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.     

Serpin-resistant mutants of human tissue-type plasminogen activator

Tissue-type plasminogen activator (t-PA) converts the inactive zymogen, plasminogen, into the powerful protease, plasmin, which then degrades the fibrin meshwork of thrombi. To prevent systemic activation of plasminogen, plasma contains several inhibitors of t-PA, the most important of which is plasminogen activator inhibitor-1 (PAI-1), a member of the serpin superfamily. As the ability to produce serpin-resistant variants of t-PA could increase the potential of this enzyme as a thrombolytic agent, we have used the known three-dimensional structure of the complex between trypsin and bovine pancreatic trypsin inhibitor (BPTI) to model the interactions between the active site of human t-PA and PAI-1. On the basis of this model we then altered by site-directed mutagenesis those amino acids of t-PA predicted to make contact with PAI-1 but not with the substrate plasminogen. We report here that although the resulting mutants have enzymatic properties similar to those of wild-type t-PA, they display significant resistance to inhibition by PAI-1. For example, following incubation with an amount of the serpin that completely inhibits the wild-type enzyme, one variant retains 95% of its initial activity. This mutant is also resistant to inhibition by the complex mixture of serpins present in human plasma.     

Discovery and Content Variation Analysis of Fibrinolytic Enzyme in Semen Sojae Praeparatum Fermentation

A strong fibrinolytic activity was demonstrated in the Semen Sojae Praeparatum(SSP), which is a famous traditional Chinese medicine. To study the activities and dynamic changes of fibrinolytic enzyme, standard fibrin plate was used to determine the fibrinolytic activity. For the first time fibrinolytic enzyme was found during the fermentation of SSP and the fibrinolytic activities of samples were shown to increase significantly over time. In the "yellow cladding" stage, the fibrinolytic activity was 619.75 IU/g. On day 6, 12 and 15 of the "secondary fermentation" stage, the fibrinolytic activity was 711.49 IU/g, 866.67 IU/g, 1 022.31 IU/g, respectively. The results indicate that fibrinolytic enzyme was generated during the fermentation of SSP and it displayed increasing activity which peaked at the "secondary fermentation" stage. The fibrinolytic enzyme was found to not only degrades fibrin directly, but also activate plasminogen to do so.     

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.     

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.     

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.     

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.     

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。     

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。     

Role of plasminogen activators and inhibitors in reproduction

The recent progress in the studies on the role of local and directed fibrinolysis controlled by plasminogen activators (PAs) and regulated by their inhibitors (PAIs) in reproduction is summarized. Hormone-induced coordinated expression of tissue type PA (tPA) and PAI type-1 (PAI-1) in the ovary is involved in the processes of ovulation and luteal regression; increases in urokinase type PA (uPA) and PAI-1 activity in the early stage of luteinized follicles may be responsible for ovarian tissue remodeling and angiogenesis; the PA system has been found to play an important role in spermatogenesis in testis and modulation of sperm maturation in epididymis. PA and matrix matalloproteanase (MMP) and their respective inhibitors have also been identified in trophoblast and uterus. The targeted proteolytic activity generated by the two systems may play an essential role in the processes of the cyclic uterine angiogenesis, implantation and placentation as well as parturition.     

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。     

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.     

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.     

蜡状芽孢杆菌Bc-05菌株纤溶酶的酶学性质

对蜡状芽孢杆菌Bc-05茵株的发酵上清液经硫酸铵分级沉淀、DEAE Sepharose Fast Flow阴离子交换层析所获得的纤溶酶进行性质研究.结果表明:经纤维蛋白平板法检测该酶有直接水解纤维蛋白和激活纤溶酶原的双重作用,最适作用温度37℃,最适pH=8.0,在pH=8.0条件下25℃和37℃放置24 h酶活力仍保持77.52%和78.96%,该酶体外对兔血凝块有明显的溶解作用;Ca2+,Mn2+离子对该酶具有激活作用,而Cu2+,Fe3+完全抑制其纤溶活性,PMSF,EDTA和DTT对该酶有抑制作用,说明活性中心含有二硫键、金属离子和丝氨酸;测其N端10个氨基酸序列为NH2-Val-Thr-Pro-Thr-Asn-Ala-Val-Asn-Thr-Gly,与其他生物来源的纤溶酶相比较没有同源性.     

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。     

蚯蚓纤溶酶的分离纯化及性质鉴定

以赤子爱胜蚓为材料，经过硫酸铵盐析、DEAE-Sepharose CL-6B离子交换层析、Sephadex G-75凝胶过滤和制备电泳分离得到四种纤溶酶组分．经PAGE鉴定四种组分为单一组分；SDS-PAGE测定其分子质量分别为27、28、23和33ku；等电点均在4．0以下；经甲苯胺蓝染色后都出现糖蛋白的阳性反应；用苯酚一硫酸法测定其中性糖含量分别为8.4％、13.5％、9．1％、6．8％；纤维平板法测得四种组分的活性存在明显差异；四种组分都具有直接溶解纤维蛋白和激活纤溶酶原的双重作用．pH值对四种组分纤溶活性稳定性的影响各不相同．四种组分对温度的适应范围较广．     

A potential basis for the thrombotic risks associated with lipoprotein(a)

Lipoprotein(a) (Lp(a)) has been strongly linked with atherosclerosis and is an independent risk factor for myocardial infarction. Distinguishing Lp(a) from other low-density lipoprotein particles is its content of a unique apoprotein, apo(a). The recently described sequence of apo(a) indicates a remarkable homology with plasminogen, the zymogen of the primary thrombolytic enzyme, plasmin. Lp(a) may contain 37 or more disulphide-looped kringle structures, which are 75-85% identical to the fourth kringle of plasminogen. Plasminogen receptors are widely distributed on blood cells and are present at extremely high density on endothelial cells. These receptors promote thrombolysis by accelerating plasminogen activation and protecting plasmin from inhibition. If, by molecular mimicry, Lp(a) competes with plasminogen for receptors, then thrombolysis would be inhibited and thrombosis promoted. Here we provide support for such a mechanism being responsible for the thrombotic risks associated with elevated Lp(a) by demonstrating that Lp(a) inhibits plasminogen binding to cells.     

蛹虫草纤溶酶酶学性质的初步研究

对蛹虫草纤溶酶的酶学性质进行了研究,结果表明,其纤溶活性随温度(≤45℃)的升高和保温时间的延长,逐渐下降,当温度超过50℃时,则随温度的升高和保温时间的延长急剧下降;蛹虫草纤溶酶的最适pH为7.0,在碱性条件下纤溶活性相对稳定;金属离子Ca2+和Fe2+对蛹虫草纤溶酶的活性有显著激活作用;抑制剂中PMSF对蛹虫草纤溶酶活性的抑制作用较小,而Aprotinin、EDAT和EGTA对其纤溶活性均有较显著的抑制作用,说明蛹虫草纤溶酶可能不同于之前报道的纳豆激酶(丝氨酸蛋白酶),而是一种全新的蛋白酶,但对这一点还需要进一步的研究验证.     

蛹虫草发酵产物新纤溶酶的分离纯化

笔者所在项目组的前期研究发现,蛹虫草深层培养可以产生至少两种纤溶酶.基于此,文中对蛹虫草深层培养产生的纤溶酶的分离纯化展开了研究.采用硫酸铵盐析、Sephadex G-25凝胶色谱、Phenyl-Sepharose HP疏水相互作用色谱、CM-Sepharose FF弱阳离子交换色谱和Superdex 75凝胶色谱对纤溶酶进行分离.最终纯化后的纤溶酶Ⅰ比活力达1467.44U/mg,纯化倍数为36.07,回收率为5.79%.纤溶酶Ⅱ比活力达1681.58U/mg,纯化倍数为41.33,回收率为4.00%.两种纤溶酶经电泳鉴定均达到电泳纯,相对分子质量分别约为28000和32000.     

中国传统发酵食品中溶栓酶活性检测方法比较

通过正交实验设计建立最佳琼脂糖-纤维蛋白平板法,并比较改进的纤维蛋白平板法和四肽底物法测定中国传统发酵食品中溶栓酶的活力的线性和精密度,以找出一个灵敏易操作、经济实惠、快速准确地标示溶栓酶效价的活性检测方法。实验结果显示纤维蛋白平板法和四肽底物法分别在50～800IU.mL-1、0.03～2.60U.mL-1范围内具有良好的线性关系,且日间和日内变异系数均小于6%。因此,纤维蛋白平板法与四肽底物法均能用于溶栓酶的活性检测,前者测定结果比较直观,但四肽底物法操作比较简单,经济成本相对较低,灵敏度高,更适合溶栓菌株的高通量筛选。