Syk-dependent Phosphorylation of CLEC-2

Séverin, Sonia; Pollitt, Alice Y.; Navarro-Núñez, Leyre; Nash, Craig; Mourão-Sá, Diego; Eble, Johannes A.; Senis, Yotis A.; Watson, Steve P.

doi:10.1074/jbc.m110.167502

Cited by 96 publications

(38 citation statements)

References 42 publications

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“…34 However, there is also a significant difference: in GPVI-stimulated platelets, SFKs initiate signaling through phosphorylation of the FcRγ-associated ITAMs, leading to binding and activation of Syk; 20, 21 in contrast, signaling through CLEC-2 depends on phosphorylation of CLEC-2 by Syk in an SFK-independent manner. 35 Because DUSP3 deficiency limits platelet activation in response to both GPVI and CLEC-2 stimulation, SFK function is likely not controlled by DUSP3, which is also supported by our data showing that pY in SFKs is not altered in Dusp3 -KO platelets. On the contrary, Syk may be directly or indirectly targeted by DUSP3.…”

Section: Discussionsupporting

confidence: 80%

Dual-Specificity Phosphatase 3 Deficiency or Inhibition Limits Platelet Activation and Arterial Thrombosis

Musumeci¹,

Kuijpers²,

Gilio³

et al. 2015

Circulation

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Background A limitation of current antiplatelet therapies is their inability to separate thrombotic events from bleeding occurrences. Better understanding of the molecular mechanisms leading to platelet activation is of importance for the development of improved therapies. Recently, protein tyrosine phosphatases (PTPs) have emerged as critical regulators of platelet function. Methods and Results This is the first report implicating the dual-specificity phosphatase 3 (DUSP3) in platelet signaling and thrombosis. This phosphatase is highly expressed in human and mouse platelets. Platelets from DUSP3-deficient mice displayed a selective impairment of aggregation and granule secretion mediated through the collagen receptor glycoprotein VI (GPVI) and the C-type lectin-like receptor 2 (CLEC-2). DUSP3-deficient mice were more resistant to collagen- and epinephrine-induced thromboembolism, compared to wild-type mice, and showed severely impaired thrombus formation upon ferric chloride-induced carotid artery injury. Intriguingly, bleeding times were not altered in DUSP3-deficient mice. At the molecular level, DUSP3 deficiency impaired Syk tyrosine phosphorylation, subsequently reducing phosphorylation of PLCγ2 and calcium fluxes. To investigate DUSP3 function in human platelets, a novel small-molecule inhibitor of DUSP3 was developed. This compound specifically inhibited collagen and CLEC-2-induced human platelet aggregation, thereby phenocopying the effect of DUSP3 deficiency in murine cells. Conclusions DUSP3 plays a selective and essential role in collagen- and CLEC-2-mediated platelet activation and thrombus formation in vivo. Inhibition of DUSP3 may prove therapeutic for arterial thrombosis. This is the first time a PTP, implicated in platelet signaling, has been targeted with a small-molecule drug.

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Section: Discussionsupporting

confidence: 80%

Dual-Specificity Phosphatase 3 Deficiency or Inhibition Limits Platelet Activation and Arterial Thrombosis

Musumeci¹,

Kuijpers²,

Gilio³

et al. 2015

Circulation

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“…23 Similar agonist and signaling pathway-dependent differences in Bl1002494 efficacy were observed in various immune cells. 20 Furthermore, GPVI and CLEC-2 signaling differ in the sequential order of Src family kinases and Syk, 6 which could explain the different potencies of BI1002494 for inhibiting GPVI and CLEC-2 signaling and thereby the unaltered tail bleeding times in inhibitor-treated mice compared with the slightly prolonged bleeding times in Syk-deficient mice. However, given previous reports that the concomitant lack of GPVI and CLEC-2 severely compromises hemostasis, 18 this could also point toward signaling-independent functions of the 2 (hem)ITAM receptors in hemostasis.…”

Section: Discussionmentioning

confidence: 99%

“…Flow cytometric analyses revealed unaltered platelet integrin activation (JON-A/PE binding) and degranulation (P-selectin exposure) in response to G-protein-coupled receptor agonists but abolished responses to GPVI and CLEC-2 agonists collagen-related peptide and rhodocytin, respectively ( Figure 1A and 1B, not shown) confirming previous results. 6,17 Lack of functional Syk also resulted in abolished platelet aggregation in response to GPVI or CLEC-2 agonists ( Figure 1C and not shown). However, outside-in signaling of integrin αIIbβ3 as assessed in a platelet-spreading assay was unaffected in Syk-deficient platelets (Figure 2).…”

Section: Sykmentioning

confidence: 99%

The Novel Oral Syk Inhibitor, Bl1002494, Protects Mice From Arterial Thrombosis and Thromboinflammatory Brain Infarction

Eeuwijk

Stegner

Lamb

et al. 2016

ATVB

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Objective— Ischemic stroke, which is mainly caused by thromboembolic occlusion of brain arteries, is the second leading cause of death and disability worldwide with limited treatment options. The platelet collagen receptor glycoprotein VI (GPVI) is a key player in arterial thrombosis and a critical determinant of stroke outcome, making its signaling pathway an attractive target for pharmacological intervention. The spleen tyrosine kinase (Syk) is an essential signaling mediator downstream of not only GPVI but also other platelet and immune cell receptors. We sought to assess whether Syk might be an effective antithrombotic target. Approach and Results— We demonstrate that mice lacking Syk in platelets specifically are protected from arterial thrombus formation and ischemic stroke but display unaltered hemostasis. Furthermore, we show that mice treated with the novel, selective, and orally bioavailable Syk inhibitor BI1002494 were protected in a model of arterial thrombosis and had smaller infarct sizes and a significantly better neurological outcome 24 hours after transient middle cerebral artery occlusion, also when BI1002494 was administered therapeutically, that is, after ischemia. Conclusions— These results provide direct evidence that pharmacological Syk inhibition might provide a safe therapeutic strategy to prevent arterial thrombosis and to limit infarct progression in acute stroke.

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“…The crystal structure of rhodocytin shows that CLEC-2 receptors are activated through clustering by this tetrameric ligand [20]. The CLEC-2 receptor plays an important role in tumor metastasis [23], hemostasis and thrombosis [16, 24–26]. Unlike GPVI, which has an ITAM, CLEC-2 has a hemi-ITAM sequence that is phosphorylated by Src and Syk tyrosine kinases[21, 26], whereas phosphorylation of the ITAM is mediated solely by Src kinases[27, 28].…”

Section: Introductionmentioning

confidence: 99%

C-type lectin like receptor 2 (CLEC-2) signals independently of lipid raft microdomains in platelets

Manne

Badolia

Dangelmaier

et al. 2015

Biochemical Pharmacology

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C-type lectin like-receptor 2 (CLEC-2) has been reported to activate platelets through a lipid raft-dependent manner. Secreted ADP potentiates CLEC-2-mediated platelet aggregation. We have investigated whether the decrease in CLEC-2-mediated platelet aggregation, previously reported in platelets with disrupted rafts, is a result of the loss of agonist potentiation by ADP. We disrupted platelet lipid rafts with methyl–β-cyclodextrin (MβCD) and measured signaling events downstream of CLEC-2 activation. Lipid raft disruption decreases platelet aggregation induced by CLEC-2 agonists. The inhibition of platelet aggregation by the disruption of lipid rafts was rescued by the exogenous addition of epinephrine but not 2-Methylthioadenosine diphosphate (2MeSADP), which suggests that lipid raft disruption effects P2Y12-mediated Gi activation but not Gz. Phosphorylation of Syk (Y525/526) and PLCγ2 (Y759), were not affected by raft disruption in CLEC-2 agonist-stimulated platelets. Furthermore, tyrosine phosphorylation of the CLEC-2 hemi-ITAM was not effected when MβCD disrupts lipid rafts. Lipid rafts do not directly contribute to CLEC-2 receptor activation in platelets. The effects of disruption of lipid rafts in in vitro assays can be attributed to inhibition of ADP feedback that potentiates CLEC-2 signaling.

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Syk-dependent Phosphorylation of CLEC-2

Cited by 96 publications

References 42 publications

Dual-Specificity Phosphatase 3 Deficiency or Inhibition Limits Platelet Activation and Arterial Thrombosis

Dual-Specificity Phosphatase 3 Deficiency or Inhibition Limits Platelet Activation and Arterial Thrombosis

The Novel Oral Syk Inhibitor, Bl1002494, Protects Mice From Arterial Thrombosis and Thromboinflammatory Brain Infarction

C-type lectin like receptor 2 (CLEC-2) signals independently of lipid raft microdomains in platelets

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