新型水蛭素嵌合抗栓剂的构建表达与功能研究

水蛭素（Hirudin,HV）作为新一代抗凝剂,它是目前已知最强的天然凝血酶抑制剂,并有望在临床上完全取代肝素。虽然水蛭素有许多优点,但出血倾向是其在临床应用上的主要副作用。目前尚没有好的解决办法。针对水蛭素在临床应用中所出现的这个问题,依据血栓形成的生理生化机制,构建出了含FXa识别序列水蛭素嵌合抗栓剂,来降低水蛭素在非血栓部位的活性从而减小出血的危险。小鼠尾部血栓模型实验表明：此新型嵌合抗栓剂能在不减少水蛭素抗凝血活性的同时,又能大幅度地降低出血副作用。具有非常重要的临床意义。     

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。     

水蛭素12肽和尿激酶B链融合基因的构建与表达

尿激酶原是一种溶栓效果好、副作用小的溶栓药物。经研究证明尿激酶B链(即pro-UK的144-411aa)具有与尿激酶原相似的溶栓性,即对血栓溶解具有特异性,引起系统性出血副作用小。水蛭素是一种分离自医用水蛭、由65个氨基酸残基组成的单链多肽,能特异、高亲和性地抑制凝血酶的活性,研究证明它各方面的性能都优于肝素。近年来,通过多肽合成方式对水蛭素结构与功能进行了大量研究,确定水蛭素C端12肽是一个具有抗栓功能的结构片段。由于它对凝血酶的高度亲和性,相对低浓度     

抗凝血蛋白的抗凝溶栓机制

自然界中存在着大量具有抗凝溶栓活性的生物大分子,如水蛭素、蚓激酶等。这些生物大分子主要是通过抑制凝血酶及凝血因子活性,水解纤维蛋白或纤溶酶原,或是抑制血小板聚集来达到抗凝溶栓的作用。该文对这些活性物质的抗凝溶栓机制进行综述。     

抗凝蛋白EH体外活性检测条件的建立

EH蛋白是一种水蛭素衍生物,它是在水蛭素的N端引入了一小段寡肽,该寡肽可被凝血因子XIa(factor XIa,FXIa)和Xa(factor Xa,FXa)裂解释放水蛭素的抗凝血酶活性.比较在不同条件下FXa裂解EH蛋白的效果,包括裂解时间(2h,4h,6h,8h,10h,20h)、裂解温度(25℃,37℃,40℃)...     

血栓调节蛋白的生物化学及病理生理学

血栓调节蛋白是分布于血管内皮表面的质膜蛋白,其理化性质极稳定。在体内参与血液凝固和纤溶过程的调节,通过与凝血酶形成复合物,改变后者作用的特异性,加速抗凝因子蛋白C的活化,并且抑制血小板在微循环中的聚集释放反应。它有较广泛的生物学效应,可作为蛋白C体外活性检测的工具酶,用于临床各疾病中高凝状态的观察。在某些血管源性恶性肿瘤中,血栓调节蛋白可作为特异性的表面标志物,指导临床诊断和鉴别诊断。     

蛇毒类凝血酶的分子生物学研究进展及其应用

蛇毒类凝血酶在体外可以作用于纤维蛋白原使其凝固,具有类似凝血酶的功能。但在体内却表现出抗凝、降纤的功能。本概述了蛇毒类凝血酶对纤维蛋白原的识别和作用、序列同源性特点、cDNA克隆的表达以及在临床中的应用。     

重组抗凝蛋白-新蛭素的原核表达研究

目的:重组新蛭素(EH)是在抗凝蛋白水蛭素的氨基末端添加3个氨基酸(EPR)的衍生物,以往EH的表达工艺沿用水蛭素的酵母表达工艺,生产周期长、目标蛋白表达效率相对较低。而水蛭素类的蛋白在大肠杆菌中往往以包涵体形式表达,后期的分离纯化收率较低,无法适应产业化。为了提高EH的生产效率,探索了EH在大肠杆菌中的可溶性表达。方法:首先通过PCR的方法获得eh的cDNA,PCR产物连接入原核表达载体pET-22或pET-24中获得重组表达质粒,将重组表达质粒转化大肠杆菌BL21(DE3)或BL21(plySs),获得重组工程菌BL21(DE3)-pET-24-eh,BL21(DE3)-pET-22-eh,BL21(plySs)-pET-22-eh。重组工程菌进行IPTG诱导,SDS-PAGE和Western blot鉴定表达产物。结果:EH在3个重组工程菌中均可实现可溶性表达。表达水平较高的为BL21(DE3)-pET-24-eh工程菌;之后通过优化诱导温度,时间,诱导剂浓度、诱导前菌种密度,确定最佳条件为:37℃,诱导6h,IPTG浓度为0.4μmol/L,诱导前菌种密度在OD₆₀₀=1左右。诱导产物经分离纯化,其纯度可达96.93%。最后通过蛋白含量测定及抗凝活性检测,确定表达的EH蛋白本身无抗凝活性,被FXa裂解后可以释放出水蛭素的抗凝活性。结论:实现了EH在大肠杆菌中的可溶性表达,表达周期短,有望提高EH的生产效率,为EH的产业化奠定了基础,也为水蛭素类产品的生产提供了新的工艺途径。     

重组水蛭素相关肽Hi-lys的表达与纯化(英文)

为开发一种新的有临床应用价值的抗血栓药物,根据水蛭素保持抗凝活性的2 0肽片段,设计并构建了水蛭素相关肽(Hi lys)与天冬酰胺酶C端的融合表达系统.为方便目的肽与融合伙伴的分离,增加了富含带电序列的8肽(KRKRKKSR)及酸敏感的天冬氨酰 脯氨酸(Asp Pro)位点,获得了表达质粒pED P8 Hi lys.将其转化E .coliBL 2 1,玉米浆培养基(kanr)培养,乳糖诱导获得融合蛋白(AnsB C P8 Hi lys)的高效表达.通过细菌裂解、包涵体洗涤、尿素溶解、乙醇沉淀、酸水解和DEAE 纤维素5 2柱层析纯化获得目的肽Hi lys ,用凝血酶测定法测得其抗凝活性为5 0ATU mg .     

重组水蛭素Ⅲ分子改建及其构效关系的初步研究

采用一种新的PCR定点诱变技术成功地对野生型水蛭素Ⅲ进行了定点诱变,这种方法快速、简便,有效。通过突变,将野生型水蛭素Ⅲ分子的活性功能非必需区的指状结构顶端第33～36位的氨基酸残基替换为RGDS序列,并进行了高效分泌表达,表达产物分泌到细胞外发酵液中。改构的水蛭素突变体与野生型水蛭素Ⅲ相比,两者的抗凝血酶活性基本一致,但体外抗血小板凝集试验结果表明,前者除原有的抗凝活性外还具有显著的抗ADP诱导的血小板凝集活性,该研究为水蛭素Ⅲ分子的结构功能关系研究以及新型抗凝药物的研制打下了基础。     

Alboserpin, a factor Xa inhibitor from the mosquito vector of yellow fever, binds heparin and membrane phospholipids and exhibits antithrombotic activity

The molecular mechanism of factor Xa (FXa) inhibition by Alboserpin, the major salivary gland anticoagulant from the mosquito and yellow fever vector Aedes albopictus, has been characterized. cDNA of Alboserpin predicts a 45-kDa protein that belongs to the serpin family of protease inhibitors. Recombinant Alboserpin displays stoichiometric, competitive, reversible and tight binding to FXa (picomolar range). Binding is highly specific and is not detectable for FX, catalytic site-blocked FXa, thrombin, and 12 other enzymes. Alboserpin displays high affinity binding to heparin (K(D) ~ 20 nM), but no change in FXa inhibition was observed in the presence of the cofactor, implying that bridging mechanisms did not take place. Notably, Alboserpin was also found to interact with phosphatidylcholine and phosphatidylethanolamine but not with phosphatidylserine. Further, annexin V (in the absence of Ca(2+)) or heparin outcompetes Alboserpin for binding to phospholipid vesicles, suggesting a common binding site. Consistent with its activity, Alboserpin blocks prothrombinase activity and increases both prothrombin time and activated partial thromboplastin time in vitro or ex vivo. Furthermore, Alboserpin prevents thrombus formation provoked by ferric chloride injury of the carotid artery and increases bleeding in a dose-dependent manner. Alboserpin emerges as an atypical serpin that targets FXa and displays unique phospholipid specificity. It conceivably uses heparin and phosphatidylcholine/phosphatidylethanolamine as anchors to increase protein localization and effective concentration at sites of injury, cell activation, or inflammation.     

Construction and characterization of a novel staphylokinase variant with thrombin-inhibitory activity

Staphylokinase (SAK) is an effective thrombolysis agent for therapy of myocardial infarction. We have constructed a fusion SAK variant (SAK-HV) with a thrombin-binding domain composed of 12 amino acids from hirudin and expressed it in Escherichia coli and purified the resultant protein. SAK-HV maintained fibrinolytic activity similar to SAK and had anticoagulant activity attributable to its hirudin segment. Measurement of thrombin-binding activity in vitro demonstrated that SAK-HV possessed binding activity with thrombin while SAK did not. SAK-HV might thus be a more potent thrombolytic agent with anticoagulation property than SAK.     

Cloning, purification and biochemical characterization of a thrombus-ditargeting thrombolytic agent, comprised of annexin B1, ScuPA-32K and fibrin-adherent peptide

The development of thrombolytic agent could provide invaluable progress for antithrombotic therapy. In this paper, we reported the cloning, purification and biochemical characterization of AnxB1ScuPAFap, a thrombus-ditargeting chimera composed of annexin B1, low molecular single-chain urokinase (ScuPA-32K) and fibrin-adherent peptide (dodecapeptide, Fap). In vitro test showed that, the chimera was a thrombolytic agent with anticoagulant activity and thrombus-ditargeting with the activated-platelet membrane binding and fibrin clot binding activity. Compared to urokinase, the chimera had less reperfusion time, higher reperfusion ratio, and less bleeding effects on coronary thrombolysis by clot lysis assay in dogs. Thus, the chimera appeared to be suitable for thrombolytic therapy of thrombus diseases.     

Anticoagulant activities of curcumin and its derivative

Curcumin, a polyphenol responsible for the yellow color of the curry spice turmeric, possesses antiinflammatory, antiproliferative and antiangiogenic activities. However, anticoagulant activities of curcumin have not been studied. Here, the anticoagulant properties of curcumin and its derivative (bisdemethoxycurcumin, BDMC) were determined by monitoring activated partial thromboplastin time (aPTT), prothrombin time (PT) as well as cell-based thrombin and activated factor X (FXa) generation activities. Data showed that curcumin and BDMC prolonged aPTT and PT significantly and inhibited thrombin and FXa activities. They inhibited the generation of thrombin or FXa. In accordance with these anticoagulant activities, curcumin and BDMC showed anticoagulant effect in vivo. Surprisingly, these anticoagulant effects of curcumin were better than those of BDMC indicating that methoxy group in curcumin positively regulated anticoagulant function of curcumin. Therefore, these results suggest that curcumin and BDMC possess antithrombotic activities and daily consumption of the curry spice turmeric might help maintain anticoagulant status.     

Anticoagulant activities of oleanolic acid via inhibition of tissue factor expressions

Oleanolic acid (OA), a triterpenoid known for its anti-inflammatory and anti-cancer properties, is commonly present in several medicinal plants but its anticoagulant activities have not been studied. Here, the anticoagulant properties of OA were determined by monitoring activated partial thromboplastin time (aPTT), prothrombin time (PT), fibrin polymerization as well as cell-based thrombin and activated factor X (FXa) generation activities. Data showed OA prolonged aPTT and PT significantly and inhibited thrombin catalyzed fibrin polymerization. In addition, OA inhibited the activities of thrombin and FXa and inhibited the generation of thrombin or FXa in human endothelial cells. OA also inhibited TNF-α-induced tissue factor expression on human endothelial cells. In accordance with these anticoagulant activities, OA showed an anticoagulant effect in vivo. These results indicate that OA possesses antithrombotic activities and suggest that daily consumption of a herb containing OA may be preventing thrombosis in pathological states.     

Anticoagulant activities of piperlonguminine in vitro and in vivo

Piperlonguminine (PL), an important component of Piper longum fruits, is known to exhibit anti-hyperlipidemic, antiplatelet and anti-melanogenic activities. Here, the anticoagulant activities of PL were examined by monitoring activatedpartial-thromboplastin-time (aPTT), prothrombin-time (PT), and the activities of thrombin and activated factor X (FXa). The effects of PL on the expressions of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) were also tested in tumor necrosis factor-α (TNF-α) activated HUVECs. The results showed that PL prolonged aPTT and PT significantly and inhibited the activities of thrombin and FXa. PL inhibited the generation of thrombin and FXa in HUVECs. In accordance with these anticoagulant activities, PL prolonged in vivo bleeding time and inhibited TNF-α induced PAI-1 production. Furthermore, PAI-1/t-PA ratio was significantly decreased by PL. Collectively, our results suggest that PL possesses antithrombotic activities and that the current study could provide bases for the development of new anticoagulant agents. [BMB Reports 2013; 46(10): 484-489]     

Design,synthesis, and structure–activity relationship of novel and effective apixaban derivatives as FXa inhibitors containing 1,2,4-triazole/pyrrole derivatives as P2 binding element

Four series of novel and potent FXa inhibitors possessing the 1,2,4-triazole moiety and pyrrole moiety as P2 binding element and dihydroimidazole/tetrahydropyrimidine groups as P4 binding element were designed, synthesized, and evaluated for their anticoagulant activity in human and rabbit plasma in vitro. Most compounds showed moderate to excellent activity. Compounds 14a, 16, 18c, 26c, 35a, and 35b were further examined for their inhibition activity against human FXa in vitro and rat venous thrombosis in vivo. The most promising compound 14a, with an IC₅₀ (FXa) value of 0.15 μM and 99% inhibition rate, was identified for further evaluation as an FXa inhibitor.     

Antithrombotic and profibrinolytic activities of eckol and dieckol

In order to develop new anticoagulant agents, two single compounds (eckol and dieckol) were isolated from Eisenia bicyclis and examined their anticoagulant activities by monitoring activated partial thromboplastin time (aPTT), prothrombin time (PT) as well as cell-based thrombin and activated factor X (FXa) generation activities. And the effects of eckol and dieckol on the expression of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) were tested in tumor necrosis factor-α (TNF-α) activated human umbilical vein endothelial cells (HUVECs). Data showed that eckol and dieckol prolonged aPTT and PT significantly and inhibited thrombin and FXa activities. They also inhibited the generation of thrombin or FXa in HUVECs. In accordance with these anticoagulant activities, eckol or dieckol showed anticoagulant effect in vivo. Furthermore, eckol and dieckol inhibited TNF-α induced PAI-1 production and the ratio between PAI-1 and t-PA was found to be significantly decreased by eckol and dieckol. Surprisingly, these anticoagulant and profibrinolytic effects of dieckol were better than those of eckol indicating that hydroxyl group in eckol positively regulated anticoagulant function of eckol. Therefore, these results suggest that eckol or dieckol possesses antithrombotic activities and provides a possibility to develop as an agent for the anticoagulation.