Direct interaction of platelet with tumor cell aggravates hepatocellular carcinoma metastasis by activating TLR4/ADAM10/CX3CL1 axis.

Metastasis is the main culprit of cancer-related death and account for the poor prognosis of hepatocellular carcinoma. Although platelets have been shown to accelerate tumor cell metastasis, the exact mechanism remained to be fully understood. Here, we found that high blood platelet counts and increased tumor tissue ADAM10 expression indicated the poor prognosis of HCC patients. Meanwhile, blood platelet count has positive correlation with tumor tissue ADAM10 expression. In vitro, we revealed that platelet increased ADAM10 expression in tumor cell through TLR4/NF-κB signaling pathway. ADAM10 catalyzed the shedding of CX3CL1 which bound to CX3CR1 receptor, followed by inducing epithelial to mesenchymal transition and activating RhoA signaling in cancer cells. Moreover, knockdown HCC cell TLR4 (Tlr4) or inhibition of ADAM10 prevented platelet-increased tumor cell migration, invasion and endothelial permeability. In vivo, we further verified in mice lung metastatic model that platelet accelerated tumor metastasis via cancer cell TLR4/ADAM10/CX3CL1 axis. Overall, our study provides new insights into the underlying mechanism of platelet-induced HCC metastasis. Therefore, targeting the TLR4/ADAM10/CX3CL1 axis in cancer cells hold promise for the inhibition of platelet-promoted lung metastasis of HCC.

Adiponectin receptor agonist AdipoRon modulates human and mouse platelet function.

Adiponectin, an adipokine secreted by adipocytes, has anti-atherosclerotic and antithrombotic activities. AdipoRon is synthetic small molecule adiponectin receptor agonist. In this study, we investigated the effect of AdipoRon on platelet activation and thrombus formation. Washed human platelets were prepared from the peripheral blood of healthy donors. In a series of in vitro platelet functional assays, pre-treatment with AdipoRon (10, 20, 40 µg/mL) dose-dependently inhibited the aggregation, granule secretion and spreading of washed human platelets. We showed that AdipoRon (20, 40 µg/mL) significantly inhibited AMPK, Syk, PLCγ2, PI3K, Akt, p38-MAPK and ERK1/2 signalling pathways in washed human platelets. In addition, we demonstrated that the phosphorylation of CKII at Tyr255 was an important mechanism of the integrin αIIbß3-mediated platelet activation. Meanwhile, AdipoR1 deficiency impaired the inhibitory effect of AdipoRon on mouse platelets. In ferric chloride-induced carotid injury model, injection of AdipoRon (5 or 12.5 mg/kg, iv) significantly attenuated arterial thrombosis. In conclusion, AdipoRon attenuates platelet function via the AdipoR1/AMPK/CKII/PI3K/AKT signalling pathways, while exerting a protective effect against arterial thrombosis. This study offers new insights into the fields of cardiovascular disease and antiplatelet drug discovery.Schematic model of AdipoRon regulating platelet activation. (BioRender.com).

Risk Assessment and Regulatory Exploration of the Lubrication Coating on Intravascular Medical Devices.

Lubricious polymer coatings are increasingly used on intravascular devices to facilitate application processes. Although increasing reports about the detachment and subsequent embolism of polymer particles, this iatrogenic polymer embolism has not been paid enough clinical attention for many years. This article reviews the hazard of coating separation and the difficulty to find it. Furthermore, this proposes the scientific evaluation concept and regulatory exploration to solve the problems.

Platelet-Derived TGF (Transforming Growth Factor)-ß1 Enhances the Aerobic Glycolysis of Pulmonary Arterial Smooth Muscle Cells by PKM2 (Pyruvate Kinase Muscle Isoform 2) Upregulation.

BACKGROUND: Metabolic reprogramming is a hallmark of pulmonary arterial hypertension. Platelet activation has been implicated in pulmonary arterial hypertension (PAH), whereas the role of platelet in the pathogenesis of PAH remains unclear. METHODS: First, we explored the platelet function of semaxanib' a inhibitor of VEGF receptor (SU5416)/hypoxia mice and monocrotaline-injected rats PAH model. Then we investigated pulmonary arterial smooth muscle cell aerobic glycolysis after being treated with platelet supernatant. TGF (transforming growth factor)-ßRI, pyruvate kinase muscle 2, and other antagonists were applied to identify the underlying mechanism. In addition, platelet-specific deletion TGF-ß1 mice were exposed to chronic hypoxia and SU5416. Cardiopulmonary hemodynamics, vascular remodeling, and aerobic glycolysis of pulmonary arterial smooth muscle cell were determined. RESULTS: Here, we demonstrate that platelet-released TGF-ß1 enhances the aerobic glycolysis of pulmonary arterial smooth muscle cells after platelet activation via increasing pyruvate kinase muscle 2 expression. Mechanistically, platelet-derived TGF-ß1 regulate spyruvate kinase muscle 2 expression through mTOR (mammalian target of rapamycin)/c-Myc/PTBP-1(polypyrimidine tract binding protein 1)/hnRNPA-1(heterogeneous nuclear ribonucleoprotein A1) pathway. Platelet TGF-ß1 deficiency mice are significantly protected from SU5416 plus chronic hypoxia-induced PAH, including attenuated increases in right ventricular systolic pressure and less pulmonary vascular remodeling. Also, in Pf4cre+ Tgfb1fl/fl mice, pulmonary arterial smooth muscle cells showed lower glycolysis capacity and their pyruvate kinase muscle 2 expression decreased. CONCLUSIONS: Our data demonstrate that TGF-ß1 released by platelet contributes to the pathogenesis of PAH and further highlights the role of platelet in PAH.

Sanguinarine Attenuates Collagen-Induced Platelet Activation and Thrombus Formation.

Sanguinarine, a benzophenanthridine alkaloid, has been described to have an antiplatelet activity. However, its antithrombotic effect and the mechanism of platelet inhibition have not thoroughly been explored. The current study found that sanguinarine had an inhibitory effect on thrombus formation. This inhibitory effect was quite evident both in the flow-chamber assays as well as in a murine model of FeCl3-induced carotid artery thrombosis. Further investigations also revealed that sanguinarine inhibited the collagen-induced human platelet aggregation and granule release. At the same time, it also prevented platelet spreading and adhesion to immobilized fibrinogen. The molecular mechanisms of its antiplatelet activity were found to be as follows: 1. Reduced phosphorylation of the downstream signaling pathways in collagen specific receptor GPVI (Syk-PLCγ2 and PI3K-Akt-GSK3ß); 2. Inhibition of collagen-induced increase in the intracellular Ca2+ concentration ([Ca2+]i); 3. Inhibition of integrin αIIbß3 outside-in signaling via reducing ß3 and Src (Tyr-416) phosphorylation. It can be concluded that sanguinarine inhibits collagen-induced platelet activation and reduces thrombus formation. This effect is mediated via inhibiting the phosphorylation of multiple components in the GPVI signaling pathway. Current data also indicate that sanguinarine can be of some clinical value to treat cardiovascular diseases involving an excess of platelet activation.

Platelets are recruited to hepatocellular carcinoma tissues in a CX3CL1-CX3CR1 dependent manner and induce tumour cell apoptosis.

The mechanisms and biological functions of migrating platelets in cancer remain largely unknown. Here, we analyzed platelet infiltration in hepatocellular carcinoma. We detected platelet extravasation in both mouse and human HCC tissues. CX3CL1 directly induced platelet migration, and hypoxia enhanced platelet migration by upregulating CX3CL1 expression. Knocking down CX3CL1 in HCC cells reduced platelet migration in vitro, as well as infiltration of HCC tissue in an orthotopic HCC mouse model. Components of the CX3CR1/Syk/PI3K pathway were essential for CX3CL1-induced platelet migration. Migrating platelets induced HCC cell apoptosis in vitro, as indicated by a reduced mitochondrial membrane potential and an increased percentage of apoptotic cells. In the orthotopic tumor implantation model, decreased platelet infiltration was associated with accelerated tumor growth. Taken together, our findings indicate that HCC cell-derived CX3CL1 contributes to tumor infiltration by platelets, which in turn promotes apoptosis of HCC cells.

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.

Cancer cell-derived immunoglobulin G activates platelets by binding to platelet FcγRIIa.

Tumor-associated thrombosis is the second leading risk factor for cancer patient death, and platelets activity is abnormal in cancer patients. Discovering the mechanism of platelet activation and providing effective targets for therapy are urgently needed. Cancer cell- derived IgG has been reported to regulate development of tumors. However, studies on the functions of cancer cell-derived IgG are quite limited. Here we investigated the potential role of cancer cell-derived IgG in platelet activation. We detected the expression of CD62P on platelets by flow cytometry and analyzed platelet function by platelets aggregation and ATP release. The content of IgG in cancer cell supernatants was detected by enzyme-linked immune sorbent assay. The distribution of cancer-derived IgG in cancer cells was analyzed by immunofluorescence assay. Western blot was performed to quantify the relative expression of FcγRIIa, syk, PLCγ2. The interaction between cancer cell-derived IgG and platelet FcγRIIa was analyzed by co-immunoprecipitation. The results showed that higher levels of CD62P were observed in cancer patients' platelets compared with that of healthy volunteers. Cancer cell culture supernatants increased platelet CD62P and PAC-1 expression, sensitive platelet aggregation and ATP release in response to agonists, while blocking FcγRIIa or knocking down IgG reduced the activation of platelets. Coimmunoprecipitation results showed that cancer cell-derived IgG interacted directly with platelet FcγRIIa. In addition, platelet FcγRIIa was highly expressed in liver cancer patients. In summary, cancer cell-derived IgG interacted directly with FcγRIIa and activated platelets; targeting this interaction may be an approach to prevent and treat tumor-associated thrombosis.

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.

Platelet releasates promote the proliferation of hepatocellular carcinoma cells by suppressing the expression of KLF6.

Platelets in the primary tumor microenvironment play crucial roles in the regulation of tumor progression, but the mechanisms underlying are poorly understood. Here, we report that platelet releasates exerted a proliferative effect on hepatocellular carcinoma (HCC) cells both in vitro and in vivo. This effect depended on a reduction of KLF6 expression in HCC cells. After incubation with either platelets or platelet granule contents, SMMC.7721 and HepG2 cells exhibited significant increases in proliferation and decreases in apoptosis. However, no effect was observed when incubating cancer cells with resuspended activated platelet pellet which exhausted of releasates. Platelet releasates also increased the population of HCC cells in the S and G2/M phases of the cell cycle and reduced the cell population in the G0/G1 phase. Moreover, knocking down KLF6 expression significantly diminished the platelet-mediated enhancement of HCC growth. In addition, blocking TGF-ß signaling with the TGF-ß receptor inhibitor SB431542 counteracted the effect of platelets on KLF6 expression and proliferation of HCC cells. Based on these findings, we conclude that platelet releasates, especially TGF-ß, promote the proliferation of SMMC.7721 and HepG2 cells by decreasing expression of KLF6. This discovery identifies a potential new therapeutic target for the prevention and treatment of hepatocellular carcinoma.

Ambient fine particles (PM2.5 ) attenuate collagen-induced platelet activation through interference of the PLCγ2/Akt/GSK3ß signaling pathway.

AIMS: It has been proven that carbon nanoparticles or diesel exhaust particles stimulate platelet activation. However, the effect of fine particle matter (PM2.5 ) on platelet activation remains unknown, which motivates this study. METHODS: PM2.5 samples were collected in an urban area of Zhengzhou, China. To study the morphological characteristics and the mass concentrations of trace elements of PM2.5 samples, a filed-emission scanning electron microscope, the Image-J software, and an inductively coupled plasma mass spectrometry were used. Washed human platelets or platelet-rich-plasma were used to study the effect of PM2.5 on platelet aggregation, P-selectin expression, or platelet signaling pathways. The cytotoxicity in platelets exposed to PM2.5 was evaluated by a lactate dehydrogenase assay kit. In addition, platelet adhesion and spreading were studied on collagen-coated surfaces in stable conditions. RESULTS: The filed-emission scanning electron microscope scanning showed that PM2.5 samples varied in shape and size distributions. The mean equivalent spherical diameter of these particles was 1.97 ± 0.04 µm, of which 82.40% were particles with equivalent spherical diameters of less than 2.5 µm. The mass concentration of Ca was higher than that of other elements. The other elements followed the trend of Al>Fe>Zn>Mg>Pb>K>Mn>Cu>Ti>Ba>As>Sr>Sn>Sb>Cd>B>Se>Mo>Ag>Ni>TI>V>Co. Furthermore, pretreatment of PM2.5 significantly inhibited rather than potentiated collagen-induced platelet aggregation and P-selectin expression, whereas it had no significant effect on ADP-induced platelet aggregation and P-selectin expression. The lactate dehydrogenase analysis showed trivial cytotoxic effect of PM2.5 exposure on platelets. Pretreatment of PM2.5 inhibited platelet adhesion on immobilized collagen-coated surfaces; however, it almost did not impact the platelet spreading. Immunoblotting analysis indicated that PM2.5 reduced collagen-induced phosphorylation of phospholipase C gamma-2 (PLCγ2) at Tyr759, Akt at Ser473, and glycogen synthase kinase 3ß (GSK3ß) at Ser9. CONCLUSIONS: PM2.5 attenuated collagen-induced platelet aggregation, α-granule secretion and adhesion, with the potential mechanism of impairing PLCγ2, Akt, and GSK3ß signaling. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 530-540, 2017.

Antiplatelet activity of chrysin via inhibiting platelet αIIbß3-mediated signaling pathway.

SCOPE: Propolis is thought to help prevent thrombotic and related cardiovascular diseases in humans. Chrysin, a bioflavonoids compound found in high levels in propolis and in honey, has been reported to possess antiplatelet activity. However, the mechanism by which it inhibits platelet function is unclear. METHODS AND RESULTS: The effects of chrysin on agonist-activated platelet-aggregation, granule-secretion, and integrin αIIbß3 activation were examined. Its effects on the phosphorylation of Akt, GSK3ß, MAPKs, and several proteins of the glycoprotein VI (GPVI) signaling pathway were also studied on collaged-activated platelets. In addition, human platelet spreading on immobilized fibrinogen was also tested. We found that chrysin dose dependently inhibited platelet aggregation and granule secretion induced by collagen, as well as platelet aggregation induced by ADP, thrombin, and U46619. Chrysin also markedly reduced the number of adherent platelets and the single platelet spreading area on immobilized fibrinogen. Biochemical analysis revealed that chrysin inhibited collagen-induced activation of Syk, PLCγ2, PKC, as well as the phosphorylation of Akt and ERK1/2. Additionally, chrysin attenuated phosphorylation of molecules such as FcγRIIa, FAK, Akt, and GSK3ß in platelet spreading on immobilized fibrinogen. CONCLUSIONS: Our findings indicate that chrysin suppresses not only integrin αIIbß3-mediated "inside-out" signaling, but also the "outside-in" signal transmission. This implies that chrysin may represent a potential candidate for an antiplatelet agent.

Pentamethylquercetin (PMQ) reduces thrombus formation by inhibiting platelet function.

Flavonoids exert both anti-oxidant and anti-platelet activities in vitro and in vivo. Pentamethylquercetin (PMQ), a polymethoxylated flavone derivative, has been screened for anti-carcinogenic and cardioprotective effects. However, it is unclear whether PMQ has anti-thrombotic effects. In the present study, PMQ (20 mg/kg) significantly inhibited thrombus formation in the collagen- epinephrine- induced acute pulmonary thrombosis mouse model and the ferric chloride-induced carotid injury model. To explore the mechanism, we evaluated the effects of PMQ on platelet function. We found that PMQ inhibited platelet aggregation and granule secretion induced by low dose agonists, including ADP, collagen, thrombin and U46619. Biochemical analysis revealed that PMQ inhibited collagen-, thrombin- and U46619-induced activation of Syk, PLCγ2, Akt, GSK3ß and Erk1/2. Therefore, we provide the first report to show that PMQ possesses anti-thrombotic activity in vivo and inhibited platelet function in vitro, suggesting that PMQ may represent a potential therapeutic candidate for the prevention or treatment of thrombotic disorders.

Antiplatelet activity of loureirin A by attenuating Akt phosphorylation: In vitro studies.

Loureirin A is a flavonoid extracted from Dragon׳s Blood that has been used to promote blood circulation and remove stasis in Chinese traditional medicine. However, the mechanisms of these effects are not fully understood. We explored the anti-platelet activity and underlying mechanism of loureirin A in vitro. Our results indicated that loureirin A negatively affected agonist-induced platelet aggregation such as collagen, collagen-related peptide (CRP), ADP and thrombin. Loureirin A inhibited collagen-induced platelet ATP secretion and thrombin-stimulated P-selectin expression in a dose-dependent manner. Platelet spreading on immobilized fibrinogen was significantly impaired in the presence of loureirin A. Immunoblotting analysis indicated that 100µM of loureirin A almost completely eliminated collagen-induced Akt phosphorylation at Ser473. Interestingly, a submaximal dose (50µM) of loureirin A had an additive inhibitory effect with the phosphoinositide 3-kinase (PI3K) inhibitor Ly294002 on collage-induced Akt phosphorylation in platelets. Taken together, loureirin A had an inhibitory effect on platelet activation, perhaps through an impairment of PI3K/Akt signaling.

2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside (THSG) attenuates human platelet aggregation, secretion and spreading in vitro.

INTRODUCTION: 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside(THSG) is a water-soluble component of the rhizome extract from the traditional Chinese herb Polygonum multiflorum. Recent studies have demonstrated that THSG has potent anti-oxidative and anti-inflammatory effects. In this study, we investigated the anti-platelet aggregation, secretion and spreading of THSG with different methods. The purpose was to explore the anti-platelet effect of THSG and the underlying mechanism. MATERIALS AND METHODS: We investigated the anti-platelet activity of THSG on platelet aggregation induced by collagen (2 µg/mL), thrombin(0.04U/mL), U46619 (3 µM) and ADP (2 µM). ATP secretion induced by collagen (2 µg/mL) was also investigated. P-selectin expression and PAC-1 binding were measured by flow cytometry. In addition, human platelet spreading on immobilized fibrinogen and immunoblotting were also tested. RESULTS: THSG dose-dependently inhibited platelet aggregation and ATP secretion induced by collagen. It inhibited platelet P-selectin expression and PAC-1 binding induced by thrombin(0.1U/mL). THSG also inhibited human platelet spreading on immobilized fibrinogen, a process mediated by platelet outside-in signaling. Western blot analysis showed that THSG could inhibit platelet Fc γ RIIa, Akt(Ser473)and GSK3ß(Ser9) phosphorylation. CONCLUSIONS: Our study indicates that THSG has potent anti-platelet activity to collagen induced aggregation. THSG is likely to exert protective effects in platelet-associated thromboembolic disorders by modulating human platelet.

Neferine exerts its antithrombotic effect by inhibiting platelet aggregation and promoting dissociation of platelet aggregates.

INTRODUCTION: Neferine, a kind of isoquinoline alkaloid, extracted from the seed embryo of Nelumbo nucifera Gaertn, has long been recognized in traditional medicine as a medicinal plant with various usages. Neferine has many biological activities, including anti-hypertensive, anti-arrhythmic, negative inotropic effect and relaxation on vascular smooth muscle. Although previous studies have reported its antithrombotic effect, the mechanisms by which it exerts antithrombotic effect have not been thoroughly studied. METHOD: Washed mice platelets and mice platelet-rich-plasma (PRP) were used to investigate the effects of neferine on platelet aggregation, secretion induced by various agonists and dissociation of agonist-formed platelet aggregates. Bioflux plates coated with collagen were used to investigate the effect of neferine on platelet adhesion and thrombosis in vitro. With collagen-epinephrine-induced acute pulmonary thrombus formation mouse model, the effect of neferine on thrombosis in vivo was also examined. RESULTS: Neferine, significantly and dose-dependently, inhibited collagen-, thrombin-, U46619-induced platelet aggregation in mice washed platelets, or ADP-induced platelet aggregation in PRP. Neferine treatment decreased platelet dense granule secretion initiated by collagen, thrombin and U46619. Also, Neferine dramatically and dose-dependently promoted the dissociation of platelet aggregates pre-formed by various agonists including collagen, thrombin, U46619 or ADP. Neferine can significantly reduce the area of mice platelets adhesion to the collagen and inhibit thrombosis in vitro. In collagen-epinephrine-induced acute pulmonary thrombus mouse model, neferine, at 6 mg/kg, significantly attenuated thrombus formation. CONCLUSIONS: Neferine remarkably prevents thrombus formation by inhibiting platelet activation, adhesion and aggregation, as well as promoting disassembly of pre-formed platelet aggregates. The inhibitory effects of neferine on platelet activation might be relevant in cases involving aberrant platelet activation where neferine could be used as an anti-platelet and antithrombotic agent.

Activation of epidermal growth factor receptor mediates reperfusion arrhythmias in anaesthetized rats.

AIMS: Epidermal growth factor receptor (EGFR) plays a critical role in the development and function of the heart. Previous studies have demonstrated that EGFR is involved in regulating electrical excitability of the heart. The present study was designed to investigate whether EGFR activation would mediate cardiac arrhythmias induced by reperfusion in anaesthetized rats. METHODS AND RESULTS: Reperfusion arrhythmias were induced by 10 min ligation of the left anterior descending coronary artery, followed by a 30 min reperfusion in anaesthetized rats. The incidence and severity of cardiac arrhythmias were significantly reduced by pre-treatment with the EGFR kinase inhibitor AG556. The phosphorylation level of myocardial EGFR was increased during ischaemia and at early reperfusion. Intramyocardial transfection of EGFR siRNA reduced EGFR mRNA and protein, and decreased the incidence of ventricular fibrillation induced by reperfusion. Interestingly, tyrosine phosphorylation levels of cardiac Na(+) channels (I(Na)) and L-type Ca(2+) channels (I(Ca,L)) were significantly increased at time points corresponding to the alteration of EGFR phosphorylation levels during reperfusion. AG556 pre-treatment countered the increased tyrosine phosphorylation level of Na(+) and L-type Ca(2+) channels induced by reperfusion. Patch-clamp studies proved that AG556 could inhibit I(Na) and I(Ca,L) in rat ventricular myocytes. No significant alteration was observed in tyrosine phosphorylation levels of cardiac Kv4.2 and Kir2.1 channels during reperfusion. CONCLUSION: These results demonstrate for the first time that EGFR plays an important role in the genesis of arrhythmias induced by reperfusion, which is likely mediated at least in part by enhancing tyrosine phosphorylation of cardiac Na(+) and L-type Ca(2+) channels.

Effects of WIN55,212-2 on voltage-gated sodium channels in trigeminal ganglion neurons of rats.

OBJECTIVES: This study was carried out to investigate the effects of WIN55,212-2, a potential cannabinoid receptor agonist, on voltage-gated sodium currents I(Na) in cultured trigeminal ganglion neurons of rats, and to investigate whether the anti-nociceptive effects of cannabinoid receptor subtype 1 (CB1) were produced through its modulation on I(Na). METHODS: Whole cell patch clamp techniques were used to record I(Na) before and after WIN55,212-2 was perfused in cultured trigeminal ganglion neurons of rats. RESULTS: WIN55,212-2 (0.01 micromol/l) could enhance I(Na) slightly by 11.5 +/- 4.7% (n=7, p<0.05), and this effect could not be blocked by AM251, the CB1 receptor antagonist. However, WIN55,212-2 could inhibit I(Na) in concentration dependent manner at concentrations from 0.1 to 100 micromol/l. The inhibitory rates were 17.4 +/- 6.0, 22.5 +/- 7.8, 43.9 +/- 9.4 and 73.9 +/- 6.7% respectively by 0.1, 1, 10, 100 micromol/l WIN55,212-2, and the EC(50) was 17.8 micromol/l (n=7, p<0.05 or p<0.01). This inhibitory effect could be blocked partly by 1 micromol/l AM251 (n=7, p<0.05). WIN55,212-2 (0.01 micromol/l) shifted the active curve of I(Na) leftward slightly (n=7, p<0.05), but had no effect on its stable inactive curve (n=7, p>0.05). WIN55,212-2 (10 micromol/l) did not affect the active and stable inactive curves of I(Na) (n=7, p>0.05). CONCLUSION: WIN55,212-2 had bidirectional (two phases) effects on I(Na) in trigeminal ganglion neurons. It might act on different receptors, and the CB1 receptor participated in its modulation on I(Na).

Role of oxidative stress in the apoptosis of hepatocellular carcinoma induced by combination of arsenic trioxide and ascorbic acid.

AIM: The present study was designed to determine the possible pathway underlying the enhancement of apoptosis induced by the combined use of arsenic trioxide (As(2)O(3)) and ascorbic acid (AA). METHODS: The level of intracellular reactive oxygen species (ROS) was detected by means of flow cytometry analysis with an oxidation-sensitive fluorescent probe (6-carboxy-2',7' dichlorodihydrofluorescein diacetate) uploading. The activity of glutathione (GSH), glutathione peroxidase (GPx), and superoxide dismutase (SOD) were detected by biochemical methods. The mitochondrial membrane potential was measured by flow cytometry analysis with rhodamine 123 staining. Bcl-2, Bax, and p17 subunit of caspase-3 were analyzed using the Western blot method. The apoptosis rate was determined by flow cytometry with annexin-V/propidium iodide staining. RESULTS: Compared with As(2)O(3) (2.0 micromol/L) treated alone, As(2)O(3) (2.0 micromol/L) in combination with AA (100 micromol/L) decreased intracellular GSH content from 101.30+/-5.76 to 81.91+/-3.12 mg/g protein, and increased ROS level from 127.61+/-5.12 to 152.60+/-5.88, which was represented by the 2, 7-dichlorofluorescein intensity. The loss of mitochondria membrane potential was increased from 1269.97+/-36.11 to 1540.52+/-52.63, which was presented by fluorescence intensity. The p17 subunit of caspase-3 expression was increased approximately 2-fold. However, SOD and GPx depletion and the ratio of Bcl-2 to Bax were equal to that of As2O3 treated alone (P>0.05). When the ROS scavenger, N-acetyl-L-cysteine, was added to As(2)O(3) and AA combined treatment group, the apoptosis rate decreased from 15.60 %+/-1.14% to 9.48%+/-0.67%, and the ROS level decreased from 152.60+/-5.88 to 102.77+/-10.25. CONCLUSION: AA potentiated As(2)O(3)-induced apoptosis through the oxidative pathway by increasing the ROS level. This may be the result of depleting intracellular GSH. It may influence the downstream cascade following ROS, including mitochondria depolarization and caspase-3 activation. However, SOD and GPx depletion and the ratio of Bcl-2 to Bax influenced by As(2)O(3) was not found to be potentiated by AA.