A covalent hybrid plasminogen activator was prepared from the sulfhydryl forms of the NH2-terminal A chain of human plasmin (Pln^) containing the fibrin-binding domain, and the COOH-terminal B chain of tissue plasminogen activator (t-PAB) containing the catalytic domain. The PlnA (SH)2 and t-PAB(SH) chains were mixed in a 1:1 molar ratio, and hybridization was allowed to proceed at 4 °C for 6 days. The covalent PlnA-t-PAB hybrid activator was isolated from the mixture by a two-step affinity chromatography method, with L-lysine-substituted Sepharose and Zn-chelated agarose. The protein yield of purified hybrid was 10% with a major component (77%) of Mr ∼92,000. The covalent PlnA-t-PAB hybrid activator, contained 1 mol of each chain; after reduction, it gave the two parent chains, PlnA and t-PAA, also shown to be present by double immunodiffusion. The specific plasminogen activator activity, with soluble fibrin, and the specific amidolytic activity, of the purified covalent hybrid activator was determined to be 200,000 IU/mg of protein, about 40% of the specific activity of the parent t-PA. In a fibrin clot lysis assay, the covalent hybrid activator and t-PA have similar specific fibrinolytic activities, 500,000 IU/mg of protein; however, the clot lysis time curves were not parallel. The binding of the covalent PlnA-t-PAB hybrid activator and t-PA to forming fibrin were found to be similar; at physiological fibrinogen concentrations, binding of both activators to forming fibrin was about 90%.
