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. 125 I-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 125 I-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 125 I-fibrin radioactivity in thrombosed saphenous veins was similar after bolus injections
of M23 and saruplase. The maximal dissolution of 125 I-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.
