紫锥菊提取物及酚酸化合物体外抗血小板聚集活性研究

为探究紫锥菊提取物及酚酸化合物体外抗血小板聚集活性,体外抗血小板聚集实验采用Born比浊法,以聚集抑制率和半数抑制浓度为指标评价。同时采用分子对接方法,选择凝血因子V(F5)、凝血因子VIII(F8)及凝血因子XI(F11)与酚酸类化合物进行虚拟对接,研究其抗血小板聚集的分子作用靶点。结果表明化合物S-1～S-10及其提取物对体外ADP诱导的血小板聚集有抑制作用,抑制率呈浓度依赖性,S-6与靶点的结合位点更多,选择性更强。紫锥菊中酚酸类化合物及其提取物在体外均显现出抗ADP诱导的血小板聚集活性,为紫锥菊体内研究提供依据。     

Thromboxane synthetase inhibitors as pharmacological tools: differential biochemical and biological effects on platelet suspensions

The comparative effects of three so called "thromboxane-synthetase-inhibitors" (imidazole, N-0164, and U-51605) on arachidonate metabolism and on platelet aggregation were studied. All three compounds blocked platelet microsomal thromboxane synthesis from prostaglandin endoperoxides without affecting platelet adenyl cyclase. Imidazole, blocked thromboxane synthesis in intact platelets either from arachidonic acid or PGH2, without affecting aggregation. U-51605 simultaneously inhibited thromboxane synthesis and platelet suspension aggregation. N-0164 inhibited aggregation probably at extracellular sites, at concentrations that did not alter arachidonate or PGH2 metabolism. High concentrations of N-0164 simultaneously inhibited PG cyclo-oxygenase and thromboxane synthetase. The lack of specificity of these compounds requires that other actions of these compound must be considered when they are used as pharmacological tools to inhibit thromboxane synthetase.     

华北绣线菊二萜生物碱抗血小板聚集活性研究

采用Born氏比浊法观察华北绣线菊小叶变种中分离得到的总碱和 9个hetisine型C2 0 二萜生物碱及其衍生物体外对血小板活化因子 (PAF)、花生四烯酸 (AA)和二磷酸腺苷 (ADP)三种诱导剂引起的血小板聚集活性的影响 ,并初步探讨了构效关系。结果表明 ,华北绣线菊小叶变种中总碱对PAF和ADP诱导的血小板聚集均有明显的抑制作用 ;9个hetisine型C2 0 二萜生物碱中 ,有 8个显著抑制PAF诱导的血小板聚集 ,其活性与分子结构明显相关 ;此外 ,hetisine型生物碱及其衍生物对ADP诱导的血小板聚集亦有一定的抑制作用 ,但总碱及生物碱对AA诱导的聚集影响不明显。提示hetisine型C2 0 二萜生物碱具有抗血小板聚集活性。     

Variation in economically and ecologically important traits in onion plant organs during reproductive development

The spatial distribution of organosulphur compounds throughout the onion (Allium cepa L.) plant body during reproduction is of ecological and horticultural interest. These secondary metabolites are associated with both pest resistance and many of the vegetable's culinary and medicinal properties, including the ability to inhibit platelet aggregation. Inhibition of platelet aggregation can be of benefit to human cardiovascular health. Organosulphur compound concentrations are associated with elemental sulphur, pungency, soluble solids and effect on human platelet aggregation. These parameters were evaluated in extracts collected separately from bulb scales, leaf blades, scapes and umbels biweekly throughout the reproductive phase of the life cycle of the onion. Significant variation in pungency, platelet inhibition, total sulphur content and soluble solids existed among samples of organs and within organs over time during reproductive growth. Furthermore, some extracts from leaf, scape and bulb induced rather than inhibited platelet aggregation.     

Inhibition of thrombocyte aggregation by antioxidants

The effects of antioxidants (3-hydroxypyridines, 5-hydroxypyrimidines, hindered phenols) on platelet aggregation were studied. All the compounds under study possessed low anti-aggregation activity against indometacin-sensitive aggregation (activation with arachidonic acid, 50 M). Half-maximal inhibition of aggregation was achieved at a concentration similar to that of the compounds used (10(-3) M in cases of indomethacin-insensitive aggregation, platelet activation by thrombine 1.5 mu/ml and Ca2+-ionophore A23187 1.5 g/ml). 4-methyl-2.6-ditretbutyl phenol (BHT) in the concentration range of 10(-5)-4 X 10(-5) M inhibited and in the concentration range of 4 X 10(-5)-10(-4) M activated indomethacin-sensitive aggregation. The latter effect was not observed in the absence of Ca2+ ions in the incubation medium. It is concluded that the effects of the antioxidants studied on platelet aggregation were due to their non-specific action on platelet membranes.     

ESR analysis with long-chain alkyl spin labels in bovine blood platelets. Relationship between the increase in membrane fluidity by alcohols and phenolic compounds and their inhibitory effects on aggregation

Four spin-labeled probes (5-doxylstearic acid (5-NS), its methyl ester (5-NMS), 16-doxylmethylstearate (16-NMS) and 4-(N,N-dimethyl-N-pentadecyl)ammonium-2,2,6,6-tetramethylpiperidine-1-ox yl (CAT-15)) were used to monitor membrane fluidity change in bovine platelets induced by three alkyl alcohols, benzyl alcohol and two phenolic compounds. The relationship between the increase in membrane fluidity induced by these compounds and their inhibitory effects on platelet aggregation was observed. Experiments with the four probes showed that n-hexyl alcohol induced decreases in the order parameter of 5-NS and apparent rotational correlation times of the other probes at the same minimal alcohol concentration. The decreases were observed in the concentration range that inhibited aggregation. n-Amyl alcohol and n-butyl alcohol decreased the values of the parameters of the above mentioned only at higher concentrations that were dependent on their hydrophobicities. Like alkyl alcohols, benzyl alcohol and phenolic compounds decreased the values of the parameters in the concentration ranges in which these compounds inhibited platelet aggregation. The concentration of these compounds causing 50% inhibition of platelet aggregation, the IC50 values, and data on 5-NS-labeled platelets indicated that they inhibited aggregation and decreased the value of the order parameter at lower concentrations relative to their Poct values in comparison to the effective concentrations of alcohols. Phenolic compounds also decreased the values of the apparent rotational correlation times of 5-NMS and 16-NMS. These results indicate that the inhibition of platelet aggregation by alcohols and phenolic compounds is due to membrane perturbation in wide range in depths within the lipid bilayer.     

PLATELET PHAGOCYTOSIS AND AGGREGATION

The addition of latex particles to native (no anticoagulant) or citrated human platelet-rich plasma (PRP), or to a once-washed platelet suspension causes platelet aggregation. This aggregation is associated with phagocytosis of the latex particles by the platelets and appears to be due to release of adenosine diphosphate (ADP) from the platelets. Adenosine and adenosine monophosphate, which are known to inhibit platelet aggregation induced by ADP, also block that induced by latex. These compounds do not prevent the phagocytosis of latex particles by the platelet. The addition of iodoacetate and 2,4-dinitrophenol in appropriate concentrations to the PRP, prior to the addition of the latex, blocks platelet aggregation and phagocytosis. This is also true for the chelating agent ethylenediaminetetraacetate (EDTA). Platelets left in contact with latex for a sufficient period of time show loss of their granules. Leucocytes phagocytose both latex and platelets that had themselves phagocytosed latex. It is concluded that phagocytosis of latex particles by platelets resembles that by white cells, and that in both processes metabolic changes appear to be involved.     

Inhibition of adenylate cyclase by adenosine analogues in preparations of broken and intact human platelets. Evidence for the unidirectional control of platelet function by cyclic AMP.

Whereas adenosine itself exerted independent stimulatory and inhibitory effects on the adenylate cyclase activity of a platelet particulate fraction at low and high concentrations respectively, 2-substituted and N6-monosubstituted adenosines had stimulatory but greatly decreased inhibitory effects. Deoxyadenosines, on the other hand, had enhanced inhibitory but no stimulatory effects. The most potent inhibitors found were, in order of increasing activity, 9-(tetrahydro-2-furyl)adenine (SQ 22536), 2',5'-dideoxyadenosine and 2'-deoxyadenosine 3'-monophosphate. Kinetic studies on prostaglandin E1-activated adenylate cyclase showed that the inhibition caused by either 2',5'-dideoxyadenosine or compound SQ 22536 was non-competitive with MgATP and that the former compound, at least, showed negative co-operativity; 50% inhibition was observed with 4 micron-2',5'-dideoxyadenosine or 13 micron-SQ 22536. These two compounds also inhibited both the basal and prostaglandin E1-activated adenylate cyclase activities of intact platelets, when these were measured as the increases in cyclic [3H]AMP in platelets that had been labelled with [3H]adenine and were then incubated briefly with papaverine or papaverine and prostaglandin E1. Both compounds, but particularly 2',5'-dideoxyadenosine, markedly decreased the inhibition by prostaglandin E1 of platelet aggregation induced by ADP or [arginine]vasopressin as well as the associated increases in platelet cyclic AMP, so providing further evidence that the effects of prostaglandin E1 on platelet aggregation are mediated by cyclic AMP. 2'-Deoxyadenosine 3'-monophosphate did not affect the inhibition of aggregation by prostaglandin E1, suggesting that the site of action of deoxyadenosine derivatives on adenylate cyclase is intracellular. Neither 2',5'-dideoxyadenosine nor compound SQ 22536 alone induced platelet aggregation. Moreover, neither compound potentiated platelet aggregation or the platelet release reaction when suboptimal concentrations of ADP, [arginine]vasopressin, collagen or arachidonate were added to heparinized or citrated platelet-rich plasma in the absence of prostaglandin E1. These results show that cyclic AMP plays no significant role in the responses of platelets to aggregating agents in the absence of compounds that increase the platelet cyclic AMP concentration above the resting value.     

The evaluation and structure-activity relationships of 2-benzoylaminobenzoic esters and their analogues as anti-inflammatory and anti-platelet aggregation agents

Forty-seven 2-benzoylaminobenzoic esters were synthesized and evaluated in anti-platelet aggregation, inhibition of superoxide anion generation, and inhibition of neutrophil elastase release assays. Most 2-benzoylamino-4-chlorobenzoic acid derivatives showed selective inhibitory effects on arachidonic acid (AA)-induced platelet aggregation. Among them, compounds 6b and 7b exhibited more potent inhibitory effects (ca. 200-fold) than aspirin. Additionally, compounds 1a and 5a showed strong inhibitory effects on neutrophil superoxide generation with IC(50) values of 0.65 and 0.17 microM, respectively. However, compounds 6d and 6e exhibited dual inhibitory effects on platelet aggregation and neutrophil elastase (NE) release; therefore, these two compounds may be new leads for development as anti-inflammatory and anti-platelet aggregatory agents.     

Inhibitory effect of sulfur-containing compounds in Scorodocarpus borneensis Becc. on the aggregation of rabbit platelets

The inhibitory effects of three pure compounds isolated from wood garlic, 2,4,5-trithiahexane (I), 2,4,5,7-tetrathiaoctane (II), and 2,4,5,7-tetrathiaoctane 2,2-dioxide (III), on rabbit platelet aggregation induced by collagen, arachidonic acid, U46619, ADP (adenosine 5'-diphosphate), PAF (platelet aggregating factor), and thrombin were studied in vitro. The anti-aggregating activity of 2,4,5,7-tetrathiaoctane 4,4-dioxide (IV) was also measured with collagen and arachidonic acid. I, II, III, and IV inhibited the platelet aggregation induced by all tested agonists. I, II, and III exhibited a stronger inhibitory effect against the thrombin-induced aggregation of GFP (gel-filtered platelets) than against the aggregation induced by the other agonists. Notably, the IC50 value for III was 4 microM, which is approximately 2.5 times stronger than MATS (methyl allyl trisulfide), a major anti-platelet compound isolated from garlic. In inhibiting collagen-induced aggregation, II was as potent as MATS and aspirin, with a marked disaggregation effect on the secondary aggregation by arachidonic acid, at the rate of 47.05%/min at a concentration of 10(-4) M. I, II, and III also suppressed U46619-induced aggregation. These results suggest that sulfur-containing compounds in wood garlic not only inhibit arachidonic acid metabolism but also suppress aggregation in association with the function of the platelet plasma membrane.     

EL-4 tumor cell-induced human and rabbit platelet aggregations

EL-4 tumor cells were assayed in vitro for their ability to aggregate two kinds of platelets. An inhibition study showed that the EL-4 tumor cell can induce platelet aggregation by at least two different mechanisms. One, mediated by thrombin, was dominant with rabbit platelets because hirudin, which specifically inhibits thrombin, considerably suppressed the rabbit platelet aggregation induced by EL-4 tumor cells. In contrast, EL-4 cells induced the aggregation of human platelets even in citrated PRP. It is the apyrase-sensitive pathway that is believed to work in human platelets. The human platelet responses to EL-4 tumor cells clearly differed from those of rabbit platelets in terms of inhibition by hirudin and apyrase and of reactivity in citrated PRP. Both phospholipase A2 and dibutyryl cAMP strongly inhibited EL-4 tumor cell-induced platelet aggregation in both rabbit and human platelets. These two compounds may block a vital step in platelet aggregation that is elicited by the EL-4 tumor cells. Our results show that human platelet response to tumor cells is not necessarily deducible from experimental data obtained with animal platelets.     

Design, synthesis, and vasorelaxant and platelet antiaggregatory activities of coumarin-resveratrol hybrids

We have synthesized the coumarin-resveratrol hybrid 4 and its dimethoxy derivative 3 by a very direct synthetic route involving a Pechmann procedure. Compound 4 has also been synthesized by an alternative route (Perkin), which also allowed the synthesis of compounds 9-13. In addition, we have evaluated the potential vasorelaxant activity of the new compounds in endothelium-containing rat aorta rings pre-contracted with noradrenaline, as well as the inhibitory effects on platelet aggregation induced by thrombin in washed human platelets. The compounds reported here relaxed vascular smooth muscle and inhibited platelet aggregation with a profile similar to that of trans-resveratrol (t-RESV) and, in some cases, showed activity higher than that of the natural compound. This is the case for compound 13, which has a vasorelaxant activity that is twice as high as that of t-resveratrol and a platelet antiaggregant activity that is six times higher. These results suggest that these novel compounds may have potential as structural templates for the design and subsequent development of new vasodilatory and platelet antiaggregatory drugs.     

Monitoring modulators of platelet aggregation in a microtiter plate assay

Platelets play a central role in maintaining biological hemostasis. Inappropriate platelet activation is responsible for thrombotic diseases such as myocardial infarction and stroke. Therefore, novel agents that can inhibit platelet activation are necessary. However, assays that monitor platelet aggregation are generally time-consuming and require high volumes of blood and specialized equipment. Therefore, a medium- to high-throughput assay that can monitor platelet aggregation would be considered useful. Such an assay should be sensitive, comparable to the "gold standard" assay of platelet aggregometry, and able to monitor multiple samples simultaneously but with low assay volumes. We have developed such a microtiter assay. It can assay an average of 60 independent treatments per 60 ml blood donation and demonstrates greater sensitivity than the current gold standard assay, namely platelet aggregation in stirring conditions in a platelet aggregometer. The microtiter plate (MTP) assay can detect known inhibitors of platelet function such as indomethacin, aspirin, and ReoPro. It is highly reproducible when using standard doses of agonists such as thrombin receptor-activating peptide (20 microM) and collagen (0.19 mg/ml). Finally, the MTP assay is rapid and sensitive and can detect unknown platelet-modulating agents from a library of compounds.     

Direct evidence for the interaction between papaverine and alpha 2-adrenergic receptors in canine platelets

The effects of papaverine on specific [3H]-yohimbine binding to canine platelet alpha 2-adrenergic receptors and on the platelet aggregation were assessed and compared with those of verapamil. Both compounds concentration-dependently inhibited [3H]-yohimbine binding with KI values for respective compounds of 0.39 +/- 0.05 microM (n = 3) and 15 +/- 0.19 microM (n = 3). In the presence of either compound KD values in Scatchard analysis of the equilibrium ligand binding increased in concentration-dependent manner, whereas Bmax did not change, indicating competitive inhibition of the ligand binding by these compounds. (-)-Epinephrine (3 microM) potentiation of adenosine diphosphate (ADP, 0.1 microM) aggregation was inhibited by papaverine with IC50 of 11 +/- 3.6 microM (n = 4). In the same experiments verapamil inhibited the platelet aggregation with lower IC50 (3.1 +/- 0.87 microM, n = 4) in comparison with that for papaverine. These results suggest that papaverine, like verapamil, inhibits physiological response of canine platelets through alpha-adrenergic receptor stimulation by direct interaction with the receptors.     

Comparison of the effects of inhibitors of cytochrome P-450-mediated reactions on human platelet aggregation and arachidonic acid metabolism

Metyrapone and SKF-525A, together with amphenone B, a structural analogue of metyrapone, which are all inhibitors of cytochrome P-450-mediated reactions, were shown to inhibit the arachidonic acid-induced aggregation of human platelets. Amphenone B, like metyrapone, exhibited a type II (ligand) binding spectrum with rat liver microsomal cytochrome P-450, in contrast to SKF 525A which is a type I (substrate) binding agent. Independently of their type of binding spectra and of their maximum spectral change, however, the affinity of the three compounds for rat liver cytochrome P-450 showed a close proportional correlation with their platelet aggregation inhibitory potency. All three compounds inhibited the formation of [1-14C]thromboxane B2 from [1-14C]arachidonic acid by human platelets aggregated with collagen. The effect of metyrapone on the remaining labelled products suggested that it is a selective thromboxane synthesis inhibitor, while amphenone B exhibited activity reminiscent of cyclo-oxygenase inhibitors. SKF 525A produced complex effects possibly attributable to cyclo-oxygenase inhibition and enhanced lipid peroxidation, since it also enhanced platelet malonaldehyde formation, which the other two compounds inhibited. These data provide further support for a role of cytochrome P-450 in thromboxane synthesis and platelet aggregation.     

Antiplatelet properties of novel N-substituted-phenyl-1,2,3-triazole-4-acylhydrazone derivatives

This paper describes the design, synthesis and pharmacological evaluation of new N-acylhydrazone (NAH) compounds, belonging to the N-substituted-phenyl-1,2,3-triazole-4-acylhydrazone class (2a-p). Classical heteroaromatic ring bioisosterism strategies were applied to the previously reported N-phenylpyrazolyl-4-acylhydrazone derivative 1, elected as lead-compound due to its important anti-aggregating profile on arachidonic acid induced platelet aggregation (IC(50)=24+/-0.5 micro M), from which emerge this new series 2. These new compounds 2a-p were readily synthesized, characterized and tested on platelet aggregation assays induced by collagen (5 micro g/mL), ADP (5 micro M) and arachidonic acid (100 micro M) in rabbit citrated platelet-rich plasma. Compounds 2b, 2d, and 2h were found to be the most potent, exhibiting a significant antiplatelet activity on arachidonic acid- and collagen-induced platelet aggregation. In addition, these new antiplatelet agents are free of gastric ulcerogenic effect and presented discrete anti-inflammatory and analgesic properties. The N-para-chlorophenyltriazolyl-4-acylhydrazone compound 2h produced the highest inhibitory effect on collagen (IC(50)=21.6+/-0.4 micro M) and arachidonic acid-induced platelet aggregation (IC(50)=2.2+/-0.06 micro M), suggesting that the nature of the substituent on the phenyl ring of the N-heteroaromatic system of NAH moiety may be an important structural requirement for the improvement of antiplatelet activity, in comparison with lead-series 1.     

Synthesis and evaluation of antiplatelet activity of trihydroxychalcone derivatives

In an effort to develop potent antiplatelet agents, a series of trihydroxychalcones was synthesized and screened in vitro for their inhibitory effects on washed rabbit platelet aggregation induced by arachidonic acid (100 microM) and collagen (10 microg/ml). Of five compounds with potent inhibitory effects on arachidonic acid- and collagen-induced platelet aggregation, compound 4e was found to be the most potent. The structure-activity relationships suggested that antiplatelet activity was governed to a greater extent by the substituent on B ring of the chalcone template, and most of the active compounds had methoxy or dimethoxy groups on B ring.     

Design, synthesis, and inhibition of platelet aggregation for some 1-o-chlorophenyl-1,2,3,4-tetrahydroisoquinoline derivatives

Based on ticlopidine active as an ADP receptor antagonist for inhibiting platelet aggregation in clinical test, and upon finding (±)-1,2-substituted-7-sulfonylamide/amide-1,2,3,4-tetrahydroisoquinoline (11–31) inhibited of platelet aggregation, a series of (±)-1-o-chlorophenyl-2-substituted-tetrahydroisoquinoline derivatives was designed and synthesized. Four analogs proved to be potential antiplatelet aggregation agents, and compound 9 (TQP-3, applying for patent) which inhibits ADP-induced human platelet aggregation with IC₅₀ values of approximately 0.206 nM was the most active. Compound 2 is more active than compound 1, which (Type I) is similar to ticlopidine. This is because there is a spacial hindrance in compound 1, and the o-chloro group of compound 2 may play the same a role as o-chloro group of ticlopidine. On the other hand, with the different substitutions at different positions on the 2-substituted phenylacyl group, their inhibition of platelet aggregation differs. These compounds with m-substituted group (5, 7, 9) showed a higher IC₅₀ value for inhibiting ADP-induced human platelet aggregation than those with o-substituted group (4, 6) or p-substituted group (3, 8). It was observed that their inhibition is bromine-substituted derivative (9), chlorine-substituted derivative (7), and nitro-substituted derivative (5) in turn. Moreover, these compounds (Type II) may be more similar to clopidogrel than to ticlopidine due to the acyl group at 2 position of the nucleus playing a role as the ester group of clopidogrel. It was conjectured that these analogs function as a potential antiplatelet aggregation role by acting as ADP receptor antagonists.     

Different effects of two thromboxane A2/prostaglandin H2 receptor ligands, U46619 and S-145, on rabbit platelets

Stimulation of rabbit platelets with U46619 induced platelet shape change, aggregation and secretion of ATP. However, S-145, which specifically binds to the thromboxane A2/prostaglandin H2 receptor like U46619, induced only shape change. Both compounds rapidly elevated cytoplasmic Ca2+ concentration although only U46619 evoked the formation of inositol phosphates. Chelating external Ca2+ with EGTA did not affect the S-145-induced platelet shape change while intracellular Ca2+ movement was severely reduced. These results suggest an essential role of phospholipase C in the induction of platelet aggregation and secretion and that some factor other than Ca2+ and phospholipase C participates in platelet shape change.     

TRP-independent inhibition of the phospholipase C pathway by natural sensory ligands

Menthol, cinnamaldehyde, and camphor are activators for temperature-sensitive transient receptor potential ion channels (thermoTRPs). Here we found that these three compounds inhibit the phospholipase C (PLC) signaling. P2Y purinoceptor-mediated or histamine receptor-mediated cytosolic calcium mobilization through the PLC pathway was significantly suppressed by menthol, cinnamaldehyde, and camphor. Experiments using a fluorescent pleckstrin homology domain of PLCδ1 and IP1 accumulation assays demonstrated that direct inhibition of PLC activity occurred upon the addition of the sensory compounds. P2Y receptor-mediated PLC activation is part of the mechanism of platelet aggregation. The three compounds inhibited ADP-induced platelet aggregation. Calcium influx studies showed that thermoTRPs do not function in platelets, suggesting that the anti-aggregation effect is independent of thermoTRP activity. These results suggest that menthol, cinnamaldehyde, and camphor are able to modify PLC signaling and that those effects may lead to changes in cellular functions. This study also identifies new types of compounds that could potentially modulate platelet-related pathological events.