Covalent molecular weight .apprx. 92,000 hybrid plasminogen activator derived from human plasmin amino-terminal and urokinase carboxyl-terminal domains

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Abstract
The preparation of a new class of covalent hybrid plasminogen activators containing the fibrin-binding domains of human plasmin(ogen) and the catalytic active center of human urokinase will be described. Hybridization of the sulfhydryl form of the NH2-terminal plasmin-derived heavy (A) chain (PlnA) with the sulfhydryl form of the COOH-terminal urokinase-derived active heavy (B) chain (u-PAB) was carried out; a covalent PlnA-u-PAB hybrid plasminogen activator was prepared. The sulfhydryl form of PlnA (PlnA(SH)2) was isolated from reduced Lys-2-plasmin by L-lysine-substituted Sepharose column chromatography. For the isolation of the sulfhydryl form of u-PAB (u-PAB(SH)), high molecular weight urokinase was adsorbed onto a benzamidine-Sepharose column and reduced with 100 mM 2-mercaptoethanol on the column. The urokinase NH2-terminal light (A) chain was washed off the column, and the u-PAB(SH) chain was eluted from the column. The specific activity of the isolated u-PAB(SH) chain was determined to be 242,000 IU/mg of protein. The PlnA(SH)2 and u-PAB(SH) chains were mixed at a molar ratio of PlnA(SH)2 to u-PAB(SH) of 3:2; the reducing agents were then removed by gel filtration. The hybridization (reoxidation) reaction was allowed to proceed for 48 h at 4.degree. C. The covalent hybrid activator, in 40% yield, was purified from the reaction mixture to homogeneity, by a sequential affinity chromatography method with L-lysine-substituted Sepharose followed by anti-low molecular weight urokinase IgG-Sepharose, and then gel filtration through Sephadex G-150. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, one molecular species of the covalent PlnA-u-PAB hybrid activator was found, Mr .apprx. 92,000, indicating that is contains 1 mol of each chain. Reduction of the covalent hybrid activator gave the two parent chains, PlnA and u-PAB. The specific activity of the covalent hybrid activator was determined to be 71,500 IU/mg of protein in an amidolytic assay with a specific tripeptide chromogenic substrate, whereas its specific fibrinolytic activity was determined to be 279,000 IU/mg of protein, in a fibrin clot lysis assay, a 4-fold increase in the presence of a fibrin clot. The Glu-plasminogen activator activity of the covalent hybrid enzyme was significantly stimulated in the presence of soluble fibrin, about 5-fold, whereas the activator activity of high molecular weight urokinase was stimulated by soluble fibrin only about 0.5-fold. The hybrid activator was also more strongly adsorbed to a fibrin clot, about 25%, than was high molecular weight urokinase, about 3%. These findings indicate that this new covalent hybrid plasminogen activator, Mr .apprx. 92,000, is a more potent plasminogen activator in the presence of fibrin than is high molecular weight urokinase, Mr .apprx. 54,000.