溶栓剂研究的新进展

近年来对溶栓剂在研究取得了很好的成果,主要集中在单链酶型纤溶酶原激活剂（scu-PA),组织型纤溶酶原激活剂（t-PA),葡萄球菌激酶（SAK）等,对分子进行设计改造,保留它们高效的溶栓功能同时赋予新的功能,在对分子空间结构域和活性功能区分析的基础上,研制出更有效的,更安全的溶栓制剂具有广阔的发展前景。     

蕲蛇酶溶栓和抗栓作用机理

蕲蛇酶以全面改善纤溶系统各组份功能,诱导血管内皮细胞释放t-PA,t-PA将纤维蛋白溶酶原变成纤溶酶,溶解血栓。此外,本品分解血纤维蛋白原,生成降解A肽纤维蛋白单体,从而促进t-PA激活纤维蛋白溶酶原转变成纤溶酶效应,促进溶栓。蕲蛇酶是目前唯一对各种诱导剂引起的血小板聚集都具有明显抑制作用的药物。蕲蛇酶对新鲜性、陈旧性,及各种模型动静脉血栓均具溶栓作用蕲蛇酶溶栓和抗栓作用机理     

t-PA F-E复合片段的三维结构模建及其结构与功能关系的研究

组织型纤溶酶原激活剂(t-PA)是一种在血栓和心梗的临床治疗中具有巨大应用前景的蛋白分子。野生型t-PA由于半衰期短,用量大,在实际治疗过程中会产生一些副作用,因此研制半衰期适当延长、血栓特异性增强的新型t-PA突变体非常重要。大量实验证明F区的C末端或F-E连接区对t-PA在血液中的半衰期及其血栓特性发挥了重要作用。1994年Smith等人报道了利用NMR研究F区-E区复合片     

尿激酶原的性质、结构、功能及其药代动力学和临床应用效果

溶栓疗法不仅已被常规地用于急性心肌梗死的治疗,而且也已用于其它血栓病的治疗中,如急性缺血性脑血栓、肺栓塞、急性周围动脉血栓等。尿激酶原是双链尿激酶的单链前体,它主要激活纤维蛋白表面的纤溶蛋白原,所以具有选择性溶栓作用。临床结果表明它是一种安全有效的溶栓药物,与t-PA、链激酶或尿激酶伍用均有协同作用。本文综述它了的特性、结构与功能,以及它的药代动力学和临床的治疗效果。     

t-PA蛋白质分子的改造

组织型纤溶酶原激活剂(t-PA)与纤维蛋白有高亲和力,具有特异地溶解血栓的功能。t-PA含有多个结构域,其分子结构和功能关系的阐明,促进了改造t-PA分子以发展更特异的血栓溶解剂的研究。     

肌注组织型纤溶酶原激活剂基因在小鼠体内表达

组织型纤溶酶原激活剂(t-PA)是人体内重要的丝氨酸蛋白酶,它能特异地激活血栓中的纤溶酶原,使之成为具有溶解纤维蛋白能力的纤溶酶,临床上将提纯的t-PA作为心血管栓塞性疾病急救和康复治疗的首选药物。由于t-PA在天然材料中含量甚微,难以大量制备,基因工程产品产量有限,临床应用货源紧缺且价格昂贵。为探索心血管疾病的基因治疗和预     

一种由藤黄微球菌产生的纤溶酶

血栓栓塞性疾病如心肌梗塞、脑栓塞等是危害人类健康、导致死亡率最高的疾病之一.全球每年约1 700万人死于心脑血管病,我国每年约有300万人死于此类疾病.治疗此类疾病的主要手段之一是溶栓疗法,即注射溶栓剂疏通血管,但传统的溶栓药物如t-PA、尿激酶等有半衰期短、使用量大、价格昂贵、易引发出血症等不良副作用,因此开发新型溶栓类药物,提供廉价高效、副作用小、使用方便的溶栓药物,对栓塞性疾病的治疗具有重要意义.     

组织型纤溶酶原激活剂（t—PA）工程细胞系遗传特性及成瘤性分析

组织型纤溶酶原激活剂(t-PA)因其在防止血栓形成中起重要作用而受到人们的重视。但由于t-PA在血液中半衰期很短,作为溶栓药,一时难于推广。为了延长半衰期、增强其特异活性,本组构建了t-PA突变体并在CHO-dhfr~-细胞中获得了高效表达。我们在细胞培养基中加入秋水仙素,通过低张、固定、染色,进行染色体分析,结果表明,t-PA工程细胞系染色体条数为20条,畸变类型有异着丝粒。四倍体、裂隙、断片,畸变率为15％,属于正常范围。同时我们对该细胞系进行成瘤性试验,选用4周龄裸鼠作为试验鼠,以Hela细胞为阳性对照,CHO-dhfr~-细胞为阴性对照,试验表明:t-PA工程细胞及表达产物对裸鼠均无成瘤性。     

人组织型纤溶酶原激活剂-水蛭素融合基因的构建及其在毕赤酵母中的表达

构建并表达兼有溶栓和抗凝活性、减少出血副作用的人组织型纤溶酶原激活剂(t-PA)和水蛭素(HV2)的融合蛋白。通过提取总RNA和RT-PCR获得t-PA基因,与HV2基因通过活化凝血因子X(Fxa)识别序列（IEGR）的对应碱基序列连接构成融合蛋白基因,融合蛋白基因经pGEM-T、pIC9克隆至表达载体pIC9K上,电转导入毕赤酵母（Pichia pastoris）GS115。转化子摇瓶内甲醇诱导表达。纤维蛋白平板溶圈法和纤维蛋白凝块法分别检测溶栓和抗凝活性。琼脂糖凝胶电泳结果显示克隆的t-PA基因片段大小为1700bp,序列测定结果表明其35位氨基酸由文献报道的精氨酸突变为色氨酸。限制性酶切和PCR鉴定结果均表明融合蛋白基因已克隆入表达载体和宿主菌。甲醇利用实验、G418抗性筛选获得多拷贝甲醇利用快型克隆。甲醇诱导表达产物具有纤溶活性并可被抗t-PA抗体抑制。完整融合蛋白无抗凝活性,但以Fxa裂解后可释放抗凝活性。同时,融合蛋白以单链和双链两种形式存在。融合蛋白在血栓部位特有的Fxa作用下靶向释放抗凝活性,具有溶栓抗凝双功能,有望降低临床出血副作用。     

t-PA突变体的构建、表达及生物学特性分析

t-PA是纤溶系统的生理性激活剂,但它在血浆中半衰期甚短及活性可被天然快速抑制剂PAI-1抑制等生理特性成为其作为临床溶栓剂的主要缺陷。为了研制新型t-PA溶栓剂,本文根据t-PA结构与功能研究的最新信息,针对延长t-PA半衰期、提高特异活性及引入PAI-1抗性等,设计并构建了8种t-PA突变体。进而在COS-7及CHO细胞中进行了表达研究,并对突变体表达产物的特性进行了分析和评价。     

Plasminogen activators: general aspects and recent developments

Considerable interest in plasminogen activators as human thrombolytic drugs has stimulated rapid biotechnologic progresses. These enzymes have been classified in two immunochemically distinct groups: "urokinase-like" activators or u-PA which do not interact with fibrin and "tissue activator-like" activators or t-PA which interact with fibrin. Plasminogen activators are widely distributed in normal and malignant tissues and they are implicated in various physiological and pathological processes. They maintain the functional integrity of the vascular system and their presence may be of importance in tissue remodeling and cell migration. Urokinase and streptokinase are used in human thrombolytic therapy. However, the properties displayed by t-PA suggest that this enzyme may be a superior fibrinolytic agent. The primary structures of urokinase and t-PA are known; both enzymes have been synthesized by DNA technology. In order to produce t-PA in large quantities by gene cloning, intensive studies are conducted by pharmaceutical industries. Clinical trials using t-PA for dissolving thrombi in coronary heart disease, strokes and pulmonary embolism are in progress. This review presents the molecular and structural properties of plasminogen activators, as well as related physiological, pathological and therapeutic aspects.     

A novel variant of t-PA resistant to plasminogen activator inhibitor-1; expression in CHO cells based on in silico experiments

Resistance to PAI-1 is a factor which confers clinical benefits in thrombolytic therapy. The only US FDA approved PAI-1 resistant drug is Tenecteplase?. Deletion variants of t-PA have the advantage of fewer disulfide bonds in addition to higher plasma half lives. A new variant was developed by deletion of the first three domains in t-PA in addition to substitution of KHRR 128-131 amino acids with AAAA in truncated t-PA. The specific activity of this new variant, 570 IU/μg, was found to be similar to those found in full length t-PA (Alteplase?), 580 IU/μg. A 65% and 85% residual activity after inhibition by rPAI-1 was observed for full length and truncated-mutant form, respectively. This new variant as the first PAI-1 resistant truncated t-PA may offer more advantages in clinical conditions in which high PAI-1 levels makes the thrombolytic system prone to re-occlusion.     

组织型纤溶酶原激活剂工程细胞系遗传特性及成瘤性分析

构建了突变体t-PA,使其在CHO-dhfr细胞中表达。获得t-PA工程细胞系。对该细胞系进行了遗传特性分析,在培养基中加入秋水仙素,通过低张、固定处理并将细胞滴片、染色,进行染色体分析,结果表明,该工程细胞系染色体条数为20条,畸变类型有异着丝粒、四倍体、裂隙、断片、微小体,畸变率为15％,属于正常范围。同时进行成瘤性检测,选用4周龄裸鼠作为试验鼠,以人宫颈癌细胞(Hela细胞)为阳性对照,CHO-dhfr细胞为阴性对照,试验表明,t-PA工程细胞及表达产物对裸鼠均无成瘤性。     

蕲蛇酶抗栓作用机理的初步分析

动物实验结果示,蕲蛇酶能裂解纤维蛋白原成为可溶性纤维蛋白,降低血中纤维蛋白原浓度,抑制血小板聚集,对抗在酶诱导的血浆凝块订的血浆凝块回缩,因而发挥防血栓形成作用。对纤维蛋白平板试验无直接溶纤作用,但能增加实验动物血中ｔ－ＰＡ活性。可能通过促使血管内皮细胞释放ｔ－ＰＡ而发挥溶栓作用。     

The potential improvement of thrombolytic therapy by targeting with bispecific monoclonal antibodies: Why they are used and how they are made

The generation of the proteolytic enzyme plasmin from its inactive precursor plasminogen, mediated by so called plasminogen activators, is the essential step in thrombolytic therapy. Plasmin is responsible for the degradation of the insoluble fibrin, the major component of a thrombus, to soluble fibrin degradation products. So far, the use of the more recently developed thrombolytic agents single-chain urokinase-type plasminogen activator (scu-PA) and tissue-type plasminogen activator (t-PA) were disappointing, mainly due to some of their negative propertiesin vivo, i.e., rapid inhibition and/or hepatic clearance. Besides some background information on the haemostatic balance; t-PA and scu-PA structure; and mechanisms of action, we here review some reported attempts to improve on these agents for thrombolytic therapy following various strategies. One of the more potential strategies, antibody-targeted thrombolytic therapy using bispecific monoclonal antibodies, is discussed somewhat more extensively, as are the several procedures that can befollowed for bispecific antibody preparation.     

Molecular mechanisms of fibrinolysis and their application to fibrin-specific thrombolytic therapy

The fibrinolytic system comprises a proenzyme, plasminogen, which can be converted to the active enzyme, plasmin, which degrades fibrin. Plasminogen activation is mediated by plasminogen activators, which are classified as either tissue-type plasminogen activators (t-PA) or urokinase-type plasminogen activators (u-PA). Inhibition of the fibrinolytic system may occur at the level of the activators or at the level of generated plasmin. Plasmin has a low substrate specificity, and when circulating freely in the blood it degrades several proteins including fibrinogen, factor V, and factor VIII. Plasma does, however, contain a fast-acting plasmin inhibitor, alpha 2-antiplasmin, which inhibits free plasmin extremely rapidly but which reacts much slower with plasmin bound to fibrin. A "systemic fibrinolytic state" may, however, occur by extensive activation of plasminogen and depletion of alpha 2-antiplasmin. Clot-specific thrombolysis therefore requires plasminogen activation restricted to the vicinity of the fibrin. Two physiological plasminogen activators, t-PA and single-chain u-PA (scu-PA) induce clot-specific thrombolysis, via entirely different mechanisms, however. t-PA is relatively inactive in the absence of fibrin, but fibrin strikingly enhances the activation rate of plasminogen by t-PA. This is explained by an increased affinity of fibrin-bound t-PA for plasminogen and not by alteration of the catalytic rate constant of the enzyme. The high affinity of t-PA for plasminogen in the presence of fibrin thus allows efficient activation on the fibrin clot, while no significant plasminogen activation by t-PA occurs in plasma. scu-PA has a high affinity for plasminogen (Km = 0.3 microM) but a low catalytic rate constant (kcat = 0.02 sec-1). However, scu-PA does not activate plasminogen in plasma in the absence of a fibrin clot, owing to the presence of (a) competitive inhibitor(s). Fibrin-specific thrombolysis appears to be due to the fact that fibrin reverses the competitive inhibition. The thrombolytic efficacy and fibrin specificity of natural and recombinant t-PA has been demonstrated in animal models of pulmonary embolism, venous thrombosis, and coronary artery thrombosis. In all these studies intravenous infusion of t-PA at sufficiently high rates caused efficient thrombolysis in the absence of systemic fibrinolytic activation. The efficacy and relative fibrinogen-sparing effect of t-PA was recently confirmed in three multicenter clinical trials in patients with acute myocardial infarction.(ABSTRACT TRUNCATED AT 400 WORDS)     

Different receptors mediate the hepatic catabolism of tissue-type plasminogen activator and urokinase.

Tissue-type plasminogen activator (t-PA) and urokinase (u-PA) are proteins with partial structural similarity and which are of importance in the therapy of thrombotic diseases. Both are known to be cleared from the circulation in vivo by uptake in the liver. The present study investigated whether the hepatic catabolism of u-PA and t-PA is mediated by a common receptor system. Four experimental protocols of increasing complexity were used: hepatocyte plasma membranes, isolated primary hepatocytes, liver perfusion and whole animals. For t-PA, a specific high-affinity binding site to hepatocytes and plasma membranes could be defined with a mean Kd of 4 +/- 3 nM, whereas the Kd for u-PA was less than 300 nM. Binding of t-PA could not be competed for by u-PA, and vice versa. Furthermore, clearance of t-PA in isolated perfused rat livers and in rabbits in vivo was 3-fold higher than that of u-PA, and a 50-100-fold molar excess of u-PA failed to inhibit clearance of t-PA in either system, and vice versa. Taken together, the results imply that hepatic elimination of t-PA and u-PA is mediated by distinct receptor systems of differing affinity.     

Inactivation of tissue plasminogen activator in plasma. Demonstration of a complex with a new rapid inhibitor

A new specific and sensitive method for determination of tissue plasminogen activator (t-PA) in plasma samples has been used to demonstrate the presence of a fast inhibitor to t-PA in plasma. By adding [35S]Met internally labeled t-PA (Mr approximately 70,000) to plasma, we were able to demonstrate the rapid formation of a stable complex with an apparent molecular weight of about 115,000 as estimated by gel filtration. The complex was partially purified by immunoadsorbtion chromatography on insolubilized antibodies against porcine t-PA, and a molecular weight of about 120,000 was found by dodecyl sulfate-polyacrylamide gel electrophoresis. From the apparent molecular weight of the complex (120,000) and the molecular weight of t-PA (70,000), a molecular weight of about 50,000 would be expected for the inhibitor. However, gel filtration of inhibitor-rich plasma resulted in the appearance of a symmetrical peak of t-PA inhibitory activity with an apparent molecular weight of about 205,000. The reason for this discrepancy is not known, but several different models are possible.     

半衰期延长获得PAI－1抗性的t—PA突变体的构建、表达及特性分析

用基因重级及定位突变技术成功地构建了ｔ－ＰＡ的Ｋ１区缺失突变体ｔ－ＰＡｄｅｌＫ１、ＰＡＩ－１结合位点缺失突变体ｔ－ＰＡｄｅｌ（２９６－３０２）及两的组合突变全ｔ－ＰＡｄｅｌ（Ｋ１,２９６－３０２）,并在ＣＯＳ－７细胞中实现三的暂时性表达,在ＣＨＯ细胞中实现了ｔ－ＰＡｄｅｌ（Ｋ１,２９６－３０２）的稳定性表达。对表达产物的生物学特性分析表明,ｔ－ＰＡｄｅｌ（２９６－３０２）及ｔ－ＰＡｄｅｌ（Ｋ１