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Role of Tyrosine Kinase Syk in Thrombus Stabilisation at High Shear.

ABSTRACT: Understanding the pathways involved in the formation and stability of the core and shell regions of a platelet-rich arterial thrombus may result in new ways to treat arterial thrombosis. The distinguishing feature between these two regions is the absence of fibrin in the shell which indicates that in vitro flow-based assays over thrombogenic surfaces, in the absence of coagulation, can be used to resemble this region. In this study, we have investigated the contribution of Syk tyrosine kinase in the stability of platelet aggregates (or thrombi) formed on collagen or atherosclerotic plaque homogenate at arterial shear (1000 s^-1). We show that post-perfusion of the Syk inhibitor PRT-060318 over preformed thrombi on both surfaces enhances thrombus breakdown and platelet detachment. The resulting loss of thrombus stability led to a reduction in thrombus contractile score which could be detected as early as 3 min after perfusion of the Syk inhibitor. A similar loss of thrombus stability was observed with ticagrelor and indomethacin, inhibitors of platelet adenosine diphosphate (ADP) receptor and thromboxane A₂ (TxA₂), respectively, and in the presence of the Src inhibitor, dasatinib. In contrast, the Btk inhibitor, ibrutinib, causes only a minor decrease in thrombus contractile score. Weak thrombus breakdown is also seen with the blocking GPVI nanobody, Nb21, which indicates, at best, a minor contribution of collagen to the stability of the platelet aggregate. These results show that Syk regulates thrombus stability in the absence of fibrin in human platelets under flow and provide evidence that this involves pathways additional to activation of GPVI by collagen.

SUBMITTER: Perrella G

PROVIDER: S-EPMC8745592 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Role of Platelet Glycoprotein VI and Tyrosine Kinase Syk in Thrombus Formation on Collagen-Like Surfaces.

Project description: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.

| S-EPMC6600290 | biostudies-literature

Tyrosine kinase SYK is a potential therapeutic target for liver fibrosis.

Project description:Spleen tyrosine kinase (SYK) plays a critical role in immune cell signaling pathways and has been reported as a biomarker for human hepatocellular carcinoma (HCC). We sought to investigate the mechanism by which SYK promotes liver fibrosis and to evaluate SYK as a therapeutic target for liver fibrosis. We evaluated the cellular localization of SYK and the association between SYK expression and liver fibrogenesis in normal, hepatitis B virus (HBV)-infected, hepatitis C virus (HCV)-infected and non-alcoholic steatohepatitis (NASH) liver tissue (n=36, 127, 22 and 30, respectively). A polymerase chain reaction (PCR) array was used to detect the changes in transcription factor (TF) expression in hepatic stellate cells (HSCs) with SYK knockdown. The effects of SYK antagonism on liver fibrogenesis were studied in LX-2 cells, TWNT-4 cells, primary human HSCs, and three progressive fibrosis/cirrhosis animal models, including a CCL4 mouse model, and diethylnitrosamine (DEN) and bile duct ligation (BDL) rat models. We found that SYK protein in HSCs and hepatocytes correlated positively with liver fibrosis stage in human liver tissue. HBV or HCV infection significantly increased SYK and cytokine expression in hepatocytes. Increasing cytokine production further induced SYK expression and fibrosis-related gene transcription in HSCs. Up-regulated SYK in HSCs promoted HSC activation by increasing the expression of specific TFs related to activation of HSCs. SYK antagonism effectively suppressed liver fibrosis via inhibition of HSC activation, and decreased obstructive jaundice and reduced HCC development in animal models. Conclusion: SYK promotes liver fibrosis via activation of HSCs and is an attractive potential therapeutic target for liver fibrosis and prevention of HCC development. (Hepatology 2018).

| S-EPMC6138581 | biostudies-literature

Dual Role of the Tyrosine Kinase Syk in Regulation of Toll-Like Receptor Signaling in Plasmacytoid Dendritic Cells.

Project description:Crosslinking of regulatory immunoreceptors (RR), such as BDCA-2 (CD303) or ILT7 (CD85g), of plasmacytoid dendritic cells (pDCs) efficiently suppresses production of type-I interferon (IFN)-?/? and other cytokines in response to Toll-like receptor (TLR) 7/9 ligands. This cytokine-inhibitory pathway is mediated by spleen tyrosine kinase (Syk) associated with the ITAM-containing adapter of RR. Here we demonstrate by pharmacological targeting of Syk that in addition to the negative regulation of TLR7/9 signaling via RR, Syk also positively regulates the TLR7/9 pathway in human pDCs. Novel highly specific Syk inhibitor AB8779 suppressed IFN-?, TNF-? and IL-6 production induced by TLR7/9 agonists in primary pDCs and in the pDC cell line GEN2.2. Triggering of TLR9 or RR signaling induced a differential kinetics of phosphorylation at Y352 and Y525/526 of Syk and a differential sensitivity to AB8779. Consistent with the different roles of Syk in TLR7/9 and RR signaling, a concentration of AB8779 insufficient to block TLR7/9 signaling still released the block of IFN-? production triggered via the RR pathway, including that induced by hepatitis B and C viruses. Thus, pharmacological targeting of Syk partially restored the main pDC function-IFN-? production. Opposing roles of Syk in TLR7/9 and RR pathways may regulate the innate immune response to weaken inflammation reaction.

| S-EPMC4892542 | biostudies-literature

Role of the protein tyrosine kinase Syk in regulating cell-cell adhesion and motility in breast cancer cells.

Project description:The expression of the Syk protein tyrosine kinase in breast cancer cells is inversely correlated with invasive growth and metastasis. The expression of Syk inhibits cell motility while supporting the formation of cell clusters by enhancing cell-cell contacts and promoting the redistribution of the adhesion proteins cortactin and vinculin to these contacts. Syk associates physically with cortactin and catalyzes its phosphorylation on tyrosine. The clustering of integrins leads to the phosphorylation of Syk and of numerous cellular proteins in a manner dependent on the activity of the kinase and on the presence of tyrosine 342 located in the linker B region. The ability of Syk to participate in integrin-mediated protein tyrosine phosphorylation correlates well with its ability to inhibit cell motility.

| S-EPMC2788757 | biostudies-literature

Spleen tyrosine kinase (Syk) regulates systemic lupus erythematosus (SLE) T cell signaling.

Project description:Engagement of the CD3/T cell receptor complex in systemic lupus erythematosus (SLE) T cells involves Syk rather than the zeta-associated protein. Because Syk is being considered as a therapeutic target we asked whether Syk is central to the multiple aberrantly modulated molecules in SLE T cells. Using a gene expression array, we demonstrate that forced expression of Syk in normal T cells reproduces most of the aberrantly expressed molecules whereas silencing of Syk in SLE T cells normalizes the expression of most abnormally expressed molecules. Protein along with gene expression modulation for select molecules was confirmed. Specifically, levels of cytokine IL-21, cell surface receptor CD44, and intracellular molecules PP2A and OAS2 increased following Syk overexpression in normal T cells and decreased after Syk silencing in SLE T cells. Our results demonstrate that levels of Syk affect the expression of a number of enzymes, cytokines and receptors that play a key role in the development of disease pathogenesis in SLE and provide support for therapeutic targeting in SLE patients.

| S-EPMC3754955 | biostudies-literature

Mast cell signaling: the role of protein tyrosine kinase Syk, its activation and screening methods for new pathway participants.

Project description:The aggregation by antigen of the IgE bound to its high affinity receptor on mast cells initiates a complex series of biochemical events that result in the release of inflammatory mediators. The essential role of the protein tyrosine kinase Syk has been appreciated for some time, and newer results have defined the mechanism of its activation. The use of siRNA has defined the relative contribution of Syk, Fyn and Gab2 to signaling and has made possible a screening study to identify previously unrecognized molecules that are involved in these pathways.

| S-EPMC2998559 | biostudies-literature

Platelet transport rates and binding kinetics at high shear over a thrombus.

Project description:Thrombus formation over a ruptured atherosclerotic plaque cap can occlude an artery with fatal consequences. We describe a computational model of platelet transport and binding to interpret rate-limiting steps seen in experimental thrombus formation over a collagen-coated stenosis. The model is used to compute shear rates in stenoses with growing boundaries. In the model, moving erythrocytes influence platelet transport based on shear-dependent enhanced diffusivity and a nonuniform platelet distribution. Adhesion is modeled as platelet-platelet binding kinetics. The results indicate that observed thrombus growth rates are limited by platelet transport to the wall for shear rates up to 6000 s(-1). Above 7000 s(-1), the thrombus growth rate is likely limited by binding kinetics (10(-4) m/s). Thrombus growth computed from these rate-limiting steps match the thrombus location and occlusion times for experimental conditions if a lag time for platelet activation is included. Using fitted parameters, the model is then used to predict thrombus size and shape at a higher Reynolds number flow consistent with coronary artery disease.

| S-EPMC3714887 | biostudies-literature

The nonreceptor tyrosine kinase SYK drives caspase-8/NLRP3 inflammasome-mediated autoinflammatory osteomyelitis.

Project description:Chronic recurrent multifocal osteomyelitis (CRMO) in humans can be modeled in Pstpip2cmo mice, which carry a missense mutation in the proline-serine-threonine phosphatase-interacting protein 2 (Pstpip2) gene. As cmo disease in mice, the experimental model analogous to human CRMO, is mediated specifically by IL-1β and not by IL-1α, delineating the molecular pathways contributing to pathogenic IL-1β production is crucial to developing targeted therapies. In particular, our earlier findings support redundant roles of NLR family pyrin domain-containing 3 (NLRP3) and caspase-1 with caspase-8 in instigating cmo However, the signaling components upstream of caspase-8 and pro-IL-1β cleavage in Pstpip2cmo mice are not well-understood. Therefore, here we investigated the signaling pathways in these mice and discovered a central role of a nonreceptor tyrosine kinase, spleen tyrosine kinase (SYK), in mediating osteomyelitis. Using several mutant mouse strains, immunoblotting, and microcomputed tomography, we demonstrate that absent in melanoma 2 (AIM2), receptor-interacting serine/ threonine protein kinase 3 (RIPK3), and caspase recruitment domain-containing protein 9 (CARD9) are each dispensable for osteomyelitis induction in Pstpip2cmo mice, whereas genetic deletion of Syk completely abrogates the disease phenotype. We further show that SYK centrally mediates signaling upstream of caspase-1 and caspase-8 activation and principally up-regulates NF-κB and IL-1β signaling in Pstpip2cmo mice, thereby inducing cmo These results provide a rationale for directly targeting SYK and its downstream signaling components in CRMO.

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Carboxamide Spleen Tyrosine Kinase (Syk) Inhibitors: Leveraging Ground State Interactions To Accelerate Optimization.

Project description:Optimization of a series of highly potent and kinome selective carbon-linked carboxamide spleen tyrosine kinase (Syk) inhibitors with favorable drug-like properties is described. A pervasive Ames liability in an analogous nitrogen-linked carboxamide series was obviated by replacement with a carbon-linked moiety. Initial efforts lacked on-target potency, likely due to strain induced between the hinge binding amide and solvent front heterocycle. Consideration of ground state and bound state energetics allowed rapid realization of improved solvent front substituents affording subnanomolar Syk potency and high kinome selectivity. These molecules were also devoid of mutagenicity risk as assessed via the Ames test using the TA97a Salmonella strain.

| S-EPMC5150694 | biostudies-literature

Entropic allostery dominates the phosphorylation-dependent regulation of Syk tyrosine kinase release from immunoreceptor tyrosine-based activation motifs.

Project description:Spleen tyrosine kinase (Syk) is an essential player in immune signaling through its ability to couple multiple classes of membrane immunoreceptors to intracellular signaling pathways. Ligand binding leads to the recruitment of Syk to a phosphorylated cytoplasmic region of the receptors called ITAM. Syk binds to ITAM with high-affinity (nanomolar Kd ) via its tandem pair of SH2 domains. The affinity between Syk and ITAM is allosterically regulated by phosphorylation at Y130 in a linker connecting the tandem SH2 domains; when Y130 is phosphorylated, the binding affinity decreases (micromolar Kd ). Previous equilibrium binding studies attribute the increase in the binding free energy to an intra-molecular binding (isomerization) step of the tandem SH2 and ITAM, but a physical basis for the increased free energy is unknown. Here, we provide evidence that Y130 phosphorylation imposes an entropy penalty to isomerization, but surprisingly, has negligible effect on the SH2 binding interactions with ITAM and thus on the binding enthalpy. An analysis of NMR chemical shift differences characterized conformational effects of ITAM binding, and binding thermodynamics were measured from isothermal titration calorimetry. Together the data support a previously unknown mechanism for the basis of regulating protein-protein interactions through protein phosphorylation. The decreased affinity for Syk association with immune receptor ITAMs by Y130 phosphorylation is an allosteric mechanism driven by an increased entropy penalty, likely contributed by conformational disorder in the SH2-SH2 inter-domain structure, while SH2-ITAM binding contacts are not affected, and binding enthalpy is unchanged.

| S-EPMC6225982 | biostudies-literature

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