Stimulation by fibrinogen of the amidolytic activity of single-chain tissue plasminogen activator

The amidolytic steady-state kinetic properties of a series of recombinant tissue plasminogen activators (rt-PA) have been examined in the presence and absence of the positive effectors fibrinogen (Fg) and native soluble (des-A)-fibrin (sFn). Two-chain (tc) native rt-PA displayed a Km value of 0.50 mM and a kcat value of 13.2 s-1 toward the substrate, H-D-Ile-Pro-Arg-p-nitroanilide (S2288) at 37 degrees. When these same assays were conducted in the presence of Fg or sFn, the Km and kcat values remained essentially the same. On the other hand, the activity of single-chain (sc) rt-PA was significantly increased in the presence of Fg or sFn, by approximately 3.4- to 4-fold, due to alterations in both the Km and kcat of the reaction. Similar results were obtained with rt-PA deletion variants, obtained by site-directed mutagenesis. With rt-PA domain-deletion derivatives, consisting of kringles 1 (K1)-2 (K2)-protease (P), and K2-P, the amidolytic activities of the scrt-PA preparations were significantly stimulated (2.0- to 2.5-fold) by Fg and sFn, a property not shared by the corresponding tcrt-PA. On the other hand, neither the single- nor two-chain derivatives of a deletion mutant containing only the finger (F)-growth factor (E)-P domains displayed stimulation by Fg or sFn, results suggestive of the importance of the K2 region in the observed Fg- and Fn-induced stimulations rt-PA amidolytic activity. With one strategically important derivative, a molecule containing the amino acid replacement, Cys264----Gly [(Cys264----Gly)-rt-PA], a change resulting in the loss of covalent attachment of the heavy and light chains of tcrt-PA, the amidolytic activities of neither the single-chain nor the two-chain form of the molecule were stimulated by the presence of the above two positive effectors. With the single-chain form of this same derivative, the kcat of the reaction was extremely low (1.5 s-1), but increased to approximately 50.5 s-1 for the two-chain form, this latter value being nearly 4-fold higher than that of any of the wild-type recombinant rt-PA preparations examined. This suggests that the latent heavy chain of rt-PA inhibits the amidolytic activity found in the trypsin-like P domain.(ABSTRACT TRUNCATED AT 250 WORDS)