THROMBOLYSIS BY RAPID DIRECT-ACTING FIBRINOLYTIC AGENTS

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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.