天麻成分对羟基苯甲醛抗血小板聚集作用及急性毒性研究

为探讨天麻成分对羟基苯甲醛的抗血小板聚集作用,本实验以二磷酸腺苷(ADP)、花生四烯酸(AA)、血小板活化因子(PAF)为诱导剂,探讨了对羟基苯甲醛的体外抗血小板聚集活性;采用ADP静脉注射致小鼠肺栓塞方法以及下腔静脉结扎致大鼠静脉血栓方法,考察了对羟基苯甲醛的体内抗血小板聚集活性;并对经口给药的对羟基苯甲醛的急性毒性进行了检测。结果表明,天麻成分对羟基苯甲醛对ADP诱导的家兔体外血小板聚集有明显的对抗作用,其半数抑制率(50%inhibitory concentration,IC50)为2mmol/L,对PAF诱导的家兔体外血小板聚集无明显影响;天麻成分对羟基苯甲醛能明显降低小鼠肺栓塞死亡率,并显著对抗大鼠下腔静脉血栓的形成。说明天麻成分对羟基苯甲醛在体外、体内均具有显著的抗血小板聚集活性。急性毒性研究结果表明天麻成分对羟基苯甲醛经口给药的小鼠半数致死量(lethal dose 50,LD50)为1.23 g/kg。     

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。     

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。     

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。     

滇产胡椒属植物醇提物抗血小板活性研究

采用Born氏比浊法观察了12种滇产胡椒属植物的乙醇提取物对血小板活化因子(PAF)、花生四烯酸(AA)和二磷酸腺苷(ADP)引起血小板活化聚集的影响.结果表明,这12种醇提物均明显抑制PAF诱导的血小板聚集,其中部分醇提物对AA和ADP引起的聚集亦有显著拮抗作用.结果提示,该12种滇产胡椒属植物醇提物具有较高的抗血小板活性     

白藜芦醇甙对血小板聚集功能及内钙水平的影响

为了研究白藜芦醇甙(polydatin,PD)的抗血小板聚集作用及对细胞内钙水平的影响,并探讨其抗血栓形成作用的机制,应用Bom比浊法和Grynkiewicz方法分别测定PD对兔血小板聚集功能和血小板内钙水平的影响。PD在体外显著抑制花生四烯酸(arachidonic acid,AA)和腺苷二磷酸(adenosine diplrasphate,ADP)诱导的富血小板血浆中的血小板聚集,其半数抑制浓度(medium inhibitory concentration,IC50)分别为5．13及10．0μmol／L;5、10和20mg／kg的PD静注均明显降低兔血小板聚集率,且呈明显的剂量一效应关系;PD明显减少兔洗涤血小板内钙释放及外钙内流,本实验说明PD体内、外均有明显的抗血小板聚集作用,其机制与其降低血小板内钙浓度密切相关。     

褐藻酸钠硫酸酯对血液流变学和血小板聚集的影响

褐藻酸钠硫酸酯(Sodium Alginate Sulfates SAS)是以褐藻酸为原料经磺化、酯化而成的硫酸酯多糖的钠盐系列物(Ⅰ、Ⅱ、Ⅲ、Ⅳ)。本文研究了SAS对兔和大鼠血液流变学和血小板聚集的影响,结果发现:SAS静注5mg/kg能够显著降低高切、低切下的全血比粘度,降低血细胞压积,降低红细胞聚集指数,提高红细胞沉降率,从而改善血液的流变特性。实验中还发现,SAS体内外均可缩短大鼠红细胞电泳时间,体内还可以缩短大鼠血小板电泳时间,而体外则可延长血小板电泳时间。SAS体外可诱导兔、大鼠血小板聚集,能够促进ADP诱导的血小板聚集,而对AA诱导的血小板聚集则无促进作用。结果提示:静注SAS能够改善血液的流变特性,增加红细胞和血小板表面电荷,抑制红细胞及血小板间的粘附和聚集,从而使其具有一定的抗血栓作用。同时还提示,SAS在体内循环和代谢的过程中,结构可能发生了变化,从而使其体内、体外的作用特征出现了相对立的结果。     

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。     

丹心Ⅲ号和丹心V号对血小板聚集功能的影响

本文观察了丹心Ⅲ号和丹心Ｖ号对血小板聚集功能的影响,实验结果：丹心Ⅲ号和丹心Ｖ号在体外均可显著抑制ＡＤＰ和花生四烯酸诱导的人血小板聚集,其ＩＣ５０分别为１．６０５ｍｇ／ｍｌ,２．５８９ｍｇ／ｍｌ,８．４ｌ６ｍｇ／ｍｌ和６．６０６ｎｇ／ｍｌ;在体内可抑制连续给药（３５０一７００ｍｇ／ｌｋｇ．ｄ）１０ｄ大鼠血小板对ＡＤＰ和花生烯酸诱导的血小板聚集,但对凝血酶诱导的血小板聚集无明显抑制作用。上述结果提示丹心Ⅲ号和丹心Ⅴ号对血小板聚集功能具有明显的抑制作用。     

血小板聚集功能障碍与梅尼埃病的关系

目的探讨血小板聚集功能障碍与梅尼埃病的关系。方法对３２例梅尼埃病患者血小板聚集功能进行检测。结果血小板最大聚集率均数为（６１．４２±１９．４）％，与对照组（４７．２７±２０．４）％比较，有统计学差异（Ｐ＜０．０１）。结论血小板聚集功能的异常，可能与梅尼埃病的发病有密切关系。     

Critical roles for the COOH-terminal NITY and RGT sequences of the integrin beta3 cytoplasmic domain in inside-out and outside-in signaling

Bidirectional signaling of integrin alphaIIbbeta3 requires the beta3 cytoplasmic domain. To determine the sequence in the beta3 cytoplasmic domain that is critical to integrin signaling, cell lines were established that coexpress the platelet receptor for von Willebrand factor (vWF), glycoprotein Ib-IX, integrin alphaIIb, and mutants of beta3 with truncations at sites COOH terminal to T741, Y747, F754, and Y759. Truncation at Y759 did not affect integrin activation, as indicated by vWF-induced fibrinogen binding, but affected cell spreading and stable adhesion. Thus, the COOH-terminal RGT sequence of beta3 is important for outside-in signaling but not inside-out signaling. In contrast, truncation at F754, Y747, or T741 completely abolished integrin activation. A point mutation replacing Y759 with alanine also abolished integrin activation. Thus, the T755NITY759 sequence of beta3, containing an NXXY motif, is critical to inside-out signaling, whereas the intact COOH terminus is important for outside-in signaling. In addition, we found that the calcium-dependent protease calpain preferentially cleaves at Y759 in a population of beta3 during platelet aggregation and adhesion, suggesting that calpain may selectively regulate integrin outside-in signaling.     

Apyrase, Ascorbic Acid and Aprotinin Ameliorate the Storage Lesion in Pelleted Platelet Preparations

To evaluate the effect of apyrase, ascorbic acid and aprotinin (AAA) in preventing platelet activation during storage, 12 sets of platelet concentrates (PCs), were treated with AAA and evaluated at days 1, 3, and 5 utilizing platelet functional and morphological assays. Platelets treated with AAA demonstrated significantly enhanced response to ADP-induced platelet aggregation, higher morphology scores, and elevated ATP levels compared to control samples after 5 days of storage. Similarly, platelet specimens treated with AAA had significantly reduced PF4 secretion and P-selectin expression compared to controls. Finally, Western blots of aggregated platelets at day 5 demonstrated that AAA-treated PCs continue to express the platelet membrane GPIb whereas specimens from control PCs do not. These results show that PCs treated with AAA have reduced platelet activation and enhanced functional platelet activity.     

Platelet lumiaggregation testing: Reference intervals and the effect of acetylsalicylic acid in healthy adults

BackgroundLight transmission aggregometry with lumiaggregometry are methods commonly recommended as a first-line test in platelet dysfunction diagnostic work-up. They are poorly standardized and usually performed in specialized laboratories. For proper interpretation, each laboratory should establish its own diagnostic approach in order to recognize abnormal aggregation patterns. The aim of this study was to measure plasma lumiaggregometry with basic agonists to establish the analyzer-reagent reference intervals (RI) for adults and to test the method response to aspirin.MethodsThe Chrono-Log Model 700 lumiaggregometer using Chrono-Par and Chrono-lume reagents (Chrono-Log Corp., Havertown, PA, USA) was used to measure the maximal aggregation and adenosine triphosphate release using adenosine diphosphate (2 μmol/L), collagen (2 μg/mL), arachidonic acid (1 μmol/L), epinephrine (5.5 μmol/L) and ristocetin (1.25 mg/mL), and thrombin (1 U/mL). The effect of aspirin on platelet aggregation and granule release was inspected.ResultsRIs derived from 40 healthy adults were calculated using the non-parametric approach. Wider intervals and low lower limits were determined for weak agonist as well as absence or impaired aggregation in up to one of 7 healthy controls. The response of platelets to aspirin shows response comparable to previously reported study.ConclusionsLocally established RI in our study enable us to investigate platelet function in patients with a high probability of bleeding disorders. Values are agonist and equipment specific. The variability of the method can be reduced by considering standardized preanalytical and analytical variables. Pathological results must be interpreted in the context of other hemostasis test results and clinical findings.     

Docosahexaenoic acid but not eicosapentaenoic acid withstands dietary cholesterol-induced decreases in platelet membrane fluidity

To determine the differenetial effects of docosahexaenoic (DHA) and eicosapentaenoic (EPA) acid on platelet membrane fluidity under hypercholesterolemic conditions. DHA and EPA were orally administered (300 mg/kg body weight^.day) to hypercholesterolemic rats for 12 weeks. Membrane fluidity, evaluated by fluorescence polarization of nonpolar 1,6-diphenyl-1,3,5-hexatriene (DPH), of the platelets of high cholesterol (HC; 1%)-fed rats decreased significantly compared with that of the platelets of normocholesterolemic rats. In HC-fed rats, dietary administration of DHA, unlike that of EPA, significantly increased platelet membrane fluidity. A high cholesterol diet significantly increased platelet aggregation, compared with the platelet aggregation of normocholesterolemic rats. DHA administration significantly decreased the aggregation, whereas EPA had no effect. Levels of EPA in the platelets of the EPA-fed HC rats and those of DHA in the platelets of the DHA-fed HC rats increased by 482 and 174%, respectively, compared with those in the platelets of the HC-fed rats. The unsaturation index and the ratio of saturated to (poly)unsaturated fatty acid of the platelet membrane increased only in the DHA-fed rats. The phospholipid content in platelet membranes remained unaltered in all groups, whereas the cholesterol content decreased significantly in DHA-fed rats, resulting in a significant decrease in the cholesterol/phospholipid molar ratio only in the platelet membranes of DHA-fed rats. These results suggest that DHA is a more potent membrane-fluidizer than EPA in withstanding cholesterol-induced decreases in platelet membrane fluidity and a stronger ameliorative modulator of platelet hyperaggregation.     

Effect of the thiol group inhibitor monobromobimane and other inhibitors on the composition of the platelet cytoskeletal core and its association with glycoprotein IIIa

SDS-polyacrylamide gel electrophoresis was used to study the effects of the thiol inhibitor monobromobimane (MB), EDTA, and prostaglandin E1 (PGE1) on the formation and composition of the platelet cytoskeletal core (Triton-insoluble residue) and its association with glycoprotein (GP) IIIa. Stimulation or aggregation of platelets in response to ADP or thrombin increased the amount of Triton-insoluble myosin. Aggregation resulted in incorporation of [125I]GP IIIa and a new band at about 210 kDa into the cytoskeletal core. EDTA and PGE1 caused little disaggregation of platelets that were aggregated in PRP with ADP and that had secreted the contents of their granules. In contrast to EDTA, PGE1 decreased the amount of Triton-insoluble residue and its association with GP IIIa. MB added after ADP-induced aggregation caused an increase in the amount of cytoskeletal core despite marked disaggregation and a substantial decrease in core-associated GP IIIa. With aspirin-treated platelets that had not secreted, EDTA, PGE1, and MB all caused disaggregation and loss of cytoskeletal GP IIIa. MB diminished, but did not reverse, thrombin-induced aggregation of washed platelets and arrested GP IIIa incorporation into the cytoskeletal core. Concanavalin A (Con A) cross-links glycoproteins on a single platelet and induces incorporation of GP IIIa into the Triton-insoluble residue in the absence of platelet aggregation. This induction was not inhibited by MB, although this reagent, as well as aspirin, inhibited Con A-induced secretion. Since GP IIIa incorporation caused by ADP-induced aggregation differs from that caused by Con A in its susceptibility to MB, it seems unlikely that thiol groups are directly involved in the association of GP IIIa with the cytoskeletal core.     

Activation of G12/G13 results in shape change and Rho/Rho-kinase-mediated myosin light chain phosphorylation in mouse platelets

Platelets respond to various stimuli with rapid changes in shape followed by aggregation and secretion of their granule contents. Platelets lacking the alpha-subunit of the heterotrimeric G protein Gq do not aggregate and degranulate but still undergo shape change after activation through thromboxane-A2 (TXA2) or thrombin receptors. In contrast to thrombin, the TXA2 mimetic U46619 led to the selective activation of G12 and G13 in Galphaq-deficient platelets indicating that these G proteins mediate TXA2 receptor-induced shape change. TXA2 receptor-mediated activation of G12/G13 resulted in tyrosine phosphorylation of pp72(syk) and stimulation of pp60(c-src) as well as in phosphorylation of myosin light chain (MLC) in Galphaq-deficient platelets. Both MLC phosphorylation and shape change induced through G12/G13 in the absence of Galphaq were inhibited by the C3 exoenzyme from Clostridium botulinum, by the Rho-kinase inhibitor Y-27632 and by cAMP-analogue Sp-5,6-DCl-cBIMPS. These data indicate that G12/G13 couple receptors to tyrosine kinases as well as to the Rho/Rho-kinase-mediated regulation of MLC phosphorylation. We provide evidence that G12/G13-mediated Rho/Rho-kinase-dependent regulation of MLC phosphorylation participates in receptor-induced platelet shape change.     

The microvesicle/CD36 complex triggers a prothrombotic phenotype in patients with non‐valvular atrial fibrillation

The mechanisms responsible for platelet activation, the prothrombotic state, in non‐valvular atrial fibrillation (NVAF) are still obscure. Microvesicles (MVs) can transfer various messages to target cells and may be helpful for exploring the detailed mechanisms. We aimed to investigate the possible mechanisms by which proatherogenic factors of NVAF contribute to platelet activation. Two hundred and ten patients with NVAF were stratified as being at ‘low to moderate risk’ or ‘high risk’ for stroke according to the CHADS2 score. Levels of platelet‐derived MVs (PMVs) and platelet activation were examined. CD36‐positive or CD36‐deficient human platelets were stimulated by MVs isolated from NVAF patients with or without various inhibitors in vitro. Levels of PMVs and platelet activation markers enhanced significantly in high‐risk patients. The MVs isolated from plasma of NVAF patients bound to platelet CD36 and activated platelets by phosphorylating the mitogen‐activated protein kinase 4/Jun N‐terminal kinase 2 (MKK4/JNK2) pathways. However, CD36 deficiency protected against MV‐induced activation of platelets. We reveal a possible mechanism of platelet activation in NVAF and suggest that the platelet CD36 might be an effective target in preventing the prothrombotic state in NVAF.     

Characterization of the LDL receptor in rat promegakaryoblasts in culture

Rat promegakaryoblasts (RPM, a precursor platelet cell line) in culture exhibited a capacity to bind, take up and degrade¹²⁵I-LDL. The low density lipoprotein (LDL) binding showed the following characteristics: (a) high affinity, (b) saturability, (c) specificity, (d) down-regulation, after exposure to 25 hydroxycholesterol. Furthermore the proteolytic degradation of¹²⁵I-LDL by RPMs was inhibited by chloroquine which interferes with the lysosomal degradation processes. These findings show LDL receptor cell biology of RPM to be of the classical type and to differ from that of platelets.