INVOLVEMENT OF THE MAP KINASE JNK1 IN THROMBUS FORMATION Alexandre Kauskot1,6 ...

24 Pages·2007·0.65 MB·English

JBC Papers in Press. Published on September 4, 2007 as Manuscript M701596200
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M701596200
INVOLVEMENT OF THE MAP KINASE JNK1
IN THROMBUS FORMATION
Alexandre Kauskot1,6§, Frédéric Adam1§, Alexandra Mazharian1,6, Nadine Ajzenberg2,5
Eliane Berrou1,6, Arnaud Bonnefoy3, Jean-Philippe Rosa1,6, Marc F Hoylaerts4, Marijke
Bryckaert1,6

1Centre de Recherche Cardiovasculaire INSERM Lariboisière, U689-E4, Hôpital Lariboisière, 8 rue Guy
Patin, 75010 Paris, France; 2U698 INSERM, Hôpital X. Bichat, Paris, France; 3U743 INSERM, Montréal,
H2X1P1, QC, Canada; 4Center for Molecular and Vascular Biology, University of Leuven, Leuven,
Belgium; 5Hématologie et immunologie, Hôpital X. Bichat, AP-HP, Paris, France; 6Université Paris VII
Denis Diderot, Paris, France

Running title: JNK1 and thrombus formation

Address correspondence to: Dr. Marijke Bryckaert, Hôpital Lariboisière, U689-E4 INSERM, 8 rue Guy
Patin, Paris 75010, France; Tel: (33) 1 53 21 67 81; Fax: (33) 1 53 21 67 39; E-mail:
[email protected]
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The involvement of the MAP kinase c-Jun Blood platelets play a key role in haemostasis fro
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NH2 terminal kinase-1 (JNK1) has never been and thrombosis. At the site of vascular injury, h
investigated in haemostasis and thrombosis. platelets roll and adhere to various components ttp
Using two JNK inhibitors (SP600125 and 6o), of the subendothelial matrix through a number of ://w
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we have demonstrated that JNK1 is involved adhesive receptors present on the platelet w
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in collagen-induced platelet aggregation surface. Several mechanisms support the initial c.o
dependent on ADP. In these conditions, JNK1 adhesion of platelets and their subsequent brg/
activation requires the coordinated signaling activation and aggregation into a thrombus (1). y g
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pathways of collagen receptors (α2β1 and Collagen is the most thrombogenic component es
glycoprotein (GP)VI) and ADP. In contrast, of the subendothelial matrix. It supports firm t on
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JNK1 is not required for platelet adhesion on platelet adhesion mediated by its two major e
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a collagen matrix in static or blood flow surface receptors, the integrin α2β1 and the m
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conditions (300 s-1-1500 s-1) involving collagen glycoprotein (GP) VI, and induces aggregation, er 1
4
receptors (α2β1 and GPVI). Importantly, at secretion and procoagulant activity (1). At higher , 2
1500 s-1, JNK1 acts on thrombus formation on shear rates found in small arteries, arterioles and 01
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a collagen matrix dependent on GPIb-von diseased vessels (2-4), initial platelet arrest
Willebrand Factor (vWF) interaction but not depends on the interaction of von Willebrand
ADP receptor activation. This is confirmed by factor (vWF) itself bound to collagen fibres
the involvement of JNK1 in shear-induced (5,6). This interaction is unstable, but firm
platelet aggregation at 4000 s-1. We also platelet adhesion and platelet recruitment occur
provide evidence during rolling and adhesion via the interaction of the collagen with its
of platelets to vWF that platelet-GPIb-vWF receptors (α2β1 and GPVI) and the interaction of
interaction triggers αIIbβ3 activation in a vWF with the integrin αIIbβ3 (6).
JNK1 dependent manner. This was confirmed Mitogen-activated protein (MAP) kinases are
with a Glanzmann Thrombastenic patient a family of serine/threonine protein kinases
lacking αIIbβ3. Finally, in vivo, JNK1 is which comprises three subgroups: Extracellular
involved in arterial but not in venular signal-Regulated Kinases (ERKs), p38 and c-Jun
thrombosis in mice. Overall, our in vitro NH -terminal Kinases (JNKs) (7). Mammalian
2
studies define a new role of JNK1 in JNKs were found to be activated by a variety of
thrombus formation in flowing blood that is environmental stresses, inflammatory cytokines
relevant to thrombus development in vivo. and growth factors (8,9). JNKs have also been
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Copyright 2007 by The American Society for Biochemistry and Molecular Biology, Inc.
characterized by their ability to phosphorylate formation but not platelet adhesion over a
regulatory sites in the N-terminus of the collagen matrix. Moreover, JNK1 was partially
transcription factor c-Jun (10) and a variety of involved in αIIbβ3-activated pathway required
additional transcription factors such as Elk-1, for shear-induced platelet aggregation, for
ATF-2 (11,12). JNK substrates are not limited to platelet rolling and for firm adhesion to vWF.
transcription factors or nuclear proteins such as Finally, in vivo, JNK1 was found to be involved
the apoptotic proteins Bcl-X(L) or the protein in a mouse model of arterial thrombosis. To our
cytoskeleton paxillin (13,14). In platelets, ERK2, knowledge, this is the first report showing the
p38 and JNK1 are present and activated by involvement of JNK1 in thrombus formation.
various agonists. The involvement of ERK2 and
p38 in hemostasis and thrombosis is still a matter
of debate. For example, Marshall et al. EXPERIMENTAL PROCEDURES
demonstrated that αIIbβ3 activation was
dependent on Src kinase pathway but not ERK2 Materials - Equine type I collagen was obtained
pathway (15). By contrast, Li et al. showed that from Kordia (Leiden, The Netherlands). Purified
activation of integrin αIIbβ3 after vWF binding von Willebrand Factor was purchased from
to GPIb, was dependent on protein kinase G and C.A.F-D.F.C (Brussels, Belgium). Leupeptin,
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ERK2 (16,17). Recently, our group aprotinin, apyrase grade VII, prostaglandin E1 o
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demonstrated a critical role for ERK2 in platelet (PGE1), Me2SO, MRS2179, monoclonal nloa
adhesion in blood flow conditions and in platelet antibody (mAb) directed against β-tubulin and de
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spreading (18,19). With other agonists, such as polyclonal antibody against vWF were from fro
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low doses of collagen (20) or thrombin (21), Sigma (St Louis, MO). Indomethacin was h
ERK2 is involved in platelet aggregation. In obtained from Cayman Chemical Company (Ann ttp
these conditions, ERK2 acts on platelet secretion Arbor, MI). JNK inhibitors: SP600125 and the ://w
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and activation of myosin light chain kinase N-(4-amino-5-cyano-6-ethoxypyridin-2-yl)-2- w.jb
(20,22). (2,5-dimethoxyphenyl)acetamide (6o) (30), d- c.o
The other MAP Kinase, p38, is involved in Phe-Pro-Arg chloromethylketone brg/
collagen-induced platelet aggregation (23), dihydrochloride (PPACK), and phenylarsine y g
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vWF-induced platelet activation (24) and oxide were from Calbiochem-VWR (Fontenay- es
procoagulant activity (25). In vivo, the sous-Bois, France). Calcein-AM and Alexa t on
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involvement of MAP kinases is largely Fluor® 488-phalloidin were purchased from e
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unexplored. Recently, in a model of thrombotic Molecular Probes (Eugene, OR). The m
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occlusion of injured arterioles and venules, P2X1 SAPK/JNK assay kit was from Cell Signaling r 1
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and ERK2 were reported to be involved in shear (Beverly, MA). Rose bengal was purchased from , 2
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stress controlled thrombosis (26). Moreover, in a Acros Organics (Halluin, France). Antibody to 18
model of artery carotid injury, p38 was also human αIIbβ3/αvβ3 (ReoPro®) was from Lilly
involved in thrombosis (27). (Leiden, The Netherlands). The mAb 6F1
Our group found that JNK1 is present in directed against the α2β1 integrin was
platelets and downregulated by αIIbβ3 generously provided by Prof. B.S. Coller
engagement after thrombin induction (28). (Rockefeller University, New York, NY). The
Moreover, JNK1 is activated by porcine vWF mAb G19H10 which blocks the binding of vWF
(29) and by thrombin (28) but its role in platelet to GPIb was produced as previously described
activation and thrombosis has never been (31). AR-C69931 MX was generously provided
investigated. In this study, we examined the by Dr. B. Humphries (Astra Zeneca, UK).
involvement of JNK1 in platelet aggregation Polyclonal antibody directed against the
induced by collagen and vWF, and platelet phosphorylated form of JNK1 (JNK1-P) was
adhesion in blood flow conditions and in a purchased from Promega (Madison, WI).
mouse model of thrombosis. We found that Horseradish peroxidase-conjugated secondary
JNK1 was involved in collagen-induced platelet antibodies were from Jackson ImmunoResearch
aggregation dependent on ADP. In blood flow Laboratories (West Grove, PA).
conditions, JNK1 was required for thrombus
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Preparation and fluorescence labeling of chemiluminescence detection reagents (Perbio
human platelets - Venous blood was collected Science, Brebières, France).
from healthy donors and from a patient with JNK1 activity - JNK1 activity was examined
Glanzmann’s Thrombasthenia (GT) after collagen stimulation in the presence or
characterized by an absence of αIIbβ3 (32). absence of various concentrations of SP600125
Written informed consent was obtained from all or 6o (5 and 10 µM) or Me SO. JNK1 activity
2
the donors. Platelet-rich plasma (PRP) was was measured according to the manufacturer’s
obtained by centrifugation (120g; 15 min; 20°C) instructions (Cell signalling). Briefly, platelets (4
and platelets were isolated by differential x 105/µl) were lysed with cell lysis buffer (20
centrifugation as previously described (20). The mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1
platelet pellet was resuspended in Tyrode’s mM EGTA, 1% (vol/vol) Triton X-100, 2.5 mM
buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 3 sodium pyrophosphate, 1 mM β-
mM KCl, 5 mM NaHCO , 0.5 mM MgCl , 1 glycerolphosphate, 1 mM Na VO , 1 μg/ml
3 2 3 4
mM CaCl and 10 mM glucose). leupeptin, 10 µg/ml aprotinin) and centrifuged at
2
To study platelet adhesion to vWF, platelets 14,000g for 10 min at 4°C. Immobilized c-Jun
in PRP were labeled with 2.5 μM calcein-AM fusion protein beads (20 µl) were added in the
and resuspended at 3 x 105/µl in autologous supernatant and incubated with gentle rocking
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plasma, while erythrocytes were maintained as a overnight at 4°C to pull down JNK. After o
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packed cell suspension. Reconstituted blood was centrifugation (14,000g; 30 s; 4°C), the pellet nlo
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obtained by gently mixing labeled-platelets and was washed twice with cell lysis buffer and de
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erythrocytes at 45% hematocrit just before twice with kinase buffer on ice (25 mM Tris pH fro
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perfusion. 7.5, 5 mM β-glycerolphosphate, 2 mM h
Platelet aggregation - Platelet aggregations dithiothreitol (DTT), 0.1 mM Na VO , 10 mM ttp
were monitored by measuring light transmission MgCl ). The pellet was suspende3d in4 50 µl of ://w
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through the stirred suspension of washed kinase buffer supplemented with 200 µM ATP w
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platelets at 37°C using a Chronolog dual-beam and incubated 30 min at 30°C. Proteins were c.o
aggregometer (Coultronics, Margency, France). separated by SDS-PAGE according to standard brg/
Platelets (4 x 105/µl) were preincubated with protocols and c-Jun-GST substrate, y g
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SP600125 (10 min, 2 to 10 µM) or with 6o (30 phosphorylated by JNK1 (c-Jun-P), was detected es
min, 2 to 10 µM) or Me SO at 37°C without by Western blotting using specific antibody. t on
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stirring. The quantity of Me SO never exceeded Platelet deposition - Platelet suspension (2 x e
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0.2% (vol/vol). Platelet aggregation was initiated 105) was preincubated with SP600125 or 6o (10 m
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by adding type I collagen (0.2 to 10 µg/ml). µM) or Me2SO in the presence or absence of r 1
Aggregation was measured and expressed as the ReoPro® (10 µg/ml) and then plated on type I 4, 2
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percent change in light transmission, with the collagen (50 µg/ml) for 60 min at 20°C. After 1
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value for the blank sample (buffer without three washes, platelet deposition was quantified
platelet) set at 100%. by an acid phosphatase assay, as previously
Immunoblotting - Samples were subjected to described (34).
immunoblotting as previously described (33). Platelet perfusion studies - Platelet adhesion
Platelets were lysed in sodium dodecyl sulfate to type I collagen under flow was evaluated
(SDS) denaturing buffer (50 mM Tris, 100 mM using a parallel-plate perfusion chamber. Glass
NaCl, 50 mM NaF, 5 mM EDTA, 40 mM β- coverslips were coated overnight at 4°C with
glycerophosphate, 100 µM phenylarsine oxide, type I collagen (50 µg/ml). Whole blood,
1% SDS, 5 µg/ml leupeptin, 10 µg/ml aprotinin, collected into 40 µM PPACK, was perfused for
pH 7.4). Proteins were subjected to SDS-PAGE 4 min, using a KD Scientific syringe pump
and transferred to nitrocellulose. Membranes (Fisher Bioblock Scientific, Illkirch, France) at
were incubated with various primary antibodies: shear rates of 300 s-1 or 1500 s-1 followed by
anti-JNK1-P (1/5000), anti-c-jun-P (1/1000), and washing in the same conditions. After fixation
anti-tubulin (1/4000). Immunoreactive bands (4% paraformaldehyde) and permeabilization
were visualized with enhanced (0.2% Triton X-100), F-actin of adherent
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platelets was detected using Alexa Fluor® 488- Xylocain® 0.5% (vol/vol) was used as a local
phalloidin (1/200). analgesic.
Platelet adhesion to vWF under flow was Mouse platelet isolation and labeling for
evaluated using glass microcapillary tubes (Fiber intravital microscopy - Blood was collected on
Optic Center, New Bedford, MA). PPACK (80 µM) with ACD buffer (1/5) (93 mM
Microcapillary tubes were coated overnight at Na -citrate, 7 mM citric acid, 0.14 M dextrose,
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4°C with purified vWF (20 µg/ml). pH 6.5) via puncture of the heart of anesthetized
Reconstituted blood was drawn through the mice. PRP was obtained by consecutive
coated glass microcapillary at a shear rate of centrifugation of the blood for 30 s at 850g and 5
1500 s-1 for 4 min, then rinsed in the same min at 150g. Upon addition of 0.2 volume of
conditions. ACD, 1 µM PGE , platelets were isolated by
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Adherent platelets were recorded using an centrifugation at 850g for 5 min. Platelets were
inverted epifluorescent microscope (Nikon resuspended at 0.5 x 106/µl and labeled with 10
Eclipse TE2000-U; Champigny sur Marne, µM calcein-AM for 20 min at 37°C.
France), coupled to Metamorph 7.0r1 software Photochemically induced thrombosis model -
(Universal Imaging Corporation). The surface Vascular injury of caecum arterioles and venules
covered by platelets was expressed as the was induced by the photoactivatable dye rose
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relative surface covered to facilitate the bengal as previously described (37). o
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comparison of different experimental conditions. Anesthetized mice were catheterized via the nlo
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Quantification of platelet surface velocity - jugular vein. Fluorescently labeled murine de
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To calculate the velocity of platelets interacting platelets (200 μl; 0.5 x 106 platelets/μl) were fro
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with the surface of vWF-coated microcapillary, injected through the catheter, followed by an h
reconstituted blood containing labeled platelets intravenous bolus of rose bengal (30 mg/kg) ttp
(3 x 103/µl) was perfused at a shear rate of 1500 which was infused (30 mg/kg/h) for 10 min, the ://w
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s-1. Real-time platelet adhesion and platelet time required for the injected platelets to reach w
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rolling were recorded at 2 frames per second. steady-state, prior to induction of the vascular c.o
Platelet velocity was determined as previously injury and then throughout the experiment. To brg/
described (35). evaluate the involvement of JNK1 in thrombus y g
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Shear-induced platelet aggregation (SIPA) - formation, a bolus plus perfusion of the inhibitor es
SIPA was measured by means of a coaxial SP600125 or Me SO was applied through the t on
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cylinder shearing device, as previously described catheterized jugular vein 10 min prior to vessel e
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(36). Briefly, washed platelets (1.8 x 105/µl) wall injury (bolus of 0.91 µmole/kg = 200 µg/kg m
b
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were preincubated with SP600125 (10 µM) or followed by an intravenous infusion at 200 r 1
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Me2SO and were exposed to a shear rate of 200 µg/kg/h). The mouse intestines were then spread , 2
s-1 or 4000 s−1 for 2 min at 20°C with vWF (40 on a microscopic glass and the arterioles and 01
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µg/ml). Samples were fixed with 1.25% venules lying on the caecum were visualized
paraformaldehyde and the number of single with the epifluorescent microscope. Injury was
platelets was measured by flow cytometry induced by exposing the selected arterioles and
(Coulter Epics XL, Beckman Coulter, Roissy, venules at 540 nm for 30 s. Recruitment of
France) before and after exposure to shear. SIPA fluorescent platelets to the lesion site was
was expressed as the percentage of registered in real time and recorded in the
disappearance of single platelets (DSP). memory of an attached computer for analysis
Animals - All experimental procedures on with the Metamorph program. We analyzed the
animals were performed according to French vessel occlusion time, defined as blood flow
legislation on the protection of animals. FVB arrest for at least 1 min.
mice were obtained from Janvier Laboratories Statistics - Results are expressed as means ±
(Le Genest-St-Isle, France) and kept in animal SEM for at least three independent experiments.
facilities. Eight- to twelve-week-old male mice Statistical significance was assessed with a one-
were anesthetized with intraperitoneal injection way ANOVA with Dunnett’s post-test or the
of sodium pentobarbital (60 mg/kg) and Mann-Whitney test, depending on the case.

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RESULTS C69931 MX; 10 µM), the P2Y1 antagonist
(MRS2179) having a weak inhibitory effect on
JNK1 is involved in collagen-induced platelet JNK1 phosphorylation (Fig. 4). Taken together,
aggregation – We have previously reported that these results indicate that JNK1 is involved in
JNK1 was present in platelets (28), but its platelet aggregation induced by low collagen
involvement in hemostasis and thrombosis concentrations dependent on ADP.
remains to be established. Therefore, we first
investigated the phosphorylation level of JNK1 Collagen-induced platelet deposition does not
in collagen-induced platelet aggregation. Platelet require JNK1 activation - First, we examined the
suspension was incubated with various doses of state of JNK1 phosphorylation in collagen-
collagen (0.2 to 10 µg/ml) under stirring induced platelet deposition. Platelet suspensions
conditions. Whereas JNK1 was not were plated on type I collagen for various times.
phosphorylated in resting platelets, JNK1-P was detected after 15 min of adhesion,
phosphorylated JNK1 (JNK1-P) was detected at followed by a decrease in the JNK1
a low collagen concentration (0.2 µg/ml) (Fig. 1) phosphorylation level (Fig. 5A). To investigate
and reached a maximum at 2 µg/ml collagen the role of JNK1 in platelet deposition on
after 2 min. These results indicate that JNK1 is collagen, a static adhesion assay was performed
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activated during platelet aggregation induced by in conditions where only platelet adhesion o
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collagen. occurred (2 x 105 platelets/µl), which was nlo
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To further investigate the role of JNK1 in confirmed by the identical adhesion observed in de
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platelets, we used two JNK inhibitors, SP600125 the presence or absence of anti-αIIbβ3, ReoPro® fro
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and 6o. We first assessed the inhibitory effect of (10 µg/ml) (Fig. 5B-C). Neither of the two JNK h
SP600125 and 6o in conditions of collagen- inhibitors (10 µM) affected platelet adhesion. ttp
induced platelet aggregation by testing JNK1 Our results show that JNK1 is not required for ://w
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activity. This activity was quantified by the collagen-induced platelet deposition. w
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phosphorylation level of the specific substrate, c- c.o
jun (10). SP600125 and 6o inhibited JNK1 JNK1 is required in thrombus formation on brg/
activity in a dose dependent manner (Fig. 2). collagen under blood flow conditions at high y g
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Next, the role of JNK1 was investigated in shear rates - We next investigated the role of es
platelet aggregation induced by collagen in JNK1 in platelet adhesion to collagen at different t on
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conditions where platelets were pretreated with shear rates. Whole blood was perfused over e
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SP600125, 6o or Me SO. At low collagen immobilized type I collagen at low (300 s-1) and m
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concentrations (0.2 and 0.5 µg/ml), platelet high (1500 s-1) shear rates. After 4 min of r 1
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aggregation was totally or partially impaired by perfusion, thrombi were smaller at 300 s-1 , 2
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both inhibitors (10 µM), whereas at a high compared with 1500 s-1 and the covered surface 18
collagen concentration (5 µg/ml), these reached 22.5 ± 0.5% and 40.6 ± 7.4%
inhibitors had no effect (Fig. 3A-B). We next respectively (Fig. 6A). At 300 s-1 and 1500 s-1,
examined the effect of various concentrations of only 15 ± 8% of platelet surface coverage and
JNK inhibitors (2 to 10 µM) on platelet aggregate size were dependent on ADP/ATP.
aggregation induced by a low collagen The addition of SP600125 or 6o, at 300 s-1, did
concentration (0.2 µg/ml). Two µM of SP600125 not modify surface coverage
or 6o impaired platelet aggregation by 28 ± 3% (adhesion/aggregation) on immobilized collagen
and 14 ± 3% respectively compared to control, (Fig 6A, 6B). In contrast, at 1500 s-1, surface
whereas 10 µM of SP600125 or 6o impaired coverage was strongly impaired by SP600125
platelet aggregation by 98 ± 1% and 98 ± 4% (71 ± 5%; p<0.01) (Fig. 6A) and 6o (52 ± 2%;
respectively (p<0.01) (Fig. 3C). p<0.01) (Fig. 6B). In addition, the size of
In these conditions, at a low collagen thrombi measured by confocal laser scanning
concentration (0.2 µg/ml), platelet aggregation microscopy was impaired by 77% in the
and JNK1 phosphorylation were mostly impaired presence of SP600125 (10 µM) at 1500 s-1 (data
by ADP/ATP scavenger apyrase (2 U/ml) and not shown). These results show that JNK1
the antagonist of ADP receptor P2Y12 (AR- engagement in thrombus formation, is dependent
5
on shear conditions. To better understand the observed. In contrast, at a higher shear rate
apparent discrepancy observed between the low (4000 s-1), DSP reached 41.0 ± 4.2% with
and high shear rates, we next examined the control platelets and 18.9 ± 2.3% in the presence
different receptors involved. Blood perfusion of JNK1 inhibitor, corresponding to an inhibitory
assays were performed in the presence or effect of 52 ± 11% (p<0.01). These results
absence of mAb 6F1 which blocks α2β1, and indicate that JNK1 is involved in SIPA.
mAb G19H10 which blocks GPIb. At 300 s-1,
6F1 inhibited surface coverage by platelets by 99 Platelet rolling and adhesion to vWF are
± 2% (p<0.01) whereas G19H10 had no effect dependent on JNK1 - To further investigate the
(Fig. 7A). VWF deposition on the collagen role of JNK1 in platelet interactions between
surface visualized by immunofluorescence vWF and its receptors, GPIb and αIIbβ3, we
microscopy after perfusion at 300 s-1 confirmed examined platelet rolling and platelet adhesion
that small amounts of vWF were deposited (Fig. on a vWF matrix. Platelet translocation velocity
7B) and that thrombus formation was only was examined after 60 s of perfusion. The
dependent on collagen receptors. In contrast, at average velocity of platelets translocating on
1500 s-1, 6F1 and G19H10 impaired surface immobilized vWF (1.36 ± 0.08 µm/s) increased
coverage (aggregation and/or adhesion) by 50 ± to 1.95 ± 0.07 µm/s (p<0.01) in the presence of
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3% and 87 ± 7% respectively (p<0.01) (Fig. 7A). ReoPro® (Fig. 9A). Moreover, an elevated o
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Larger amounts of vWF were deposited on velocity was observed in the presence of nlo
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immobilized type I collagen (Fig. 7B) showing SP600125 alone (1.91 ± 0.08 µm/s) (p<0.01), de
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that in conditions where JNK1 is involved, which did not increase after the addition of fro
thrombus formation is mostly dependent on ReoPro®. These data strongly suggest that JNK1 hm
GPIb-vWF interaction. Finally, to further is involved in platelet rolling and in αIIbβ3 ttp
investigate the role of JNK1 at 1500 s-1, adhesion engagement. ://w
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without thrombi was examined in control To confirm the involvement of JNK1 in the w
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platelets in the presence of anti-αIIbβ3 αIIbβ3 engagement, we examined the effect of c.o
(ReoPro®) and in platelets from GT patient SP600125 on firm platelet adhesion to brg/
characterized by an absence of αIIbβ3. When immobilized vWF at 1500 s-1. SP600125 y g
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thrombi were inhibited in the presence of treatment partially impaired platelet adhesion (37 es
ReoPro®, only platelet adhesion was observed ± 2%; p<0.001) after 4 min of perfusion (Fig. t on
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and addition of SP600125 did not affect 9B). In parallel, ReoPro® inhibited platelet e
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adhesion (Fig. 8A). Importantly, in absence of adhesion by 60 ± 1% (p<0.001). This was m
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thrombi, platelet adhesion was inhibited only by confirmed by using platelets from the GT patient r 1
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6F1 (98 ± 1%), and not by G19H10 (8 ± 5%), where platelet adhesion was impaired by 58%. In , 2
indicating that platelet adhesion was dependent both conditions (ReoPro® and GT) adhesion was 01
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on collagen receptors (α2β1 and GPVI) but not not modified by the addition of SP600125 (55 ±
on GPIb-vWF interaction (Fig. 8B) as previously 1% and 59% respectively). Moreover, the
described (37). Our results show that, at a high addition of the ATP/ADP scavenger apyrase or
shear rate, thrombus formation dependent on indomethacin did not affect platelet adhesion on
GPIb-vWF interaction requires JNK1 activation. vWF (Fig. 9C). By contrast, addition of PGE ,
1
which inhibits platelet activation, impaired
JNK1 is involved in shear-induced platelet surface coverage by 56 ± 4%. To confirm the
aggregation - To further study the involvement inhibitory effect of SP600125 on platelet
of JNK1 in thrombus formation in relation to adhesion to vWF at high (1500 s-1) shear rate, the
GPIb-vWF interaction, we examined the effect second JNK inhibitor, 6o, was assessed. In the
of SP600125 on shear-induced platelet presence of 6o (10 µM), platelet adhesion was
aggregation (SIPA), by measuring the inhibited but in a lesser potency than SP600125
disappearance of single platelets (DSP) in the (data not shown), confirming the involvement of
presence of soluble vWF (40 µg/ml). SIPA is JNK1. Our results show that αIIbβ3-activation
dependent on vWF-GPIb interaction. At a low which is required for firm platelet adhesion to
shear rate (200 s-1), no significant DSP was vWF is partially dependent on JNK1.
6
JNK1 is involved in arteriolar thrombosis - and p38 in platelet aggregation and secretion
The role of JNK1 was investigated in a model of induced by collagen (20,23,38). We found, with
arteriolar and venular thrombosis in mice. The others, that ERK2 was involved in low
development of the thrombus and vessel concentration collagen-induced platelet
occlusion was monitored in real time under a aggregation which was dependent on ADP (20)
microscope. Figure 10 shows a chronological and/or ATP via the P2X1 ion channel,
series of images of developing thrombosis in the previously reported as contributing to the
caecum arterioles and venules of mice treated amplification of platelet granule release (38).
with Me SO or SP600125. The mean venous Here, we have shown for the first time that JNK1
2
occlusion time was not impaired in mice treated was activated in conditions of collagen-induced
with SP600125 (36.7 ± 2.6 min) compared with platelet aggregation. The level of phosphorylated
controls treated with Me SO (27.5 ± 3.5 min). JNK1 argues in favor of different pathways
2
By contrast, in arterioles, occlusion times were involved at low and high concentrations of
significantly longer in mice treated with collagen. We found that platelet aggregation
SP600125, reaching 50.2 ± 2.0 min compared induced by only low doses of collagen were
with 26.8 ± 3.9 min in controls (p<0.001). Taken dependent on the JNK1 pathway. In these
together, these results show that JNK1 is conditions, identical inhibition of JNK1
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primarily involved in arteriolar thrombosis but phosphorylation was observed with the o
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not in venular thrombosis. ADP/ATP scavenger apyrase and the antagonist nlo
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of P2Y12 receptor of ADP. Surprisingly, ADP de
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alone, in conditions of platelet aggregation, was fro
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DISCUSSION not sufficient to induce full JNK1 activation h
(data not shown). This provides strong evidence ttp
This study demonstrates that the MAP kinase that, in conditions of collagen activation, JNK1 ://w
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JNK1 is involved in platelet adhesion and in phosphorylation like ERK2 phosphorylation (20) w
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thrombus formation. JNK1 plays a role in requires the P2Y12 receptor of ADP and an c.o
platelet aggregation induced by low additional pathway working in combination with brg/
concentrations of collagen. We found that a ADP. The relationship between ERK and JNK y g
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coordinated pathway between collagen receptors remains to be established. Moreover, the link es
(α2β1 and GPVI) and ADP release is required between ADP via its P2Y12/P2Y1 receptors and t on
D
for JNK1 activation. Moreover, the involvement JNK1 is still unknown. PI3 kinase is a potential e
c
e
of JNK1 in platelet adhesion and thrombus candidate for the role of signaling intermediate. m
b
e
formation over a collagen matrix is dependent on In proliferative cells, JNK activation has been r 1
4
shear stress. JNK1 is not required for static reported to be dependent on PI3 kinase (39). If , 2
0
adhesion or adhesion with small thrombi in this is the case, different pathways are involved 1
8
blood flow conditions obtained at a low shear in ERK2 and JNK1 activation in platelets since
rate (300 s-1). Conversely, JNK1 is required for inhibitors of PI3 kinase did not significantly
thrombus growth but not adhesion at a high affect collagen-induced ERK2 activation (results
shear rate (1500 s-1), i.e. where GPIb-vWF not shown). The Ras family GTP binding protein
interaction is crucial. This is confirmed by the Rap1b and/or Rap2b reported to require a Gi-
involvement of JNK1 in SIPA. Moreover, dependent pathway in platelets (40-42) could be
platelet rolling and firm platelet adhesion on the other candidates. Indeed, Rap2, sharing 60%
vWF matrix at a shear rate of 1500 s-1 strongly homology with Rap1, was described as
suggest that JNK1 acts on αIIbβ3 engagement enhancing MAP4K4-induced activation of JNK
after GPIb-vWF interaction. Finally, in a model (43). Futher studies are required to answer to
of arteriolar and venular thrombosis in mice, these questions.
only the mean arterial occlusion time is
significantly longer in mice treated with JNK1 We show here, for the first time, that in blood
inhibitor compared with control mice. flow conditions on collagen, JNK1 is involved in
In recent years, a lot of progresses have been thrombus formation but not in adhesion and is
made concerning the role of MAP kinases ERK2 dependent on shear stress. In static adhesion or at
7
a low shear rate (300 s-1) involving collagen independent of ADP and thromboxane A2. In
receptors only (GPVI and α2β1) and supporting these conditions, JNK1, involved in “inside-out”
small thrombi, JNK1 was not involved. In these pathway, could directly activate αIIbβ3 required
conditions, small thrombi were independent of for firm adhesion. In this case, greater inhibition
ADP (data not shown). The fact that JNK1 was of adhesion observed with an anti-αIIbβ3
involved in collagen-induced aggregation but not (Reopro®) or with GT platelets suggests that
in small thrombi in blood flow could be JNK1 is partially involved in the activation of
explained by the involvement of ADP at the low αIIbβ3. The other possibility is that JNK1,
concentration of collagen in conditions of involved in “inside-out” pathway, acts
platelet aggregation. downstream of αIIbβ3 activation on integrin
In contrast, at a high shear rate (1500 s-1), the clustering or on interaction of αIIbβ3 with the
vWF was readily detected bound to the collagen cytoskeleton. Staining of the αIIbβ3 antibody
matrix. In these conditions, vWF-GPIb PAC1, which recognized only the active form of
interaction which can secondarily activate α2β1 integrin, did not provide an answer to this
(44) was essential to form large thrombi, question (results not shown). The mechanism of
strongly suggesting that much of the effect of αIIbβ3 activation by GPIb-IX-V is still
JNK1 on thrombus formation depended on vWF. controversial. Li et al. suggested that αIIbβ3
D
These data are in agreement with the inhibitory activation is mediated through activation of o
w
effect of JNK inhibitor observed in SIPA and cyclic guanosine monophosphate (cGMP)- nlo
a
with previous reports showing a crucial role of dependent protein kinase, protein kinase G and de
d
GPIb-vWF interaction in platelet recruitment in ERK2 pathway (17). In contrast, Marshall et al. fro
m
arterial thrombus (45,46). In contrast, platelet confirmed a crucial role of Src, and showed an h
adhesion, observed in the presence of ReoPro® inhibitory effect of PKG (15). Src could be a ttp
or with platelets from the GT patient, was candidate upstream of JNK1 since in ://w
w
dependent on collagen receptors but not GPIb- proliferative cells Src has been reported to be w
.jb
vWF interaction as previously described (6) and involved in JNK1 activation (49,50). Further c.o
independent of JNK1. studies are required to elucidate the signaling brg/
How does JNK1 function in thrombus formation pathway of JNK1 activation. The fact that JNK1 y g
u
under blood flow conditions? The role of JNK1 participates in platelet rolling and adhesion in es
in relation to GPIb-vWF-mediated platelet blood flow conditions (1500 s-1) on vWF t on
D
activation in thrombus formation is in supports the hypothesis that JNK1 plays a major e
c
e
accordance with previous work (47) showing role in platelet adhesion to the arterial m
b
e
that, at high shear rates, vWF is predominantly subendothelium, which was confirmed by in vivo r 1
4
distributed at the outer areas of thrombi whereas studies. , 2
0
fibrinogen is present in the inner area. In Finally, in arterial and venular murine 1
8
contrast, at low shear rates, only fibrinogen thrombosis, we found that JNK1 is required for
appears in thrombi. Also, in blood flow (1500 s- arterial but not venular occlusion in relation to
1) over a collagen matrix, platelet surface shear stress controlled signaling processes. The
coverage and aggregate size were largely relatively smaller prolongation of occlusion
independent of ADP (15%) as previously times by the inhibitor of JNK in venules can be
described (48). This confirms that JNK1 is explained by a weaker effect of vWF in venular
involved in collagen-induced formation of thrombosis than in arterial thrombosis. In fact,
packed large thrombi under physiological inhibition of GPIb-vWF interaction by the
conditions and supports the hypothesis that recombinant murine vWF-A1 domain increased
JNK1 acts via an independent ADP/ATP the time of arterial thrombosis but not the time of
pathway, but a dependent GPIb-vWF pathway venular thrombosis (26). Our results show for
that participates in thrombi formation. the first time that JNK1 contributes to thrombus
formation and thrombosis in a shear-controlled
Furthermore, platelet adhesion in blood flow manner coupled to the interaction of vWF-GPIb-
conditions (1500 s-1) on a vWF matrix was mediated signaling pathway.

8
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