5-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-3,7-dimethoxy-4H-chromen-4-one (MSF-2) suppresses fMLP-mediated respiratory burst in human neutrophils by inhibiting phosphatidylinositol 3-kinase activity.

Respiratory burst mediates crucial bactericidal mechanism in neutrophils. However, undesirable respiratory burst leads to pathological inflammation and tissue damage. This study investigates the effect and the underlying mechanism of 5-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-3,7-dimethoxy-4H-chromen-4-one (MSF-2), a lignan extracted from the fruit of Melicope Semecarprifolia, on fMLP-induced respiratory burst in human neutrophils and suggests a possible therapeutic approach to ameliorate disease associated with neutrophil hyperactivation. MSF-2 inhibited fMLP-induced neutrophil superoxide anion production, cathepsin G release and migration in human neutrophils isolated from healthy volunteers, reflecting inhibition of phosphatidylinositol 3-kinase (PI3K) activation. Specifically, PI3K/AKT activation results in migration, degranulation and superoxide anion production in neutrophils. MSF-2 suppresses PI3K activation and phosphatidylinositol (3,4,5)-trisphosphate (PIP3) production, and consequently inhibits downstream activation of PDK1 and AKT. Further, PI3K also stimulates respiratory burst via PLC-dependent elevation of intracellular calcium. MSF-2 reduces fMLP-mediated PLCγ2 activation and intracellular calcium accumulation notably through extracellular calcium influx in a PI3K and PLC-dependent manner. However, MSF-2 is not a competitive or allosteric antagonist of fMLP. Additionally, in an in vivo study, MSF-2 prevents fMLP-induced neutrophil infiltration and inflammation in mice. In conclusion, MSF-2 opposes fMLP-mediated neutrophil activation and inflammation by inhibiting PI3K activation and subsequent activation of AKT and PLCγ2.

Measurement of the binding parameters of annexin derivative-erythrocyte membrane interactions.

Erythrocyte ghosts prepared from fresh blood expressed phosphatidylserine (PS) on the membrane surfaces in a rather stable fashion. The binding of fluorescein-5-isothiocyanate (FITC)-labeled annexin V (ANV) derivatives to these membranes was studied by titration with proteins and with calcium. Whereas the preaddition of ethylenediaminetetraacetic acid (EDTA) to reaction mixtures totally prevented membrane binding, Ca(2+)-dependent binding was only partially reversed by EDTA treatment, consistent with an initial Ca(2+)-dependent binding that became partially Ca(2+) independent. Data derived from saturation titration with ANV derivatives poorly fit the simple protein-membrane equilibrium binding equation and showed negative cooperativity of binding with increasing membrane occupancy. In contrast, calcium titration at low binding site occupancy resulted in excellent fit into the protein-Ca(2+)-membrane equilibrium binding equation. Calcium titrations of FITC-labeled ANV and ANV-6L15 (a novel ANV-Kunitz protease inhibitor fusion protein) yielded a Hill coefficient of approximately 4 in both cases. The apparent dissociation constant for ANV-6L15 was approximately 4-fold lower than that of ANV at 1.2-2.5mM Ca(2+). We propose that ANV-6L15 may provide improved detection of PS exposed on the membrane surfaces of pathological cells in vitro and in vivo.

Anti-inflammatory Biphenyls and Dibenzofurans from Rhaphiolepis indica.

Bioassay-guided fractionation of the methanolic extract of the roots of Rhaphiolepis indica var. tashiroi afforded four new dibenzofurans, 2-hydroxy-3,4,6-trimethoxydibenzofuran (1), 2-hydroxy-3,4,9-trimethoxydibenzofuran (2), 2-hydroxy-3,4,6,9-tetramethoxydibenzofuran (3), and 1,2-methylenedioxy-3,4,6-trimethoxydibenzofuran (4), two new biphenyls, 3-hydroxy-2',5-dimethoxybiphenyl (5) and 2',3-dihydroxy-5-methoxybiphenyl (6), and 3-hydroxy-5-methoxybiphenyl (7). Among the isolates, 3, 5, and 6 exhibited inhibitory effects on N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced superoxide production, with in vitro IC50 values < 8.36 µM.

Thrombin regulates matrix metalloproteinase-9 expression in human monocytes.

We investigated whether thrombin, the final activator of coagulation cascade, regulates expression of matrix metalloproteinases (MMP)-9 in human monocytes. We show that thrombin stimulation induced MMP-9 secretion of monocytes dose- and time-dependently as revealed by gelatin zymography. Real-time RT-PCR and Western blot analysis demonstrated that thrombin up-regulated mRNA and protein levels of MMP-9. Pre-incubation with anti-protease-activated receptor (PAR)-1 or anti-PAR-3 antibody partially inhibited the thrombin-induced MMP-9 secretion. Simultaneous incubation with both showed synergistic effect, indicating the involvement of both receptors in this thrombin effect. BAPTA, a Ca(2+) chelator, abolished the thrombin-induced MMP-9 secretion, indicating the requirement of Ca(2+) mobilization in this process. Inhibition of thrombin-induced MMP-9 secretion by either MEK inhibitor or p38 kinase inhibitor revealed that the thrombin effect was mediated by both ERK1/2 and p38 pathways. The activation of NFkappaB by thrombin as demonstrated by electromobility shift assay was also shown to be critical to the thrombin-induced MMP-9 up-regulation.

Hemeoxygenase-1 upregulation is critical for sirtinol-mediated attenuation of lung injury after trauma-hemorrhage in a rodent model.

BACKGROUND: Hemeoxygenase-1 induction in response to adverse circulatory conditions is protective. Our recent study has shown that administration of sirtinol attenuates hepatic injury in male Sprague-Dawley rats after trauma-hemorrhage; however, the mechanism by which sirtinol produces the salutary effects remains unknown. We hypothesized that sirtinol administration in male Sprague-Dawley rats after trauma-hemorrhage decreases cytokine production and protects against lung injury through a hemeoxygenase-1 related pathway. METHODS: Male Sprague-Dawley rats (n = 8 per group) underwent trauma-hemorrhage (mean arterial blood pressure 40 mm Hg for 90 min, then resuscitation). A single dose of sirtinol (1 mg/kg of body weight) with or without a hemeoxygenase enzyme inhibitor (chromium-mesoporphyrin) or vehicle was administered IV during resuscitation. Twenty-four hours thereafter, myeloperoxidase activity (a marker of neutrophil sequestration) and tumor necrosis factor alpha, interleukin-6, and interleukin-10 levels in the lung, protein concentrations in bronchoalveolar lavage fluid and tissue histology were measured. Lung hemeoxygenase-1 protein level was also determined. RESULTS: In the sirtinol-treated rats subjected to trauma-hemorrhage, there were significant improvements in lung myeloperoxidase activity (4.68 +/- 0.31 vs 9.36 +/- 1.03 U/mg protein, P < 0.05), tumor necrosis factor alpha levels (710.7 +/- 28 vs 1288 +/- 40.69 pg/mg protein, P < 0.05), interleukin-6 levels (343.6 +/- 18.41 vs 592.7 +/- 22.3 pg/mg protein, P < 0.05), and protein concentrations (303.8 +/- 24.54 vs 569.6 +/- 34.82 microg/mL, P < 0.05) and lesser damage in histology. There was no statistically significant difference in interleukin-10 levels in the lung between sirtinol-treated trauma-hemorrhaged rats and vehicle-treated trauma-hemorrhaged rats (842.5 +/- 54.18 vs 756.2 +/- 41.34 pg/mg protein, respectively). Lung hemeoxygenase-1 protein levels were increased in rats receiving sirtinol treatment as compared with vehicle-treated trauma-hemorrhaged rats (5.18 +/- 0.25 vs 2.70 +/- 0.16, P < 0.05). Administration of the hemeoxygenase inhibitor chromium-mesoporphyrin prevented the sirtinol-induced attenuation of shock-induced lung damage. CONCLUSION: The salutary effects of sirtinol administration on attenuation of lung inflammation after trauma-hemorrhage are mediated via upregulation of hemeoxygenase-1 expression.

A new dibenzofuran and further constituents from the stems of Pourthiaea lucida with inhibitory activity on superoxide generation by neutrophils.

A new dibenzofuran, lucidafuran (1), was isolated from the stems of Pourthiaea lucida, together with eight known compounds. The structure of this new compound was determined through NMR and mass-spectrometric analyses. Among the isolated compounds, lucidafuran (1) and aucuparin (3) exhibited potent inhibitory activity against fMLP-induced superoxide (O(*-)(2)) production by human neutrophils with IC(50) values of 18.7+/-4.4 and 17.0+/-6.8 microM, resp.

(2R,3R)-2-(3',4'-dihydroxybenzyl)-3-(3'',4''-dimethoxybenzyl)butyrolactone suppresses fMLP-induced superoxide production by inhibiting fMLP-receptor binding in human neutrophils.

This study investigated the mechanism underlying the inhibiting effect of (2R,3R)-2-(3',4'-dihydroxybenzyl)-3-(3'',4''-dimethoxybenzyl) butyrolactone (PP-6), a lignan from Piper philippinum, on superoxide anion production induced by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP) in human neutrophils. Human neutrophils were stimulated with fMLP (1 microM), PMA (100 nM) or leukotriene B(4) (LTB(4); 1 microM) and induced superoxide anion release. PP-6 specifically inhibited fMLP-induced superoxide anion production in a concentration-dependent manner with an IC(50) value of 0.3+/-0.1 microM. Intracellular signaling caused by fMLP, PMA or LTB(4) were evaluated. PP-6 specifically inhibited fMLP-induced intracellular calcium mobilization and ERK (p42/p44), Akt and p38 phosphorylation. Moreover, PP-6 specifically inhibited fMLP-induced Mac-1 expression without affecting this caused by LTB(4) or PMA. PP-6 did not increase cAMP level in human neutrophils. PP-6 did not inhibit superoxide anion production by NaF (20 mM), a direct activator of G-protein, the target of the inhibitory action of PP-6 appears to be a component of the signal transduction pathway upstream of G-protein. PP-6 inhibited FITC-fMLP binding to neutrophils in a concentration-dependent manner with an IC(50) of 1.5+/-0.2 microM. PP-6 did not bring a parallel shift in the concentration response of fMLP-induced superoxide anion. Additionally, the inhibiting effect of PP-6 on fMLP-induced superoxide anion was reversed when PP-6 was washed out. These experimental results suggest that PP-6 exerts non-competitive and reversible antagonistic effect on fMLP receptor.

Induction of cytosolic phospholipase A2 by lipopolysaccharide in canine tracheal smooth muscle cells: involvement of MAPKs and NF-kappaB pathways.

Cytosolic phospholipase A2 (cPLA2) plays a pivotal role in mediating agonist-induced arachidonic acid (AA) release for prostaglandins (PG) synthesis induced by bacterial lipopolysaccharide (LPS) and cytokines. However, the intracellular signaling pathways mediating LPS-induced cPLA2 expression and PGE2 synthesis in canine tracheal smooth muscle cells (TSMCs) remains unknown. LPS-induced expression of cPLA2 and release of PGE2 was attenuated by inhibitors of tyrosine kinase (genistein), phosphatidylcholine-phospholipase C (D609), phosphatidylinositol-phospholipase C (U73122), PKC (GF109203X and staurosporine), removal of Ca2+ by BAPTA/AM plus EDTA, MEK1/2 (PD98059), p38 (SB202190), JNK (SP600125), and phosphatidylinositol 3-kinase (PI3-K; LY294002 and wortmannin). The involvement of MPAKs in LPS-induced responses was further confirmed by transfection of TSMCs with dominant negative mutants of ERK2 and p38. LPS-induced cPLA2 expression and PGE2 synthesis was inhibited by a selective NF-kappaB inhibitor (helenalin) and transfection with dominant negative mutants of NF-kappaB inducing kinase (NIK), IkappaB kinase (IKK)-alpha, and IKK-beta, consistent with that LPS-stimulated both IkappaB-alpha degradation and NF-kappaB translocation into nucleus in these cells. LPS-stimulated cPLA2 phosphorylation was inhibited by PD98059, GF109203X, and staurosporine, indicating the regulation by p42/p44 MAPK and PKC. Moreover, LPS-induced up-regulation of cPLA2 and COX-2 linked to PGE2 synthesis was inhibited by AACOCF3 (a selective cPLA2 inhibitor), implying the involvement of cPLA2 in these responses. These findings suggest that phosphorylation and expression of cPLA2 correlates with the release of PGE2 from LPS-challenged TSMCs, at least in part, mediated through MAPKs and NF-kappaB signaling pathways. LPS-mediated responses were modulated by PLC, Ca2+, PKC, tyrosine kinase, and PI3-K in TSMCs.

Lignans, an amide and anti-platelet activities from Piper philippinum.

Investigation of the stem extract of Piper philippinum led to isolation of eight compounds, piperphilippinins I-VI (1-6), philippinamide (7), and (+)-bornyl caffeate (8), together with 26 known compounds. Among the isolates, (-)-3',4'-O,O-demethylenehinokinin (10) and 3,4-methylenedioxycinnamaldehyde (23) showed anti-platelet activities in vitro.

Effects of rosiglitazone on native low-density-lipoprotein-induced respiratory burst in circulating monocytes and on the leukocyte-endothelial interaction in cholesterol-fed rats.

Low-density lipoprotein (LDL) has been implicated in the initiation and progression of atherosclerotic vascular disease. But whether LDL can elicit similar effects in the microcirculation remain unexplored. To approach this issue, the hypothesis that LDL promotes oxidative stress in monocytes and results in microvascular inflammation was tested. Native LDL was capable of stimulating respiratory burst in rat monocytes, and this was blocked by BAPTA, cytochalasin B, apocynin, and diphenyliodonium. In monocytes from rats on a high-cholesterol (4%) diet, increased intracellular calcium, actin polymerization, respiratory burst, and surface CD18 expression were found. Concurrently, leukocyte-endothelial interaction was enhanced in the cremaster microcirculation. Rosiglitazone, an insulin-sensitizing agent with antiinflammatory properties, was found to suppress native-LDL-induced actin polymerization and respiratory burst in monocytes. It also improved leukocytes activation and leukocyte-endothelial interaction due to the high cholesterol intake. Hence, native LDL stimulation of monocytes contributed to hypercholesterolemia-associated microvascular inflammation, which could be treated by rosiglitazone.

Modulation of human monocyte-derived dendritic cells maturation by a soluble guanylate cyclase activator, YC-1, in a cyclic nucleotide independent manner.

This study evaluated how YC-1, a guanylate cyclase activator, affects the maturation of human monocyte-derived dendritic cells. Maturation markers and intracellular signaling pathways were evaluated. YC-1 inhibited the lipopolysaccharide up-regulation of mature markers, including CD40, CD80 or CD86 in a concentration-dependent manner with IC(50) values of 4.6+/-0.4, 4.9+/-0.6 or 4.5+/-0.5 microM, respectively. YC-1, at a higher concentration, inhibited lipopolysaccharide-induced HLADR expression. These effects of YC-1 were not reversed by ODQ (10 microM), which is a soluble guanylate cyclase inhibitor, nor by KT5823 (1 microM), which is a PKG inhibitor. Additionally, YC-1 did not increase levels of cyclic nucleotides in dendritic cells, supporting the claim that YC-1 affects dendritic cells maturation in a cGMP-independent manner. YC-1, in a cGMP-independent manner, inhibited lipopolysaccharide-induced Akt activation, IkappaBalpha degradation and NF-kappaB translocation, all of which are associated with co-stimulatory molecules expression. YC-1 inhibited the capacity of dendritic cell to activate allogenic T cells with an IC(50) value of 1.2+/-0.3 microM. YC-1-treated dendritic cells have mature phenotypes that exhibit up-regulated CCR7, enhanced IL-10 release and low phagocytosis activity in the presence of lipopolysaccharide. In conclusion, YC-1 inhibited the lipopolysaccharide-induced co-stimulatory molecular expression of dendritic cells by inhibiting Akt activation, IkappaBalpha degradation and NF-kappaB translocation. These inhibitory effects on co-stimulatory molecules suppressed the capacity of dendritic cells to activate allogenic T cells. Additionally, YC-1 treated dendritic cells exhibit the up-regulation of CCR7, enhanced IL-10 release and the down-regulation of phagocytosis in the presence of lipopolysaccharide. Accordingly, YC-1 might be a useful tool for evaluation of dendritic cells on autoimmune or allergic disease.

Amides with anti-platelet aggregation activity from Piper taiwanense.

Three new amides, taiwanamides A-C (1-3), and two known amides, 1-cinnamoylpyrrolidine (4) and 1-(m-methoxycinnamoyl)pyrrolide (5), have been isolated from the stem of Piper taiwanense. The structures of the new amides were determined by spectral analyses. These five amides exhibited inhibitory activity of platelet aggregation in vitro. Taiwanamide C (3) owned the most potent inhibition activity of platelet aggregation induced by collagen showing IC(50) value as 8.9 microM. Taiwanamide B (2) and 1-(m-methoxycinnamoyl)pyrrolide (5) exhibited IC(50) values of 17.3 and 17.4 microM, respectively.

Interference of neutrophil-platelet interaction by YC-1: a cGMP-dependent manner on heterotypic cell-cell interaction.

N-Formyl-Met-Leu-Phe (fMLP) activated neutrophils and then induced neutrophil-platelet complex formation in co-incubation condition. In addition, fMLP induce intracellular calcium mobilization in platelets, only when it is incubated along with neutrophils. This data established that fMLP-stimulated neutrophils activate platelets. 9E1, a monoclonal antibody of P-selectin, significantly blocks the formation of neutrophil-platelet complex induced by fMLP, indicating the involvement of P-selectin in the neutrophil-platelet complex formation. 3-(5'-hydroxymethyl-2'-furyl-1-benzylindazole (YC-1), an unique nitric oxide-independent activator of soluble guanylate cyclase, was evaluated for its effect on neutrophil-platelet complex. YC-1 inhibits fMLP-induced neutrophil-platelet complex formation in a concentration-dependent manner with an IC50 value of 15.3+/-3.5 microM. However, this effect of YC-1 is partially reversed by pre-treatment of 1H-(1,2,4)oxadiazolo[4,3-a]quinozalin-1-one (ODQ; 10 microM), which is a soluble guanylate cyclase inhibitor. Pre-treatment of either neutrophils or platelets with YC-1 (50 microM) prevent the fMLP-induced neutrophil-platelet complex formation, indicating that YC-1 could potentially exert its effects individually on either neutrophils or platelets alone. Cathepsin G released from fMLP-stimulated neutrophil activates the nearby platelets. YC-1 was also shown to inhibit this release of cathepsin G in a concentration-dependent manner. The IC50 value was 6.2+/-0.2 microM. This inhibitory effect of YC-1 on cathepsin G release is reversed by ODQ (10 microM) and a protein kinase G inhibitor [1-oxo-9.12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-l][1,6]benzodiazocine-10-carbooxylic acid methyl ester (KT5835); 1 microM]. YC-1 inhibits cathepsin G-induced P-selectin expression on human platelet at the IC50 value of 32.5+/-2.6 microM. A further study showed that YC-1 inhibits fMLP-induced neutrophil-platelet complex formation in whole blood at the IC50 value of 35.8+/-8.1 microM in a concentration-dependent manner. According to these data, it was hypothesized that fMLP stimulates neutrophils to release cathepsin G, which subsequently activates the nearby platelets, creating neutrophil-platelet complexes. YC-1 inhibits fMLP-induced neutrophil from releasing cathepsin G via a cGMP-dependent pathway. This inhibitory effect of YC-1 on cathepsin G release is a major mechanism for affecting fMLP-induced neutrophil-platelet complex. YC-1's inhibition P-selectin expression on platelet may potentiate its effects. These inhibitory effects may contribute to the inhibition of neutrophil-platelet complex formation in whole blood.

Effects of phenolic acid esters and amides on stimulus-induced reactive oxygen species production in human neutrophils.

BACKGROUND: Phenolic acids and their derivatives are widely distributed in plants. A series of phenolic acid esters and amides have been synthesized. METHODS: We determined the effects of phenolic acid derivatives on antiinflammatory activity against phorbol 12-myristate 13-acetate (PMA) and N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced production of superoxide anion, an inflammatory mediator produced by neutrophils. RESULTS: When the cells were preincubated with phenolic acids and their derivatives, the superoxide generation induced by fMLP (1.0 micromol/l) and PMA (0.16 micromol/l) was inhibited to various degrees with compounds 1, 2 and 4 significantly suppressing such generation in a concentration-dependent manner. CONCLUSION: Phenolic acid derivatives may exert their antiinflammatory action through inhibiting superoxide generation.

Synthesis, and cytotoxic and antiplatelet activities of oxime- and methyloxime-containing flavone, isoflavone, and xanthone derivatives.

A series of oxime- and methyloxime-containing flavone, isoflavone, and xanthone derivatives (1-12) were synthesized (Scheme) and evaluated for their cytotoxic (Table 1) and antiplatelet activities (Table 2). The in vitro anticancer assay indicated that the cytotoxicity of structurally related compounds decreases in the order isoflavones (7a-7c) > flavones (8a-8c) > xanthones (9a-9c), electron-releasing substituents (R) on the Ph ring being favorable (mean GI50 values of 2.84, 12.3, and 20.9 microM for 7c, 8c, and 9c, resp.). The inhibition of platelet aggregation induced by arachidonic acid (AA) similarly decreased from the isoflavone 1 (IC50 = 2.97 microM) to the flavone 2 (7.70 microM) to the xanthone 3 (inactive). Thereby, compound 1 seems to be a promising lead, since it was not only the most-potent aggregation inhibitor (IC50 = 2.97 microM), but was also found to be noncytotoxic at a concentration of 100 microM.

Antioxidant and antiplatelet effects of dang-gui-shao-yao-san on human blood cells.

Dang-Gui-Shao-Yao-San (DGSYS) is a mixture of medicinal herbs, which has long been used in traditional Chinese medicine for treating anemia and ovulary disorders. Its preparation comprises Angelicae sinensis (Oliv.) Diels, Ligustucum chuanxiong Hort, Paeonia lactiflora pall, Poria cocos (Schw.) Wolf, Atractylodis macrocephala Koidz and Alisma orientalis (Sam.) Juzep. The present study examined the anti-superoxide formation, free radical scavenging and anti-lipid peroxidation activities of DGSYS by xanthine oxidase inhibition, cytochrome C system with superoxide anion released by the fMLP or PMA activating pathway in human neutrophils, and FeCl2 ascorbic acid-induced lipid peroxidation effects on lipids in rat liver homogenate, respectively. DGSYS showed anti-superoxide formation and free radical scavenging activity in a concentration-dependent manner. It also inhibited PMA- but not fMLP-induced superoxide anion released from human neutrophils. These antioxidant actions of DGSYS showed beneficial cytoprotective effects against lipid peroxidation in rat liver homogenate, human platelet aggregation induced by arachidonic acid (AA) and adenosine diphosphate (ADP) and mitomycin C-mediated hemolytic in human erythrocytes.

Modulation of platelet aggregation by areca nut and betel leaf ingredients: roles of reactive oxygen species and cyclooxygenase.

There are 2 to 6 billion betel quid (BQ) chewers in the world. Areca nut (AN), a BQ component, modulates arachidonic acid (AA) metabolism, which is crucial for platelet function. AN extract (1 and 2 mg/ml) stimulated rabbit platelet aggregation, with induction of thromboxane B2 (TXB2) production. Contrastingly, Piper betle leaf (PBL) extract inhibited AA-, collagen-, and U46619-induced platelet aggregation, and TXB2 and prostaglandin-D2 (PGD2) production. PBL extract also inhibited platelet TXB2 and PGD2 production triggered by thrombin, platelet activating factor (PAF), and adenosine diphosphate (ADP), whereas little effect on platelet aggregation was noted. Moreover, PBL is a scavenger of O2(*-) and *OH, and inhibits xanthine oxidase activity and the (*)OH-induced PUC18 DNA breaks. Deferoxamine, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and neomycin prevented AN-induced platelet aggregation and TXB2 production. Indomethacin, genistein, and PBL extract inhibited only TXB2 production, but not platelet aggregation. Catalase, superoxide dismutase, and dimethylthiourea (DMT) showed little effect on AN-induced platelet aggregation, whereas catalase and DMT inhibited the AN-induced TXB2 production. These results suggest that AN-induced platelet aggregation is associated with iron-mediated reactive oxygen species production, calcium mobilization, phospholipase C activation, and TXB2 production. PBL inhibited platelet aggregation via both its antioxidative effects and effects on TXB2 and PGD2 production. Effects of AN and PBL on platelet aggregation and AA metabolism is crucial for platelet activation in the oral mucosa and cardiovascular system in BQ chewers.

Moderate-intensity exercise suppresses platelet activation and polymorphonuclear leukocyte interaction with surface-adherent platelets under shear flow in men.

The reciprocal modulation of platelet and polymorphonuclear leukocyte (PMN) activities is important in the pathogenesis of thrombosis and inflammation. This study investigated how moderate exercise affects shear-induced platelet activation and subsequent PMNs interaction with platelet-related thrombi under shear flow. Sixteen sedentary healthy men engaged in moderate exercise (about 60% VO(2max)) on a bicycle ergometer. Platelet activation, PMNs interaction with surface-adherent platelets, and PMN-dependent inhibition of platelet activation under shear flow were measured both before and immediately after exercise. The results of this study can be summarized as follows: (1) moderate exercise was associated with lower extents of shear-induced platelet adhesion and aggregation, binding of von Willbrand factor (vWF) to platelets, and glyco-protein IIb/IIIa activation and P-selectin expression on platelet than at rest; (2) the velocity and percentage of rolling PMNs increased while the number of PMNs remaining bound to surface-adherent platelets decreased after moderate exercise; (3) although treating the PMNs with oxidized-low density lipoprotein (Ox-LDL) enhanced PMNs interaction with surface-adherent platelets, moderate exercise suppressed the enhancement of platelet-PMN interaction by Ox-LDL; (4) moderate exercise decreased platelet [Ca (2+)](i) elevation induced by ADP and platelet [Ca(2+)](i) levels mediated by PMN and Ox-LDL-treated PMN; and (5) plasma and PMN-derived nitric oxide metabolites and plasma vWF antigen and activity increased after moderate exercise, whereas plasma and platelet-derived soluble P-selectin levels remained unchanged in response to exercise. Therefore, we conclude that moderate-intensity exercise suppresses shear-induced platelet activation and subsequent PMNs adhesion to platelets deposited at sites of vascular injury under flow, thereby reducing the risks of vascular thrombosis and inflammation.

The role of PAR4 in thrombin-induced thromboxane production in human platelets.

There are two protease-activated receptors (PARs), PAR1 and PAR4, in human platelets. It has been suggested that PAR1 mediates platelet responses to low concentrations of thrombin, whereas PAR4 mediates signaling only at high concentrations. In the present study, we used a selective PAR4 blocker, YD-3, to investigate the role of PAR4 in thrombin-induced thromboxane formation in human platelets. YD-3 completely prevented thromboxane production by either a low concentration of thrombin (0.1 U/ml) or the PAR4 agonist peptide GYPGKF. In contrast, YD-3 did not affect thromboxane production caused by the PAR1 agonist peptide SFLLRN, collagen or arachidonic acid. YD-3 also decreased [(3) H]arachidonic acid release from thrombin-stimulated platelets. Moreover, desensitization of platelets with GYPGKF prevented low thrombin-induced thromboxane formation. The decreased thromboxane production by YD-3 is linked to inhibition of calcium influx in thrombin-stimulated platelets. These results suggest that PAR4 plays an important role in the regulation of thromboxane formation in platelets responding to thrombin through prolonged elevation of [Ca(2+)](i) and activation of phospholipase A(2). These data also indicate that PAR4 can be activated by relatively low concentrations of thrombin in human platelets. The selective inhibition of thrombin-induced thromboxane production by YD-3 may be of therapeutic benefit for thrombotic diseases.

Selective inhibition of protease-activated receptor 4-dependent platelet activation by YD-3.

In the present study, the antiplatelet effect and its mechanism of a new synthetic compound YD-3 [1-benzyl-3-(ethoxycarbonylphenyl)-indazole] were examined. YD-3 inhibited the aggregation of washed human platelets caused by protease-activated receptor (PAR) 4 agonist peptide GYPGKF (IC50 = 0.13 +/- 0.02 microM), but had no or little effect on that by thrombin, PAR1 agonist peptide SFLLRN, collagen or U46619. YD-3 produced a parallel, rightward shift of the concentration-response curve for GYPGKF without decreasing of the maximum platelet aggregation, indicating a competitive antagonism. In contrast to human platelets, both thrombin- and GYPGKF-induced mouse platelet shape change and aggregation were completely inhibited by YD-3. YD-3 also selectively prevented GYPGKF-induced intracellular Ca2+ mobilization in human platelets. Furthermore, in the PAR1-desensitized human platelets, thrombin induced a relatively slow rise and decay of calcium mobilization that was significantly inhibited by YD-3. In addition, the synergistic effect of SFLLRN and GYPGKF on platelet activation was prevented by YD-3. YD-3 also inhibits both fMLP-stimulated neutrophil- and purified cathepsin G-induced platelet aggregation, which has been demonstrated to be PAR4-dependent. Taken together, our results suggest that YD-3 selectively inhibits PAR4-dependent platelet activation through blockade of PAR4. To the best of our knowledge, it is the first non-peptide PAR4 antagonist.