The importance of the preactivation peptide in the two-stage mechanism of human plasminogen activation.

The two stages in the activation of human plasminogen by urokinase have been examined kinetically in order to evaluate the significance of each stage in the activation process. The cleavage of the preactivation peptide from the NH2 terminus of native plasminogen (NH2-terminal glutamic acid) is clearly catalyzed by urokinase and is the rate-limiting first step in activation (Stage 1); this reaction is 20-fold slower than the conversion of the intermediate plasminogen (NH2-terminal lysine) to plasmin (Stage 2). Both lysine and its analogoue, epsilon-aminocaproic acid, exert two effects on the activation of native plasminogen. At low concentrations of these agents, activation is greatly accelerated. Analysis of activation in the presence and absence of these agents by sodium dodecyl sulfate gel electrophoresis indicates that the activation pathway is the same in both cases with the formation of a transient intermediate plasminogen; only the kinetics of proteolysis are altered. This enhancement in the rate of activation results solely from acceleration of the Stage 1 reaction; Stage 2 is essentially unaffected at low concentrations. Stage 1 is maximally enhanced (75-fold) at either 0.0025 M epsilon-aminocaproic acid or 0.025 M lysine and occurs 4 times more rapidly than Stage 2, which becomes the rate-limiting step at these concentrations. Plasmin also cleaves the preactivation peptide from native plasminogen and this reaction rate is enhanced by the same concentrations of lysine and epsilon-aminocaproic acid. These data suggest that lysine and epsilon-aminocaproic acid, which are known to bind to plasminogen and significantly alter its conformation, may thereby enhance preactivation peptide cleavage and consequently, plasminogen activation. At high concentrations, both Stages 1 and 2 are similarly inhibited by these agents, which suggests that this effect may be exerted by the direct inhibition of urokinase. The relative rates of preactivation peptide cleavage by the enzymes urokinase, plasmin, thrombin, and ancrod were also determined. Urokinase is 10 times more effective than plasmin in catalyzing this reaction and 1.8 X 10(4) times more effective than thrombin, while ancrod does not exert an effect. No plasmin is formed by either thrombin or ancrod.     

Kinetic studies of the urokinase catalysed conversion of NH2-terminal lysine plasminogen to plasmin.

A method for determining initial velocities of the urokinase (EC 3.4.99.26) catalysed converstion of NH2-terminal lysine plasminogen to plasmin (EC 3.4.21.7) is presented. This reaction has been coupled with the hydrolysis of alpha-N-benzyoly-L-arginine ethyl ester, which is catalysed by plasmin, and its rate has been determined from the time course of the overall reaction. The proenzyme-enzyme conversion was found to obey the Michaelis-Menten rate equation. The following values of the kinetic parameters were obtained: the apparent Michaelis constant, Km = 40.7 +/- 6.2 muM; the catalytic constant, kc = 2.59+/-0.31 s(-1), and kc/Km = 6.36-10(4) +/- 0.24-10(4) M(-1)-s(-1).     

Identification of the actin and plasminogen binding regions of group B streptococcal phosphoglycerate kinase

Phosphoglycerate kinase (PGK), present on the surface of group B streptococcus (GBS), has previously been demonstrated to bind the host proteins actin and plasminogen. The actin and plasminogen binding sites of GBS-PGK were identified using truncated GBS-PGK molecules, followed by peptide mapping. These experiments identified two actin and plasminogen binding sites located between amino acids 126-134 and 204-208 of the 398-amino acid-long GBS-PGK molecule. Substitution of the lysine residues within these regions with alanine resulted in significantly reduced binding to both actin and plasminogen. In addition, conversion of the glutamic acid residue at amino acid 133 to proline, the amino acid found at this position for the PGK protein of Streptococcus pneumoniae, also resulted in sign ificantly reduced binding to actin and plasminogen. These results demonstrate that the lysine residues at amino acid positions 126, 127, 130, 204, and 208 along with the glutamic acid residue at amino acid position 133 are necessary for actin and plasminogen binding by GBS-PGK.     

Kinetic studies on novel plasminogen activators. Demonstration of fibrin enhancement for hybrid enzymes comprising the A-chain of plasmin (Lys-78) and B-chain of tissue-type plasminogen activator (Ile-276) or urokinase (Ile-159).

The activation of plasminogen by two novel hybrid enzymes, constructed from the A-chain of plasmin and the B-chains of tissue-type plasminogen activator (t-PA) or urokinase, was compared with the activation by the parent enzymes. Basal kinetic constants for 'Lys-plasminogen' (human plasminogen with N-terminal lysine) and 'Glu-plasminogen' (human plasminogen with N-terminal glutamic acid) activation were similar to those of the parent activators. The Km for plasminogen turnover for both hybrid enzymes was considerably decreased in the presence of both soluble fibrin and a mimic, a CNBr digest of fibrinogen. These enhancements and the related apparent negative co-operativity are similar to the behaviour of t-PA itself. The results are discussed with regard to the molecular features involved in the mechanism of fibrin stimulation.     

Structural relationship between "glutamic acid" and "lysine" forms of human plasminogen and their interaction with the NH2-terminal activation peptide as studied by affinity chromatography.

Urokinase digestion of maleinated plasminogen results in cleavage of the single peptide bond Arg-68-Met-69, which is one of the bonds normally cleaved during the first step of the activation procedure. The inactive intermediate compound formed in this way was subjected to NH2-terminal amino acid sequence analysis, which clearly demonstrates the structural relationship between the forms of plasminogen with different NH2-terminal amino acids. It is thus shown that lysine-78 and valine-79 in the "glutamic acid" plasminogen actually are the NH2-terminal amino acids in "lysine" and "valine" plasminogen respectively. The forms with glutamic acid in NH2-terminal position are called plasminogen A, while all other forms lacking the NH2-terminal part of the molecule and which can be activated in a single step are called plasminogen B. By affinity chromatographic studies of the NH2-terminal activation peptide on insolubilized plasminogen B, it was demonstrated that this peptide has specific affinity for plasminogen B. It was also shown that this noncovalent interaction is broken by 6-aminohexanoic acid in two concentration. The tryptic heptapeptide (Ala-Phe-Gln-Tyr-His-Ser-Lys) which occupies the positions number 45 to 51 in the NH2-terminal activation peptide (as well as in the intact plasminogen molecule) is importance for the conformational state of the plasminogen molecule.     

The mechanism of activation of rabbit plasminogen by urokinase. Lack of a preactivation peptide

We have obtained direct evidence which we interpret to prove that an amino terminal peptide need not be released from rabbit plasminogen prior to its conversion to plasmin by urokinase. The single chain plasminogen molecule possesses an amino terminal amino acid sequence of NH₂-glu-pro-leu-asp-asp. When this plasminogen is activated to plasmin by urokinase in the presence of the Kunitz bovine trypsin-plasmin-kallikrein inhibitor (BTI), a two chain disulfide linked molecule of plasmin is obtained. The heavy chain of this plasmin is directly derived from the original amino terminus of plasminogen since it possesses the identical amino terminal sequence as does native plasminogen. When the same plasminogen activation is carried out in the absence of BTI, the heavy chain of the plasmin obtained has a molecular weight of 6,000–8,000 less than the heavy chain of the plasmin obtained in the presence of this inhibitor. In addition, the heavy chain of this latter plasmin has an amino terminal sequence which differs from the original native plasminogen. These data show, in agreement with others, that the activation of plasminogen by urokinase is accompanied by the loss of an amino terminal peptide from plasminogen but also show, in contrast to the human plasminogen system, that cleavage of the internal peptide bond, leading to plasmin formation, can occur without cleavage of the amino terminal peptide.     

A functionally active heavy chain derived from human high molecular weight urokinase

Human high molecular weight urokinase, a plasminogen activator, when minimally reduced with 0.01 M 2-mercaptoethanol for 10 h at pH 8.0 and 25 degrees C and then carboxymethylated with sodium iodoacetate, gave two chains, a functionally active heavy chain with about 80% of the original activity and a light chain. These two chains were found to be linked by a single interchain disulfide bond. The functionally active heavy chain can be isolated by an affinity chromatography method with [N alpha-(epsilon-aminocaproyl)-DL-homoarginine hexylester]-Sepharose. The light chain, which has no enzyme activity, is not adsorbed to the affinity matrix, whereas the active heavy chain was adsorbed and subsequently eluted. The active heavy chain was further purified by gel filtration on Sephadex G-100. This preparation was found to be homogeneous by both analytical and sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. The molecular weight of the active heavy chain was determined to be 33,000 by Sephadex G-100 gel filtration and 31,000 by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Its specific activity, with L-pyroglutamyl-glycyl-L-arginine-p-nitroanilide, was determined to be 208,000 IU/mg of protein. Approximately 87% active sites were found by p-nitrophenyl-p'-guanidino-benzoate titration with a molar activity of 7.41 X 10(9) IU/mmol of active site. The active heavy chain when compared to low molecular weight urokinase has a similar molecular weight, specific activity, and amino acid composition. The NH2-terminal residue found in the active heavy chain was lysine which was the same as that found in low molecular weight urokinase, whereas the NH2-terminal residues found in high molecular weight urokinase were serine and lysine. Serine is the NH2-terminal residue of the light chain of high molecular weight urokinase. The steady state kinetic parameters of activation of human Glu-plasminogen by the active heavy chain were also similar to low molecular weight urokinase, as were the amidase parameters of these enzymes. The Michaelis constants of activation (Kplg) were 2.11 and 2.21 microM, respectively; the catalytic rate constants of activation (kplg) were 51.7 and 44.1 min-1, respectively, with second order rate constants, kplg/Kplg of 24.5 and 20.2 microM-1 min-1, respectively.     

Kinetic studies of urokinase-catalysed hydrolysis of 5-oxo-L-prolylglycyl-L-arginine 4-nitroanilide

The enzymic properties of urokinase (EC 3.4.21.31) were studied. The kinetic parameters of hydrolysis of 5-oxo-Pro-Gly-Arg-NA were determined in the pH range 5-9, at 25 degrees C and 37 degrees C. The reaction is affected by only one ionizing group of urokinase with pK 7.15 (25 degrees C) and pK 6.82 (37 degrees C). The results indicate that 5-oxo-Pro-Gly-Arg-NA is a good model substrate for studies of the conversion of plasminogen to plasmin. The Km values of the urokinase-catalysed hydrolysis of plasminogen and 5-oxo-Pro-Gly-Arg-NA are of the same order of magnitude. Plasmin catalyses the hydrolysis of 5-oxo-Pro-Gly-Arg-NA, but the Km value is several hundred times that of urokinase. Urokinase is shown not to react with good plasmin substrates, such as Bz-Arg-OEt and D-Val-Leu-Lys-NA, but is linearly competitively inhibited by 6-amino-hexanoic acid and trans-4-aminomethylcyclohexane-1-carboxylic acid.     

Plasminogen activator from cells transformed by an oncogenic virus: inhibitors of the activation reaction.

This paper describes an assay for direct measurement of plasminogen activation and its application for determining the kinetic constants and for screening potential inhibitors of the reaction. The assay is based on the conversion of the single chain of 125I-labelled plasminogen to the two chains of 125I-labelled plasmin (EC 3.4.21.7), the latter then being separated from each other and from the plasminogen substrate by electrophoresis under reducing conditions in SDS-polyacrylamide gels. The Km of activator from transformed murine cells for human plasminogen was 180 nM. A broad range of compounds was tested as potential inhibitors of plasminogen activation and of plasmin-catalyzed fibrinolysis respectively, and the two reactions differed qualitatively and quantitatively in their response to previous agents. The principal qualitative difference was in the susceptibility of the reactions to a spectrum of naturally-occurring macromolecular inhibitors: all of the macromolecular inhibitors that blocked the action of plasmin were without effect on murine activator or human urokinase (EC 3.4.99.26). A variety of small molecules inhibited both of the reactions tested, and showed significant quantitative differences; some of these were active at micron concentrations. The exacting specificity of plasminogen activators for macromolecules, both substrates and inhibitors, encourages the expectation that effective inhibitors of great specificity may be isolated from as yet undiscovered natural sources.     

Activation of human plasminogen by equimolar levels of streptokinase.

Native Glu-human plasminogen (Mr approximately 92,000 with NH2-terminal glutamic acid) is able to combine directly with streptokinase in an equivalent molar ratio, to yield a stoichiometric complex. The plasminogen moiety in the complex then undergoes streptokinase-induced conformational changes. As a result of such, an active center develops in the plasminogen moiety of the complex. This proteolytically active complex then activates plasminogen in the complex to plasmin and at least two peptide bonds are cleaved in the process. The data presented in this paper reveal that initially an internal peptide bond of plasminogen (in the complex) is cleaved to yield a two-chain, disulfide-linked plasmin molecule. The heavy chain (Mr approximately 67,000 with NH2-terminal glutamic acid) of this plasmin molecule has an identical NH2-terminal amico acid as the native plasminogen. The light chain (Mr approximately 25,000 with NH2-terminal valine) of plasmin is known to be derived from the COOH-terminal portion of the parent plasminogen molecule. A second peptide is then cleaved from the NH2-terminal end of the heavy chain of plasmin producing a proteolytically modified heavy chain (Mr =60.000 with NH2-terminal lysine). This cleavage of the NH2-terminal peptide from the heavy chain of plasmin is shown to be mediated by the dissociated free plasmin present in the activation mixture. Plasmin in the streptokinase-plasmin complex is unable to cleave this NH2-terminal peptide. This same NH2-terminal peptide can also be cleaved from native Glu-plasminogen or from the Glu-plasminogen-streptokinase complex by free plasmin and not by a complex of streptokinase-plasmin. From these studies we conclude (a) in the streptokinase-plasminogen complex, the NH2-terminal peptide need not be released prior to the cleavage of the essential Arg-Val peptide bond which leads to the formation of a two chain plasmin molecule and (b) that this peptide is cleaved from the native plasminogen or from the heavy chain of the initially formed plasmin in the streptokinase complex by free plasmin and not by the plasmin associated with streptokinase. In agreement with this, plasmin associated with streptokinase was unable to cleave the NH2-terminal peptide from the isolated native heavy chain possessing glutamic acid as the NH2-terminal amino acid; whereas free plasmin readily cleaved this peptide from the same isolated Glu-heavy chain.     

Binding of the NG2 proteoglycan to kringle domains modulates the functional properties of angiostatin and plasmin(ogen)

Interactions of the developmentally regulated chondroitin sulfate proteoglycan NG2 with human plasminogen and kringle domain-containing plasminogen fragments have been analyzed by solid-phase immunoassays and by surface plasmon resonance. In immunoassays, the core protein of NG2 binds specifically and saturably to plasminogen, which consists of five kringle domains and a serine protease domain, and to angiostatin, which contains plasminogen kringle domains 1-3. Apparent dissociation constants for these interactions range from 12 to 75 nm. Additional evidence for NG2 interaction with kringle domains comes from its binding to plasminogen kringle domain 4 and to miniplasminogen (kringle domain 5 plus the protease domain) with apparent dissociation constants in the 18-71 nm range. Inhibition of plasminogen and angiostatin binding to NG2 by 6-aminohexanoic acid suggests that lysine binding sites are involved in kringle interaction with NG2. The interaction of NG2 with plasminogen and angiostatin has very interesting functional consequences. 1) Soluble NG2 significantly enhances the activation of plasminogen by urokinase type plasminogen activator. 2) The antagonistic effect of angiostatin on endothelial cell proliferation is inhibited by soluble NG2. Both of these effects of NG2 should make the proteoglycan a positive regulator of the cell migration and proliferation required for angiogenesis.     

Characterization of an antibody-urokinase conjugate. A plasminogen activator targeted to fibrin

The plasminogen activator urokinase was linked covalently to a monoclonal antibody specific for the amino terminus of the beta chain of human fibrin by means of the unidirectional cross-linking reagent N-succinimidyl-3-(2-pyridyldithio)propionate. N-Succinimidyl-3-(2-pyridyldithio)propionate allowed the amino groups on urokinase to be coupled to the sulfhydryl groups on iminothiolane (which had been introduced into the antibody before the coupling reaction). The inter-heavy chain sulfhydryl of the Fab' of this antibody was also linked to N-succinimidyl-3-(2-pyridyldithio)propionate-substituted urokinase. The antibody- or Fab'-urokinase complexes were purified by two affinity chromatography steps. In the first, benzamidine was used as ligand for urokinase, in the second, a heptapeptide consisting of the 7 amino-terminal residues of the beta chain of fibrin (beta peptide) was used as ligand for the antibody. The activity of the purified conjugates was compared with that of urokinase alone in an assay measuring lysis of 125I-fibrin monomer covalently linked to Sepharose CL-4B. For any concentration of either urokinase alone or urokinase-antifibrin antibody conjugate, an equivalent amount of lysis (release of labeled peptide from fibrin monomer-Sepharose) was obtained with 1/250 the concentration (with respect to urokinase content) of antifibrin antibody-urokinase conjugate. The antifibrin Fab'-urokinase conjugate exhibited a similar enhancement of activity in comparison with urokinase. Enhanced fibrinolysis was fully inhibited by beta peptide. These results suggest that antibody targeting enhances the concentration of urokinase in the vicinity of immobilized fibrin monomer, thereby also increasing the local conversion of plasminogen to plasmin, which in turn degrades its substrate, fibrin. Univalent antigen-antibody binding is sufficient for optimal efficiency.     

Purification and characterization of human liver "high Km" aldehyde dehydrogenase and its identification as glutamic gamma-semialdehyde dehydrogenase

The enzyme previously considered as an isozyme (E4, ALDH IV) of human liver aldehyde dehydrogenase (NAD+) (EC 1.2.1.3) has been purified to homogeneity by the use of ion exchange chromatography on CM-Sephadex and affinity chromatography on Blue Sepharose CL-6B and 5'-AMP Sepharose 4B and identified as glutamic gamma-semialdehyde dehydrogenase, or more precisely 1-pyrroline-5-carboxylate dehydrogenase (EC 1.5.1.12). Glutamic gamma-semialdehyde dehydrogenase was never previously purified to homogeneity from any mammalian species. The homogeneous enzyme is seen on isoelectric focusing gels as two fine bands separated by 0.12 pH units: pI = 6.89 and 6.77. In addition, the enzyme also appears as two bands in gradient gels; however, in polyacrylamide gels containing sodium dodecyl sulfate the enzyme migrates as one band, indicating that its subunits are of identical size. Because the enzyme molecule is considerably smaller (Mr approximately 142,000-170,000) than that of aldehyde dehydrogenases (EC 1.2.1.3) (Greenfield, N. J., and Pietruszko, R. (1977) Biochim. Biophys. Acta 483, 35-45; Mr approximately 220,000) and its subunit weight is different (70,600 versus approximately 54,000 for E1 and E2 isozymes), the enzyme is not an isozyme of aldehyde dehydrogenase previously described. The Michaelis constants for glutamic gamma-semialdehyde dehydrogenase with acetaldehyde and propionaldehyde are in the millimolar range. Its substrate specificity within the straight chain aliphatic aldehyde series is essentially confined to that of acetaldehyde and propionaldehyde with butyraldehyde and longer chain length aldehydes being considerably less active. Other substrates include succinic, glutaric, and adipic semialdehydes in addition to glutamic gamma-semialdehyde. The reaction velocity with glutamic gamma-semialdehyde is at least an order of magnitude larger than with carboxylic acid semialdehydes. Aspartic beta-semialdehyde is not a substrate. The reaction catalyzed appears to be irreversible. Although NADP can be used, NAD is the preferred coenzyme. The enzyme also exhibits an unusual property of being subject to substrate inhibition by NAD.     

Kinetics of the inhibition of plasminogen activators by the plasminogen-activator inhibitor. Evidence for ''second-site'' interactions.

The reactions between plasminogen-activator inhibitor (PAI) and different plasminogen activators were studied in the presence of chromogenic peptide substrates for the enzymes. Our findings suggest that the rate constants for the reactions of PAI with single-chain tissue plasminogen activator (tPA), two-chain tPA, high-Mr urokinase and low-Mr urokinase are high and quite similar (1.6 X 10(7)-3.9 X 10(7) M-1.s-1). A free active site in the enzymes seems to be necessary for their reaction with PAI. Amino acids with antifibrinolytic properties did not interfere with the reactions. However, di-isopropyl phosphorofluoridate-inactivated tPA inhibited the reaction between PAI and all plasminogen activators in a similar way. These findings clearly demonstrated that a 'second-site' interaction, in addition to that between the enzyme active site and the inhibitor 'bait' peptide bond, is of importance for the high reaction rate. The reaction rate between PAI and single-chain tPA in the presence of an activator substrate (D-Ile-Pro-Arg p-nitroanilide) was decreased in the presence of fibrin. Fibrin caused a decrease in the Km for the single-chain tPA-substrate reaction. As a consequence, the 'free' concentration of single-chain tPA in the system decreased in the presence of fibrin, affecting the reaction rate between PAI and single-chain tPA. The phenomenon might be of physiological relevance, in the sense that single-chain tPA bound to fibrin in the presence of plasminogen would be protected against inactivation by PAI.     

The mechanism of activation of rabbit plasminogen by urokinase.

The data presented in this paper show that when rabbit plasminogen is activated to plasmin by urokinase at least two peptide bonds are cleaved in the process. Urokinase first cleaves an internal peptide bond in plasminogen, leading to two-chain disulfide-linked plasmin molecule. The plasmin heavy chain of molecular weight 66,000 to 69,000 possesses an NH2-terminal amino acid sequence identical with the original plasminogen (molecular weight 88,000 to 92,000). The plasmin light chain of molecular weight 24,000 to 26,000 is known to be derived from the COOH-terminal portion of plasminogen. The plasmin generated during the activation of plasminogen is capable, by a feedback process, of cleaving a peptide of molecular weight 6,000 to 8,000 from the NH2 terminus of the heavy chain, producing a proteolytically modified heavy chain of molecular weight 58,000 to 62,000. Plasmin also can cleave this same peptide from the original plasminogen, yielding an altered plasminogen of molecular weight 82,000 to 86,000. This plasmin-altered plasminogen and the plasmin heavy chain derived from it by urokinase activation process NH2-terminal amino acid sequences which are identical with each other and with the plasminolytic product of the original plasmin heavy chain. These studies support a mechanism of activation of plasminogen by urokinase which involves loss of a peptide located on the NH2 terminus of plasminogen. However, these same results show that this NH2-terminal peptide need not be released from rabbit plasminogen prior to the cleavage of the internal peptide bond which leads to the two-chain plasmin molecule. Furthermore, these studies show that urokinase cannot remove this peptide from either the original rabbit plasminogen molecule or from the heavy chain of the initial plasmin formed.     

Involvement of aspartic and glutamic residues in kringle-2 of tissue-type plasminogen activator in lysine binding, fibrin binding and stimulation of activity as revealed by chemical modification and oligonucleotide-directed mutagenesis

Modification of glutamic and aspartic acid residues of tissue-type plasminogen activator (t-PA) with 1-ethyl-3(3-dimethyl-aminopropyl)-carbodiimide leads to a decrease in affinity for lysine and fibrin, to a decrease of plasminogen activation activity in the presence of a fibrin mimic, but leaves amidolytic activity and plasminogen activation without fibrin mimic unaffected. Experiments with kringle-2 ligands and a deletion mutant of t-PA (K2P) suggests that glutamic or aspartic acid residues in K2 of t-PA are involved in stimulation of activity, lysine binding and fibrin binding. Mutant t-PA molecules were constructed by site-directed mutagenesis in which one or two of the five aspartic or glutamic acid residues in K2 were changed to asparagine or glutamine respectively. Mutation of Asp236 and/or Asp238 leads to t-PA molecules with 3- to 4-fold lower specific activity in the presence of fibrin mimic and having no detectable affinity for lysine analogs. However, fibrin binding was not influenced. Mutation of Glu254 also leads to a 3- to 4-fold lower activity, but to a much smaller reduction of lysine or fibrin binding. Residues Asp236 and Asp238 are both essential for binding to lysine derivatives, while Glu254 might be involved but is not essential. Residues Asp236, Asp238 and Glu254 are all three involved in stimulation of activity. Remarkably, mutation of residues Asp236 and/or Asp238 appears not to influence fibrin binding of t-PA whereas that of Glu254 does.     

The blockage of the high-affinity lysine binding sites of plasminogen by EACA significantly inhibits prourokinase-induced plasminogen activation

Prourokinase-induced plasminogen activation is complex and involves three distinct reactions: (1) plasminogen activation by the intrinsic activity of prourokinase; (2) prourokinase activation by plasmin; (3) plasminogen activation by urokinase. To further understand some of the mechanisms involved, the effects of epsilon-aminocaproic acid (EACA), a lysine analogue, on these reactions were studied. At a low range of concentrations (10-50 microM), EACA significantly inhibited prourokinase-induced (Glu-/Lys-) plasminogen activation, prourokinase activation by Lys-plasmin, and (Glu-/Lys-) plasminogen activation by urokinase. However, no inhibition of plasminogen activation by Ala158-prourokinase (a plasmin-resistant mutant) occurred. Therefore, the overall inhibition of EACA on prourokinase-induced plasminogen activation was mainly due to inhibition of reactions 2 and 3, by blocking the high-affinity lysine binding interaction between plasmin and prourokinase, as well as between plasminogen and urokinase. These findings were consistent with kinetic studies which suggested that binding of kringle 1-4 of plasmin to the N-terminal region of prourokinase significantly promotes prourokinase activation, and that binding of kringle 1-4 of plasminogen to the C-terminal lysine158 of urokinase significantly promotes plasminogen activation. In conclusion, EACA was found to inhibit, rather than promote, prourokinase-induced plasminogen activation due to its blocking of the high-affinity lysine binding sites on plasmin(ogen).     

Mechanism of the urokinase-catalyzed activation of human plasminogen.

When human plasminogen (Glu-Pga) is activated by urokinase in the presence of pancreatic trypsin inhibitor, the plasmin produced (Glu-Pma) exclusively contains a heavy chain (Glu-Ha) derived intact from the original NH2 terminus of Glu-Pga. Similar activations, utilizing a low molecular weight synthetic plasmin acylating agent, p-nitrophenyl-p-(pyridiniummethyl) benzoate, still result in a plasmin molecule with approximately 50% of the plasmin heavy chain containing the intact NH2 terminus of the original Glu-Pga. Activations performed at high levels of urokinase in the absence of any inhibitors initially produce Glu-Pma. However, the final stable plasmin, Lys-Pmb, which is obtained contains a heavy chain (Lys-Hb) which arises by plasminolysis of a small peptide from the NH2 terminus of Glu-Ha. Alternatively, Lys-Pmb can be formed in a separate series of reactions initially involving plasminolysis of Glu-Pga to yield Lys-Pgb. The peptide removed in this step is identical to the peptide removed in the Glu-Ha to Lys-Hb reaction. Next, urokinase catalyzes the conversion of Lys-Pgb to Lys-Pmb without further loss of peptide material. This latter pathway involving Lys-Pgb is probably the major pathway for human Lys-Pmb generation. These studies support a mechanism of activation of human plasminogen which involves at least two bond cleavages in Glu-Pga. However, these same studies strongly indicate that the Nh2-terminal peptide need not be released from Glu-Pga prior to plasmin formation. Further, we feel that plasmin and not urokinase catalyzes cleavage of the NH2-terminal peptide bond from Glu-Pga and the Glu-Ha heavy chain of Glu-Pma.     

Activation of fibrin-bound plasminogen by pro-urokinase and its complementariness with that by tissue plasminogen activator

Single chain urokinase (SC-UK) is a precursor of 55 kd two-chain UK (TC-UK). Treatment with catalytic proportions of plasmin or kallikrein converts SC-UK to TC-UK as a consequence of cleavage of its Lys158-Ile159 peptide bond. This plasmin-mediated activation of SC-UK induces a positive feedback secondary reaction and complicates measurement of its activity against its natural substrate, Glu-plasminogen. The fibrin-selective effect of pro-UK-induced clot lysis is not related to fibrin binding. Rather, a conformational change in Glu-plasminogen, conferred when it binds to certain carboxy-terminal lysine residues on fibrin, has been implicated in this mechanism. This is complementary to t-PA. Fibrin-bound t-PA was found to exclusively activate plasminogen bound to certain internal lysine residues. Their complementariness is believed to explain their synergism in fibrinolysis.     

Studies on the lysine-binding sites of human plasminogen. The effect of ligand structure on the binding of lysine analogs to plasminogen

A method is described for measuring relative binding constants of lysine and analogs of lysine to plasminogen and plasminogen 'kringle' fragments. Plasminogen or kringle fragments adsorbed to lysine-Sepharose are eluted with increasing concentrations of lysine or other ligands, the concentration of ligand required to elute 50% of the protein being taken as a measure of the binding constant. The method is simple and is not dependent on monitoring conformational changes. We confirm earlier reports that the best ligands for the lysine binding sites of plasminogen are omega-amino acids containing five or six carbons. We show further that both Glu-plasminogen (the native form with N-terminal glutamic acid) and Lys-plasminogen (a degraded form with N-terminal lysine), as well as the heavy chain fragments, kringle 4 and kringle 1+2+3, have very similar properties with regard to binding specificity for omega-amino acids. For all species optimal binding is observed when the distance between the amino and carboxyl carbon is about 0.68 nm. The finding of ligands is decreased by the presence of polar atoms on the alpha and beta positions of the carbon chain of amino acids. Arginine binds relatively weakly at the lysine site and there does not appear to be a separate arginine binding site in plasminogen.