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

Abstract 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 K m value of 0.50 m m and a K cat value of 13.2 s −1 toward the substrate, HDIleProArg- p -nitroanilide (S2288) at 37 °. When these same assays were conducted in the presence of Fg or sFn, the K m and K cat 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 K m and K cat 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 (K 1 )2 (K 2 )- protease (P), and K 2 -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 K 2 region in the observed Fg- and Fn-induced stimulations of rt-PA amidolytic activity. With one strategically important derivative, a molecule containing the amino acid replacement, Cys 264 → Gly [(Cys 264 → 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 K cat 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. However, with another variant rt-PA, containing a Arg 275 → Ser [(Arg 275 → Ser)-rt-PA)] substitution, a change that leads to a loss of ability of plasmin-like enzymes to catalyze conversion of scrt-PA to tcrt-PA, a much larger stimulation by Fg and sFn of its amidolytic activity toward S2288 occurred, due to an approximate 10-fold decrease in the K m of the reaction. These results indicate major differences in the enzymatic properties of scrt-PA and tcrt-PA, regarding their respective abilities to be stimulated in their amidolytic properties by Fg and sFn. Additionally, the capacity for scrt-PA to be enhanced in this manner is dependent on the integrity of the two covalent bonds i.e., Cys 264 Cys 395 and Arg 275 Ile 276 , that stabilize the latent heavy and light chains of tPA. Finally, the nature of the amino acid residue located at the cleavage site of scrt-PA is of great importance to its ability to be stimulated by Fg and Fn.