纤溶酶产生菌的鉴定及产酶优化

目的对纤溶酶产生菌——LZ-01菌株进行种属鉴定并优化其产酶能力。方法对LZ-01菌株的形态特征进行观察,并进行生理生化试验和phoR基因的序列相似性分析以确定菌株种属,通过单因素试验考察碳源、氮源等对菌株发酵产酶活力的影响,采用正交实验确定菌株最佳产酶工艺条件。结果经鉴定,LZ-01菌株与解淀粉芽孢杆菌标准菌株FZ42的相似度达99.78%,鉴定LZ-01菌株为解淀粉芽孢杆菌。经工艺优化后的酶活力是未经优化酶活力的3.63倍。结论本研究为进一步开发纤溶酶制剂溶栓药物提供了一定的理论依据。     

急性心肌梗死患者溶栓治疗前后凝血与纤溶系统的变化

目的探讨急性心肌梗死(AMI)患者溶栓治疗前后不同时间段凝血与纤溶系统的变化情况.方法对41例经溶栓治疗的AMI患者分别于治疗前及治疗后4、8、12、48 h和3、7 d共7次抽取静脉血,分别检测凝血酶原时间(PT)、活化部分凝血活酶时间(APTT)、纤维蛋白原(Fg)、D-二聚体(DD)、纤溶酶原(PLG)、α2-纤溶酶抑制物(α2-PI)、组织型纤溶酶原激活物(t-PA)和纤溶酶原激活物抑制剂-1(PAI-1)等指标的活性或含量.结果所有患者经溶栓治疗后,均导致PT、APTT的明显延长,t-PA活性、DD含量的明显增高,PLG、α2-PI、PAI-1活性和Fg含量的明显降低(与溶栓前比较,P均<0.01).但这种变化为时较短,至溶栓后12 h,各项指标已出现不同程度的恢复,t-PA与PAI-1已回复至溶栓前水平.结论凝血与纤溶活性的变化与溶栓疗效关系密切,应用时监测PLG、α2-PI、t-PA、PAI-1、Fg和DD等指标,对判断溶栓疗效有重要价值.     

溶栓药物的研究进展

心脑血管疾病是严重危害人类生命健康的常见病,其死亡率一直居于前3位,其中以血管栓塞性疾病的发病率、致残率和死亡率最高,而溶栓治疗则是目前最主要的治疗方法。现将溶栓药物的发展现状作一综述:1溶栓药物的发展现状1.1第1代溶栓药物第1代溶栓药物以链激酶(SK)和尿激酶(UK)为代表。链激酶应用最早,目前临床上应用最为广泛,它是C组β溶血性链球菌产生的一种蛋白质,能与纤溶酶原结合,形成SK-纤溶酶原复合物,促使游离的纤溶酶原转变成纤溶酶,溶解纤维蛋白;尿激酶则能直接激活纤溶酶原形成纤溶酶。二者共性是溶栓力强但缺乏溶栓特异性,易导…     

下肢深静脉血栓形成患者溶栓前后凝血、抗凝和纤溶活性改变的研究

目的观察下肢深静脉血栓（LDVT）患者溶栓前后凝血、抗凝和纤溶指标的变化，以探讨出凝血、纤溶活性改变与LDVT患者溶栓后高血栓发生率的相关性。方法测定50例LDVT患者溶栓前后抗凝血浆标本的抗凝血酶活性（AT：A）、纤维蛋白原（FIB）、纤溶酶原活性（PLG：A）、组织型纤溶酶原激活物抗原性（t—PA：Ag）、纤溶酶原活性抑制剂-1抗原性（PAI：Ag）、血浆D-二聚体（D-D）、血管性血友病因子（VWF）、Ⅷ促凝活性（Ⅷ：C）、凝血因子Ⅻ活性（Ⅻ：C）；并取32例健康体检者作对照。结果AT：A、PLG：A、t—PA：Ag、Ⅻ：C在溶栓前明显低于对照组（P〈0．01），溶栓后升高（P〈0．05或P〈0．01），其中AT：A、PIG：A、Ⅻ：C仍低于对照组（P〈0．05或P〈0．01），而t-PA：Ag无显著性差异（P〉0．05）；FIB、PAI：A、D-D、Ⅷ：C、VWF在溶栓前显著高于对照组（P〈0．01），溶栓后下降（P〈0．05或P〈0．01），其中PAI：A、D-D、Ⅷ：C、VWF下仍明显高于对照组（P〈0．05或P〈0．01），而FIB无显著性差异（P〉0．05）。结论出凝血系统机能紊乱、纤溶-抗纤溶系统失衡与LDVT患者溶栓后血栓的发生可能有密切的关系。     

急性心肌梗死患者溶栓治疗前后凝血与纤溶系统的变化

目的　探讨急性心肌梗死 (AMI)患者溶栓治疗前后不同时间段凝血与纤溶系统的变化情况。方法　对 4 1例经溶栓治疗的AMI患者分别于治疗前及治疗后 4、8、12、4 8h和 3、7d共 7次抽取静脉血 ,分别检测凝血酶原时间 (PT)、活化部分凝血活酶时间 (APTT)、纤维蛋白原 (Fg)、D 二聚体 (DD)、纤溶酶原 (PLG)、α2 纤溶酶抑制物 (α2 PI)、组织型纤溶酶原激活物 (t PA)和纤溶酶原激活物抑制剂 1(PAI 1)等指标的活性或含量。结果　所有患者经溶栓治疗后 ,均导致PT、APTT的明显延长 ,t PA活性、DD含量的明显增高 ,PLG、α2 PI、PAI 1活性和Fg含量的明显降低 (与溶栓前比较 ,P均 <0 .0 1)。但这种变化为时较短 ,至溶栓后 12h ,各项指标已出现不同程度的恢复 ,t PA与PAI 1已回复至溶栓前水平。结论　凝血与纤溶活性的变化与溶栓疗效关系密切 ,应用时监测PLG、α2 PI、t PA、PAI 1、Fg和DD等指标 ,对判断溶栓疗效有重要价值。     

重组织型纤溶酶原激活剂治疗急性心梗58例溶栓疗效分析

重组织型纤溶酶原激活剂(rt—PA)是直接激活纤维蛋白溶酶原(plasminogen，PLASMG)转变为纤维蛋白溶酶(纤溶酶)，使纤维蛋白或纤维蛋白原降解以达到溶栓效果溶栓剂。是近年临床广泛应用的静脉溶栓药物。     

地龙溶栓酶对家兔血液凝固与纤溶活性的影响

本文通过给家兔静脉注射地龙溶栓酶观察了该酶对家兔血液凝固与纤溶活性的影响。实验结果表明，该酶可使家兔血浆的凝 血酶时间延长，血浆纤维蛋白原浓度降低，纤维蛋白降解产生增多，上述变化均具有显著意义，但优球蛋白溶解时间无明显改变。     

1例尿激酶溶栓治疗中致严重不良反应护理

尿激酶是从健康人尿中分离,或从人肾组织培养中获得的一种酶蛋白。尿激酶应用于血栓栓塞性疾病的溶栓治疗内源性纤维蛋白溶解系统,能催化裂解纤溶酶原成纤溶酶,后者不仅能降解纤维蛋白凝块,亦能降解血循环中的纤维蛋白原、凝血因子V和凝血因子Ⅷ等,从而发挥溶栓作用。     

复发性缺血性脑卒中患者血纤溶系统的变化及其临床意义

目的：探讨复发性缺血性脑卒中患者血纤溶系统变化的临床意义。方法：观察41例复发缺血性脑卒中、58例初发缺血性脑卒中患者及42例健康对照组血液组织型纤溶酶原激活物（tPA)，纤溶酶原激活物抑制剂（PAI－1），纤溶酶的（Plg)，纤溶酶（Plm)及纤维蛋白原（Fig) 含量。结果：复发性缺血性脑卒中与初发缺血性脑卒中患者血tPA-1水平均明显高于对照组，初发缺血性脑卒中患者Plg明显升高，与对照组比较有显著差异；血纤维蛋白原水平在复发中风组明显高于初发中风组和对照组。TPA－1与房颤和中风家族史呈正相产；Plm与冠心病心绞痛呈正相关。结论：缺血性脑卒中的复发与多种危险因素有关，其中血纤维蛋白原水平的增加和纤溶功能的降低在复发中起着重要作用；降低血纤维蛋白原的水平和纠正降低的血纤溶功能在预防缺血性脑卒中的复发方面有重要意义。     

刺参酸性粘多糖对纤维蛋白凝胶结构及其溶解性的影响

为探讨刺参酸性粘多糖（Ｓｊａｍｐ）促纤溶的机制，体外用浊度法和发色底物法观察了Ｓｊａｍｐ对纤维蛋白凝胶聚集和溶解以及对纤溶酶活性的影响，同时在电镜下也观察了Ｓｊａｍｐ对纤维蛋白凝胶结构的改变。结果显示Ｓｊａｍｐ以浓度依赖的方式明显抑制纤维蛋白单体的聚集，提高纤溶酶的活性；在Ｓｊａｍｐ存在下形成的纤维蛋白凝胶被纤溶酶溶解的速度明显较对照组为快；在Ｓｊａｍｐ存在下形成的纤维蛋白凝块中纤维蛋白丝的质量－长度比（μ）及直径明显增加，从而增加了凝块对纤溶酶的敏感性；此外Ｓｊａｍｐ还以浓度依赖的方式提高纤溶酶的活性。Ｓｊａｍｐ可通过影响纤维蛋白的聚集而改变纤维蛋白凝胶的结构以及增强纤溶酶的活性而促进纤溶。     

Development of a new test for the global fibrinolytic capacity in whole blood.

BACKGROUND: The development of global tests for the fibrinolytic capacity in blood is hampered by the low base-line fibrinolytic activity in blood, by the involvement of both plasmatic components and blood cells in the fibrinolytic system and by the loss of fibrinolytic activity as a result of the action of plasminogen activator inhibitor-1 (PAI-1). OBJECTIVE: To develop a new test for the global fibrinolytic capacity (GFC) of whole blood samples. METHODS AND RESULTS: Collection of blood in thrombin increased the subsequent generation of fibrin degradation products. This was ascribed to rapid clot formation and concomitant reduction of in vitro neutralization of tissue-type plasminogen activator (tPA) by PAI-1. On the basis of this observation, the following test was designed: blood samples were collected in thrombin with and without aprotinin and clots were incubated for 3 h at 37 degrees C. The GFC was assessed from the difference between the fibrin degradation products in the two sera. The assay was applied to blood samples from patients and healthy subjects. Other hemostasis parameters were determined in plasma samples taken simultaneously. The GFC varied considerably (normal range 0.13-13.6 microg mL(-1)); physical exercise strongly increased the GFC. Statistically significant correlations were found with tPA activity, PAI-1 activity and fibrinogen level. A mixture of antibodies against tPA and urokinase-type plasminogen activator (uPA) completely inhibited the GFC. An inhibitor of activated thrombin-activatable fibrinolysis inhibitor (TAFI) accelerated fibrinolysis 8-fold. CONCLUSION: The new test represents a global assessment of the main fibrinolytic factors in plasma and potentially those associated with blood cells.     

Isolation of bacteria which produce yeast cell wall-lytic enzymes and their characterization

To isolate bacteria capable of producing enzymes that lyse cell wall, we screened soil samples from Rokko Mountain where is situated in west Japan. The isolated strains KH1, KH2 and KH3 that secrete yeast cell wall lytic enzymes are described herein. The activity of the enzymes, after isolation from culture supernatants, were examined. The lytic enzyme produced by strain KH3 was the most active in both cell wall degradation and beta-1,3 glucanase activity in comparison with commercially available enzymes. This enzyme also had the highest thermo- and pH-stability. On the baisis of partial 16S rDNA sequence analyses, KH1 and KH3 were identified as Cellulosimicrobium sp. and KH2 as Bacillus sp. KH3 was tested by DNA-DNA hybridization and GC content in an attempt to precisely classify it, because the 16S rDNA sequence similarities to other strains were very high at 99.4%. These chemotaxonomic experiments indicate that KH3 belongs to Cellulosimicrobium cellulans.     

Fibrinolysis in health and disease: severe abnormalities in systemic lupus erythematosus

Methods are described to measure fibrinolysis in healthy persons and in patients with systemic lupus erythematosus. Using the fibrin plate method, total fibrinolytic activity and vascular plasminogen activator were measured. (Total fibrinolytic activity expresses the fibrinolytic potential and consists of both the intrinsic [factor XII-dependent and independent] activities and the extrinsic activities [vascular or tissue type]. Vascular plasminogen activator, assessed in a separate assay, refers to the endothelium-derived component only.) In addition, the degree of inhibition by plasma of both urokinase-induced and of plasmin-induced fibrinolysis were analyzed. Vascular plasminogen activator levels were low in 63% of plasma samples from 55 patients with systemic lupus erythematosus. The level of an inhibitor of plasminogen activation was significantly elevated in 87% of patients and levels of an inhibitor of plasmin were significantly elevated in 29%. The nonspecific serine protease inhibitors, including alpha 2-macroglobulin, were within the normal range in all patients. The natures of inhibitor of plasminogen activation and plasmin inhibitor were studied further. Using both the fibrin plate and the lysis time methods, the data indicated that the urokinase-inhibiting activity increased with time of incubation of plasma-enzyme mixtures, whereas the plasmin inhibiting activity did not. Elevated levels of plasmin inhibitor measured with the fibrin plate method correlated well with prolonged lysis times. Results using the chromogenic substrate S-2251, commonly used as a simple and specific assay for antiplasmin, agreed reasonably well with those using the fibrin plate method, but elevated plasmin inhibitor levels could be quantitated with greater accuracy and sensitivity by the fibrin plate method. Studies with an antiserum directed against alpha 2-antiplasmin showed that inhibitor of plasminogen activation and plasmin inhibitor were different inhibitors, and that plasmin inhibitor was identical to alpha 2-antiplasmin. The abnormalities are discussed in the light of current knowledge on fibrinolysis and as possible mediators in the pathogenesis and perpetuation of lupus glomerulonephritis.     

A simple spectrophotometric assay of plasminogen activator: comparison with the fibrinolytic method

We describe a simple assay of plasminogen activator in which the enzyme reacts with a mixture of plasminogen and H-D-valyl-L-leucyl-L-lysine-p-nitroanilide for 1 h at 37 degrees C after which the absorbance is measured at 405 nm. The method detects as little as 2 CTA milliunits of activator and is linear over a 100-fold range of enzyme concentration. The new procedure has been used successfully for the assay of activator in breast tumor cytosols, cell culture supernatants, and pleural or ascitic fluids. Thirty-two biological samples have been assayed for plasminogen activator activity with both the spectrophotometric method and a classical fibrinolytic technique using radiolabeled fibrin. Although the two series of results are significantly correlated, the activities measured with the former assay are significantly different from those determined with the latter. It is shown that the spectrophotometric method is, in many respects, superior to the fibrinolytic procedure.     

Dextran sulfate-dependent fibrinolysis in whole human plasma

This study shows that Flu-beta-Ala can reduce the ability of human plasma to inhibit plasmin. This observation was utilized to develop a method for generating detectable fibrinolytic activity in whole human plasma as assessed on a radiolabeled fibrin plate. Plasma was pretreated with Flu-beta-Ala to remove inhibitors of fibrinolysis: then dextran sulfate was added and the mixture was further incubated at 4 degrees C. When normal plasma was treated in this manner, the rate of generation of fibrinolytic activity after 0.75 hr incubation with radiolabeled fibrin was equivalent to that of 35 ng/ml plasmin. The plasminogen dependence of this activity was tested by pretreating plasma with antibodies against plasminogen. The generation of fibrinolytic activity was totally blocked by this treatment, indicating that the observed fibrinolytic activity was plasminogen-dependent. When plasmas deficient in prekallikrein, factor XII, or high-molecular-weight kininogen were treated with Flu-beta-Ala and dextran sulfate, the initial rate of fibrinolytic activity was less than normal. But after 3 hr incubation with radiolabeled fibrin, the rate of fibrinolytic activity in these deficient plasmas approached that of normal plasma. Thus this dextran sulfate-dependent fibrinolytic activity is dependent on factor XII, prekallikrein, and high-molecular-weight kininogen, but the requirement is not absolute.     

产妇DIC中凝血与纤溶系统水平临床分析

目的 探讨产妇弥漫肉性血管内凝血(DIC)时凝血与纤溶系统水平检测并分析.方法 本院2004年1月至2009年1月产科DIC52例为DIC组,同期与本科健康体检的门诊患者60例为对照组,检测内容:凝血酶原时间(PT)、活化的部分凝血活酶时间(APTT);D2二聚体(D-D)、纤维蛋白/纤维蛋白原降解产物(FDP)、3 P试验,进行比较并观察各指标间的相关性.结果 DIC组与对照组进行观察检测内容比较P<0.05,有显著差异性.结论 产妇患者出现明显的凝血功能指标如D-D、PT、FDPAPTT等明显异常时,应特别警惕D IC的可能.如存在D IC的诱发因素和凝血、纤溶异常但尚未达到D IC的确诊标准,可能正处于D IC的前状态,应引起临床高度的重视,笔者的观察显示,联合D-D,FDP和APTT检测是早期诊断DIC的最好策略.     

产妇DIC中凝血与纤溶系统水平临床分析

目的 探讨产妇弥漫肉性血管内凝血(DIC)时凝血与纤溶系统水平检测并分析.方法 本院2004年1月至2009年1月产科DIC52例为DIC组,同期与本科健康体检的门诊患者60例为对照组,检测内容:凝血酶原时间(PT)、活化的部分凝血活酶时间(APTT);D2二聚体(D-D)、纤维蛋白/纤维蛋白原降解产物(FDP)、3 P试验,进行比较并观察各指标间的相关性.结果 DIC组与对照组进行观察检测内容比较P<0.05,有显著差异性.结论 产妇患者出现明显的凝血功能指标如D-D、PT、FDPAPTT等明显异常时,应特别警惕D IC的可能.如存在D IC的诱发因素和凝血、纤溶异常但尚未达到D IC的确诊标准,可能正处于D IC的前状态,应引起临床高度的重视,笔者的观察显示,联合D-D,FDP和APTT检测是早期诊断DIC的最好策略.     

Acute effect of smoking on fibrinolysis: increase in the activity level of circulating extrinsic (tissue-type) plasminogen activator*

Abstract. The acute effect of cigarette smoking on the fibrinolytic enzyme system in blood was studied. It was found imperative to have an initial 30 min rest period, after venipuncture, to obtain a stable baseline in the fibrinolytic studies.
The average heart rate, in inhaling smokers, increased from 64 to a peak of 79 beats min^-1 5–10 min after commencement of smoking. A peak in fibrinolytic activity was found to occur later, at 22·5 min. Analysis of the increase in fibrinolytic activity revealed no demonstrable activation of intrinsic systems via factor XII, nor changes in plasminogen, prekallikrein and C1-inactivator. No plasmin- α ₂-antiplasmin complexes were detectable. The increase ( P <0·01) was found to be due to extrinsic (tissue-type) plasminogen activator, revealed as C1-inactivator-resistant plasminogen activator activity, and further identified by quenching with anti-tissue plasminogen activator IgG.
Thus, smoking appears to elicit a significant increase in the level of activity of circulating extrinsic plasminogen activator.     

Enhanced fibrinolytic activity during cardiopulmonary bypass in open-heart surgery in man is caused by extrinsic (tissue-type) plasminogen activator

The nature of the enhanced blood fibrinolytic activity which is known to occur during cardiopulmonary bypass is not understood. We show here that the cause is an increase in extrinsic (tissue-type) plasminogen activator. In six patients, the nature of the enhanced blood fibrinolytic activity that evolved during cardiopulmonary bypass was characterized by differential inhibition using the fibrin plate method and was shown to be C1-inactivator-resistant (extrinsic-activator activity). The C1-inactivator-resistant-activator activity was completely quenched by an antibody against extrinsic (tissue-type) plasminogen activator but not by antiurokinase, proving that the activity was due to the presence of extrinsic (tissue-type) plasminogen activator. The concentration of extrinsic (tissue-type) plasminogen activator increased during cardiopulmonary bypass and disappeared rapidly thereafter. Fibrinogen, plasminogen and alpha 2-antiplasmin were not consumed during cardiopulmonary bypass, while no increase or occasionally a moderate one in fibrinogen degradation products occurred. This is in accord with the property of extrinsic (tissue-type) plasminogen activator which activates plasminogen predominantly at sites where fibrin is present and not in the free circulation.     

In vitro characterization of A-315675, a highly potent inhibitor of A and B strain influenza virus neuraminidases and influenza virus replication

A-315675 is a novel, pyrrolidine-based compound that was evaluated in this study for its ability to inhibit A and B strain influenza virus neuraminidases in enzyme assays and influenza virus replication in cell culture. A-315675 effectively inhibited influenza A N1, N2, and N9 and B strain neuraminidases with inhibitor constant (K(i)) values between 0.024 and 0.31 nM. These values were comparable to or lower than the K(i) values measured for oseltamivir carboxylate (GS4071), zanamivir, and BCX-1812, except for the N1 enzymes that were found to be the most sensitive to BCX-1812. The time-dependent inhibition of neuraminidase catalytic activity observed with A-315675 is likely due to its very low rate of dissociation from the active site of neuraminidase. The half times for dissociation of A-315675 from B/Memphis/3/89 and A/Tokyo/3/67 (H3N2) influenza virus neuraminidases of 10 to 12 h are significantly slower than the half times measured for oseltamivir carboxylate (33 to 60 min). A-315675 inhibited the replication of several laboratory strains of influenza virus in cell culture with potencies that were comparable or superior to those for oseltamivir carboxylate and BCX-1812, except for the A/H1N1 viruses that were found to be two- to fourfold more susceptible to BCX-1812. A-315675 and oseltamivir carboxylate exhibited comparable potencies against a panel of A/H1N1 and A/H3N2 influenza virus clinical isolates, but A-315675 was found to be significantly more potent than oseltamivir carboxylate against the B strain isolates. The favorable in vitro results relative to other clinically effective agents provide strong support for the further investigation of A-315675 as a potential therapy for influenza virus infections.