Properties of chimeric (tissue-type/urokinase-type) plasminogen activators obtained by fusion at the plasmin cleavage site
Colucci, M.; Cavallo, L.G.; Agnelli, G.; Mele, A.; Bürgi, R.; Heim, J.; Semeraro, N.
Thrombosis and Haemostasis 69(5): 466-472
1993
ISSN/ISBN: 0340-6245 PMID: 8391727 Document Number: 414597
Two hybrid plasminogen activators (K-2tu-PA and FK-2tu-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 in two peptide bonds (Glu-143-Leu-144 and Arg-156-Phe-157) that led to low molecular weight scu-PA and to thrombin-inactivated tcu-PA. The specific activities of K-2tu-PA and FK-2tu-PA, as measured by fibrin plate were 2.5 times 10-6 and 1.0 times 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 mu-g/ml for K-2tu-PA and 1 mu-g/ml for EK-2tu-PA, as compared to 0.5 mu-g/ml and gt 2 mu-g/ml for t-PA and scu-PA, respectively. Plasminogen and alpha-2-antiplasmin consumption induced by the hybrid PAs in clot-free plasma was comparable to (K-stu-PA) or lower than (FK-2tu-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.