Anti-platelet effects of yuzu extract and its component

In this study, we investigated whether the methanolic extract of yuzu (yuzu ME) and its components hesperidin and naringin, have anti-platelet activities. Yuzu ME and hesperidin inhibited collagen-, arachidonic acid (AA)-, ADP- and thrombin-induced rat platelet aggregation in vitro and ex vivo. Naringin also inhibited platelet aggregation induced by collagen, AA, or thrombin, but not aggregation induced by ADP. The oral administration of yuzu ME or hesperidin prolonged mouse tail vein bleeding time in a dose-dependent manner in vivo. These results suggest that yuzu ME and hesperidin have anti-platelet activity, and that intake of yuzu, which includes various flavonoids such as hesperidin, may be beneficial for individuals at high risk of cardiovascular diseases.     

Antiplatelet effects of piplartine,an alkamide isolated from Piper tuberculatum: possible involvement of cyclooxygenase blockade and antioxidant activity

Objectives Piplartine (piperlongumine; 5,6‐dihydro‐1‐[1‐oxo‐3‐(3,4,5‐trimethoxyphenyl]‐2(1H) pyridinone) is an alkaloid amide isolated from Piper species (Piperaceae). It has been reported to show multiple pharmacological activities in vitro and in vivo. Methods We evaluated the in‐vitro antiplatelet effect of piplartine isolated from the roots of P. tuberculatum, on human platelet aggregation induced in platelet‐rich plasma by the agonists collagen, adenosine 5′‐diphosphate (ADP), arachidonic acid (AA) and thrombin. Key findings Piplartine (100μg/ml) caused a 30% inhibition in platelet aggregation when collagen was the agonist. At 200 μg/ml, piplartine significantly inhibited the aggregation induced by arachidonic acid (100%), collagen (59%) or ADP (52%) but not that induced by thrombin. The highest concentration of piplartine (300 μg/ml) inhibited thrombin‐ (37%), ADP‐ (71%) and collagen‐ (98%) induced aggregation. The inhibitory effect of piplartine on ADP‐induced platelet aggregation was not modified by pretreatment with pentoxifylline (a phosphodiesterase inhibitor), l ‐arginine (a substrate for nitric oxide synthase) or ticlopidine (a P2Y₁₂ purinoceptor antagonist). However, aspirin, a well‐known inhibitor of cyclooxygenase, greatly increased the inhibitory effect of piplartine on arachidonic‐acid‐induced platelet aggregation. Conclusions The mechanism underlying the piplartine antiplatelet action is not totally clarified. It could be related to the inhibition of cyclooxgenase activity and a decrease in thromboxane A₂ formation, similar to that occurring with aspirin. This and other possible mechanisms require further study.     

酸枣仁皂苷A的抗血小板聚集活性研究

目的 研究酸枣仁皂苷A的抗血小板聚集活性。方法 采用兔体外血小板聚集实验方法,结合酶联免疫吸附法测定血清血小板活化因子（platelet activating factors,PAF）、P-选择素和血小板因子-4（platelet factor-4,PF-4）的含量,探讨不同浓度的酸枣仁皂苷A对二磷酸腺苷（adenosine diphosphate,ADP）、花生四烯酸（arachidonic acid,AA）和PF-4诱导血小板聚集的抑制活性。结果 酸枣仁皂苷A能有效抑制AA、ADP和PF-4诱导的血小板聚集,能显著降低PAF、P-选择素和PF-4的含量。结论 酸枣仁皂苷A具有明显的抗血小板聚集活性。     

The mechanism of anti-platelet activity of davallialactone: involvement of intracellular calcium ions, extracellular signal-regulated kinase 2 and p38 mitogen-activated protein kinase

This study was designed to investigate the effect of davallialactone, which was isolated from the mushroom Inonotus xeranticus, on platelet aggregation induced by collagen, thrombin and ADP. We found that davallialactone dose-dependently inhibited platelet aggregation that was stimulated either by collagen (2.5 microg/ml), a potent ligand of integrin alpha2beta1 and glycoprotein VI, or by thrombin (0.1U/ml), a potent agonist of the protease-activated receptors (PARs) PAR1 and PAR3. In addition, davallialactone inhibited platelet aggregation induced by ADP, an agonist of P2Y receptor. To understand the mechanism of anti-platelet activity, we determined whether davallialactone affected the downstream signaling in collagen-activated platelets. Using the fura-2/AM fluorometric assay, we found that davallialactone dose-dependently inhibited intracellular calcium concentration levels ([Ca2+]i). Moreover, davallialactone inhibited the phosphorylation of extracellular signal-regulated protein kinase (ERK)-2 and p38 mitogen-activated protein kinase (MAPK), in a dose-dependent manner. The tyrosine phosphorylation of 60 and 85kDa proteins, which were activated by collagen, were differentially inhibited by davallialactone. Taken together, these data suggest that davallialactone may have potential anti-platelet aggregation activity via suppression of intracellular downstream signaling pathways.     

Ginsenoside-Rp1 inhibits platelet activation and thrombus formation via impaired glycoprotein VI signalling pathway, tyrosine phosphorylation and MAPK activation

BACKGROUND AND PURPOSE

Ginsenosides are the main constituents for the pharmacological effects of Panax ginseng. Such effects of ginsenosides including cardioprotective and anti-platelet activities have shown stability and bioavailability limitations. However, information on the anti-platelet activity of ginsenoside-Rp1 (G-Rp1), a stable derivative of ginsenoside-Rg3, is scarce. We examined the ability of G-Rp1 to modulate agonist-induced platelet activation.

EXPERIMENTAL APPROACH

G-Rp1 in vitro and ex vivo effects on agonist-induced platelet-aggregation, granule-secretion, [Ca²⁺]_i mobilization, integrin-α_IIbβ₃ activation were examined. Vasodilator-stimulated phosphoprotein (VASP) and MAPK expressions and levels of tyrosine phosphorylation of the glycoprotein VI (GPVI) signalling pathway components were also studied. G-Rp1 effects on arteriovenous shunt thrombus formation in rats or tail bleeding time and ex vivo coagulation time in mice were determined.

KEY RESULT

G-Rp1 markedly inhibited platelet aggregation induced by collagen, thrombin or ADP. While G-Rp1 elevated cAMP levels, it dose-dependently suppressed collagen-induced ATP-release, thromboxane secretion, p-selectin expression, [Ca²⁺]_i mobilization and α_IIbβ₃ activation and attenuated p38^MAPK and ERK2 activation. Furthermore, G-Rp1 inhibited tyrosine phosphorylation of multiple components (Fyn, Lyn, Syk, LAT, PI3K and PLCγ2) of the GPVI signalling pathway. G-Rp1 inhibited in vivo thrombus formation and ex vivo platelet aggregation and ATP secretion without affecting tail bleeding time and coagulation time, respectively.

CONCLUSION AND IMPLICATIONS

G-Rp1 inhibits collagen-induced platelet activation and thrombus formation through modulation of early GPVI signalling events, and this effect involves VASP stimulation, and ERK2 and p38^-MAPK inhibition. These data suggest that G-Rp1 may have therapeutic potential for the treatment of cardiovascular diseases involving aberrant platelet activation.     

Characterization of a unique dihydropyrimidinone, ethyl 4-(4'-heptanoyloxyphenyl)-6-methyl-3,4-dihydropyrimidin-2-one-5-carboxylate, as an effective antithrombotic agent in a rat experimental model

Objectives To evaluate the potential of a novel dihydropyrimidinone, ethyl 4‐(4′‐heptanoyloxyphenyl)‐6‐methyl‐3,4‐dihydropyrimidin‐2‐one‐5‐carboxylate (H‐DHPM), as a calcium channel blocker, endowed with the ability to inhibit platelet aggregation effectively. Methods In‐vitro and in‐vivo studies were conducted for the determination of antiplatelet activity using adenosine diphosphate (ADP), collagen or thrombin as inducers. Calcium channel blocking activity and nitric oxide synthase (NOS) activity were monitored. Lipopolysaccharide (LPS)‐mediated prothrombotic conditions were developed in rats to study the efficacy of H‐DHPM to suitably modulate the inflammatory mediators such as inducible NOS (iNOS) and tissue factor. The cGMP level and endothelial NOS (eNOS) expression were checked in aortic homogenate of LPS‐challenged rats pretreated with H‐DHPM. The effect of H‐DHPM on FeCl₃‐induced thrombus formation in rats was examined. Key findings The concentrations of H‐DHPM required to give 50% inhibition (IC50) of in‐vitro platelet aggregation induced by ADP, collagen or thrombin were 98.2 ± 2.1, 74.5 ± 2.3 and 180.7 ± 3.4 µm , respectively. H‐DHPM at a dose of 52.0 ± 0.02 mg/kg (133 µmol/kg) was found to optimally inhibit ADP‐induced platelet aggregation in‐vivo. The level of nitric oxide was found to be up to 9 ± 0.08‐fold in H‐DHPM‐treated platelets in‐vitro and 8.2 ± 0.05‐fold in H‐DHPM‐pretreated rat platelets in‐vivo compared with control. OH‐DHPM, the parent compound was found to be ineffective both in‐vitro and in‐vivo. H‐DHPM‐pretreated rats were able to resist significantly the prothrombotic changes caused by LPS by blunting the expression of iNOS, tissue factor and diminishing the increased level of cGMP to normal. H‐DHPM enhanced the eNOS expression in aorta of rats treated with LPS. H‐DHPM displayed synergy with antiplatelet activity of aspirin even at lower doses. H‐DHPM was found to inhibit the LPS‐induced platelet aggregation in younger as well as older rats. H‐DHPM exhibited the ability to markedly decrease FeCl₃‐induced thrombus formation in rats. Conclusions H‐DHPM has the attributes of a promising potent antiplatelet candidate molecule that should attract further study. H‐DHPM displayed antiplatelet activity both in vivo and in vitro, which was due partially by lowering the intraplatelet calcium concentration.     

The protective effects of paclitaxel on platelet aggregation through the inhibition of thromboxane A<Subscript>2</Subscript> synthase

Paclitaxel is an anticancer drug used in the treatment of ovarian, breast, head and neck, lung, and prostate cancer. We investigated the anti-platelet activity of paclitaxel in vitro as well as a possible anti-platelet mechanism. Paclitaxel inhibited washed rabbit platelet aggregation induced by collagen in a concentration-dependent manner, with an IC₅₀ of 59.7 ± 3.5. However, it had little effect on platelet aggregation mediated by arachidonic acid, U46619, a thromboxane (TX) A₂ mimic, or thrombin, suggesting that paclitaxel may strongly inhibit collagenmediated signal transduction. In accordance with these findings, paclitaxel blocked collageninduced cytosolic calcium mobilization, arachidonic acid liberation, and serotonin secretion. In addition, it inhibited arachidonic acid-mediated platelet aggregation by about 37% by interfering with TXA₂ synthase as measured by the formation of arachidonic acid-mediated TXA₂ and prostaglandin D₂, as well as cyclooxygenase-1 and TXA₂ synthase activity assays. Taken together, these results point to a cellular mechanism for the anti-platelet activity of paclitaxel through the inhibition of TXA₂ synthase and cytosolic calcium mobilization. This may contribute to the beneficial effects of paclitaxel on the cardiovascular system.     

Effect of 2(1-piperazinyl)-4H-pyrido[1,2-a]pyrimidin-4-one (AP155) on human platelets in vitro

The effect on human platelets of 2-(1-piperazinyl)-4H-pyrido[1,2-a]pyrimidin-4-one (AP155) was tested in vitro by measuring cyclic adenosine monophosphate (cAMP) level, cytosolic Ca⁺⁺, [¹²⁵I]fibrinogen binding as well as aggregation induced by several agonists. AP155 dose-dependently inhibited aggregation both in platelet rich plasma (PRP) and in washed platelets (WP), exerting its maximal power in the presence of collagen, ADP and platelet activating factor (PAF). It specifically inhibited the activity of cAMP high affinity phosphodiesterase (PDE), resulting in a sufficient increase in cAMP levels to activate cAMP-dependent protein kinase. AP155 was able to inhibit aggregation, the increase in cytosolic Ca⁺⁺ induced by thrombin, and fibrinogen binding to ADP or thrombin-stimulated platelets. Thus, this new pyridopyrimidine derivative exerts its antiplatelet activity by increasing cAMP intracellular concentration.     

Cardiotoxin from Naja naja atra snake venom: A potentiator of platelet aggregation

Cardiotoxin, isolated from Naja naja atra snake venom, potentiates platelet aggregation induced by ADP, thrombin, collagen and venom phospholipase A₂. The malondialdehyde formation caused by ADP, thrombin and venom phospholipase A₂ were also increased in the presence of cardiotoxin. Both potentiation of aggregation and increase in malondialdehyde were blocked by indomethacin or Ca²⁺ (5 mM or 0.05 mM). Cardiotoxin did not potentiate thrombin-induced aggregation of p-bromophenacyl bromide-modified platelets. Thromboxane B₂ formation induced by thrombin or collagen was also increased by cardiotoxin, while that by arachidonate was not affected. As a membrane-active polypeptide, cardiotoxin might augment the Ca²⁺-flux during the activation of the platelet membrane by aggregation inducers and then increase the activation of endogenous phospholipase A₂.     

Inhibition by picotamide of thromboxane production in vitro and ex vivo

Summary The effect of picotamide on platelet function has been studied in vitro and ex vivo.Picotamide at micromolar concentrations inhibited platelet aggregation induced by ADP, arachidonic acid and collagen, and it also inhibited the production of thromboxane A₂ (TxA₂). Unlike aspirin, picotamide did not affect the synthesis of prostacyclin by blood vessels.In eight healthy subjects who took picotamide 1200 mg/d platelet aggregation and TxA₂ production were inhibited.Picotamide appears to be an antiplatelet drug that reduces TxA₂ synthesis without affecting cyclooxygenase activity.     

Antiplatelet Effects of Some Aporphine and Phenanthrene Alkaloids in Rabbits and Man

Two aporphines (boldine and laurolitsine) and five phenanthrene alkaloids (litebamine, secoboldine, N-cyanosecoboldine, N-methylsecoglaucine and N-methylsecopredicentrine) were evaluated in-vitro for their ability to inhibit platelet aggregation. All seven alkaloids inhibited aggregation of rabbit platelets and inhibited the release of ATP induced by arachidonic acid and collagen in rabbit platelets. Those aggregations induced by platelet-activating factor (PAF), thrombin, U46619 and ADP were inhibited by the three N-substituted secoboldine derivatives only. Thromboxane B₂ formation caused by arachidonic acid was also suppressed by these compounds. They did not affect the generation of [³H]inositol monophosphate caused by collagen, PAF and thrombin in the presence of indomethacin. Platelet cyclic AMP level was unaffected by litebamine, but was increased by N-methyl-secoglaucine. Litebamine suppressed the secondary aggregation, but not the primary aggregation, induced by ADP and adrenaline in platelet-rich plasma from man, whereas N-methylsecoglaucine inhibited both primary and secondary aggregation. It is concluded that the antiplatelet effect of these seven aporphine and phenanthrene alkaloids is mainly a result of inhibition of thromboxane A₂ formation; N-methylsecoglaucine has additional antiplatelet activity as a result of increasing the levels of platelet cyclic AMP.     

Inhibition of rat platelet aggregation by a Prudhoe Bay crude oil and its aliphatic,aromatic, and heterocyclic fractions

Wasjed platelets isolated from rats 24 hr after oral treatment with a Prudhoe Bay crude oil (PBCO) showed a substantial inhibition of aggregation induced by ADP, arachidonic acid, or epinephrine. In vitro addition of a dimethyl sulfoxide extract of PBCO or its aliphatic, aromatic, or heterocyclic fractions to washed platelets also resulted in an inhibition of aggregation. ADP release was inhibited in platelets to which an extract of PBCO or its fractions were added in vitro or in platelets isolated from rats treated in vivo with PBCO. Thromboxane B₂ release was increased in platelets isolated from rats intubated with PBCO or in platelets to which a dimethyl sulfoxide extract of the aromatic or heterocyclic fraction was added. However, thromboxane B₂ release was inhibited in platelets to which PBCO or the aliphatic fraction extracts were added. The results indicate that PBCO inhibits platelet aggregation presumably by bringing about alterations in the platelet plasma membrane. Inhibition of ADP release could contribute to the inhibition of aggregation but thromboxane B₂ is believed not to play a significant role.     

A platelet function inhibitor purified from Vipera russelli siamensis (Smith) snake venom

Y.-S. Li, K.-F. Liu, Q.-C. Wang, Y.-L. Rpan and G.-C. Tu. A platelet function inhibitor purified from Vipera russelli siamensis (Smith) snake venom. Toxicon23, 895–903, 1985. — By means of CM-Sephadex C-50 column chromatography and gel filtration on Sephadex G — 75, a potent platelet function inhibitor was purified from Vipera russelli siamensis venom. It appeared as a single protein band on polyacrylamide gel electrophoresis in the presence or absence of SDS, and consists of 123 amino acid residues. Its NH₃-terminal residue is serine. It showed the following characteristics: molecular weight, 13,800; isoelectric point, 10.4; ld(In50), 0.5 ± 0.12 mg/kg (i.v.). The platelet inhibitor exhibited phospholipase A₂ activity with a specific activity of 35 μmoles/min/mg. From 2 g of the venom, 70 mg of the purified inhibitor was obtained. Inhibition of human platelet aggregation induced by ADP or adrenaline was dose-dependent, with id(In50) of 1.14μg/ml or 0.37 μg/ml, respectively. The platelet aggregation induced by thrombin or collagen was also inhibited and the inhibitory activity on platelet aggregation was heat stable (at 100°C, 20 min) in an acidic medium (pH 5.8), while its phospholipase A₂ activity was relatively heat labile under the same condition. The release of ³H-serotonin in platelets stimulated by ADP was also inhibited and this was positively correlated with inhibition of platelet aegregation induced by ADP (r = 0.998, P < 0.002).     

莽草酸对血小板聚集和凝血的抑制作用

目的：研究莽草酸(SA)对血小板聚集性的影响及其机制,并研究其对凝血时间的影响。 方法：采用比浊法测定血小板聚集功能,放射免疫法测定血小板TXB2和血浆6-keto-PGF_1α水平,玻管法测定凝血时间。结果：SA体外呈浓度依赖性抑制ADP、胶原诱导的兔血小板聚集,IC₅₀分别为9.25, 3.56 mmol.L^-1;在体内(ip)抑制脑血栓模型鼠由ADP诱导的血小板聚集; SA促进大鼠血浆6-keto-PGF_1α的生成并延长小鼠凝血时间。结论：SA对血小板聚集及凝血系统有显著的抑制作用。     

Inhibition of Platelet Thromboxane Formation and Phosphoinositides Breakdown by Diisoeugenol

Abstract— Diisoeugenol inhibited the platelet aggregation and ATP release of rabbit platelets caused by ADP, arachidonic acid, platelet-activating factor (PAF), collagen and thrombin. Prolongation of the incubation time of platelets with diisoeugenol did not cause further inhibition and the aggregability of platelets could not be restored after washing. In human platelet-rich plasma, diisoeugenol inhibited the biphasic aggregation and ATP release induced by adrenaline and ADP in a concentration-dependent manner. Thromboxane B₂ formation caused by arachidonic acid, collagen and thrombin was markedly inhibited by diisoeugenol in a concentration-dependent manner. Diisoeugenol also inhibited the formation of inositol monophosphate caused by collagen, PAF and thrombin. The cAMP level of washed platelets was not changed by diisoeugenol. It is concluded that the antiplatelet effect of diisoeugenol is due to the inhibition of thromboxane formation and phosphoinositides breakdown.     

Inhibition of platelet aggregation by papaverine-like drugs: evidence for a novel mechanism of action.

Trimethoquinol [6,7-dihydroxy-1-(3′,4′,5′-trimethoxybenzyl)-1,2,3,4-tetrahydroisoquinoline] (TMQ) was chosen as a model compound for studying inhibition of platelet aggregation in vitro, because of its β-adrenoceptor agonist properties and structural resemblance to the anti-aggregatory agent, papaverine. TMQ inhibited collagen-induced aggregation of human platelet-rich plasma (I₅₀, 2 μM), the second wave of aggregation induced by 2.5 μM ADP (I₅₀, 0.9 μM), and the second wave of aggregation induced by 45 μM epinephrine (I₅₀, 2.5 μM). Collagen-induced aggregation of human washed platelets was inhibited by TMQ (I₅₀, 1 μM). TMQ was a better inhibitor than aspirin and papaverine and had an inhibitory activity similar to indomethacin in all of the systems studied. TMQ retained inhibitory activity in the presence of both β-adrenoceptor antagonist: propranolol (50 μM), and α-adrenoceptor antagonist: phentolamine (2.5 μM). Platelet adenylate cyclase was not activated and neither cAMP nor cGMP-phosphodiesterase activities were inhibited by TMQ, PGF_2α, biosynthesis by aggregating platelets during the coagulation of blood obtained from rats pretreated with aspirin (10 mg/kg, p.o.) or indomethacin (1 mg/kg, p.o.) was inhibited. However, similar pretreatment with TMQ (100 mg.kg, p.o.) and papaverine hydrochloride (100 mg/kg, p.o.) had no effect. TMQ acted synergistically with the aggregation inhibitors: papaverine, aspirin, and PGE₁. The in vitro inhibitory action of papaverine, aspirin, and TMQ was enhanced by increasing calcium concentration. These data indicate that the platelet anti-aggregation activity of TMQ, in contrast to its myocardial stimulating and bronchodilating mechanism, is independent of adrenergic activation. Cyclic AMP accumulation or prostaglandin biosynthesis also seem not to be involved in TMQ action. Therefore, it appears that TMQ may have a novel anti-aggregatory mechanism of action.     

Inhibition of platelet aggregation by 5′-nucleotidase purified from Trimeresurus gramineus snake venom

By means of DEAE-Sephadex A-50 column chromatography and gel filtrations on Sephadex G-75, Sephacryl S-300 and Sephadex G-100, successively, a potent 5′-nucleotidase was purified from Trimeresurus gramineus venom. The venom 5′-nucleotidase is a single polypeptide chain and homogeneous as judged by SDS-polyacrylamide gel electrophoresis. It is a thermostable glycoprotein consisting of 589 amino acid residues. Its molecular weight was estimated to be 74,000 by SDS-polyacrylamide gel electrophoresis. It possessed nucleotidase activities toward adenosine monophosphate and adenosine diphosphate. The specific activities toward AMP and ADP were 504 ± 28 and 101 ± 8 μg P_i/min per mg, respectively. Pre-incubation of this venom's 5′-nucleotidase with ADP resulted in the cleavage of ADP and formation of adenosine. The 5′-nucleotidase activity was inhibited by EDTA. Both Zn²⁺ and Co²⁺ reversed the inhibitory effect of EDTA.In rabbit platelet-rich plasma, it inhibited completely the ADP (2 × 10^-5 g/ml)-induced platelet aggregation. It also inhibited the platelet aggregations induced by sodium arachidonate (100 μM), collagen (20 μg/ml) and ionophore A-23187 (5 μM). In rabbit platelet suspensions, it inhibited the platelet aggregation induced by ADP (2 × 10^-5 g/ml), sodium arachidonate (100 μM) and low concentration of thrombin (0.03 U/ml). The collagen (20 μg/ml)- and ionophore A-23187 (5 μM)-induced platelet aggregations were not affected significantly by this venom 5′-nucleotidase. In ADP-refractory platelet-rich plasma, the venom 5′-nucleotidase inhibited the platelet aggregations induced by collagen (20 μg/ml) or sodium arachidonate (100 μM). The venom 5′-nucleotidase showed a more pronounced inhibitory effect on sodium arachidonate-induced platelet aggregation than creatine phosphate/creatine phosphokinase and apyrase did. No lactate dehydrogenase was released by this venom 5′-nucleotidase, indicating that no platelet lysis occurred. It is concluded that removal of ADP, which is released by these platelet aggregation inducers, and the subsequent accumulation of adenosine are responsible for the inhibitory effect of the venom 5′-nucleotidase on platelet aggregations.     

Inhibition of collagen- and ADP-induced platelet aggregation by substance P in vivo: involvement of endothelium-derived relaxing factor.

We examined the effect of substance P, a potent stimulator of endothelium-derived relaxing factor (EDRF) release, on responses to collagen and adenosine 3',5'-diphosphate (ADP) in an in vivo model of platelet aggregation. Substance P inhibited platelet aggregation induced in vivo by both collagen and ADP. This anti-platelet effect was particularly pronounced against collagen-induced aggregation and was prevented by prior administration of haemoglobin (Hb), a known inhibitor of EDRF-mediated responses. Collagen-induced platelet aggregation in vitro was unaffected by a concentration of substance P equivalent to that achieved in plasma following in vivo administration. This study provides a clear demonstration of the anti-platelet activity of EDRF in vivo and an indication that its effectiveness may depend on the aggregating agent used.     

香草酸抗血小板聚集作用的体内外研究

目的香草酸是一种酚酸类化合物,具有抗氧化、抗炎等药理作用,其抗血栓作用尚未有报道。本实验室前期通过筛选发现香草酸具有良好的抗血小板聚集作用,因此本研究通过体内外实验对香草酸抗血小板聚集的作用进行系统评价。方法采用花生四烯酸(arachidonic acid,AA)、二磷酸腺苷(adenosine diphosphate,ADP)、凝血酶(thrombin,THR)诱导体内外血小板聚集模型,结合凝血四项检测评价香草酸抗血小板聚集及抗血栓的作用。结果体外实验中,香草酸对ADP和AA诱导的血小板聚集具有显著抑制作用,并剂量依赖性抑制ADP诱导的血小板聚集;体内试验中,香草酸(10、30和100 mg/kg)能够剂量依赖性抑制ADP和AA诱导的血小板聚集;同时,香草酸(100 mg/kg)能显著降低纤维蛋白原、增加凝血酶原时间,对活化部分凝血活酶时间和血浆凝血酶时间无明显影响。结论本研究首次发现香草酸对ADP和AA诱导的血小板聚集具有抑制作用,为研发具有自主知识产权的抗血小板聚集药物提供了实验依据。     

Effects of thromboxane synthetase inhibitors on aggregation of rabbit platelets

Thromboxane (TX) synthetase activity was selectively inhibited by (E)-3-[4-(1-imidazolylmethyl)phenyl]-2-propenoic acid hydrochloride monohydrate (OKY-046) and sodium (E)-3-[4-(3-pyridylmethyl)phenyl]-2-methyl-propenoate (OKY-1581) (OKYs). Their IC₅₀ for the rabbit platelet enzyme were found to be 11nM and 3nM respectively. Arachidonic acid (AA) or collagen induced platelet aggregation, and generated TXA₂ and prostaglandins (PGs) in rabbit platelets. OKYs inhibited platelet aggregation and TXA₂ generation without affecting PGs generation, while aspirin inhibited platelet aggregation, and TXA₂ and PGs generation. There was a parallel relation between the degree of inhibition of platelet aggregation and TXA₂ generation by OKYs, but the inhibitory effects of aspirin on platelet aggregation was related to that on both TXA₂ and PGs generation. However, OKYs and aspirin did not inhibit ADP-induced platelet aggregation which did not involve the generation of TXA₂ and PGs. These results suggested that TXA₂ generation is related to platelet aggregation induced by AA or collagen, and that the inhibitory effect of OKYs on platelet aggregation is due to the inhibition of TX synthetase.