THE ACTIVITY OF A SINGLE CHAIN rt-PA MUTANT IN A PRIMATES AND RABBITS
Recombinant tissue-type plasminogen activator(rt-PA) contains a plasmin sensitive peptide bond domain between amino acids (AAs) 275 and 276. Since the single chain form of rt-PA is rapidly clipped to the two chain form during thrombolysis, one way to determine the relative activities of single chain and two chain rt-PA is to produce a mutant which no longer has this clip site; therefore, a single AA substitution was made at position 275, replacing Glu for Arg (Tate, et al., Biochemistry 1987, in Press). The thrombolytic activity of this mutant (Glu275) was compared to the activity of the wild type rt-PA (Arg275) in a new primate thrombolysis model.An arterio-venous shunt model (Hotchkiss et al, this meeting) was used. Various doses of the rt-PAs were infused over a 90 min period with 10% of the total dose administered as an initial bolus. The rate and extent of thrombolysis were quantitated by the measurement of radioactivity remaining in the thrombus at 15, 30, 60 and 90 min, using an external probe. Data were collected from 5 cyncmolgus monkeys (male 5 to 6 kg). The dose response curves were used to determine the ED40 (dose for 40% clot lysis). The ED40 for Arg275 and Glu275 wire 870 and 680 (ugAg)r respectively. The plasna concentrations of rt-PA were measured at plateau for various doses and the regression of the plasma concentrations (Y,ng/ml) versus the dose rates (X, ug/kg/min) was calculated. The regression equations were; Y=28+51X and Y=39+113X for Arg275 and Glu275, respectively. This indicates that the clearance rate of Glu275 is 2.2 fold slower than Arg275.These experiments were also performed in rabbits. The ED40 for Arg275 and Glu275 were 740 and 660 (ug/kg) respectively. The Glu275 was cleared more slowly, having a 1.7 fold slower clearance rate.These results support the conclusion, that the "one chain" Glu275 rt-PA is still active in vivo and that it is slightly more potent than the wild type in these animal models. Some of this increase in potency is apparently due to a slower clearance rate for the Glu275 rt-PA.