Antiplatelet Activity of Tussilagone via Inhibition of the GPVI Downstream Signaling Pathway in Platelets.

Tussilagone is a sesquiterpenoid extracted from Tussilago farfara and is used as an oriental medicine for asthma and bronchitis. Although previous studies have shown that tussilagone has an inhibitory effect on platelet aggregation, no studies have been performed to investigate its precise effect on platelets, and the underlying mechanism remains unclear. In the present study, we showed that tussilagone inhibited platelet aggregation induced by collagen, thrombin and ADP, as well as platelet release induced by collagen and thrombin, in mice. Tussilagone decreased P-selectin expression and αIIbß3 activation (JON/A binding) in activated platelets, which indicated that tussilagone inhibited platelet activation. Moreover, tussilagone suppressed platelet spreading on fibrinogen and clot retraction. The levels of phosphorylated Syk, PLCγ2, Akt, GSK3ß, and MAPK (ERK1/2 and P38) and molecules associated with GPVI downstream signaling were downregulated in the presence of tussilagone. In addition, tussilagone prolonged the occlusion time in a mouse model of FeCl3-induced carotid artery thrombosis and had no effect on mouse tail bleeding time. These results indicate that tussilagone inhibits platelet function in vitro and in vivo and that the underlying mechanism involves the Syk/PLCγ2-PKC/MAPK and PI3K-Akt-GSK3ß signaling pathways downstream of GPVI. This research suggests that tussilagone is a potential candidate antiplatelet drug for the prevention of thrombosis.

Pharmacological actions of neferine in the modulation of human platelet function.

Neferine has long been recognized as a medicinal herbal ingredient with various physiological and pharmacological activities. Although previous studies have reported its antithrombotic effect, the underlying mechanisms have not been thoroughly investigated. Since platelets play a key role in thrombosis, we investigated the effects of neferine on human platelet function and the potential mechanisms. Platelet aggregation, adhesion and spreading were performed to investigate the effect of neferine on inhibition of platelet function. Flow cytometry was used to determine platelet alpha granule secretion and integrin IIb/IIIa activation, as detected by CD62P (P-selectin) expression, PAC-1 and fibrinogen binding. Western blotting was utilized to investigate the effect of neferine on intracellular signaling of activated platelet. We found that neferine significantly suppressed platelet aggregation and remarkably promoted the dissociation of platelet aggregates induced by collagen, thrombin, U46619, ADP and adrenaline in a dose-dependent manner. Flow cytometry analysis showed that neferine inhibited thrombin-induced platelet P-selectin expression, PAC-1 and fibrinogen binding. In addition, neferine reduced the adhesion of human platelets on coated collagen under both static and shearing condition at an arterial shear rate of 40 dyne/cm2. Neferine also inhibited the spreading of human platelets on immobilized fibrinogen. Western blot analysis showed that neferine inhibited PI3K activation, and decreased the levels of phosphorylation of Akt, GSK3ß and p38 MAPK in platelets. In summary, neferine has the potential to be an antiplatelet and antithrombotic agent by inhibiting the PI3K-Akt-GSK3ß/p38 MAPK signaling pathway.

Pharmacological actions of miltirone in the modulation of platelet function.

Salvia miltiorrhiza Bunge contains various active constituents, some of which have been developed as commercially available medicine. Moreover, some other ingredients in Salvia miltiorrhiza play roles in anti-platelet activity. The aim of the present study was to investigate the effects and the underlying mechanism of miltirone, a lipophilic compound of Salvia miltiorrhiza Bunge. The ability of miltirone to modulate platelet function was investigated by a variety of in vitro and in vivo experiments. Platelet aggregation and dense granule secretion induced by various agonists were measured with platelet aggregometer. Clot retraction and spreading were imaged by digital camera and fluorescence microscope. Ferric chloride-induced carotid injury model and pulmonary thromboembolism model were used to check miltirone antithrombotic effect in vivo. To elucidate the mechanisms of anti-platelet activity of miltirone, flow cytometry and western blotting were performed. Miltirone (2, 4, 8 µM) was shown to suppress platelet aggregation, dense granule, and α granule secretion in a dose-dependent manner. Meanwhile, miltirone inhibited the clot retraction and spreading of washed platelets. It reduced the phosphorylation of PLCγ2, PKC, Akt, GSK3ß and ERK1/2 in the downstream signal pathway of collagen receptor. It also reduced the phosphorylation of Src and FAK in the integrin αIIbß3-mediated "outside-in" signaling, while it did not suppress the phosphorylation of ß3. In addition, miltirone prolonged the occlusion time and reduced collagen/epinephrine-induced pulmonary thrombi. Miltirone suppresses platelet "inside-out" and "outside-in" signaling by affecting PLCγ2/PKC/ERK1/2, PI3K/Akt, and Src/FAK signaling. Therefore, miltirone might represent a potential anti-platelet candidate for the prevention of thrombotic disorders.

Platelet derived TGF-ß promotes cervical carcinoma cell growth by suppressing KLF6 expression.

Platelets in the primary tumor microenvironment play crucial roles in regulating tumor growth, metastasis, and angiogenesis, but the underlying mechanisms are unclear. Here, we show that platelet releasates exhibited a proliferative effect on HeLa cells, and this effect correlated with a reduction of KLF6 expression. After incubation with either washed human platelets or collagen-related peptide (CRP) activated platelet releasates, expression of KLF6 in the HeLa cervical tumor cell line was markedly reduced. However, no significant difference was observed between control HeLa cells and HeLa cells incubated with resuspended activated platelet pellet. Moreover, the platelets' promoting effect on HeLa cell growth was significantly abolished in KLF6 silenced HeLa cells. In addition, blocking TGF-ß signaling with SB431542, a TGF-ß receptor inhibitor, also counteracted the effect of platelets on proliferation and KLF6 expression in HeLa cells. From these findings, we conclude that platelet derived TGF-ß promotes proliferation of HeLa cells by decreasing the expression of KLF6. The discovery that KLF6 is a key target of platelet-derived TGF-ß signaling in HeLa cells identifies a potential new therapeutic target for the prevention and treatment of cervical carcinoma.

Genotypic Variation in Yield, Yield Components, Root Morphology and Architecture, in Soybean in Relation to Water and Phosphorus Supply.

Water shortage and low phosphorus (P) availability limit yields in soybean. Roots play important roles in water-limited and P-deficient environment, but the underlying mechanisms are largely unknown. In this study we determined the responses of four soybean [Glycine max (L.) Merr.] genotypes [Huandsedadou (HD), Bailudou (BLD), Jindou 21 (J21), and Zhonghuang 30 (ZH)] to three P levels [applied 0 (P0), 60 (P60), and 120 (P120) mg P kg-1 dry soil to the upper 0.4 m of the soil profile] and two water treatment [well-watered (WW) and water-stressed (WS)] with special reference to root morphology and architecture, we compared yield and its components, root morphology and root architecture to find out which variety and/or what kind of root architecture had high grain yield under P and drought stress. The results showed that water stress and low P, respectively, significantly reduced grain yield by 60 and 40%, daily water use by 66 and 31%, P accumulation by 40 and 80%, and N accumulation by 39 and 65%. The cultivar ZH with the lowest daily water use had the highest grain yield at P60 and P120 under drought. Increased root length was positively associated with N and P accumulation in both the WW and WS treatments, but not with grain yield under water and P deficits. However, in the WS treatment, high adventitious and lateral root densities were associated with high N and P uptake per unit root length which in turn was significantly and positively associated with grain yield. Our results suggest that (1) genetic variation of grain yield, daily water use, P and N accumulation, and root morphology and architecture were observed among the soybean cultivars and ZH had the best yield performance under P and water limited conditions; (2) water has a major influence on nutrient uptake and grain yield, while additional P supply can modestly increase yields under drought in some soybean genotypes; (3) while conserved water use plays an important role in grain yield under drought, root traits also contribute to high nutrient uptake efficiency and benefit yield under drought.

[Effects of potato/soybean intercropping on photosynthetic characteristics and yield of three soybean varieties].

The potato/soybean intercropping trials using three soybean varieties including Zhonghuang 30 (early-maturing variety) , Jidou 17 (mid maturing variety) and Qihuang 34 (late maturing variety) with the sole cropping potato as control were carried out to determine the dynamic changes of leaf area index (LAI) of soybean, accumulation of dry matter, photosynthetic characteristics, yield and yield components. The results showed that the LAI, dry matter accumulation, net photosynthesis (Pn), transpiration rate (Tr) and stomatal conduction (g(s)) of soybean in all intercropping systems were lower than in monoculture because of the influence of intensified lower light during earlier growing stage, and the duration from planting to flowering was extended. When the potato was harvested, the LAI, dry matter accumulation, Pn, Tr and g(s) of soybean in all intercropping systems increased, especially for mid-maturing and late-maturing varieties, which became much closer to those in the monoculture. Compared with sole cropping, the pods per plant, seeds per plant and seeds per pod in intercropping system significantly decreased by 22.0%, 36.0% and 17.6% for early-maturing soybean, 5.1%, 13.1% and 8.9% for mid-maturing soybean, 5.7%, 7.6% and 2.1% for late-maturing soybean, respectively. The yields of mid-maturing and late-maturing varieties in intercropping systems were higher than that of the early-maturing, which increased by 92.4% and 163.4%, with the land equivalent ratio (LER) of 1.81 and 1.84, respectively. This suggested that mid-maturing and late-maturing soybean varieties were suitable for intercropping with the potato to improve photosynthetic efficiency, dry matter accumulation and yield of intercropping soybean.