PROPERTIES OF CHIMERIC (TISSUE-TYPE UROKINASE-TYPE) PLASMINOGEN ACTIVATORS OBTAINED BY FUSION AT THE PLASMIN CLEAVAGE SITE

Two hybrid plasminogen activators (K2tu-PA and FK2tu-PA), linking the kringle 2 domain or the finger plus the kringle 2 domains of tissue-ty pe plasminogen activator (t-PA) to the catalytic domain of single-chai n urokinase-type plasminogen activator (scu-PA) were studied. At varia nce with similar constructs previously reported, they were obtained by fusion of the t-PA and scu-PA derived portions at their plasmin cleav age site (between Arg275 of t-PA and Ile159 of scu-PA), thus eliminati ng from scu-PA the two peptide bonds (Glu143-Leu 144 and Arg156-Phe157 ) that lead to low molecular weight scu-PA and to thrombin-inactivated tcu-PA. The specific activities of K2tu-PA and FK2tU-PA, as measured by fibrin plate were 2.5 X 10(6) and 1.0 x 10(6) t-PA equivalent units /mg, respectively. Activation of plasminogen by hybrid PAs was stimula ted by both CNBr-digested fibrinogen (40- and 80-fold) and Des-A-fibri n monomers (6- and 12-fold). The relatively weak stimulation of chimer ic PAs by minimally degraded fibrin monomers was consistent with their reduced fibrin binding capacity. Like scu-PA, the chimeric PAs, in th e single-chain form, were insensitive to inhibition, as they retained full activity after prolonged incubation in plasma and did not interac t with SDS-reactivated recombinant PAI-1. The concentration producing 50% lysis of blood clots in 3 h was 0.5 mug/ml for K2tu-PA and 1 mug/m l for FK2tu-PA, as compared to 0.5 mug/ml and >2 mug/ml for t-PA and s cu-PA, respectively. Plasminogen and alpha2-antiplasmin consumption in duced by the hybrid PAs in clot-free plasma was comparable to (K2tu-PA ) or lower than (FK2tu-PA) that induced by either t-PA or scu-PA. When exposed to plasmin, the hybrids were completely converted into two-ch ain molecules with full enzymatic activity. At variance with u-PA, how ever, the two-chain recombinant activators still required fibrin for f ull expression of activity. These data indicate that the products of s uch ''artificial'' fusion behave like true chimeras without loss of bi ological activity. The insensitivity to thrombin inactivation and to t he proteolytic cleavage leading to low molecular weight scu-PA might c onfer enhanced stability to the molecules, especially at thrombus leve l. Moreover, if the thrombolytic activity observed in vitro is maintai ned in vivo, the prolonged half life of these hybrids should result in higher plasma levels of activator and thus in more extensive and rapi d lysis.