平原人进驻高原后凝血和纤溶功能的改变及其意义

目的:探讨高原低氧环境下凝血及纤溶功能的变化.方法:对从平原(海拔1 400 m)进驻3700 m和5 380 m高原第7 d及半年的40名健康青年血浆抗凝血酶Ⅲ(AT-M)、纤溶酶原含量(PLG)、D-二聚体含量(DD)、组织纤溶酶原激活物活性(t-PA)、纤溶酶原激活抑制物活性(PAI)、纤溶蛋白原(Fg)、α2-抗纤溶酶抑制物活性(α2-PI)进行检测,并与20名平原健康青年作对照.结果:高原低氧环境AT-Ⅲ、t-PA明显低于平原(P<0.05或P<0.01),且随海拔高度的升高而降低(P<0.01),随居住时间的延长而升高(P<0.05或P<0.01).PLG、DD、PAI、α2-PI及Fg在海拔3 700 m第7 d和海拔5 380 m第7d及半年均较平原增高显著((P<0.05或P<0.01),且随海拔高度的升高而增高,居住时间的延长而降低(P<0.05或P<0.01);3 700 m居住半年时较平原无显著性差异(P>0.05).结论:高原低氧存在凝血功能紊乱,表现为凝血与纤溶被激活的同时伴有纤溶受抑,凝血及纤溶的平衡被破坏而使血液呈高凝和低纤溶状态.     

Ⅱ型糖尿病视网膜病变与纤溶系统的关系探讨

目的:探讨2型糖尿痛视网膜病变与纤溶系统的关系。方法:随机选取2型糖尿病视网膜痛变患者30例,无并发症患者30例,健康人30例,测定血浆纤溶酶原、纤溶酶原激活抑制物,纤溶酶原(PLG)、纤溶酶原激活抑制物(PAI-1)均采用发色底物法检测。结果:与对照组相比2型糖尿病患者血浆PLG、PAI-1水平升高(P＜0．05),糖尿病视网膜病变组升高更明显(P＜0．01)。结论:糖尿病患者血液存在低纤状态,当发生视网膜并发症者更为明显,检测2型糖尿病患者血浆PLG、PAI-1水平对糖尿痛视网膜病变的预防及治疗具有一定的临床意义。     

纤溶酶原激活剂抑制物2型抑制细胞凋亡的研究进展

纤溶酶原激活剂抑制物２型（ＰＡＩ－２）是一种多功能蛋白质,除了能有效抑制尿激酶（ｕＰＡ）和双链组织型纤溶酶原激活物（ｔＰＡ）而调节纤溶活性外,还参与了很多其它的生理病理过程,例如组织重建、胚胎发育、感染、免疫系统发育、肿瘤浸润和迁移等。更有研究表明胞内型ＰＡＩ－２在抑制细胞凋亡方面也发挥着重要作用。本文就近年来ＰＡＩ－２抑制细胞凋亡的研究进展作一综述。     

重组纤溶酶原激活剂抑制剂－2基因在HT1080亚克隆中的表达

重组纤溶酶原激活剂抑制剂－２基因在ＨＴ１０８０亚克隆中的表达曹祥荣（南京师范大学生物学系,２１００２４）关键词纤溶酶原激活剂,基因重组,纤溶酶原激活剂抑制剂活性表达目前认为纤溶酶系统是肿瘤细胞浸润和转移蛋白水解过程中重要的酶,纤溶酶能直接水解细胞外间...     

t—PA cDNA基因转移与基因治疗

组织型纤溶酶原激活剂（ｔ－ＰＡ）能够特异、高效地溶解血栓,在纤溶系统中起着重要作用,ｔ－ＰＡ与其抑制物（ＰＡＩ）平衡失调,可导致多种疾病的发生。基因转移技术使外源ｔ－ＰＡ ｃＤＮＡ成功地在血管内皮细胞中得以表达,为防治以血栓形成为主要病理变化的血管性疾病开辟了广阔的前景。     

纤溶酶原激活物抑制因子—1蛋白分子的结构与功能

纤溶酶原激活物抑制因子－１（ＰＡＩ－１）是组织型纤溶酶原激活物（ｔ－ＰＡ）和尿型纤溶酶原激活物（ｕ－ＰＡ）的特异性抑制剂。对ＰＡＩ－１分子的结构与功能的认识,有助于了解ＰＡＩ－１作用的机理。本综述了近年来对ＰＡＩ－１蛋白分子结构与功能研究的进展,介绍了ＰＡＩ－１分子中一些区域的作用以及影响ＰＡＩ－１抑制活性的一些因子。     

蚯蚓体内一种纤溶酶原激活剂(e-PA)的部分性质研究

从赤子爱胜蚓（Ｅｉｓｅｎｉａｆａｅｔｉｄａ）中纯化出的一种纤溶酶原激活剂（ｅ－ＰＡ）在纤维蛋白平板上可表现出三种活性,分别记为：ＣＦＰｇ,ｕＣＦＰｇ和ｕＣＦ．为更好了解各种活性与ｅ－ＰＡ的纤溶能力的关系,考察了在ＳＤＳ和不同抑制剂存在下各种活性的变化．结果表明,ＳＤＳ可以增强ＣＦＰｇ活性且使得ｅ－ＰＡ变得对一些抑制剂更敏感;ｌｅｕｐｅｐｔｉｎ,ｃｈｙｍｏｓｔａｔｉｎ,ｐｅｐｓｔａｔｉｎ,ａｐｒｏ－ｔｉｎｉｎ,ｐｈｅｎｙｌｍｅｔｈｙｌｓｕｌｆｏｎｙｌｆｌｕｏｒｉｄｅ（ＰＭＳＦ）和ｄｉｔｈｉｏｔｈｒｅｉｔｏｌ（ＤＴＴ）对ｕＣＦ没有影响;ｐｅｐ－ｓｔａｔｉｎ能增强ＣＦＰｇ和ｕＣＦＰｇ活性,Ｅ－６４（一种巯基抑制剂）能增强ｕＣＦＰｇ和ｕＣＦ活性．这些现象说明不能简单将ｅ－ＰＡ归结为丝氨酸蛋白酶或巯基蛋白酶．此外又以纤溶酶原为底物,分析了ｅ－ＰＡ在体外降解天然蛋白质的肽键特异性,结果表明：ｅ－ＰＡ可以切割碱性氨基酸,小的中性氨基酸及Ｍｅｔ的羧基端,同时ｅ－ＰＡ确能将纤溶酶原切割为纤溶酶;这一结论为ｅ－ＰＡ有可能成为新型溶栓药物提供了生化基础．     

表达人PAI-1的重组腺病毒的制备及检测

人纤溶酶原激活剂抑制物1（PAI-1）基因与复制缺陷型腺病毒载体AdCMVHSgD重组,与pJM17质粒共转染293细胞,采用不铺琼脂的方法产生重组病毒.PCR证实PAI-1基因重组进入腺病毒.进一步感染B16（F10）细胞,细胞表面洗提物和上清分别经纤维显示胶和反向纤维蛋白显示胶检测PAI-1抑制纤溶酶原激活剂（PA）的活性.     

同型半胱氨酸对人脐静脉血管内皮细胞tPA 及PAI-1基因表达的影响

目的探讨同型半胱氨酸(Hcy)对纤溶系统的影响,观察Hcy在转录水平对人脐静脉血管内皮细胞(HUVEC)表达组织型纤溶酶原激活物(tPA)和纤溶酶原激活物抑制剂1(PAI1)的影响。方法将体外培养的HUVEC分为生理浓度(10μmol/LHcy)组,病理浓度(50、200、500μmol/L)Hcy组及单纯培养基组(0μmol/LHcy),培养24h后,提取RNA,反转录聚合酶链反应分析(RTPCR)法分析各组tPA及PAI1基因表达水平。结果500μmol/LHcy组与10μmol/LHcy组相比,tPAmRNA基因表达明显下调(P<0.05),PAI1mRNA表达则明显上调(P<0.05)。而与单纯培养基组相比,10μmol/LHcy组tPAmRNA表达明显增高(P<0.05)。结论生理浓度Hcy可以增加纤溶系统活性,减少血栓性疾病的发生。高Hcy(病理浓度)则抑制纤溶系统活性,促进缺血性心脑血管疾病的发生。     

睾酮对人血管内皮细胞产生NO、tPA和PAI-1的影响

目的:观察不同浓度睾酮对人血管内皮细胞生长、产生舒张因子及纤溶活性的影响.方法:体外培养人脐静脉内皮细胞(HUVEC),分为五个浓度睾酮组及单纯培养基对照组.做MTT实验观察睾酮对HUVEC生长的影响;还原酶法测定各组HUVEC释放NO量;ELISA法测定各组培养基中纤溶酶原激活物(tPA)及其抑制物(PAI-1)含量.结果:3×10-10mol/L-3×10-8mol/L睾酮组与对照组相比细胞生长良好,无明显差别;而大于生理剂量的两组(3×10-6～3×10-1mol/L)3 d后细胞生长明显受到抑制(P＜0.05).各浓度睾酮组产生NO量与对照组无明显区别.而3×10-10 mol/L～3×10-8 mol/L睾酮组tPA含量明显高于对照组(P＜0.01);大剂量组tPA产生明显减少(P＜0.01).所有实验组的PAI-1含量均明显降低.结论:生理及略低于生理剂量的睾酮对HUVEC生长及释放NO无不利影响,且增加纤溶活性.说明生理剂量睾酮对血管内皮功能、心血管系统有一定的保护作用,有利于防止动脉粥样硬化的发生、发展.     

Function of the central domain of streptokinase in substrate plasminogen docking and processing revealed by site-directed mutagenesis

The possible role of the central beta-domain (residues 151-287) of streptokinase (SK) was probed by site-specifically altering two charged residues at a time to alanines in a region (residues 230-290) previously identified by Peptide Walking to play a key role in plasminogen (PG) activation. These mutants were then screened for altered ability to activate equimolar "partner" human PG, or altered interaction with substrate PG resulting in an overall compromised capability for substrate PG processing. Of the eight initial alanine-linker mutants of SK, one mutant, viz. SK(KK256.257AA) (SK-D1), showed a roughly 20-fold reduction in PG activator activity in comparison to wild-type SK expressed in Escherichia coli (nSK). Five other mutants were as active as nSK, with two [SK(RE248.249AA) and SK(EK281.282AA), referred to as SK(C) and SK(H), respectively] showing specific activities approximately one-half and two-thirds, respectively, that of nSK. Unlike SK(C) and SK(H), however, SK(D1) showed an extended initial delay in the kinetics of PG activation. These features were drastically accentuated when the charges on the two Lys residues at positions 256 and 257 of nSK were reversed, to obtain SK(KK256.257EE) [SK(D2)]. This mutant showed a PG activator activity approximately 10-fold less than that of SK(D1). Remarkably, inclusion of small amounts of human plasmin (PN) in the PG activation reactions of SK(D2) resulted in a dramatic, PN dose-dependent rejuvenation of its PG activation capability, indicating that it required pre-existing PN to form a functional activator since it could not effect active site exposure in partner PG on its own, a conclusion further confirmed by its inability to show a "burst" of p-nitrophenol release in the presence of equimolar human PG and p-nitrophenyl guanidino benzoate. The steady-state kinetic parameters for HPG activation of its 1:1 complex with human PN revealed that although it could form a highly functional activator once "supplied" with a mature active site, the Km for PG was increased nearly eightfold in comparison to that of nSK-PN. SK mutants carrying simultaneous two- and three-site charge-cluster alterations, viz., SK(RE24249AA:EK281.282AA) [SK(CH)], SK(EK272.273AA;EK281.282AA) [SK(FH)], and SK(RE248.249AA;EK272.273AA:EK281.282AA+ ++) [SK(CFH)], showed additive/synergistic influence of multiple charge-cluster mutations on HPG activation when compared to the respective "single-site" mutants, with the "triple-site" mutant [SK(CFH)] showing absolutely no detectable HPG activation ability. Nevertheless, like the other constructs, the double- and triple-charge cluster mutants retained a native like affinity for complexation with partner PG. Their overall structure also, as judged by far-ultraviolet circular dichroism, was closely similar to that of nSK. These results provide the first experimental evidence for a direct assistance by the SK beta-domain in the docking and processing of substrate PG by the activator complex, a facet not readily evident probably because of the flexibility of this domain in the recent X-ray crystal structure of the SK-plasmin light chain complex.     

组织型纤溶酶原活化物的纯化及性质研究

新鲜猪心组织制成丙酮粉后,用0.45mol/L,pH4.2醋酸钾抽提组织型纤溶酶原活化物(t-PA)。抽提液经硫酸铵盐析,Benzamidine和血纤维蛋白亲和层析,Sephadex G-150凝胶过滤,纯化得到t-PA。比活11000IU/mg,经SDS-聚丙烯酰胺凝胶电泳鉴定,分子量为67000。 本文比较了t-PA、高分子量尿激酶(H-UK)和低分子量尿激酶(L-UK)的热稳定性及抑制剂对它们的抑制作用。结果表明,抑制剂对H-UK的抑制作用最强,L-UK次之,t-PA最弱;三者的热稳定性相似。     

Characterization of the Binding Sites for Plasminogen and Tissue-Type Plasminogen Activator in Cytokeratin 8 and Cytokeratin 18

Cytokeratin 8 (CK8) is an intermediate filament protein that penetrates to the external surfaces of breast cancer cells and is released from cells in the form of soluble heteropolymers. CK8 binds plasminogen and tissue-type plasminogen activator (t-PA) and accelerates plasminogen activation on cancer cell surfaces. The plasminogen-binding site is located at the C-terminus of CK8. In this study, we prepared GST-fusion proteins which contained either 174 amino acids from the C-terminus of CK8 (CK8f) or 134 amino acids from the C-terminus of CK18 (CK18f). A third GST-CK fusion protein was identical to CK8f except that the C-terminal lysine was mutated to glutamine (CK8f_K483Q). CK8f bound plasminogen; the K _D was 0.5 M. Binding was completely inhibited by ACA. CK8f_K483Q also bound plasminogen, albeit with decreased affinity (K _D 1.5 M). CK18f did not bind plasminogen at all. All three fusion proteins bound t-PA equivalently, providing the first evidence that CK18 may function as a t-PA receptor. t-PA and plasminogen cross-competed for binding to CK8f. Thus, t-PA and plasminogen cannot bind to the same CK8f monomer simultaneously. Nevertheless, CK8f still promoted plasminogen activation, probably reflecting the fact that CK8f was purified in dimeric or tetrameric form. These studies demonstrate that CK8 may promote plasminogen activation by t-PA only when present in an oligomerized state. CK18 may participate in the oligomer, together with CK8, based on its ability to bind t-PA.     

Expression of plasminogen activator and plasminogen activator inhibitor mRNA in human fibroblasts grown on different substrates

mRNA levels for urokinase type plasminogen activator (uPA), tissue type plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator inhibitor-2 (PAI-2) were examined in human diploid (neonatal foreskin) fibroblasts grown in 200-ml microcarrier suspension culture. Four different substrates were used. These included gelatin-coated polystyrene plastic, DEAE-dextran, glass-coated polystyrene plastic and uncoated polystyrene plastic. Our previous studies have shown that culture fluids from diploid fibroblasts grown on DEAE-dextran contained higher levels of plasminogen-dependent fibrinolytic activity than culture fluids from the same cells grown on other substrates. The increased plasminogen activator activity was due largely to elevated amounts of tPA (In Vitro Cell. Develop. Biol. 22: 575–582, 1986). The present study shows that there is a corresponding elevation of tPA mRNA in diploid fibroblasts cultured on DEAE-dextran relative to the other substrates. There does not appear to be any difference in uPA mRNA or in mRNA for PAI-1 or PAI-2 produced by the same cells on the four substrates. These data suggest that the influence of the substrate on plasminogen activator production is mediated at the genetic level.     

Moderate Exercise and Fibrinolytic Potential in Obese Sedentary Men with Metabolic Syndrome

Objective: To investigate the impact of 30‐minute walking exercise at 70% Vo _2max on tissue plasminogen activator (t‐PA) Ag and plasminogen activator inhibitor type 1 (PAI‐1) Ag in obese sedentary males. Research Methods and Procedures: A controlled observational study of the effect of a 30‐minute acute exercise bout at 70% Vo _2max on plasma t‐PA antigen and PAI‐1 antigen in 10 obese sedentary males matched for age, ethnic origin, and smoking status with 10 nonobese sedentary male controls. Results: The obese group remained hypofibrinolytic compared with the nonobese group at all time‐points before, during, and after exercise. t‐PA increased in both groups with exercise before returning to baseline values 30 minutes after exercise. PAI‐1 did not significantly change in either group with exercise but rose significantly 30 minutes after exercise in the obese group. Discussion: The reduction in fibrinolytic potential in the obese group represents an increase in acute thrombotic risk and could account for the increased incidence of exercise‐associated myocardial infarction observed in sedentary obese groups.     

Role of the amino-terminal region of streptokinase in the generation of a fully functional plasminogen activator complex probed with synthetic peptides.

The mechanism whereby fragments of streptokinase (SK) derived from its N terminus (e.g., SK1-59 or SK1-63) enhance the low plasminogen (PG)-activating ability of other fragments, namely SK64-386, SK60-414, SK60-387, and SK60-333 (reported previously), has been investigated using a synthetic peptide approach. The addition of either natural SK1-59, or chemically synthesized SK16-59, at saturation (about 500-fold molar excess) generated amidolytic and PG activation capabilities in equimolar mixtures of human plasminogen (HPG) and its complementary fragment (either SK60-414 or SK56-414, prepared by expression of truncated SK gene fragments in Escherichia coli) that were approximately 1.2- and 2.5-fold, respectively, of that generated by equimolar mixtures of native SK and HPG. Although in the absence of SK1-59 equimolar mixtures of SK56-414 and HPG could generate almost 80% of amidolytic activity, albeit slowly, less than 2% level of PG activation could be observed under the same conditions, indicating that the contribution of the N-terminal region lay mainly in imparting in SK56-414 an enhanced ability for PG activation. The ability of various synthetic peptides derived from the amino-terminal region (SK16-51, SK16-45, SK37-59, SK1-36, SK16-36, and SK37-51) to (1) complement equimolar mixtures of SK56-414 and HPG for the generation of amidolytic and PG activation functions, (2) inhibit the potentiation of SK56-414 and HPG by SK16-59, and (3) directly inhibit PG activation by the 1:1 SK-HPG activator complex was tested. Apart from SK16-59, SK16-51, and 16-45, the ability to rapidly generate amidolytic potential in HPG in the presence of SK56-414 survived even in the smaller SK-peptides, viz., SK37-59 and SK37-51. However, this ability was abolished upon specifically mutating the sequence -LTSRP-, present at position 42-46 in native SK. Although SK16-51 retained virtually complete ability for potentiation of PG activation in comparison to SK16-59 or SK1-59, this ability was reduced by approximately fourfold in the case of SK16-45, and completely abolished upon further truncation of the C-terminal residues to SK16-36 or SK1-36. Remarkably, however, these peptides not only displayed ability to bind PG, but also showed strong inhibition of PG activation by the native activator complex in the micromolar range of concentration; the observed inhibition, however, could be competitively relieved by increasing the concentration of substrate PG in the reaction, suggesting that this region in SK contains a site directed specifically toward interaction with substrate PG. This conclusion was substantiated by the observation that the potentiation of PG activating ability was found to be considerably reduced in a peptide (SK25-59) in which the sequence corresponding to this putative locus (residues 16-36) was truncated at the middle. On the other hand, fragments SK37-51 and SK37-59 did not show any inhibition of the PG activation by native activator complex. Taken together, these findings strongly support a model of SK action wherein the HPG binding site resident in the region 37-51 helps in anchoring the N-terminal domain to the strong intermolecular complex formed between HPG and the region 60-414. In contrast, the site located between residues 16 and 36 is qualitatively more similar to the previously reported PG interacting site (SK254-273) present in the core region of SK, in being involved in the relatively low-affinity enzyme-substrate interactions of the activator complex with PG during the catalytic cycle.     

纤维蛋白对尿激酶原激活纤溶酶原反应动力学的影响

反应体系中存在的纤维蛋白（fibrin）对尿激酶（UK）、scu-PA以及组织型纤溶酶原激活剂（t-PA）激活纤溶酶原（plasminogen）的反应有不同的作用：UK、t-PA激活plasminogen的反应可被反应体系中存在的fibrin所加强;fibrin对scu-PA激活plasminogen反应的动力学常数无明显影响;但对小分子质量scu-PA与单链抗体的嵌合分子激活plasminogen的反应起明显的抑制作用.为确定反应体系中存在的fibrin对scu-PA的K区插入突变体-InB激活plasminogen反应的影响,测定了在反应体系中存在fibrin的情况下的InB激活plasminogen反应的K_m^fibrin以及k_cat^fibrin.K_m^fibrin＝4.2 μmol·L^-1,远远大于无fibrin时的K_m＝0.379 μmol·L^-1,说明有fibrin存在时突变体InB与天然底物plasminogen的亲和性降低了.k_cat^fibrin＝0.107 s^-1,也远远大于无fibrin时k_cat＝0.0165 s^-1,说明有fibrin存在时突变体InB对plasminogen的反应活性增强了.原因可能是：与fibrin结合的plasminogen的构象发生了有利于被纤溶酶原激活剂水解的变化.     

人子宫内膜纤蛋白溶酶元激活因子及其抑制因子的分布与调控

人子宫内膜中存在组织型(tPA)及尿激酶型(uPA)两类纤蛋白溶酶元激活因子,其含量在增殖期高于分泌期。本文应用免疫组织化学定位证实uPA及tPA两类抗原存在于子宫内膜的腺体细胞和间质细胞中。应用SDS-PAGE分高蛋白质,继而应用纤蛋白-琼脂糖铺盖技术测得离体培养下间质细胞仅释放tPA,腺体细胞仅释放uPA,但两种细胞均分泌PA的抑制因子(PAI)。培液中加入孕酮,明显抑制PA和刺激PAI生成。雌二醇作用与孕酮相反。某些肽类激素hCG、PRL、GnRH及cAMP作用基本与雌二醇相同。但福司克林(FK)则刺激间质、腺体两种细胞产生tPA及少量uPA,抑制PAI生成。本工作表明人子宫内膜中存在PA及PAI作用相反的酶,受激素调控,其生理意义尚待进一步探讨。     

Suppression of tumor growth and invasion in 9,10 dimethyl benz(a) anthracene induced mammary carcinoma by the plant bioflavonoid quercetin

Administration of quercetin, a common polyphenolic component of many vascular and edible plants including vegetables, fruits and tea significantly reduced the tumor volume in rats induced for mammary carcinoma using dimethyl benz (a) anthracene (DMBA). Dose response was assessed, by treating the animals with different doses (15-45 mg/kgbw) of quercetin and 25 mg/kgbw was taken as effective dose. Quercetin was administered as an intra tumoral injection once a week for 4 weeks. Serum levels of carcino embryonic antigen (CEA), a potent marker for tumor growth and invasion was significantly decreased on quercetin treatment. Quercetin caused a significant decrease in the activities of acid phosphatase and Cathepsin D in serum of experimental animals. Activities of lysosomal enzymes- (beta-D galactosidase, beta-D glucuronidase, beta-D glucosidase and sialidase), in serum and tissue were significantly altered in DMBA animals compared to control animals. However, quercetin treatment caused no significant change in lysosomal enzyme activities in tissues, whereas the activities were significantly lowered in serum. Partial purification of tissue type plasminogen activator (t-PA) from the tumor and kidney showed increased activity in the DMBA induced animals. Serum urokinase, -like plasminogen activator (u-PA) was also increased in animals with tumor, indicating tumor invasion. Administration of quercetin caused a significant decrease of both t-PA and u-PA. In conclusion, the present study suggests the possible role of quercetin in primary and invasive mammary tumor treatment. The above observations in vivo warrant further studies, due to the easy availability, common occurrence and low toxicity of this dietary bioflavonoid.