Che-Ming Teng

YC-1 inhibited human platelet aggregation through NO-independent activation of soluble guanylate cyclase.

1 Our previous study demonstrated that YC‐1, a derivative of benzylindazole, is a novel activator of soluble guanylate cyclase (sGC) in rabbit platelets. This work investigated whether the antiplatelet effect of YC‐1 was mediated by a nitric oxide (NO)/sGC/cyclic GMP pathway in human platelets. 2 In human washed platelets, YC‐1 inhibited platelet aggregation and ATP released induced by U46619 (2 μm), collagen (10 μg ml−1) and thrombin (0.1 u ml−1) in a concentration‐dependent manner with IC50 values of (μm) 2.1 ± 0.3, 11.7 ± 2.1 and 59.3 ± 7.1, respectively. 3 In a 30,000 g supernatant fraction from human platelet homogenate, YC‐1 (5–100 μm) increased sGC activity in a concentration‐dependent manner. At the same concentration‐range, YC‐1 elevated cyclic GMP levels markedly, but only slightly elevated cyclic AMP levels in the intact platelets. 4 MY‐5445, a selective inhibitor of cyclic GMP phosphodiesterase, potentiated the increases in cyclic GMP caused by YC‐1, and shifted the concentration‐anti‐aggregation curve of YC‐1 to the left. In contrast, HL‐725, a selective inhibitor of cyclic AMP phosphodiesterase, did not affect either the increases in cyclic nucleotides or the anti‐aggregatory effect caused by YC‐1. 5 Methylene blue, an inhibitor of sGC, blocked the increases of cyclic GMP caused by YC‐1, and attenuated markedly the anti‐aggregatory effect of YC‐1. The adenylate cyclase inhibitor, 2′,5′‐dideoxyadenosine (DDA) did not affect YC‐1‐induced inhibition of platelet aggregation. 6 Haemoglobin, which binds NO, prevented the activation of sGC and anti‐aggregatory effect caused by sodium nitroprusside, but did not affect YC‐1 responses. 7 These results would suggest that YC‐1 activates sGC of human platelets by a NO‐independent mechanism, and exerts its antiplatelet effects through the sGC/cyclic GMP pathway.

Fibrinogenolytic enzymes of Trimeresurus mucrosquamatus venom

By means of CM-Sephadex C-50 column chromatography, Trimeresurus mucrosquamatus venom was separated into twenty fractions. The fibrinogenolytic activity was concentrated in Fractions 8, 10, 12, 13 and 14. Fractions 8 adn 13 had the highest ratio of fibrinogenolytic and caseinolytic activities. Fraction 8 possessed tosyl-L-arginine methyl esterase activity, while the others did not. The caseinolytic activities of Fractions 10, 12, 13 and 14 were inhibited by EDTA, while that of Fraction 8 was not. Fractions 8 and 13 were further purified by CM-cellulose and gel filtration and were homogeneous as judged by electrophoresis on polyacrylamide gel and cellulose acetate membrane. The molecular weights of the purified Fractions 8 and 13 were 26 000 and 22 400, respectively. Both were single peptide chains. The specific fibrinogenolytic activity of Fraction 8 was 17 mg fibrinogen/min/mg protein, while that of Fraction 13was 100 mg fibrinogen/min/mg protein. Fraction 13 digested specifically the alpha(A) chain of monomeric fibrinogen to yield two cleavage products. Fraction 8 digested the beta(B) chain first to yield four cleavage products. When the incubation time was prolonged, the alpha(A) chain was also partially digested by Fraction 8 to yield two cleavage products.

Inhibition of platelet aggregation by some flavonoids

The inhibitory effects of five flavonoids on the aggregation and secretion of platelets were studied. These flavonoids inhibited markedly platelet aggregation and ATP release of rabbit platelets induced by arachidonic acid or collagen, and slightly those by platelet-activating factor. ADP-induced platelet aggregation was also suppressed by myricetin, fisetin and quercetin. The IC50 on arachidonic acid-induced platelet aggregation was: fisetin, 22 microM; kaempferol, 20 microM; quercetin, 13 microM; morin, 150 microM less than IC50 less than 300 microM. The thromboxane B2 formations were also inhibited by flavonoids in platelets challenged with arachidonic acid. Fisetin, kaempferol, morin and quercetin antagonized the aggregation of washed platelets induced by U46619, a thromboxane A2/prostaglandin endoperoxides mimetic receptor agonist. In human platelet-rich plasma, quercetin prevented the secondary aggregation and blocked ATP release from platelets induced by epinephrine or ADP. These results demonstrate that the major antiplatelet effect of flavonoids tested may be due to both the inhibition of thromboxane formation and thromboxane receptor antagonism.

TWO ANTIPLATELET AGENTS FROM MAGNOLIA OFFICINALIS

Magnolol and honokiol are two position isomers isolated from the bark of Magnolia officinalis. Both inhibited the aggregation and ATP release of rabbit platelet-rich plasma induced by collagen and arachidonic acid without affecting that induced by ADP, PAF or thrombin. Aggregation of washed platelets was more markedly inhibited than that of platelet-rich plasma, while the aggregation of whole blood was least affected by both inhibitors. Thromboxane B2 formation caused by collagen, arachidonic acid or thrombin was in each case inhibited by magnolol and honokiol. The rise of intracellular calcium caused by arachidonic acid or collagen was also suppressed by both agents. Collagen-induced intracellular calcium increase in the presence of indomethacin was suppressed by magnolol. It is concluded that the antiplatelet effect of magnolol and honokiol is due to an inhibitory effect on thromboxane formation and also an inhibition of intracellular calcium mobilization.

Inhibitory effect of YC-1 on the hypoxic induction of erythropoietin and vascular endothelial growth factor in Hep3B cells.

YC-1 is a newly developed agent that inhibits platelet aggregation and vascular contraction. Although its effects are independent of nitric oxide (NO), it mimics some of the biological actions of NO. For example, it stimulates soluble guanylate cyclase (sGC) and increases intracellular cGMP concentration. Here, we tested the possibility that YC-1 inhibits hypoxia-inducible factor (HIF)-1-mediated hypoxic responses, as does NO. Hep3B cells were used during the course of this work to observe hypoxic induction of erythropoietin (EPO) and vascular endothelial growth factor (VEGF), and the effects of YC-1 were compared with those of a NO donor, sodium nitropurruside (SNP). In hypoxic cells, YC-1 blocked the induction of EPO and VEGF mRNAs, and inhibited the DNA-binding activity of HIF-1. It suppressed the hypoxic accumulation of HIF-1alpha, but not its mRNA level. It also reduced HIF-1alpha accumulation induced by cobalt and desferrioxamine. Treatment with antioxidants did not recover the HIF-1alpha suppressed by YC-1. We examined whether these effects of YC-1 are related to the sGC/cGMP signal transduction system. Two sGC inhibitors examined failed to block the effects of YC-1, and 8-bromo-cGMP did not mimic actions of YC-1. The effects of YC-1 on the hypoxic responses were comparable with those of SNP. These results suggest that YC-1 and SNP suppressed the hypoxic responses by post-translationally inhibiting HIF-1alpha accumulation. The YC-1 effect may be linked with the metal-related oxygen sensing pathway, and is not due to the stimulation of sGC. This observation implies that the inhibitory effects of YC-1 on hypoxic responses can be developed to suppress EPO-overproduction by tumor cells and tumor angiogenesis.

YC-1 inhibits HIF-1 expression in prostate cancer cells: Contribution of Akt/NF-κB signaling to HIF-1α accumulation during hypoxia

Hypoxia-inducible factor 1 (HIF-1), a transcription factor that is critical for tumor adaptation to microenvironmental stimuli, represents an attractive chemotherapeutic target. YC-1 is a novel antitumor agent that inhibits HIF-1 through previously unexplained mechanisms. In the present study, YC-1 was found to prevent HIF-1α and HIF-1β accumulation in response to hypoxia or mitogen treatment in PC-3 prostate cancer cells. Neither HIF-1α protein half-life nor mRNA level was affected by YC-1. However, YC-1 was found to suppress the PI3K/Akt/mTOR/4E-BP pathway, which serves to regulate HIF-1α expression at the translational step. We demonstrated that YC-1 also inhibited hypoxia-induced activation of nuclear factor (NF)-κB, a downstream target of Akt. Two modulators of the Akt/NF-κB pathway, caffeic acid phenethyl ester and evodiamine, were observed to decrease HIF-1α expression. Additionally, overexpression of NF-κB partly reversed the ability of wortmannin to inhibit HIF-1α-dependent transcriptional activity, suggesting that NF-κB contributes to Akt-mediated HIF-1α accumulation during hypoxia. Overall, we identify a potential molecular mechanism whereby YC-1 serves to reduce HIF-1 expression.

Characterization of snake venom components acting on blood coagulation and platelet function

Snake venoms can affect blood coagulation and platelet function in various ways. The physicochemical properties and the mechanisms of actions of the snake venom components affecting blood coagulation and platelet function are discussed.

EDRF-release and Ca+(+)-channel blockade by magnolol, an antiplatelet agent isolated from Chinese herb Magnolia officinalis, in rat thoracic aorta.

Magnolol is an antiplatelet agent isolated from Chinese herb Magnolia officinalis. It inhibited norepinephrine (NE, 3 microM)-induced phasic and tonic contractions in rat thoracic aorta. At the plateau of the NE-induced tonic contraction, addition of magnolol caused two phases (fast and slow) of relaxation. These two relaxations were concentration-dependent (10-100 micrograms/ml), and were not inhibited by indomethacin (20 microM). The fast relaxation was completely antagonized by hemoglobin (10 microM) and methylene blue (50 microM), and disappeared in de-endothelialized aorta while the slow relaxation was not affected by the above treatments. Magnolol also inhibited high potassium (60 mM)-induced, calcium-dependent (0.03 to 3 mM) contraction of rat aorta in a concentration-dependent manner. 45Ca(+)+ influx induced by high potassium or NE was markedly inhibited by magnolol. Cyclic GMP, but not PGI2, was increased by magnolol in intact, but not in de-endothelialized aorta. It is concluded that magnolol relaxed vascular smooth muscle by releasing endothelium-derived relaxing factor (EDRF) and by inhibiting calcium influx through voltage-gated calcium channels.

Antiplatelet effect of butylidenephthalide

Butylidenephthalide inhibited, in a dose-dependent manner, the aggregation and release reaction of washed rabbit platelets induced by collagen and arachidonic acid. Butylidenephthalide also inhibited slightly the platelet aggregation induced by PAF and ADP, but not that by thrombin or ionophore A23187. Thromboxane B2 formation caused by collagen, arachidonic acid, thrombin and ionophore A23187 was in each case markedly inhibited by butylidenephthalide. Butylidenephthalide inhibited the aggregation of ADP-refractory platelets, thrombin-degranulated platelets, chymotrypsin-treated platelets and platelets in the presence of creatine phosphate/creatine phosphokinase. Its inhibition of collagen-induced aggregation was more marked at lower Ca2+ concentrations in the medium. The aggregability of platelets inhibited by butylidenephthalide could be recovered after the washing of platelets. In human platelet-rich plasma, butylidenephthalide and indomethacin prevented the secondary aggregation and blocked ATP release from platelets induced by epinephrine. Prostaglandin E2 formed by the incubation of guinea-pig lung homogenate with arachidonic acid could be inhibited by butylidenephthalide, indomethacin and aspirin. It is concluded that the antiplatelet effect of butylidenephthalide is mainly due to an inhibitory effect on cyclo-oxygenase and may be due partly to interference with calcium mobilization.

Soluble decoy receptor 3 induces angiogenesis by neutralization of TL1A, a cytokine belonging to tumor necrosis factor superfamily and exhibiting angiostatic action

TL1A is a member of the tumor necrosis factor superfamily and plays an important role in regulating endothelial cell apoptosis. A previous study shows TL1A is able to interact with death receptor 3 and decoy receptor 3 (DcR3). Here, we demonstrate that DcR3 is able to induce angiogenesis in human umbilical vein endothelial cells (HUVECs). DcR3 promotes HUVEC proliferation and migration and up-regulates matrix metalloproteinase-2 mRNA expression and enzyme activity. Furthermore, DcR3 enhances EC differentiation into cord vascular-like structures in vitro, as well as neovascularization in vivo. The effects of DcR3 on HUVECs are also mimicked by anti-TL1A and antideath receptor 3 antibodies. In contrast, human aortic endothelial cells, which do not express TL1A, are not responsive to DcR3 treatment, including cell proliferation, migration, and angiogenic differentiation. These data demonstrate DcR3 might not only help tumor cells to escape immune surveillance but also induce angiogenesis by blocking TL1A action in endothelial cells. The pathological role of DcR3 in promoting cancer progress raises the possibility to target DcR3 for antiangiogenic therapy in the future.