,
B and D ). Thus, lack of platelet talin impairs ? 2 ? 1 integrin –
dependent adhesion to collagen in fl ow and integrin ? IIb ? 3 –
dependent platelet thrombus formation.
Platelet talin is required for integrin-mediated platelet
adhesion to collagen and platelet aggregation
We examined platelet adhesion and aggregation in vitro to
directly assess the eff ects of talin defi ciency on these integrin-
dependent processes. In static adhesion assays, the talin-defi -
cient platelets showed a marked defect in adhesion to soluble
type I col lagen (Fig. S1 A, available at http://www.jem.org/
cgi/content/full/jem.20071800/DC1). Furthermore, like
? 3(L746A) platelets ( 18 ), talin-defi cient platelets manifested
profoundly impaired aggregation in response to stimulation with
ADP or PAR4 peptide (Fig. S1 B). Thus, the talin-defi cient
platelets manifest virtual absence of platelet functions mediated
by both ? 2 ? 1 and ? IIb ? 3 integrins, thus accounting for their
profound hemostatic defect.
Talin is required for activation of platelet ? 2 ? 1
and ? IIb ? 3 integrins
Agonist-induced increase in integrin ? IIb ? 3 affi nity (activa-
tion) is required for platelet aggregation ( 19 ). Indeed, ? 3(L746A)
mice, in which ? IIb ? 3 – talin interactions are disrupted, have
impaired ? IIb ? 3 integrin activation and platelet aggrega-
tion ( 18 ). To test the requirement of talin for the activation
of ? IIb ? 3, we measured binding of FITC-labeled fi brinogen
to the surface of washed Tln1 fl /fl Cre + and Tln1 fl /fl Cre ? plate-
lets by fl ow cytometry. Stimulation of Tln1 fl /fl Cre ? platelets
with a combination of ADP/epinephrine (100 ? M each)
or PAR4 peptide (1 mM) led to an increase in the amount of
bound fi brinogen. In contrast, the amount of agonist-induced
fi brinogen binding was greatly reduced in Tln1 fl /fl Cre + plate-
lets ( Fig. 4 A and Fig. S3, which is available at http://www
.jem.org/cgi/content/full/jem.20071800/DC1). In the pres-
ence of 0.5 mM MnCl2, however, Tln1 fl /fl Cre + and Tln1 fl /fl
Cre ? platelets bound similar amounts of fi brinogen, indicat-
ing that the ? IIb ? 3 present on Tln1 fl /fl Cre + platelets is capa-
ble of binding fi brinogen if activated exogenously ( Fig. 4 B and
Fig. S3). Thus, with regards to ? IIb ? 3 activation, platelet ta-
lin defi ciency phenocopies the ? 3(L746A) mutation in which
the ? 3 integrin – talin interaction is disrupted. Collectively,
Figure 4. Impaired agonist-induced activation of b3 and b1 inte-
grins in Tln1 fl /fl Cre + platelets. (A) The amount of FITC-labeled fi brinogen
bound to platelets from Tln1 fl /fl Cre + or Tln1 fl /fl Cre 2 mice was measured by
fl ow cytometry and expressed as the amount of fi brinogen bound to
platelets in each group relative to the amount of fi brinogen bound to
platelets in the presence of 0.5 mM MnCl 2 . *, P < 0.001. (B) Fibrinogen
binding to Tln1 fl /fl Cre + and Tln1 fl /fl Cre 2 platelets was similar in the pres-
ence of 0.5 mM MnCl 2 . (C) Activation of b1 integrin in Tln1 fl /fl Cre + , Tln1 fl /fl
Cre 2 , and b3(L746A) platelets after stimulation with 1 mM PAR4, 100 mm
ADP, and 100 mM epinephrine was measured by binding of the confor-
mation-sensitive antibody 9EG7 and expressed relative to total b1 integ-
rin surface expression measured by the conformation-insensitive antibody
HMb1-1. *, P < 0.05; **, P < 0.0005.
TALIN IS REQUIRED FOR PLATELET INTEGRIN FUNCTION | Petrich et al.
cytometry, was not signifi cantly diff erent in Tln1 fl /fl Cre + and
Tln1 fl /fl Cre ? platelets ( Fig. 5 C ). Of particular importance,
the quantity of surface P-selectin was similar on both resting
and stimulated Tln1 fl /fl Cre + and Tln1 fl /fl Cre ? platelets, con-
fi rming the presence of platelet ? granules in the absence of
talin. Furthermore, both platelet genotypes exhibited a similar
fourfold increase in P-selectin surface expression in response
to ADP/epinephrine/PAR4 peptide stimulation, confi rming
that the mutant platelets were capable of responding to plate-
let agonists. Collectively, these data demonstrate that talin is
dispensable for the formation of platelets that can respond to
platelet agonists and manifest a normal complement of granule
Thus, the principle that talin is required for activation of
? 1 and ? 3 integrins, which was suggested by in vitro studies
( 20 ), applies in vivo. Furthermore, the central role of talin in
integrin function in invertebrates ( 11, 12, 21 ) extends to ver-
tebrates. A ? 3(L746A) mutation that selectively disrupts the
ability of ? 3 integrin to bind talin leads to impaired agonist-
induced activation of platelet ? IIb ? 3 ( 18 ). Together with the
present fi nding of impaired agonist-induced activation of
? IIb ? 3 in talin-defi cient platelets, these data show that talin
binding to integrin ? cytoplasmic domains is a fi nal common
step in integrin activation in vivo, and that disruption of this
interaction has a profound impact on integrin-dependent ad-
hesive functions in mammals.
Talin-defi cient platelets are nearly completely defi cient in
hemostatic function. It is noteworthy that the pathological
bleeding observed in Tln1 fl /fl Cre + mice is absent in ? 3(L746A)
mice despite having comparable impairments in ? IIb ? 3
activation and platelet aggregation ( 18 ). One obvious ex-
planation for this more severe phenotype in the platelet
talin-defi cient animals is the impairment in the activation and
function of platelet ? 1 integrins. Furthermore, in Drosophila ,
talin deletion results in marked weakening of the connections
of integrins with the actin cytoskeleton; hence, a defect in
the connection of ? 1 and ? 3 integrins to the actin cytoskel-
eton may also contribute to the severe phenotype observed.
Deletion of platelet ? 1 integrins or point mutations that would
disrupt ? 1 – talin interactions can result in defects in platelet
function and in hemostasis ( 4, 22 ). Given the hemostatic de-
fects that result from lack of platelet ? 3 or ? 1 integrins, and
our fi nding that platelet integrin function is virtually com-
pletely dependent on talin, it is likely that the hemostatic de-
fect in Tln1 fl /fl Cre + mice is ascribable largely to the lack of
Morphology and surface receptor expression is normal
in talin-defi cient platelets
To examine the eff ect of deleting talin on platelet structure,
we examined Tln1 fl /fl Cre + and Tln1 fl /fl Cre ? platelet mor-
phology by electron microscopy. Platelet shape, ? granules,
mitochondria, open canalicular system, and microtubule coils
appeared similar in Tln1 fl /fl Cre + and Tln1 fl /fl Cre ? platelets
( Fig. 5 A ). Tln1 fl /fl Cre + mice had slightly larger platelets than
Tln1 fl /fl Cre ? mice as judged by fl ow cytometry (forward
scatter, 13.9 ± 0.2 vs. 15.3 ± 0.4 arbitrary units, Tln1 fl /fl Cre ?
vs. Tln1 fl /fl Cre + , P < 0.005) and by measurement of the area
of at least 140 randomly selected electron microscopic plate-
let profi les (0.67 ± 0.03 ? m 2 vs. 0.97 ± 0.4 ? m 2 , Tln1 fl /fl
Cre ? vs. Tln1 fl /fl Cre + , P < 0.005). As noted above, the Tln1 fl /fl
Cre + mice have active gastrointestinal bleeding, suggesting
that the slightly increased platelet size could be due to an in-
creased proportion of circulating young platelets. In contrast
to ? 3 integrin null platelets ( 17 ), talin-defi cient platelets had
normal fi brinogen content, suggesting that talin-dependent
activation of ? IIb ? 3 in mature megakaryocytes is not required
for fi brinogen uptake ( Fig. 5 B ). In addition, the surface ex-
pression of several adhesion receptors, as measured by fl ow
Figure 5. Platelets from Tln1 fl /fl Cre + mice are structurally normal
and express normal levels of surface receptors. (A) Electron micro-
graphs showing normal structural features of Tln1 fl /fl Cre + platelets. Plate-
lets from Tln1 fl /fl Cre + (top left) and Tln1 fl /fl Cre 2 (bottom left) mice both
display a discoid shape characteristic of resting platelets and similar gran-
ular contents. Insets show microtubule coils in platelets from both Tln1 fl /fl
Cre + and Tln1 fl /fl Cre 2 mice. Equatorial section of a Tln1 fl /fl Cre + platelet (top
right) shows circumferential microtubule coil (arrow heads). Bars, 1 mm.
(B) Platelet fi brinogen content was determined by Coomassie blue staining
of platelet lysates separated by SDS-PAGE. 5 mg of purifi ed human fi brinogen
served as a marker for the prominent protein band corresponding to
fi brinogen in the platelet lysate samples. Consistent with previous reports
(reference 17 ), fi brinogen content is reduced in platelets from b3 integrin
null mice. (C) Surface expression of integrin aIIb, a2, b1, b3, and P-selectin
was measured by flow cytometry. For P-selectin expres sion, platelets
were incubated with or without PAR4/ADP/epinephrine (1 mm/100 mM/
100 mM) for 5 min before the addition of an FITC-conjugated
JEM VOL. 204, December 24, 2007
BRIEF DEFINITIVE REPORT
Thus, talin is necessary for the activation of ? 2 ? 1 and ? IIb ? 3
integrins in vivo, and platelet talin is absolutely required for
hemostasis because it is necessary for the adhesive functions
of these integrins.
MATERIALS AND METHODS
Generation of mice. Conditional talin1 knockout mice were generated by
introducing loxP sites fl anking coding exons 1 – 4 of the Tln1 gene by gene
targeting. Targeting of the Tln1 locus was confi rmed by Southern blot of
EcoR1-digested genomic DNA hybridized with a 5 ? cDNA probe. Mice
were genotyped by PCR using the following primers indicated in Fig. 1 A :
primer a: 5 ? -aagcaggaacaaaagtaggtctcc-3 ? and primer b: 5 ? -gcatcgtcttcacca-
cattcc-3 ? . Mice homozygous for the Tln1 fl oxed allele ( Tln1 fl /fl ) on a mixed
C57BL/6-Sv129 genetic background were crossed with PF4-Cre (Cre + )
mice on a C57BL/6 background ( 15 ). To obtain mice with talin1-defi cient
platelets, Tln1 fl /fl Cre + males were bred with Tln1 fl /fl Cre ? females. In all
experiments, Tln1 fl /fl Cre + mice were compared with Tln1 fl /fl Cre ? sex-
matched littermates. The generation of ? 3(L746A) mice has been recently
described ( 18 ). Mice were housed in the University of California, San Diego,
animal facility, and experiments were approved by the university ’ s Institu-
tional Animal Care and Use Committee.
SDS-PAGE. For examination of platelet talin protein content, washed
platelets were lysed by adding 1 vol of 2X modifi ed RIPA buff er (300 mM
NaCl, 100 mM Tris, pH 7.4, 0.2% SDS, 2% Triton X-100, 2% sodium de-
oxycholate, 2 mM PMSF, 2 mM NaVO4, 2 mM NaF, 2 mM EDTA, and
complete protease inhibitor; Roche), and samples were clarifi ed by centrifu-
gation at 13,000 g for 10 min at 4 ° C. Laemmli buff er containing 10 mM
DTT was added to 10 ? g of protein lysates, and samples were boiled for
5 min before being separated on 6% Tris-glycine gels (Invitrogen) and stained
with Coomassie blue. For analysis of platelet fi brinogen content, platelets
were lysed in Laemmli buff er in the absence of any reducing agent and sepa-
rated on a 6% Tris-glycine gel. Fibrinogen was identifi ed as a Coomassie
blue – stained band migrating with an apparent molecular mass of 340,000.
Immunofl uorescence. After lysing red blood cells with RBC lysis buff er
(155 mM NH4Cl, 10 mM KHCO 3 , and 0.1 mM EDTA) bone marrow cells
from the femurs of 6 – 9-wk-old mice were fi xed in 3.7% formaldehyde/PBS
and applied to fi brinogen-coated (100 ? g/ml) glass slides by Cytospin prep-
aration (Thermo Fisher Scientifi c). Cells were permeabilized with 0.1% Triton
X-100/PBS containing 5% BSA for 1 h at room temperature and incubated
with talin antibody 8d4 (1:50 dilution; Sigma-Aldrich) overnight at 4 ° C.
After washing with PBS, cells were incubated with 2.5 ? g/ml FITC-conjugated
anti-CD41 (BD Biosciences) and 5 ? g/ml Alexa-568 goat anti – mouse IgG
for 2 h at room temperature. After washing, slides were mounted with coverslips
using Vectashield anti-fade media (Vector Laboratories) and observed on a
Leica DM LS fl uorescence microscope. Images were captured with a spot
color digital camera (National Instruments) using manual exposure settings
that were identical for Tln1 fl /fl Cre + and Tln1 fl /fl Cre ? samples.
Hemostasis assays. The presence of fecal blood was detected with a
guaiac-based hemoccult detection assay (Helena Laboratories) on freshly
obtained stool samples.
Tail bleeding assays were performed by resecting 1 mm of the tail, fol-
lowed by immersion in 37 ° C isotonic saline as described previously ( 17 ). All
experiments were terminated at 10 min by cauterizing the tail.
Platelet isolation and functional assays. Washed platelets were obtained
as described previously ( 30 ). Soluble fi brinogen binding was measured by
incubating platelets for 20 min with 150 ? g/ml FITC-labeled fi brinogen,
followed by fi xation with 1% formaldehyde for 10 min at room tempera-
ture. Specifi c fi brinogen binding was determined by subtracting the amount
of fi brinogen bound in the presence of 5 mM EDTA. Bound fi brinogen
constituents and adhesion receptors. Thus, the observed adhe-
sion defects in Tln1 fl /fl Cre + mice are ascribable to loss of talin-
mediated integrin functions and not a general disruption of
platelet structure and function.
Platelet shape has been proposed to depend on the corti-
cal actin cytoskeleton. Platelet fi lamin and spectrin play im-
portant roles in this cortical cytoskeleton ( 23 ), whereas talin
is cytoplasmic in resting platelets and only recruited to the
cortical cytoskeleton after platelet activation ( 24, 25 ). The
normal shape of the talin-defi cient platelets provides direct
proof that platelet talin makes little if any contribution to
the integrity of the cortical cytoskeleton in resting platelets.
Furthermore, the talin-defi cient platelets contained normal-
appearing ? granules and comparable contents of fi brinogen
and P-selectin to littermate control mice. P-selectin is syn-
thesized by megakaryocytes, whereas the bulk of platelet fi -
brinogen is taken up from the plasma; that uptake depends
on integrin ? IIb ? 3 ( 26 ). The ability of talin-defi cient mega-
karyocytes to package fi brinogen into ? granules is notewor-
thy in light of the defect in activation of integrin ? IIb ? 3 in
these platelets. It is possible that residual talin in early mega-
karyocytes may permit normal fi brinogen uptake; however,
we have also observed normal platelet fi brinogen in the
? 3(L746A) platelets that have a similar defect in ? IIb ? 3
integrin activation (unpublished data), and human platelets
with a ? 3(S752P) mutation also contain normal quantities
of fibrinogen in spite of manifesting defective ? IIb ? 3 acti-
vation ( 27 ). Thus, even though integrin ? IIb ? 3 ligand bind-
ing function is required for normal fibrinogen uptake in
megakaryocytes ( 26 ), activation of the integrin is not needed.
In addition, talin-defi cient platelets increased their surface
expression of P-selectin in response to platelet agonists, indi-
cating that the platelets could respond to the agonists, a
conclusion supported by the normal shape change in the
platelet aggregation tracings. Similarly, the increased surface
P-selectin suggests that ? granule secretion does not depend
on talin; notably, this process is PIP2 dependent ( 28 ), and ta-
lin can recruit and regulate one isoform of PI5 kinase ( 29 ), a
rate-limiting step in PIP2 synthesis. In summary, the talin-
defi cient platelets exhibit normal morphology and respond to
Here, we have shown that platelet talin is essential for
platelet-dependent hemostasis because it is required for the
function and activation of ? 1 and ? IIb ? 3 integrins. Despite
Tln1 fl /fl Cre + mice having normal platelet counts, these ani-
mals exhibited both lethal spontaneous bleeding and resis-
tance to induced thrombosis. These in vivo findings were
ascribable to profound defects in the function of multiple
platelet integrins, as platelets from Tln1 fl /fl Cre + mice failed
to adhere to collagen or to form platelet-rich thrombi ex vivo.
In vitro studies documented the profound impairment of
integrin-mediated adhesion and platelet aggregation in talin1-
defi cient platelets and showed that these platelets were de-
fi cient in the agonist-induced activation of both ? 2 ? 1 and
? IIb ? 3 integrins, despite maintaining the capacity to respond
to the agonists as indicated by surface display of P-selectin.
TALIN IS REQUIRED FOR PLATELET INTEGRIN FUNCTION | Petrich et al.
Submitted: 22 August 2007
Accepted: 7 November 2007
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? 1 integrin activation was measured by the binding of an FITC-labeled
confi rmation-sensitive ? 1 integrin antibody 9EG7 (BD Biosciences), or the con-
fi rmation-insensitive PE-conjugated ? 1 antibody HM ? 1-1 (BD Biosciences).
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FITC – anti-CD61, FITC – anti-CD41 (BD Biosciences), and PE – anti- ? 2
For analysis of static adhesion of platelets to collagen, 96-well plates (Im-
mulon HB2; Dynex Technologies) were coated with 2 ? g of acid-soluble
type I collagen from rat tail (Sigma-Aldrich) in 100 ? l PBS overnight at 4 ° C.
After two washes with PBS and blocking with 5% BSA/PBS for 2 h at room
temperature, 5 × 10 6 washed platelets suspended in platelet incubation buff er
were added to each well and allowed to adhere for 1 h at room temperature.
Wells were then washed three times with platelet incubation buff er, and
adherent platelets were quantifi ed by acid-phosphatase assay ( 18 ). Percent plate-
let adhesion was calculated as the number of adherent platelets relative to the
number of platelets in wells that were not washed (total platelets per well).
Platelet aggregation was performed as described previously ( 18 ) using platelet-
rich plasma (PRP) diluted to a platelet concentration of 3 × 10 8 platelets/ml
with platelet-poor plasma.
Ex vivo adhesion to collagen. Adhesion and thrombus formation in fl ow-
ing blood was performed and analyzed as described previously ( 31, 32 ).
Transmission electron microscopy. Blood was drawn by cardiac puncture
into 0.1 vol of 0.13 M sodium citrate. After adding 1 vol modifi ed Tyrode ’ s
buff er (140 mM NaCl, 2.7 mM KCl, 0.4 mM NaH 2 PO 4 , 10 mM NaHCO 3 ,
5 mM dextrose, and 10 mM Hepes) samples were centrifuged for 5 min at 200 g
at room temperature to obtain PRP. The PRP was incubated for 30 min at
37 ° C before fi xing by the addition of 1 vol of 2X fi xative (3% gluteraldehyde
and 6% paraformaldehyde in 0.2 M cacodylate buff er plus 10% sucrose, pH 7.4)
and incubated for 15 min at room temperature. Platelets were centrifuged at
700 g for 5 min and resuspended and stored overnight in 1X fi xative. Samples
were processed as described previously ( 33 ), and images were obtained with a
JEOL 1200 EX II electron microscope.
Ferric chloride – induced thrombosis. Ferric chloride – induced thrombosis
was performed as described previously ( 18 ) by applying a 1.2 X 1.2 – mm piece
of fi lter paper soaked in 10% ferric chloride to each side of the common
carotid artery of a mouse under isofl urane anesthesia.
Statistics. Statistical analyses of mouse survival and spontaneous death were
performed with ? 2 and Fisher ’ s exact tests, respectively. The statistical signifi -
cance of all other data were determined using Student ’ s t test. A p-value of
< 0.05 was considered statistically signifi cant. All error bars represent standard
error of the mean.
Online supplemental material. Video S1 shows adhesion of fl uorescently
labeled platelets to fi brillar type I collagen in fl owing blood. Time shown is
from the beginning of fl ow over the collagen-coated surface. Video S1 is avail-
able at http://www.jem.org/cgi/content/full/jem.20071800/DC1.
We gratefully acknowledge Timo Meerloo and Dr. Marilyn Farquhar for help
with electron microscopy. We are grateful to Dr. Catrin Pritchard for advice in
This work was supported by grants from the National Institutes of Health
(HL31950 and HL078784), the Cell Migration Consortium, NIH (U54 GM064346),
and the Wellcome Trust (077532). B.G. Petrich is a fellow of the American
The authors have no confl icting fi nancial interests.
JEM VOL. 204, December 24, 2007 Download full-text
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