Ming Yi Shen

Inhibitory activity of kinetin on free radical formation of activated platelets in vitro and on thrombus formation in vivo

Kinetin has been shown to have anti-aging effects on several different systems, including plants and human cells. Recently, we demonstrated that kinetin markedly inhibited platelet aggregation in washed human platelets. In the present study, an electron spin resonance (ESR) method was used to further evaluate the scavenging activity of kinetin on the free radicals formed. Kinetin (70 and 150 microM) concentration dependently reduced the ESR signal intensity of hydroxyl radicals in collagen (1 microg/ml)-activated platelets. Furthermore, kinetin was effective in reducing the mortality of ADP-induced acute pulmonary thromboembolism in mice when administered intravenously at doses of 4 and 6 mg/kg. In addition, intravenous injection of kinetin (4 and 6 mg/kg) significantly prolonged the bleeding time by approximately 1.9- and 2.1-fold as compared with normal saline in severed mesenteric arteries of rats. A continuous infusion of kinetin (0.6 mg/kg/min) for 10 min also significantly increased the bleeding time by about 2.3-fold, and the bleeding time returned to baseline within 120 min after cessation of kinetin infusion. Platelet thrombi formation was induced by irradiation of mesenteric venules with filtered light in mice pretreated intravenously with fluorescein sodium. When kinetin was administered at 13 and 14 mg/kg in mice pretreated with fluorescein sodium (5 mg/kg), the occlusion time was significantly prolonged. In conclusion, these results suggest that kinetin has effective free radical-scavenging activity in vitro and antithrombotic activity in vivo. Treatment with kinetin may lower the risk of thromboembolic-related disorders. Therefore, kinetin may be a potential therapeutic agent for arterial thrombosis, but its toxicity must be further assessed.

Mechanisms involved in the antiplatelet activity of magnesium in human platelets

Summary. In this study, magnesium sulphate dose‐dependently (0·6–3·0 mmol/l) inhibited platelet aggregation in human platelets stimulated by agonists. Furthermore, magnesium sulphate (3·0 mmol/l) markedly interfered with the binding of fluorescein isothiocanate‐triflavin to the glycoprotein (GP)IIb/IIIa complex in platelets stimulated by collagen. Magnesium sulphate (1·5 and 3·0 mmol/l) also inhibited phosphoinositide breakdown and intracellular Ca+2 mobilization in human platelets stimulated by collagen. Magnesium sulphate (3·0 mmol/l) significantly inhibited thromboxane A2 formation stimulated by collagen in platelets. Moreover, magnesium sulphate (1·5 and 3·0 mmol/l) obviously increased the fluorescence of platelet membranes tagged with diphenylhexatriene. In addition, magnesium sulphate (1·5 and 3·0 mmol/l) increased the formation of cyclic adenosine monophosphate (AMP) in platelets. Phosphorylation of a protein of Mr 47 000 (P47) was markedly inhibited by magnesium sulphate (1·5 mmol/l). In conclusion, the antiplatelet activity of magnesium sulphate may involve the following two pathways. (1) Magnesium sulphate may initially induce membrane fluidity changes with resulting interference of fibrinogen binding to the GPIIb/IIIa complex, followed by inhibition of phosphoinositide breakdown and thromboxane A2 formation, thereby leading to inhibition of both intracellular Ca2+ mobilization and phosphorylation of P47. (2) Magnesium sulphate might also trigger the formation of cyclic AM, ultimately resulting in inhibition of the phosphorylation of P47 and intracellular Ca+2 mobilization.

Mitochondrial mechanisms in amyloid beta peptide-induced cerebrovascular degeneration

Prevailing evidence suggests that amyloid beta peptide (Abeta), a key mediator in age-dependent neuronal and cerebrovascular degeneration, activates death signaling processes leading to neuronal as well as non-neuronal cell death in the central nervous system. A major cellular event in Abeta-induced death of non-neuronal cells, including cerebral endothelial cells, astrocytes and oligodendrocytes, is mitochondrial dysfunction. The death signaling cascade upstream of mitochondria entails Abeta activation of neutral sphingomyelinase, resulting in the release of ceramide from membrane sphingomyelin. Ceramide then activates protein phosphatase 2A (PP2A), a member in the ceramide-activated protein phosphatase (CAPP) family. PP2A dephosphorylation of Akt and FKHRL1 plays a pivotal role in Abeta-induced Bad translocation to mitochondria and transactivation of Bim. Bad and Bim are pro-apoptotic proteins that cause mitochondrial dysfunction characterized by excessive ROS formation, mitochnondrial DNA (mtDNA) damage, and release of mitochondrial apoptotic proteins including cytochrome c, apoptosis inducing factor (AIF), endonuclease G and Smac. The cellular events activated by Abeta to induce death of non-neuronal cells are complex. Understanding these death signaling processes will aid in the development of more effective strategies to slow down age-dependent cerebrovascular degeneration caused by progressive cerebrovascular Abeta deposition.

A novel antioxidant, octyl caffeate, suppression of LPS/IFN-γ-induced inducible nitric oxide synthase gene expression in rat aortic smooth muscle cells

In the present study, we investigated the effects and mechanisms of a novel potent antioxidant, octyl caffeate, on the induction of iNOS expression by lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) in cultured primary rat aortic smooth muscle cells (RASMCs) in vitro and LPS-induced hypotension in vivo. Octyl caffeate (0.1-1.0 microM) exerted a concentration-dependent inhibition of iron-catalyzed lipid peroxidation in rat brain homogenates. Furthermore, octyl caffeate (20, 50, and 100 microM) concentration-dependently diminished the initial rate of superoxide-induced NBT reduction and the enzymatic activity of xanthine oxidase. It also concentration-dependently (1-50 microM) inhibited the NO production, iNOS protein and messenger RNA expressions upon stimulation by LPS (100 microg/mL)/IFN-gamma (100U/mL) in RASMCs. In addition, we found that octyl caffeate did not significantly affect IkappaBalpha degradation stimulated by LPS/IFN-gamma in RASMCs. On the other hand, octyl caffeate (10 and 50 microM) significantly suppressed activation of c-Jun-N-terminal kinase and extracellular signal-regulated kinase. Moreover, octyl caffeate (10mg/kg, i.v.) significantly inhibited the fall in mean arterial pressure stimulated by LPS (7.5mg/kg) in rats. In conclusion, we demonstrate that a novel potent antioxidant, octyl caffeate, significantly ameliorates circulatory failure of endotoxemia in vivo by a mechanism involving suppression of iNOS expression through inactivation of mitogen-activated protein kinases in RASMCs.

Mechanisms Involved in the Antiplatelet Activity of Ketamine in Human Platelets

The aim of this study was to systematically examine the inhibitory mechanisms of ketamine in platelet aggregation. In this study, ketamine concentration-dependently (100–350 µM) inhibited platelet aggregation both in washed human platelet suspensions and platelet-rich plasma stimulated by agonists. Ketamine inhibited phosphoinositide breakdown and intracellular Ca2+ mobilization in human platelets stimulated by collagen. Ketamine (200 and 350 µM) significantly inhibited thromboxane (Tx) A2 formation stimulated by collagen. Moreover, ketamine (200 and 350 µM) increased the fluorescence of platelet membranes tagged with diphenylhexatriene. Rapid phosphorylation of a platelet protein of Mr 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12,13-dibutyrate (100 nM). This phosphorylation was markedly inhibited by ketamine (350 µM). These results indicate that the antiplatelet activity of ketamine may be involved in the following pathways. Ketamine may change platelet membrane fluidity, with a resultant influence on activation of phospholipase C, and subsequent inhibition of phosphoinositide breakdown and phosphorylation of P47, thereby leading to inhibition of intracellular Ca2+ mobilization and TxA2 formation, ultimately resulting in inhibition of platelet aggregation.

Expression of amyloid beta peptide in human platelets: Pivotal role of the phospholipase Cγ2-protein kinase C pathway in platelet activation

The amyloid beta peptide (Abeta), a mediator of neuronal and vascular degeneration in the pathogenesis of Alzheimers disease and cerebral amyloid angiopathy may have peripheral actions. Platelets are enriched with Abeta and have been shown to enhance platelet actions. However, the detailed signaling pathways through which Abeta activates platelets have not been previously explored. In this study, we examined the intra-platelet Abeta distribution using a gold labeling technique and noted that Abeta was predominantly localized in the cytoplasm of resting platelets. A marked increase in Abeta-gold labeling in an open canalicular system was observed in collagen-activated platelets. Exogenous Abeta (2-10 microM) stimulated platelet aggregation accompanied by phospholipase Cgamma2 (PLCgamma2) phosphorylation, phosphoinositide breakdown, and [Ca(2+)]i mobilization as well as protein kinase C (PKC) activation. Ro318220, an inhibitor of PKC, suppressed Abeta-induced platelet aggregation, PKC activation, and [Ca(2+)]i mobilization in platelets, suggesting that the PLCgamma2-PKC pathway is involved in Abeta-induced platelet aggregation. In the electron spin resonance study, Abeta (2 and 10 microM) markedly triggered hydroxyl radical formation in platelets. In an in vivo study, Abeta (2mg/kg) significantly shortened the latency for inducing platelet plug formation in the mesenteric venules of mice. In conclusion, we are the first to demonstrate (1) the distribution of Abeta in human platelets; and that (2) Abeta activation of platelets is mediated, at least partially, by the PLCgamma2-PKC pathway; and (3) Abeta triggers thrombus formation in vivo.

Involvement of the antiplatelet activity of magnesium sulfate in suppression of protein kinase C and the Na+/H+ exchanger

Magnesium sulfate is widely used to prevent seizures in pregnant women with hypertension. The aim of this study was to examine the inhibitory mechanisms of magnesium sulfate in platelet aggregation in vitro. In this study, magnesium sulfate concentration-dependently (0.6–3.0 mM) inhibited platelet aggregation in human platelets stimulated by agonists. Magnesium sulfate (1.5 and 3.0 mM) also concentration-dependently inhibited phosphoinositide breakdown and intracellular Ca+2 mobilization in human platelets stimulated by thrombin. Rapid phosphorylation of a platelet protein of Mr 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12-13-dibutyrate (PDBu, 50 nM). This phosphorylation was markedly inhibited by magnesium sulfate (3.0 mM). Magnesium sulfate (1.5 and 3.0 mM) further inhibited PDBu-stimulated platelet aggregation in human platelets. The thrombin-evoked increase in pHi was markedly inhibited in the presence of magnesium sulfate (3.0 mM). In conclusion, these results indicate that the antiplatelet activity of magnesium sulfate may be involved in the following two pathways: (1) Magnesium sulfate may inhibit the activation of protein kinase C, followed by inhibition of phosphoinositide breakdown and intracellular Ca+2 mobilization, thereby leading to inhibition of the phosphorylation of P47. (2) On the other hand, magnesium sulfate inhibits the Na+/H+ exchanger, leading to reduced intracellular Ca+2 mobilization, and ultimately to inhibition of platelet aggregation and the ATP-release reaction.

Morphine-Potentiated Platelet Aggregation in in vitro and Platelet Plug Formation in in vivo Experiments

The detailed mechanisms underlying morphine-signaling pathways in platelets remain obscure. Therefore, we systematically examined the influence of morphine on washed human platelets. In this study, washed human platelet suspensions were used for in vitro studies. Furthermore, platelet thrombus formation induced by irradiation of mesenteric venules with filtered light in mice pretreated with fluorescein sodium was used for an in vivo thrombotic study. Morphine concentration dependently (0.6, 1, and 5 µM) potentiated platelet aggregation and the ATP release reaction stimulated by agonists (i.e., collagen and U46619) in washed human platelets. Yohimbine (0.1 µM), a specific α2-adrenoceptor antagonist, markedly abolished the potentiation of morphine in platelet aggregation stimulated by agonists. Morphine also potentiated phosphoinositide breakdown and intracellular Ca2+ mobilization in human platelets stimulated by collagen (1 µg/ml). Moreover, morphine (0.6–5 µM) markedly inhibited prostaglandin E1 (10 µM)-induced cyclic AMP formation in human platelets, while yohimbine (0.1 µM) significantly reversed the inhibition of cyclic AMP by morphine (0.6 and 1 µM) in this study. The thrombin-evoked increase in pHi was markedly potentiated in the presence of morphine (1 and 5 µM). Morphine (2 and 5 mg/g) significantly shortened the time require to induce platelet plug formation in mesenteric venules. We concluded that morphine may exert its potentiation in platelet aggregation by binding to α2-adrenoceptors in human platelets, with a resulting inhibition of adenylate cyclase, thereby reducing intracellular cyclic AMP formation followed by increased activation of phospholipase C and the Na+/H+ exchanger. This leads to increased intracellular Ca2+ mobilization, and finally potentiation of platelet aggregation and of the ATP release reaction.

Involvement of p38 MAPK phosphorylation and nitrate formation in aristolochic acid-mediated antiplatelet activity

Aristolochic acid (AsA) is produced from Aristolochia fangchi, and has been used as a Chinese herbal medicine. AsA possesses various biological activities including antiplatelet, antifungal, and anti-inflammatory properties. The aim of this study was to examine the mechanisms of AsA in inhibiting platelet aggregation. AsA (75 - 150 microM) exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen (1 microg/mL) than other agonists. AsA (115 and 150 microM) inhibited collagen-induced platelet activation accompanied by [Ca+2)]i mobilization, thromboxane A2 (TxA2) formation and phosphoinositide breakdown. On the other hand, AsA also markedly increased levels of NO/cyclic GMP, and cyclic GMP-induced vasodilator-stimulated phosphoprotein phosphorylation. AsA inhibited p38 MAPK but not ERK1/2 phosphorylation in washed platelets. In conclusion, the most important findings of this study suggest that the inhibitory effects of AsA possibly involve the (1) inhibition of the p38 MAPK-cytosolic phospholipase A2-arachidonic acid-TxA2-[Ca+2)]i cascade, and (2) activation of NO/cyclic GMP, resulting in inhibition of phospholipase C. These results imply that Aristolochia fangchi treatment alone or in combination with other antiplatelet drugs, may result in alteration of hemostasis in vivo.

Sesamol reduces the atherogenicity of electronegative L5 LDL in vivo and in vitro

Highly electronegative low-density lipoprotein (LDL) L5 induces endothelial cell (EC) apoptosis, which leads to the development of atherosclerosis. We examined the effects of sesamol (1), a natural organic component of sesame oil, on plasma L5 levels and atherosclerosis development in a rodent model and on the L5-induced apoptosis of ECs. Syrian hamsters, which have an LDL profile similar to that of humans, were fed a normal chow diet (control), a high-fat diet (HFD), or a HFD supplemented with the administration of 50 or 100 mg/kg of 1 via oral gavage (HFD+1) for 16 weeks (n = 8 per group). Hamsters in the HFD+1 groups had reduced plasma L5 levels when compared with the HFD group. Oil Red O staining showed that atherosclerotic lesion size was markedly reduced in the aortic arch of hamsters in the HFD+1 groups when compared with that in the HFD group. In human aortic ECs, 0.3-3 μM 1 blocked L5-induced apoptosis in a dose-dependent manner. Further mechanistic studies showed that 1 inhibited the L5-induced lectin-like oxidized LDL receptor-1 (LOX-1)-dependent phosphorylation of p38 MAPK and activation of caspase-3 and increased phosphorylation of eNOS and Akt. Our findings suggest that sesamol (1) protects against atherosclerosis by reducing L5-induced atherogenicity.