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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 degrees 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Sedimentation velocity and sedimentation equilibrium studies have been carried out on the Glu- and Lys-plasminogen-streptokinase complexes as well as on the complexes formed by Val442-plasmin and the light (B) chain of plasmin. Sedimentation equilibrium molecular weights are consistent with a 1 to 1 molar complex in all cases and give values consistent with the differences in size of the plasminogen moieties. Sedimentation velocity determinations in the presence of protease inhibitors give values consistent with the conformational differences already reported for the Glu- and Lys-plasminogen molecules. However, unlike Glu-plasminogen, the addition of epsilon-aminocaproic acid or lysine does not alter the conformation of the Glu-plasminogen complex. The values of the sedimentation coefficient and the molecular weight of the plasmin and the Val442-plasmin-streptokinase complexes increase to those of a dimer when determined in the absence of active-site inhibitors but return to monomer values when these inhibitors are added. Thus, dimer formation requires the presence of an available active site in at least one of the two molecules involved and is reversible.

mRNA was isolated from total RNA of monkey liver by oligo(dT)-cellulose chromatography and was translated in a rabbit reticulocyte cell-free system. Analysis of the translation products immunoprecipitated with specific antibodies to monkey plasma plasminogen revealed a molecule with characteristics similar to those of native plasminogen. The purification of the mRNA by centrifugation on sucrose gradients indicated the presence of plasminogen mRNAs in both the 23S and 18S RNA fractions. Both plasminogen mRNAs can be further purified by chromatography on Sepharose 4B. Affinity chromatography of the proteins synthesized in vitro by total mRNA from liver, as well as by the purified mRNAs, on L-lysine-substituted Sepharose revealed that both major plasma plasminogen forms (1 and 2) are synthesized, as precursors, in the system. The in vitro synthesized plasminogen is similar in its physical and chemical properties to native plasma plasminogen as determined by its ability to bind to L-lysine-substituted Sepharose and its molecular interaction with streptokinase. The purified mRNAs were also translated in the presence of dog pancreas microsomal membranes, and and fractionated on concanavalin A-Sepharose. The 23S mRNA directed the synthesis of a plasminogen molecule similar to the circulating plasma plasminogen form 1, whereas the 18S mRNA directed the synthesis of a molecule similar to the circulating plasma plasminogen form 2. Our evidence indicates that the synthesis of the two major circulating plasma plasminogen forms is directed in the liver by separate mRNAs.

The kinetic parameters of three activator species of Glu1-plasminogen (Glu1-Plg) were compared in their reaction at pH 7.4 and 37 degrees C, in the presence and absence of CNBr-digested fibrinogen (CNBr-Fg). The urokinase- (u-PA-) derived covalent hybrid activator PlnA-u-PAB had an apparent Michaelis constant (Kplg) of 7.44 microM, a catalytic rate constant (kplg) of 51.1 min-1, and a second-order rate constant (kplg/Kplg) of 6.87 microM-1 min-1. The tissue plasminogen activator (t-PA) derived covalent hybrid activator PlnA-t-PAB was characterized by a Kplg of 3.33 microM, a kplg of 1.03 min-1, and a kplg/Kplg of 0.309 microM-1 min-1. The kplg/Kplg values for the parent u-PA and t-PA activators were 6- and 16-fold higher than the respective hybrids, mainly due to an approximately 10-fold increase in the apparent Kplg for the hybrids. In the presence of CNBr-Fg, the increase of the kplg/Kplg values for u-PA and its hybrid was 1.1-fold, but for t-PA and its hybrid, the increases were 7- and 12-fold, respectively. In both the absence and presence of CNBr-Fg, activator t-PAB had an apparent Kplg of 19.1 and 27.6 microM and a kplg of 2.9 and 5.0 min-1, respectively. The increase in the kplg/Kplg value with CNBr-Fg was 1.2-fold. The streptokinase- (SK-) derived activators Glu1-plasmin.SK (Glu1-Pln.SK), Val442-Pln.SK, and Val561-Pln.SK had apparent Kplg values of 0.458, 0.268, and 0.121 microM and kplg values of 20.0, 126.0, and 63.3 min-1, respectively. In the presence of CNBr-Fg, the first two activators showed an approximately 1.4-fold increase and the last showed a 1.4-fold decrease in their kplg/Kplg values. The catalytic efficiency (kplg/Kplg) of the various activator species fell in the decreasing order SK greater than u-PA greater than t-PA, in either the presence or absence of CNBr-Fg. CNBr-Fg enhanced significantly the activities of only two activators, t-PA and PlnA-t-PAB.

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 degrees 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 approximately 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.(ABSTRACT TRUNCATED AT 250 WORDS)