红花黄色素抑制血小板激活因子诱导的内皮细胞炎性因子蛋白表达升高作用的研究

目的:观察红花黄色素(SY)抑制血小板激活因子(PAF)诱发的内皮细胞炎性因子蛋白表达升高及抑制PAF与其受体特异性结合的作用。方法:用酶联免疫吸附测定法(ELISA)检测SY缓解1μmol/L PAF刺激脐静脉内皮细胞株Eahy926培养液中的炎症因子白细胞介素(IL)-1β、IL-6和肿瘤坏死因子(TNF)-α的蛋白表达水平的变化;以放射配基结合试验观察SY抑制[3H]标记的PAF与家兔血小板膜和膜蛋白PAF受体特异性结合的作用。结果:与正常组比较,PAF损伤组内皮细胞培养上清液中IL-1β、IL-6及TNF-α蛋白表达水平明显升高(P0.001),SY处理组(浓度为4.5×10-5,4.5×10-4,4.5×10-3g/L)时可明显抑制Eahy926培养液中IL-1β、IL-6及TNF-α蛋白表达水平的升高(P0.05,0.01,0.001);受体结合实验结果表明浓度为16.49g/L的SY可抑制0.17 nmol/L[3H]PAF与家兔血小板膜的特异性结合,浓度为11.17和22.08g/L的SY分别可抑制0.083及0.25nmol/L[3H]PAF与兔血小板膜蛋白的特异性结合。结论:SY通过抑制PAF与其受体的特异性结合缓解了PAF诱发的血管内皮细胞炎症介质IL-1β、IL-6及TNF-α蛋白的高表达。     

血小板活化因子致豚鼠气道高反应性作用机制的研究

为探讨血小板活化因子 (PAF)导致气道高反应性的作用机制 ,我们建立PAF导致的气道高反应性的豚鼠模型 ,来研究非肽类长效神经激肽 1(NK1)和NK2 受体拮抗剂SR140 333和SR4896 8,分别在PAF导致气道高反应性中的作用 ,结果 :吸入PAF组组胺的PC2 0 值明显小于对照组 (P <0 .0 1) ,表明吸入PAF后可导致气道高反应性 ;辣椒素预处理组组胺的PC2 0 值与对照组相比无显著性差异 (P >0 .0 5 ) ,表明辣椒素预处理可完全抑制PAF导致气道高反应性 ;NK1受体拮抗剂组组胺的PC2 0 值与吸入PAF组相比无显著性差异 (P >0 .0 5 ) ,而NK2 受体拮抗剂组组胺的PC2 0 值明显大于吸入PAF组 (P <0 .0 1) ,表明NK2 受体拮抗剂能完全抑制PAF导致气道高反应性。结果 :速激肽的释放是PAF导致气道高反应性的关键步骤 ,而且NK2 受体的激活在PAF导致的气道高反应性中起主要作用     

血小板活化因子拮抗剂对抗血小板活化研究

目的：用全血流式细胞术方法,研究血小板活化因子（PAF）激活血小板的病理-生理过程,为PAF特异性拮抗剂的临床应用,提供理论依据。方法：将PAF受体拮抗剂银杏内酯B（GB）加入全血中,以阻断PAF对血小板的活化作用;用PAF及二磷酸腺苷（ADP）分别加入全血中,激活血小板,流式细胞术（FCM）检测活化后血小板P-选择素（CD62P）的表达。结果：①PAF组、ADP组和对照组CD62P百分率分别为76.08&#177;7.36、64.74&#177;9.92、24.92&#177;4.49,各组之间差异有统计学意义（P〈0.01）。②PAF组、PAF加GB组和对照组CD62P百分率分别为76.08&#177;7.36、57.83&#177;7.04、24.92&#177;4.49,各组之间差异有统计学意义（P〈0.01）。③ADP组、ADP加GB组和对照组CD62P百分率分别为64.74&#177;9.92、52.67&#177;8.93、24.92&#177;4.49,各组之间差异有统计学意义（P〈0.01）。结论：①PAF及ADP均增加CD62P的表达,但PAF较ADP有更强的血小板活化作用。②GB明显抑制PAF诱导的CD62P的表达,但不能完全抑制血小板的活化,表明许多细胞因子及递质参与其中。③PAF受体拮抗剂（GB）也抑制ADP诱导的CD62P的表达,但不能完全抑制血小板的活化,表明PAF的自分泌作用及多种细胞之间的相互作用。④GB有明显拮抗PAF的作用,在血体形成具有广泛的应用前景。     

血小板糖蛋白Ⅱ_b/Ⅲ_a受体拮抗剂研究现状

糖蛋白Ⅱｂ/Ⅲａ(ＧＰⅡｂ/Ⅲａ)受体拮抗剂的药理作用是对抗ＧＰⅡｂ/Ⅲａ 受体与黏附分子 [纤维蛋白原、ＶｏｎＷｌｌｅ ｂｒａｎｄ因子 (ｖＷＦ)等 ]结合 ,抑制血小板聚集。目前批准用于临床的已有 3种静脉用ＧＰⅡｂ/Ⅲａ 受体拮抗剂 ,现对几种药物的作用机制、临床疗效和安全性综述如下。1　ＧＰⅡｂ/Ⅲａ 受体拮抗剂作用机制1.1　血小板黏附和聚集的模式　正常血管内皮下存在黏附糖蛋白 ,内皮将其与循环血小板隔开。当血管内皮受损或动脉粥样硬化斑块破裂 ,导致黏附糖蛋白 (如ｖＷＦ)和胶原的暴露 ,血小板对这些糖蛋白有许多受体 ,如…     

心力衰竭患者血小板活化因子及其受体表达的变化

目的 :探讨心力衰竭患者血浆血小板活化因子 (PAF)、血小板活化因子乙酰水解酶 (PAF AH)活性的变化 ,以及外周血中单个核细胞 (PBMC)PAF受体 (PAF R)的表达。方法 :对 30例心力衰竭患者采用生物法检测血浆中PAF含量 ,酶水解底物显色法测定PAF AH活性 ,流式细胞仪检测PBMC上PAF R的表达。并与 30例健康体检者对照。结果 :心力衰竭患者血浆中PAF含量 (11.12± 1.80 ) μg L ,PAF AH活性 (2 4 .5 0± 7.86 ) μmol (min·L)、PBMC上PAF R的表达与健康体检者比较差异有非常显著性意义 (P <0 .0 1)。结论 :PAF、PAF AH、PAF R三者在心力衰竭发生和发展中起重要作用     

红花黄色素抑制血小板聚集和缓解心肌细胞缺氧缺糖损伤的作用

目的:本文以整体动物实验,观察红花黄色素(SY)抑制二磷酸腺苷(ADP)诱发的家兔血小板聚集,及降低血小板激活因子(PAF)致小鼠死亡的作用;体外细胞模型实验,观察SY缓解乳鼠心肌细胞缺氧缺糖损伤的作用。方法:家兔耳缘静脉注射SY,给药前后各取动脉血,比浊法测定ADP诱发的血小板聚集率;小鼠腹腔注射PAF和SY,观察SY降低小鼠死亡的药效;体外培养乳鼠心肌细胞,以通入高纯氮气造成心肌细胞缺氧缺糖损伤模型,Fura-2/AM荧光光度法测定胞内游离Ca2+浓度、NAD动力学法,检测心肌细胞上清液LDH活性以反映细胞损伤的程度。结果:SY整体给药可明显抑制ADP诱发的家兔血小板聚集(与对照组比较P<0.05)、降低PAF致小鼠中毒的死亡(与PAF组比较P<0.01);SY还可降低乳鼠心肌细胞缺氧缺糖损伤时胞内Ca2+浓度的升高(与损伤组比较P<0.01),并可缓解心肌细胞缺氧缺糖损伤时LDH的漏出。结论:SY有抗血小板聚集、缓解心肌缺血损伤的作用。     

血小板膜糖蛋白GPIb受体拮抗剂的研究进展

血小板膜糖蛋白GPIb受体与血管性血友因子(vWF)相互作用是血小板在血管损伤部位发生黏附和动脉血栓形成的始动环节,同时也是高剪切力下血小板在血管病理性狭窄部位发生黏附和聚集的重要机制,因此,GPIb受体被认为是抗血栓治疗的重要靶点.GPIb受体拮抗剂通过阻GPIb复合物与vWF相互作用而抑制血小板黏附和聚集,达到抗血小板血栓形成的目的,对于心、脑血管疾病防治有着重要的意义.本文综述当前一些GPIb受体拮抗剂研究进展,将要探讨的有:6B4-Fab、SZ2,属于抗GPIbα鼠源单克隆抗体;RAM.1,属于抗GPIbβ的鼠源单克隆抗体;Agkisacucetin、Jerdonibitin、Rhodocetin-αβ,均属蛇毒蛋白.     

血小板糖蛋白Ⅱb／Ⅲa受体拮抗剂在直接经皮冠状动脉介入治疗中的应用

斑块破裂导致血栓形成过程中,血小板首先在血管壁损伤部位黏附、激活,随后通过纤维蛋白原与血小板糖蛋白（GP）Ⅱb／Ⅲa受体结合,使相邻的血小板连在一起,这是血小板聚集的共同最后通路。血小板GPⅡb／Ⅲa受体拮抗剂通过阻断纤维蛋白原与GPⅡb／Ⅲa受体结合,抑制血小板聚集,被认为是目前最强的抗血小板聚集药物。     

血小板糖蛋白Ⅱb/Ⅲa受体拮抗剂在急性冠脉综合征应用研究的进展

血小板糖蛋白Ⅱb/Ⅲa受体拮抗剂(GPI)通过占据GP Ⅱb/Ⅲa受体的结合位点,阻碍纤维蛋白原与其结合,进而抑制血小板聚集,是迄今最强、最具应用价值的抗血小板药物。本文综述其在ACS应用的安全性和有效性研究进展。     

松胞菌素D对血小板聚集和血小板—中性粒细胞间相互作用的影响

目的探讨松胞菌素D对血小板聚集及血小板—中性粒细胞间相互作用的影响。方法应用玫瑰花结试验和Born方法观察松胞菌素D对血小板—中性粒细胞之间相互作用及体内外对血小板聚集功能的影响。结果松胞菌素D在体内外对花生四烯酸（AA）、血小板活化因子（PAF）诱导的血小板聚集无明显抑制作用,而对腺苷二磷酸（ADP）诱导的血小板聚集则有明显的抑制作用;松胞菌素D可以阻抑血小板与中性粒细胞间的黏附作用;松胞菌素D明显抑制PAF（1μmol/L）激活的中性粒细胞悬液引起的洗涤血小板聚集。结论松胞菌素D在体内外均具有选择性抑制ADP诱导的血小板聚集作用,且明显阻抑血小板与中性粒细胞间的相互作用。     

Synthesis of platelet-activating factor and its receptor expression in Kupffer cells in rat carbon tetrachloride-induced cirrhosis

AIM： To determine the platelet-activating factor （PAF） synthesis and its receptor expression in Kupffer cells in rat carbon tetrachloride-induced cirrhosis.
METHODS： Kupffer cells, isolated from the livers of control and CCl4-induced cirrhotic rats, were placed in serum-free medium overnight. PAF saturation binding, ET-1 saturation and competition binding were assayed. ET-1 induced PAF synthesis, mRNA expression of PAF, preproendothelin-1, endothelin A （ETA） and endothelin B （ETB） receptors were also determined.
RESULTS： A two-fold increase of PAF synthesis （1.42 ± 0.14 vs 0.66 ± 0.04 pg/μg DNA） and a 1.48-fold increase of membrane-bound PAF （1.02 ± 0.06 vs 0.69 ± 0.07 pg/μg DNA） were observed in activated Kupffer cells of cirrhotic rats. The application of ET-1 to Kupffer cells induced PAF synthesis in a concentration-dependent manner in both cirrhotic and normal rats via ETB receptor, but PAF synthesis in the activated Kupffer cells was more effective than that in the normal Kupffer cells. In activated Kupffer cells, PAF receptor expression and PAF binding capacity were markedly enhanced. Activated Kupffer cells raised the [^125I]-ET-1 binding capacity, but changed neither the affinity of the receptors, nor the expression of ETA receptor.
CONCLUSION： Kupffer cells in the course of CCl4- induced cirrhosis are the main source of increased PAF. ET-1 is involved endogenously in stimulating the PAF synthesis in activated Kupffer cells via ETB receptor by paracrine. ETA receptor did not appear in activated Kupffer cells, which may exacerbate the hepatic and extrahepatic complications of cirrhosis.     

Effect of increased hepatic platelet activating factor and its receptor portal hypertension in CCl4-induced liver cirrhosis

AIM: To evaluate the changes in hepatic platelet activating factor (PAF) and its receptors and their effect on portal pressure of cirrhotic rats induced by CCl4. METHODS: A model of liver cirrhosis was replicated in rats by intra-peritoneal injection of CCl4 for 8 wk. We determined the effect of hepatic PAF and its receptor level on portal and arterial pressure by EIA, saturation binding and RT-PCR technique. RESULTS: Compared to control rats, cirrhotic rats had higher hepatic PAF levels and output as well as higher plasma PAF levels (P<0.01, P<0.01, P<0.05, respectively). Both hepatic PAF receptor mRNA levels and PAF binding were nearly 3-fold greater in cirrhotic rats (P<0.01). Portal injection of PAF (1 g/kg WT) increased the portal pressure by 22% and 33% in control and cirrhotic rats, respectively. In contrast, the arterial pressure was decreased in the both groups (54% in control rats and 42% in cirrhotic rats). Injection of the PAF antagonist BN52021 (5 mg/kg WT) decreased the portal pressure by 16% in cirrhotic rats but had no effect in the control rats. CONCLUSION: The upregulation of the PAF system contributes to hepatic hemodynamic and metabolic abnormalities in cirrhosis, and the increased release of PAF into the circulation has impacts on the systemic hemodynamics.     

Thromboxane A2/prostaglandin endoperoxide (TXA2/PG-END) receptor binding properties in human platelets of ridogrel, a combined TXA2 synthase inhibitor--TXA2/PG-END receptor antagonist.

Ridogrel, a potent thromboxane A2 (TXA2) synthase inhibitor, also has thromboxane A2 prostaglandin endoperoxide (TXA2/PG-END) receptor antagonistic properties as documented in functional studies of human platelets. In the present study, the binding affinities of the TXA2 synthase inhibitors, ridogrel, dazoxiben, dazmegrel and pirmagrel, and the TXA2/PG-END receptor antagonists, GR32191, L670596, SQ29548, ICI159995, AH69212 and sulotroban, for the TXA2/PG-END receptor labelled with [3H]SQ29548 on intact human platelets were assessed. The potencies of the TXA2/PG-END receptor antagonists to inhibit specific [3H]SQ29548 binding to intact human platelets ranged between 1.2 nM and 6,200 nM and corresponded to the ability of the drugs to suppress human platelet aggregation induced by TXA2/PG-END receptor stimulation with U46619 and collagen. The TXA2 synthase inhibitors dazoxiben, dazmegrel and pirmagrel could not inhibit specific [3H]SQ29548 binding to intact human platelets, tested up to 10(-5) M, nor suppress human platelet aggregation, indicating lack of any receptor antagonistic properties. Ridogrel, however, directly bound to the TXA2/PG-END receptor with micromolar affinity (IC50 = 5.2 microM) and inhibited U46619-27, or collagen-induced platelet aggregation, with ED50-values of 27 microM and 4.7 microM respectively. The present study thus demonstrates that antagonism by ridogrel of TXA2/PG-END receptor activation on platelets as defined in functional tests, coincides with inhibition of specific ligand binding to the receptors.     

Induction of functional lipoxin A4 receptors in HL-60 cells

The appearance of [11,12-3H]lipoxin A4 (LXA4) specific binding sites was examined with human acute promyelocytic leukemic cell line 60 (HL- 60) cells exposed to either retinoic acid, phorbol 12-myristate 13- acetate (PMA), or dimethyl sulfoxide (DMSO). All three agents induced a threefold to fivefold increase in the expression of specific [11,12- 3H]LXA4 binding. Similar results were obtained in parallel with [14,15- 3H]leukotriene (LT) B4. For both 3H-ligands, homologous displacement curves were similar and independent of the agent used to induce differentiation. Specific binding of [11,12-3H]LXA4 to differentiated HL-60 cells gave a kd = 0.6 +/- 0.3 nmol/L. The appearance of both [11,12-3H]LXA4 and [14,15-3H]LTB4-specific binding sites was inhibited by actinomycin D, and LXA4 binding was sensitive to protease treatment. Specific binding of [11,12-3H]LXA4 was not evident with human platelets, red blood cells (RBCs) or the cultured B-cell (Raji), T-cell (Jurkat) lines save human endothelial cells (kd = 11.0 +/- 0.3 nmol/L). The structural specificity of induced [11,12-3H]-LXA4 recognition sites was assessed with LXB4, LTC4, LTB4, and trihydroxyhepatanoic methyl ester. Only LTC4, at 3-log molar excess, competed for 3H-LXA4-specific binding with HL-60 cells and gave a 30% reduction. The leukotriene D4 receptor antagonist SKF 104353 was ineffective in blocking [11,12- 3H]LXA4-specific binding with HL-60 cells while it competed for specific [11,12-3H]LXA4 binding with endothelial cells where LTD4 binding appears to be virtually identical to that of LXA4 binding. In addition, the LTB4 receptor antagonist ONO 4057 was ineffective at competing for [11,12-3H]LXA4 binding. When phospholipase D activation was monitored in human polymorphonuclear leukocytes (PMN) and HL-60 cells, a correlation was shown between activation and specific 3H-LXA4 binding. LXA4-induced phospholipase D (PLD) activation gave a biphasic concentration-dependent response comprised of at least two components: one phase being islet-activating protein (IAP)-sensitive (LXA4 10(-9) mol/L peak activity) and the other was staurosporine-sensitive (LXA4 10(-7) mol/L peak activity). Results indicate that HL-60 cells exposed to differentiating agents express [11,12-3H]LXA4 recognition sites also present in PMN. In addition, specific LXA4 recognition sites of myeloid cells can be distinguished by competition binding with SKF 104353 and 3H-LXA4 cross-reactivity with putative LTD4 receptors present on human endothelial cells. Moreover, they provide evidence indicating that binding of LXA4 to its recognition sites confers functional responses.     

Ligand-specific glucocorticoid receptor activation in human platelets

Few studies have addressed the effects of classical anti-inflammatory glucocorticoids on platelet function. Here, we report for the first time that human platelets contain the glucocorticoid receptor (GR) as identified by a combination of biochemical and functional techniques. Ligand-binding studies revealed the presence of a high- and low-affinity binding site for [3H]-dexamethasone in platelets. The 2 GR ligands prednisolone and dexamethasone competed for [3H]-dexamethasone binding, as did the mineralocorticoid aldosterone. However, while prednisolone (1-10 microM) reduced adenosine diphosphate (ADP, 4 microM) and thromboxane A2 receptor agonist U46619 induced platelet aggregation (up to 75%), dexamethasone had no effect. The inhibition produced by prednisolone was reversed by preincubation with the GR antagonist mifepristone (10 microM; RU486), suggesting the functional importance of the ligand-receptor complex. In addition, prednisolone caused a marked (approximately 50%) reduction in thromboxane B2 levels, whereas dexamethasone was without effect. The apparently anomalous binding data were clarified by the fact that washed platelets (1) contained mineralocorticoid receptor and that (2) it was associated with GR. Taken together, our data suggest that platelet GR forms a heterodimeric complex with the mineralocorticoid receptor that is susceptible to differential activation by specific receptor ligands.     

Lipoxin A4 induces hyperadhesiveness in human endothelial cells for neutrophils

Lipoxin A4 (LXA4), but not lipoxin B4, induced in vitro a dose- dependent, slowly emerging hyperadhesiveness in human umbilical vein endothelial cells (HUVEC), leading to a 1.9-fold increase in the binding of neutrophils (polymorphonuclear neutrophil granulocytes [PMN]). The maximal response to LXA4 occurred at 1 nmol/L and after 30 minutes of treatment of HUVEC. These response kinetics were intermediate in comparison with those of fast-acting inducers of HUVEC adhesivity (eg, thrombin, leukotriene B4 [LTB4] or platelet activating factor [PAF]), needing 5 to 15 minutes, or to the slow inducer interleukin-1 (IL-1 beta), which requires hours. The maximal LXA4 effect was slightly lower than that of LTB4 (100 nmol/L) and thrombin (1 U/mL), and less than that of PAF (100 nmol/L) or IL-1 beta (2.5 U/mL) (2.2-, 2.0-, 2.4-, or 13.6-fold increases, respectively). The LXA4 effect was inhibited by the PAF receptor antagonist WEB-2086; however, it could not be blocked by pertussis toxin. LXA4 conferred a slow, sustained increase in HUVEC cytosolic calcium ion concentrations, whereas thrombin did so rapidly and transiently. LXA4 also caused PMN to become hyperadhesive. Thus, this novel effect of LXA4 on HUVEC appears to be associated with endogenous PAF expression and slow increases of cytosolic calcium concentrations but not pertussis- sensitive G proteins.     

The influence of platelet activating factor on the effects of platelet agonists and antiplatelet agents in vitro

We assessed the effect of the intercellular mediator of inflammation, platelet activating factor (PAF), on platelet function. The interaction between PAF and the platelet agonists ADP, thrombin and convulxin was analyzed in vitro in whole blood with the use of flow cytometry and was further characterized with the use of receptor antagonists to PAF (ABT-491), P2Y1 (MRS-2179), and P2Y12 (cangrelor) as well as a monoclonal anti-PSGL-1 antibody (anti-CD162). Low concentrations of PAF (0.1 nM) synergistically augmented platelet activation induced by other agonists (P < 0.01). Augmentation by PAF was receptor mediated and did not require platelet–leukocyte interaction. With >99% inhibition of P2Y receptor-mediated platelet activation, greater than additive activation was still observed with the combination of ADP plus PAF. Accordingly, PAF synergistically augments platelet activation in response to ADP and thrombin, and the extent of inhibition exerted by P2Y receptor antagonists is decreased in the presence of PAF.     

Characterization of the vascular thromboxane A2/prostaglandin endoperoxide receptor in rabbit aorta. Regulation by dexamethasone

Recently, we have shown that dexamethasone treatment of rabbits specifically reduces vascular smooth muscle responsiveness to agonists that interact with the vascular thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor. One potential site at which dexamethasone can influence prostanoid-mediated vasoconstriction may be at the level of the vascular TXA2/PGH2 receptor. Therefore, we characterized the vascular TXA2/PGH2 receptor in rabbit aortic membranes and examined the influence of dexamethasone treatment on vascular TXA2/PGH2 receptor affinity and number. The binding of [125I][1S-(1 alpha,2 beta(5Z),3 alpha(1E,3R)4 alpha)]-7-[3-(3- hydroxy-4-(p-iodophenoxy)-1-butenyl)-7-oxabicyclo[2.2.1] heptan-2-yl]-5-heptanoic acid ([125I]BOP), a potent TXA2/PGH2 receptor agonist, to rabbit aortic membranes was saturable, displaceable, and dependent on protein concentration. Scatchard analysis of equilibrium binding data disclosed one class of high affinity binding sites with a Kd of 0.44 +/- 0.13 nM and a Bmax of 114.4 +/- 5.2 fmol/mg protein (n = 7). Removal of the endothelium before membrane preparation did not significantly alter the affinity or number of binding sites for [125I]BOP. Kinetic analysis of the rates of [125I]BOP association/dissociation yielded a Kd of 0.62 nM. The ability of various agonists at the TXA2/PGH2 receptor to displace [125I]BOP from vascular membranes correlated well with their contractile potencies in rabbit aortic rings. Moreover, stereospecific displacement of [125I]BOP binding in aortic membranes and inhibition of U46619-mediated aortic contractions were obtained with the stereoisomers L657925(-) and L657926(+). Collectively, these data suggest that this binding site represents the functionally relevant vascular TXA2/PGH2 receptor. In functional experiments, [127I]BOP induced concentration-dependent contractions of the rabbit aorta, which were reduced by 52% in vessels from dexamethasone-treated rabbits.(ABSTRACT TRUNCATED AT 250 WORDS)