A NOVEL CHIMERIC DERIVATIVE OF SARUPLASE, RSCU-PA-40KDA HIR, BINDS TOTHROMBIN AND EXERTS THROMBUS-SPECIFIC FIBRINOLYSIS IN ARTERIAL AND VENOUS THROMBOSIS IN DOGS/

The chimaeric molecule rscu-PA-40kDA/Hir (M23) comprises the kringle a nd protease domain of saruplase (rscu-PA) and a thrombin inhibitory do main fused to the C-terminus of the protease domain. The 27 amino acid long thrombin inhibitory domain contains a sequence directed to the a ctive site of thrombin and a fragment from the C-terminal region of hi rudin. I-125-radiolabelled M23 (0.03 mu M) bound to thrombin that was immobilised onto CNBr-activated sepharose beads. Unlabelled M23 (0.01- 10 mu M) and hirudin (0.001-10 mu M) concentration-dependently displac ed I-123-M23 from its binding to thrombin. Saruplase (up to 10 mu M) d id not influence the thrombin binding of M23. The fibrinolytic propert ies of M23 and saruplase were compared in anaesthetized dens with femo ral artery and saphenous vein thrombosis. Under concomitant hepariniza tion, the intravenous bolus injections of 1 mg/kg M23 or saruplase ind uced reperfusion of thrombotically occluded femoral arteries in 4 out of 5 treated animals in each case. There was one reocclusion in the M2 3-treated group. Time to reperfusion (23 +/- 4 vs 25 +/- 11 min) and m aximal height of reperfusion blood flow (98 +/- 21 vs 108 +/- 15 % of baseline flow) did not differ significantly between the treatment grou ps. The time course of the lysis of incorporated I-125-fibrin radioact ivity in thrombosed saphenous veins was similar after bolus injections of M23 and saruplase. The maximal dissolution of I-125-fibrin in the venous thrombosis model was 91 +/- 1 % in M23- and 88 +/- 5 % in sarup lase-treated animals. plasma levels of fibrinogen were not influenced and alpha(2)-antiplasmin levels were slightly reduced (-27 +/- 3 %) af ter bolus injection of M23. In contrast, bolus injection of saruplase was accompanied by a significant decrease of fibrinogen (-55 +/- 19 %) and alpha(2)-antiplasmin (-75 +/- 11 %) plasma levels. Template bleed ing 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 wit h saruplase resulted in a significant prolongation of template bleedin g time from 2.6 +/- 0.2 min to 28 +/- 13 min. It is concluded that the saruplase derivative M23, while inducing equieffective thrombolysis a fter intravenous bolus injection in dogs, causes much fewer haemostati c side effects than its parent molecule. The high thrombus-specific ac tivity of M23 is tentatively attributed to its affinity to clot-bound thrombin.