海洋假单胞菌碱性蛋白酶的纤溶和溶栓作用研究

目的：为进一步确认海洋假单胞菌碱性蛋白酶(MPAP)的纤溶性质，并观察其抗栓作用。方法：通过体外试凝块实验，观察MPAP对纤维蛋白和纤维蛋白原的降解作用；建立家兔肺动脉血栓模型，观察MPAP的溶栓作用以及对组织型纤溶酶原激活剂(t—PA)和组织型纤溶酶原激活荆抑制剂(PAI)纤溶功能的影响。结果：MPAP有直接降解纤维蛋白和纤维蛋白原的作用，而无纤溶激酶活性；MPAP能明显溶解肺动脉血栓，MPAP用药后t—PA／PAl明显升高。结论：MPAP具有明显的溶栓和提高纤溶系统活性的作用。     

海洋微生物代谢产物FGFC1的纤溶促进作用

目的:研究海洋微生物产生的吡喃并异吲哚酮化合物FGFC1体外和实验动物体内促纤溶作用.方法:采用异硫氰酸荧光素(F1TC)纤维蛋白降解法和急性肺血栓大鼠模型法研究FGFC1体外和体内的纤溶促进作用.结果:在体外FITC-纤维蛋白降解实验中,FGFC1在0.5～ 25 μmol/L浓度范围内,其纤溶促进作用随着浓度升高逐渐增强;当FGFC1浓度≥25 μmol/L时,其纤溶促进作用维持在最高水平,EC50为5 μmol/L.在大鼠急性肺血栓实验中,FGFC1的剂量为5或10 mg/kg能溶解肺血栓,达到与5 nmol/L剂量单链尿激酶型纤溶酶原激活剂相当的溶栓效果.结论:溶栓药物先导化合物FGFC1具有优良的纤溶促进作用和溶栓效果.     

重组人组织型纤溶酶原激活剂及其突变体的溶血栓研究

目的 比较重组人组织型纤溶酶原激活剂(rht-PA)及其突变体FsGGI和FrGGI的溶血栓作用,验证突变体的构建思想。方法 采用兔颈静脉溶栓模型和体外溶栓试验对纯化的rht-PA及其突变体FsGGI和FrGGI进行家兔体内外溶栓研究。结果 rht-PA、FsGGI及FrGGI三者均有体内外溶栓作用,体外溶栓能力基本相似;体内溶栓活力突变体FsGGI与FrGGI类似,均显著高于rht-PA,溶栓度分别为20.1％、49.1％和46.6％。在体内溶栓作用的同时均未引起血浆纤维蛋白原滴度下降。结论 溶栓作用是血栓特异性的,两种突变体是优于野生型t-PA的溶栓剂,上游的构建思想是正确的。     

蛇毒纤溶酶原激活剂的研究进展

蛇毒是含有多种生物活性蛋白质和多肽的混合物,其中一些蛋白可作用于人的凝血系统。蛇毒纤溶酶原激活剂是具独特活性的蛇毒丝氨酸蛋白酶,具有较长的半衰期,可专一性地裂解人Glu-纤溶酶原的Arg^561-Val^562肽键,使之变成有活性的纤溶酶,从而发挥溶栓作用。该文主要对蛇毒纤溶酶原激活剂的结构特点和作用机制进行了阐述,旨在为新型溶栓剂的研究提供方向。     

海洋微生物来源吲哚酮纤溶化合物影响纤溶因子构象特性的研究

摘 要:目的 研究FGFC1对各纤溶因子构象的影响及FGFC1促进纤溶反应的机制。方法 采用圆二色谱法研究FGFC1对各纤溶因子结构的影响,采用发色底物法研究FGFC1对各纤溶因子活性的影响,采用SDS-PAGE电泳法研究FGFC1在纤溶酶原和单链尿激酶型纤溶酶原激活剂构成的相互活化反应体系中的作用。结果 远紫外区的CD结果表明FGFC1在23 μmol?L?1~115 μmol?L?1的浓度范围内均使各纤溶因子的二级结构发生变化,这变化都导致酶分子柔性增加、更易发生反应;近紫外区的CD结果表明,FGFC1使纤溶酶原在285 nm处的摩尔旋光度增大。CD研究结果表明FGFC1对单链尿激酶型纤溶酶原激活剂和尿激酶型纤溶酶原激活剂结构的影响较为微弱,对纤溶酶及纤溶酶原结构的影响复杂而深刻。运用发色底物法检测加入不同浓度FGFC1后各纤溶因子的酶活力,排除了FGFC1对纤溶因子的结构影响而导致的纤溶因子失活的可能性。在体外构筑的纤溶反应体系中,SDS-PAGE的结果显示FGFC1的加入促进FITC-纤维蛋白原的降解,表明FGFC1加速纤溶酶原和单链尿激酶型纤溶酶原的相互活化作用。结论 FGFC1作用于纤溶酶原并辅以改变单链尿激酶型纤溶酶原激活剂的二级结构发挥纤溶促进作用。     

海洋假单胞菌纤溶酶的体外溶栓实验研究

目的确证血纤维蛋白溶酶的体外溶栓作用。方法用血纤维蛋白平板法、体外血块溶解实验研究该酶的纤溶特点及对血块的溶解作用。结果可直接水解血纤维蛋白 ,溶栓活性同蝮蛇抗栓酶类似 ,但对红细胞形态没有影响。结论血纤维蛋白溶酶是一种直接纤溶酶 ,有较强的纤溶活性     

蚯蚓提取液纤溶及抗凝作用的实验研究

通过纤维蛋白平板法和纤溶酶原激活剂活力测定试剂盒测定，证实蚯蚓提取液具有较强的纤溶活性和较高的纤溶酶原激活剂活力。动物实验结果显示，给SD大鼠静往蚯蚓提取液，能迅速提高大鼠血浆总纤溶酶原激活剂活力；口服给药也能明显减轻13％高分子葡聚糖溶液诱导大鼠实验性弥漫性血管内凝血的严重程度。     

化学修饰对组织型纤溶酶原激活剂的纤溶活性和体内半衰期的影响

用肝素、右旋糖酐和聚乙二醇对重组组织型纤溶酶原激活剂(rt-PA)进行化学修饰。不同的化学修饰物对rt-PA体外纤维蛋白溶解活性的影响不同。肝素修饰小PA的纤维蛋白溶解活性提高63％;右旋糖酐和聚乙二醇修饰rt-PA的纤维蛋白溶解活性则分别降低了36％和63％。rt-PA经化学修饰后半衰期均有所延长,其中聚乙二醇修饰的小PA的半衰期由修饰前的3．4min延长至6．3mim。     

活血化瘀中药的纤溶和纤溶抑制作用

分别用尿激酶型纤溶酶原激活剂，组织型纤溶酶原激活剂激活纤溶酶原的纤溶试验，观察了２０余种常用活血化瘀药对纤溶的影响，结果表明，１１味中药的水浸液在含纤溶酶原和不含纤溶酶原的纤维蛋白板上都出现同样程度的纤溶活性，按由强到弱的排列是益母草，三棱，水蛭，五灵脂，炮山甲、姜黄、川芎、红花、土鳖虫，桃仁，元胡，但未发现对ｔ－ＰＡ，ｕ－ＰＡ激活纤溶的增强作用。部分药物不同程度抑制ｔ－ＰＡ，ｕ－ＰＡ激活纤溶。其     

蚯蚓纤溶酶的亲和层析纯化及部分性质

用牛血纤溶酶原Ｓｅｐａｒｏｓｅ４Ｂ亲和层析方法从蚯蚓粗提物丙酮粉中分离纯化一种纤维蛋白溶解酶。该酶为糖蛋白,含糖量约为１．４３％,Ｍｒ为３００００。实验结果表明此酶具有直接溶解纤维蛋白和激活纤溶酶原的间接溶解纤维蛋白的双重作用,并对人血凝块有明显的溶解作用。１％ＰＭＳＦ对酶有显著的抑制作用,说明此蚯蚓纤溶酶为典型的丝氨酸蛋白酶类酶。     

rAcAP5的溶栓作用及作用机制研究(英文)

犬钩虫抗凝肽（Ancylostoma caninum anticoagulant peptide,AcAP5）是一种已报道的FXa抑制剂,它对防止血栓溶解的TAFIa具有很强的抑制作用,本文进一步研究rAcAP5的纤溶活性和溶栓活性。采用发色产物法测定rAcAP5对TAFIa的抑制作用,用比浊法测定rAcAP5对尿激酶（UK）诱导的纤维蛋白溶解时间的影响,通过血栓称重法进行体外溶栓实验和体内动静脉旁路溶栓实验。采用常规方法测定正常大鼠优球蛋白溶解时间（ELT）,血浆纤维蛋白原含量和纤维蛋白降解产物（FDP）含量。rAcAP5浓度依赖地抑制TAFIa活性,其IC₅₀为63.7 nmol/L,为一个强的TAFIa抑制剂。rAcAP5（5-40 nmol/L）能够显著地加速尿激酶诱导纤维蛋白的溶解,缩短纤溶时间。rAcAP5能够提高尿激酶诱导的血栓减重（P<0.05）,单独使用rAcAP5也能显著增加体外实验的血栓减重（P<0.05）。在大鼠体内的动静脉旁路模型中,单次静脉给予rAcAP5（50-200μg/kg）能够剂量依赖地增加血栓减重（P<0.01）。rAcAP5在有效剂量对正常大鼠的ELT、血浆纤维蛋白原含量和FDP含量均无明显影响。研究结果提示:rAcAP5是一个强效溶栓多肽,它可能通过抑制TAFIa的活性而在血栓表面具有溶栓活性,而不影响循环血液中的纤溶活性和纤维蛋白原含量。     

Purification and biochemical characterization of F II(a), a fibrinolytic enzyme from Agkistrodon acutus venom.

A fibrinolytic enzyme, F II(a), was isolated from Agkistrodon acutus venom by ion-exchange chromatography and gel filtration. F II(a) consisted of a single polypeptide chain with a molecular weight of 26,000 and an isoelectric point of 4.6. F II(a) was shown to solubilize fibrin and fibrinogen. F II(a) cleaved, primarily, the alpha chain of fibrinogen and fibrin followed by the beta chain, while the gamma chain was minimally affected. Thus, the enzyme was an alpha,beta-fibrinogenase. The cleavage pattern of fibrinogen clearly varied from plasmin cleavage of the same molecule. In vivo, F II(a) had no influence on the rat's tissue-type plasminogen activator and plasminogen activator inhibitor-1 activities in plasma. At the dosage of 5mg/kg, histological examination of heart, liver and lung tissue showed no hemorrhage. F II(a) is an enzyme that hydrolyzed fibrin directly without hemorrhagic activity.     

The usefulness of systemic administration of recombinant human tissue-type plasminogen activator in femoral arterial thrombolysis of rabbits

The thrombolytic effect of locally or systemically administered recombinant human tissue-type plasminogen activator (rt-PA) was investigated in comparison with the effect of tissue culture urokinase (TCUK), using a model of femoral artery thrombosis in rabbits. An 125I-labeled fibrinogen thrombus was formed in the femoral artery following injury of the intima by diluted sulfuric acid, and thrombolytic activity was evaluated one hour after the end of infusion of the agents. Local infusion of rt-PA (500-10,000 IU/kg) and TCUK (10,000 IU/kg) induced a marked thrombolysis. When rt-PA and TCUK were injected systemically in a high dose (200,000 IU/kg), rt-PA but not TCUK had a significant thrombolytic activity. In these cases, rt-PA was not accompanied by systemic activation of the fibrinolytic system, as evaluated by unaltered levels of alpha 2-antiplasmin in the plasma, while TCUK led to a substantial decrease in the alpha 2-antiplasmin level. These results suggest that systemically administered rt-PA but not TCUK induce a significant thrombolysis without systemic activation of the fibrinolytic system in cases of peripheral artery thrombosis.     

低分子量肝素对体内、体外血栓的溶解作用

目的：探讨低分子量肝素（ＬＭＷＨ）对体内、外血栓的溶解作用。方法：（１）取雄性家兔动脉血，制备人工血栓，放入贫血小板血浆中，观察ＬＭＷＨ对血栓的溶解作用；（２）于家兔一侧颈外静脉制备血栓，观察耳缘静脉用药（８００，４００，２００ＩＵ·ｋｇ－１）后静脉血栓溶解情况；同时测定ＡＰＴＴ、ＥＬＴ和血浆纤维蛋白原的含量。结果：不同于尿激酶，ＬＭＷＨ体外无溶栓作用；体内给药２００～８００ＩＵ·ｋｇ－１能有效地溶解体内血栓，降低纤维蛋白原的含量，缩短优球蛋白溶解时间（ＥＬＴ），并呈剂量依赖性，４００ＩＵ·ｋｇ－１溶栓作用与尿激酶５０００Ｕ·ｋｇ－１相似。结论：ＬＭＷＨ通过激活体内纤溶系统发挥溶栓作用。     

一种体内溶栓活性优于总蚯蚓纤溶酶的组分

目的筛选高效低毒的蚯蚓纤溶酶单一组分。方法通过亲和色谱从赤子爱胜蚓获得含多组分的总蚯蚓纤溶酶(EFE)，经离子交换分离得到3个主要溶栓组分EfP-0-2，EfP-I-1和EfP-I-2，与EFE比较体内外溶栓效果和毒副作用。结果与其他组分比较，EfP-I-1的体外溶栓作用较强；当静脉注射4.5 mg·kg^-1时，各组分对纤维蛋白原的影响均不明显，只有EfP-I-1能显著溶解动静脉旁路血栓；当静脉注射达6 mg·kg^-1时，只有该组分对延长出血时间的影响不明显；急性毒性实验表明，该组分的LD₅₀是EFE的2.17倍。结论EfP-I-1有较高的体内外溶栓活性和较低的毒副作用。由于该组分在总EFE中所占比例较高，并易于分离纯化，故适于作为单一组分的溶栓药物进行开发。     

不同分子量仿刺参糖胺聚糖对大鼠纤溶系统活性的影响研究D

目的 以不同分子量仿刺参糖胺聚糖为研究对象,考察其对大鼠纤溶系统的影响。方法 运用kurz法,经FeCl3刺激,诱导大鼠颈总动脉血栓形成,考察4种不同分子量仿刺参糖胺聚糖（分子量由大到小依次为93 200、43 600、12 800、5 400 Da）对血栓形成的影响,以及对t-PA、u-PA、PAI-1及Plg含量的影响。结果 4种不同分子量仿刺参糖胺聚糖在不同给药剂量时,均能不同程度地抑制血栓形成（P < 0.05或P < 0.01）;在20 mg/kg的给药剂量下,体内t-PA含量显著升高（P < 0.05）,同时PAI-1和Plg含量降低（P < 0.05）,u-PA含量均无明显变化（P > 0.05）;实验结果表明仿刺参糖胺聚糖具有剂量依赖性和分子量依赖性,其中AHG（93 200 Da）作用最强,DAHG-III（5 400 Da）作用最弱。结论 不同分子量仿刺参糖胺聚糖可通过增强t-PA活性、抑制PAI-1活性,以及促进Plg转化为纤溶酶,增强机体的纤溶活性,发挥溶栓作用;四种不同分子量糖胺聚糖中,AHG对纤溶系统的作用最强。     

Current developments in thrombolytic therapy using novel plasminogen activators

As a result of intensive recent research, the molecular mechanisms governing the human fibrinolytic system could be elucidated. Clinical trials using streptokinase and urokinase demonstrated that therapeutic thrombolysis is a valuable regimen in the treatment of patients with thromboembolic diseases. After unsuccessful attempts to increase the clot specificity of these agents, efforts were increased towards developing fibrin-specific substances with high thrombolytic potency and negligible effects on systemic hemostasis. Tissue-type plasminogen activator manufactured by recombinant DNA technology (rt-PA) is currently the most comprehensively investigated fibrin-specific thrombolytic agent with the most clearly understood mechanism of action in vivo. Pro-urokinase has also become available through biotechnology. A large number of clinical trials studying thrombolytic therapy of myocardial infarction have proved that intravenous rt-PA posesses superior efficacy compared to conventional fibrinolytic agents. Comparatively few clinical studies have been performed using pro-urokinase. At therapeutic doses, the fibrin specificities of these agents differ. So far it has not been possible to correlate the incidence of bleeding complications during fibrinolytic therapy with alterations in hemostasis parameters. For routine laboratory monitoring of thrombolysis the determination of fibrinogen and thrombin clotting time can be recommended. New developments with potential for yielding the next generation of thrombolytic agents are mutants and chimeras of t-PA and pro-urokinase, and conjugates of these substances with fibrin specific antibodies.     

Selective fibrinolytic activity of recombinant non-glycosylated human pro-urokinase (single-chain urokinase-type plasminogen activator) from bacteria

To assess their therapeutic value recombinant non-glycosylated human pro-urokinase (cPUK) and recombinant non-glycosylated human low molecular mass urokinase (cLUK) both obtained from genetically transformed bacteria were compared with respect to their thrombolytic efficacy and their potential to induce systemic plasminogen activation which impairs the coagulation system. The investigations were performed in in vitro and in vivo test systems. In vitro, both substances significantly and concentration-dependently lysed radiolabelled human thrombi in rotating loops of tubes (Chandler-loops) with equivalent efficacy. Fibrinolytic activity of cLUK was accompanied by a decrease in alpha 2-antiplasmin, plasminogen and fibrinogen. In contrast, cPUK did not change the plasminogen and fibrinogen levels and induced a substantially smaller decline in alpha 2-antiplasmin than cLUK. In the lysis of pulmonary embolized radiolabelled blood clots in anesthetized rabbits cPUK and cLUK dose-dependently exerted significant effects of similar extent. Whereas cLUK significantly decreased plasma levels of alpha 2-antiplasmin, plasminogen and fibrinogen, cPUK caused only a marginal decrease in alpha 2-antiplasmin and left the plasminogen and fibrinogen levels unchanged. The results indicate that cPUK exerts fibrinolytic efficacy without systemic plasminogen activation and breakdown of fibrinogen. Therefore, cPUK might be expected to be a more specific and safer thrombolytic agent than urokinase and other traditional fibrinolytics.     

The effect of 17 beta-oestradiol on variables of coagulation and fibrinolysis in postmenopausal women with type 2 diabetes mellitus

Type 2 diabetes mellitus is frequently accompanied by hypercoagulability and hypofibrinolysis. Both are related to increased cardiovascular risk, but possibly with endothelial injury as well. Studies with nondiabetic persons indicate that unopposed oestrogen replacement therapy (oERT) decreases cardiovascular risk, possibly mediated in part by effects on coagulation and fibrinolysis. In a double-blind, randomised placebo-controlled trial, we assessed the effect of oral 17 beta-oestradiol daily during 6 weeks on indicators of coagulation and of fibrinolysis in postmenopausal women with type 2 diabetes mellitus. We observed significant increases of Factor VII (FVII) and von Willebrand factor (vWF) after oERT and no change in the already high fibrinogen. Prothrombin fragment 1 + 2 (F1 + 2) increased after oERT, whereas thrombin-antithrombin (TAT) complexes was unchanged, but increments of F1 + 2 and TAT correlated. Soluble fibrin (SF) levels remained stable. In fibrinolysis, a clear reduction in plasminogen activator inhibitor 1 (PAI-1) was observed, but no significant change in tissue-type plasminogen activator antigen (t-PA-Ag) or activity was found, although fibrinolytic activity assessed as t-PA activity (t-PA-Act) tended to increase after oERT. Indicators of fibrinolytic activity (plasmin-antiplasmin complexes and fibrin degradation products) however did not change. oERT increased C-reactive protein (CRP) but none of the coagulation or fibrinolysis changes significantly associated with the CRP changes. It is concluded that oERT increases the coagulation potency as well as the fibrinolytic potency raising the question of the net effect in their balance. Increase in F1 + 2 suggests that in diabetic women oERT effectively increases the chronic, continuous activation of coagulation, which appears to be compensated for or not effective in the blood compartment as judged from the unchanged levels of SF. Suspected increased fibrin formation in the vascular wall is at least not followed by increases in fibrinogen degradation products (TDP), which suggests the possibility of accumulation and increased cardiovascular risk. The results indicate that specific attention should be paid to fibrin turnover in studying other categories of women and the effects of the addition of progesterone.