Abstract
Summary
Two hybrid plasminogen activators (K
2
tu-PA and FK
2
tu-PA), linking the kringle 2 domain or the finger plus the kringle 2 domains of tissue-type plasminogen activator (t-PA) to the catalytic domain of single-chain urokinase-type plasminogen activator (scu-PA) were studied. At variance with similar constructs previously reported, they were obtained by fusion of the t-PA and scu-PA derived portions at their plasmin cleavage site (between Arg
275
of t-PA and Ile
159
of scu-PA), thus eliminating from scu-PA the two peptide bonds (Glu
143
-Leu
144
and Arg
156
-Phe
157
) that lead to low molecular weight scu-PA and to thrombin-inactivated tcu-PA. The specific activities of K
2
tu-PA and FK
2
tu-PA, as measured by fibrin plate were 2.5 × 10
6
and 1.0 × 10
6
t-PA equivalent units/mg, respectively. Activation of plasminogen by hybrid PAs was stimulated by both CNBr-digested fibrinogen (40- and 80-fold) and Des-A-fibrin monomers (6- and 12-fold). The relatively weak stimulation of chimeric PAs by minimally degraded fibrin monomers was consistent with their reduced fibrin binding capacity. Like scu-PA, the chimeric PAs, in the single-chain form, were insensitive to inhibition, as they retained full activity after prolonged incubation in plasma and did not interact with SDS-reactivated recombinant PAI-1. The concentration producing 50% lysis of blood clots in 3 h was 0.5 μg/ml for K
2
tu-PA and 1 μg/ml for FK
2
tu-PA, as compared to 0.5 μg/ml and >2 μg/ml for t-PA and scu-PA, respectively. Plasminogen and α
2
-antiplasmin consumption induced by the hybrid PAs in clot-free plasma was comparable to (K
2
tu-PA) or lower than (FK
2
tu-PA) that induced by either t-PA or scu-PA. When exposed to plasmin, the hybrids were completely converted into two-chain molecules with full enzymatic activity. At variance with u-PA, however, the two-chain recombinant activators still required fibrin for full expression of activity. These data indicate that the products of such “artificial” fusion behave like true chimeras without loss of biological activity. The insensitivity to thrombin inactivation and to the proteolytic cleavage leading to low molecular weight scu-PA might confer enhanced stability to the molecules, especially at thrombus level. Moreover, if the thrombolytic activity observed in vitro is maintained in vivo, the prolonged half life of these hybrids should result in higher plasma levels of activator and thus in more extensive and rapid lysis.