家兔组织浸液促凝及其抑制物活性的实验研究

目的研究家兔脏器组织促凝及其抑制物活性的分布特点。方法用气栓法致死家兔．取其脏器组织，以每克湿重组织加0．01moL／L pH7．2的PBS 2ml的比例用细胞匀浆器或研钵充分研碎，用血浆复钙时间（PRT）、抗凝血酶（AT）和肝素辅因子Ⅱ（HCⅡ）检测组织浸出液中的活性量。结果PRT高促凝活性（〈30s）有髓上腺、脾、肾皮质、脑、胃、肺、心肌和横纹肌。显著高于相对低促凝活性（〉30s）的大肠、肝、肾髓质（P〈0．01）。AT为肝、肺、胃和脑组织。明显高于大肠、肾髓质、肾皮质、横纹肌、脾和心肌组织（P〈0．05）。HCⅡ活性为肾髓质、脑、大肠和横纹肌显著高于其他组织（P〈0．05-0．01）。结论不同组织以及同一组织的不同解剖部位有着不同的促凝及其抑制物活性，对了解脏器组织的出凝血特点以及易于发生血栓形成或出血倾向有重要的意义。     

硫代磷酸酯寡核苷酸的合成及其对内皮细胞组织因子(Tissue factor,TF)基因表达及TF促凝活性的作用

为合成与 TF基因启动子区切应力反应元件 ( Shear stress responsive element,SSRE)形成三链 DNA的硫代磷酸酯寡核苷酸 ( Triple helix- forming phosphorothioate oligodeoxynucleotides,TFO- ps) ,探讨其对内皮细胞TF基因表达及 TF促凝活性的影响。我们设计反向 TFO ( T2 1GTa)序列 ,采用固相亚磷酰胺三酯固相法合成TFO。在 TFO的 3′末端进行硫代磷酸酯修饰。应用电泳迁移分析 ( Electrophoretic mobility shift assays,EMSA)观察寡核苷酸和硫代脱氧寡核苷酸的亲和性。在 ECV3 0 4内皮细胞株观察 γ- 32 P标记硫代磷酸酯 TFO的细胞摄取及其对 TF基因表达的影响。结果显示 ,TFO- ps( T2 1Gta- ps)与靶序列能形成三链 DNA,其 Kd值为 3 .6× 10 - 1 0 M。在内皮细胞株 ECV3 0 4细胞 ,TFO- ps( T2 1GTa- ps)的细胞吸收率为 11.65 % ,主要分布在核沉淀 ,约占吸收总量的77.2 5 % ,显著降低内皮细胞 TF基因 m RNA表达及 TF蛋白合成的平均光密度 ( AOD)值 ,显著降低 TF的促凝活性。以上结果表明 ,TFO- ps( T2 1GTa- ps)具有较好的抗凝活性 ,其机制与抑制 TF基因表达有关     

脑梗死患者血清同型半胱氨酸、超敏C反应蛋白水平和全血组织因子促凝活性的检测分析

目的:探讨血清同型半胱氨酸(Hcy)、超敏C反应蛋白(hs-CRP)和全血组织因子促凝活性(TF-PCA)对脑梗死临床诊断及治疗的意义。方法:使用仪器法对88例脑梗死患者(脑梗死组)及100例正常对照人群(对照组)血清Hcy、hs-CRP水平及全血TF-PCA进行检测,并比较不同梗死容积组上述三指标的差异。结果:脑梗死组Hcy、hs-CRP和TF-PCA水平均显著高于对照组(P<0.01)。不同梗死容积组比较,梗死容积越大,其Hcy、hs-CRP及TF-PCA水平越高。结论:常规监测血清Hcy、hs-CRP和全血TF-PCA水平可为脑梗死患者病情变化和临床治疗提供参考依据。     

2型糖尿病肾病患者外周血单个核细胞组织因子活性检测的意义

目的探讨2型糖尿病肾病患者外周血单个核细胞组织因子促凝活性（TF-PCA）的改变及其临床意义。方法 63名2型糖尿病患者按照尿白蛋白/肌酐比共分为三组：比值〈30mg/g为正常蛋白尿组（n=22）,30mg/g≤比值〈300mg/g为微量白蛋白尿组（n=26）,比值≥300mg/g为大量白蛋白尿组（n=15）;同时选择21名健康体检者作为对照组。采用一期凝固法测定外周血单个核细胞TF-PCA。常规检测空腹血糖、糖化血红蛋白、超敏CRP（Hs-CRP）、尿酸（UA）、胱抑素C（CYSC）,并进行相关分析。结果糖尿病组外周血TF-PCA随尿蛋白水平升高而升高,同时单个核细胞TF-PCA与空腹血糖、Hs-CRP、UA等呈正相关,相关系数分别为0.419、0.293、0.232（P〈0.05）。结论 2型糖尿病患者外周血单个核细胞TF-PCA明显升高,且与多种因素相关,TF-PCA升高可能与DN的发病机制、病程进展相关。     

血液循环中白细胞组织因子活性的测定方法及临床意义

目的:建立一种用于辅助临床诊断血栓性疾病的白细胞组织因子促凝活性(TF-PCA)测定方法。方法:利用动态监测法测定脂多糖(LPS)刺激后单个核白细胞(MLC)的TF-PCA的变化,利用流式细胞技术检测其TF抗原的表达,以鉴定检测方法的特异性,并进行临床验证。结果:凝血动力学指标变化与细胞数、LPS浓度、反应时间等有不同程度的相关性,TF-PCA在内源性乏凝血因子Ⅷ、Ⅸ血浆凝固中显示明显的促凝活性,而外源性乏凝血因子Ⅶ、Ⅹ则未显示TF-PCA;TF-PCA与凝血动力学的凝固延迟时间和纤维蛋白单体聚合速率呈良好的负、正相关(r=-0.986、r=0.928);LPS对MLC刺激上调TF表达具有较高的特异性,由此印证了本检测方法的可行性。结论:凝血动力学检测能较简单、快捷的检测MLC的TF-PCA,并有较高的特异性和敏感性,对血栓性疾病的诊断及防治有应用价值。     

组织因子途径抑制物

组织因子途径抑制物（ＴＥＰＩ）是一种新发现的依赖Ｘａ的Ⅶａ／组织因子复合物的抑制物，属Ｋｕｎｉｔｚ－类型丝氨酸蛋白酶抑制物家族成员。本文就ＴＥＰＩ的组成、结构、基因定位、合成、代谢、分布以及ＴＥＰＩ的作用机理、生理及病理意义等作一评述。     

慢性脑血管病治疗前后血液流变学指标和全血组织因子活性的变化

目的:观察血流变学指标和组织因子促凝活性(TF-PAC)检测对慢性脑血管病(CCVD)临床诊断和疗效监测的作用。方法:采用全自动血流变检测仪测定全血粘度等6项指标;采用全血复钙凝血时间法检测TF-PAC;比较CCVD患者与正常健康人群之间两类指标的变化情况。结果:CCVD组男女性患者血液变6项指标中5项指标以及TF-PAC均较健康对照组升高(红细胞变形指数降低)(P<0.05或P<0.01)。治疗后血流变指标明显改善,其中以红细胞聚集指数、纤维蛋白原(Fg)和TF-PAC降低尤为明显(P<0.01)。结论:应用血液流变学指标和TF-PAC对CCVD进行临床诊断及治疗监测有重要意义。     

奥扎格雷钠联合低分子肝素钙对短暂性脑缺血发作的疗效及内皮素和组织因子促凝活性的影响

目的:回顾性分析奥扎格雷钠联合低分子肝素钙对短暂性脑缺血发作(TIA)患者血浆内皮素-1(ET-1)及全血组织因子促凝活性(TF-PCA)的影响。方法:选取108例经临床确诊的TIA患者,按随机数字表法分为奥扎格雷钠联合低分子肝素钙治疗组(观察组,n=54)和低分子肝素钙治疗对照组(治疗组,n=54),两组均给予降压、降脂、控制血糖等常规措施,另选同期体检健康者为对照组(n=56)。1个疗程后(7天)观察两组TIA患者临床疗效及不良反应,并分析治疗前后两组血浆ET-1及全血TF-PCA水平变化。结果:观察组的治愈率和总有效率均明显高于治疗组(70.37%vs 46.30%,94.44%vs 72.22%,P均0.05)。治疗前,两组TIA患者ET-1及TFPCA水平无明显差异,但均显著高于对照组(P0.01);治疗后,两组患者ET-1及TF-PCA均较治疗前明显降低(P0.01),观察组较治疗组降低更明显(P0.05)。治疗期间两组均未发生脑及其它脏器出血。结论:奥扎格雷钠联合低分子肝素钙治疗TIA效果良好,且无不良反应。     

人类组织因子途径抑制因子的研究进展

人类组织因子途径抑制因子属于库尼(Kunitz)型丝氨酸蛋白酶抑制剂家族蛋白,分为组织因子途径抑制因子-1(tissuefactorpathwayinhibitor-1,TFPI-1)和组织因子途径抑制因子-2(tissuefactorpathwayinhibitor-2,TFPI-2)。TFPI-1以抗凝血作用为主,而TFPI-2是广谱丝氨酸蛋白酶抑制剂。二者在结构上有部分同源性,均由3个重复的Kunitz结构域组成。但二者在基因序列、组织来源、分布和作用机理上的很大差异,导致二者在多种生理和病理过程中发挥的作用也大不相同。就有关研究进展做一综述。     

人组织因子途径抑制因子重组蛋白表达菌株的建立

组织因子途径抑制因子是机体凝血过程的主要抑制因子，在血栓形成性疾病的防治中具有广阔的应用前景。本研究采用pGEX—2T为表达载体、大肠杆菌细胞为表达宿主进行了人组织因子途径抑制因子重组蛋白的制备。制备的重组蛋白产量较高，纯化过程简单，且具有良好的抑制组织因子的功能。     

The source of urinary epidermal growth factor in humans

Summary To clarify the source of human urine EGF, we studied EGF renal clearance in 20 healthy, young adult subjects. Immunoreactive EGF was measured hourly in EDTA plasma, heparin plasma, serum and urine of 12 males and 8 females during a 3 h study period. Plasma and urine creatinine and creatinine clearance were measured and calculated hourly. Mean (and SEM) creatinine clearance was similar in males and females (118±12 vs 105±6 ml/min). EGF was not detectable in plasma, whereas relatively high levels were measured in serum (2.5±0.25 vs 1.5±0.18 ng/ml in males and females respectivelyp<0.05). Urine EGF excretion averaged 1641±233 ng/h in males and 1507±191 ng/h in females (p>0.05). A significant correlation was observed between urine creatinine and urine EGF concentrations in both male (r = 0.98,p<0.01) and female (r=0.94,p< 0.01) subjects. EGF immunoreactivity in urine and serum eluted from G-75 sephadex columns similarly to recombinant 6000 Mr hEGF. Urine excretion of EGF approximated 1.5 g/h or 25 ng/mg creatine. The high concentrations of EGF found in urine in the face of non-detectable levels of EGF in plasma favor the hypothesis that EGF in urine is derived from kidney synthesis and secretion. The significant positive correlation between urine creatinine and urine EGF suggests a functional correlation between glomerular filtration and the process of tubular EGF excretion.Abbreviations EGF epidermal growth factor - hEGF human epidermal growth factor - IGF insulin-like growth factor - TGF alpha transforming growth factor - TGF beta transforming growth factor - NGF nerve growth factor - PDGF platelet derived growth factor - CPDA citrate phosphate dextrose adenine buffer - EDTA ethylenedinitrilotetraacetic acid - PBS phosphate saline buffer     

Development and validation of an assay for urinary tissue factor activity.

BACKGROUND: Activation of blood coagulation is a common complication of cancer and inflammation in both humans and experimental animals. Increased production of tissue factor--the principal initiator of the coagulation process--by endothelial cells, monocytes, and macrophages has been implicated in these conditions. AIM: To investigate whether urinary tissue factor (uTF) might reflect the state of monocyte/macrophage activation and be a useful diagnostic test. METHODS: Urine was centrifuged at 51,000 g to sediment tissue factor containing membrane vesicles. The tissue factor was then solubilised in beta-octyl-glucopyranoside and assayed in a specific chromogenic assay adapted for use in microtitre plates. RESULTS: The assay proved to be sensitive, specific, and reproducible. The normal range of uTF was relatively narrow and unaffected by age, sex, or cigarette smoking. Levels were not significantly influenced by storage of urine samples before assay or by the presence of fresh blood in the urine sample. CONCLUSIONS: This method may have diagnostic application in the study of haemostatic activation in patients with cancer and other disease states.     

comparison of clot-based vs chromogenic factor Xa procoagulant phospholipid activity assays

We compared 2 commercial plasma procoagulant phospholipid activity (PPA) assays, chromogenic, using bound annexin V to capture phosphatidylserine-containing microparticles, and clot-based. In both, anionic phospholipids accelerated activation of prothrombin by factor Xa. PPA levels were lower in the chromogenic vs the clot-based assay, with poor correlation between methods: normal samples, mean ± SD, 27 ± 17 vs 590 ± 414 nmol/L (n = 24; r(2) = 0.29) and patient samples, mean ± SD, 45 ± 44 vs 401 ± 330 nmol/L (n = 51; r(2) = 0.26). Recovery of phosphatidylserine added to normal, heparinized, and warfarin plasma samples averaged 109% ± 39% using the chromogenic assay but was higher and more varied (mean ± SD, 176% ± 59%) in the clot-based assay. Lupus anticoagulants caused low recovery in both assays. Removal of microparticles by 0.22-μm filtration reduced PPA by 91% in the clot-based and 65% in the chromogenic assay. The clot-based assay showed higher correlation (r(2) = 0.82 vs 0.23) with flow cytometric platelet microparticle counts. The 2 assays measure different aspects of PPA in plasma, with the chromogenic assay primarily measuring smaller microparticles.     

Co-adsorbed fibrinogen and von Willebrand factor augment platelet procoagulant activity and spreading

Previously we observed that platelets adherent to surfaces preadsorbed with blood plasma exhibited 1.3 to 2.4 times greater procoagulant activity than platelets on surfaces adsorbed with fibrinogen (Fg) only. These observations suggested that the adhesion proteins adsorbed from plasma may activate platelets in a cooperative, or synergistic manner. In the present study, polystyrene surfaces adsorbed with both Fg and vWF induced up to three times greater procoagulant activity than surfaces adsorbed with Fg or vWF only. The amounts of Fg and vWF adsorbed from binary mixtures that resulted in increased procoagulant activity were found to be similar to the amounts that adsorbed to PS from 100% plasma. The effect of adsorbed adhesion proteins on platelet spreading was also investigated. The proportion of fully spread platelets increased, depending on the adhesion protein preadsorbed to the surface, in the following order: vWF < Fg < Fn < (vWF + Fg) < Vn < plasma.     

Platelet-activating factor modulates endotoxin-induced macrophage procoagulant activity by a protein kinase C-dependent mechanism.

Macrophage procoagulant activity is an important mediator of extravascular fibrin deposition at sites of infection and appears to contribute to the pathogenesis of several infectious disease processes. Previous studies have shown that the inflammatory mediator platelet-activating factor was able to prime macrophages for induction of procoagulant activity by bacterial lipopolysaccharide. The present studies were designed to examine the mechanism of this priming effect. Platelet-activating factor (100 nM) primed macrophages for procoagulant activity generation in response to endotoxin at concentrations as low as 100 ng/ml and also following exposure to Escherichia coli, Bacteroides fragilis, and Staphylococcus aureus. The priming effect occurred following a pretreatment with platelet-activating factor for as short as 1 min, suggesting a rapid activation event. Two different doses of the calcium ionophore ionomycin were used to mimic the peak and sustained effects of platelet-activating factor on cytoplasmic calcium levels (1 microM and 100 nM, respectively). Neither dose was able to mimic the priming effect. However, extracellular calcium was necessary for induction of procoagulant activity and the priming effect. By contrast, the protein kinase C agonist phorbol myristate acetate reproduced the priming phenomenon observed for platelet-activating factor. In further support of the concept that protein kinase C activation mediated the effect of platelet-activating factor, the specific protein kinase C inhibitor staurosporine reversed the ability of platelet-activating factor to augment induction of macrophage procoagulant activity by endotoxin. These data suggest mechanisms by which inflammatory mediators within the microenvironment of infection might modulate the host response to bacterial pathogens.     

Monocyte procoagulant activity in Whipple's disease

We have studied the expression of procoagulant activity by the circulating mononuclear cells of four patients with Whipple's disease. There was a spontaneous expression of procoagulant activity in two patients with active untreated Whipple's disease. This activity was shown to originate in the monocyte fraction of the mononuclear cells and was demonstrated to cleave prothrombin directly. This prothrombinase activity was not Factor Xa, because it was not neutralized by anti-Factor X serum and was not inhibited by an established panel of Factor Xa inhibitors. The prothrombinase activity was not expressed by the monocytes of these patients following 8 weeks of antibiotic therapy, by which time the patients' symptoms resolved, and was not found in two patients previously treated for Whipple's disease who were in clinical remission or in normal subjects. Serial studies in one patient with active disease showed that monocytes failed to express increased prothrombinase within 2 weeks of antibiotic therapy. A second procoagulant activity was produced in response to endotoxin (LPS) by cells from controls and patients with Whipple's disease and was identified as thromboplastin. These observations suggest that circulating monocytes of patients with active Whipple's disease are endogenously stimulated to express prothrombinase activity, which may contribute, at least in part, to the pathophysiology of this condition.     

微小病变性肾病早期差异尿蛋白质组分析

目的筛选微小病变性肾病早期尿蛋白标志物。方法以阿霉素肾病大鼠作为该病动物模型,采用LC-MS/MS蛋白质组学技术,非标记法蛋白质相对定量方法,分别分析尿全蛋白质组及ConA偶联琼脂糖珠富集的尿糖蛋白组的变化。结果尿全蛋白质组分析得到25个差异蛋白,分别来源于血浆蛋白、免疫炎性细胞分泌蛋白及泌尿道特异分泌蛋白等,参与不同的致病过程,如血流动力学改变、足细胞损伤及免疫功能紊乱等;尿糖蛋白质组分析得到21个差异蛋白,其中12个蛋白(57%)与未进行富集实验得到的差异蛋白不同,说明两种方法具有互补性,可以从不同角度对肾脏病变进行刻画。结论这些差异蛋白可作为微小病变性肾病早期诊断的候选标志物,对其功能的进一步验证将有助于其发病机制的解析。