The calcium sensor STIM1 is an essential mediator of arterial thrombosis and ischemic brain infarction.

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© 2008 Varga-Szabo et al.
The Rockefeller University Press $30.00
J. Exp. Med. Vol. 205 No. 7 1583-1591 www.jem.org/cgi/doi/10.1084/jem.20080302
1583
BRIEF DEFINITIVE REPORT
Platelet activation and aggregation at sites of ves-
sel wall injury is crucial to prevent posttraumatic
blood loss, but it also causes precipitate diseases
such as myocardial infarction and stroke, which
are still leading causes of death and disability in
industrialized countries ( 1 ). Inhibition of platelet
function is an important strategy for the preven-
tion and treatment of myocardial infarction ( 2 )
and, possibly, stroke ( 2, 3 ). Platelet activation is
triggered by subendothelial collagens, throm-
boxane A 2 (TxA 2 ) and ADP released from acti-
vated platelets, and thrombin generated by the
coagulation cascade ( 4 ). Although these agonists
trigger diff erent signaling pathways, all activate
phospholipase Cs (PLCs), leading to the produc-
tion of diacylglycerol (DAG) and inositol 1,4,5-
triphosphate (IP 3 ). IP 3 induces the release of Ca 2+
from the sarcoplasmatic reticulum (SR), which is
thought to trigger the infl ux of extracellular Ca 2+
by a mechanism known as store-operated Ca 2+
entry (SOCE) ( 5, 6 ). In addition, DAG and some
of its metabolites have been shown to induce
non-SOCE ( 7 ). Stromal interaction molecule 1
(STIM1) is an SR/endoplasmic reticulum (ER) –
resident protein necessary for the detection of
ER Ca 2+ depletion and the activation of SOC
channels in T cells ( 8 – 10 ) and mast cells ( 11 ). In
human T cells, the four transmembrane – domain
protein Orai1 (Ca 2+ release – activated channel
modulator) appears to be the predominant SOC
channel ( 12 ), but the C-terminal region of
STIM1 also interacts with other SOC channel
candidates, such as transient receptor potential
channels (TRPCs) 1, 2, and 4 ( 13 ). In platelets,
STIM1 is expressed at high levels ( 14 ) and may
CORRESPONDENCE
Bernhard Nieswandt:
bernhard.nieswandt@
virchow.uni-wuerzburg.de
D. Varga-Szabo and A. Braun contributed equally to this paper.
The online version of this article contains supplemental material.
The calcium sensor STIM1 is an essential
mediator of arterial thrombosis and ischemic
brain infarction
David Varga-Szabo , 1 Attila Braun , 1 Christoph Kleinschnitz , 2
Markus Bender , 1 Irina Pleines , 1 Mirko Pham , 3,5 Thomas Renn é , 4
Guido Stoll , 2 and Bernhard Nieswandt 1,4
1 Rudolf Virchow Center, DFG Research Center for Experimental Biomedicine, 2 Department of Neurology, 3 Department
of Neuroradiology, and 4 Institute of Clinical Biochemistry and Pathobiochemistry, University of W ü rzburg,
97078 W ü rzburg, Germany
5 Department of Neuroradiology, University of Heidelberg, 69120 Heidelberg, Germany
Platelet activation and aggregation are essential to limit posttraumatic blood loss at sites
of vascular injury but also contributes to arterial thrombosis, leading to myocardial infarc-
tion and stroke. Agonist-induced elevation of [Ca 2+ ] i is a central step in platelet activation,
but the underlying mechanisms are not fully understood. A major pathway for Ca 2+ entry in
nonexcitable cells involves receptor-mediated release of intracellular Ca 2+ stores, followed
by activation of store-operated calcium (SOC) channels in the plasma membrane. Stromal
interaction molecule 1 (STIM1) has been identifi ed as the Ca 2+ sensor in the endoplasmic
reticulum (ER) that activates Ca 2+ release – activated channels in T cells, but its role in
mammalian physiology is unknown. Platelets express high levels of STIM1, but its exact
function has been elusive, because these cells lack a normal ER and Ca 2+ is stored in a
tubular system referred to as the sarcoplasmatic reticulum. We report that mice lacking
STIM1 display early postnatal lethality and growth retardation. STIM1-defi cient platelets
have a marked defect in agonist-induced Ca 2+ responses, and impaired activation and
thrombus formation under fl ow in vitro. Importantly, mice with STIM1-defi cient platelets
are signifi cantly protected from arterial thrombosis and ischemic brain infarction but have
only a mild bleeding time prolongation. These results establish STIM1 as an important
mediator in the pathogenesis of ischemic cardio- and cerebrovascular events.

1584 STIM1 AND THROMBOSIS | Varga-Szabo et al.
Mice heterozygous for the STIM1-null mutation developed
normally, whereas a majority ( � 70%) of mice lacking STIM1
( Stim1 � / � ) died within a few hours after birth. Marked cyano-
sis was noted before death, suggesting a cardiopulmonary
defect. Surviving Stim1 � / � mice exhibited marked growth re-
tardation, achieving � 50% of the weight of wild-type litter-
mates at 3 and 7 wk of age ( Fig. 1, A and B ). Western blot
analyses confi rmed the absence of STIM1 in platelets ( Fig. 1 C ,
top) and other tissues (not depicted). Blood platelet counts
( Fig. 1 D ), mean platelet volume, and expression levels of ma-
jor platelet surface receptors, including glycoprotein (GP) Ib-
V-IX, GPVI, CD9, and � 1 and � 3 integrins (not depicted)
contribute to SOCE by interacting with TRPC1 ( 15 ). We re-
cently reported that mice expressing an activating EF-hand
mutant of STIM1 have elevated [Ca 2+ ] i levels in platelets, mac-
rothrombocytopenia, and a bleeding disorder, indicating a role
for STIM1-dependent SOCE in platelet function ( 14 ). The
importance of SOCE for platelet activation, hemostasis, and
thrombosis, however, remains unknown, and the mechanisms
underlying the process are not defi ned.
RESULTS AND DISCUSSION
To address the function of STIM1 in vivo, the Stim1 gene was
disrupted in mice by insertion of an intronic gene trap cassette.
Figure 1. Defective SOCE in Stim1-defi cient platelets. (A) 5-wk-old wild-type and Stim1 � / � littermates. (B) Body weights of wild-type (+/+) and
Stim1 � / � ( � / � ) mice. Values are mean ± SD. ***, P < 0.001. (C) Western blot analyses of platelet lysates from mice with the indicated genotypes (top) or
of mice transplanted with the indicated bone marrow (bottom). Stim1 was assessed using an antibody that can recognize the N-terminal region of the
protein (GOK/Stim1; reference 11 ). An antibody to � 3 integrin served as control. Results from two individuals per group are shown. (D) Peripheral platelet
counts in wild-type and Stim1 � / � mice. Values are mean ± SD. (E) Fura-2 – loaded platelets were stimulated with 5 μ M TG for 10 min, followed by the
addition of extracellular Ca 2+ and monitoring of [Ca 2+ ] i . Representative measurements (left) and maximal increase in intracellular Ca 2+ concentrations
compared with baseline levels ( � [Ca 2+ ] i ) ± SD ( n = 4 mice per group) before and after addition of 1 mM Ca 2+ (right) are shown. **, P < 0.01; ***, P < 0.001.

JEM VOL. 205, July 7, 2008 1585
BRIEF DEFINITIVE REPORT
low concentrations of these agonists (not depicted). In con-
trast, responses to collagen and CRP ( Fig. 2 C ) and the strong
GPVI agonist convulxin (not depicted) were signifi cantly di-
minished. The activation defect was confi rmed by fl ow cyto-
metric analysis of integrin � IIb � 3 activation using the JON/A-PE
antibody, and of degranulation-dependent P-selectin surface
exposure ( Fig. 2 D ). Therefore, loss of STIM1-dependent
SOCE impairs GPVI-induced integrin activation and degran-
ulation, whereas G protein – coupled agonists are still able to
induce normal activation in Stim1 � / � platelets in these assays,
despite the defect in [Ca 2+ ] i signaling.
In vivo, platelet activation on the extracellular matrix or a
growing thrombus occurs in fl owing blood, where locally pro-
duced soluble mediators are rapidly cleared. Under these con-
ditions, reduced potency of platelet activators may become
limiting, particularly at the high fl ow rates found in arteries and
arterioles. Therefore, we analyzed the ability of Stim1 � / � plate-
lets to form thrombi on collagen-coated surfaces in a whole-
blood perfusion system ( 16 ). Under high shear conditions
(1,700 s � 1 ), wild-type platelets adhered to collagen fi bers and
formed aggregates within 2 min that consistently grew into
large thrombi by the end of the perfusion period ( Fig. 2 E ). In
sharp contrast, Stim1 � / � platelets exhibited reduced adhesion,
and three-dimensional growth of thrombi was markedly im-
paired. As a consequence, the surface area covered by platelets
and the total thrombus volume were reduced � 42 and � 81%,
respectively. Similar results were obtained at intermediate shear
rates (1,000 s � 1 ; not depicted). These fi ndings indicate that
STIM1-mediated SOCE is required for effi cient platelet acti-
vation on collagen and on the surface of growing thrombi un-
der conditions of high shear.
Unstable arterial thrombi in Stim1 � / � mice
Because platelet aggregation may contribute to pathologi-
cal occlusive thrombus formation, we studied the eff ects of
STIM1 defi ciency on ischemia and infarction by in vivo
fl uorescence microscopy after ferric chloride – induced mes-
enteric arteriole injury. In all wild-type chimeras, the forma-
tion of small aggregates was observed � 5 min after injury,
with progression to complete vessel occlusion in 8 out of
10 mice within 30 min (mean occlusion time = 16.5 ± 2.8
min; Fig. 3, A – C ). In contrast, aggregate formation was sig-
nifi cantly delayed in � 50% of the Stim1 � / � chimeras (10.6 ±
5.8 min in wild-type and 17.1 ± 7.3 min in Stim1 � / � chi-
meras until the fi rst thrombus > 20 μ m in diameter appeared;
P < 0.05), and formation of stable thrombi was almost com-
pletely abrogated. This defect was caused by the release of
individual platelets from the surface of the thrombi (Videos
1 and 2, available at http://www.jem.org/cgi/content/
full/jem.20080302/DC1) and not by embolization of large
thrombus fragments. Blood fl ow was maintained throughout
the observation period in 9 out of 10 vessels, demonstrating a
crucial role for STIM1 during occlusive thrombus formation.
This was confi rmed in a second arterial thrombosis model
in which the abdominal aorta was mechanically injured and
blood fl ow was monitored with an ultrasonic fl ow probe.
were normal, indicating that STIM1 is not essential for mega-
karyopoiesis or platelet production. Similarly, no diff erences
were found in red blood cell counts, hematocrit, or the acti-
vated partial thromboplastin time, a method for the assessment
of plasma coagulation ( Table I ). To determine if STIM1 has a
role in platelet SOCE, we induced SOC infl ux in wild-type
and Stim1 � / � platelets with the SR/ER Ca 2+ ATPase (SERCA)
pump inhibitor thapsigargin (TG). Interestingly, TG-induced
Ca 2+ store release was reduced � 60% in Stim1 � / � platelets
compared with wild-type controls ( Fig. 1 E ). Furthermore,
subsequent TG-dependent SOC infl ux was almost completely
absent in Stim1 � / � cells ( Fig. 1 E ). This demonstrates for the
fi rst time that STIM1 is essential for SOCE in platelets and
suggests that STIM1-dependent processes contribute to the
regulation of Ca 2+ store content in these cells.
Defective SOC infl ux in Stim1 � / � platelets
Because of the early mortality and pronounced growth retar-
dation in Stim1 � / � mice, all subsequent studies were per-
formed with lethally irradiated wild-type mice transplanted
with Stim1 � / � or wild-type bone marrow. 4 wk after trans-
plantation, platelet counts were normal and STIM1 defi ciency
in platelets was confi rmed by Western blotting ( Fig. 1 C , bot-
tom). To determine the signifi cance of defective SOCE for
agonist-induced platelet activation, we assessed changes in
[Ca 2+ ] i in response to ADP, thrombin, a collagen-related pep-
tide (CRP) that stimulates the collagen receptor GPVI ( Fig. 2,
A and B ), and the TxA 2 analogue U46619 (not depicted). Ca 2+
release from intracellular stores was reduced in Stim1 � / � plate-
lets compared with control for all agonists, indicating reduced
Ca 2+ levels in stores in Stim1 � / � cells. In the presence of extra-
cellular calcium, Ca 2+ infl ux was dramatically reduced in
Stim1 � / � platelets. Thus, STIM1-dependent SOCE is a crucial
component of the Ca 2+ signaling mechanism in platelets for all
major agonists, and non-SOCE makes only a minor contribu-
tion, at least under the conditions tested.
STIM1 in platelet activation and thrombus formation
To test the functional consequences of this defect, we per-
formed ex vivo aggregation studies. Stim1 � / � platelets aggre-
gated normally to the G protein – coupled agonists ADP,
thrombin ( Fig. 2 C ), and U46619 (not depicted), even at very
Table I. Hematology and hemostasis in Stim1 � / � chimeras
Stim1 +/+ Stim1 � / �
Erythrocytes 8,450 ± 139 8,250 ± 264
HCT (%) 40.8 ± 0.4 41.7 ± 1.9
aPTT (sec) 37.7 ± 5.1 38.7 ± 3.1
PT (sec) 9.4 ± 0.5 9.8 ± 0.7
TCT (sec) 19.2 ± 2.6 21.8 ± 1
Fibrinogen 2.2 ± 0.1 2.8 ± 0.6
Erythrocyte counts per nanoliter and coagulation parameters for control and
Stim1 � / � chimeras are shown. Values are mean ± SD of fi ve mice for each genotype.
aPTT, activated partial thromboplastin time; HCT, hematocrit; PT, prothrombin time;
TCT, thrombin clotting time.

1586 STIM1 AND THROMBOSIS | Varga-Szabo et al.
Although 10 out of 11 control chimeras formed irreversible
occlusions within 16 min (mean occlusion time = 4.4 ± 4.1
min), occlusive thrombus formation did not occur in 6 out of
8 Stim1 � / � chimeras during the 30-min observation period
(P < 0.005; Fig. 3, D and E ). These results demonstrate that
STIM1 is required for the propagation and stabilization of
platelet-rich thrombi in small and large arteries, irrespective
of the type of injury.
To test whether the defect in Stim1 � / � platelets impaired
hemostasis, we measured tail bleeding times. Although bleed-
ing stopped in 28 out of 30 (93.3%) control mice within
10 min, bleeding was highly variable in Stim1 � / � chimeras,
with 11 out of 31 (35.5%) mice bleeding for > 10 min (P <
0.02; Fig. 3 F ). These results show that STIM1 is required
for normal hemostasis.
STIM1 is an essential mediator of ischemic brain infarction
Ischemic stroke is the third leading cause of death and disability
in industrialized countries ( 17 ). Although it is well established
that microvascular integrity is disturbed during cerebral ische-
mia ( 18 ), the signaling cascades involved in intravascular throm-
bus formation in the brain are poorly understood. To determine
the importance of STIM1-dependent SOCE in this process,
we studied the development of neuronal damage in Stim1 � / �
chimeras after transient cerebral ischemia in a model that de-
pends on thrombus formation in microvessels downstream
from a middle cerebral artery (MCA) occlusion ( 19 ). To initi-
ate transient cerebral ischemia, a thread was advanced through
the carotid artery into the MCA and allowed to remain for 1 h
(transient MCA occlusion [tMCAO]), reducing regional cere-
bral fl ow by > 90% ( 3 ). In Stim1 � / � chimeras, infarct volumes
24 h after reperfusion, as assessed by 2,3,5-triphenyltetrazolium
chloride (TTC) staining, were reduced to < 30% of the infarct
volumes in control chimeras (17 ± 4.4 vs. 62.9 ± 19.3 mm 3 ;
P < 0.0001; Fig. 4 A ). Reductions in infarct size were function-
ally relevant, as the Bederson score assessing global neurological
function (1.86 ± 0.48 vs. 3.07 ± 0.35, respectively; P < 0.0001)
and the grip test, which specifi cally measures motor function
and coordination (3.71 ± 0.39 vs. 2 ± 0.65, respectively; P <
0.0001), were signifi cantly better in Stim1 � / � chimeras com-
pared with controls ( Fig. 4, B and C ). Serial magnetic reso-
nance imaging (MRI) on living mice was used to confi rm the
protective eff ect of STIM1 defi ciency on infarct development.
Hyperintense ischemic infarcts on T2-weighted (T2-w) MRI
Figure 2. Defective agonist-induced Ca 2+ signaling and aggregate
formation under fl ow in Stim1 � / � platelets. Fura-2 – loaded wild-type
(black line) or Stim1 � / � (gray line) platelets were stimulated with 0.1 U/ml
thrombin, 10 μ M ADP, or 10 μ g/ml CRP in the presence of extracellular
1 mM EGTA or 0.5 mM Ca 2+ , and [Ca 2+ ] i was monitored. Representative
measurements (A) and maximal increase in intracellular Ca 2+ concentra-
tions compared with baseline levels before stimulus ( � [Ca 2+ ] i ) ± SD ( n =
4 mice per group; B) are shown. (C) Impaired aggregation of Stim1 � / �
platelets (gray lines) in response to CRP and collagen but not ADP and
thrombin (recording time = 10 min). (D) Flow cytometric analysis of inte-
grin � IIb � 3 activation (binding of JON/A-PE; left) and degranulation-
dependent P-selectin exposure (right) in response to 0.1 U/ml thrombin,
10 μ M ADP, 10 μ g/ml CRP, and 1 μ g/ml convulxin. Results are means ± SD
( n = 6 mice per group). (E) Stim1 � / � platelets in whole blood fail to form
stable thrombi when perfused over a collagen-coated (0.2 mg/ml) surface
at a shear rate of 1,700 s � 1 . (top) Representative phase-contrast images.
(bottom) Mean surface coverage (left) and relative platelet deposition as
measured by the integrated fl uorescent intensity per square millimeter
(right) ± SD ( n = 4 mice). ***, P < 0.001. Bar, 100 μ m.

JEM VOL. 205, July 7, 2008 1587
BRIEF DEFINITIVE REPORT
Moreover, no intracranial hemorrhage was detected on T2-w
gradient echo images, a highly sensitive MRI sequence for the
detection of blood ( Fig. 4 D ), indicating that STIM1 defi ciency
in hematopoietic cells is not associated with an increase in
in Stim1 � / � chimeras were < 10% of the size of infarcts in
control chimeras 24 h after tMCAO (P < 0.0001; Fig. 4 D ).
Importantly, infarct volume did not increase between days 1 and
7, indicating a sustained protective eff ect for STIM1 defi ciency.
Figure 3. In vivo analysis of thrombosis and hemostasis. (A – C) Mesenteric arterioles were treated with FeCl 3 , and adhesion and thrombus formation
of fl uorescently labeled platelets were monitored by in vivo video microscopy. Representative images (A), the time to appearance of the fi rst thrombus
> 20 μ m (B), and the time to vessel occlusion (C) are shown. Each symbol represents one individual. The asterisk in A indicates occlusion of the vessel.
Horizontal bars in B represent means. Bar, 50 μ m. (D and E) The abdominal aorta was mechanically injured, and blood fl ow was monitored for 30 min or
until complete occlusion occurred (blood fl ow stopped > 5 min). (D) Representative cross sections of the abdominal aorta of mice with wild-type or
Stim1 � / � platelets 30 min after injury. Bar, 250 μ m. (E) Time to vessel occlusion. Each symbol represents one individual. (F) Tail bleeding times in wild-type
and Stim1 � / � chimeras. Each symbol represents one individual. Videos 1 and 2 are available at http://www.jem.org/cgi/content/full/jem.20080302/DC1.