牛血凝血酶的制备工艺

目的研究1种高纯度牛凝血酶的制备工艺。方法采用不同终浓度的Ba2+吸附、硫酸铵分段盐析从牛血浆中提取凝血酶原,用Ca2+激活,活化的凝血酶用CM-Sepharose Fast Flow离子交换柱纯化。结果用冷冻1周血浆制备的凝血酶粗品产量、比活明显高于来自新鲜血浆的粗品;Ba2+终浓度在0.107 mol/L以上时凝血酶的产量较高;凝血酶原的最佳激活条件为:Ca2+终浓度0.05 mol/L,37℃,30~60 min;CM-Sepharose Fast Flow柱制备的凝血酶比活达到2 000 U/mg以上。结论该工艺适用于高纯度凝血酶的制备。     

超滤法从藏牦牛血中分离纯化凝血酶

目的从藏牦牛血中分离纯化凝血酶。方法根据牛凝血酶原的相对分子质量,采用超滤法,截留相对分子质量60000以上的物质。结果所得凝血酶平均比活为38.24IU/mg,比传统方法所得比活提高了2倍。结论此工艺适于中型规模的牛凝血酶生产。     

高纯度凝血酶的分离纯化

目的 利用猪血浆制备高纯度凝血酶.方法 使用DEAE-Sepharose FF阴离子交换层析分离猪血浆获得凝血酶原后,用CaCl2激活凝血酶原生成凝血酶.获得的凝血酶粗制品经2次SP-Sepharose FF阳离子交换层析获得高纯度凝血酶.结果 2次层析后的凝血酶的比活为2 546 IU/mg,是凝血酶粗制品的8倍,得率为54％.快速蛋白液相层析检测表明,2次层析后的凝血酶纯度达89％.结论 猪血浆通过1次DEAE-Sepharose FF和2次SP-Sepharose FF离子交换层析可得到高纯度凝血酶.     

猪凝血因子V的制备及其活性分析

从猪血浆中制备猪凝血因子V,并分析凝血因子V加速凝血酶原活化的促凝活性。采用聚乙二醇沉淀、柠檬酸钡吸附、DEAE纤维素离子交换层析等方法,制备了猪凝血因子V。经SDSPAGE检测,猪凝血因子V得以成功制备,收率为每升血浆收90.2mg,纯度为64.4%。经纤维蛋白凝结实验分析,采用上述方法制备的猪凝血因子V具有较高的促凝血酶原活化活性。     

六月雪水提取物促凝血作用及机制的研究

目的 研究六月雪水提取物促凝血的作用及机制.方法 将清洁级SD大鼠60只随机分为生理盐水组、低剂量SSD组（1.5 g/kg六月雪水提取物组）、高低剂量SSD组（3.0 g/kg六月雪水提取物组）.采用体外血浆法测定体外凝血时间,采用凝血因子分析仪测定凝血酶时间、凝血酶原时间、优球蛋白溶解时间.结果 与生理盐水对照组比较,低、高剂量SSD组的六月雪水提取物能明显缩短体外凝血时间（P＜0.05,P＜0.01）、血浆凝血酶时间（P＜0.05,P＜0.01）、凝血酶原时间（P＜0.05,P＜0.01）,且明显延长优球蛋白溶解时间（P＜0.05,P＜0.01）.结论 1.5、3.0 g/kg的六月雪水提取物具有明显的促凝血作用,其作用机制可能与缩短血浆凝血酶时间、凝血酶原时间及升高优球蛋白溶解时间有关.     

纳豆激酶抗凝血作用的研究

目的研究纳豆激酶 (NK)的活性和抗凝血作用。方法采用纤维蛋白平板法测定NK的活性 ;采用试管法测定家兔全血凝固时间、血浆复钙时间、凝血酶时间、大鼠凝血活酶时间、凝血酶引起的纤维蛋白凝固时间 ;采用Quick法测定大鼠凝血酶原时间。结果固体粉末NK的活性为 5 6 5 5 10U/g ,是尿激酶的 3.13倍 ;液体NK的活性为 15 2 34U/ml;NK能显著延长家兔全血凝固时间、血浆复钙时间、凝血酶时间、大鼠凝血活酶时间、凝血酶引起的纤维蛋白凝固时间、大鼠凝血酶原时间。结论NK对凝血过程的三阶段都有影响。     

凝血酶制备工艺的研究

目的从猪血中分离纯化凝血酶。方法采用离子交换树脂法对从猪血制备获得的凝血酶进行纯化 ,并考察凝血酶原的激活条件。结果离子交换树脂法纯化的凝血酶平均比活为 36 .5u/mg ,凝血酶原的最佳激活条件为 :CaCl2 浓度 1 .0 % ,时间 1 5min ,温度 37℃。结论离子交换树脂法分离纯化凝血酶的工艺是可行的 ,适用于规模化生产。     

水蛭注射液抗血栓与抗凝血作用

目的研究水蛭注射液的抗血栓和抗凝血作用。方法采用体外血栓法、纤维蛋白平板法及全血血块法,观察水蛭注射液的抗血栓作用;采用家兔血浆复钙时间法、凝血酶时间法及凝血酶原时间法,观察水蛭注射液的抗凝血作用。结果水蛭注射液高剂量(1.0 kg.L-1)、中剂量(0.5 kg.L-1)和低剂量(0.25 kg.L-1)均能明显溶解体外血栓(P<0.001)、纤维蛋白平板(P<0.001)和全血凝块(作用显著);能显著延长家兔血浆复钙时间、凝血酶时间和凝血酶原时间(P<0.001)。结论水蛭注射液具有较强的抗血栓和抗凝血作用。     

龙胆多糖的抗凝血作用的研究

目的:研究龙胆多糖的抗凝血作用.方法:采用体外实验的方法,考查龙胆多糖对大鼠部分凝血活酶时间(PTT)、大鼠凝血酶原时间(PT)以及对凝血酶的作用.结果:浓度为12.5、25、50、100mg/ml的龙胆多糖对大鼠部分凝血活酶时间(PTT)大鼠凝血酶原时间(PT)有抑制作用,血浆凝固时问显著延长,同时对凝血酶也有抑制作用,使纤维蛋白凝固时间显著延长.结论:龙胆多糖具有抗凝血作用.     

萱藻多糖的制备及抗凝血抗血栓活性研究

目的制备萱藻多糖并对家兔体外凝血及小鼠体内血栓形成的抑制作用进行研究。方法采用热水浸提-乙醇沉淀法制备萱藻多糖;检测家兔心脏动脉血活化部分凝血酶时间(APTT),凝血酶原时间(PT)和凝血酶时间(TT)。以角叉菜胶诱导小鼠尾部血栓模型,检测萱藻多糖体内抗血栓作用。结果萱藻多糖可显著延长家兔血浆APTT、PT和TT;对角叉菜胶诱导的小鼠尾部血栓具有显著的抑制作用(P<0.01)。结论萱藻多糖具有抗凝和抑制实验性血栓形成的作用。     

Procoagulant Adaptation of a Blood Coagulation Prothrombinase-like Enzyme Complex in Australian Elapid Venom

The macromolecular enzyme complex prothrombinase serves an indispensable role in blood coagulation as it catalyzes the conversion of prothrombin to thrombin, a key regulatory enzyme in the formation of a blood clot. Interestingly, a virtually identical enzyme complex is found in the venom of some Australian elapid snakes, which is composed of a cofactor factor Va-component and a serine protease factor Xa-like subunit. This review will provide an overview of the identification and characterization of the venom prothrombinase complex and will discuss the rationale for its powerful procoagulant nature responsible for the potent hemostatic toxicity of the elapid venom.     

Origin of a fluorescence increase accompanying the limited proteolysis of fluorescein-labeled human prothrombin by Factor Xa

In a search for a probe which would report its proteolysis to thrombin, the human blood coagulation zymogen prothrombin was covalently labeled with fluorescein. Fluorescein isothiocyanate (FITC) and dichlorotriazinylamino-fluorescein (DCTAF) both introduced ^? 1 molecule of dye, but labeling occurred at different locations, as FITC had no effect on clotting activity whereas DCTAF caused 95% inactivation. At pH 9.0 DCTAF, but not FITC, could induce labeling up to 4 mol/mol. All derivatives were activated normally by prothrombinase (the activating complex of Factor Xa, Factor V(a), Ca²⁺ and phospholipids), as indicated by the pattern of bands on SDS gel electrophoresis and an unaltered yield of activity toward a chromogenic substrate for thrombin. Upon undergoing this limited proteolysis, the most heavily labeled derivative showed a 40% increase in fluorescence of the fluorescein at 520 nm (Λ_ex 480 nm). In contrast, the fluorescence of lightly labeled forms was more intense but increased by only 0–5% upon activation. The data suggest that the lower fluorescence of the most labeled form is due to an intramolecular quenching effect between the dye molecules on individual polypeptide chains that is partly relieved when activation occurs.     

Determinants of specificity of factor xa interaction with its physiological inhibitors

Factor Xa (fXa) is the vitamin K-dependent serine protease of the prothrombinase complex (fXa, factor Va, negatively charged membrane, and calcium) which is responsible for the conversion of prothrombin to thrombin in the final stage of the coagulation cascade. The proteolytic activity of fXa in plasma is primarily regulated by three physiological inhibitors, antithrombin (AT), protein Z-dependent protease inhibitor (ZPI) and tissue factor pathway inhibitor (TFPI). The first two inhibitors belong to the serpin family of plasma inhibitors, both of which require cofactors for their effective interaction with fXa. Thus, the AT interaction with the heparin-like glycosaminoglycans on the surface of the endothelium, and the ZPI complex formation with protein Z on membrane phospholipids is required for the physiological regulation of fXa by both serpins. On the other hand, TFPI is a slow and tight-binding, Kunitz type inhibitor that is capable of rapidly inhibiting fXa independent of a cofactor. This article will review the structural features that enable fXa to specifically interact with these three inhibitors under different conditions.     

Factor Xa inhibitors: today and beyond

Serine proteases play an important role in thrombogenesis, the process that leads to blood clotting and conditions such as heart attack, stroke and other cardiovascular disorders. In the coagulation network, the activation of various serine proteases facilitates the formation of the serine protease Factor Xa, which plays a central role in the process of coagulation and platelet activation. Factor Xa is an essential component of the prothrombinase complex, from which thrombin is formed, which then directly leads to fibrin clot formation. Thus, the inhibition of Factor Xa and its generation is an important strategy in the development of new antithrombotic drugs.     

The race to an orally active Factor Xa inhibitor: recent advances

Factor Xa (fXa) is a key enzyme in the coagulation cascade and an essential component of the prothrombinase complex, which activates prothrombin to thrombin, leading to fibrin clot formation. In the search for a more effective and safer orally active anticoagulant, fXa has emerged as a major target for potential therapeutic applications in the treatment and prevention of thrombosis. This review focuses on recent advances in the chemistry of drug design and lead optimization of orally bioavailable fXa inhibitors. Many of these orally active fXa inhibitors are currently in clinical trials and are anticipated to change the landscape of thrombosis therapy.     

Structure-function relationships and mechanism of anticoagulant phospholipase A2 enzymes from snake venoms.

Phospholipase A₂ (PLA₂) enzymes from snake venom are toxic and induce a wide spectrum of pharmacological effects, despite similarity in primary, secondary and tertiary structures and common catalytic properties. Thus, the structure–function relationships and the mechanism of this group of small proteins are subtle, complex and intriguing challenges. This review, taking the PLA₂ enzymes from spitting cobra (Naja nigricollis) venom as examples, describes the mechanism of anticoagulant effects. The strongly anticoagulant CM-IV inhibits both the extrinsic tenase and prothrombinase complexes, whereas the weakly anticoagulant PLA₂ enzymes (CM-I and CM-II) inhibit only the extrinsic tenase complex. CM-IV binds to factor Xa and interferes in its interaction with factor Va and the formation of prothrombinase complex. In contrast, CM-I and CM-II do not affect the formation of prothrombinase complex. In addition, CM-IV inhibits the extrinsic tenase complex by a combination of enzymatic and nonenzymatic mechanisms, while CM-I and CM-II inhibit by only enzymatic mechanism. These functional differences explain the disparity in the anticoagulant potency of N. nigricollis PLA₂ enzymes. Similarly, human secretory enzyme binds to factor Xa and inhibits the prothrombinase complex. We predicted the anticoagulant region of PLA₂ enzymes using a systematic and direct comparison of amino acid sequences. This region between 54 and 77 residues is basic in the strongly anticoagulant PLA₂ enzymes and neutral or negatively charged in weakly and non-anticoagulant enzymes. The prediction is validated independently by us and others using both site directed mutagenesis and synthetic peptides. Thus, strongly anticoagulant CM-IV binds to factor Xa (its target protein) through the specific anticoagulant site on its surface. In contrast, weakly anticoagulant enzymes, which lack the anticoagulant region fail to bind specifically to the target protein, factor Xa in the coagulation cascade. Thus, these studies strongly support the target model which suggests that protein–protein interaction rather than protein–phospholipid interaction determines the pharmacological specificity of PLA₂ enzymes.     

Clinical pharmacology of direct and indirect factor Xa inhibitors

The limitations of conventional anticoagulants have stimulated the development of new anticoagulants. The central position of factor Xa (FXa) at the junction of the intrinsic and extrinsic pathways in the coagulation cascade means that direct and indirect FXa inhibitors have increasingly changed antithrombotic strategies. FXa inhibitors potently and selectively inhibit thrombin formation rather than thrombin activity. Direct FXa inhibitors may directly bind to FXa, whereas indirect inhibitors are dependent on antithrombin. Direct inhibitors may bind free FXa and, in contrast to indirect inhibitors, FXa within the prothrombinase complex or within clots as well. Fondaparinux is the prototype indirect FXa inhibitor and has been extensively studied in the prevention and treatment of thromboembolic diseases, including acute coronary syndromes. Due to a favourable efficacy and safety profile and convenient once-daily dosing without the need for monitoring, fondaparinux is preferentially recommended in recent guidelines dealing with antithrombotic treatment. A number of small-molecule direct FXa inhibitors are currently at different stages of clinical development. After an extensive clinical trial programme demonstrating superior efficacy without a significant increase in major bleeds compared with enoxaparin, rivaroxaban is now available for the prevention of thromboembolic events in patients undergoing orthopaedic surgery. Rivaroxaban also offers the convenience of oral once-daily dosing without the need for monitoring. Whereas most direct FXa inhibitors are orally active, otamixaban is administered intravenously, offering rapid on-off anticoagulant activity. Other compounds under development may offer additional options for tailored antithrombotic strategies according to differing indications, clinical situations and patient variables.     

Effects of fondaparinux and a direct factor Xa inhibitor TAK-442 on platelet-associated prothrombinase in the balloon-injured artery of rats

Endothelial damage triggers platelet adhesion and platelet-associated prothrombinase formation at the point of injury, resulting in the progression of thrombus formation. The present study compared the inhibitory effects of fondaparinux, an indirect factor Xa (FXa) inhibitor, and TAK-442, a direct FXa inhibitor, on platelet-associated prothrombinase activity in the balloon-injured rat artery. TAK-442 and fondaparinux inhibited endogenous FXa activity in platelet-poor human [half-maximal inhibitory concentration (IC(50)): 53 nM, TAK-442; 11 nM, fondaparinux] and rat (IC(50): 32 nM, TAK-442; 19 nM, fondaparinux) plasma. TAK-442 inhibited in vitro reconstituted human prothrombinase (system included FXa, calcium, and washed platelets) with an IC(50) value of 51 nM, whereas fondaparinux exhibited only weak inhibition (IC(50): 1700 nM). In a rat model of balloon injury, thrombin activity on the surface of injured vessels increased to 3.2-, 22-, and 5.8-fold the activity on the surface of the intact aorta at 5 minutes, 1 hour, and 24 hours after the injury, respectively. At approximately 1 hour after the injury, TAK-442 blocked platelet-associated thrombin generation on the surface of injured aortas with an IC(50) value of 19 nM, whereas fondaparinux showed no significant inhibition at the highest concentration tested (IC(50): >300 nM). These results suggest a possible limitation of fondaparinux in inhibiting platelet-associated prothrombinase activity and resultant thrombus formation as compared with TAK-442.