Antiplatelet actions of aporphinoids from Formosan plants

Seventeen aporphines were tested for antiplatelet activity. L-(+)-hemovine HCl and 7-hydroxydehydrothalicsimidine strongly inhibited platelet aggregation induced by adenosine 5'-diphosphate (ADP), arachidonic acid (AA), collagen, and platelet-activating factor (PAF). The latter showed the strongest antiplatelet activity with an IC50 of 70.4 microM against AA-induced platelet aggregation.     

Beta-amyrin from Ardisia elliptica Thunb. is more potent than aspirin in inhibiting collagen-induced platelet aggregation

Ardisia elliptica Thunberg (Myrsinaceae) is a medicinal plant traditionally used for alleviating chest pains, treatment of fever, diarrhoea, liver poisoning and parturition complications. The objectives of the study were to investigate the effect of A. elliptica on collagen induced platelet aggregation and to isolate and purify potential antiplatelet components. Fresh A. elliptica leaves were extracted using methanol (70% v/v) by Soxhlet extraction and the extract was analysed for its inhibition of collagen-induced platelet aggregation. Inhibition of platelet aggregation was assessed by incubating the extracts with rabbit blood and collagen in a whole blood aggregometer and measuring the impedance. The leaf extract was found to inhibit platelet aggregation with an IC50 value of 167 microg/ml. Using bioassay guided fractionation, beta-amyrin was isolated and purified. The IC50 value of beta-amyrin was found to be 4.5 microg/ml (10.5 microM) while that of aspirin was found to be 11 microg/ml (62.7 microM), indicating that beta-amyrin was six times as active as aspirin in inhibiting platelet aggregation. This paper is the first report that beta-amyrin isolated from A. elliptica is more potent than aspirin in inhibiting collagen-induced platelet aggregation. In conclusion, A. elliptica leaves were found to inhibit collagen-induced platelet aggregation and one of the bioactive components responsible for the observed effect was determined to be beta-amyrin.     

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.     

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.     

Effects of a novel non-carboxylic thromboxane A2 receptor antagonist (BM-531) derived from torasemide on platelet function

In this study we examined the thromboxane A(2)(TXA(2)) receptor antagonist property of BM-531 (N-tert -butyl- N'-[(2-cyclohexylamino-5-nitrobenzene)sulfonyl]urea), a torasemide derivative, on platelet function. The drug affinity for human washed platelet TXA(2)receptors labelled with [(3)H]SQ-29,548 has been determined (IC50: 0.0078 microM) and demonstrated to be higher than sulotroban (IC50: 0.93 microM) and SQ-29,548 (IC50: 0.021 microM). The antiaggregatory potency has been confirmed since we demonstrated that BM-531 prevented platelet aggregation in human citrated platelet-rich plasma induced by arachidonic acid (600 microM) (ED100: 0.125 microM), U-46619, a stable TXA(2)agonist (1 microM) (ED50: 0.482 microM) and collagen (1 microg mL(-1)) (% of inhibition: 42.9% at 10 microM) and inhibited the second wave of ADP (2 microM). Moreover, when BM-531 was incubated in whole blood from healthy donors, the closure time measured by the recently developed platelet function analyser (PFA-100(trade mark)) was significantly prolonged. These results suggest that BM-531 can be regarded as a novel non-carboxylic TXA(2)antagonist with a powerful antiplatelet potency.     

Antiplatelet activity of synthetic pyrrolo-benzylisoquinolines

Pyrrolo-benzylisoquinolines were prepared as target compounds and their antiplatelet aggregation activity, adreno-receptor affinity, and cytotoxicity were screened. Compounds 1d-9d showed specific antiplatelet aggregation activity induced by arachidonic acid and collagen. Among them, 8d and 9d exhibited better activity than the reference drug, aspirin and 9d also showed inhibition of platelet aggregation by all four inducers.     

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.     

Synthesis, antiproliferative, and antiplatelet activities of oxime- and amide-containing quinolin-2(1H)-one derivatives

Certain oxime- and amide-containing quinolin-2(1H)-one derivatives were synthesized and evaluated for their antiproliferative and antiplatelet activities. These compounds were synthesized via alkylation of hydroxyl precursors followed by the reaction with NH(2)OH or NaN(3) (Schmidt reaction). The preliminary assays indicated that amide derivatives are either weakly active or inactive while the oxime counterparts exhibited potent inhibitory activities against platelet aggregation induced by collagen, AA (arachidonic acid), and U46619 (the stable thromboxan A(2) receptor agonist). Among them, (Z)-6-[2-(4-methoxyphenyl)-2-hydroxyiminoethoxy]quinolin-2(1H)-one (7c) was the most active against AA induced platelet aggregation with an IC(50) of 0.58microM and was inactive against cell proliferation. For the inhibition of U46619 induced aggregation, 7a and 8a-c exhibited very potent activities with IC(50) values in a range between 0.54 and 0.74microM. For the antiproliferative evaluation, N-(biphenyl-4-yl)-2-(2-oxo-1,2-dihydroquinolin-7-yloxy)acetamide (11d) was the most potent with GI(50) values of <10, 10.8, and <10microM against the growth of MT-2, NCI-H661, and NPC-Tw01, respectively, and possessed only a weak antiplatelet activity. Further evaluation of 11d as a potential anticancer agent is on-going.     

An inhibitor of collagen-stimulated platelet activation from the salivary glands of the Haementeria officinalis leech. I. Identification, isolation, and characterization.

A protein that blocks collagen-stimulated platelet aggregation has been identified and isolated from the soluble fraction of salivary glands from Haementeria officinalis leeches. We have named this protein leech antiplatelet protein (LAPP). LAPP was isolated from soluble crude salivary gland extract by heparin-agarose, size exclusion, and C18 reverse phase high-performance chromatography. Its molecular weight is approximately 16,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under both reduced and nonreduced conditions. The sequences of peptides generated by V8 digestion of LAPP as well as its amino acid composition suggested no homology to other known proteins. The IC50 for LAPP to inhibit platelet aggregation was approximately 60 nM. This inhibitory activity is specific for collagen-induced aggregation. Platelet aggregation in response to ADP, arachidonic acid, U46619, thrombin, and ionophore A23187 was not inhibited by LAPP at a concentration that blocked platelet aggregation to collagen by 100%. In contrast, crude salivary gland-soluble extract contained activity(ies) which inhibited aggregation to all these agonists except thrombin at 1 unit/ml and 2 microM A23187. Thus, the H. officinalis leech has evolved multiple mechanisms to prevent hemostasis, including an inhibitor of collagen-stimulated platelet aggregation. The identification and isolation of LAPP demonstrates the existence of a new type of platelet inhibitor that should be useful to better understand the mechanism of collagen stimulation of platelets.     

Beclobrinic acid--a new hypolipidemic agent--inhibits in vitro human platelet activation by blocking prostaglandin synthesis

Effects and the mechanism of the antiplatelet actions of beclobrinic acid, free acid form of a new hypolipidemic agent beclobrate [(+)-2-[d-(P-chlorophenyl)p-tolyl)oxy)-2-methyl-butyrate), were examined using human platelets. Platelet-rich plasma (PRP) which has been prelabeled with (14C)-serotonin was incubated with beclobrinic acid (BBA) for one minute before the addition of various agonists. BBA (0.1-1.5 mM) inhibited platelet aggregation and serotonin secretion induced by ADP, epinephrine, arachidonic acid and collagen in a concentration dependent manner. BBA also inhibited arachidonic acid-induced production of malondialdehyde (MDA), a byproduct of prostaglandins, in a concentration dependent manner. However, up to 1.0 mM BBA did not inhibit platelet aggregation induced by U46619, a stable analog of prostaglandin H2. In other experiments BBA also blocked thrombin-induced release of (3H)-arachidonic acid from platelet phospholipids. These findings suggest that: (a) BBA inhibits platelet aggregation and serotonin secretion by inhibiting prostaglandin synthesis at two steps. First by interfering in the release of arachidonic acid from platelet phospholipids and second by inhibiting its conversion into prostaglandins; and (b) BBA does not inhibit the action of prostaglandins on human platelets.     

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.     

Spice active principles as the inhibitors of human platelet aggregation and thromboxane biosynthesis

Spice active principles are reported to have anti-diabetic, anti-hypercholesterolemic, antilithogenic, anti-inflammatory, anti-microbial and anti-cancer properties. In our previous report we have shown that spices and their active principles inhibit 5-lipoxygenase and also formation of leukotriene C4. In this study, we report the modulatory effect of spice active principles viz., eugenol, capsaicin, piperine, quercetin, curcumin, cinnamaldehyde and allyl sulphide on in vitro human platelet aggregation. We have demonstrated that spice active principles inhibit platelet aggregation induced by different agonists, namely ADP (50 μM), collagen (500 μg/ml), arachidonic acid (AA) (1.0 mM) and calcium ionophore A-23187 (20 μM). Spice active principles showed preferential inhibition of arachidonic acid-induced platelet aggregation compared to other agonists. Among the spice active principles tested, eugenol and capsaicin are found to be most potent inhibitors of AA-induced platelet aggregation with IC50 values of 0.5 and 14.6 μM, respectively. The order of potency of spice principles in inhibiting AA-induced platelet aggregation is eugenol>capsaicin>curcumin>cinnamaldehyde>piperine>allyl sulphide>quercetin. Eugenol is found to be 29-fold more potent than aspirin in inhibiting AA-induced human platelet aggregation. Eugenol and capsaicin inhibited thromboxane B2 (TXB2) formation in platelets in a dose-dependent manner challenged with AA apparently by the inhibition of the cyclooxygenase (COX-1). Eugenol-mediated inhibition of platelet aggregation is further confirmed by dose-dependent decrease in malondialdehyde (MDA) in platelets. Further, eugenol and capsaicin inhibited platelet aggregation induced by agonists—collagen, ADP and calcium ionophore but to a lesser degree compared to AA. These results clearly suggest that spice principles have beneficial effects in modulating human platelet aggregation.     

Pharmacological evaluation of the novel thromboxane modulator BM-567 (I/II). Effects of BM-567 on platelet function

The aim of this work was to evaluate the effects of BM-567 (N-pentyl-N'-[(2-cyclohexylamino-5-nitrobenzene)sulfonyl]urea), a torasemide derivative, on both thromboxane A(2) (TXA(2)) receptors (TP) and thromboxane synthase of human platelets. The drug affinity for TP receptors of human washed platelets has been determined. In this test, BM-567 showed a high affinity (IC(50): 1.1+/-0.1nM) for the TP receptors in comparison with BM-531 (IC(50): 7.8+/-0.7nM) and sulotroban (IC(50): 931+/-85nM), two TXA(2) antagonists. We also demonstrated that BM-567 prevented platelet aggregation induced by arachidonic acid (AA) (600 microM) (ED(100): 0.20+/-0.10 microM), U-46619, a stable TXA(2) agonist (1 microM) (ED(50): 0.30+/-0.04 microM) and collagen (1microgram ml(-1)) (% of inhibition: 44.3+/-4.3% at 10 microM) and inhibited the second wave of ADP (2microM). Moreover, when BM-567 was incubated in whole blood from healthy donors, the closure time measured by the Platelet Function analyzer (PFA-100((R))) was significantly prolonged (closure time: 215+/-21s) by using collagen/epinephrine cartridges. Finally, at the concentration of 1 microM, BM-567 completely reduced the TXB(2) production from human platelets stimulated with AA (600 microM). These results indicate that BM-567 is a novel combined TXA(2) receptor antagonist and thromboxane synthase inhibitor characterized by a powerful antiplatelet potency.     

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.     

Activity of a novel dual thromboxane A(2)receptor antagonist and thromboxane synthase inhibitor (BM-573) on platelet function and isolated smooth muscles.

The pharmacomodulation of sulfonylureas structurally related to torasemide and characterized by a TXA(2)antagonism led to the synthesis of BM-573. This original molecule showed a high affinity (IC(50)1.3 nM) for the TXA(2)receptor of human platelets in comparison with both reference compounds, SQ-29548 (IC(50)21 nM) and sulotroban (IC(50)930 nM). Moreover, this torasemide derivative was found to be a potent inhibitor of human platelet aggregation induced by arachidonic acid (ED(100)=0.13 microM) or by U-46619 (ED(50)=0.24 microM), a TXA(2)agonist. BM-573 relaxed the isolated rat thoracic aorta (ED(50)=28.4 nM) and guinea-pig trachea (ED(50)=17.7 nM) contracted by U-46619. BM-573 (1 microM) completely reduced the platelet production of TXB(2)induced by arachidonic acid. Finally, BM-573 (30 mg/kg, per os) lost the diuretic properties of torasemide in rats.     

Novel furfurylidene N-acylhydrazones derived from natural safrole: discovery of LASSBio-1215, a new potent antiplatelet prototype

We describe herein the discovery of (E)-N-methyl-N'-((5-nitrofuran-2-yl)methylene)benzo[d]( 1 , 3 ) dioxole-5-carbohydrazide (9e), named LASSBio-1215, as a novel antiplatelet agent belonging to the N-methyl-N-acylhydrazone class, which exert their antiaggregating actions on human and rabbit platelets induced by different agonists, through cyclooxygenase-1 (COX-1) or thromboxane synthase inhibition. This compound was elected after screening of a series of functionalized furyl N-acylhydrazone derivatives, synthesized from natural safrole 10. In vitro assays showed that compound 9e presents platelet-aggregating activity in rabbit platelet-rich plasma (PRP) induced by arachidonic acid (IC(50)?=?0.7 μM) and collagen (IC(50)?=?4.5 μM). Moreover, LASSBio-1215 also inhibited almost completely the second wave of adenosine diphosphate-induced platelet aggregation in human PRP, and this effect was correlated with their ability to block the production of pro-aggregating autacoid thromboxane A(2).     

Comparison of in vitro platelet aggregation and its inhibition by three antithrombotic drugs between human and guinea pig

Platelets of guinea pigs are frequently used to evaluate the effect of new antiplatelet agents. Although several studies have compared the platelet aggregation between humans and guinea pigs, but so far the information is still limited. In this study, we compare the inhibitory effect of aspirin, dipyridamole and pentoxifylline on the platelet aggregation induced by adenosine diphosphate (ADP), collagen, arachidonic acid and thrombin between humans and guinea pigs. The results for humans and guinea pigs were compared and analysed by two-way analysis of variance (ANOVA). Our results showed: 1. The trends wherein these three drugs suppressed collagen-induced platelet aggregation was very similar in humans and guinea pigs. 2. In ADP-induced aggregation, the trend of inhibition caused by the three drugs was also similar in humans and guinea pigs except that a difference in platelet disaggregation at a late phase of platelet aggregation was noted. 3. In arachidonic acid- and thrombin-induced aggregations, the trend of inhibition caused by the three drugs was somewhat different in humans and guinea pigs. 4. Considering all activators as a whole, it was found that the status of platelet disaggregation at the late phase of platelet aggregation was different in humans and guinea pigs. Therefore, we concluded that: 1. Collagen was the most appropriate platelet activator when we used platelets of guinea pigs to study the effect of new antiplatelet agents. 2. When platelets of guinea pigs were used to study platelet aggregation, no matter which activator was used, we should avoid using the late phase of aggregation as the control index for comparison, because the results thus obtained might not be applicable to human platelets.     

一种快速、灵敏的血小板释放功能检测方法及其在抗血小板药物研究中的应用

本文报道了一种快速、灵敏的血小板释放功能检测方法:利用荧光素-荧光素酶在有ATP、Mg~(2+)、O_2存在时产生的生物发光素测定血小板ATP的释放量,以反映血小板的释放功能;研究了ADP、AA、胶原、凝血酶等四种诱导剂对血小板释放功能的作用,发现ADP的诱导释放能力较其他三者为弱;观察在不同剂量ADP和AA的诱导下,血小板聚集强度和释放能力之间的关系,研究了血小板数等因素对ATP释放功能测定的影响。应用该方法研究了Aspirin及活血化淤药物川芎嗪,毛冬青甲素对血小板释放功能的影响,发现Aspirin对AA诱导的释放反应有强烈的抑制作用。在以ADP诱导的释放反应中,川芎嗪的抑制作用较毛冬青甲素更为强烈。     

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.     

Antiplatelet effect of green tea catechins: a possible mechanism through arachidonic acid pathway

We have previously reported that green tea catechins (GTC) showed an antithrombotic activity, which might be due to antiplatelet effect rather than anticoagulation. The present study was performed to investigate the effect of GTC on the arachidonic acid (AA) metabolism in order to elucidate a possible antiplatelet mechanism. GTC inhibited the collagen-, AA- and U46619-induced rabbit platelet aggregation in vitro in a concentration-dependent manner, with IC50 values of 61.0+/-2.5, 105.0+/-4.9 and 67.0+/-3.2 microg/ml, respectively. Moreover, GTC administered orally into rats inhibited the AA-induced platelet aggregation ex vivo by 46.9+/-6.1% and 95.4+/-2.2% at the doses of 25 and 50 mg/kg, respectively. [3H]AA liberation induced by collagen in [3H]AA incorporated rabbit platelets was significantly suppressed by GTC compared to the control. GTC also significantly inhibited the thromboxane A2 (TXA2) and prostaglandin D2 (PGD2) generations induced by addition of AA in intact rabbit platelets. GTC significantly inhibited TXA2 synthase activity in a concentration-dependent manner. Moreover, adenosine triphosphate (ATP) release from dense granule was inhibited by GTC in washed platelets. These results suggest that the antiplatelet activity of GTC may be due to the inhibition of TXA2 formation through the inhibition of AA liberation and TXA2 synthase.