磷脂酰肌醇3激酶通过mDial参与调控凝血酶诱导的血小板聚集

目的 探讨mDial(mammalian diaphanous 1)在人血小板中的表达和血小板聚集过程中的作用以及磷脂酰肌醇3激酶(PI3K)对该过程的调控作用.方法 采用血小板聚集仪检测PI3K抑制剂和抗mDial抗体导入后对人血小板聚集率的影响;Western blot法检测mDial在血小板静止及活化过程中的表达及其与肌动蛋白细胞骨架的关系.结果 mDial在血小板静止、凝血酶诱导的铺展或聚集血小板内表达水平没有明显差异;凝血酶诱导血小板聚集过程中,mDial从Triton-X100可溶性(胞质)部分向Triton-X100不可溶性(细胞骨架)部分转位;抗mDial抗体导入血小板后能够抑制凝血酶诱导的血小板聚集;PI3K抑制剂渥曼青霉素及Ly294002能够抑制血小板聚集,抑制mDial从Triton-X100可溶性部分向Triton-X100不可溶性部分的转位.结论 PI3K通过mDial参与调控凝血酶诱导的血小板聚集过程中肌动蛋白细胞骨架的重构.     

Metabolism of bradykinin In vivo in humans: identification of BK1-5 as a stable plasma peptide metabolite

Studies investigating the role of bradykinin in disease states such as hypertension, sepsis, and asthma have been confounded by difficulties in measuring the concentration of this short-lived peptide. The purpose of this study was to determine a stable metabolite of bradykinin in the systemic circulation of humans. Bradykinin (containing trace concentrations of [(3)H]bradykinin) was administered i.v. into three human volunteers in increasing amounts up to a maintenance rate of 200 ng/kg/min until a total dose of 1 mg was given. Metabolic products were purified and identified by HPLC and by electrospray ionization mass spectrometry. Infused bradykinin was rapidly degraded, such that no exogenous bradykinin was detected in venous plasma sampled during infusion. BK1-5 (Arg-Pro-Pro-Gly-Phe), the 1-to-5 amino acid fragment of bradykinin, was identified as a major stable plasma metabolite of bradykinin. Plasma concentrations of BK1-5 correlated with dose of bradykinin infused and concentrations at the end of bradykinin infusion were 1510 to 4600 fmol/ml of blood. BK1-5 was cleared from blood with a terminal half-life of 86 to 101 min. Thus, in humans, bradykinin is rapidly degraded in vivo to BK1-5, a stable metabolite. Measurement of this metabolite could provide a tool to assess pathophysiologic and pharmacologic alterations in systemic bradykinin generation associated with human disease.     

Ingestion of quercetin inhibits platelet aggregation and essential components of the collagen-stimulated platelet activation pathway in humans

BACKGROUND: Quercetin, a flavonoid present in the human diet, which is found in high levels in onions, apples, tea and wine, has been shown previously to inhibit platelet aggregation and signaling in vitro. Consequently, it has been proposed that quercetin may contribute to the protective effects against cardiovascular disease of a diet rich in fruit and vegetables. OBJECTIVES: A pilot human dietary intervention study was designed to investigate the relationship between the ingestion of dietary quercetin and platelet function. METHODS: Human subjects ingested either 150 mg or 300 mg quercetin-4'-O-beta-D-glucoside supplement to determine the systemic availability of quercetin. Platelets were isolated from subjects to analyse collagen-stimulated cell signaling and aggregation. RESULTS: Plasma quercetin concentrations peaked at 4.66 microm (+/- 0.77) and 9.72 microm (+/- 1.38) 30 min after ingestion of 150-mg and 300-mg doses of quercetin-4'-O-beta-D-glucoside, respectively, demonstrating that quercetin was bioavailable, with plasma concentrations attained in the range known to affect platelet function in vitro. Platelet aggregation was inhibited 30 and 120 min after ingestion of both doses of quercetin-4'-O-beta-D-glucoside. Correspondingly, collagen-stimulated tyrosine phosphorylation of total platelet proteins was inhibited. This was accompanied by reduced tyrosine phosphorylation of the tyrosine kinase Syk and phospholipase Cgamma2, components of the platelet glycoprotein VI collagen receptor signaling pathway. CONCLUSIONS: This study provides new evidence of the relatively high systemic availability of quercetin in the form of quercetin-4'-O-beta-D-glucoside by supplementation, and implicates quercetin as a dietary inhibitor of platelet cell signaling and thrombus formation.     

血小板聚集试验及其临床应用

目的探讨血小板聚集试验及其临床应用.方法以APACT2型血小板聚集仪用ADP为诱聚剂,探讨血小板聚集试验的影响因素,测定66例正常人及12例急性白血病患者的血小板聚集率,并观察阿斯匹林对血小板聚集试验的影响.用流式细胞术检测6例正常人及12例急性白血病患者的血小板膜糖蛋白Ⅱb/Ⅲa,比较血小板聚集试验与血小板膜糖蛋白Ⅱb/Ⅲa的结果.结果ADP应用的终浓度为5μgmol/L测定正常健康成人血小板聚集试验正常参考范围(χ±1.96s)为30.3%～86.3%.阿斯匹林对血小板聚集有抑制作用,与生理盐水对照组有显著差异(P＜0.02)急性白血病患者的血小板聚集率均值为5.4%,明显低于PRP为相同血小板浓度的正常人(28.7%)(P＜0.005).经等级相关分析血小板聚集试验与血小板膜糖蛋白Ⅱb/Ⅲa的检测有正相关(P＜0.0001).结论血小板聚集试验简便、实用可作为评价血小板聚集功能的初筛试验.     

Influence of platelet volume on the ability of prostacyclin to inhibit platelet aggregation and the release reaction

The influence of mean platelet volume (MPV) and platelet count on in vitro platelet sensitivity to prostacyclin (PGI2) was studied with human size-dependent platelet subpopulations prepared by counterflow centrifugation. The original unfractionated platelet suspension and each of five size-dependent platelet fractions were suspended in buffer at a platelet count of 2 X 10(8)/ml. The percent decrease in the extent of platelet aggregation and adenosine triphosphate (ATP) release in response to 10 micrograms/ml collagen was determined over a range of PGI2 concentrations in a Lumi-Aggregometer. A significant positive correlation between MPV and the concentration required to give 50% inhibition for both platelet aggregation and ATP release (r = 0.99, p less than 0.001 and r = 0.99, p less than 0.001, respectively) was observed. In separate experiments, the effect of platelet count on the ability of a given dose of PGI2 to inhibit platelet aggregation and ATP release was determined, and a significant inverse correlation was noted (r = 0.99, p less than 0.01 and r = 0.98, p less than 0.01, respectively). Our data indicate that the sensitivity of human platelets to the inhibitory effects of PGI2 is dependent on both the platelet volume and the platelet count. Thus, the presence of a greater platelet mass, resulting from either an increased MPV or an increased platelet count, decreases the inhibitory effectiveness of PGI2 on both platelet aggregation and the release reaction.     

部分蔬菜所含环氧化酶抑制物的抗血小板花生四烯酸代谢研究

为了研究部分蔬菜的环氧化酶抑制剂抗血小板花生四烯酸(arachidonic acid,AA)代谢的作用,将不同种类的蔬菜汁(以阿司匹林作为阳性对照)分别与正常人富血小板血浆(PRP)孵育,加入AA,观察各种蔬菜汁对血小板聚集的影响;血红素和环氧化酶-l(cyclooxygenase-I,COX1)或环氧化酶-2(cyclooxygenase-2,COX2)加入含有COX反应缓冲液的试管,漩涡混匀,与阿司匹林和各种蔬菜汁进行孵育,加入AA,然后利用盐酸终止反应,再加入氯化亚锡,通过酶免疫试剂盒测定COX抑制剂浓度;将不同种类的蔬菜汁(以阿司匹林作为阳性对照)分别与正常人全血进行孵育,加入AA进行反应,再加入消炎痛终止反应,离心,取上清,放射免疫法测定血浆血栓烷素B2(thromboxane B2,TXB2).结果表明,在AA诱导下,菠菜汁、蒜苔汁、蒜黄汁和韭菜汁与对照组相比对人血小板聚集的抑制显著,均在80％以上.4种蔬菜中均含有一定量的COX抑制物,其中菠菜的COX1和COX2抑制物浓度均高于阿司匹林;与生理盐水(对照组)比较,4种蔬菜汁均能显著降低AA诱导下人血浆TXB2的浓度(p＜0.05).结论:4种生蔬菜均含有COX抑制物,抑制了TXA2( thromboxane A2,TXA2)的产生,从而抑制了血小板聚集.因此,生菠菜、蒜黄、韭菜、蒜苔在体外具有显著抑制血小板聚集功能的作用,在抗血栓预防和治疗中可能有潜在的应用前景.     

部分蔬菜抗血小板聚集功能及其作用机制的初步研究

目的对部分具有抗血小板聚集功能蔬菜的作用机制进行初步探讨。方法将不同种类的蔬菜汁（以西红柿汁作为阳性对照）分别与正常人富血小板血浆（PRP）孵育，加入血小板聚集诱导剂，观察各种蔬菜汁对血小板聚集的影响；用诱导剂激活血小板，流式细胞仪分析蔬菜汁对活化血小板纤维蛋白原受体（Fib-R）和P-选择素（CD62P）的表达水平及血小板纤维蛋白原结合量的影响；利用动物试验对血小板抑制作用进行体内验证，普通白兔随机分为5组，分别定量饲以生理盐水、熟西红柿汁、蒜黄汁、大白菜汁及菠菜汁，观察不同时间后血小板聚集情况。结果在花生四烯酸（AA）诱导下，未发现熟蔬菜汁有明显抑制体外人血小板聚集的作用，但在二磷酸腺苷（ADP）、胶原或肾上腺素诱导下，熟蒜黄汁、大白菜汁、菠菜汁能够明显抑制血小板聚集，并且随着蔬菜汁浓度的增加，对血小板聚集的抑制率增高。熟蒜黄汁、大白菜汁、菠菜汁不能抑制ADP、胶原或肾上腺素诱导的血小板Fib-R、CD62P表达，但能够显著抑制由ADP或胶原诱导的血小板与纤维蛋白原结合量。普通白兔在饲以熟大白菜汁、蒜黄汁、菠菜汁后，ADP诱导的兔血小板聚集率分别在第3、5、8周末时开始降低，熟大白菜汁喂养组血小板聚集的抑制率在第3、5、8周末分别为：45．7％、53．6％、48．5％，熟蒜黄汁喂养组分别为：10．7％、66．7％、46．3％，熟菠菜汁喂养组分别为：8．7％、21．0％、42．6％；饲以熟大白菜汁、蒜黄汁后，胶原诱导的兔血小板聚集率分别在第5、8周末时开始降低，熟大白菜汁喂养组血小板聚集的抑制率在第5、8周末分别为：54．9％、56．3％，熟蒜黄汁喂养组分别为：28．4％、86．7％。在8周内，熟西红柿汁对兔血小板未产生抗聚集作用。结论在ADP或胶原诱导下，熟蒜黄汁、大白菜汁及菠菜汁能明显抑制活化血小板与纤维蛋白原的结合，能够显著抑制血小板的聚集，具有潜在的预防或治疗血栓性疾病的前景。     

西红柿汁体外抑制血小板聚集功能及其机制探讨

目的:观察西红柿汁在体外对血小板聚集、血小板活化标志物的表达及对血小板纤维蛋白原结合量的影响,初步探讨西红柿汁对血小板聚集功能的影响机制。方法:将不同浓度的西红柿汁与正常人富血小板血浆(PRP)孵育,加入5-腺苷二磷酸二钠盐(ADP)和胶原,观察西红柿汁对血小板聚集的影响。用ADP及胶原激活血小板,流式细胞仪(FCM)分析西红柿汁对活化血小板纤维蛋白原受体(Fib-R)和P-选择素(CD62P)的表达水平及血小板纤维蛋白原结合量的影响。结果:西红柿汁能够明显抑制由ADP、胶原诱导的血小板聚集,且随着西红柿汁浓度的增加,对血小板聚集的抑制率增高。西红柿汁不能抑制ADP、胶原诱导的血小板Fib-R、CD62P表达,但能显著抑制由ADP、胶原诱导的血小板与纤维蛋白原结合量。结论:西红柿汁在体外能够显著抑制血小板聚集,其抗血小板聚集的作用机制与阻碍纤维蛋白原与血小板Fib-R结合有关。西红柿汁具有潜在的抗血小板治疗前景。     

丁胺卡那霉素对EDTA依赖性凝集血小板的解离及其机制

目的研究丁胺卡那霉素对EDTA抗凝剂依赖的聚集血小板的解离作用和机制,为血常规标本中血小板凝聚提供可靠的解决方法。方法在EDTA依赖的假性血小板减少症(PTCP)患者的EDTA-K2抗凝血样本中,于不同时间段加入不同浓度丁胺卡那霉素进行凝集血小板的解离试验,通过血小板计数和涂片观察解离效果;用流式细胞仪检测血小板膜表面CD41、CD61、CD62p、PAC-1和IgG的表达百分率。结果抽血后1h内加入丁胺卡那霉素对血小板凝集的解离作用明显,血小板计数可恢复到即时检测的水平,血小板CD62p、PAC-1和IgG的表达量被显著抑制,而CD41和CD61未受明显影响。结论PTCP患者血常规样品抽血后1h内加入丁胺卡那霉素能有效解离凝集的血小板,作用机制可能与抑制患者血小板膜表面CD62p、PAC-1和IgG的表达有关;此法有助于解决EDTA所致的血小板计数的假性减少。     

New metabolic and pharmacokinetic characteristics of thiocolchicoside and its active metabolite in healthy humans

Thiocolchicoside (TCC) has been prescribed for several years as a muscle relaxant drug, but its pharmacokinetic (PK) profile and metabolism still remain largely unknown. Therefore, we re-investigated its metabolism and PK, and we assessed the muscle relaxant properties of its metabolites. After oral administration of 8 mg (a therapeutic dose) of 14C-labelled TCC to healthy volunteers, we found no detectable TCC in plasma, urine or faeces. On the other hand, the aglycone derivative obtained after de-glycosylation of TCC (M2) was observed and, in addition, we identified, as the major circulating metabolic entity, 3-O-glucuronidated aglycone (M1) obtained after glucuro-conjugation of M2. One hour after oral administration, M1 plus M2 accounted for more than 75% of the circulating total radioactivity. The pharmacological activity of these metabolites was assessed using a rat model, the muscle relaxant activity of M1 was similar to that of TCC whereas M2 was devoid of any activity. Subsequently, to investigate the PK profile of TCC in human PK studies, we developed and validated a specific bioanalytical method that combines liquid chromatography and ultraviolet detection to assay both active entities. After oral administration, TCC was not quantifiable with an lower limit of quantification set at 1 ng/mL, whereas its active metabolite M1 was detected. M1 appeared rapidly in plasma (tmax=1 h) and was eliminated with an apparent terminal half-life of 7.3 h. In contrast, after intramuscular administration both active entities (TCC and M1) were present; TCC was rapidly absorbed (tmax=0.4 h) and eliminated with an apparent terminal half-life of 1.5 h. M1 concentration peaked at 5 h and this metabolite was eliminated with an apparent terminal half-life of 8.6 h. As TCC and M1 present an equipotent pharmacological activity, the relative oral pharmacological bioavailability of TCC vs. intramuscular administration was calculated and represented 25%. Therefore, to correctly investigate the PK and bioequivalence of TCC, the biological samples obtained must be assayed with a bioanalytical method able to specifically analyse TCC and its active metabolite M1.     

Pain, tenderness, wheal and flare induced by substance-P, bradykinin and 5-hydroxytryptamine in humans

The algesic effect of substance-P with and without the addition of bradykinin or 5-hydroxytryptamine was studied in 13 healthy volunteers. Test substances dissolved in saline were injected into the temporal muscle and the forearm skin and the effects compared with those of saline. In the temporal muscle, none of the test substances induced more pain than saline, but substance-P with bradykinin lowered the pressure pain threshold by 18% (p less than 0.02). All test substances induced pain wheal and flare in the forearm skin. Substance-P induced a more pronounced flare reaction than bradykinin, whereas the latter induced more pain than substance-P. This dissociation between pain and flare may indicate that C-fibres in the human skin represent more than one type of nociceptor.     

Melatonin secretion in humans assessed by measuring its metabolite, 6-sulfatoxymelatonin

Comparing a direct radioimmunoassay for 6-sulfatoxymelatonin (aMT6s) with an established gas chromatographic/mass spectrometric method for 6-hydroxymelatonin, we found a good correlation r = 0.94 (P less than 0.001, n = 100). aMT6s was stable, both in urine and plasma samples, without preservative, for at least two years at -20 degrees C and for five days at room temperature. Urinary excretion of aMT6s showed considerable inter-individual differences; however, the aMT6s excretion of any one individual was consistent over a four-day period, as assessed by continuous collection from 18 normal volunteers. Total 24-h urinary excretion of aMT6s was significantly correlated with the area under the curve of the respective profiles for plasma melatonin (r = 0.75, P = 0.0002) and plasma aMT6s (r = 0.70, P = 0.0005) for 22 healthy volunteers. At 24:00 h and 03:00 h, sampling plasma at 30-s intervals provided no evidence for episodic secretion (in short pulses) of either melatonin or aMT6s.     

Aspirin kinetics and platelet aggregation in man

Our aims were (1) to determine the effect of six commercially available aspirin (ASA) preparations on in vitro platelet aggregation, and (2) to relate changes in platelet function to ASA kinetics. Each of six subjects took a single dose of one of the following preparations--600 mg Asproclear, 600 mg Bufferin, 600 mg Bi-prin, 600 mg compressed ASA, 650 mg Ecotrin, or 650 mg S.R.A.--in random order every 3 wk. Venous blood was drawn before and at 2, 4, 6, and 24 hr after ASA dosage to measure platelet aggregation in response to collagen and adenosine diphosphate and, at more frequent intervals, to characterize ASA kinetics. Asproclear, Bufferin, Bi-prin, and compressed ASA yielded peak plasma ASA levels of 28 to 56 mumol/l (5 to 10 mg/l) within 15 to 60 min and peak salicylic acid (SA) levels of 72 to 290 mumol/l (10 to 40 mg/l) within 2 hr. Ecotrin and S.R.A. yielded plasma SA levels of 14 to 87 mumol/l (2-12 mg/l) within 4 to 24 hr and no measurable ASA at any time after dosing. Platelet aggregation was inhibited to an equal extent by all preparations. The time course for this inhibition was the same for all preparations but Ecotrin (which led to a more delayed effect). There was significant recovery of collagen-induced platelet aggregation at 24 hr with all preparations but Ecotrin. With Ecotrin and S.R.A. there was inhibition of platelet aggregation in the absence of measurable circulating ASA. We postulate that this was due to acetylation of cyclooxygenase in the portal circulation and that inhibition of peripheral cyclooxygenase may be spared.     

Interactions between integrin alphaIIbbeta3 and the serotonin transporter regulate serotonin transport and platelet aggregation in mice and humans

The essential contribution of the antidepressant-sensitive serotonin (5-HT) transporter SERT (which is encoded by the SLC6A4 gene) to platelet 5-HT stores suggests an important role of this transporter in platelet function. Here, using SERT-deficient mice, we have established a role for constitutive SERT expression in efficient ADP- and thrombin-triggered platelet aggregation. Additionally, using pharmacological blockers of SERT and the vesicular monoamine transporter (VMAT), we have identified a role for ongoing 5-HT release and SERT activity in efficient human platelet aggregation. We have also demonstrated that fibrinogen, an activator of integrin alphaIIbbeta3, enhances SERT activity in human platelets and that integrin alphaIIbbeta3 interacts directly with the C terminus of SERT. Consistent with these findings, knockout mice lacking integrin beta3 displayed diminished platelet SERT activity. Conversely, HEK293 cells engineered to express human SERT and an activated form of integrin beta3 exhibited enhanced SERT function that coincided with elevated SERT surface expression. Our results support an unsuspected role of alphaIIbbeta3/SERT associations as well as alphaIIbbeta3 activation in control of SERT activity in vivo that may have broad implications for hyperserotonemia, cardiovascular disorders, and autism.     

Blood platelet level and platelet aggregation in adult respiratory distress syndrome

39 patients after surgery for generalized peritonitis with adult respiratory distress syndrome (ARDS) in the postoperative period have been examined. It is suggested that platelets play a certain role in the pathogenesis of ARDS and may serve as one of the mechanisms triggering disturbances in the function of both pulmonary vessels and respiratory airways. The degree of changes in platelet number and aggregation "below" and "above" the lungs may predict the severity of ARDS.