Role of Tyrosine Kinase Syk in Thrombus Stabilisation at High Shear

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.     

Inhibitory effects of flavonoid glycosides isolated from the peel of Japanese persimmon (Diospyros kaki Fuyu) on antigen-stimulated degranulation in rat basophilic leukaemia RBL-2H3 cells

We found that two distinct flavonoid glycosides isolated from the peel of Japanese persimmon (Diospyros kaki Fuyu), isoquercitrin (Isq) and hyperin (Hyp), are capable of inhibiting antigen-stimulated degranulation in rat basophilic leukaemia RBL-2H3 cells. In order to elucidate the underlying mechanisms, we examined effects of Isq and Hyp on cellular responses induced by antigen stimulation. Treatment with both Isq and Hyp markedly inhibited antigen-stimulated elevation of intracellular free Ca²⁺ concentration and reactive oxygen species (ROS). Isq and Hyp did not affect NADPH oxidase (NOX) activity, but they possessed DPPH radical-scavenging activity similar to that of epigallocatechin gallate, a potent anti-oxidant, Finally, Isq and Hyp showed little or no effects on Ag-stimulated Syk activation or phosphorylation of signalling molecules. These results indicate that inhibition of antigen-stimulated degranulation by Isq and Hyp is mainly due to suppression of intracellular Ca²⁺ elevation, which is caused by direct scavenging of ROS that are generated by NOX. Our findings suggest that Isq and Hyp, isolated from the peel of persimmon, would be beneficial for alleviating symptoms of type I allergy.     

Ficus carica Polysaccharides Promote the Maturation and Function of Dendritic Cells

Various polysaccharides purified from plants are considered to be biological response modifiers and have been shown to enhance immune responses. Ficus carica L. is a Chinese traditional plant and has been widely used in Asian countries for its anti-tumor properties. Ficus carica polysaccharides (FCPS), one of the most essential and effective components in Ficus carica L., have been considered to be a beneficial immunomodulator and may be used in immunotherapy. However, the immunologic mechanism of FCPS is still unclear. Dectin-1 is a non-toll-like pattern recognition receptor, predominately expressed on dendritic cells (DCs). Activation of DCs through dectin-1 signaling can lead to the maturation of DC, thus inducing both innate and adaptive immune responses against tumor development and microbial infection. In our study, we found that FCPS could effectively stimulate DCs, partially through the dectin-1/Syk pathway, and promote their maturation, as shown by the up-regulation of CD40, CD80, CD86, and major histocompatibility complex II (MHCII). FCPS also enhanced the production of cytokines by DCs, including IL-12, IFN-γ, IL-6, and IL-23. Moreover, FCPS-treated DCs showed an enhanced capability to stimulate T cells and promote T cell proliferation. Altogether, these results demonstrate that FCPS are able to activate and maturate DCs, thereby up-regulating the immunostimulatory capacity of DCs, which leads to enhanced T cell responses.     

Syk/JNK/AP-1 Signaling Pathway Mediates Interleukin-6-Promoted Cell Migration in Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma (OSCC) typically migrates and metastasizes. Interleukin-6 (IL-6) is a multifunctional cytokine associated with disease status and cancer outcomes. The effect of IL-6 on human OSCC cells, however, is unknown. Here, we showed that IL-6 increased cell migration and Intercellular adhesion molecule-1 (ICAM-1) expression in OSCC cells. Pretreatment of OSCC cells with IL-6R monoclonal antibody (mAb) significantly abolished IL-6-induced cell migration and ICAM-1 expression. By contrast, IL-6-mediated cell motility and ICAM-1 upregulation were attenuated by the Syk and c-Jun N-terminal kinase (JNK) inhibitors. Stimulation of OSCC cells with IL-6 promoted Syk and JNK phosphorylation. Furthermore, IL-6 enhanced AP-1 activity, and the IL-6R mAb, Syk inhibitor, or JNK inhibitor all reduced IL-6-mediated c-Jun phosphorylation, c-Jun binding to the ICAM-1 promoter, and c-Jun translocation into the nucleus. Our results indicate that IL-6 enhances the migration of OSCC cells by increasing ICAM-1 expression through the IL-6R receptor and the Syk, JNK, and AP-1 signal transduction pathways.     

Antithrombotic Effects of Fostamatinib in Combination with Conventional Antiplatelet Drugs

New antithrombotic medications with less effect on haemostasis are needed for the long-term treatment of acute coronary syndromes (ACS). The platelet receptor glycoprotein VI (GPVI) is critical in atherothrombosis, mediating platelet activation at atherosclerotic plaque. The inhibition of spleen tyrosine kinase (Syk) has been shown to block GPVI-mediated platelet function. The aim of our study was to investigate if the Syk inhibitor fostamatinib could be repurposed as an antiplatelet drug, either alone or in combination with conventional antiplatelet therapy. The effect of the active metabolite of fostamatinib (R406) was assessed on platelet activation and function induced by atherosclerotic plaque and a range of agonists in the presence and absence of the commonly used antiplatelet agents aspirin and ticagrelor. The effects were determined ex vivo using blood from healthy volunteers and aspirin- and ticagrelor-treated patients with ACS. Fostamatinib was also assessed in murine models of thrombosis. R406 mildly inhibited platelet responses induced by atherosclerotic plaque homogenate, likely due to GPVI inhibition. The anti-GPVI effects of R406 were amplified by the commonly-used antiplatelet medications aspirin and ticagrelor; however, the effects of R406 were concentration-dependent and diminished in the presence of plasma proteins, which may explain why fostamatinib did not significantly inhibit thrombosis in murine models. For the first time, we demonstrate that the Syk inhibitor R406 provides mild inhibition of platelet responses induced by atherosclerotic plaque and that this is mildly amplified by aspirin and ticagrelor.     

TULA-Family Regulators of Platelet Activation

The two members of the UBASH3/TULA/STS-protein family have been shown to critically regulate cellular processes in multiple biological systems. The regulatory function of TULA-2 (also known as UBASH3B or STS-1) in platelets is one of the best examples of the involvement of UBASH3/TULA/STS proteins in cellular regulation. TULA-2 negatively regulates platelet signaling mediated by ITAM- and hemITAM-containing membrane receptors that are dependent on the protein tyrosine kinase Syk, which currently represents the best-known dephosphorylation target of TULA-2. The biological responses of platelets to collagen and other physiological agonists are significantly downregulated as a result. The protein structure, enzymatic activity and regulatory functions of UBASH3/TULA/STS proteins in the context of platelet responses and their regulation are discussed in this review.     

The Serine/Threonine Protein Phosphatase 2A (PP2A) Regulates Syk Activity in Human Platelets

Distinct membrane receptors activate platelets by Src-family-kinase (SFK)-, immunoreceptor-tyrosine-based-activation-motif (ITAM)-dependent stimulation of spleen tyrosine kinase (Syk). Recently, we reported that platelet activation via glycoprotein (GP) VI or GPIbα stimulated the well-established Syk tyrosine (Y)-phosphorylation, but also stoichiometric, transient protein kinase C (PKC)-mediated Syk serine(S)297 phosphorylation in the regulatory interdomain-B, suggesting possible feedback inhibition. The transient nature of Syk S297 phosphorylation indicated the presence of an unknown Syk pS297 protein phosphatase. In this study, we hypothesize that the S-protein phosphatase 2A (PP2A) is responsible for Syk pS297 dephosphorylation, thereby affecting Syk Y-phosphorylation and activity in human washed platelets. Using immunoblotting, we show that specific inhibition of PP2A by okadaic acid (OA) alone leads to stoichiometric Syk S297 phosphorylation, as analyzed by Zn²⁺-Phos-tag gels, without affecting Syk Y-phosphorylation. Pharmacological inhibition of Syk by PRT060318 or PKC by GF109203X only minimally reduced OA-induced Syk S297 phosphorylation. PP2A inhibition by OA preceding GPVI-mediated platelet activation induced by convulxin extended Syk S297 phosphorylation but inhibited Syk Y-phosphorylation. Our data demonstrate a novel biochemical and functional link between the S-protein phosphatase PP2A and the Y-protein kinase Syk in human platelets, and suggest that PP2A represents a potential enhancer of GPVI-mediated Syk activity caused by Syk pS297 dephosphorylation.