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An attempt will be made in this article to classify known abnormal Plgs based only on studies of the isolated highly purified proteins

A new abnormal plasminogen, Frankfurt I, has been identified in the plasma of a 42 year-old male patient who had recurring thromboses, thrombophlebitis and pulmonary embolism since his age of 29. Reduced functional and also slightly reduced antigen plasminogen concentrations were found in both the proposituts and his mother. Plasmin generation rates carried out by Streptokinase and Urokinase were also abnormal. The plasmin generated was very unstable in the absence of stabilizing ligands and/or substrates. Crossed immunoelectrophoresis of the purified Frankfurt I revealed a peak with normal size and shape, but displaced with respect to normal Glu-plasminogen toward the anode. Isoelectric focusing followed by zymography on an agarose-fibrin plate proved this observation but did not indicate a separation of the normal from the abnormal plasminogen molecular species, also, fewer bands were found in the abnormal plasminogen isozyme pattern. Kinetic studies of Frankfurt I Glu-plasminogen and plasmin showed that most of the functional abnormality is related to absence of active sites in half of the molecules.

A comparison was made of the esterase and activator activities of the various activated forms of human plasminogen and their streptokinase complexes with Nalpha-Cbz-L-lysine-p-nitrophenyl ester as the substrate. The steady state kinetic properties of Glu- and Lys-plasmins, and Glu- and Lys-plasminogen-streptokinase complexes were identical, while the Lys-plasmin-streptokinase complex showed a 2-fold increase in Km with the same kcat and a 3-fold increase in Ki for the competitive inhibitor leupeptin. Lys-plasminogen (zymogen with an active site) was prepared which incorporated 0.7 mol of [3H]idisopropyl phosphorofluoridate and 0.43 mol of p-nitrophenyl-p'-guanidinobenzoate/mol of protein. The Km for Lys-plasminogen was 3-fold higher than that of Lys-plasmin, and its maximum velocity 10-fold lower. The steady state kinetic parameters of a plasmin-derived light (B) chain (CmCys)3, and a derived equimolar light (B) chain-streptokinase complex (CmCys)3, isolated from human plasmin and equimolar plasmin-streptokinase, or plasminogen-streptokinase, complexes, respectively, were determined. When the light (B) chain-streptokinase complex is isolated from its parent complexes, there is a complete retention of the original parent's esterase activities, with respect to Km and kcat, and interaction with the competitive inhibitors benzamidine and leupeptin. The plasmin-derived light (B) chain does not retain its parent esterase activities. This chain has very similar kinetic properties to Lys-plasminogen except that streptokinase, in an equal molar amount, does not impart full esterase activity to the light (B) chain whereas the zymogen can be completely activated by streptokinase. The kcat of the plasmin-derived light (B) chain, and its streptokinase complex can be enhanced by 50 and 30%, respectively, in the presence of 10(-4) M leupeptin, a competitive inhibitor of plasmin, attesting to the increased structural flexibility within the active site of this enzyme species. Urokinase hydrolyzes Nalpha-Cbz-L-lysine p-nitrophenyl ester efficiently with a kcat/Km of one-third that of plasmin. The human plasminogen activator activities of various activated forms of human plasminogen and their equimolar streptokinase complexes were compared in a kinetic assay. The Lys-plasmin-streptokinase complex, and streptokinase were the least active of the activator species and were approximately equal in their activator activities. Glu- and Lys-plasminogen-streptokinase complexes had approximately 1.5 times the activity of streptokinase, whereas the equimolar light (B) chain-streptokinase complexes had approximately 2- to 3-times the activator activity of streptokinase. Since the esterase activity remained unchanged, this indicates a greater degree of specificity in the active site of the equimolar light (B) chain-streptokinase activator complex. Urokinase proved to be a poor activator species...

SummarySpecific assay methods for the human plasmin-derived light (B) chain · streptokinase (B·SK) complex, in terms of both streptokinase (SK) and urokinase (UK) International Units, are described. The kinetic properties of various SK activator complexes with plasminogen, Val 442-plasmin, and the plasmin-derived light (B) chain were compared to SK in terms of their catalytic efficiencies and Lineweaver-Burk plots. Similar kinetic data, and Lineweaver-Burk plots, are described for both highly purified high-molecular weight UK and low-molecular weight UK, including different clinical UK preparations. The B·SK complex has the highest catalytic efficiency of all the activator species studied. The Lineweaver-Burk plots of each of the various activator species are “fingerprints” of the enzymatic character of the activator. The B-SK complex is more like UK than SK, as an activator, in activating non-human plasminogen species. The biological halflife of the B·SK complex, in a dog model, was determined to be about 4 hr which is longer than the biological half-life(s) of SK in the same animal model, namely 0.6 hr (47%) and 2.8 hr (53%). This new second-generation activator complex may prove to be a useful thrombolytic agent in the treatment of thromboembolic diseases.

Highly purified human (Lys-forms), cat, dog, and rabbit plasminogens showed significantly different sensitivities to activation by highly purified streptokinase. The most sensitive plasminogen is the human zymogen, followed by the cat, dog, and rabbit zymogens, respectively. These human, cat, dog, and rabbit zymogens reacted with streptokinase to form homogeneous equimolar plasminogen-streptokinase complexes. The electrophoretic mobilities of the human and cat complexes on cellulose acetate were different from their respective zymogens, but the electrophoretic mobilities of the dog and rabbit complexes were similar to their respective zymogens. The human, cat, and dog complexes showed different mobilities from their respective zymogens in acrylamide gel electrophoretic systems and appeared to show multiple electrophoretic forms. With these species, all of the multiple isoelectric forms of the zymogens reacted with streptokinase. Analyses of the human (Glu- and Lys-forms), cat, dog, and rabbit equimolar plasminogen-streptokinase complexes, incubated for various time intervals, in an acrylamide gel-dodecyl sulfate-urea electrophoretic system, showed the gradual conversion, or transformation, of the plasminogen-streptokinase complexes into plasmin-streptokinase complexes. These transformations occurred at different rates in each of the mammalian plasminogen-streptokinase complexes. This acrylamide gel system also permitted a comparison between the plasmin-derived carboxymethyl heavy (A) and carboxymethyl light (B) chains produced from these mammalian zymogens by two different methods of activation, namely with equal molar ratios of streptokinase to zymogen and with low molar ratios of urokinase to zymogen (1:1000). The carboxymethyl heavy (A* and A') chains produced by streptokinase activation of the human (Glu-forms), cat, and rabbit plasminogens in the complex appeared to have higher molecular weights than the major carboxymethyl heavy (A and A1) chains derived from each of these zymogens by urokinase activation; the human (Lys-forms) and dog carboxymethyl heavy (A and A') chains appeared to be similar in both activation systems. The carboxymethyl heavy (A* and A') chains of the human (Glu- and Lys-forms), cat, dog, and rabbit plasmins found in the complexes were similar but not identical with each other in molecular weight. The plasmin-derived carboxymethyl light (B') chains produced by streptokinase activation of the human (Glu- and Lys-forms), cat, dog, and rabbit plasminogens in the complex appeared to have molecular weights similar to the carboxymethyl light (B) chains derived from each of these plasminogens by urokinase activation. But, the plasmin-derived carboxymethyl light (B') chains of the four species obtained from the equimolar complexes appeared to have somewhat different molecular weights. Streptokinase fragmentation within the complexes occurs within a few seconds after the complexes are formed, and intact streptokinase disappears after several minutes of incubation. Four major streptokinase fragments (SK1, SK2, SK3, and SK4), having molecular weights between 47,600 and 25,700, are produced in each of the equimolar human (Glu- and Lys-forms), cat, dog, and rabbit complexes. They appear to be the same fragments in each of the mammalian plasminogen-streptokinase and plasmin-streptokinase complexes; each of the complexes contains varying amounts of streptokinase fragments, SK1, SK2, SK3, and SK4. But, the rate of degradation of streptokinase into these major fragments differed with each species. Plasmin and streptokinase moieties prepared by dissociation of an equimolar plasminogen (Lys-forms)-streptokinase complex at pH 3.0 (Summaria, L., Robbins, K. C., and Barlow, G. H. (1971) J. Biol. Chem. 246, 2136–2142) were also analyzed in the acrylamide gel-dodecyl sulfateurea electrophoretic system. The isolated plasmin moiety gave carboxymethyl heavy and carboxymethyl light chains, both of which were of lower molecular weight than the plasmin-derived carboxymethyl heavy (A) and carboxymethyl light (B) chains produced by urokinase activation. The isolated streptokinase moiety contains approximately equal amounts of streptokinase fragments SK2 and SK4, and a third smaller fragment.

The equimolar human light (B) chain·streptokinase activator complex was shown to be an effective fibrinolytic agent in the dissolution of human cross-linked plasma fibrin clots, in vitro; its activator activity was similar to that of the equimolar human plasmin·streptokinase complex. 125 I-radiolabelled preparations of the human plasmin-derived light(B) and heavy(A) chains, and Lys-plasminogen were shown to be adsorbed to cross-linked plasma fibrin clots invitro to the same extent. Clot lysis by the light(B) chain·streptokinase complex, the plasmin·streptokinase complex, streptokinase and urokinase was enhanced by a factor of at least 10-fold when the fibrin clots were pretreated with either the light(B) chain or Lys-plasminogen, but was not enhanced when the fibrin clots were pretreated with the heavy(A) chain. The activator activities of the light(B) chain·streptokinase complex, the plasmin·streptokinase complex and streptokinase were similar on these pretreated cross-linked fibrin clots. Limited proteolysis of fibrinogen by the light(B) chain and by plasmin showed the same specificity giving Fragments X, Y, D and E, but the rate of hydrolysis of fibrinogen by the light(B) chain was much slower.

The isolated equimolar human plasmin-streptokinase activator complex was found to be an excellent fibrinolytic agent in an experimental dog model. It will dissolve artificially produced human clots placed in the femoral vein of the animals. There appears to be a critical dose level for fibrinolytic activity which may be approximately 0.1 mg per kg body weight. At the critical dose level, the activator complex appeared to be more effective than equivalent individual doses of either streptokinase or human plasmin.

Bovine plasminogen activator preparations were isolated at 0° and 25° from a mixture of streptokinase and human plasminogen. The degree of incorporation and inhibition with radioactive 32P-labeled diisopropyl phosphofluoridate (DFP) demonstrated a definite difference between these two preparations. Incorporation of approximately 1 mole of DFP-32P per mole of bovine plasminogen activator prepared at 25° and per mole of human plasmin indicated that bovine plasminogen activator and human plasmin each contains a single "active site" which may be identical. Incorporation of 0.50 mole of DFP-32P per mole of bovine plasminogen activator prepared at 0° and its retention of partial activity indicated that this activator preparation was partly a human plasminogen-streptokinase complex. Acid treatment of this preparation and inhibition studies with e-aminocaproic acid also indicated that this preparation contains a plasminogen-streptokinase complex. Complex formation also occurred when streptokinase was allowed to react with DFP-32P-treated human plasminogen and plasmin. Starch gel electrophoretic analyses indicated that all the complexes formed under these conditions possessed the same mobility. Dissociation of the 25° activator complex inhibited with DFP-32P after its isolation revealed that the radioactivity was located in the plasmin moiety of the activator complex.

Specific rabbit precipitating antibodies have been preparted to human plasminogen and plasmin. The preoenzyme-enzyme pair were found to be indentical in gel diffusion. In immunoelectrophoresis, the proenzyme-enzyme pair differ in their electrophoretic mobilities. Antibody γ-globulin preparations to either human plasminogen or plasmin were found to neutralize the proteolytic activity of plasmin with maximum inhibitions of 80 percent. There were some differences in the ability of antibody γ-globulin to neutralize plasminogen activated in situ, with either streptokinase or urikinase, as compared to plasmin. The antibodies also partially inhibit the activation of proenzyme to enzyme. Trypsin inhibitors which inhibit plasmin, appear to be different, in certain respects, from specific antibodies.

Bovine plasminogen activator was isolated from a mixture of streptokinase and human plasminogen. The method of preparation was based on the difference in solubility between the activator and its precursors. The activator can be prepared from either crude or highly purified streptokinase and highly purified human plasminogen. The activator complex gave a single component in starch gel electrophoresis, in either lysine or e-aminocaproic acid buffers, and in immunochemical studies with a specific rabbit antibody to human plasminogen. However, the mobility of the activator was different from human plasmin, human plasminogen, or streptokinase both in starch gel electrophoresis and in immunoelectrophoresis. The activator can be dissociated into two components, apparently corresponding to streptokinase and human plasmin, by electrophoresis in starch gels. Gel diffusion analyses with specific antibody revealed that the activator was immunologically identical with both human plasmin and human plasminogen. In sedimentation velocity experiments, the activator showed a single component in e-aminocaproic acid buffers with a sedimentation coefficient of approximately 5 S. In lysine buffers, activator showed some heterogeneity, but the sedimentation coefficient of the major component was found to be approximately 11 S. The apparent molecular weight of activator by sedimentation equilibrium methods, in e-aminocaproic acid buffers, was found to be approximately 150,000. In lysine buffers the apparent molecular weight in dilute solutions was found to be approximately 138,000, whereas in concentrated solutions the molecular weight was found to be approximately 308,000. These data suggest a possible monomer-dimer relationship which is concentration dependent. The dimer form appeared to be present when activator was dissolved in lysine buffers, in concentrated solutions only, whereas the monomer form was always found when activator was dissolved in e-aminocaproic acid buffers.

The activation of human plasminogen in a 25% glycerol medium was carried out with low molar ratios of urokinase, streptokinase, trypsin, and pig heart activator. Starch gel electrophoresis in 8 m urea at pH 3.2 showed that each of the four plasmins possessed the same electrophoretic mobility. Upon reduction with 2-mercaptoethanol in 8 m urea, each plasmin was found to contain two major, electrophoretically unique components. Determination of the aminoterminal and the carboxyl-terminal amino acid residues of these plasmins indicated, in accordance with our previous studies, that the activation of human plasminogen proceeds primarily through the cleavage of a single arginyl-valine bond. Each of the plasmins contains two polypeptide chains connected by a disulfide bond.

Five native mammalian plasminogen species, namely, cat, dog, bovine, rabbit and horse, were studied and compared to native human plasminogen with respect to their substrate and enzymatic properties in various activated forms. These studies are an extension of previous work and were designed to confirm our previously proposed mechanism of plasminogen activation, using a series of native, but different, plasminogen substrates. The plasminogen activator species used were high molecular weight urokinase, streptokinase, human Glu-plasminogen-streptokinase complex, human plasmin-derived light(B)-chain-streptokinase complex, and the equimolar streptokinase activator complexes prepared from cat and dog plasmins. The peptidase parameters of the plasmins, plasmin-streptokinase and plasminogen-streptokinase complexes were determined with H-D-valyl-L-leucyl-L-lysyl-p-nitroanilide and Tos-glycyl-L-prolyl-L-lysyl-p-nitroanilide. Activation kinetics were measured with the same substrates. The peptidase parameters of all plasmin species were found to be similar, but with minor variations. The equimolar streptokinase mixtures of bovine, rabbit and horse plasminogens and plasmins did not form complexes and did not form active sites with plasminogen, under the conditions used. The second-order rate constants of activation revealed great differences (as much as 1400-fold), presumably expressing differences in the tertiary structure of the various plasminogen scissile bonds. The catalytic rate constants of activation, kplg, varied by as much as a 100-fold, while differences in Kplg were relatively small. The results of this study confirm the activation mechanism we have postulated previously, namely, that rapid-equilibrium rather than steady-state conditions prevail and that k2 (acylation) is the catalytic rate constant and the rate-determining step, while KS is a true dissociation constant. Calculations of the free energy of interaction of the peptidase and plasminogen activation reactions showed -4.4 to -5.6 kcal/mol for peptidase and -6.5 to -10 kcal/mol for the activation reaction. These values indicate 1-3 subsite binding interactions for the peptidase activity and 3-5 subsite binding interactions for the activation catalytic event. Streptokinase activator complexes have at least one more interacting subsite than the urokinase active site.

Methods have been developed to quantitatively measure the major plasma components of the human fibrinolytic system. Plasminogen is measured functionally with a 9M excess of streptokinase and immunochemically by rocket immunoelectrophoresis; the normal range was found to be 16.7–23.8 mg/dl and 17.4–21.6 mg/dl, respectively. α 2 -plasmin inhibitor is measured functionally and immunochemically; the normal range for the major plasma plasmin inhibitor was found to be 5.30–6.60 mg/dl by both methods. Plasminogen activator concentrations, as well as, free, and complexed, protease activities are measured along with plasmin generation rates by spectrophotometric assays with chromogenic substrates. Both activator and free protease activities are zero in plasma samples from normal human subjects. Plasmin generation rates are 0.25–0.47% with urokinase and 5.30–9.70% with streptokinase; these values are the percentages of the respective initial velocities of activation in purified systems.

The factors involved in the regulation and control of the human plasma fibrinolytic system at the cellular level are unknown at this time. The physiological regulation of plasmin formation in plasma depends primarily on the nature of the circulating zymogen, plasminogen, the physiological activators formed both in the blood and in the vascular endothelium, and the specific plasmin inhibitors found both in plasma and in certain of the cellular elements of the blood. The biosynthesis of the zymogen must be under genetic control, and the activators are probably released, after thrombus and clot formation, from components involved in the surface-mediated initiation of the coagulation system, and from the vascular endothelium. Activation of plasminogen can occur both in the fluid phase surrounding the thrombus and probably at thrombus surfaces, involving both the fibrin clot and the platelet membrane. The plasmin inhibitors act to control the system in order to prevent proteolytic degradation of important physiological trace proteins of the coagulation, complement and kallikrein-kinin systems by the enzyme.

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.

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.

Human plasminogen and diisopropyl phosphorofluoridate or l-1-chloro-3-tosylamido-7-amino-2-heptanone-treated plasmin, prepared from plasma Fraction III2,3 by ion exchange and gel filtration methods, and the equimolar human plasminstreptokinase complex show multiple molecular forms in acrylamide gel electrophoresis at pH 8.4 in either 0.3 m e-aminocaproic acid or 8 m urea. Eight similar bands were found in the gel patterns of the zymogen and enzyme. The S-carboxymethyl heavy (A) chain derivative of plasmin did not show multiple molecular forms in acrylamide gel electrophoresis in either 0.3 m e-aminocaproic acid or 8 m urea. However, the S-carboxymethyl light (B) chain derivative showed multiple forms in acrylamide gel electrophoresis but in 8 m urea only. Plasminogen prepared from either pooled plasma, or plasma Fractions III or III2,3, by affinity chromatography on lysine-substituted-Sepharose columns showed the same multiple molecular forms in acrylamide gel electrophoresis in 0.3 m e-aminocaproic acid as were found in our highly purified plasminogen preparations but with different quantitative distributions. Affinity chromatography of only plasma Fraction III2,3 gave preparations with specific potential proteolytic activities which were equivalent in activity to our standard preparations. The multiple molecular forms of plasminogen and plasmin prepared from plasma Fraction III2,3 by ion exchange and gel filtration methods were isolated by isoelectric focusing methods. Eight plasminogen forms were obtained with isoelectric points ranging from pH 6.4 to pH 8.5. All of the forms had similar specific potential proteolytic activities. Each isoelectric form contained two closely related acrylamide gel electrophoretic components (doublet bands), both common to either adjacent isoelectric forms. Eight diisopropylphosphoryl-plasmin forms were isolated with isoelectric points ranging from pH 7.0 to pH 8.5. Radiolabeling and specific antibody were used to determine specific plasmin forms. Each plasmin isoelectric form also contained two closely related acrylamide gel electrophoretic components (doublet bands), both common to either adjacent isoelectric forms. Plasminogen isoelectric forms pH values 7.2 and 7.5 and diisopropylphosphoryl-plasmin isoelectric forms pH values 7.4 and 7.7 show the same acrylamide gel electrophoretic mobilities, respectively; the zymogen and enzyme forms differ in isoelectric point by approximately 0.2 pH unit. Plasminogen form pH 7.2 and its derived diisopropylphosphoryl-plasmin form show the same acrylamide gel electrophoretic mobilities. The S-carboxymethyl heavy (A) and light (B) chain derivatives of plasmin were studied in isoelectric focusing. Three major light (B) chain fractions were isolated with isoelectric points of pH values 5.8, 5.9, and 6.0. The amount of each of the three major acrylamide gel electrophoretic forms in each isoelectric fraction was different; none of the fractions showed a single pure form. The heavy (A) chain derivative gave a single form with an isoelectric point of pH 4.9. The plasminogen isoelectric forms pH values 7.5 and 7.8, after conversion to plasmin, followed by reduction and alkylation, each gave light (B) chain preparations containing all of the major acrylamide gel electrophoretic forms. No changes in acrylamide gel electrophoretic mobility could be found in any of the multiple molecular forms of zymogen, enzyme, or light (B) chain derivative following isoelectric focusing or other preparative procedures.

SummaryHuman plasminogen was treated with either DFP, TLCK, or SBTI under conditions which completely inhibited the plasmin contaminant in our preparation. Each treated plasminogen preparation could be completely activated with low molar ratios of streptokinase to plasminogen in 24 hr, in 25% glycerol. Activation of DFP-treated plasminogen with either streptokinase or a DFP-treated equimolar human plasmin-streptokinase complex could also be carried out in 10-2M DFP, in 25% glycerol; however, much longer periods of time were required to obtain nearly complete activation. Under these conditions for activation, the plasmin contaminant in our human plasminogen preparation is therefore not essential for streptokinase-activation of the proenzyme. These data indicate that streptokinase, alone or in the complex, acts directly on human plasminogen. A new electrophoretic method for differentiating human plasminogen from human plasmin is described.