Structural basis of latency in plasminogen activator inhibitor-1.

Human plasminogen activator inhibitor-1 (PAI-1) is the fast-acting inhibitor of tissue plasminogen activator and urokinase and is a member of the serpin family of protease inhibitors. Serpins normally form complexes with their target proteases that dissociate very slowly as cleaved species and then fold into a highly stable inactive state in which the residues that flank the scissile bond (P1 and P1';) are separated by about 70 A. PAI-1 also spontaneously folds into a stable inactive state without cleavage; this state is termed 'latent' because inhibitory activity can be restored through denaturation and renaturation. Here we report the structure of intact latent PAI-1 determined by single-crystal X-ray diffraction to 2.6 A resolution. The three-dimensional structure reveals that residues on the N-terminal side of the primary recognition site are inserted as a central strand of the largest beta sheet, in positions similar to the corresponding residues in the cleaved form of the serpin alpha 1-proteinase inhibitor (alpha 1-PI). Residues C-terminal to the recognition site occupy positions on the surface of the molecule distinct from those of the corresponding residues in cleaved serpins or in the intact inactive serpin homologue, ovalbumin, and its cleavage product, plakalbumin. The structure of latent PAI-1 is similar to one formed after cleavage in other serpins, and the stability of both latent PAI-1 and cleaved serpins may be derived from the same structural features.     

Development of venous occlusions in mice transgenic for the plasminogen activator inhibitor-1 gene

The fibrinolytic potential of the vasculature is modulated primarily by the availability and activity of plasminogen activators, which convert the zymogen plasminogen into the active fibrin-degrading enzyme plasmin. The activities of these key regulatory enzymes are directly neutralized by their primary endogenous inhibitor, plasminogen activator inhibitor-1 (PAI-1). Although some individuals with a tendency to develop thrombotic disorders exhibit elevated levels of PAI-1 in their plasma, the cause-and-effect relationship between increased PAI-1 and thrombosis is still unclear. Specifically, it is not known whether chronic depression of fibrinolytic activity results in the development of thrombosis. To address this question we developed transgenic mice in which the contribution of PAI-1 to thrombus formation could be evaluated. The results presented in this report indicate that elevated levels of PAI-1 contribute to the development of venous but not arterial occlusions.     

Mobile reactive centre of serpins and the control of thrombosis

Two protease inhibitors in human plasma play a key part in the control of thrombosis: antithrombin inhibits coagulation and the plasminogen activator inhibitor PAI-1 inhibits fibrinolysis, the dissolving of clots. Both inhibitors are members of the serpin family and both exist in the plasma in latent or inactive forms. We show here that the reactive centre of the serpins can adopt varying conformations and that mobility of the reactive centre is necessary for the function of antithrombin and its binding and activation by heparin; the identification of a new locked conformation explains the latent inactive state of PAI-1. This ability to vary conformation not only allows the modulation of inhibitory activity but also protects the circulating inhibitor against proteolytic attack. Together these findings explain the retention by the serpins of a large and unconstrained reactive centre as compared to the small fixed peptide loop of other families of serine protease inhibitors.     

Lipoprotein(a) modulation of endothelial cell surface fibrinolysis and its potential role in atherosclerosis

Endothelial cells play a critical role in thromboregulation by virtue of a surface-connected fibrinolytic system. Cultured endothelial cells synthesize and secrete tissue-type plasminogen activator (t-PA) which can bind to at least two discrete sites on the cell surface. These binding sites preserve the catalytic activity of t-PA and protect it from its physiological inhibitor (PAI-1). N-terminal glutamic acid plasminogen (Glu-PLG), the main circulating fibrinolytic zymogen, also interacts specifically with the endothelial cell surface. Binding is associated with a 12-fold increase in catalytic efficiency of plasmin generation by t-PA which may reflect conversion of Glu-PLG to its plasmin-modified form, N-terminal lysine plasminogen (Lys-PLG). Lipoprotein(a) is an atherogenic lipoprotein particle which contains the plasminogen-like apolipoprotein(a) bound to low density lipoprotein. We report here that lipoprotein(a) interferes with endothelial cell fibrinolysis by inhibiting plasminogen binding and hence plasmin generation. In addition, we demonstrate lipoprotein(a) accumulation in atherosclerotic lesions. These findings may provide a link between impaired cell surface fibrinolysis and progressive atherosclerosis.     

复杂性疾病血栓前状态指标变化的共性研究

为研究复杂性疾病患者在血栓前状态的共性并探讨其意义,采用流式细胞仪、ELASA法、放射免疫法、全自动血凝分析仪等技术方法,检测复杂性疾病患者血小板表面P-选择素(CD62p)的表达,血浆组织型纤溶酶原激活剂(t-PA)及血浆组织型纤溶酶原激活抑制剂(PAI-1)、血清内皮素(ET-1)及凝血4项即血浆纤维蛋白原(FIB)水平、活化部分凝血活酶时间(APTT)、凝血酶原时间(PT)、凝血酶时间(TT)。结果表明:与正常对照组比较,复杂性疾病组血小板表面CD62p的表达及血浆PAI-1水平明显升高(P<0.05);复杂性疾病组血浆t-PA的含量明显降低(P<0.05);与正常对照组比较,肿瘤组血清ET-1水平明显升高(P<0.05)。上述指标与疾病组间比较差异无显著性(P>0.05);与正常对照组比较,各疾病组APTT时间明显缩短(P<0.05);与正常对照组比较各疾病组血浆FIB浓度明显升高(P<0.05)。其中肿瘤组血浆FIB浓度的变化比其他3个疾病组更为明显;与正常对照组比较,肿瘤组、糖尿病组及高血压组PT时间明显缩短(P<0.05);APTT,PT,TT进行疾病组间比较差异无显著性(P>0.05)。由此得出结论:复杂性疾病患者血小板活化、凝血功能增强、纤溶功能减退,存在明显的血栓前状态。血栓前状态可能是贯穿于复杂性疾病发生、发展过程的共同病理生理机制之一。     

Kunitz抑制剂家族成员抑肽酶对纤溶酶原活化的抑制作用

抑肽酶属Kunitz抑制剂家族成员，能够抑制激肽释放酶、纤溶酶及胰蛋白酶的蛋白水解活性．研究表明，抑肽酶能够抑制尿激酶型-纤溶酶原激活刹（u—PA）和组织型纤溶酶原激活剂（t—PA）对纤溶酶原的激活，但不影响u—PA和t—PA对小分子底物的酰胺水解活性．用u—PA研究了上述作用的机制，发现抑肽酶与u—PA的丝氨酸蛋白酶功能区特异性结合，而与纤溶酶原没有相互作用．抑肽酶与uPA的结合并不阻断u—PA的活性位点，因为u—PA对小分子底物的水解活性仍然保持．上述发现提示抑肽酶可能存在另一种抑制作用模式，该模式不同于以前报道的关于Kunitz抑制剂或纤溶酶原激活酶抑制剂的作用．由于人体内的Kunitz抑制剂与抑肽酶在结构上非常相似，根据研究结果，推测体内纤溶酶原的激活作用并非仅受丝氨酸蛋白酶抑制剂的控制。     

pEGFP-N3-t-PA真核表达质粒的构建及其在成纤维细胞中的表达

目的利用基因工程技术克隆人组织纤溶酶原激活物（t—PA）基因并构建一种能高效、安全表达t—PA的pEGFP—N3-t—PA真核表达重组质粒,为进一步研制转基因动物奠定基础．方法采用高效Trizol试剂快速从黑色素瘤细胞中提取总RNA,RT—PCR获得t—PAcDNA,并将真核表达质粒pEGFP—N3和t—PA基因片段分别双酶切,将回收的pEGFP—N3大片段（4．7kb）与t—PA基因片段（1．1kb）重组．对pEGFP—N3-t—PA质粒进行酶切鉴定和基因测序鉴定．脂质体介导pEGFP—N3-t—PA转染成纤维细胞（NIn313）,并用RT—PCR法从mRNA水平检测t—PA的表达情况,用倒置荧光显微镜检测、分析其在NH 3T3细胞中的表达．结果成功地从黑色素瘤细胞中克隆了t—PA基因,并构建了以pEGFP—N3为载体的真核表达质粒载体,并能在真核细胞中表达、分泌t—PA．结论含t—PA基因真核表达质粒构建成功．     

异常黑胆质性哮喘血栓前状态的变化特点

 为探讨新疆维吾尔族异常黑胆质型哮喘患者血栓前状态的变化特点,对76 例哮喘患者按维医体液论进行辨证分型（其中异常黑胆质型哮喘组30 例,非异常黑胆质型哮喘组46 例）,并与健康对照组89 名进行对照,检测血小板膜表面糖蛋白CD41 和CD62P、内皮素（ET-1）、组织型纤溶酶原激活物（t-PA）、组织型纤溶酶原激活物抑制剂-1（PAI-1）和凝血4 项指标,即血浆纤维蛋白原（FIB）水平、活化部分凝血活酶时间（APTT）、凝血酶原时间（PT）、凝血酶时间（TT）。研究发现,与正常对照组相比,异常黑胆质性哮喘组和非异常黑胆质性哮喘组CD62P、ET-1、PAI-1 水平均升高,差异有统计学意义（P<0.05）,且异常黑胆质性哮喘组CD62P、ET-1、PAI-1 水平高于非异常黑胆质性哮喘组,差异有统计学意义（P<0.05）。与正常对照组相比,异常黑胆质性哮喘组和非异常黑胆质性哮喘组t-PA 含量均降低,差异有统计学意义（P<0.05）,且以异常黑胆质性哮喘组下降较明显,差异有统计学意义（P<0.05）。与正常对照组相比,异常黑胆质性哮喘组和非异常黑胆质性哮喘组FIB 含量明显升高,差异有统计学意义（P<0.01）,且异常黑胆质性哮喘组FIB 含量高于非异常黑胆质性哮喘组,差异有统计学意义（P<0.05）。APTT、PT 时间较正常对照组偏短,差异有统计学意义（P<0.05）,APTT、PT 时间在异常黑胆质性哮喘组最短,其次为非异常黑胆质性哮喘组。研究表明,异常黑胆质性哮喘患者的血管内皮细胞、血小板、凝血和纤溶功能都发生紊乱,提示异常黑蛋质性哮喘处于血栓前状态,且这种状态较非异常黑胆质性哮喘更为突出。     

Role of plasminogen activator and plasminogen activator inhibitor type-1 in luteolysis—a minireview

This minireview summarized our recent studies on the role of plasminogen activator (PA) and inhibitor type-1 (PAI-1) in luteolysis. We have demonstrated that (1) both tissue type and urokinase type plasminogen activators (tPA and uPA) and a plasminogen activator inhibitor type-1 (PAI-1) were present in the corpus luteum of rat and rhesus monkey; (2) decrease in progesterone production in corpus luteum was well correlated with a sharp increase in tPA (but not uPA) and PAI-1 secretion; (3) exogenous tPA decreased luteal progesterone synthesis while monoclonal antibodies increased progesterone production; (4) interferony inhibited luteal progesterone synthesis and stimulated tPA production while LH plus prolactin increased progesterone production and decreased tPA (hut not uPA) activity in cultured luteal cells; (5) increase in proteolysis in the corpus luteum was also correlated with decrease in progesterone production in mouse. These data suggest that local degradation of extracellular matrix controlled by plasminogen activator and inhibitor is involved in the processes of luteolysis.     

Role of plasminogen activator and plasminogen activator inhibitor type-1 in luteolysis——a minireview

This minireview summarized our recent studies on the role of plasminogen activator (PA) and inhibitor type-1 (PAI-1) in luteolysis. We have demonstrated that (1) both tissue type and urokinase type plasminogen activators (tPA and uPA) and a plasminogen activator inhibitor type-1 (PAI-1) were present in the corpus luteum of rat and rhesus monkey; (2) decrease in progesterone production in corpus luteum was well correlated with a sharp increase in tPA (but not uPA) and PAI-1 secretion; (3) exogenous tPA decreased luteal progesterone synthesis while monoclonal antibodies increased progesterone production; (4) interferon y inhibited luteal progesterone synthesis and stimulated tPA production while LH plus pro-lactin increased progesterone production and decreased tPA (but not uPA) activity in cultured luteal cells; (5) increase in proteolysis in the corpus luteum was also correlated with decrease in progesterone production in mouse. These data suggest that local degradation of extracellular matrix controlled by plasminogen activator and inhibitor is involved in the processes of luteolysis.     

糖尿病患者血栓前状态研究

采用流式细胞仪、ELISA法、全自动血凝分析仪,检测糖尿病患者血小板表面P-选择素(CD62P)的表达、血浆组织型纤溶酶原激活剂(t-PA)、抑制剂(PAI-1),以及凝血4项,即血浆纤维蛋白原(Fib)水平、活化部分凝血活酶时间(APTT)、凝血酶原时间(PT)、凝血酶时间(TT)。结果显示,糖尿病组血小板表面CD62P的表达、血浆PAI-1、Fib 3项指标均较正常组增高(P<0.05),而t-PA水平较正常组降低(P<0.05);糖尿病组APTT、PT及TT时间明显缩短(P<0.05);糖尿病伴血管病变组血小板表面CD62P的表达、血浆PAI-1、Fib 3项指标均较不伴血管病变组增高(P<0.05);糖尿病伴血管病变组APTT及TT较不伴血管病变组明显缩短(P<0.05);糖尿病伴血管病变组血浆t-PA水平、PT与不伴血管病变组指标比较没有显著性差异(P>0.05)。研究表明,糖尿病及糖尿病伴血管病变患者血小板活化、凝血功能增强、纤溶功能减退,存在明显的血栓前状态;测定血小板表面CD62P的表达、血浆t-PA、PAI-1,以及凝血4项,即血浆Fib水平、APTT、PT、TT等有助于早期发现血栓前状态及血管病变,并可以指导临床早期干预治疗。     

单关节类风湿性关节炎滑膜组织中uPA、uPAR、PAI-1蛋白表达及意义

为探讨尿激酶型纤溶酶原激活物(urokinase-type plasminogen activator,uPA)及其受体(urokinase-type plas-minogen activator receptor,uPAR)、纤溶酶原激活物抑制剂(plasminogen activator inhibitor,PAI-1)在单关节发病类风湿关节炎(rheumatoid arthritis,RA)的表达。采用免疫组化方法检测13例单关节发病RA、19例典型RA和20例正常滑膜组织中uPAu、PAR、PAI-1蛋白表达情况。结果显示:13例单关节RA滑膜组织中uPA、u PAR、PAI-1阳性表达主要分布在滑膜衬里细胞、滑膜下层单核细胞及血管内皮细胞,阳性表达部位与典型RA相同,但表达强度明显低于典型RA(P<0.05)。与正常滑膜组织相比,单关节RA滑膜中uPA、uPAR、PAI-1蛋白表达明显增高(P<0.05)。由此可知:单关节RA滑膜组织中uPA、uPAR、PAI-1表达明显低于典型RA滑膜组织,可能与单关节RA为典型RA病程早期阶段,滑膜中uPA、uPAR、PAI-1蛋白尚处于低水平表达阶段有关。     

酰化纤溶酶原-链激酶 (APSAC)的制备和生物学性质

建立在Lys-Sepharose4B柱上用人纤溶酶原(HPg),链激酶(SK)和对氨基苯甲酸对脒基苯酚酯合成新型溶栓药物酰化纤溶酶原-链激酶(APSAC)的一种新方法,该法使APSAC的纯度和收率大为提高,分子量为130000,体外有明显溶栓效果,在兔体内半衰期8.8h.用同方法制备的未经修饰的纤溶酶原-链激酶复合物(PSAC)在兔血浆内半衰期仅为12min.     

冠心病患者血栓前状态分子标志物检测

通过采用流式细胞仪、酶联免疫吸附双抗体夹心法(ELISA)法、放射免疫法、全自动血凝分析仪等,检测冠心病患者血小板膜表面P-选择素(CD62p)、血浆组织型纤溶酶原激活剂(t-PA)及其抑制剂(PAI-1)、血清内皮素(ET-1),以及凝血4项即血浆纤维蛋白原(FIB)水平、活化部分凝血活酶时间(APTT)、凝血酶原时间(PT)、凝血酶时间(ITT),研究冠心病患者血栓前状态相关指标改变并探讨其意义.结果表明,冠心病患者血小板膜表面CD62p的表达、血浆PAI-1、FIB 3项指标均较正常组显著增高(P<0.05),而t-PA水平较正常组显著降低(P<0.05);冠心病患者APTT时间显著缩短IP<0.05).由此可知,冠心病患者血小板活化、凝血功能增强、纤溶功能减退,存在明显的血栓前状态;测定血小板表面CD62p的表达、血浆t-PA和PAI-1及凝血4项有助于早期发现冠心病患者血栓前状态并以指导临床早期干预治疗.     

骨关节炎滑膜uPA、uPAR、PAI-1表达及意义

探讨尿激酶型纤溶酶原激活物(uPA)及其受体(uPAR)和其抑制剂(PAI-1)在OA的发生发展中的意义。方法:采用免疫组化方法检测OA滑膜组织中uPA,uPAR,PAI-1的表达。结果显示:36例OA滑膜中阳性表达uPA 29例(80.56%)、uPAR 25例(69.44%)P、AI-1 27例(75.00%)。21例对照滑膜中阳性表达uPA 6例(28.57%)、uPAR 6例(28.57%)、PAI-1 3例(14.29%)。实验组与对照组间3种蛋白表达差异均有统计学意义(P<0.05);再将实验组按年龄及软骨破坏程度分组,按年龄分组3种蛋白表达差异均无统计学意义(P>0.05);按软骨破坏程度分组3种蛋白表达差异均有统计学意义(P<0.05)。阳性表达主要分布在滑膜衬里层细胞。结论:uPA系统可能参与介导软骨降解、促进OA的发生发展;而PAI-1则通过抑制uPA活性延缓OA的发生发展。     

鼠肾纤维蛋白溶酶原激活酶的纯化和部分鉴定

采用肝素—Sepharose亲和层析和凝胶过滤自鼠肾提取纤维蛋白溶酶原激活酶,产率5.1×10~(-5)％。在还原和非还原状态下SDS—聚丙烯酰胺凝胶电泳及电泳酶谱分析均呈单一区带,证实产物仍保持单链状态。测得其分子量为70000。纤维平板试验和无纤维蛋白溶酶原纤维平板试验证实其活性依赖于纤维蛋白溶酶原。     

Localization and the possible role of plasminogen activators and inhibitors in early stages of placentation

The distribution of mRNAs of tissue type (t) and urokinase type (u) plasminogen activator (PA) plus their corresponding inhibitors, type-1 (PAI-1) and type-2 (PAI-2) have been studied in the tissues of human first and second trimester placentae by in situ hybridization. The results show that: (ⅰ) All the molecules, tPA, uPA, PAI-1 and PAI-2, were identified in the blood vessels, the majority of extravillous trophoblastic cells of the decidual layer between Rohr’s and Nitabuch’s stria and in the trophoblast cells lining the chorionic plate, basal plate, intercotyledonary septae and cytotrophoblast cells of the chorionic villous tree. (ⅱ) No expression of such probes was observed in the basal and chorionic plate, glandular cells of the decidua, the septal tissues or the villous core mesenchyme. The co-distribution of the molecules observed suggests that the co-ordinated expression of the activators and inhibitors in various cells of the placental tissue may play a role in angiogenesis related to conversion of spiral arteries into utero-placental arteries and establishment of a chorio-decidual blood flow during early stages of placentation.     

大批量制备人血浆纤维蛋白溶酶原

血浆经大豆胰蛋白酶抑制物、苯甲基磺酰氟和苯甲脒预处理,保持低温操作,以及赖氨酸—Sepharose 4B 亲和载体合成手续的改进,可以从近千毫升血浆中以140mg/L 血浆的产率制得低纤维蛋白溶解酶活性的纤维蛋白溶酶原制剂。     

Increase Renaturation Yield of Reteplase Using the Recombinant Human Protein Disulfide Isomerase

Reteplase, the recombinant type of novel tissue plasminogen activator (t-PA) variant, is a promising thrombolytics in clinics. Expressed in the form of an inclusion body, reteplase consists of about 40% of the total intracellular proteins of Escherichia coli. The recombinant human protein disulfide isomerase (rhPDI) is used to increase the chance for the correct matching of the 18 hydrosulfide groups of the reteplase molecule in the renaturation process and it increase is the reteplase renaturation yield from 1%-2% to 15% - 20% with a the purity about 99% and the specific activity of 5(105 IU/mg is reached.This novel method can reduce significantly the cost of production.