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 Impaired Glycoprotein VI-Mediated Signaling and Platelet Functional Responses in CD45 Knockout Mice

2019 ◽  
Vol 119 (08) ◽  
pp. 1321-1331
Author(s):  
Vaishali V. Inamdar ◽  
John C. Kostyak ◽  
Rachit Badolia ◽  
Carol A. Dangelmaier ◽  
Bhanu Kanth Manne ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Tyrosine Phosphatase ◽  
Receptor Protein ◽  
Functional Responses ◽  
Glycoprotein Vi ◽  
Extracellular Region ◽  
C Terminus

Background and Objective CD45 is a receptor protein tyrosine phosphatase present on the surface of all hematopoietic cells except for erythrocytes and platelets. Proteomics studies, however, have demonstrated the presence of a CD45 c-terminal catalytic peptide in platelets. Therefore, we investigated the functional role of this truncated isoform of CD45 in platelets, which contains the c-terminal catalytic domain but lacks the extracellular region. Methods and Results We used an antibody specific to the c-terminus of CD45 to confirm the presence of a truncated CD45 isoform in platelets. We also examined ex vivo and in vivo platelet function using CD45 knockout (KO) mice. Aggregation and secretion mediated by the glycoprotein VI (GPVI) receptor was impaired in CD45 KO platelets. Consequently, CD45 KO mice had impaired hemostasis indicated by increased tail bleeding times. Also, using a model of pulmonary embolism we showed that CD45 KO mice had defective in vivo thrombus formation. Next, we investigated whether or not the truncated isoform of CD45 had a role in GPVI signaling. The full-length isoform of CD45 is known to regulate Src family kinase (SFK) activation in lymphocytes. We find a similar role for the truncated isoform of CD45 in platelets. SFK activation was impaired downstream of the GPVI receptor in the CD45 KO murine platelets. Consequently, Syk, PLCγ2, and pleckstrin phosphorylations were also impaired in CD45 KO murine platelets. Conclusion We conclude that the truncated CD45 isoform regulates GPVI-mediated signaling and platelet functional responses by regulating SFK activation.

scholarly journals The low-frequency allele of the platelet collagen signaling receptor glycoprotein VI is associated with reduced functional responses and expression

Blood ◽  
2003 ◽  
Vol 101 (11) ◽  
pp. 4372-4379 ◽  
Author(s):  
Lotta Joutsi-Korhonen ◽  
Peter A. Smethurst ◽  
Angela Rankin ◽  
Elaine Gray ◽  
Martin IJsseldijk ◽  
...  
Keyword(s):  
Platelet Function ◽  
Polyclonal Antibodies ◽  
Low Frequency ◽  
Direct Interaction ◽  
Annexin V ◽  
Functional Difference ◽  
Functional Responses ◽  
Platelet Surface ◽  
Glycoprotein Vi ◽  
The Difference

Abstract Interaction of platelets with collagen under conditions of blood flow is a multi-step process with tethering via glycoprotein IbIXV (GPIbIXV) over von Willebrand factor, adhesion by direct interaction with the integrin GPIaIIa, and signaling via GPVI. GPVI can be specifically agonized by cross-linked collagen-related peptide (CRP-XL), which results in a signaling cascade very similar to that evoked by native collagen. The GPVI gene has 2 common alleles that differ by 3 replacements in the glycosylated stem and 2 in the cytoplasmic domain. We used CRP-XL to elucidate the variation in responses observed in platelet function in different individuals. We observed a 3-fold difference in the response to CRP-XL in platelet aggregation when comparing platelets from 10 high-frequency allele homozygotes with 8 low-frequency ones (2-way analysis of variance [ANOVA], P < .0001). The difference in functional responses was reflected in fibrinogen binding and in downstream signaling events as measured by tyrosine phosphorylation, the expression of P-selectin, and the binding of annexin V and the generation of thrombin on the platelet surface (2-way ANOVA, P < .001). Platelets homozygous for the low-frequency allele tended to be less able to form a thrombus on a collagen surface in flowing whole blood or in the platelet function analyzer–100 (t test, P = .065 and P = .061, respectively). The functional difference was correlated to a difference in total and membrane-expressed GPVI measured by monoclonal and polyclonal antibodies. This study demonstrates for the first time that platelet function may be altered by allelic differences in GPVI.

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.

scholarly journals No Obvious Abnormality in Mice Deficient in Receptor Protein Tyrosine Phosphatase β

2000 ◽  
Vol 20 (20) ◽  
pp. 7706-7715 ◽  
Author(s):  
S. Harroch ◽  
M. Palmeri ◽  
J. Rosenbluth ◽  
A. Custer ◽  
M. Okigaki ◽  
...  
Keyword(s):  
Nervous System ◽  
Neurite Outgrowth ◽  
Tyrosine Phosphatase ◽  
Protein Tyrosine Phosphatases ◽  
Structural Features ◽  
Receptor Protein ◽  
Protein Tyrosine ◽  
Deficient Mice

ABSTRACT The development of neurons and glia is governed by a multitude of extracellular signals that control protein tyrosine phosphorylation, a process regulated by the action of protein tyrosine kinases and protein tyrosine phosphatases (PTPs). Receptor PTPβ (RPTPβ; also known as PTPζ) is expressed predominantly in the nervous system and exhibits structural features common to cell adhesion proteins, suggesting that this phosphatase participates in cell-cell communication. It has been proposed that the three isoforms of RPTPβ play a role in regulation of neuronal migration, neurite outgrowth, and gliogenesis. To investigate the biological functions of this PTP, we have generated mice deficient in RPTPβ. RPTPβ-deficient mice are viable, are fertile, and showed no gross anatomical alterations in the nervous system or other organs. In contrast to results of in vitro experiments, our study demonstrates that RPTPβ is not essential for neurite outgrowth and node formation in mice. The ultrastructure of nerves of the central nervous system in RPTPβ-deficient mice suggests a fragility of myelin. However, conduction velocity was not altered in RPTPβ-deficient mice. The normal development of neurons and glia in RPTPβ-deficient mice demonstrates that RPTPβ function is not necessary for these processes in vivo or that loss of RPTPβ can be compensated for by other PTPs expressed in the nervous system.

Assessment of the effects of Syk and BTK inhibitors on GPVI-mediated platelet signaling and function

2021 ◽  
Author(s):  
Tony J. Zheng ◽  
Elizabeth R. Lofurno ◽  
Alexander R. Melrose ◽  
Hari Hara Sudhan Lakshmanan ◽  
Jiaqing Pang ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Platelet Glycoprotein ◽  
Critical Systems ◽  
Functional Responses ◽  
Platelet Surface ◽  
Glycoprotein Vi ◽  
Platelet Signaling ◽  
Granule Secretion ◽  
And Function ◽  
Btk Inhibitors

Spleen tyrosine kinase (Syk) and Bruton's tyrosine kinase (BTK) play critical roles in platelet physiology, facilitating ITAM-mediated signaling downstream of platelet glycoprotein VI (GPVI) and GPIIb/IIIa receptors. Small molecule tyrosine kinase inhibitors (TKIs) targeting Syk and BTK have been developed as anti-neoplastic and anti-inflammatory therapeutics and have also gained interest as anti-platelet agents. Here, we investigate the effects of 12 different Syk and BTK inhibitors on GPVI-mediated platelet signaling and function. These inhibitors include, four Syk inhibitors, Bay 61-3606, R406 (fostamatinib), entospletinib, TAK-659, four irreversible BTK inhibitors, ibrutinib, acalabrutinib, ONO-4059 (tirabrutinib), AVL-292 (spebrutinib), and four reversible BTK inhibitors, CG-806, BMS-935177, BMS-986195, and fenebrutinib. In vitro, TKIs targeting Syk or BTK reduced platelet adhesion to collagen, dense granule secretion, and alpha granule secretion in response to the GPVI agonist CRP-XL. Similarly, these TKIs reduced the percentage of activated integrin αIIbβ3 on the platelet surface in response to CRP-XL, as determined by PAC-1 binding. While all TKIs tested inhibited PLCγ2 phosphorylation following GPVI-mediated activation, other downstream signaling events proximal to PI3K and PKC were differentially affected. In addition, reversible BTK inhibitors had less pronounced effects on GPIIb/IIIa-mediated platelet spreading on fibrinogen and differentially altered the organization of PI3K around microtubules during platelets spreading on fibrinogen. Select TKIs also inhibited platelet aggregate formation on collagen under physiological flow conditions. Together, our results suggest that TKIs targeting Syk or BTK inhibit central platelet functional responses but may differentially affect protein activities and organization in critical systems downstream of Syk and BTK in platelets.

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 Receptor-Like Protein Tyrosine Phosphatase α Homodimerizes on the Cell Surface

2000 ◽  
Vol 20 (16) ◽  
pp. 5917-5929 ◽  
Author(s):  
Guoqiang Jiang ◽  
Jeroen den Hertog ◽  
Tony Hunter
Keyword(s):  
Biological Activity ◽  
Cell Surface ◽  
Protein Tyrosine Phosphatase ◽  
Intracellular Signaling ◽  
Catalytic Domain ◽  
Transmembrane Domain ◽  
Tyrosine Phosphatase ◽  
Receptor Protein ◽  
Dependent Manner ◽  
Protein Tyrosine

ABSTRACT We reported previously that the N-terminal D1 catalytic domain of receptor protein-tyrosine phosphatase α (RPTPα) forms a symmetrical, inhibited dimer in a crystal structure, in which a helix-turn-helix wedge element from one monomer is inserted into the catalytic cleft of the other monomer. Previous functional studies also suggested that dimerization inhibits the biological activity of a CD45 chimeric RPTP and the catalytic activity of an isolated RPTPς D1 catalytic domain. Most recently, we have also shown that enforced dimerization inhibits the biological activity of full-length RPTPα in a wedge-dependent manner. The physiological significance of such inhibition is unknown, due to a lack of understanding of how RPTPα dimerization is regulated in vivo. In this study, we show that transiently expressed cell surface RPTPα exists predominantly as homodimers, suggesting that dimerization-mediated inhibition of RPTPα biological activity is likely to be physiologically relevant. Consistent with our published and unpublished crystallographic data, we show that mutations in the wedge region of D1 catalytic domain and deletion of the entire D2 catalytic domain independently reduced but did not abolish RPTPα homodimerization, suggesting that both domains are critically involved but that neither is essential for homodimerization. Finally, we also provide evidence that both the RPTPα extracellular domain and the transmembrane domain were independently able to homodimerize. These results lead us to propose a zipper model in which inactive RPTPα dimers are stabilized by multiple, relatively weak dimerization interfaces. Dimerization in this manner would provide a potential mechanism for negative regulation of RPTPα. Such RPTPα dimers could be activated by extracellular ligands or intracellular binding proteins that induce monomerization or by intracellular signaling events that induce an open conformation of the dimer.

A Novel Histidine Tyrosine Phosphatase, TULA-2, Associates with Syk and Negatively Regulates GPVI Signaling in Platelets.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1843-1843
Author(s):  
Dafydd H Thomas ◽  
Carol Dangelmaier ◽  
Jianguo Jin ◽  
Alexander Tsygankov ◽  
Satya P. Kunapuli ◽  
...  
Keyword(s):  
T Cell ◽  
Protein Tyrosine Kinase ◽  
Tyrosine Phosphatase ◽  
Dense Granule ◽  
Negative Regulation ◽  
Negative Regulator ◽  
Signaling Cascade ◽  
Glycoprotein Vi ◽  
Platelet Receptor

Abstract Glycoprotein VI (GPVI) is the primary platelet receptor for collagen signaling. Following damage to the vascular endothelium, the GPVI receptor interacts with the exposed sub-endothelial collagen. This interaction initiates a signaling cascade involving phosphorylation of the dual ITAM motif of the FcRγ chain by Fyn and Lyn, followed by the recruitment, phosphorylation and activation of Syk. This leads to the eventual activation of PLCγ2 and the release of calcium from intracellular stores to cause platelet activation. While a lot is known about the activation processes involved in GPVI signaling less is known about its negative regulation. The T-cell ubiquitin ligand (TULA) family of proteins has been implicated in the negative regulation protein tyrosine kinase (PTK)-dependent signaling pathways. More recently, it has been shown the TULA family member, TULA-2, exhibits phosphatase activity towards PTKs, including Syk, and this activity is responsible for the negative regulation of T-cell receptor signaling (Mikhailik et. al. 2007, Agrawal et. al. 2008). Thus, we investigated the role of TULA-2 in the negative regulation of the GPVI signaling cascade. We show that TULA-2 is expressed in both human and murine platelets. Deletion of TULA-2 in murine platelets manifests itself functionally as enhanced aggregation in response to the GPVI agonist convulxin as well as enhanced dense granule secretion when compared to wild type platelets. No difference was witnessed in response to the PAR4 agonist AYPGKF. TULA-2-deficient platelets also exhibit sustained hyperphosphorylation of Syk at tyrosines 525 and 526 as well as hyperphosphorylation of PLCγ2 at tyrosines 753 and 759, indicative of enhanced kinase and phospholipase activity respectively. GST-pulldown experiments suggest that Syk and TULA- 2 are able to associate in resting and convulxin stimulated platelets and in-vitro phosphatase assays demonstrate that TULA-2 can dephosphorylate Syk at tyrosines 525 and 526. Taken together, these data suggest that TULA-2 is a negative regulator of GPVI signaling and this regulation is mediated by an association of TULA-2 with Syk, allowing the dephosphorylation of Syk at catalytically important tyrosine residues.

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