Hinokitiol (4-isopropyl-tropolone) is a bioactive compound with various pharmacological activities that is found in the wood of cupressaceous plants. Platelet activation plays an important role in thrombogenesis. In our previous study, hinokitiol specifically inhibited collagen-induced platelet aggregation ex vivo and prolonged thrombogenesis in vivo. The glycoprotein (GP) VI and integrin alpha(2)beta(1) are major collagen receptors that mediate platelet adhesion and aggregation. In our current study, we investigated which of these collagen receptors is involved in the hinokitiol-mediated inhibition of platelet activation. Treatment with 2-100 mu M hinokitiol caused a dose-dependent right, parallel shift in the collagen concentration- response curve (0.5-10 mu g/ml), with no change in the maximal responses. Furthermore, hinokitiol inhibited platelet aggregation and relative [Ca2+]i mobilization stimulated by convulxin, an agonist of GP VI, but not by aggretin, an agonist of integrin alpha(2)beta(1), indicating that hinokitiol mediates the inhibition of platelet activation through GP VI, rather than through integrin alpha(2)beta(1). Hinokitiol also specifically inhibited the convulxin-mediated activation of protein kinase C, phospholipase C gamma 2, Akt, mitogen-activated protein kinases, and Lyn. Hinokitiol markedly diminished the co-immunoprecipitation of GP VI-bound Lyn after convulxin stimulation. In conclusion, hinokitiol, an antagonist of collagen GP VI may represent a novel antiplatelet drug for the prevention of thrombi associated with coronary and cerebral artery diseases.

The purpose of the present study was to elucidate the possible signal transduction pathway involved in the underlying mechanism of glucosamine (GLN)’s influence on the gene expression of matrix metalloproteinases (MMPs) in chondrocytes stimulated with IL-1β. Using chondrosarcoma cells stimulated with IL-1β, the effects of GLN on the mRNA and protein levels of MMP-3, the activation of JNK, ERK, p38, NF-κB, and AP-1, the nuclear translocation of NF-κB/Rel family members, and PI3-kinase/Akt activation were studied. GLN inhibited the expression and the synthesis of MMP-3 induced by IL-1β, and that inhibition was mediated at the level of transcription involving both the NF-κB and AP-1 transcription factors. Translocation of NF-κB was reduced by GLN as a result of the inhibition of IκB degradation. A slightly synergistic effect on the activation of AP-1 induced by IL-1β was shown in the presence of GLN. Among MAPK pathways involved in the transcriptional regulation of AP-1, phosphorylation of JNK and ERK was found to increase with the presence of GLN under IL-1β treatment, while that for p38 decreased. It was also found that GLN alone, but also synergistically with IL-1β, was able to activate the Akt pathway. The requirements of NF-κB translocation and p38 activity are indispensably involved in the induction of MMP-3 expression in chondrosarcoma cells stimulated by IL-1β. Inhibition of the p38 pathway in the presence of GLN substantially explains the chondroprotective effect of GLN on chondrocytes that regulate COX-2 expression, PGE2 synthesis, and NO expression and synthesis. The chondroprotective effect of GLN through the decrease in MMP-3 production and stimulation of proteoglycan synthesis may follow another potential signaling pathway of Akt.