Fibrinolytic Enzyme from Aspergillus Oryzae) in the Dog
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J Clin Pathol: first published as 10.1136/jcp.25.7.635 on 1 July 1972. Downloaded from Problems related to fibrinolysis 635 Thrombolytic properties and side effects of brinase (fibrinolytic enzyme from Aspergillus oryzae) in the dog WALTER H. E. ROSCHLAU From the Department ofPharmacology, University of Toronto, Ontario, Canada The thrombolytic properties of 'brinase' were in- Since the mean protease resistance of a large dog vestigated in the dog on standard 24-hour-old population was 85 TTR units with a range from 50 thrombi produced in the femoral artery and jugular to 120 TTR units, the inhibitor titre was always vein by scarification of the intima (Roschlau, 1964). measured before treatment for the prediction of a These thrombi measured about 1-5 to 2-5cmadhering systemic dose, and during an infusion for the firmly to the intima and occluding the vessels as monitoring of progressive inhibitor neutralization. confirmed by zero flow and pressure downstream Occluding arterial and venous thrombi were from the thrombus. They resembled in structure rapidly lysed within a few hours following single and composition those commonly associated with infusions of doses of brinase that reduced protease thromboembolic disease. Since spontaneous lysis did resistance to 30 TTR units (Roschlau, 1964). not occur within appropriate observation times, clot Inhibitor recovery to pretreatment levels occurred resolution after fibrinolytic treatment of the animals within 24 hours following single brinase infusions. was ascribed to the direct effects of therapy. Sustained inhibitor reduction to 40 TTR units by repeated administrations of smaller enzyme doses copyright. Systemic Administration over two to three days (Roschlau, 1965) resulted in somewhat slower but equally complete thrombo- Systemic intravenous infusions of 'brinase' were lysis. standardized to deliver 25000 C2 units (=2-5 mg) When examined at necropsy, formerly thrombosed in 10 ml of physiological saline per minute. Throm- vessel segments did not differ from non-thrombosed bolysis was assessed by direct observation and by the vessels. except for the lesions caused by scarification http://jcp.bmj.com/ measurement of blood flow and pressure beyond the of the intima during thrombus production. All vessel occlusion, followed after varying observation times segments examined for thrombus remnants which, by detailed necropsy studies and histopathological when present, consisted of white platelet deposits examinations. without red clot. Long-term survival studies The thrombolytic efficacy and safety of systemic following brinase thrombolysis showed no rethrom- brinase infusions were found to depend on the bosis in vessels bearing primary intima lesions, individual pretreatment inhibitor titre, which was despite the intentional omission of anticoagulants termed 'protease resistance'. A quickly performed during the posttreatment period. on September 27, 2021 by guest. Protected and simple inhibitor test was developed for bedside Side effects were usually absent if protease use. It employed a series of small test tubes con- resistance was prevented from falling below the 30 taining a constant amount of thrombin and in- TTR units target level. However, intentional or creasing amounts of brinase, with which 1 ml accidental overdose with excessive reduction or aliquots of the animal's blood were allowed to react depletion of inhibitors resulted in a number of during a 10-minute incubation time. There was untoward reactions that can be summarized as initial clotting, followed by lysis in those tubes follows: (Roschlau and Tosoni, 1965; Roschlau, whose concentration of brinase exceeded the Freedman, and Miller, 1969; Freedman and inhibitor content of the blood. The end point was Roschlau, 1970). expressed as the number of units of brinase required Haematological to liquefy a 1 ml blood clot within 10 minutes (reported as the 'test tube requirement' or TTR A moderate transient elevation of whole blood units). From the pretreatment TTR value a total clotting time was observed with all doses of brinase systemic dose was calculated by a correlation table that exceeded the 30 TTR units threshold of protease (Roschlau, 1964; Roschlau and Tosoni, 1965). resistance reduction. This was attributed to the J Clin Pathol: first published as 10.1136/jcp.25.7.635 on 1 July 1972. Downloaded from 636 Problems related to fibrinolysis action of fibrinogen degradation products. A segment. Although subsequent doses of enzyme reversible haemoconcentration was frequently noted, overbalanced this initial clot promotion, pretreat- which seemed to be unrelated to the size of a given ment with heparin (or the admixture of small dose. amounts of heparin to the brinase solution) was desirable to obtain prompt lysis with minimal Haemodynamic enzyme doses. After pretreatment with heparin arterial and Slight hypotensive fluctuations occurred in the venous thrombi lysed within minutes of injection of majority of cases, becoming progressively severe as brinase. Two doses of 2 000 units usually canalized brinase inhibitors were reduced. Intentional depletion the thrombi, and two or three injections resulted in of inhibitors was always accompanied by profound complete removal of thrombotic material. The hypotension, which occasionally resulted in the death duration of treatment rarely exceeded 30 minutes, of the animal from acute cardiovascular collapse. the vessels remaining patent under moderate anti- The haemodynamic effects were attributed to the coagulation. kininogenic properties of the proteolytic enzyme, The instillation of small amounts of brinase into leading to progressive activation of vasodepressor isolated vessel segments was entirely without polypeptides. systemic side effects. At necropsy 24 hours after brinase treatment, both arteries and veins were found to be free of thrombus, and the appearance of Systemic the vessels was essentially normal, excepting the scars at the primary thrombus site. An elevation of liver enzymes (transaminases) was regularly associated with excessive brinase adminis- Conclusions tration. This was considered to be an indication of free proteolysis following the depletion of brinase These experiments showed that systemic administra- inhibitors. Animals showing high elevations of tion of brinase required the partial neutralization of transaminase values during therapy usually dis- circulating inhibitors, which exist in a variety ofcopyright. played varying degrees of internal extravasation at mammalian species including man. Inhibitor titres necropsy. vary between species and between individuals. The appearance of side effects was without Prediction and titration of individual dosage, exception related to the drug-induced depression of therefore, was recognized as a requisite for the protective brinase inhibitors. In the dog, the critical attainment of optimal thrombolytic activity and the level of protease resistance was found to be near 30 prevention of overdosage and side effects. Optimal http://jcp.bmj.com/ TTR units, at which level optimal thrombolytic inhibitor neutralization has been defined experi- activity was obtained without major side effects. We mentally, and methods of dose prediction and have therefore adopted the target of 30 TTR units monitoring of inhibitor levels have been developed. as the non-toxic thrombolytic endpoint of inhibitor The side effects that may occur from over- neutralization during systemic brinase therapy. treatment with brinase appear all to be related to the progressive depletion of protective inhibitor sub- Local Administration stances. Provided that basic mechanisms ofaction of on September 27, 2021 by guest. Protected brinase are recognized and incorporated in the Local thrombolysis of artificial vascular occlusions therapeutic approach, brinase may be used as a in dogs was assessed by injecting brinase via a potent and safe systemic agent in clinical thrombo- cannula directly to the thrombus head (Roschlau and lysis. Fisher, 1966). A solution containing 1 000 C2 units The doses required to effect thrombolysis with of brinase per ml of physiological saline was local administration were found to be substantially administered in 2 ml aliquots in intervals of five to lower than systemic requirements. The effects on 10 minutes. Thrombolysis was assessed by saline inhibitors were hardly perceptible, conferring to the filling of the vessel and transillumination. Following local administration an unusually great margin of thrombolysis, the animals received systemic heparin safety and obviating the laboratory control of in order to maintain an elevated clotting time for an systemic therapy. This has resulted in the adoption additional 24 hours. of brinase as the agent of choice for the treatment of The first injection of 2 000 units of brinase clotting disorders during long-term haemodialysis frequently caused additional clotting in the injected therapy (Roschlau, 1968)..