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INTRODUCTION |
Platelet activation via cleavage of the protease-activated
receptors by thrombin leads to formation of a platelet-rich thrombus, which can severely impair blood flow to vital organs, including the
brain, heart, lungs, and kidneys (1). Since the serine protease
thrombin is the most potent activator of platelet-mediated coagulation,
a better understanding of the signaling pathways regulating
thrombin-induced platelet activation may provide the basis for new and
effective strategies for prevention and/or treatment of thromboembolism.
Recent studies have revealed important roles for several
protein-tyrosine kinases in platelet physiology (2-5). Notably, JAK3,1 a member of the Janus
family of protein-tyrosine kinases (6-8), was shown to be
constitutively active in human platelets, but its potential physiologic
role in agonist-induced platelet activation or aggregation remains
unknown. The purpose of this study was to examine the role of JAK3 in
thrombin-induced platelet activation and aggregation. Here we show
genetic and biochemical evidence that implicates JAK3 as one of the
regulators of platelet function. Furthermore, our study uniquely
identifies a small molecule chemical inhibitor of JAK3 as a novel
antiplatelet agent for prevention of potentially fatal thromboembolic events.
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EXPERIMENTAL PROCEDURES |
Tyrosine Kinase Inhibitors--
The JAK3 inhibitor
WHI-P131 (4-(4'-hydroxylphenyl)amino-6,7-dimethoxyquinazoline) was
rationally designed using a homology model for the kinase domain of
JAK3, synthesized, and characterized as previously described in detail
(9, 10). WHI-P131 inhibits JAK3, but not JAK1 or JAK2, the ZAP/SYK
family tyrosine kinase SYK, the TEC family tyrosine kinase BTK, the SRC
family tyrosine kinase LYN, or the receptor family tyrosine kinase
insulin receptor kinase (9, 10). The physical data for WHI-P131 were as
follows: m.p. 245.0-248.0 °C; 1H NMR
(Me2SO-d6)
11.21 (s, 1H, -NH),
9.70 (s, 1H, -OH), 8.74 (s, 1H, 2-H), 8.22 (s, 1H, 5-H), 7.40 (d, 2H,
J = 8.9 Hz, 2',6'-H), 7.29 (s, 1H, 8-H), 6.85 (d, 2H,
J = 8.9 Hz, 3',5'-H), 3.98 (s, 3H, -OCH3),
and 3.97 (s, 3H, -OCH3); IR (KBr) 3428, 2836, 1635, 1516, 1443, and 1234 cm
1; gas chromatography/mass
spectrometry, m/z 298 (M+ + 1, 100),
297 (M+, 27), and 296 (M+
1, 12); and
analyzed
(C16H15N3O3·HCl), C,
H, N. The x-ray crystallographic data for WHI-P131 were recently
published (10). The physical data for the parent compound, WHI-P258
(4-(phenyl)-amino-6,7-dimethoxyquinazoline), were as follows: yield of
88.26%; m.p. 258.0-260.0 °C; 1H NMR
(Me2SO-d6)
11.41 (s, 1H, -NH),
8.82 (s, 1H, 2-H), 8.32 (s, 1H, 5-H), 7.70-7.33 (m, 5H,
2',3',4',5',6'-H), 7.36 (s, 1H, 8-H), 4.02 (s, 3H, -OCH3),
and 4.00 (s, 3H, -OCH3); IR (KBr) 2852, 1627, 1509, 1434, and 1248 cm
1; gas chromatography/mass
spectrometry, m/z 282 (M+ + 1, 11), 281 (M+, 55), 280 (M+
1, 100), 264 (16), and 207 (9); analyzed
(C16H15N3O2), C, H, N.
Mice--
Control C57BL/6 mice were purchased from Taconic Farms
Inc. (Germantown, NY). A breeder pair of Jak3
knockout mice (Jak3
/
,
C57BL/6 × 129/Sv, H-2b (11), A011 (male) and A1038
(female)) were obtained from Dr. J. N. Ihle (St. Jude
Children's Research Hospital, Memphis, TN). These mice were created by
the targeted disruption of the Jak3 gene through homologous
recombination using the hygromycin resistance gene (Hyg)
cassette (11). These founder
Jak3
/
mice were bred with
C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME), and the offspring of
the F1 generation were back-crossed to C57BL/6 mice. After three
generations of back-crossing to C57BL/6 mice, the offspring were
intercrossed to produce homozygous
Jak3
/
and wild-type
Jak3+/+ mice. The genotype of mice was confirmed
by multiplex polymerase chain reaction (PCR) tests. In brief, a
0.5-inch (1.27-cm) tail tissue section was taken from each mouse and
digested at 55 °C in 600 µl of lysis buffer (50 mM
Tris (pH 8.0), 100 mM EDTA, 100 mM NaCl, and
1% SDS) with 50 µl of proteinase K (10 mg/ml). Genomic DNA was
purified by phenol/chloroform extractions and ethanol precipitation
(12). Three primers were employed in the PCR tests: the 30-base primer
Jak3-S (5'-ACC TAG TCC CCA GCT TGG CTG TCA CTT GGG-3'), the
30-base primer Jak3-AS (5'-CAA AGC GGT GAC ATG TCT CCA GCC
CAA ACC-3'), and the 30-base primer Jak3-Hyg
(5'-ATG GTT TTT GGA TGG CCT GGG CAT GGA CCG-3') (Biosynthesis
Inc., Lewisville, TX). The
Jak3-AS/Jak3-Hyg PCR primer pair
yielded a 620-bp mutant PCR product in tissues from
Jak3
/
mice. The
Jak3-AS/Jak3-S PCR primer pair yielded a 720-bp
wild-type PCR product in tissues from homozygous
Jak3+/+ or heterozygous
Jak3+/
mice. The homozygous
Jak3+/+ genotype was documented by a single
720-bp PCR product, and the homozygous
Jak3
/
genotype was documented by
a single 620-bp PCR product (Fig. 1). The
heterozygous Jak3+/
genotype was documented by
the presence of both 720- and 620-bp PCR products. Each 50-µl PCR
medium consisted of 1× PCR buffer II containing 2.5 mM
MgCl2 (AmpliTaq Gold kit, PerkinElmer Life Sciences), 0.2 mM dNTP (Roche Molecular Biochemicals), 0.4 µM each primer, 6% Me2SO, and 2.5 units of
AmpliTaq Gold enzyme. The PCR conditions were 94 °C for 10 min, 30 cycles (94 °C for 1 min, 57 °C for 1 min, 72 °C for 1 min with
a 5 sec extension), then 72 °C for 10 min in a Touchdown
thermocycler (Hybaid, Potomac, MD). The PCR products were cloned
into the original TA cloning vector (Invitrogen, Carlsbad, CA).
Sequence analysis was accomplished by ThermoSequenase PCR (Amersham
Pharmacia Biotech) using Cy5-labeled T3 and T7 sequencing primers
(Integrated DNA Technologies, Inc., Coralville, IA). DNA
sequences were analyzed against published Jak3 DNA sequence
using Lasergene software (DNASTAR, Madison, WI).

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Fig. 1.
Jak3 knockout mice. The
homozygous wild-type Jak3+/+ genotype was
documented by detection of a single 720-bp multiplex PCR product, and
the homozygous knockout Jak3 /
genotype was documented by detection of a single 620-bp multiplex PCR
product. Neg Con, negative control.
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Platelet Aggregation Assays--
Platelet-rich plasma (PRP) was
purchased from the Memorial Blood Bank (Minneapolis, MN) and used
according to the guidelines of the Parker Hughes Institute Human
Subjects Committee. The PRP samples were treated with varying
concentrations of WHI-P131 for 20 min at 37 °C. Control PRP samples
were treated with vehicle alone. The treated PRP samples were diluted
1:4 with sterile normal saline, and platelets were stimulated with
thrombin (0.1 unit/ml; Chronolog Inc., Philadelphia, PA) under
stirring. Platelet aggregation was monitored using the Born
method of turbidimetric aggregation in a Whole Blood Platelet
Aggregometer (Model 560 dual chamber instrument, Chronolog Inc.) for 5 min. When the aggregating agent is added to the platelet-rich plasma
according to this method, the formation of the large platelet aggregate
is accompanied by a clearing of the plasma. The IC50 values
for WHI-P131-mediated inhibition of agonist-induced platelet
aggregation were calculated by nonlinear regression analysis using
GraphPAD Prism Version 2.0 software. In impedance aggregation studies,
the increase in electrical impedance caused by adherence and
aggregation of platelets on an electrode is measured. For these
studies, blood was extracted from Jak3 knockout and control
C57BL/6 mice by eye bleeds into tubes containing 15% (v/v) buffer
containing 0.8% (w/v) citric acid, 2.2% (w/v) trisodium citrate, and
2.45% (w/v) dextrose and mixed gently to prevent coagulation. Citrated
blood was diluted with an equal volume of saline and prewarmed at
37 °C for 5 min. The platelet agonist thrombin (0.1 unit/ml) was
added at 1 min to induce aggregation. Thrombin-induced platelet
aggregation was measured in wild-type and knockout mice
(n = 3 for each type) in the whole blood platelet aggregometer.
Immunoprecipitation and Western Blot Analysis--
Platelets
were isolated from PRP as previously described (13) and resuspended at
a concentration of 3 × 109 cells/ml in modified
Tyrode's buffer (137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.5 mM glucose, 3.3 mM NaH2PO4, and 3.8 mM
Hepes (pH 7.4)). Platelets were incubated with the indicated
concentrations of WHI-P131 or vehicle (PBS supplemented with 1%
Me2SO) for 30 min at 37 °C. Platelets were then
stimulated at 37 °C with 2 or 10 µg/ml collagen or 0.1 unit/ml
thrombin. Stimulation was stopped, and platelets were lysed at the
indicated time points by adding ice-cold 3× Triton X-100 lysis buffer
(150 mM NaCl, 15 mM EGTA, 3% Triton X-100, 3%
sodium deoxycholate, 0.3% SDS, 3 mM phenylmethylsulfonyl fluoride, 3 mM Na3VO4, 60 µg/ml
leupeptin, 60 µg/ml aprotinin, and 50 mM Tris-HCl (pH
7.4)) and incubating for 1 h on ice. Following removal of the
membranous fraction by centrifugation (12,000 × g, 30 min), the samples were subjected to immunoprecipitation utilizing
antibodies raised against JAK3 and STAT1 (E23, Santa Cruz
Biotechnology, Santa Cruz, CA) or STAT3 (Transduction Laboratories, Lexington, KY) (14). E23 recognizes both the
(p91) and
(p84) isoforms of STAT1. Similarly, the anti-STAT3 antibody recognizes both
the
(p92) and
(p83) isoforms of STAT3.
Immunoprecipitations, immune complex protein kinase assays, and
immunoblotting on polyvinylidene difluoride membranes (Millipore Corp.,
Bedford, MA) using the ECL chemiluminescence detection system (Amersham
Pharmacia Biotech) were conducted as described previously (9, 14-17).
For immunoblotting, we used antibodies against phosphotyrosine, JAK3,
STAT1, STAT3, phospho-STAT1 (recognizes both STAT1
and STAT1
),
and phospho-STAT3 (recognizes both STAT3
and STAT3
) (New England
Biolabs Inc., Beverly, MA). Horseradish peroxidase-conjugated sheep
anti-mouse and donkey anti-rabbit secondary antibodies were purchased
from Transduction Laboratories. Horseradish peroxidase-conjugated sheep
anti-goat antibodies were purchased from Santa Cruz Biotechnology.
Following electrophoresis, kinase gels were dried onto Whatman No.
3 Filter Paper and subjected to phosphorimaging on a molecular
imager (Bio-Rad) as well as autoradiography on film. Similarly, all
chemiluminescent JAK3 Western blots were subjected to three-dimensional
densitometric scanning with the molecular imager and an imaging
densitometer using Molecular Analyst/Macintosh Version 2.1 software
following the specifications of the manufacturer (Bio-Rad). A JAK3
kinase activity index was determined by comparing the ratios of the
kinase activity in PhosphorImager units (PIU) and density of the
protein bands in densitometric scanning units (DSU) with those of the base-line sample using the following formulas: activity index = (PIU of kinase band/DSU of JAK3 protein band)test sample; and stimulation index = (PIU of kinase band/DSU of JAK3 protein band)test sample/(PIU of kinase band/DSU of JAK3 protein band)base-line control sample.
Cytoskeletal Fractionation--
Platelets (1 × 108/ml) were treated with WHI-P131 (100 µM,
30 min, 37 °C) or vehicle (1% Me2SO) and stimulated
with thrombin (0.1 unit/ml) or collagen (10 µg/ml). Isolation of the
cytoplasmic and Triton X-100-soluble and -insoluble fractions was
performed as previously described (18, 19). Fractions were analyzed by
Western blot analysis utilizing antibodies raised against JAK3, STAT1,
SYK (Santa Cruz Biotechnology), STAT3, tubulin, and actin (Sigma).
Serotonin Release--
Platelet samples were prepared as
previously described (20). Release of serotonin from thrombin (0.1 unit/ml)-stimulated platelets was measured using a serotonin detection
kit (Immunotech, Marseilles, France) according to the manufacturer's
specifications. Sonicated platelets were used for measurement of the
total serotonin content of platelets.
High-resolution Low-voltage Scanning Electron Microscopy
(HR-LVSEM)--
HR-LVSEM was utilized for topographical imaging of the
platelet surface membrane as previously reported (21). Aliquots of human platelets were incubated with 100 µM WHI-P131 or
vehicle alone for 30 min. Treated platelets were then stimulated with thrombin (0.1 unit/ml) for 10 s. 3% glutaraldehyde was added to stop the reaction. Samples were prepared for HR-LVSEM as previously described (21) and analyzed using a Hitachi S-900 SEM instrument at an
accelerating voltage of 2 kV.
Transmission Electron Microscopy (TEM)--
Aliquots of human
platelets were incubated with 100 µM WHI-P131 or vehicle
alone for 30 min and then stimulated with thrombin (0.1 unit/ml) for
10 s. Samples were prepared for TEM as previously described (22).
Briefly, 0.1% glutaraldehyde was added to stop the reaction. Following
a brief centrifugation, the sample pellets were layered with 3%
glutaraldehyde for 40 min at room temperature. The samples were then
post-fixed in 1% OsO4 for 1 h at 4 °C, rinsed three times in distilled water at room temperature, and dehydrated in a
graded ethanol series (25, 50, 75, 90, 95, and 100%) and 100%
propylene oxide. The samples were embedded in Embed 812 (Electron Microscopy Sciences, Fort Washington, PA). Silver sections were picked
up on mesh grids and stained for 10 min in 1% uranyl acetate and 70%
ethanol and for 10 min in Reynold's lead citrate. Sections were viewed
in a Jeol 100× electron microscope at 60 kV. True magnifications were
determined by photographing a calibration grid at each magnification
step on the microscope and using this scale to determine final print enlargements.
Measurement of Bleeding and Clotting Times in Mice--
Mice
were treated intravenously with a single intraperitoneal bolus
injection of 200 µl of vehicle (PBS supplemented with 10%
Me2SO) or varying doses of WHI-P131 in 200 µl of vehicle
30 min prior to and with an intravenous bolus of the same dose 5 min
before the bleeding time measurements. Mice were placed in a tube
holder, and tail bleeding was performed with a 2-mm cut from the
protruding tail tip. The tail was placed vertically into 10 ml of
normal saline in a 37 °C water bath, and bleeding times were
determined at 30 min post-intraperitoneal injection of WHI-P131 as
previously described (1).
Thromboplastin-induced Thromboembolism Model--
4-6-week-old
male ICR mice were treated intravenously with 200 µl of vehicle (PBS
supplemented with 10% Me2SO) or varying doses of WHI-P131
in 200 µl of vehicle administered in two bolus injections 30 min
(intraperitoneal bolus) and 5 min (intravenous bolus) prior to the
thromboplastin challenge. The mice were challenged with 25 mg/kg
thromboplastin (Sigma) via an intravenous bolus injection into the tail
vein as previously described (23). At the time of
thromboembolism-related death after the thromboplastin injection or
elective sacrifice at 48 h using ketamine/xylazine, all mice were
perfused with PBS, followed by 4% phosphate-buffered Formalin. PBS and
Formalin were pumped through the left ventricle of the heart and
allowed to exit through a 3-mm incision through the anterior wall of
the right ventricle. During necropsy, several selected tissues (brain,
heart, liver, and lungs) were harvested, fixed in 10% neutral buffered
Formalin, dehydrated, and embedded in paraffin by routine methods for
histopathological examination. Glass slides with affixed 6-µm tissue
sections were prepared and stained with hematoxylin and eosin or
Masson's trichrome.
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RESULTS |
JAK3-dependent Tyrosine Phosphorylation of STAT1 and
STAT3 Proteins in Thrombin-stimulated Platelets--
We first set out
to examine the effects of thrombin stimulation on the phosphorylation
status of STAT1 and STAT3 proteins in platelets from wild-type C57BL/6
mice. Notably, treatment of platelets with 0.1 unit/ml thrombin induced
tyrosine phosphorylation of the
isoform (p91) of STAT1 (Fig.
2A) and the
isoform (p83) of STAT3 (Fig. 2B). Thrombin-induced tyrosine
phosphorylation of STAT1 and STAT3 was JAK3-dependent
because thrombin stimulation failed to induce tyrosine phosphorylation
of these STAT proteins in JAK3-deficient platelets from Jak3
knockout mice (Jak3
/
).
Similarly, stimulation of human platelets with 0.1 unit/ml thrombin
enhanced the tyrosine phosphorylation of STAT1 and STAT3 proteins (Fig.
2, C and D). Pretreatment of human platelets with the JAK3 inhibitor WHI-P131 (100 µM) decreased the
base-line enzymatic activity of constitutively active JAK3 by 81% as
measured by autophosphorylation (Fig. 2E) and abolished the
thrombin-induced tyrosine phosphorylation of STAT1 and STAT3
(F and G). It is noteworthy that at 60 s
after thrombin stimulation, the enzymatic activity of JAK3 was reduced by 41% (activity index: 0.35 = 59% of the activity index before thrombin stimulation) (Fig. 2E, first and
second lanes). The significance of this observation is
currently unknown.

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Fig. 2.
JAK3-dependent tyrosine
phosphorylation of STAT1 and STAT3 in thrombin-stimulated
platelets. A and B, whole cell lysates from
control and JAK3-deficient mouse platelets stimulated with 0.1 unit/ml
thrombin were subjected to Western blot analysis utilizing antibodies
raised against phosphorylated STAT1 (A, upper
panels), STAT1 (A, lower panels),
phosphorylated STAT3 (B, upper panels), and STAT3
(B, lower panels). C, STAT1 was
immunoprecipitated from human platelets stimulated with 0.1 unit/ml
thrombin. The immunoprecipitates were subjected to Western blot
analysis utilizing antibodies raised against phosphotyrosine
(PO4Y; upper panel) and STAT1
(lower panel). D, STAT3 was immunoprecipitated
from human platelets that were stimulated with 0.1 unit/ml thrombin.
The immunoprecipitates were subjected to Western blot analysis
utilizing antibodies raised against phosphorylated STAT3 (upper
panel) and STAT3 (lower panel). E, JAK3 was
immunoprecipitated from platelets that were stimulated with thrombin
(0.1 unit/ml) after treatment with vehicle (1% dimethyl sulfoxide
(DMSO) in phosphate-buffered saline) or WHI-P131 (100 µM). The immunoprecipitates were subjected to
quantitative kinase assays (upper panel) and immunoblotting
with an anti-JAK3 antibody (lower panel) as described under
"Experimental Procedures." F and G, human
platelets were pretreated with vehicle or WHI-P131 (100 µM) prior to thrombin stimulation. In F, STAT1
was immunoprecipitated from platelets that were stimulated with 0.1 unit/ml thrombin. The immunoprecipitates were subjected to Western blot
analysis utilizing antibodies raised against phosphotyrosine
(upper panel) and STAT1 (lower panel). In
G, whole cell lysates from platelets stimulated with 0.1 unit/ml thrombin were subjected to Western blot analysis utilizing
antibodies raised against phosphorylated STAT3 (upper panel)
and STAT3 (lower panel). PIU, PhosphorImager
units; DSU, densitometric scanning units.
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Effects of the JAK3 Inhibitor WHI-P131 on Thrombin-induced Platelet
Activation--
Activation of platelets after exposure to thrombin is
associated with actin polymerization and rapid translocation of the tyrosine kinase SYK (24, 25) as well as tubulin to the Triton X-100-insoluble fraction that is associated with the actin filament network. As shown in Fig. 3A,
Western blot analysis of the cytoplasmic and Triton X-100-soluble and
-insoluble fractions from unstimulated platelets confirmed the presence
of abundant amounts of actin in the Triton X-100-insoluble fraction and
of SYK as well as tubulin in the Triton X-100-soluble (but not
-insoluble) fraction. Within 60 s after thrombin stimulation, a
significant amount of SYK and tubulin translocated to the
membrane-associated cytoskeleton as evidenced by the Western blot
detection of SYK and tubulin in the actin-containing Triton
X-100-insoluble fractions. Notably, thrombin stimulation also induced
the translocation of JAK3, STAT1
/
, and STAT3
proteins to the
Triton X-100-insoluble fraction. As shown in Fig. 3B,
pretreatment of platelets with the JAK3 inhibitor WHI-P131 prevented
the thrombin-induced relocalization of SYK, tubulin, JAK3, STAT1, and
STAT3 to the Triton X-100-insoluble fractions.

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Fig. 3.
Effects of WHI-P131 on thrombin-induced
translocation of Triton X-100-soluble proteins to the
membrane-associated cytoskeleton. Human platelets were pretreated
with vehicle (A) or WHI-P131 (100 µM)
(B) for 30 min and then stimulated with thrombin (0.1 unit/ml). Subsequently, platelets were fractionated into cytoplasmic
and Triton X-100 (TX-100)-soluble and -insoluble fractions
as described under "Experimental Procedures." Fractions were
subjected to Western blot analysis utilizing antibodies raised against
JAK3, tubulin, actin, STAT1, STAT3, and SYK.
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Platelet activation after thrombin stimulation was accompanied by
marked changes in platelet shape and ultrastructural organization. Topographical imaging of the surface membrane of thrombin (0.1 unit/ml)-stimulated human platelets by HR-LVSEM at 40× magnification showed development of pseudopodious extensions indicative of activation (Fig. 4, A and B).
WHI-P131 (100 µM) inhibited thrombin-induced pseudopod
formation (Fig. 4, C and D). Examination of
thrombin-stimulated platelets by TEM at 40,000× magnification
showed a rapid shape change from discoidal cells to spheres with
pseudopods extending from the surface and coalescence of granules as
well as canalicular cisternae in the center of the platelet as a
prelude to degranulation (Fig. 5,
A and B). In contrast, no pseudopods were
observed, and the granules remained uniformly dispersed after thrombin
stimulation of WHI-P131-treated platelets (Fig. 5, C and
D). In accordance with its inhibitory effects on
activation-associated shape changes and granule migration in
thrombin-stimulated platelets, WHI-P131 inhibited platelet
degranulation after thrombin stimulation as evidenced by a markedly
reduced amount of serotonin secreted from WHI-P131-treated platelets
after thrombin challenge (Fig. 5E). The measured serotonin
values in platelet supernatants were 157 ± 26 nM for
vehicle-treated control platelets (n = 4), 907 ± 20 nM for vehicle-treated, thrombin-stimulated platelets
(n = 4), and 313 ± 19 nM for
WHI-P131-treated, thrombin-stimulated platelets (n = 4). Taken together, these results provide unprecedented evidence that
JAK3 plays a critical role during the earliest events of thrombin-induced platelet activation.

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Fig. 4.
Effects of WHI-P131 on thrombin-induced shape
changes in platelets. HR-LVSEM was utilized for topographical
imaging of the platelet surface membrane as previously reported (21).
Aliquots of human platelets were incubated with 100 µM
WHI-P131 or vehicle alone for 30 min and then stimulated with thrombin
(0.1 unit/ml). Samples were prepared for HR-LVSEM as previously
described (21) and analyzed at an accelerating voltage of 2 kV.
A, resting platelets with a discoid appearance and smooth
contours; B, vehicle-pretreated control platelets stimulated
with thrombin; C, WHI-P131-pretreated unstimulated
platelets; D, WHI-P131-pretreated platelets stimulated with
thrombin.
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Fig. 5.
Effects of WHI-P131 on thrombin-induced
ultrastructural changes and degranulation in platelets.
A-C, aliquots of human platelets were incubated with 100 µM WHI-P131 or vehicle alone for 30 min and then
stimulated with thrombin (0.1 unit/ml) for 10 s. Samples were
prepared for TEM as described under "Experimental Procedures."
Sections were viewed in a Jeol 100× electron microscope at 60 kV.
A, TEM images of untreated unstimulated control
(CON) platelets with a typical discoid appearance and
disperse distribution of granules; B, TEM images of
vehicle-treated, thrombin-stimulated platelets with spike-like
pseudopodia and coalescence of granules in the center; C,
TEM images of WHI-P131-treated unstimulated platelets; D,
TEM images of WHI-P131-treated, thrombin-stimulated platelets with the
largely discoid appearance of resting platelets; E,
serotonin release from platelets stimulated with 1 unit/ml thrombin for
60 s measured using the serotonin detection kit according to the
manufacturer's specifications.
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Role of JAK3 in Thrombin-induced Platelet Aggregation--
We next
sought to examine the role of JAK3 in thrombin-induced platelet
aggregation. To this end, we first compared the thrombin-induced aggregatory responses of platelets from wild-type and Jak3
knockout mice. As shown in Fig. 6, the
magnitude of the thrombin (0.1 unit/ml)-induced aggregatory response of
Jak3+/+ platelets from wild-type mice was
greater than that of Jak3
/
platelets from Jak3 knockout mice. In accordance with these
results, pretreatment of human platelets with the JAK3 inhibitor
WHI-P131 for 30 min inhibited thrombin (0.1 unit/ml)-induced platelet
aggregation in a concentration-dependent fashion, with an
average IC50 value of 1.5 µM (Fig.
7, A and B). By
comparison, WHI-P258, a structurally similar compound that does not
inhibit JAK3, did not affect the thrombin-induced aggregation of
platelets even at 100 µM (Fig. 7, A and
C).

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Fig. 6.
Role of JAK3 in thrombin-induced platelet
aggregation. Shown are representative traces of aggregation
curves of platelets from Jak3 knockout mice and wild-type
(WT) C57BL/6 mice. Thrombin (0.1 unit/ml)-induced platelet
aggregation in citrated whole blood was measured by electrical
impedance.
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Fig. 7.
Effects of the JAK3 inhibitor WHI-P131 on
thrombin-induced platelet aggregation. A, composite
concentration effect curve of WHI-P131. In two independent experiments,
triplicate platelet-rich plasma samples were treated with varying
concentrations of the JAK3 inhibitor WHI-P131, the parent compound
WHI-P258, or vehicle (1% Me2SO in phosphate-buffered
saline) and then stimulated with thrombin (0.1 unit/ml). Platelet
aggregation was monitored in a platelet aggregometer. Results are
expressed as the percent control of thrombin-induced maximum platelet
aggregation as a function of the applied WHI-P131 concentration.
B and C, representative traces of aggregation
curves of platelets treated with WHI-P131 (100 µM; shown
in B), WHI-P258 (shown in C), or vehicle and then
stimulated with thrombin (0.1 unit/ml). Platelet aggregation was
monitored in a platelet aggregometer.
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WHI-P131 Prolongs Bleeding Time in Vivo and Protects Mice against
Thromboplastin-induced Fatal Thromboembolism--
WHI-P131 is not
toxic to mice or monkeys when administered systemically at dose levels
ranging from 1 to 100 mg/kg. WHI-P131 prolonged the tail bleeding times
of mice in a dose-dependent manner: the average tail
bleeding times were 1.5 ± 0.1 min for vehicle-treated controls
(n = 12), 9.4 ± 0.6 min for 20 mg/kg WHI-P131
(n = 5; p < 0.001), >10 min for 40 mg/kg WHI-P131 (n = 10; p < 0.001),
and >10 min for 80 mg/kg WHI-P131 (n = 10;
p < 0.001).
Notably, WHI-P131 also improved the survival outcome in a mouse model
of thromboplastin-induced generalized and invariably fatal
thromboembolism (Fig. 8). In this model,
100% of the challenged mice develop dyspnea, ataxia, and
seizures and die within 10 min after the thromboplastin challenge from
widespread thrombosis in multiple organs and massive pulmonary
thromboembolism. All of the 20 vehicle-treated mice died after the
thromboplastin challenge, with a median survival time of 2.5 min.
WHI-P131 more than doubled the median survival time and produced an
event-free survival outcome of 30 ± 15% (Fig. 8). The cause of
death in WHI-P131-pretreated thromboplastin-challenged mice was
generalized thromboembolism. No drug-related toxic lesions were
detected in any of the organs of these mice. All of the 20 control mice
treated with 80 mg/kg WHI-P131 without a subsequent thromboplastin
challenge survived beyond the 48-h observation period without any
evidence of impaired health status or bleeding.

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Fig. 8.
Protective effects of WHI-P131 in a mouse
model of fatal thromboembolism. Mice were treated intravenously
with 200 µl of vehicle (PBS supplemented with 10% Me2SO;
n = 20) or WHI-P131 (20 mg/kg twice;
n = 10) in 200 µl of vehicle. The mice were
challenged with 25 mg/kg thromboplastin via an intravenous bolus
injection into the tail vein. Shown are the cumulative proportions of
mice surviving event-free 3 min, 6 min, and 48 h after the
injection of thromboplastin. Error bars represent the S.E.
values. *, p < 0.05, log-rank test.
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DISCUSSION |
In summary, our findings reveal an essential role for JAK3 in
thrombin-induced platelet activation and aggregation. As a serine protease, thrombin activates protease-activated receptors 1 and 4 (26)
by cleaving the N-terminal portion of the receptor. The cleaved peptide
then acts as a tethered ligand that activates the G-protein-coupled
receptor independent of receptor cleavage (27). JAK3 may bind to the
cytoplasmic C-terminal portion of the protease-activated receptor(s)
and play a pivotal role in transduction of the thrombin-induced
biochemical signal once the receptor is cleaved. Further studies are
needed to decipher the molecular mechanism of JAK3-mediated regulation
of platelet function.
WHI-P131 inhibited thrombin-induced tyrosine phosphorylation of STAT1
and STAT3 proteins as well as activation-associated translocation of
SYK and tubulin to the Triton X-100-insoluble fraction. In agreement
with these results, platelets from JAK3-deficient mice displayed a
decrease in thrombin-induced platelet aggregation and tyrosine
phosphorylation of STAT1 and STAT3. Following thrombin stimulation,
WHI-P131-treated platelets did not undergo shape changes indicative of
activation such as pseudopod formation. WHI-P131 inhibited
thrombin-induced degranulation/serotonin release as well as platelet
aggregation. Highly effective platelet inhibitory plasma concentrations
(
10 µM) of WHI-P131 were achieved in mice without
toxicity. WHI-P131 prolonged the bleeding time of mice in a
dose-dependent manner and improved event-free survival in a
mouse model of thromboplastin-induced generalized and fatal thromboembolism, involving the lungs, liver, heart, and central nervous
system. Thus, this study uniquely identifies WHI-P131 as a novel
antiplatelet agent targeting JAK3 for prevention of potentially fatal
thromboembolic events. To our knowledge, WHI-P131 is the first
anti-thrombotic agent that prevents platelet aggregation by inhibiting
JAK3. WHI-P131 is also being developed as an apoptosis-promoting anticancer agent (28). JAK3 inhibitors such as WHI-P131 may be useful
as a new class of anticoagulants for treatment of hypercoagulable metastatic cancer patients as well as patients with a primary cardiovascular, cerebrovascular, or hematologic disease at risk for
thromboembolic complications.