Fibrolase

MA M12 M12.133 Fibrolase (Agkistrodon contortrix) Potential target for vaccine development. 

Direct Fibrinolytics Alfimeprase is a recombinant tmncated form of fibrolase, a fibrinolytic zinc metalloproteinase isolated from the venom of the Southern copperhead snake. It degrades fibrin directly and achieves thrombolysis independent of plasmin formation. This may result in faster recanalization and a decreased risk of hemorrhagic conversion. The initial data on the safety and efficacy of alfimeprase in peripheral arterial occlusion disease appeared very promising, but recent communication from the sponsor revealed that the phase III trials of the drug in peripheral arterial disease and catheter obstruction (NAPA-2 and SONOMA-2) failed to meet their primary and key secondary endpoints of revascularization. A trial for I AT in acute stroke (CARNEROS-1) is planned to begin soon. 

FIBROLASE, AN ACTIVE THROMBOLYTIC ENZYME IN ARTERIAL AND VENOUS THROMBOSIS MODEL SYSTEMS... 

Snake venoms, particularly those from North American pit vipers, contain direct-acting fibrinolytic proteinases (13). Fibrolase is the fibrinolytic enzyme from southern copperhead venom (14). Fibrolase is a non-glycosylated metalloproteinase with a molecular weight of 23,000 it contains one mole of zinc per mole of protein (14). The enzyme has an isoelectric point of approximately pH 6.8. The amino acid sequence of the enzyme has been determined... 

The present report outlines studies with fibrolase in several different animal thrombosis model systems. Infusion of fibrolase proximal to an occlusive thrombus produces rapid and effective thrombolysis in carotid (22) and renal arterial, and iliac venous thrombosis model systems (23). Recently the recombinant form of fibrolase has been purified from a yeast expression system (24). It appears to be identical in all respects to the natural enzyme and has been used successfully in the carotid arterial thrombosis model system (22). Both natural and recombinant forms of the enzyme have effective thrombolytic activity in the different animal models employed there are no observable side effects nor toxicity, and minimal or no observable hemorrhaging. 

A four-step open column procedure was originally developed for purification of fibrolase from crude snake venom (14). Recently, we developed a rapid two-step, analytical... 

Rabbit acute renal arterial thrombosis model In vivo evaluation of purified fibrolase was initially performed, in collaboration with Joseph Bookstein, using an acute rabbit renal arterial thrombosis model. Fresh thrombi were made by adding bovine thrombin to rabbit blood. The clot was allowed to set and retract at room temperature for one hour. Rabbits... 

Figure 1. Thrombolysis produced by natural fibrolase in the rabbit acute renal arterial thrombosis model. The arteriograms show A, contrast medium injected through the catheters into both normal renal arteries B, arterial occlusion after introducing thrombus in both renal arteries C, after infusion of fibrolase into the occluded left renal artery there is almost complete clearance of the clot at 30 min. Note the lack of clearance in the non-infused (right) kidney. After 4 hours the right renal artery remained occluded, but was cleared after a 20 min infusion of fibrolase directly into the right renal artery.

Figure 2. Thrombolytic effectiveness of natural fibrolase in a subacute iliac venous thrombosis model in the rabbit. Occluding spring coils were introduced retrograde from the jugular vein into both iliac veins. After 48 hours cutdowns on each iliac vein were performed below the coils and a catheter was introduced. Standard heparin infusion was initiated via the ear vein. A control angiogram was taken. Infusion of venom enzyme was then begun and repeat venograms were taken to document the extent of lysis. A, venogram at 0 time B, following 1 hour of fibrolase infusion C, following 2 hours of enzyme infusion.

These model studies revealed that selective application of highly purified fibrolase produced rapid thrombolysis. The autologous thrombus lysed promptly whereas there was no detectable lysis of the contralateral thrombus. The lack of physiologic alterations attributable to fibrolase and the absence of demonstrable histologic changes, further suggest the favorable therapeutic potential of the enzyme. These studies indicate that the venom fibrinolytic enzyme appears to function independently of the native fibrinolytic system and offers promise as a safe and effective agent for selective thrombolysis. 

In the iliac venous thrombosis model system six animals were studied with good lysis observed venographically in five (23). Figure 2 presents representative venograms illustrating the effectiveness of thrombolysis induced by selective application of natural fibrolase. No toxicity, little or no hemorrhaging, and no evidence of other side effects were observed in the animals studied. These studies again demonstrate the favorable therapeutic potential of native fibrolase. 

Figure 3. Carotid blood flow velocity in treated and control groups. A, carotid blood flow velocity in groups treated either with APSAC alone or APSAC plus 7E3. Thirty min after thrombotic occlusion of the vessel, APSAC was infiised immediately proximal to the thrombus. Five min after restoration of the left carotid artery blood flow velocity, the 7E3 F(ab )2 monocloanl antibody was administered intravenously to only one of the groups receiving APSAC. B, carotid blood flow velocity in both the right and left carotid arteries after occlusive thrombus formation. The right carotid artery served as the control vessel and remained occluded throughout the course of the experimental procedure. Thirty min after occlusion of the left carotid artery, r-fibrolase was infused immediately proximal to the occlusive thrombus. Clot lysis was achieved in each of the five animals in the group. Five min after restoration of blood flow in the left carotid artery, the 7E3 antibody was administered intravenously. Blood flow velocity was maintained in the left carotid artery in four of the five vessels that received the combined treatment.

The administration of r-fibrolase (4 mg/kg), immediately proximal to the occlusive carotid artery thrombus, did not alter the aPTT from its baseline value of 111 6 sec vs 113 18 sec after r-fibrolase. Platelet aggregation in response to either arachidonic acid or ADP was decreased by approximately 60%. Complete inhibition of ex vivo platelet aggregation resulted following the administration of the 7E3 F(ab )2 monoclonal antibody. 

Lack of Hemorrhagic Effect of r-Fibrolase. The chest was opened in all r-fibrolase treated dogs and the pericardium exposed for evidence of excessive blood infiltration. The appearance of the heart and surrounding pericardium appeared normal in each dog. The intestines, liver, and kidneys were examined and found to be normal in appearance. There was no evidence of local or internal hemorrhaging at any site in any of the dogs which received r-fibrolase. 

The present results demonstrate that natural fibrolase exhibits effective in vivo thrombolytic activity in several animal thrombosis model systems. Infusion of the enzyme proximal to an occlusive thrombus induced rapid and specific thrombolysis in rabbit renal arterial and iliac venous thrombosis model systems. No evidence of hemorrhaging or alterations to the hemostatic system were observed in these studies. Additionally, no toxicity was observed and no angiographic, histologic, or functional evidence of side effects were obtained. The enzyme rapidly lysed 48 hr aged thrombi in the venous thrombosis model. This suggests that one of the primary mechanisms of thrombus resistance to PA-based agents. 

The primary intent of the study using the canine carotid arterial thrombosis model was to determine if the recombinant enzyme could achieve thrombolysis in an experimental animal model in which a platelet-rich, occlusive arterial thrombus, develops in response to deep arterial wall injury. We found that fibrolase produced rapid thrombolysis. The experimental model is similar to one originally developed by Lucchesi in the coronary artery (30) and subsequently adapted to the carotid artery to minimize experimental loss due to ventricular fibrillation (31). The use of the carotid has the additional advantage of greater yield of experimental data, and when necessary allows the animal to serve as its own internal control, thereby facilitating the interpretation of results and simplifying statistical evaluation. This was an important consideration in the present study where r-fibrolase was in limited supply thus accounting for the need to employ relatively small animals and to administer the enzyme by local injection immediately proximal to the occlusive platelet-rich arterial thrombus. 

The present investigations, although limited to the local application of fibrolase, demonstrate that under these conditions the enzyme lyses venous or arterial thrombi rapidly and with no observable systemic or hematologic side effects. In these thrombosis model systems the enzyme, either alone or in combination with antiplatelet therapy, offers a unique, safe, and specific mechanism for clot dissolution and may prove useful as a clinically effective alternative to, or for use in synergistic combination with, presently used thrombolytic agents. 

The authors would like to acknowledge Drs. Patricio Riquelme and Pablo Valenzuela, Chiron Corporation, Emeryville, CA, for providing r-fibrolase. This work was supported in part by NIH Grant HL 31389. Figure 3 was reproduced with permission of the American Heart Association from F.S. Markland, G.S. Friedrichs, S.R. Pewitt andB.R. Lucchesi, 1994, Thrombolytic Effects of Recombinant Fibrolase or APSAC in a Canine Model of Carotid Artery Thrombosis, Circulation 90 2448-2456. 

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