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.

A congenital CMV infection model for follow-up studies of neurodevelopmental disorders, neuroimaging abnormalities, and treatment.

Congenital cytomegalovirus (cCMV) infection is the leading infectious cause of neurodevelopmental disorders. However, the neuropathogenesis remains largely elusive due to a lack of informative animal models. In this study, we developed a congenital murine CMV (cMCMV) infection mouse model with high survival rate and long survival period that allowed long-term follow-up study of neurodevelopmental disorders. This model involves in utero intracranial injection and mimics many reported clinical manifestations of cCMV infection in infants, including growth restriction, hearing loss, and impaired cognitive and learning-memory abilities. We observed that abnormalities in MRI/CT neuroimaging were consistent with brain hemorrhage and loss of brain parenchyma, which was confirmed by pathological analysis. Neuropathological findings included ventriculomegaly and cortical atrophy associated with impaired proliferation and migration of neural progenitor cells in the developing brain at both embryonic and postnatal stages. Robust inflammatory responses during infection were shown by elevated inflammatory cytokine levels, leukocyte infiltration, and activation of microglia and astrocytes in the brain. Pathological analyses and CT neuroimaging revealed brain calcifications induced by cMCMV infection and cell death via pyroptosis. Furthermore, antiviral treatment with ganciclovir significantly improved neurological functions and mitigated brain damage as shown by CT neuroimaging. These results demonstrate that this model is suitable for investigation of mechanisms of infection-induced brain damage and long-term studies of neurodevelopmental disorders, including the development of interventions to limit CNS damage associated with cCMV infection.

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.

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.

Simvastatin prevents lipopolysaccharide-induced septic shock in rats.

Simvastatin is a hypolipidemic drug that inhibits hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase to control elevated cholesterol, or hypercholesterolemia. Previous studies have shown that simvastatin may attenuate inflammation in ischemia-reperfusion injury and sepsis. Herein, we hypothesized that simvastatin may prevent rats from lipopolysaccharide (LPS)-induced septic shock. In our study, rats were divided into a saline group, an LPS group and an LPS plus simvastatin group. Male Sprague-Dawley (SD) rats were pretreated with simvastatin (1 mg/kg) for 30 min before the addition of LPS (8 mg/kg), with variations in left ventricular pressure recorded throughout. Ninety min after LPS injection, whole blood was collected from the inferior vena cava, and neutrophils were separated from the whole blood using separating medium. The neutrophils were then lysed for Western blotting to detect the levels of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1). In addition, mesentery microcirculations of inlet diameter, outlet diameter and blood flow rate were measured in all three groups. The results indicated that simvastatin significantly promoted heart systolic function and increased the level of uPA while simultaneously inhibited the expression of PAI-1 as compared with LPS group. Moreover, simvastatin reversed the LPS-induced inhibition of mesentery microcirculation. Taken together, it was suggested that simvastatin can effectively protect the rats from LPS-induced septic shock.

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.

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.