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Developing a reliable method to predict thrombocytopenia is imperative in drug discovery. Here, we establish an assay using a microphysiological system (MPS) to recapitulate the in-vivo mechanisms of platelet aggregation and adhesion. This assay highlights the role of shear stress on platelet aggregation and their interactions with vascular endothelial cells. Platelet aggregation induced by soluble collagen was detected under agitated, but not static, conditions using a plate shaker and gravity-driven flow using MPS. Notably, aggregates adhered on vascular endothelial cells under gravity-driven flow in the MPS, and this incident increased in a concentration-dependent manner. Upon comparing the soluble collagen-induced aggregation activity in platelet-rich plasma (PRP) and whole blood, remarkable platelet aggregate formation was observed at concentrations of 30 µg/mL and 3 µg/mL in PRP and whole blood, respectively. Moreover, ODN2395, an oligonucleotide, induced platelet aggregation and adhesion to vascular endothelial cells. SYK inhibition, which mediated thrombogenic activity via glycoprotein VI on platelets, ameliorated platelet aggregation in the system, demonstrating that the mechanism of platelet aggregation was induced by soluble collagen and oligonucleotide. Our evaluation system partially recapitulated the aggregation mechanisms in blood vessels and can contribute to the discovery of safe drugs to mitigate the risk of thrombocytopenia., (© 2024. The Author(s).)

The pathophysiology of osteoarthritis (OA) includes the destruction of subchondral bone tissue and inflammation of the synovium. Thus, an effective disease-modifying treatment should act on both of these pathogenetic components. It is known that cSrc kinase is involved in bone and cartilage remodeling, and SYK kinase is associated with the inflammatory component. Thus the aim of this study was to characterize the mechanism of action and efficacy of a small molecule multikinase inhibitor MT-SYK-03 targeting SYK and cSrc kinases among others in different in vitro and in vivo arthritis models. The selectivity of MT-SYK-03 kinase inhibition was assayed on a panel of 341 kinases. The compound was evaluated in a set of in vitro models of OA and in vivo OA and RA models: surgically-induced arthritis (SIA), monosodium iodoacetate-induced arthritis (MIA), collagen-induced arthritis (CIA), adjuvant-induced arthritis (AIA). MT-SYK-03 inhibited cSrc and SYK with IC 50 of 14.2 and 23 nM respectively. Only five kinases were inhibited > 90% at 500 nM of MT-SYK-03. In in vitro OA models MT-SYK-03 reduced hypertrophic changes of chondrocytes, bone resorption, and inhibited SYK-mediated inflammatory signaling. MT-SYK-03 showed preferential distribution to joint and bone tissue (in rats) and revealed disease-modifying activity in vivo by halving the depth of cartilage erosion in rat SIA model, and increasing the pain threshold in rat MIA model. Chondroprotective and antiresorptive effects were shown in a monotherapy regime and in combination with methotrexate (MTX) in murine and rat CIA models; an immune-mediated inflammation in rat AIA model was decreased. The obtained preclinical data support inhibition of cSrc and SYK as a viable strategy for disease-modifying treatment of OA. A Phase 2 clinical study of MT-SYK-03 is to be started., (© 2021. The Author(s).)

Cell survival is facilitated by the maintenance of mitochondrial membrane potential (MMP). B cell activating factor (BAFF) plays a role in survival, differentiation, and maturation of B cells. In the present study, we examined whether BAFF could attenuate oxidative stress-induced B cell death by the regulation of MMP collapse via spleen tyrosine kinase (Syk) activation using WiL2-NS human B lymphoblast cells. BAFF binds to receptors on WiL2-NS cells. When the cells were incubated in serum-deprived conditions with 1% fetal bovine serum (FBS), BAFF reduced the percentage of dead cells as determined through trypan blue staining and caspase 3 activity. BAFF also inhibited MMP collapse with 1% FBS, as indicated by a decrease in the number of cells with high-red fluorescence of MitoProbe™ JC-1 reagent or a decrease in the percentage of DiOC 6 -stained cells. Reactive oxygen species (ROS) production was reduced by incubation with BAFF in the presence of 10% or 1% FBS. BAFF inhibited MMP collapse, cell growth retardation, dead cell formation, and caspase 3 activation caused by treatment with H 2 O 2 . Syk phosphorylation on tyrosine (Y) 525/526 was increased in cells incubated with 1% FBS in the presence of BAFF than cells incubated with 1% FBS or BAFF alone. BAY61-3606, a Syk inhibitor reduced the effect of BAFF on MMP collapse, caspase 3 activation, cell growth retardation, and dead cell formation. Together, these data demonstrate that BAFF might attenuate oxidative stress-induced B cell death and growth retardation by the maintenance of MMP through Syk activation by Y525/526 phosphorylation. Therefore, BAFF and Syk might be therapeutic targets in the pathogenesis of B cell-associated diseases such as autoimmune disease.

We previously showed that overexpression of Thy-1 inhibited and knock-down of Thy-1 enhanced endothelial cell migration. Here, we used phorbol-12-myristate-13-acetate (PMA) as an inducer for Thy-1 expression to investigate molecular mechanisms underlying Thy-1 up-regulation. Our data showed that increased levels of Thy-1 mRNA and protein in endothelial cells were observed at 14-18 hours and 20-28 hours after PMA treatment, respectively. Treatment with PMA for 32 hours induced Thy-1 up-regulation and inhibited capillary-like tube formation and endothelial cell migration. These effects were abolished by Röttlerin (a PKC-δ inhibitor), but not Gö6976 (a PKC-α/β inhibitor). Moreover, pre-treatment with Bay 61-3606 (a Syk inhibitor) or Bay 11-7082 (a NF-κB inhibitor) abolished the PMA-induced Thy-1 up-regulation and migration inhibition in endothelial cells. Using the zebrafish model, we showed that PMA up-regulated Thy-1 and inhibited angiogenesis through the PKC-δ-mediated pathway. Surprisingly, we found that short-term (8-10 hours) PMA treatment enhanced endothelial cell migration. However, this effect was not observed in PMA-treated Thy-1-overexpressed endothelial cells. Taken together, our results suggest that PMA initially enhanced endothelial cell migration, subsequently activating the PKC-δ/Syk/NF-κB-mediated pathway to up-regulate Thy-1, which in turn inhibited endothelial cell migration. Our results also suggest that Thy-1 might play a role in termination of angiogenesis.