scholarly journals TULA-2 Deficiency Enhances Platelet Functional Responses to CLEC-2 Agonists

TH Open ◽  
2018 ◽  
Vol 02 (04) ◽  
pp. e411-e419 ◽  
Author(s):  
John Kostyak ◽  
Benjamin Mauri ◽  
Carol Dangelmaier ◽  
Akruti Patel ◽  
Yuhang Zhou ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Tyrosine Phosphatase ◽  
Negative Regulator ◽  
Functional Responses ◽  
Protease Activated Receptors ◽  
Surface Receptors ◽  
Downstream Signaling ◽  
Glycoprotein Vi ◽  
Thromboxane Production ◽  
Activation Motif

AbstractPlatelet activation is essential for hemostasis. Central to platelet activation are the signals transmitted through surface receptors such as glycoprotein VI, the protease-activated receptors, and C-type lectin-like receptor 2 (CLEC-2). CLEC-2 is a HemITAM (hem-immunoreceptor tyrosine activation motif)-bearing receptor that binds podoplanin and signals through spleen tyrosine kinase (Syk). T-cell ubiquitin ligand-2 (TULA-2) is a protein tyrosine phosphatase that is highly expressed in platelets and targets phosphorylated Y352 of Syk. We wanted to determine whether TULA-2 regulates Syk phosphorylation and activity downstream of CLEC-2. To that end, we used TULA-2 knockout mice and wild-type (WT) littermate controls. We found that TULA-2 deficiency enhances the aggregation and secretion response following stimulation with an excitatory CLEC-2 antibody or the CLEC-2 agonist rhodocytin. Consistently, Syk phosphorylation of Y346 is enhanced, as well as phosphorylation of the downstream signaling molecule PLCγ2, in TULA-2 knockout platelets treated with either CLEC-2 antibody or rhodocytin, compared with WT control platelets. Furthermore, the kinetics of Syk phosphorylation, as well as that of PLCγ2 and SLP-76, is enhanced in TULA-2 knockout platelets treated with 2.5-μg/mL CLEC-2 antibody compared with WT platelets. Similarly, thromboxane production was enhanced, in both amount and kinetics, in TULA-2−/− platelets treated with 2.5-μg/mL CLEC-2 antibody. TULA-2 acts as a negative regulator of CLEC-2 signaling by dephosphorylating Syk on Y346 and restraining subsequent Syk-mediated signaling.

scholarly journals Diacylglycerol kinase ζ is a negative regulator of GPVI-mediated platelet activation

2019 ◽  
Vol 3 (7) ◽  
pp. 1154-1166 ◽  
Author(s):  
Alyssa J. Moroi ◽  
Nicole M. Zwifelhofer ◽  
Matthew J. Riese ◽  
Debra K. Newman ◽  
Peter J. Newman
Keyword(s):  
Platelet Activation ◽  
Bleeding Time ◽  
Thrombus Formation ◽  
Negative Regulator ◽  
Platelet Surface ◽  
Rotational Thromboelastometry ◽  
Surface Receptors ◽  
Glycoprotein Vi ◽  
Granule Secretion

Abstract Diacylglycerol kinases (DGKs) are a family of enzymes that convert diacylglycerol (DAG) into phosphatidic acid (PA). The ζ isoform of DGK (DGKζ) has been reported to inhibit T-cell responsiveness by downregulating intracellular levels of DAG. However, its role in platelet function remains undefined. In this study, we show that DGKζ was expressed at significant levels in both platelets and megakaryocytes and that DGKζ-knockout (DGKζ-KO) mouse platelets were hyperreactive to glycoprotein VI (GPVI) agonists, as assessed by aggregation, spreading, granule secretion, and activation of relevant signal transduction molecules. In contrast, they were less responsive to thrombin. Platelets from DGKζ-KO mice accumulated faster on collagen-coated microfluidic surfaces under conditions of arterial shear and stopped blood flow faster after ferric chloride–induced carotid artery injury. Other measures of hemostasis, as measured by tail bleeding time and rotational thromboelastometry analysis, were normal. Interestingly, DGKζ deficiency led to increased GPVI expression on the platelet and megakaryocyte surfaces without affecting the expression of other platelet surface receptors. These results implicate DGKζ as a novel negative regulator of GPVI-mediated platelet activation that plays an important role in regulating thrombus formation in vivo.

scholarly journals A novel histidine tyrosine phosphatase, TULA-2, associates with Syk and negatively regulates GPVI signaling in platelets

Blood ◽  
2010 ◽  
Vol 116 (14) ◽  
pp. 2570-2578 ◽  
Author(s):  
Dafydd H. Thomas ◽  
Todd M. Getz ◽  
Tiffanny N. Newman ◽  
Carol A. Dangelmaier ◽  
Nick Carpino ◽  
...  
Keyword(s):  
T Cell ◽  
Phospholipase C ◽  
Tyrosine Phosphatase ◽  
Negative Regulator ◽  
Platelet Glycoprotein ◽  
Functional Responses ◽  
Glycoprotein Vi ◽  
Downstream Effector

Abstract T-cell ubiquitin ligand-2 (TULA-2) is a recently discovered histidine tyrosine phosphatase thought to be ubiquitously expressed. In this work, we have investigated whether TULA-2 has a key role in platelet glycoprotein VI (GPVI) signaling. This study indicates that TULA-2 is expressed in human and murine platelets and is able to associate with Syk and dephosphorylate it. Ablation of TULA-2 resulted in hyperphosphorylation of Syk and its downstream effector phospholipase C-γ2 as well as enhanced GPVI-mediated platelet functional responses. In addition, shorter bleeding times and a prothrombotic phenotype were observed in mice lacking TULA-2. We therefore propose that TULA-2 is the primary tyrosine phosphatase mediating the dephosphorylation of Syk and thus functions as a negative regulator of GPVI signaling in platelets.

Fyn and Lyn phosphorylate the Fc receptor γ chain downstream of glycoprotein VI in murine platelets, and Lyn regulates a novel feedback pathway

Blood ◽  
2000 ◽  
Vol 96 (13) ◽  
pp. 4246-4253 ◽  
Author(s):  
Lynn S. Quek ◽  
Jean-Max Pasquet ◽  
Ingeborg Hers ◽  
Richard Cornall ◽  
Graham Knight ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Fc Receptor ◽  
Inhibitory Pathway ◽  
Functional Responses ◽  
Glycoprotein Vi ◽  
Granule Secretion ◽  
Activation Motif ◽  
Additional Role ◽  
Feedback Pathway

Abstract Activation of platelets by collagen is mediated by the complex glycoprotein VI (GPVI)/Fc receptor γ (FcRγ chain). In the current study, the role of 2 Src family kinases, Fyn and Lyn, in GPVI signaling has been examined using murine platelets deficient in one or both kinases. In the fyn−/−platelets, tyrosine phosphorylation of FcRγ chain, phopholipase C (PLC) activity, aggregation, and secretion are reduced, though the time of onset of response is unchanged. In the lyn−/−platelets, there is a delay of up to 30 seconds in the onset of tyrosine phosphorylation and functional responses, followed by recovery of phosphorylation and potentiation of aggregation and α-granule secretion. Tyrosine phosphorylation and aggregation in response to stimulation by collagen-related peptide is further attenuated and delayed in fyn−/−lyn−/−double-mutant platelets, and potentiation is not seen. This study provides the first genetic evidence that Fyn and Lyn mediate FcR immune receptor tyrosine-based activation motif phosphorylation and PLCγ2 activation after the ligation of GPVI. Lyn plays an additional role in inhibiting platelet activation through an uncharacterized inhibitory pathway.

scholarly journals The tyrosine phosphatase CD148 is an essential positive regulator of platelet activation and thrombosis

Blood ◽  
2009 ◽  
Vol 113 (20) ◽  
pp. 4942-4954 ◽  
Author(s):  
Yotis A. Senis ◽  
Michael G. Tomlinson ◽  
Stuart Ellison ◽  
Alexandra Mazharian ◽  
Jenson Lim ◽  
...  
Keyword(s):  
G Protein ◽  
Platelet Activation ◽  
Drug Target ◽  
Tyrosine Phosphatase ◽  
Underlying Mechanism ◽  
G Protein Coupled Receptors ◽  
Bleeding Tendency ◽  
Platelet Surface ◽  
Surface Receptors ◽  
G Protein Coupled

Abstract Platelets play a fundamental role in hemostasis and thrombosis. They are also involved in pathologic conditions resulting from blocked blood vessels, including myocardial infarction and ischemic stroke. Platelet adhesion, activation, and aggregation at sites of vascular injury are regulated by a diverse repertoire of tyrosine kinase–linked and G protein–coupled receptors. Src family kinases (SFKs) play a central role in initiating and propagating signaling from several platelet surface receptors; however, the underlying mechanism of how SFK activity is regulated in platelets remains unclear. CD148 is the only receptor-like protein tyrosine phosphatase identified in platelets to date. In the present study, we show that mutant mice lacking CD148 exhibited a bleeding tendency and defective arterial thrombosis. Basal SFK activity was found to be markedly reduced in CD148-deficient platelets, resulting in a global hyporesponsiveness to agonists that signal through SFKs, including collagen and fibrinogen. G protein–coupled receptor responses to thrombin and other agonists were also marginally reduced. These results highlight CD148 as a global regulator of platelet activation and a novel antithrombotic drug target.

scholarly journals Impaired GPVI Mediated Platelet Functional Responses and Signaling in CD45 Knockout Mice

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1355-1355
Author(s):  
Vaishali Inamdar ◽  
John Kostyak ◽  
Rachit Badolia ◽  
Carol Dangelmaier ◽  
Soochong Kim ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Conflicts Of Interest ◽  
Tyrosine Phosphatase ◽  
Dense Granule ◽  
Receptor Protein ◽  
Functional Responses ◽  
Platelet Surface ◽  
Glycoprotein Vi ◽  
Terminal Domain ◽  
Deficient Mice

Abstract Background: CD45 is a Receptor Protein Tyrosine Phosphatase C (PTPRC) and regulates Src Family Kinases activation in Lymphocytes. Although it is known to be absent from the platelet surface, proteomics studies prove that the CD45 c-terminal catalytic domain is present in platelets. Thus the aim of this study is to identify presence of CD45 c-terminal domain in platelets and characterize the functional implications of CD45 deficiency in platelets using a global CD45 knockout mouse. Results: Platelets from CD45-deficient mice displayed a selective impairment of aggregation and dense granule secretion mediated by the collagen receptor Glycoprotein VI. CD45 deficient mice show increased bleeding times, indicating an important role for CD45 in hemostasis. However, there was no difference observed in thrombus generation and thrombus stability using the ferric chloride-induced carotid artery injury model. Signaling downstream of the GPVI receptor, indicated by Src Family Kianse (SFK), Syk and Phospholipase C_2 (PLCg2) tyrosine phosphorylation, was also impaired. In order to establish the presence of CD45 in platelets we used an established primary antibody that recognizes the c-terminal domain of CD45. We observed that this antibody recognized a protein of approximately 65 kDa, which is the expected size of the c-terminal 1 and 2 domains of CD45, in wild type (WT) mice but not in knockout (KO)mice. Conclusion: Thus we conclude that CD45 is expressed in platelets as a truncated form, possibly generated by proteolytic cleavage, and regulates GPVI signaling, through regulation of Src Family Kinase activation. Figure Figure. Disclosures No relevant conflicts of interest to declare.

scholarly journals In-depth PtdIns(3,4,5)P3 signalosome analysis identifies DAPP1 as a negative regulator of GPVI-driven platelet function

2017 ◽  
Vol 1 (14) ◽  
pp. 918-932 ◽  
Author(s):  
Tom N. Durrant ◽  
James L. Hutchinson ◽  
Kate J. Heesom ◽  
Karen E. Anderson ◽  
Len R. Stephens ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Platelet Function ◽  
Binding Proteins ◽  
Thrombus Formation ◽  
Negative Regulator ◽  
Valuable Resource ◽  
Future Studies ◽  
Glycoprotein Vi ◽  
Key Points ◽  
Depth Analysis

Key Points We present the first in-depth analysis of platelet PtdIns(3,4,5)P3-binding proteins, providing a valuable resource for future studies. The PtdIns(3,4,5)P3-binding protein, DAPP1, negatively regulates glycoprotein VI–driven platelet activation and thrombus formation.

Fyn and Lyn phosphorylate the Fc receptor γ chain downstream of glycoprotein VI in murine platelets, and Lyn regulates a novel feedback pathway

Blood ◽  
2000 ◽  
Vol 96 (13) ◽  
pp. 4246-4253 ◽  
Author(s):  
Lynn S. Quek ◽  
Jean-Max Pasquet ◽  
Ingeborg Hers ◽  
Richard Cornall ◽  
Graham Knight ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Fc Receptor ◽  
Inhibitory Pathway ◽  
Functional Responses ◽  
Glycoprotein Vi ◽  
Granule Secretion ◽  
Activation Motif ◽  
Additional Role ◽  
Feedback Pathway

Activation of platelets by collagen is mediated by the complex glycoprotein VI (GPVI)/Fc receptor γ (FcRγ chain). In the current study, the role of 2 Src family kinases, Fyn and Lyn, in GPVI signaling has been examined using murine platelets deficient in one or both kinases. In the fyn−/−platelets, tyrosine phosphorylation of FcRγ chain, phopholipase C (PLC) activity, aggregation, and secretion are reduced, though the time of onset of response is unchanged. In the lyn−/−platelets, there is a delay of up to 30 seconds in the onset of tyrosine phosphorylation and functional responses, followed by recovery of phosphorylation and potentiation of aggregation and α-granule secretion. Tyrosine phosphorylation and aggregation in response to stimulation by collagen-related peptide is further attenuated and delayed in fyn−/−lyn−/−double-mutant platelets, and potentiation is not seen. This study provides the first genetic evidence that Fyn and Lyn mediate FcR immune receptor tyrosine-based activation motif phosphorylation and PLCγ2 activation after the ligation of GPVI. Lyn plays an additional role in inhibiting platelet activation through an uncharacterized inhibitory pathway.

scholarly journals Braking platelet activation: deactivating the receptors

Blood ◽  
2008 ◽  
Vol 111 (1) ◽  
pp. 3-3
Author(s):  
Koneti A. Rao
Keyword(s):  
Platelet Activation ◽  
Cytoplasmic Tail ◽  
Proteolytic Cleavage ◽  
Fc Receptor ◽  
Down Regulation ◽  
Platelet Surface ◽  
Surface Receptors ◽  
Activation Motif

Glycoprotein (GP) VI and Fc receptor FcγRIIa are 2 ITAM (immunoreceptor tyrosine-activation motif)–bearing platelet surface receptors. In this issue of Blood, Gardiner and colleagues show that activation of either receptor results in simultaneous proteolytic cleavage of GPVI ectodomain and FcγRIIa cytoplasmic tail, providing distinct mechanisms for their down-regulation on platelet activation.

scholarly journals Src family kinases: at the forefront of platelet activation

Blood ◽  
2014 ◽  
Vol 124 (13) ◽  
pp. 2013-2024 ◽  
Author(s):  
Yotis A. Senis ◽  
Alexandra Mazharian ◽  
Jun Mori
Keyword(s):  
Platelet Activation ◽  
Src Family Kinases ◽  
Cytoskeletal Proteins ◽  
Receptor Complex ◽  
Platelet Surface ◽  
Surface Receptors ◽  
Von Willebrand ◽  
Activation Motif ◽  
Willebrand Factor ◽  
Primary Activation

Abstract Src family kinases (SFKs) play a central role in mediating the rapid response of platelets to vascular injury. They transmit activation signals from a diverse repertoire of platelet surface receptors, including the integrin αIIbβ3, the immunoreceptor tyrosine–based activation motif–containing collagen receptor complex GPVI-FcR γ-chain, and the von Willebrand factor receptor complex GPIb-IX-V, which are essential for thrombus growth and stability. Ligand-mediated clustering of these receptors triggers an increase in SFK activity and downstream tyrosine phosphorylation of enzymes, adaptors, and cytoskeletal proteins that collectively propagate the signal and coordinate platelet activation. A growing body of evidence has established that SFKs also contribute to Gq- and Gi-coupled receptor signaling that synergizes with primary activation signals to maximally activate platelets and render them prothrombotic. Interestingly, SFKs concomitantly activate inhibitory pathways that limit platelet activation and thrombus size. In this review, we discuss past discoveries that laid the foundation for this fundamental area of platelet signal transduction, recent progress in our understanding of the distinct and overlapping functions of SFKs in platelets, and new avenues of research into mechanisms of SFK regulation. We also highlight the thrombotic and hemostatic consequences of targeting platelet SFKs.

scholarly journals CEACAM1 negatively regulates platelet-collagen interactions and thrombus growth in vitro and in vivo

Blood ◽  
2009 ◽  
Vol 113 (8) ◽  
pp. 1818-1828 ◽  
Author(s):  
Cyndi Wong ◽  
Yong Liu ◽  
Jana Yip ◽  
Rochna Chand ◽  
Janet L. Wee ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Tyrosine Phosphatase ◽  
Negative Regulator ◽  
Surface Glycoprotein ◽  
Type I ◽  
Wild Type ◽  
Glycoprotein Vi ◽  
Selective Ligand

Abstract Carcinoembryonic antigen cell adhesion molecule-1 (CEACAM1) is a surface glycoprotein expressed on various blood cells, epithelial cells, and vascular cells. CEACAM1 possesses adhesive and signaling properties mediated by its intrinsic immunoreceptor tyrosine-based inhibitory motifs that recruit SHP-1 protein-tyrosine phosphatase. In this study, we demonstrate that CEACAM1 is expressed on the surface and in intracellular pools of platelets. In addition, CEACAM1 serves to negatively regulate signaling of platelets by collagen through the glycoprotein VI (GPVI)/Fc receptor (FcR)–γ-chain. ceacam1−/− platelets displayed enhanced type I collagen and GPVI-selective ligand, collagen-related peptide (CRP), CRP-mediated platelet aggregation, enhanced platelet adhesion on type I collagen, and elevated CRP-mediated alpha and dense granule secretion. Platelets derived from ceacam1−/− mice form larger thrombi when perfused over a collagen matrix under arterial flow compared with wild-type mice. Furthermore, using intravital microscopy to ferric chloride-injured mesenteric arterioles, we show that thrombi formed in vivo in ceacam1−/− mice were larger and were more stable than those in wild-type mice. GPVI depletion using monoclonal antibody JAQ1 treatment of ceacam1−/− mice showed a reversal in the more stable thrombus growth phenotype. ceacam1−/− mice were more susceptible to type I collagen–induced pulmonary thromboembolism than wild-type mice. Thus, CEACAM1 acts as a negative regulator of platelet-collagen interactions and of thrombus growth involving the collagen GPVI receptor in vitro and in vivo.

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