Identification of the platelet ADP receptor targeted by antithrombotic drugs

Platelets have a crucial role in the maintenance of normal haemostasis, and perturbations of this system can lead to pathological thrombus formation and vascular occlusion, resulting in stroke, myocardial infarction and unstable angina. ADP released from damaged vessels and red blood cells induces platelet aggregation through activation of the integrin GPIIb-IIIa and subsequent binding of fibrinogen. ADP is also secreted from platelets on activation, providing positive feedback that potentiates the actions of many platelet activators. ADP mediates platelet aggregation through its action on two G-protein-coupled receptor subtypes. The P2Y1 receptor couples to Gq and mobilizes intracellular calcium ions to mediate platelet shape change and aggregation. The second ADP receptor required for aggregation (variously called P2Y(ADP), P2Y(AC), P2Ycyc or P2T(AC)) is coupled to the inhibition of adenylyl cyclase through Gi. The molecular identity of the Gi-linked receptor is still elusive, even though it is the target of efficacious antithrombotic agents, such as ticlopidine and clopidogrel and AR-C66096 (ref. 9). Here we describe the cloning of this receptor, designated P2Y12, and provide evidence that a patient with a bleeding disorder has a defect in this gene. Cloning of the P2Y12 receptor should facilitate the development of better antiplatelet agents to treat cardiovascular diseases.     

三七叶甙对兔血小板聚集功能的影响

 应用Born氏比浊法分别测定三七叶甙(Panax notoginseng folium saponins)对花生四烯酸(arachidonic acid,AA)、血小板活化因子(platelet activating factor,PAF)和腺苷二磷酸(adenosine diphosphate,ADP)诱导的兔血小板聚集功能的影响.实验结果表明:三七叶甙在体外显著抑制ADP诱导的血小板聚集,其半数抑制浓度(medium inhibitory concentration,IC₅₀)为89.4mg/L;三七叶甙静脉注射也能明显降低ADP诱导的兔血小板聚集率,且呈剂量-效应关系.提示三七叶甙体内外均明显抑制ADP诱导的兔血小板聚集,而对AA和PAF诱导的兔血小板聚集无明显影响.     

Do human platelets have opiate receptors?

In their study of prostaglandin E1 (PGE1)-sensitive adenylate cyclase (AC) in rat brain homogenates, Collier and Roy claimed that the activity of this enzyme is inhibited by opiates. They also proposed that opiates exert their analgesic and allied effects by inhibiting AC of neurones that are normally stimulated by E prostaglandins. Studies using neuroblastoma x glioma hybrid cells supported this hypothesis. However, subsequent studies with mammalian brain and rat brain tissue slices yielded conflicting results. PGE1 also inhibits platelet aggregation, probably through activation of platelet AC. Gryglewski et al. showed that morphine inhibits the anti-aggregating effect of PGE1 on ADP- and adrenaline-induced platelet aggregation, and suggested that the inhibition by morphine is mediated through platelet AC activity. We report here our attempts to reproduce the results of Gryglewski et al. and our examination of the effect of morphine on PGE1-sensitive AC activity in platelet lysates and on PGE1-induced accumulation of cyclic AMP in intact platelets. The possible existence of opiate receptors in platelets was also assessed by direct binding studies with 3H-etorphine. In contrast to Gryglewski et al., we could not detect any effect of opiates on the aggregation of human platelets, nor did we find any other evidence supporting the presence of opiate receptors in these cells. Thus we conclude that the presence of opiate receptors in human platelets is unlikely.     

红花黄色素抗凝血作用及对血小板聚集影响的研究

目的研究红花黄色素抗凝血作用及对血小板聚集的影响。方法大鼠心脏采血,取血浆检测凝血酶原时间(PT),凝血酶时间(TT),活化部分凝血活酶时间(APTT),血浆纤维蛋白原(Fib)含量;家兔颈动脉采血,取富血小板血浆(PRP)及穷血小板血浆(PPP),描记聚集曲线,计算聚集百分率,最大聚集百分率及药物的聚集抑制百分率。结果红花黄色素明显延长大鼠血浆凝血酶时间、凝血酶原时间、活化部分凝血活酶时间,明显降低大鼠血浆纤维蛋白原含量,显著抑制由二磷酸腺苷引起的家兔血小板聚集。结论红花黄色素具有抗凝血作用。     

Role of thrombin signalling in platelets in haemostasis and thrombosis.

Platelets are critical in haemostasis and in arterial thrombosis, which causes heart attacks and other events triggered by abnormal clotting. The coagulation protease thrombin is a potent activator of platelets ex vivo. However, because thrombin also mediates fibrin deposition and because multiple agonists can trigger platelet activation, the relative importance of platelet activation by thrombin in haemostasis and thrombosis is unknown. Thrombin triggers cellular responses at least in part through protease-activated receptors (PARs). Mouse platelets express PAR3 and PAR4 (ref. 9). Here we show that platelets from PAR4-deficient mice failed to change shape, mobilize calcium, secrete ATP or aggregate in response to thrombin. This result demonstrates that PAR signalling is necessary for mouse platelet activation by thrombin and supports the model that mouse PAR3 (mPAR3) does not by itself mediate transmembrane signalling but instead acts as a cofactor for thrombin cleavage and activation of mPAR4 (ref. 10). Importantly, PAR4-deficient mice had markedly prolonged bleeding times and were protected in a model of arteriolar thrombosis. Thus platelet activation by thrombin is necessary for normal haemostasis and may be an important target in the treatment of thrombosis.     

芪丹天胶囊对老年血瘀大鼠血小板聚集功能及血浆TXB2、6-ketO-PGF1a含量的影响

目的探讨芪丹天胶囊对血小板聚集功能的影响及作用机理.方法以老年雄性大鼠为血瘀模型,采用血小板聚集法和放免法观察芪丹天胶囊对血小板聚集功能及血浆TXB2、6-keto PGFla 含量的影响.结果芪丹天胶囊能明显抑制ADP诱导的血小板聚集,降低血浆中TXB2的含量及TXB2/6-keto-PGFla 的比值.结论芪丹天胶囊具有良好的抑制血小板聚集作用,其作用机理与降低TXB2有关.     

丹龙胶囊抗血栓形成的实验研究

目的研究丹龙胶囊抗血栓形成的作用。方法手术结扎大鼠下腔静脉使大鼠下腔静脉内形成血栓,称量血栓湿重和干重;用血栓生成仪观察记录电刺激5min内颈总动脉平均阻塞率(%);用血小板聚集凝血因子分析仪测定受试药对二磷酸二腺苷(ADP)诱导血小板聚集的抑制作用。结果丹龙胶囊能够减少下腔静脉血栓形成;降低颈总动脉平均阻塞率;抑制由二磷酸二腺苷引起的家兔血小板聚集。结论研究结果证明丹龙胶囊具有抑制血栓形成的作用。     

Human blood platelets showing no response to collagen fail to express surface glycoprotein Ia

The interaction of blood platelets with collagen is generally considered to be of primary importance in the arrest of bleeding and to have a role in the pathogenesis of thrombosis and atherosclerosis. Following damage to the vascular endothelium, circulating platelets come into contact with exposed collagen fibrils in the subendothelium and spread along it; this is followed by the secretion of several biologically active substances and by aggregation of platelets. The glycoproteins of the platelet plasma membrane have an important role in the mechanisms underlying these processes. So far, two specific defects of platelet function in patients with a bleeding disorder are known to be associated with a glycoprotein defect and the study of these patients has contributed significantly to present concepts of platelet function. The glycoprotein (GP) IIB-III complex, absent or deleted in the aggregation-defective Glanzmann's thrombasthenia, has been identified as the platelet fibrinogen receptor. GPIb, which is absent in the adhesion-defective Bernard-Soulier syndrome, has been identified as the von Willebrand factor receptor on platelets. We now report a defect of the platelet plasma membrane glycoprotein composition in a patient whose platelets are totally unresponsive to collagen.     

罗汉果黄酮的活血化瘀药理作用研究

为了观察罗汉果黄酮的活血化瘀药理作用,采用水提和树脂吸附方法提取罗汉果黄酮,采用尾静脉注射胶原蛋白和肾上腺素造成小鼠体内血栓,测定药物对小鼠脑血栓的保护率;用腺苷-5-二磷酸钠盐（ADP）诱导大鼠血小板聚集,计算药物对血小板聚集的抑制率; 腹腔注射75%蛋黄乳液造成实验性高血脂症,测定血清总胆固醇（TC）、甘油三酯（TG）、高密度脂蛋白含量; 采用毛细玻璃管法测定小鼠凝血时间,并进行了的急性毒性试验.结果表明,罗汉果黄酮对血栓形成有一定的保护作用;能抑制ADP诱导大鼠血小板聚集;明显降低高胆固醇血症小鼠的TC和TG含量,提高HDL-C的水平;以及延长小鼠的凝血时间.说明罗汉果黄酮具有一定的抗血栓形成、抗血小板聚集、降血脂、抗凝血等活血化瘀药理作用.急性毒性试验结果表明,罗汉果黄酮对小鼠的最大给药量为60.85g/kg.     

Vascular endothelial and smooth muscle cells in culture selectively release adenine nucleotides.

Endothelial cells in culture can modulate platelet aggregation and vascular tone, in part by producing prostacyclin (PGI2), a powerful vasodilator and inhibitor of platelet aggregation, but also by their ecto-ADPase activity, which initiates the conversion of pro-aggregating ADP to adenosine, a potent vasodilator and platelet inhibitor. We have now demonstrated that cultured aortic endothelial cells exposed to trypsin, thrombin or other stimuli can liberate a high proportion of their adenine nucleotides without substantial loss of lactate dehydrogenase. ADP rapidly accumulates extracellularly, reaching biologically active concentrations before there is further breakdown to adenosine. Whether this selective release of nucleotides is a response to damage, or whether it represents a specific secretory mechanism remains to be resolved. Cultured aortic smooth muscle cells can secrete adenine nucleotides in a similar manner, but extracellular conversion to adenosine occurs much faster.     

Experimental simulation of model platelet adhesion to a semi-permeable wall exposed to flow disturbance

A sudden tubular expansion with a semipermeable wall was constructed from a tubular dialysis membrane to investigate the effects of rdtration flow and flow disturbance on particle deposition. The expansion was perfused with a dilute, neutrally buoyant suspension of 1.10μm diameter polystyrene latex spheres (as models of platelets) in Tris buffer solution containing 10% Dextran T70 and 2% bovine serum albumin. The results showed that adhesion of particles correlated positively with the filtration rate and inversely with the wall shear rate. In the vortex flow region distal to the expansion, particle adhesion was significantly elevated with a maximum at the reattachment point where the wall shear rate was the lowest and particles were constantly carried toward the vessel wall along the curved streamlines. In conclusion, rdtration flow has a profound impact on the interaction of blood cells such as platelets with blood vessel walls, and the disturbed flow with a low wall shear rate can enhance the deposits of platelet thrombi to the vessel wall.     

晚期头颈恶性肿瘤组织含促血小板聚集物

为探讨头颈恶性肿瘤组织提取液是否含有聚集血小板的物质，将15例头颈恶性肿瘤和14例非恶性肿瘤组织匀浆取组织提取液，分别致聚于头颈恶性肿瘤21例。非肿瘤，非恶性肿瘤33例及健康人16例的血小板，发现，头颈恶性肿瘤组织提取液致聚于头颈恶性肿瘤21例，非肿瘤，非恶性肿瘤33例及健康人16例血小板，发现，头颈恶性肿瘤组织提取液致聚的阳性率比非恶性头颈肿瘤组织提取液致聚的高；头颈恶性肿瘤组织提取液对血小板的聚集作用随病期进展而增强。头颈部恶性肿瘤病人的血小板易受组织提取液激素，其血小板最大聚集率随病期进展的增强，表明，晚期头颈恶性肿瘤组织含促血小板聚集物。这可能是直接引起恶性肿瘤机体血小板聚集增强的原因之一。     

去氢紫堇碱对兔血小板cAMP质量浓度的影响

目的 研究去氢紫堇碱（DHC）对兔血小板cAMP质量浓度的影响，探讨其抑制血小板聚集作用机制。方法 应用放射免疫分析法观察DHC对体外血小板cAMP质量浓度的影响。结果 与对照组相比，DHC显著增加兔血小板中cAMP的质量浓度。结论 DHC可能通过增加血小板中cAMP的质量浓度而且抑制血小板聚集。     

Activation of sodium-proton exchange is a prerequisite for Ca2+ mobilization in human platelets

Stimulated platelets take up sodium ions and release hydrogen ions due to activation of Na+/H+ exchange resulting in cytoplasmic alkalinization. Suppression of Na+/H+ exchange either by removal of extracellular Na+ or by application of amiloride inhibits shape change, secretion of granule contents and aggregation. The data we present here indicate that inhibition of this transport by ethylisopropyl-amiloride or by lowering extracellular sodium reduces or even completely suppresses the rise in cytoplasmic free Ca2+ concentration that is essential for platelet aggregation in response to thrombin. We also demonstrate that cytoplasmic alkalinization produced by exposure to the ionophore monensin sensitizes the human platelet response to stimulation by thrombin resulting in enhanced Ca2+ mobilization and aggregability. We conclude that an increase in intracellular pH evoked by activation of Na+/H+ counter transport is an important signal in stimulus-response coupling and forms an essential step in the cascade of events required to increase cytoplasmic free Ca2+ in platelets.     

芘荧光探针跟踪纳米复合水凝胶的形成过程

采用阳离子型芘荧光探针芘甲胺基盐酸盐跟踪聚N-异丙基丙烯酰胺(PNIPAm)/锂藻土Laponite原位聚合制备纳米复合水凝胶的过程.首先通过提高Laponite分散液的离子强度,采用离心分离和紫外-可见光吸收光谱验证了荧光探针在Laponite片层上的吸附;然后通过跟踪吸附荧光探针的激基缔合物的荧光光谱,发现反应液在聚合反应开始20min后出现的乳白色不透明现象与体系中形成的PNIPAm链的不均匀分布相关.聚合生成的高分子链吸附在Laponite片层周围,占据了片层上一定的空间,使得吸附在Laponite片层上的荧光分子被挤压而靠近,造成荧光激基缔合物的出现,因此可以通过荧光光谱变化来推测纳米复合(NC)凝胶聚合过程中透光率的变化及生成的聚合物链与交联剂Laponite之间的相互作用,这为深入认识NC凝胶的交联结构提供了一种实验方法.     

Cloning and expression of cDNA for a human thromboxane A2 receptor.

Thromboxane A2 is a very unstable arachidonate metabolite, yet a potent stimulator of platelet aggregation and a constrictor of vascular and respiratory smooth muscles. It has been implicated as a mediator in diseases such as myocardial infarction, stroke and bronchial asthma. Using a stable analogue of this compound we recently purified the human platelet thromboxane A2 receptor to apparent homogeneity. Using an oligonucleotide probe corresponding to its partial amino-acid sequence, we have obtained a complementary DNA clone encoding this receptor from human placenta and a partial clone from cultured human megakaryocytic leukaemia cells. The placenta cDNA encodes a protein of 343 amino acids with seven putative transmembrane domains. The protein expressed in COS-7 cells binds drugs with affinities identical to those of the platelet receptor, and that in Xenopus oocytes opens Ca2(+)-activated Cl- channel on agonist stimulation. Northern blot analysis and nucleotide sequences of the two clones suggest that an identical species of the thromboxane A2 receptor is present in platelets and vascular tissues. This first report on the molecular structure of an eicosanoid receptor will promote the molecular pharmacology and pathophysiology of these bioactive compounds.     

A discrete sequence in a platelet integrin is involved in ligand recognition

Platelet membrane glycoprotein IIb-IIIa (gpIIb-IIIa; alpha IIb-beta 3), the most prominent member of the integrin family of adhesion receptors on these cells, mediates platelet aggregation by binding fibrinogen and is critical in thrombosis and haemostasis. A short amino-acid sequence at the carboxy terminus of the gamma chain of fibrinogen is recognized by gpIIb-IIIa and peptides containing this sequence are selectively crosslinked to residues 294-314 of gpIIb. Here we show that an 11-residue peptide from this region of gpIIb inhibits platelet aggregation and binding of fibrinogen to platelets and to purified gpIIb-IIIa, and that it interacts directly with fibrinogen. These results implicate this segment of gpIIb-IIIa in the ligand-binding function of the receptor. Moreover, as this region is highly conserved among integrins, it may have a general function in ligand recognition by this broadly distributed family of adhesion receptors.     

Diacylglycerol in large alpha-actinin/actin complexes and in the cytoskeleton of activated platelets

The interaction of the cytoskeleton with plasma membranes may be mediated by vinculin, alpha-actinin and other proteins; alpha-actinin can interact specifically with model membranes only if they contain diacylglycerol and palmitic acid. On stimulation of platelets by thrombin, which leads to a reorganization of the cytoskeleton, diacylglycerol is produced rapidly, simultaneously with the disappearance of phosphatidylinositol. One important function of the diacylglycerol produced in platelets may be the activation of the Ca2+-and phospholipid-dependent protein kinase C. We show here that, in the presence of diacylglycerol and palmitic acid, a supramolecular complex between alpha-actinin and actin is formed in vitro. In the electron microscope, this complex displays substructures similar to those of microfilament bundles in vivo. Furthermore, such alpha-actinin/lipid complexes can also be formed in situ during the stimulation of blood platelet aggregation. Thus, alpha-actinin may be one of the proteins directly involved in structures connecting the cytoskeleton to cell membranes.     

Optimization study on the rehydration process of lyophilized human platelets

Long-term preservation of human platelets will greatly reduce the risk of their shortage. Lyophilization has been proved feasible for this purpose. For the recovery of lyophilized platelets, rehydration is an important process. In this paper, the rehydration processes for 1 mL and 2 mL samples were studied. The effects of prehydration duration (15, 30, 60, 90, 120 and 150 min) in 37°C water vapor and the concentration of rehydration solution (25%, 50%, 75%, 100% platelet-poor plasma) on the recovery rate, MPV (mean platelet volume) and PDW (platelet distribution width) were investigated. The mass changes during the prehydration process were weighed. The optimized rehydration conditions are as follows: (1) for 1 mL sample, the prehydration duration was 15 min and for 2 mL sample the prehydration duration was 90 min; (2) the rehydration solution was 75% platelet-poor plasma. Under optimized conditions, the morphology of the rehydrated platelets kept normal and their ultrastructures kept intact, their aggregation capacity to thrombin (1 U/mL) was 82.8% of the fresh ones. These results will be helpful for designing the freeze-drying protocols for human platelets.     

斑马鱼血栓模型在中药活性成分筛选中的适用性

为了考察斑马鱼血栓生成模型在中药成分活性筛选中的适应性,利用体外血小板聚集实验和体内斑马鱼血栓形成实验,分别检测中药单体对血小板聚集和血栓形成的影响。筛选模型选择3~5 d的野生型AB系斑马鱼以及转基因TG系（integrin αIIb: EGFP）血小板标记绿色荧光斑马鱼,利用三氯化铁诱导血栓形成,以凝血时间为指标,用20种中药单体成分处理斑马鱼,考察对斑马鱼血栓形成的影响。同时,采集大鼠全血制备富血小板血浆和贫血小板血浆,利用血小板聚集仪测定血小板聚集率,检测中药单体成分活性。结果显示：有7种中药单体成分具有抑制斑马鱼血栓形成活性,首次发现桃叶珊瑚苷具有明显的抑制斑马鱼血栓形成作用;体外血小板聚集实验结果显示有12种中药材成分具有抑制血小板聚集的作用,其中100 μg/mL 红景天苷的血小板聚集抑制率88.22%;利用斑马鱼血栓模型检测出的7种中药单体与体外血小板聚集实验结果一致。斑马鱼血栓生成模型对中药抗血栓活性筛选具有良好的适应性。