THROMBOLYSIS BY RAPID DIRECT-ACTING FIBRINOLYTIC AGENTS

快速直接作用纤溶剂的溶栓作用

基本信息

批准号：
3342470
负责人：
FRANCIS S MARKLAND
金额：
$ 14.23万
依托单位：
UNIVERSITY OF SOUTHERN CALIFORNIA
依托单位国家：
美国
项目类别：
财政年份：
1984
资助国家：
美国
起止时间：
1984-01-01 至 1987-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3342470
关键词：
blood coagulation tests blood tests cardiovascular disorder diagnosis disease /disorder model drug adverse effect drug metabolism fibrinogen fibrinolysis fibrinolytic agents gel electrophoresis gel filtration chromatography histology human therapy evaluation human tissue ion exchange chromatography plasminogen activator reptile poison thromboembolism

项目摘要

Thromboembolic vascular disease comprises a significant fraction of all morbidity and mortality in the United States. Thrombolytic therapy, using systemically or selectively administered plasminogen activators, has gained increasing acceptance for the treatment of thromboembolic disease. However, further improvements in thrombolytic therapy are still required. Our search for a more effective and safer agent, led us to investigate snake venoms. In 1956, several investigators characterized fibrinolytic properties of various snake venoms and mentioned that purified venom fractions may be clinically useful as thrombolytic agents. Studies have, therefore, been initiated in our laboratories to purify and characterize fibrinolytic snake venom enzymes. These investigations employ a new method for purifying fibrinolytic enzymes and a new in vivo angiographic model for evaluating their therapeutic effectiveness. We have purified a fibrinolytic enzyme from southern copperhead venom by combination of gel filtration on Sephadex G-100 and ion exchange chromatography on carboxymethylcellulose and diethylaminoethylcellulose. The enzyme proved to be homogeneous by polyacrylamide gel electrophoresis in sodium dodecylsulfate. The enzyme does not appear to activate plasminogen, rather it has a direct lytic effect on fibrin. In vivo activity will be assessed against fresh thromboemboli introduced into rabbit kidneys. Rabbit renal arteries will be cateterized and following control arteriograms, standard volumes of clot (rabbit or human), will be injected into each kidney of fully heparinized rabbits. The test fibrinolytic enzyme will be infused into one renal artery only, while the contralateral renal artery serves as control. Thrombolysis will be assessed arteriographically at intervals up to 5 hours. Once significant in vivo thrombolytic activity of the test agent has been demonstrated in the rabbit, adverse effects will be assessed. Acute and chronic renal toxicity will be evaluated arteriographically, histologically, and functionally, following infusion into non-embolized kidneys. Cardiovascular effects will be evaluated by EKG and manometry following intrarenal, and then intracoronary, infusion. Possible alterations of coagulation parameters (thrombin time, fibrinogen concentration, clotting time) will be determined. Acute and chronic neurotoxicity will be tested after intracarotid infusion. In this way, the therapeutic ratio of several enzyme preparations will be compared. Those enzymes with the highest therapeutic ratios will be further studied to characterize their biochemical properties and in vitro activity on human fibrinogen, fibrin and blood coagulation proteins. If these investigations confirm our preliminary in vivo findings that purified snake venom enzymes are potent and safe fibrinolytic agents, these enzymes may find widespread application in the treatment of human thromboembolic disease.

血栓栓塞性血管疾病占所有疾病的很大一部分美国的发病率和死亡率。溶栓治疗，使用全身或选择性施用纤溶酶原激活剂，已获得血栓栓塞性疾病治疗的接受度不断提高。然而，溶栓治疗仍需要进一步改进。为了寻找更有效、更安全的药物，我们进行了调查蛇毒。 1956 年，几位研究人员描述了纤溶各种蛇毒的特性，并提到纯化的毒液级分可在临床上用作血栓溶解剂。研究表明，因此，我们的实验室开始纯化和表征纤溶蛇毒酶。这些调查采用了新方法用于纯化纤溶酶和新的体内血管造影模型评估其治疗效果。我们纯化了一个南方铜头蛇毒液中的纤维蛋白溶解酶与凝胶的组合 Sephadex G-100 过滤和离子交换层析羧甲基纤维素和二乙氨基乙基纤维素。酶证明通过钠聚丙烯酰胺凝胶电泳得到均质十二烷基硫酸盐。该酶似乎不会激活纤溶酶原，而是它对纤维蛋白有直接的溶解作用。将评估体内活性对抗引入兔肾的新鲜血栓栓塞。兔肾动脉将被分类并遵循控制动脉图、标准一定量的凝块（兔子或人），将被注射到每个肾脏中完全肝素化的兔子。将输注测试纤溶酶仅进入一侧肾动脉，而对侧肾动脉则充当控制。将每隔一段时间对溶栓进行动脉造影评估至5小时。一次显着的体内溶栓活性测试药物已在兔子身上得到证实，不良反应将会评估。将评估急性和慢性肾毒性输注后的动脉造影、组织学和功能进入非栓塞肾脏。心血管作用将通过以下方式评估肾内、冠状动脉内输注后进行心电图和测压。凝血参数可能发生改变（凝血酶时间、纤维蛋白原浓度、凝血时间）将被确定。急性和慢性颈动脉内输注后将测试神经毒性。这样，将比较几种酶制剂的治疗率。那些将进一步研究具有最高治疗率的酶表征它们的生化特性和对人体的体外活性纤维蛋白原、纤维蛋白和凝血蛋白。如果这些调查证实我们的初步体内研究结果，即纯化的蛇毒酶是有效且安全的纤溶剂，这些酶可能会广泛使用在治疗人类血栓栓塞性疾病中的应用。