The chimaeric molecule
rscu-PA-40 kDA/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 cid 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 microM) bound to
thrombin that was immobilised onto CNBr-activated
sepharose beads. Unlabelled M23 (0.01-10 microM) and
hirudin (0.001-10 microM) concentration-dependently displaced 125I-M23 from its binding to
thrombin.
Saruplase (up to 10 microM) 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 alpha 2-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 alpha 2-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.