مجال
التميز
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التميز الدراسي والبحثي
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البحوث المنشورة
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البحث (1):
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عنوان البحث:
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Activation of glycoprotein VI (GPVI) and C-type lectin-like
receptor-2 (CLEC-2) underlies platelet activation by diesel exhaust particles
and other charged/hydrophobic ligands
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رابط إلى البحث:
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تاريخ النشر:
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07-04-2015
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موجز عن البحث:
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Platelets are activated by a range of stimuli that share
little or no resemblance in structure to each other or to recognized ligands,
including diesel exhaust particles (DEP), small peptides [4N1-1, Champs
(computed helical anti-membrane proteins), LSARLAF
(Leu-Ser-Ala-Arg-Leu-Ala-Phe)], proteins (histones) and large polysaccharides
(fucoidan, dextran sulfate). This miscellaneous group stimulate aggregation
of human and mouse platelets through the glycoprotein VI (GPVI)–FcR γ-chain
complex and/or C-type lectin-like receptor-2 (CLEC-2) as shown using
platelets from mice deficient in either or both of these receptors. In
addition, all of these ligands stimulate tyrosine phosphorylation in
GPVI/CLEC-2-double-deficient platelets, indicating that they bind to
additional surface receptors, although only in the case of dextran sulfate
does this lead to activation. DEP, fucoidan and dextran sulfate, but not the
other agonists, activate GPVI and CLEC-2 in transfected cell lines as
shown using a sensitive reporter assay confirming a direct interaction with
the two receptors. We conclude that this miscellaneous group of ligands bind
to multiple proteins on the cell surface including GPVI and/or CLEC-2,
inducing activation. These results have pathophysiological significance in a
variety of conditions that involve exposure to activating charged/hydrophobic
agents.
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البحث (2):
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عنوان البحث:
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CLEC-2 expression is maintained on activated platelets
and on platelet microparticles
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تاريخ النشر:
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22-08-2014
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موجز عن البحث:
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The C-type lectin-like
receptor CLEC-2 mediates platelet activation through a hem-immunoreceptor
tyrosine-based activation motif (hemITAM). CLEC-2 initiates a Src- and
Syk-dependent signaling cascade that is closely related to that of the 2
platelet ITAM receptors: glycoprotein (GP)VI and FcγRIIa. Activation of
either of the ITAM receptors induces shedding of GPVI and proteolysis of the
ITAM domain in FcγRIIa. In the present study, we generated monoclonal
antibodies against human CLEC-2 and used these to measure CLEC-2 expression
on resting and stimulated platelets and on other hematopoietic cells. We show
that CLEC-2 is restricted to platelets with an average copy number of ∼2000 per cell and that activation of CLEC-2 induces
proteolytic cleavage of GPVI and FcγRIIa but not of itself. We further show
that CLEC-2 and GPVI are expressed on CD41+ microparticles in megakaryocyte
cultures and in platelet-rich plasma, which are predominantly derived from
megakaryocytes in healthy donors, whereas microparticles derived from
activated platelets only express CLEC-2. Patients with rheumatoid arthritis,
an inflammatory disease associated with increased microparticle production,
had raised plasma levels of microparticles that expressed CLEC-2 but not
GPVI. Thus, CLEC-2, unlike platelet ITAM receptors, is not regulated by
proteolysis and can be used to monitor platelet-derived microparticles.
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البحث (3):
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عنوان البحث:
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Fucoidan is a novel platelet agonist for the C-type
lectin-like receptor 2 (CLEC-2)
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رابط إلى البحث:
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تاريخ النشر:
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22-01-2013
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موجز عن البحث:
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Fucoidan, a sulfated
polysaccharide from Fucus vesiculosus,
decreases bleeding time and clotting time in hemophilia, possibly through
inhibition of tissue factor pathway inhibitor. However, its effect on
platelets and the receptor by which fucoidan induces cellular processes has
not been elucidated. In this study, we demonstrate that fucoidan induces
platelet activation in a concentration-dependent manner. Fucoidan-induced
platelet activation was completely abolished by the pan-Src family kinase
(SFK) inhibitor, PP2, or when Syk is inhibited. PP2 abolished
phosphorylations of Syk and Phospholipase C-γ2. Fucoidan-induced platelet
activation had a lag phase, which is reminiscent of platelet activation by
collagen and CLEC-2 receptor agonists. Platelet activation by fucoidan was
only slightly inhibited in FcRγ-chain null mice, indicating that fucoidan was
not acting primarily through GPVI receptor. On the other hand,
fucoidan-induced platelet activation was inhibited in platelet-specific
CLEC-2 knock-out murine platelets revealing CLEC-2 as a physiological target
of fucoidan. Thus, our data show fucoidan as a novel CLEC-2 receptor agonist
that activates platelets through a SFK-dependent signaling pathway.
Furthermore, the efficacy of fucoidan in hemophilia raises the possibility
that decreased bleeding times could be achieved through activation of
platelets.
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البحث (4):
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عنوان البحث:
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Fibrin activates GPVI in human and mouse platelets
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رابط إلى البحث:
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تاريخ النشر:
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24/09/2015
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موجز عن البحث:
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The
glycoprotein VI (GPVI)-Fc receptor γ (FcRγ) chain is the major platelet
signaling receptor for collagen. Paradoxically, in a FeCl3 injury model,
occlusion, but not initiation of thrombus formation is delayed in
GPVI-deficient and GPVI-depleted mice. In this study, we demonstrate that
GPVI is a receptor for fibrin and speculate that this contributes to
development of an occlusive thrombus. We observed a marked increase in
tyrosine phosphorylation, including the FcRγ chain and Syk, in human and
mouse platelets induced by thrombin in the presence of fibrinogen and the
αIIbβ3 blocker eptifibatide. This was not seen in platelets stimulated by a
protease activated receptor (PAR)-4 peptide, which is unable to generate
fibrin from fibrinogen. The pattern of tyrosine phosphorylation was similar
to that induced by activation of GPVI. Consistent with this, thrombin did not
induce tyrosine phosphorylation of Syk and the FcRγ chain in GPVI-deficient
mouse platelets. Mouse platelets underwent full spreading on fibrin but not
fibrinogen, which was blocked in the presence of a Src kinase inhibitor or in
the absence of GPVI. Spreading on fibrin was associated with
phosphatidylserine exposure (procoagulant activity), and this too was blocked
in GPVI-deficient platelets. The ectodomain of GPVI was shown to bind to
immobilized monomeric and polymerized fibrin. A marked increase in
embolization was seen following FeCl3 injury in GPVI-deficient mice,
likely contributing to the delay in occlusion in this model. These results
demonstrate that GPVI is a receptor for fibrin and provide evidence that this
interaction contributes to thrombus growth and stability.
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المؤتمرات العلمية:
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المؤتمر (1):
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عنوان المؤتمر:
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2nd EUPLAN
Conference
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تاريخ الإنعقاد:
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24-26 September 2014
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مكان
الإنعقاد:
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Le Bischenberg, France
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طبيعة المشاركة:
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Poster
presentation
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عنوان المشاركة:
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Diesel particles activate platelets through GPVI and
CLEC-2
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ملخص المشاركة:
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Introduction:
We are
exposed to a number of pollutants on a daily basis. A major contributor to
these pollutants is diesel exhaust. Diesel exhaust contains both gasses and
nano-particulate matter, referred to as diesel exhaust particles (DEP). Such
particles have been shown to cross the lung epithelium and enter the blood
stream and have been proposed to cause inflammation and to be cytotoxic.
Several studies have shown that DEP induce aggregation of washed platelets.
Our aim was to understand the mechanism of platelet activation.
Methods:
We have
monitored aggregation and protein phosphorylation in human and mouse washed
platelets, and used transgenic mouse platelets and transfected cell lines to
investigate the mechanism of platelet activation by DEP.
Results:
DEP
stimulated aggregation of human and mouse platelets, predominantly via the
GPVI pathway, but with CLEC-2 playing a minor role as shown by measurement of
receptor phosphorylation and use of CLEC-2 and GPVI-deficient platelets.
Further, DEP achieved aggregation solely through Syk and Src tyrosine
kinases. Nevertheless, DEP stimulated protein phosphorylation in platelets
doubledeficient in GPVI and CLEC-2. DEP was unable to stimulate CLEC-2 or
GPVI in transfected DT40 cells.
Conclusions:
This
study demonstrates that DEP activate platelets through the GPVI and/or CLEC-2
independent of direct receptor binding.
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المؤتمر (2):
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عنوان المؤتمر:
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2nd EUPLAN Conference
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تاريخ الإنعقاد:
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24-26 September 2014
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مكان
الإنعقاد:
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Le
Bischenberg, France
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طبيعة المشاركة:
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Poster
presentation
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عنوان المشاركة:
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Mechanism of platelet activation
by fucoidan and dextran
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ملخص المشاركة:
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Introduction:
A
diverse range of non-endogenous stimuli, including oxidised proteins and
nanoparticles, have been found to induce platelet activation and aggregation
via the collagen receptor complex, the GPVI-FcR γ-chain. Fucoidan, a
sulphated polysaccharide, is believed to cause activation via the Podoplanin
receptor, CLEC-2, whilst the receptor for dextran (a complex branched polysaccharide)
remains unidentified. This study aims to further clarify the pathway of
activation by fucoidan and dextran, and to describe associated receptors.
Methods:
Fucoidan
and dextran induced aggregation and activation of human and mouse model
platelets was measured using light transmission aggregometry and Western
blotting, and direct activation of GPVI and CLEC-2 receptors via an NFAT
assay and surface plasmon resonance.
Results:
Fucoidan
stimulated aggregation of human and mouse platelets predominantly via the
CLEC-2 pathway, but also, to a lesser extent, via the GPVI pathway. Further,
fucoidan achieved aggregation solely through Syk and Src tyrosine kinases.
Interestingly, fucoidan stimulated protein phosphorylation in GPVI/CLEC-2
deficient platelets. In human platelets, dextran-induced aggregation was
mediated through a Src kinase pathway, independent of Syk, whereas in mouse
platelets, activation was dependent on Src and Syk tyrosine kinases, and also
CLEC-2. Neither fucoidan nor dextran stimulated CLEC-2 or GPVI transfected
DT40 cells or bound directly to either of the recombinant receptors.
Conclusions:
This
study demonstrates that fucoidan and dextran have the common ability to
activate the CLEC-2 receptor and, to a lesser extent, the GPVI-FcR γ-chain
complex in the absence of direct binding to either receptor. While both
polysaccharides can activate other, unidentified Src kinase coupled
receptors, only dextran causes aggregation via this mechanism (in human
platelets). Given the absence of direct binding, it is likely that activation
is mediated by alterations in receptor location or interactions of the
stimuli with the membrane bilayer.
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المؤتمر (3):
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عنوان المؤتمر:
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ISTH 2015 Congress
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تاريخ الإنعقاد:
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20/06/2015
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مكان
الإنعقاد:
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Toronto, Canada
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طبيعة المشاركة:
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Paper presentation
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عنوان المشاركة:
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Fibrin supports thrombus growth through activation
of GPVI
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ملخص المشاركة:
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Background:
Fibrin is formed from soluble fibrinogen by the
action of thrombin. As the final product of the coagulation cascade, it plays
a crucial role in the formation of stable thrombi. Glycoprotein (GP) VI, the
major collagen receptor, has been shown to play a role in embolization of
thrombi, which is paradoxical as collagen is only found at the base of a
thrombus.
Aims:
We hypothesise that GPVI may be a novel fibrin
receptor.
Methods:
Protein tyrosine phosphorylation, and spreading of
human and mouse platelets was investigated using established techniques.
Results:
Thrombin stimulated a marked in tyrosine
phosphorylation in washed human platelets that were allowed to aggregate.
Tyrosine phosphorylation was reduced in the presence of the aIIbb3 blocker, eptifibatide,
and blocked in the presence of GPRP (single amino acid code), which blocks
fibrin formation. Immunoprecipitation and Western blotting studies identified
Syk and the FcR g-chain as major phosphorylated proteins. Similar results
were observed in washed mouse platelets. An increase in tyrosine
phosphorylation including in response to thrombin was also observed in GPIIb
deficient mice platelets which was also blocked by GPRP. This increase was
absent in GPVI-FcR g-chain-deficient mouse platelets. Moreover, platelets
spread on fibrin-coated surfaces to a greater extent than those on
fibrinogen-coated surfaces. This was not the case for GPVI-deficient
platelets or in the presence of a Src kinase inhibitor.
Conclusion:
These results show that fibrin activates GPVI
independently of aIIbb3. This may explain the increase in embolization in the
absence of GPVI at arterial rates of shear.
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