A Novel Chimaeric Derivative of Saruplase, rscu-PA-40kDA/Hir, Binds to Thrombin and Exerts Thrombus-specific Fibrinolysis in Arterial and Venous Thrombosis in Dogs

Summary

The chimaeric molecule rscu-PA-40kDA/Hir (M23) comprises the kringle and protease domain of saruplase (rscu-PA) and a thrombin inhibitory domain fused to the C-terminus of the protease domain. The 27 amino acid long thrombin inhibitory domain contains a sequence directed to the active site of thrombin and a fragment from the C-terminal region of hirudin. ¹²⁵I-radiolabelled M23 (0.03 µM) bound to thrombin that was immobilised onto CNBr-activated sepharose beads. Unlabelled M23 (0.01-10 |xM) and hirudin (0.001-10 µµM) concentra-tion-dependently displaced ¹²⁵I-M23 from its binding to thrombin. Saruplase (up to 10 (iM) did not influence the thrombin binding of M23. The fibrinolytic properties of M23 and saruplase were compared in anaesthetized dogs with femoral artery and saphenous vein thrombosis. Under concomitant heparinization, the intravenous bolus injections of 1 mg/kg M23 or saruplase induced reperfusion of thrombotically occluded femoral arteries in 4 out of 5 treated animals in each case. There was one reocclusion in the M23-treated group. Time to reperfusion (23 ± 4 vs 25 ± 11 min) and maximal height of reperfusion blood flow (98 ± 21 vs 108 ± 15 % of baseline flow) did not differ significantly between the treatment groups. The time course of the lysis of incorporated ¹²⁵I-fibrin radioactivity in thrombosed saphenous veins was similar after bolus injections of M23 and saruplase. The maximal dissolution of ¹²⁵I-fibrin in the venous thrombosis model was 91 ± 1 % in M23-and 88 ± 5 % in saruplase-treated animals. Plasma levels of fibrinogen were not influenced and a₂-antiplasmin levels were slightly reduced (-27 ± 3 %) after bolus injection of M23. In contrast, bolus injection of saruplase was accompanied by a significant decrease of fibrinogen (-55 ± 19 %) and a₂-antiplasmin (-75 ±11%) plasma levels. Template bleeding times virtually did not differ before (2.8 ± 0.3 min) and 60 min after bolus injection of M23 (3.1 ± 0.3 min), whereas treatment with saruplase resulted in a significant prolongation of template bleeding time from 2.6 ± 0.2 min to 28 ± 13 min. It is concluded that the saruplase derivative M23, while inducing equieffective thrombolysis after intravenous bolus injection in dogs, causes much fewer haemostatic side effects than its parent molecule. The high thrombus-specific activity of M23 is tentatively attributed to its affinity to clot-bound thrombin.

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