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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。溶栓疗法，使用系统或选择性地使用纤溶酶原激活剂，已经获得了越来越多的人接受血栓栓塞症的治疗。然而，溶栓治疗仍需进一步改进。我们寻找一名更有效、更安全的特工，导致我们调查蛇的毒液。1956年，几名研究人员描述了纤溶各种蛇毒的性质，并提到提纯的蛇毒组分可能在临床上用作溶栓剂。研究表明，因此，我们的实验室已经开始提纯和表征蛇毒纤溶酶。这些调查采用了一种新的方法用于纯化纤溶酶和一种新的体内血管造影术模型评估他们的治疗效果。我们已经提纯了一种凝胶法提取南方铜鱼蛇毒纤溶酶 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。这种酶证明了钠溶液中的聚丙烯酰胺凝胶电泳法均一十二烷基硫酸盐。这种酶似乎并不能激活纤溶酶原，相反它对纤维蛋白有直接的溶解作用。体内活性将被评估对抗新鲜血栓栓子进入兔肾脏。兔肾动脉将被分流，并遵循对照动脉造影标准体积的凝块(兔或人)，将被注射到每个肾脏完全肝素化的兔子。测试的纤溶酶将被注入仅进入一条肾动脉，而对侧肾动脉作为控制力。溶栓治疗将每隔一段时间进行动脉造影术。到5个小时。一次显着的体内溶栓活性试验该制剂已在兔体内得到证实，将产生不良反应评估过了。将评估急性和慢性肾毒性。输液后的动脉造影、组织学和功能进入未经栓塞者的肾脏。心血管影响将通过以下方式进行评估肾内、冠脉内灌注后进行心电图和测压。凝血参数(凝血酶时间、纤维蛋白原)可能的改变浓度、凝血时间)将被确定。急慢性神经毒性将在颈动脉注射后进行测试。通过这种方式，比较几种酶制剂的治疗比例。那些治疗比率最高的酶将进一步研究以它们的生化特性及其对人体的体外活性纤维蛋白原、纤维蛋白和凝血蛋白。如果这些调查证实了我们在体内的初步发现，纯化的蛇毒酶是有效和安全的纤溶剂，这些酶可能会发现广泛的在人类血栓栓塞病治疗中的应用。