A covalent molecular weight .apprx.92,000 hybrid plasminogen activator derived from human plasmin fibrin-binding and tissue plasminogen activator catalytic domains

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Abstract
A covalent hybrid plasminogen activator was prepared from the sulfhydryl forms of the NH2-terminal heavy (A) chain of human plasmin (PlnA) containing the fibrin-binding domain and the COOH-terminal B chain of tissue plasminogen activator (t-PAB) containing the catalytic domain. The sulfhydryl form of PlnA [PlnA(SH)2] was isolated from reduced Lys-2-plasmin on an L-lysine-substituted Sepharose column, and the sulfhydryl form of t-PAB [t-PAB(SH)] was prepared from reduced two-chain tissue plasminogen activator (t-PA) by removing the tissue plasminogen activator NH2-terminal A chain (t-PAA) on an L-lysine-substituted Sepharose column from the chain mixture. The specific plasminogen activator activity, with soluble fibrin, of the isolated t-PAB(SH) chain was determined to be 62,700 international units (IU)/mg of protein, about 13% of the specific plasminogen activator activity of the parent t-PA. The PlnA(SH)2 and the t-PAB(SH) chains were mixed in a 1:1 molar ratio, and hybridization (reoxidation) was allowed to proceed by first dialyzing out the reducing agent at 4.degree.C and then concentrating the mixture. The time for maximum hybridization, or formation of the covalent hybrid activator, was 6 days, as determined by both specific plasminogen activator activity, with soluble fibrin, and specific amidolytic activity; sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the continual formation of an Mr.apprx.92,000 hybrid. The covalent PlnA-t-PAb hybrid activator was isolated from the 6-day hybridization mixture by a two-step affinity chromatography method. It was first adsorbed on an L-lysine-substituted Sepharose column and eluted with .epsilon.-aminocaproic acid and then adsorbed on a Zn-chelated agarose column with subsequent elution by imidazole. The protein yield of purified hybrid was 10%, with a major component (77%) of Mr .apprx.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-PAB, also shown to be present by double immunodiffusion. The specific plasminogen activator activity, with soluble fibrin, and the sepcific amidolytic ativity of the purified covalent hybrid activator were determined to be .apprx.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, .apprx.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 was found to be similar; at physiological fibrinogen concentraitons, binding of both activators to forming fibrin was about 90%.