We have created a novel thrombolytic agent by the combination of mutation with partial deletion of tissue-type plasminogen activator (t-PA). We constructed Escherichia coli expression vectors for (i) native t-PA (nt-PA) and its derivatives; (ii) K1K2P, consisting of kringle 1 (K1), kringle 2 (K2), and protease (P) domains; (iii) K2P, consisting of K2 and P domains; (iv) D-nt-PA; (v) D-K1K2P; and (vi) D-K2P. The latter three are point mutants of nt-PA, K1K2P, and K2P, respectively, in which Arg(275) (number corresponds to that of nt-PA) has been mutated to Asp. The production of nt-PA and its derivatives was remarkably improved by (i) removal of the 3’ noncoding region of nt-PA cDNA from expression vectors and (ii) expression in mutant E. coli derived from E. coli HB101, which is insensitive to heat-shock inductions. The proteins produced were precipitated as insoluble aggregates in the cells and were renatured to active forms by extraction with 8 M urea followed by dialysis against a redox buffer containing GSH and GSSG. The renaturation yield depended on the pH of the buffer and the number of disulfide bonds of the proteins (nt-PA << K1K2P < K2P). The mutation of Arg(275) (the plasmin cleavage site) caused an increase in the catalytic enhancement by fibrin and a decrease of the interaction with plasminogen activator inhibitors. Among them, D-K2P was selected as a candidate for a new thrombolytic agent because of (i) its facility of renaturation and purification to homogeneity and (ii) its high catalytic enhancement by fibrin, as compared with nt-PA or other derivatives produced by the same system. It was produced in large amounts and characterized by structural analysis, as well as by biological and pharmacological activities. The disulfide linkages of D-K2P were found to be identical to those of nt-PA (Bowes) on the kringle 2 and protease domains. Although its specific activity is approximately one-fourth that of nt-PA (Bowes), it shows higher fibrin acceleration and a longer half-life in blood than nt-PA (Bowes). In the preliminary pharmacological studies, D-K2P exhibited more potential thrombolytic effect than recombinant nt-PA (alteplase) by both bolus and infusional administrations in the rabbit thrombus model. It was also more effective in canine coronary thrombosis. These results may indicate that plasma half-life and fibrin acceleration play important roles in the thrombolytic agent.