Interaction of Linker for Activation of T Cells with Multiple Adapter Proteins in Platelets Activated by the Glycoprotein VI-selective Ligand, Convulxin
Naoki AsazumaJonathan I. WildeOscar BerlangaMireille LeducAlbrecht LeoEdina SchweighofferVictor L. J. TybulewiczCassian BonStanley K. LiuC. Jane McGladeBurkhart SchravenSteve P. Watson
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The binding of von Willebrand factor (VWF) to the platelet membrane glycoprotein 1b-IX (GP1b-IX) leads to activation of platelets. GP1b was shown to signal via the FcRγ-ITAM (Fc Receptor γ-Immunoreceptor tyrosine-based activation motif) pathway, activating spleen tyrosine kinase (Syk) and other tyrosine kinases. However, there have been conflicting reports regarding the role of Syk in GP1b signaling. In this study, we sought to resolve these conflicting reports and clarify the role of Syk in VWF-induced platelet activation. The inhibition of Syk with the selective Syk inhibitors, OXSI-2 and PRT-060318, did not inhibit VWF-induced platelet adhesion, agglutination, aggregation, or secretion. In contrast, platelets stimulated with the Glycoprotein VI (GPVI) agonist, collagen-related peptide (CRP), failed to cause any aggregation or secretion in presence of the Syk inhibitors. Furthermore, GP1b-induced platelet signaling was unaffected in the presence of Syk inhibitors, but GPVI-induced signaling was abolished under similar conditions. Thus, we conclude that Syk kinase activity does not play any functional role downstream of GP1b-mediated platelet activation.
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Bruton's tyrosine kinase (Btk) and spleen tyrosine kinase (Syk) are major signaling proteins in human platelets that are implicated in atherothrombosis and thrombo-inflammation, but the mechanisms controlling their activities are not well understood. Previously, we showed that Syk becomes phosphorylated at S297 in glycoprotein VI (GPVI)-stimulated human platelets, which limits Syk activation. Here, we tested the hypothesis that protein kinases C (PKC) and A (PKA) and protein phosphatase 2A (PP2A) jointly regulate GPVI-induced Btk activation in platelets. The GPVI agonist convulxin caused rapid, transient Btk phosphorylation at S180 (pS180↑), Y223 and Y551, while direct PKC activation strongly increased Btk pS180 and pY551. This increase in Btk pY551 was also Src family kinase (SFK)-dependent, but surprisingly Syk-independent, pointing to an alternative mechanism of Btk phosphorylation and activation. PKC inhibition abolished convulxin-stimulated Btk pS180 and Syk pS297, but markedly increased the tyrosine phosphorylation of Syk, Btk and effector phospholipase Cγ2 (PLCγ2). PKA activation increased convulxin-induced Btk activation at Y551 but strongly suppressed Btk pS180 and Syk pS297. PP2A inhibition by okadaic acid only increased Syk pS297. Both platelet aggregation and PLCγ2 phosphorylation with convulxin stimulation were Btk-dependent, as shown by the selective Btk inhibitor acalabrutinib. Together, these results revealed in GPVI-stimulated platelets a transient Syk, Btk and PLCγ2 phosphorylation at multiple sites, which are differentially regulated by PKC, PKA or PP2A. Our work thereby demonstrated the GPVI-Syk-Btk signalosome as a tightly controlled protein kinase network, in agreement with its role in atherothrombosis.
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Introduction Src family kinases (SFKs), spleen tyrosine kinase (Syk) and Bruton´s tyrosine kinase (Btk) play central roles in the activation of immune cells and platelets. Glycoprotein VI (GPVI) stimulation activates platelet SFKs, Syk and Btk resulting in phospholipase Cγ (PLCγ) and protein kinase C (PKC) activation. However, their functional hierarchy and cross-talk in platelets are not well understood. Recently, selective Syk and Btk inhibitors showed potency to treat thrombosis, cancers and immuno-inflammatory diseases. Using such inhibitors, we investigated hierarchy of Syk, Btk and PKC with respect to their downstream effectors in GPVI-stimulated human platelets.
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Receptor-induced tyrosine phosphorylation of spleen tyrosine kinase (Syk) has been studied extensively in hematopoietic cells. Metabolic mapping and high-resolution mass spectrometry, however, indicate that one of the most frequently detected phosphorylation sites encompassed S297 (S291 in mice) located within the linker B region of Syk. It has been reported that Protein kinase C (PKC) phosphorylates Syk S297, thus influencing Syk activity. However, conflicting studies suggest that this phosphorylation enhances as well as reduces Syk activity. To clarify the function of this site, we generated Syk S291A knock-in mice. We used platelets as a model system as they possess Glycoprotein VI (GPVI), a receptor containing an immunoreceptor tyrosine-based activation motif (ITAM) which transduces signals through Syk. Our analysis of the homozygous mice indicated that the knock-in platelets express only one isoform of Syk, while the wild-type expresses two isoforms at 69 and 66 kDa. When the GPVI receptor was activated with collagen-related peptide (CRP), we observed an increase in functional responses and phosphorylations in Syk S291A platelets. This potentiation did not occur with AYPGKF or 2-MeSADP, although they also activate PKC isoforms. Although there was potentiation of platelet functional responses, there was no difference in tail bleeding times. However, the time to occlusion in the FeCl
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GPVI activates platelets through an ITAM pathway by activation of Src and Syk kinases leading to activation of PLC\(_y\)2. CLEC-2 has been shown to activate platelets using an ITAM-like sequence in its cytoplasmic tail that is also dependent on Src and Syk kinases, but shows a partial rather than an absolute dependence on adapter SLP-76 for activation of PLC\(_y\)2. The aim of this thesis is to understand some of the key differences in these signalling pathways. GPVI is in complex with FcRwhich contains the ITAM sequence (Yxx(L/I)x\(_{6-12}\)Yxx(L/I)). These two tyrosines provide a docking site for the tandem-SH2 domains of Syk. In this thesis I show that CLEC-2 signalling through Syk is mediated by phosphorylation of the CLEC-2 YxxL sequence, receptor dimerisation and cross-linking by the Syk SH2 domains. I also show that the differential requirement for SLP-76 is not mediated by Gads. Both signalling pathways also show partial dependency for LAT. I also show that a novel protein, G6f, is not able to substitute for LAT in this signalling pathway and also exclude the LAT-family proteins PAG, LIME, LAX and NTAL as potential LAT replacements in platelet activation by GPVI. These results extend our understanding of platelet activation by CLEC-2.
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Platelet interaction with collagens, via von Willebrand factor, is a potent trigger of shear-dependent thrombus formation mediated by subsequent engagement of the signaling collagen receptor glycoprotein (GP)VI, enforced by integrin α2β1. Protein tyrosine kinase Syk is central in the GPVI-induced signaling pathway, leading to elevated cytosolic Ca2+. We aimed to determine the Syk-mediated thrombogenic activity of several collagen peptides and (fibrillar) type I and III collagens. High-shear perfusion of blood over microspots of these substances resulted in thrombus formation, which was assessed by eight parameters and was indicative of platelet adhesion, activation, aggregation, and contraction, which were affected by the Syk inhibitor PRT-060318. In platelet suspensions, only collagen peptides containing the consensus GPVI-activating sequence (GPO)n and Horm-type collagen evoked Syk-dependent Ca2+ rises. In whole blood under flow, Syk inhibition suppressed platelet activation and aggregation parameters for the collagen peptides with or without a (GPO)n sequence and for all of the collagens. Prediction models based on a regression analysis indicated a mixed role of GPVI in thrombus formation on fibrillar collagens, which was abolished by Syk inhibition. Together, these findings indicate that GPVI-dependent signaling through Syk supports platelet activation in thrombus formation on collagen-like structures regardless of the presence of a (GPO)n sequence.
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<b><i>Introduction:</i></b> Ligands acting at the platelet collagen receptor, glycoprotein (GP)VI, induce intracellular FcRγ/Syk-dependent signaling pathways and Syk-dependent or Syk-independent generation of intracellular reactive oxygen species (ROS). Additional signaling-dependent or signaling-independent pathways lead to metalloproteinase-mediated shedding of GPVI. <b><i>Aim:</i></b> Analysis of platelet GPVI expression and signaling in a patient with a collagen-selective defect associated with myelodysplastic syndrome (MDS) uniquely demonstrates divergent pathways leading to ROS generation and Syk phosphorylation in human platelets. <b><i>Methods:</i></b> Surface expression of GPVI and ligand-induced ROS generation was quantitated by flow cytometry. GPVI shedding and Syk phosphorylation were analyzed by Western blot. <b><i>Results:</i></b> Despite platelet count/size and GPVI surface expression within normal ranges, platelet-rich plasma showed no aggregation in response to collagen or GPVI-selective agonist collagen-related peptide, but aggregated in response to other agonists, consistent with dysfunctional GPVI signaling. We observed rapid GPVI-dependent Syk-independent ROS generation and disulfide-dependent GPVI homodimerization, but not Syk-dependent ROS or ligand-induced shedding. Temporal analysis showed a gradual decline in platelet count and the appearance of ligand-induced phosphorylation of an ∼40-kDa Syk fragment. <b><i>Conclusions:</i></b> These studies show that GPVI ligation in platelets induces intracellular ROS production independent of either Syk activation or divergent pathways leading to platelet aggregation or ectodomain shedding.
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