L—精氨酸L—门冬氨酸盐对血小板功能的抑制

用血小板聚集、粘附、释放实验和出血时间测定观察L-精氨酸*L-门冬氨酸盐(DR)对血小板功能的作用。实验结果显示DR15mg/kg静脉给药,可明显抑制腺苷二磷酸(ADP)诱导的大鼠血小板聚集(P<0.01);15mg/kg单次口服给药可明显抑制ADP诱导的家兔血小板聚集;其药效可持续8h以上(P<0.01);DR7.5、15、30mg/kg灌胃给药(Bid×3.5d),可明显抑制ADP、胶原或凝血酶诱导的大鼠血小板聚集(P<0.01),并延长出血时间(P<0.05)。DR30mg/kg可明显抑制大鼠血小板粘附,并促进血管内皮释放前列环素(PGI2),但对活化的血小板释放血拴素(TXA2)无明显影响。本研究发现,DR可抑制血小板聚集和粘附功能,其作用机制不同于阿司匹林。这些作用部分是由于DR增加了血管内皮PGI2的释放。此结果为血小板功能的调节提供了新线索。     

Spiramine N-6,一种新的抗血小板和抗血小板-中性粒细胞相互作用的物质

Spiramine N-6属粉花秀线菊植物中提取分离的二十碳二萜生物碱。本实验采用Born,Shen和Hamburger等方法分别观察了spiramine N-6在体外和体内对兔血小板聚集功能的影响。应用荧光分光光度法测定其对血小板5-羟色胺释放反应的作用,同时评价spiramine N-6对激活的血小板与中性粒细胞之间粘附反应的影响。结果表明：spiramine N-6在体外选择性抑制血小板活化因子(PAF)诱导的血小板聚集,并呈量效关系,其IC50=26μmol／L,对花生四烯酸(AA)或腺苷二磷酸(ADP)引起的血小板聚集无明显作用;spiramine N-6静注后明显抑制PAF、AA和ADP诱导的血小板聚集。Spiramine N-6呈浓度依赖性减少AA和PAF引起血小板5-羟色胺的释放,其IC50分别为64．7和33．5μmol／L。Spiramine N-6明显阻抑激活的血小板与中性粒细胞间的粘附率,其IC50为78．6μmol／L。结果提示spiramine N-6作为二十碳二萜生物碱具有较强的抗血小板和阻抑血小板一中性粒细胞相互作用的生物活性。     

滇丹参注射液对兔血小板功能的影响

观察云南产滇丹参对血小板聚集功能的影响.方法采用Bom氏比浊法,测定滇丹参体内、体外对抗ADP、PAF、AA诱导的兔血小板聚集的作用.体外每种药物浓度分别为40 g/L、20 g/L、10 g/L、5 g/L、2.5 g/L,体内实验分为7组,即生理盐水组、两种丹参低中高剂量组分别为5 g/kg、10 g/kg、20 g/kg,每组6只.结果与对照组相比,滇丹参体外显著抑制ADP、AA诱导的血小板聚集,抑制效应呈浓度-效应关系(P＜0.05,0.01),IC50为33.7 g/L(ADP)、18.1 g/L(AA).滇丹参也显著抑制PAF诱导的血小板聚集,其最大抑制率为36.8%;体内实验结果显示,滇丹参在高剂量时可显著抑制ADP、PAF和AA诱导的血小板聚集(P＜0.05,0.01),且均具有剂量依赖性.滇丹参在药后20 min开始显效,40 min达到最大抑制作用,抑制率分别为95.6%(ADP)、91.5%(PAF)和88.5%(AA).结论以上结果表明,滇丹参体外、体内显著抑制血小板聚集,且其抗血小板聚集作用优于丹参,为进一步开发和利用滇丹参提供了依据.     

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。     

川芎嗪对血小板聚集功能的影响及其作用机制的探讨

川芎嗪是从中药川芎根中提取的一种生物碱。祖国医学对其活血化瘀作用早有所知。其化学结构已查明,并有合成制品在市上销售。 临床试用表明,川芎嗪治疗冠心病、心绞痛和急性闭塞性脑血管病有效。本文研究证明,川芎嗪在体外对由诱导剂二磷酸腺苷、胶原、凝血酶诱导所致的家兔血小板聚集有强烈抑制作用,同时也能抑制血小板丙二醛的生成。对外源性花生四烯酸诱导的血小板聚集则无抑制作用。川芎嗪能加强家兔动脉环保温液对花生四烯酸诱导的血小板聚集的抑制作用。 给家兔静脉注射大剂量花生四烯酸钠盐,可使动物突然死亡。但如预先注射川芎嗪,则可使动物得到一定保护。     

剪切应力诱导血小板聚集

剪切应力诱导血小板聚集（shear-induced platelet aggregation, SIPA）是指在高剪切流场诱导下血小板表面的膜糖蛋白（GPⅠb/Ⅸ/Ⅴ和GPⅡb/Ⅲa）与血浆中的von Willebrand因子（vWF）相结合,介导血小板的活化、黏附和聚集,是动脉血栓的重要成因.SIPA还需要Ca2+,ADP/ATP等生化因素的参与,因而SIPA现象是生化因素和力学因素偶合作用的结果.细胞外Ca2+是高剪切应力诱导血小板发生聚集的必需条件,Ca2+的跨膜内流引起细胞骨架结构的改变和GPⅡb/Ⅲa的活化.近来对ADP/ATP位于血小板膜上的P2受体的研究表明,P2受体与细胞内Ca2+协同作用通过多种生化途径调控血小板的活化过程在SIPA的信号传导中起着关键的作用.从力学环境与生化反应的偶合关系入手研究SIPA现象的触发机制,深入研究SIPA现象中的信号转导通路是今后的研究热点之一.     

四种圆斑蝰蛇蛇毒对血小板聚集抑制作用的比较研究

对四种不同来源的RVV对ADP、Col和Ris诱导的血小板聚集抑制作用进行比较研究。RVV-B,RVVS和RVV-Styp对三种诱导剂的血小板聚集均有抑制作用,而RVV-T虽有ADP和Col抑制作用,但缺乏对Ris的抑制活性。预先与RVV孵育的血小板经洗涤后重新悬浮于自身PPP中,与RVV-StyP孵育的血小板完全恢复对Ris的凝集反应,而与RVV-B和RVV-S孵育的血小板仅部分恢复其反应。上述结果表明,在RVV中可能含有二种不同的血小板聚集抑制活性,而其抑制机理可能牵涉到血小板、血浆等因素。     

四种圆斑蝰蛇毒对血小板聚集抑制作用的比较研究

对四种不同来源的RVV对ADP、Col和Ris诱导的血小板聚集抑制作用进行比较研究。RVV-B、RVVS和RVV-Styp对三种诱导剂的血小析聚集均有抑制作用,而RVV-T虽有ADP和Col抑制作用,但缺乏对Ris的抑制活性。预先与RVV孵育的血小板经洗涤后重新悬浮于自身PPP中,与RVV-Styp孵育的血小板完全恢复对Ris的凝集反应,而与RVV-B和RVV-S孵育的血小板仅部分恢复其反应。上述结果表明,在RVV中可能含有二种不同的血小板聚集抑制活性,而其抑制机理可能牵涉到血小板、血浆等因素。     

长角血蜱唾液腺中胶原特异性血小板聚集抑制因子的纯化及机制的研究

从长角血蜱唾液腺匀浆纯化出一种新的胶原特异性血小板聚集抑制因子 ,命名为“longicornin” ,它能特异性地抑制胶原诱导的血小板聚集反应 ,而对其他激动剂如ADP、花生四烯酸、凝血酶、瑞斯托霉素、A2 31 87、U46 6 1 9、乙酸豆寇佛波醇所诱导的纯化的血小板聚集均无抑制作用 .longicornin还能抑制血小板激活过程 ,包括血小板颗粒的释放和细胞间质游离Ca ²⁺浓度的升高 ,对胶原与血小板的粘附无抑制作用 .longicornin可能与胶原在血小板上有同一受体 ,并且与血小板受体的结合是可逆的 .     

大豆磷脂对老龄大鼠单胺氧化酶,血小板,动脉内膜的影响

本文观察了饲料添加剂大豆磷脂对老龄大鼠脑组织单胺氧化酶(MAO)活力、血小板聚集功能及主动脉内膜的影响。结果表示喂大豆磷脂24月龄大鼠组的大脑皮层、小脑、海马的MAO活力较对照组高,接近3月龄组;二磷酸腺苷(ADP)诱导血小板释放三磷酸腺苷(ATP)的量高于3月龄组,低于对照组;主动脉内膜扫描电镜观察内皮细胞轮廓清晰可见,有少数桥样结构,表面未见斑块样附着物,对照组则呈粥样硬化形态特征。上述结果提示,大豆磷脂作为饲料添加剂对某些衰老现象有明显的改善。     

Effect of prostaglandin E1 alone and in combination with theophylline or aspirin on collagen-induced platelet aggregation and on platelet nucleotides including adenosine 3′:5′-cyclic monophosphate

1. Human platelet nucleotides were labelled by incubating platelet-rich plasma with [U-(14)C]adenine. With such platelets, the effects of prostaglandin E1, theophylline and aspirin were determined on collagen-induced platelet aggregation and release of platelet ATP and ADP. Intracellular changes of platelet radioactive nucleotides, particularly 3':5'-cyclic AMP, were also determined both with and without collagen treatment. 2. Prostaglandin E1, theophylline and aspirin inhibited collagen-induced aggregation of platelets in a dose-dependent manner. Collagen-induced release of ATP and ADP and breakdown of radioactive ATP were also inhibited in a dose-dependent manner. 3. Prostaglandin E1 stimulated the formation of platelet radioactive 3':5'-cyclic AMP in a dose-dependent manner. With a given dose of prostaglandin E1, maximum formation of radioactive 3':5'-cyclic AMP occurred by 10-30s and thereafter the concentrations declined. The degree of inhibition of aggregation produced by prostaglandin E1, however, increased with its time of incubation in platelet-rich plasma before addition of collagen, so that there was an inverse relationship between the radioactive 3':5'-cyclic AMP concentration measured at the time of collagen addition and the subsequent degree of inhibition of aggregation obtained. 4. Neither theophylline nor aspirin at a concentration in platelet-rich plasma of 1.7mm altered platelet radioactive 3':5'-cyclic AMP contents. In the presence of prostaglandin E1, theophylline increased the concentration of radioactive 3':5'-cyclic AMP over that noted with prostaglandin E1 alone, but aspirin did not. 5. Mixtures of prostaglandin E1 and theophylline had a synergistic effect on inhibition of platelet aggregation. The same was true to a lesser extent with mixtures of prostaglandin E1 and aspirin. Such mixtures also inhibited collagen-induced release of platelet ATP and ADP and breakdown of platelet radioactive ATP. 6. Certain concentrations of either theophylline or aspirin and mixtures of small concentrations of prostaglandin E1 with either theophylline or aspirin caused little or no increase of radioactive 3':5'-cyclic AMP at the time of collagen addition, but inhibited aggregation to a marked degree, whereas higher concentrations of prostaglandin E1 alone caused a much greater increase of radioactive 3':5'-cyclic AMP at the time of collagen addition but inhibited aggregation to a lesser extent. With these compounds there does not appear to be a correlation between these parameters.     

Guanine nucleotides inhibit agonist-stimulated arachidonic acid release in both intact and saponin-permeabilized human platelets

The effects of guanine nucleotides on arachidonic acid (AA) release were studied in intact and saponin-permeabilized human platelets. While GTP[S] itself caused a stimulation of AA release in permeabilized cells, GTP[S], GDP[S], GTP, ATP and other nucleotides inhibited AA release in response to thrombin and other agonists in intact, as well as permeabilized platelets. Inhibition of agonist-stimulated AA release by nucleotides was partially attenuated by addition of ADP, and was abolished by prior stimulation of platelets to discharge the ADP-containing dense granules. These results suggest: (i) that released ADP plays an important contributory role in agonist-stimulated platelet AA release, and (ii) that guanine nucleotides can modulate platelet activation through an extracellular action which is distinct from their effects on G-proteins.     

Role of arachidonic acid in platelet aggregation induced by S. typhimurium endotoxin

The platelet aggregation reaction was used to assess the influence of arachidonic acid (AA), endotoxin (E) S. typhimurium and ADP on platelet aggregation properties. All the three substances induced platelet aggregation. A higher degree of aggregation was attained by the application of E combined with AA and ADP as compared with the effects produced by E and ADP alone. Prolonged incubation of platelet-rich plasma (PRP) samples with E led to an essential decrease of the aggregation degree on ADP addition. Incubation of PRP samples with E and ADP did not evoke any analogous decrease in the platelet aggregation degree. The data obtained indicate that AA stimulates platelet aggregation induced by E and ADP.     

Dual regulation of arachidonic acid release by P2U purinergic receptors in dibutyryl cyclic AMP-differentiated HL60 cells.

ATP promoted biphasic effects on both basal and fMLP-stimulated arachidonic acid (AA) release in neutrophil-like HL60 cells: stimulation in the micromolar range (EC50 = 3.2 +/- 0.9 microM) and inhibition at higher concentrations (EC50 = 90 +/- 11 microM). ATP also inhibited UTP- and platelet activating factor-stimulated AA release. Only stimulatory effects of ATP on basal or fMLP-stimulated phospholipase C were observed. The inhibitory effect of ATP on AA release was not due to reacylation of released AA, chelation of extracellular Ca2+, cell permeabilization, or changes in the rise of [Ca2+]i induced by agonist. The inhibition was rapid, being detected within 5-15 s. The inhibitory effect of ATP on fMLP-stimulated AA release could be desensitized by pretreatment of the cells with 2 mM ATP, but not 20 microM ATP, the concentration that resulted in maximal release of AA and inositol phosphates. The inhibition by ATP was neither dependent on generation of adenosine by ATP hydrolysis nor the result of direct interaction of ATP with P1 purinergic receptors. Among other nucleotides tested (CTP, GTP, ITP, TTP, XTP, adenosine 5'-(beta,gamma-methylene)triphosphate (AMP-PCP), adenyl-5'-yl imidodiphosphate (AMP-P(NH)P), ADP, adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), and UTP), only UTP and ATP gamma S displayed biphasic effects with potencies and efficacies almost identical to those of ATP. The other nucleotides only exhibited stimulatory effects (EC50 = 60-300 microM). The results are consistent with a model of dual regulation of AA release by two distinct subtypes of P2U receptors in HL60 cells.     

Comparison of Aggregometry with Flow Cytometry for the Assessment of Agonists′-Induced Platelet Reactivity in Patients on Dual Antiplatelet Therapy

Data on the agreement between aggregometry and platelet activation by flow cytometry regarding the measurement of on-treatment platelet reactivity to arachidonic acid (AA) and adenosine diphosphate (ADP) are scarce. We therefore sought to compare three platelet aggregation tests with flow cytometry for the assessment of the response to antiplatelet therapy. Platelet aggregation in response to AA and ADP was determined by light transmission aggregometry (LTA), the VerifyNow assays, and multiple electrode aggregometry (MEA) in 316 patients receiving aspirin and clopidogrel therapy after angioplasty with stent implantation. AA- and ADP-induced P-selectin expression and activated glycoprotein (GP) IIb/IIIa were determined by flow cytometry. LTA, the VerifyNow P2Y₁₂ assay and MEA in response to ADP correlated significantly (all p<0.001), and the best correlation was observed between LTA and the VerifyNow P2Y₁₂ assay (r = 0.63). ADP-induced platelet reactivity by all aggregation tests correlated significantly with ADP-induced P-selectin expression and activated GPIIb/IIIa (all p<0.001). The best correlation was seen between the VerifyNow P2Y₁₂ assay and activated GPIIb/IIIa (r = 0.68). The platelet surface expressions of P-selectin and activated GPIIb/IIIa in response to ADP were significantly higher in patients with high on-treatment residual platelet reactivity (HRPR) to ADP by all test systems (all p<0.001). A rather poor correlation was observed between AA-induced platelet reactivity by LTA and the VerifyNow aspirin assay (r = 0.15, p = 0.007), while both methods did not correlate with MEA. AA-induced platelet reactivity by all aggregation tests correlated significantly, but rather poorly with AA-induced P-selectin expression (all p<0.05), while only AA-induced platelet reactivity by LTA correlated significantly with AA-induced activated GPIIb/IIIa (r = 0.21, p<0.001). The platelet surface expression of P-selectin in response to AA was significantly higher in patients with HRPR by LTA AA and MEA AA (both p<0.02). In contrast, P-selectin expression in response to AA was similar in patients without and with HRPR by the VerifyNow aspirin assay (p = 0.5), and platelet surface activated GPIIb/IIIa in response to AA did not differ significantly between patients without and with HRPR to AA by all test systems (all p>0.1). In conclusion, ADP-induced platelet reactivity by aggregometry translates partly into flow cytometry. In contrast, AA-induced platelet reactivity correlates poorly between different platelet aggregation tests, and between aggregometry and flow cytometry. Overall, both approaches capture different aspects of platelet function and are therefore not interchangeable in the assessment of agonists´-induced platelet reactivity. Clinical outcome data are needed to determine which test systems and settings are associated with different in vivo consequences.     

广西眼镜蛇毒L-氨基酸氧化酶体内外抗血小板聚集实验研究

目的 研究广西眼镜蛇中提取的L-氨基酸氧化酶(L-amino acid oxidase)在人体外及家兔体内的抗血小板聚集作用.方法 用比浊法测定广西眼镜蛇毒L-氨基酸氧化酶对二磷酸腺苷(ADP)、胶原、凝血酶、花生四烯酸(AA)在人体外及家兔体内引起的血小板聚集率的影响.结果 实验中,能明显抑制二磷酸腺苷(ADP)、胶原、凝血酶、花生四烯酸(AA)引起的血小板聚集,并呈明显的正相关.结论 广西眼镜蛇毒L-氨基酸氧化酶在体内外均有较强的抗血小板聚集活性.     

Effects of antimycin and 2-deoxyglucose on adenine nucleotides in human platelets. Role of metabolic adenosine triphosphate in primary aggregation, secondary aggregation and shape change of platelets

1. Human platelet-rich plasma prelabelled with [(3)H]adenine was incubated at 37 degrees C with antimycin A and 2-deoxy-d-glucose. Variations in the amounts of ATP, ADP and P(i), and in the radioactivity of ATP, ADP, AMP, IMP, hypoxanthine+inosine and adenine were determined during incubation. Adrenaline- and ADP-induced platelet aggregation and the ADP-induced shape change of the platelets were determined concurrently. 2. 2-Deoxyglucose caused conversion of [(3)H]ATP to [(3)H]hypoxanthine+inosine. The rate of this conversion increased with increasing 2-deoxyglucose concentration and was markedly stimulated by addition of antimycin, which had no effect alone. At maximal ATP-hypoxanthine conversion rates, the IMP radioactivity remained at values tenfold higher than control, whereas [(3)H]ADP and [(3)H]AMP radioactivity gave variations typical for product/substrates in consecutive reactions. The specific radioactivityof ethanol-soluble platelet ATP decreased during incubation to less than one-tenth of its original value. The amounts and radioactivity of ethanol-insoluble ADP did not vary during incubation with the metabolic inhibitors. 3. The rate of ADP- and adrenaline-induced primary aggregation decreased as the amount of radioactive ATP declined, and complete inhibition of aggregation was obtained at a certain ATP concentration (metabolic ATP threshold). This threshold decreased with increasing concentration of inducer ADP. 4. Secondary platelet aggregation (release reaction) had a metabolic ATP threshold markedly higher than that of primary aggregation. 5. Shape change was gradually inhibited as the ATP radioactivity decreased, and had a metabolic ATP threshold distinctly lower than that of primary aggregation, and which decreased with increasing concentration of ADP. 6. A small but distinct fraction of [(3)H]ATP disappeared rapidly during the combined shape change-aggregation process induced by ADP in platelets incubated with metabolic inhibitors, whereas no ATP disappearance occurred during aggregation in their absence.     

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.