溶栓剂的研发现状及展望

由血栓引起的血栓性疾病是一种常见的心脑血管疾病。溶栓剂是预防与治疗心脑血管疾病的主要抗血栓药物。本文总结性地概述了目前国内外各种新型溶栓剂的溶栓机理、临床应用效果及其副作用等。其中,第三代溶栓剂组织型纤溶酶原激活剂变异体、导向溶栓剂、嵌合体溶栓剂,以及部分天然溶栓剂包括蝙蝠唾液纤溶酶原激活剂、蛇毒纤溶酶原激活剂、水蛭素及其变异体等均通过将纤溶酶原激活为纤溶酶溶解血栓。临床实验结果表明它们在延长体内半寿期、增强对血纤维蛋白选择性和溶栓效率等方面有较大的改进,但溶栓治疗后的冠状动脉再栓塞,颅内出血等问题仍是目前未解决的难题;而纳豆激酶及豆豉纤溶酶由于溶栓原理的不同,引起内出血的几率较低。溶栓剂未来的发展方向为如何综合不同药物的优点,使其更加安全与廉价。     

用静息细胞培养法研究影响组织型纤溶酶原激活剂生物合成的因素

建立了基因工程菌株里氏木霉(Trichoderma reesei)306生物合成组织型纤溶酶原激活剂(t—PA)的静息细胞培养系统。确定静息细胞培养基的成分为：葡萄糖1．0g／L，尿素1．0g／L，K2HPO41．0g／L；静息细胞培养的条件为：选择发酵30h的菌体，10％的接种量，pH5．4，150r／min，28℃静息培养12h。并在该静息细胞培养系统中，研究了糖类、有机酸、氨基酸和金属离子等因素对里氏木霉306生物合成组织型纤溶酶原激活剂的影响。     

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。     

重组人组织型纤溶酶原激活剂缺失变体的工业化制备与性质研究

将含有人组织型纤溶酶原激活剂缺失变体(K2tPA)重组表达质粒的工程菌经10L种子罐培养及100L发酵,IPTG诱导表达,其表达量为占菌体总蛋白的20％,表达产物经体外变复性、TI—Sepharose亲和层析、SP-Sepharose离子交换层析,每100L发酵液得重组人组织型纤溶酶原激活剂缺失变体(K2tPA)纯品4g,纯度达95％以上,比活大于500000IU／mg,内毒素及热源含量、宿主蛋白残留量、宿主DNA残留量等均达到临床使用标准．其分子量(质谱测定)、氨基酸组成、N、C-端氨基酸序列分析等均与理论值相符．同时进行了等电点测定及肽图分析等性质研究．     

重组人组织型纤溶酶原激活剂缺失变体基因的克隆及工程菌的构建与鉴定

利用RT-PCR技术,从人脐带静脉上皮细胞中克隆人组织型纤溶酶原激活剂基因,将其接入TA克隆载体PCR2．1中,经DNA序列测定后,以该重组质粒DNA为模板,用PCR方法获得了人组织型纤溶酶原激活剂缺失变体(K2tPA)基因,将其转入pET29a,构建了重组表达质粒pET29a／K2tPA,并转化至大肠杆菌BL21(DE3)中,构成工程菌．经IPTG诱导表达,在40kDa处有一明显表达条带,表达量为约占菌体总蛋白的20％．该菌种在贮存与复苏及传代过程中具有良好的质粒稳定性和表达稳定性．     

安舒克栓酶和水蛭素融合基因在枯草杆菌中的表达(简报)

安舒克栓酶(Antithromboase,AT)是本实验室从枯草杆菌N18中分离得到的具有强烈水解纤维蛋白活性的蛋白酶.研究表明,该酶具有直接纤溶和纤溶酶原激活剂的双重作用,且有较强的纤维蛋白亲和性,是极具开发价值的溶栓药物.来源于医用水蛭的水蛭素(hirudin,Hir)是凝血酶的最强有力的天然抑制剂,是极具优势的抗凝药物.但仍各有不足:安舒克栓酶只有溶栓功能,不能抗栓,不能解决血栓治疗中再栓塞的问题;水蛭素只有抗凝作用,不具溶栓功能.如果将这两种从不同途径治疗血栓的药物.通过融合     

尿激酶型纤溶酶原激活剂(uPA)系统在子宫内膜癌中的研究进展

尿激酶型纤溶酶原激活系统是纤溶系统的重要组成部分，它通过水解细胞外间质，参与组织改造和细胞迁移，在肿瘤细胞的侵袭和转移中发挥作用；讨论了uPA系统的结构、作用机理及与子宫内膜癌的关系，旨在为子宫内膜癌的诊断和治疗提供新方法。     

急性缺血性脑卒中rt-PA溶栓现状

rt-PA是重组组织型纤溶酶原激活剂,经过数年的临床试用,以及多个中心的临床试验,结果表明该药可能具有很大应用前景.结合文献对该药在急性缺血性脑卒中的应用现状进行阐述.     

碳源及氮源Trichoderma reesei306对组织型纤溶酶原激活剂生物合成的影响

本文对不同种类的碳．氮源对Trichoderma reesei306组组织型纤溶酶原激活剂(t—PA)生物合成的影响进行了研究。结果表明：在所试的各种碳氮源中，碳源以玉米粉和纤维素，氮源以尿素和胰蛋白胨效果最好，通过正交试验确定了液体发酵培养基中碳氮源的最佳组成为：玉米粉20g／l、纤维素30g／l、尿素10g／l、胰蛋白胨2g／l，优化碳氮源后t—PA酶活提高了26％。     

重组纤溶酶原激活剂及其突变体的表达研究

利用PCR技术 ,获得了人体组织型纤溶酶原激活剂tPAcDNA的缺失突变体———rPA ;在此基础上 ,运用定点突变技术 ,得到rPA的定点突变体———rPA(KHRR2 96～ 2 99AAAA) ,rPA(A473S)和二者的复合突变体rPA(KHRR2 96～2 99AAAA A473S) ;将rPA及其 3个突变体分别亚克隆至原核表达载体pET 2 8a( )中 ,获得表达载体pErA ,pErA(K) ,pErA(A)和pErA(KA) .酶切鉴定和序列分析结果均表明实现了实验设计的氨基酸突变 .表达载体转化大肠杆菌 ,经IPTG诱导、菌体裂解及SDS PAGE电泳分析发现 ,只缺失而无突变的pErA和突变的pErA(A)都未表达目的蛋白 ,突变体pErA(K)与pErA(KA)则获高水平表达 ;蛋白产量分别占菌体总蛋白的 35 .97%与 37.71% ,分子量均为 39.6kD .Westernblotting显示 ,表达产物与抗tPA抗体呈特异性阳性反应 .该产物经初步纯化后进行复性与活性 (mFAPA)测定 ,结果表明其复性产物具有明显的体外纤溶活性 .以上结果为rPA突变体的进一步纯化、体内活性研究以及规模化制备提供了基础     

UROKINASE MUTANT WITH BETTER FIBRIN SPECIFICITY

ＵＲＯＫＩＮＡＳＥＭＵＴＡＮＴＷＩＴＨＢＥＴＴＥＲＦＩＢＲＩＮＳＰＥＣＩＦＩＣＩＴＹＭａＺｈｏｎｇＹｕＲｕｉｒｏｎｇＨｕａＺｉｃｈｕｎＺｈｕＤｅｘｕ（ＤｅｐａｒｔｍｅｎｔｏｆＢｉｏｃｈｅｍｉｓｔｒｙ，ＳｔａｔｅＫｅｙＬａｂｏｒａｔｏｒｙｏｆＰｈａｒ...     

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.     

Modeling of the fibrin agarose plate assay and its application for thrombolytic analysis

The fibrin agarose plate assay is widely used in the detection of thrombolysis efficacy. However, a rigorous mathematical model for analyzing data or comparing activities of different thrombolytics has been absent. This study investigated the relationship between thrombolysis radius, R, and diffusion time, t, of molecular medicines in an agarose hydrogel system by deriving a model based on Fick’s law and experimental verification by the fibrin agarose plate assay method. The theoretical results showed that a plot of log(R) versus log(t) has a linear curve with the slope of 1/2 and this was verified by experimental results using urokinase as a modeling agent. Moreover, it was found that R÷t is constant for a specific thrombolytic and can be used as a parameter for evaluating activities of different thrombolytics. The theoretical model has potential for improving the understanding of mecha-nisms involved in molecular medicine diffusion and offers benefits for thrombolytic therapy.     

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.     

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.     

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.     

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.     

利用氨基苄脒为配基的亲和层析法纯化t—PA

利用氨基苄脒修饰的硅胶为亲和吸附剂纯化组织纤溶酶原激活剂（ｔ－ＰＡ），讨论了亲和柱体积对纯化效率的影响。由于氨基苄脒对ｔ－ＰＡ的高度特异性，根据ｔ－ＰＡ的上柱量适当调整柱体积，可使纯化倍数达到１４０以上，远高于利用其它亲和吸附剂的结果。     

抗栓肽和尿激酶原（B链）嵌合体的构建与表达

通过基因工程重组技术,将抗栓肽（decorsin）基因与尿激酶的B链即scu-PA(B)基因用柔性Linker((Gly₄Ser)₃)融合在一起,构建新的嵌合体基因dscuPA(B),在大肠杆菌Rosetta(DE3)plysS中通过IPTG诱导表达, 并考察了重组质粒在Rosetta(DE3)plysS在传代中的分离稳定性。该融合蛋白在大肠杆菌中是以包涵体的形式存在,对包涵体进行变性及复性后,并通过Zn²⁺螯合柱和SP阳离子交换柱进行纯化。质谱分析表明分子量为33.735kD,与理论值相符。目的蛋白质的纯度可达90%。纤维蛋白平板法测得嵌合分子的比活为90000IU/mg,与scuPA-32k的比活相近。体外血小板聚集实验表明融合蛋白有较强的抑制血小板聚集的功能,抑制常数IC50为0.31μmol/L。以上这些结果表明,该融合蛋白不但具有较强的溶栓功能,而且具有抗栓功能,可能是一种具有很好发展前景的双功能溶栓抗栓药物。