Influence of SK-potentiator and fibrinogen degradation products on the activation of human plasminogen by streptokinase

When fibrinogen was degraded by plasmin, very early degradation products (FgDP-5′) enhanced the activity of SK-activator (SK-plasminogen or SK-plasmin) to the largest extent, and further degradation, such as extentsive degradation of β-chain (FgDP-20′), resulted in less enhancement of SK-activator activity than fibrinogen itself which also enhanced it. Fibrin enhanced the activation rate of plasminogen by SK to the largest extent, more than SK-potentiator. The action of thrombin on SK-potentiator was as effective as fibrin in the enhancement, thus removal of fibrinopeptides from fibrinogen or SK-potentiator is important in the activator activity of the trimolecular complex of SK-plasminogen-potentiator. SDS-PAGE indicated that native plasminogen was converted to plasmin faster in the presence of SK and SK-potentiator than SK alone.     

Kinetic analyses of the activation of Glu-plasminogen by urokinase in the presence of fibrin, fibrinogen or its degradation products

The kinetics of the activation of Glu-plasminogen (Glu-plg) and Lys-plasminogen (Lys-plg) by urokinase (UK) were studied in purified systems. The activation of plasminogen by UK in the purified systems followed Michaelis-Menten kinetics with a Michaelis constant (Km) of 1.45 microM and a catalytic rate constant (kcat) of 0.93/sec for Glu-plg as compared to 0.25 microM (Km) and 0.82/sec (kcat) for Lys-plg. In the presence of fibrin and fibrinogen or its plasmin degradation products (fragment D and fragment E), Km for Glu-plg hardly changed, whereas kcat for Glu-plg increased. Effect on increase in kcat was in the order of fibrin greater than fibrinogen greater than D greater than E. Fibrin, fibrinogen, D and E did not influence the activation of Lys-plg by UK. These results indicate that Glu-plg bound to fibrin, fibrinogen, D or E becomes easily activatable by UK. The activation of Lys-plg, however, is not influenced in the presence of fibrin, fibrinogen, D or E.     

The conversion of streptokinase-plasminogen complex to SK-plasmin complex in the presence of fibrin or fibrinogen

When equimolar amounts of Glu-plasminogen (Glu-plg) and streptokinase (SK) were mixed in the presence of S-2251, SK-plg complex was formed and only gradually converted to SK-plasmin complex. When equimolar amounts of Glu-plg and SK were mixed with fibrinogen or fibrin, Glu-plg was converted faster to plasmin suggesting that Glu-plg molecule in the complex was converted to plasmin. It is thus concluded that SK-plg complex is converted to SK-plasmin complex slowly in the absence of fibrin or fibrinogen. When SK-plg-fibrin(ogen) complex was formed, plasminogen moiety was converted to plasmin faster inside a trimolecular complex of SK, plasminogen and fibrin(ogen).     

Effects of fibrinogen and fibrin on the activation of glu- and lys-plasminogen by urokinase

Glu- and Lys-plasminogen (plg) were mixed with fibrinogen or fibrinogen plus thrombin in the presence of various units of urokinase (UK) and S-2251 (H-D-Val-Leu-Lys-pNA). Time course of the hydrolysis of S-2251 and the increment of OD₄₀₅/min were monitored by using a spectrophotometer. Glu-plg was activated better by UK in the presence of fibrinogen and fibrin than in their absence. The effects of fibrin were larger than those of fibrinogen in the activation of Glu-plg. When Lys-plg was used for the similar experiments, the activation rate of Lys-plg was slightly increased in the presence of fibrinogen and fibrin (fibrin > fibrinogen), but their effects were smaller than in the case of the activation of Glu-plg.     

Enzymatic properties of plasmins converted from acid-treated and native Glu- and Lys-plasminogens by urokinase

Glu- and Lys-plasminogens (plg) had been acidified to pH 2. Glu-plg acidified had higher extent of hydrolysis of S-2251 after the activation with urokinase (UK) than non-acidified Glu-plg. Acidified Glu-plg increased hydrolysis of S-2251 after UK activation at 1 mM of tranexamic acid. Further increase in the concentration of tranexamic acid decreased the extent of its hydrolysis. Lys-plg after UK activation in the presence of tranexamic acid up to 1 mM did not change S-2251 hydrolysis. Further increase in the concentration of tranexamic acid also resulted in the decrease of S-2251 hydrolysis. Acidified Lys-plg had largest extent of S-2251 hydrolysis after UK activation in the absence of tranexamic acid. Kinetic studies indicated that both Glu- and Lys-plgs after UK activation had the same Km values, but acidified plgs had higher Vmax values. Lys-plasmin seemed to have the same Vmax value as Glu-plasmin, but Km value of Glu-plasmin was larger than Lys-plasmin. It can be concluded that plasmins formed from acidified plgs (Glu- or Lys-plg) formed products from enzyme substrate complex faster than plasmins from non-acidified plgs.     

Effects of SK-potentiator and fibrinogen on SK-plasminogen complex in the presence of tranexamic acid

The addition of tranexamic acid to the mixture of plasminogen and streptokinase (called SK-activator activity) resulted in decreased extent of hydrolysis of TAMe (tosyl arginine methyl ester). The addition of SK-potentiator to the mixture of SK (streptokinase), plasminogen and tranexamic acid prevented the decrease in SK-activator activity caused by tranexamic acid, thus SK-potentiator counteracting with the effects of tranexamic acid. Fibrinogen potentiated SK-activator activity, but did not prevent the decrease of the activity caused by tranexamic acid. Fibrinogen added to SK and plasminogen prior to tranexamic acid prevented the decrease in SK-activator activity. From these data it is suggested that SK-potentiator and fibrinogen bind with lysine binding sites of plasminogen part of SK-activator, and SK-potentiator binds with SK-plasminogen (or -plasmin) complex faster than fibrinogen.     

Effects of tranexamic acid on fibrinolysis, fibrinogenolysis and amidolysis

When Glu-plasminogen (Glu-plg) was activated by urokinase (UK) in the presence of fibrinogen or fibrinogen plus tranexamic acid (1 mM), or else tranexamic acid (1 mM), the activation as measured by the hydrolysis of S-2251 was enhanced by tranexamic acid or fibrinogen or both. When plasma or clotted plasma was activated by UK in the presence of 1 mM tranexamic acid, fibrinolysis was completely inhibited. When Lys-plg was activated by UK in the presence of tranexamic acid and fibrin or fibrinogen, fibrinolysis was completely inhibited by 1 mM tranexamic acid, but some inhibition of fibrinogenolysis was observed. The release of B beta 15-42 from fibrin in clotted plasma activated by UK was inhibited to some extent by 1 mM tranexamic acid. The release of B beta 15-42 from fibrin after UK-activation of Lys-plg was partly inhibited by tranexamic acid. In conclusion, tranexamic acid in the concentration of 1 mM enhanced amidolysis, but inhibted fibrinolysis measured by the generation of fibrin-degradation products. Fibrinogenolysis and the release of B beta 15-42 from fibrin were partly inhibited.     

Kinetic analyses of the enhancement of the activities of t-PA induced by the presence of monoclonal antibody (C9-5)

Nine monoclonal antibodies (Mab) against two chain tissue plasminogen activator (t-PA(W] were obtained. The effects of these Mabs on the enzymatic activities of one chain t-PA (t-PA(TD] and two chain t-PA (t-PA(W] were examined by incubating t-PA and Mabs with S-2288, or with plasminogen (plg) and S-2251 in the presence or absence of fibrin. One of Mabs called C9-5 significantly enhanced the activities of t-PA, which were kinetically analyzed. The kinetic analyses of the hydrolysis of S-2288 by t-PA showed increase in kcat from 5.17/sec to 7.75/sec in the presence of 75 micrograms/ml of C9-5 without change in Km. When Glu-plg was activated by t-PA(TD) in the absence of fibrin, Km decreased from 2 microM to 0.17 microM in the presence of C9-5 without change in Vmax. The addition of fibrin resulted in further decrease in Km from 0.133 microM to 0.077 microM. When Lys-plg was activated by t-PA(TD) in the absence of fibrin, Km decreased from 0.408 microM to 0.185 microM in the presence of C9-5, and kcat increased from 0.131 sec-1 to 0.465 sec-1. The presence of fibrin further increased kcat of the activation of Lys-plg. Similar results were obtained when plasminogen was activated by t-PA(W). Mab, C9-5, was shown to bind to B-chain of t-PA by immunoblotting. These results suggest that a dimolecular complex of t-PA and C9-5 has a higher affinity to plasminogen in the presence or absence of fibrin.     

Differences in the activation rates of plasminogen by tissue plasminogen activator and urokinase

The activation of a native form of plasminogen (Glu-plg) by tissue plasminogen activator(t-PA) was enhanced when the plasma was clotted by the addition of thrombin or thrombin plus Ca++. Cross-linking of fibrin in the clotted plasma did not inhibit the fibrin-associated enhancement of the activation of plasminogen by t-PA. When fibrinolysis induced by t-PA in the clotted plasma was measured using enzyme immunoassay, lysis of non cross-linked fibrin in the clotted plasma was faster than lysis of cross-linked fibrin, however such decrease in the extent of fibrinolysis was observed in cross-linked fibrin even in the absence of alpha 2antiplasmin (alpha 2AP) in a purified system. When Glu- or Lys-plg (modified plg) was activated by t-PA, the presence of fibrin enhanced significantly the extent of activation of both Glu- and Lys-plg, but the activation of Glu-plg by urokinase (UK) was enhanced in the presence of fibrin. The activation of Lys-plg by UK was rather inhibited in the presence of fibrin.     

Effects of pH on the conformation and activation of plasminogen

Glu-plasminogen (plg) or Lys-plg solution was kept at pH 2.0 for various time intervals, and then readjusted to pH 7.0. The activation rate and conformational changes of such acid-treated plg were measured using the hydrolysis of S-2251, spectrofluorometry and spectropolarimetry (circular dichroism). The activation rate of acid-treated Glu-plg increased after readjustment to pH 7.0. The increase was not reversible even after keeping acid-treated Glu-plg for 24 hrs at neutral pH. The activation rate of acid-treated Lys-plg rather decreased. The fluorescence intensity at 340 nm of acid-treated plg decreased, and the intensity of fluorescence induced by the interaction of plasminogen with ANS (1-anilino-8-naphthalene sulfonate) increased after keeping plasminogen for longer than 24 hrs at pH 2.0. Circular dichroism spectra indicated that the secondary and tertiary structure of Glu-plg changed after acidification, and that only the tertiary structure of Lys-plg changed.     

Effects of fibrin on the enhanced activation of plasminogen by urokinase and tissue plasminogen activator: role of cross-link

When human plasma was activated by urokinase (UK) in the presence of thrombin, thrombin plus Ca++, Ca++ or in their absence and the plasmin activity was measured by the hydrolysis of S-2251, plasmin activity was higher in the presence of cross-linked or non cross-linked plasma clot. The results of similar experiments utilizing plasma after severe exercise indicated that the hydrolysis of S-2251 by plasma containing tissue plasminogen activator (t-PA) was also higher in cross-linked or non cross-linked plasma clot. Fibrinolysis was faster in thrombin-induced plasma clot, but was later shown significantly in plasma clot induced by thrombin and Ca++, whereas practically no fibrinogenolysis was shown in plasma. When Glu-plasminogen (Glu-plg) was activated by UK in the presence of cross-linked or non cross-linked fibrin and alpha 2 antiplasmin (alpha 2AP), fibrinolysis was faster in cross-linked fibrin than non cross-linked fibrin in the presence of alpha 2AP. No fibrinogenolysis was shown either. Plasmin activity measured by the hydrolysis of S-2251 was also higher in cross-linked or non cross-linked fibrin than in fibrinogen in the presence of alpha 2AP. These results indicate that enhanced activation of Glu-plg by UK or t-PA in the presence of fibrin was a more significant event than the inactivation of plasmin in the plasma clot or purified clot by alpha 2AP cross-linked to fibrin.     

Glu-plasminogen I and II: their activation by urokinase and streptokinase in the presence of fibrin and fibrinogen

Two isozymes of a native form of human plasminogen (plg), Glu-plg I and II, were isolated. Glu-plg I or II was activated by urokinase (UK) or streptokinase (SK) in the presence of fibrinogen or fibrin. The activation of Glu-plg I was enhanced more than that of Glu-plg II in the presence of fibrin. Fibrin caused better activation of both Glu-plg I and II than fibrinogen. When fibrinolysis or fibrinogenolysis was measured, fibrin was degraded faster than fibrinogen after the activation of Glu-plg I and II by UK. These results suggest that the activation of Glu-plg I was enhanced more than that of Glu-plg II in the presence of fibrin or to less extent fibrinogen.     

The activation of two isozymes of glu-plasminogen (I and II) by urokinase and streptokinase

Glu-plasminogen (Glu-plg) was eluted through lysine-Sepharose by using a gradient of 6 aminohexanoic acid, and two peaks corresponding to Glu-plg I and II were obtained. Glu-plg I has a molecular weight of 93,000 and Glu-plg II has a molecular weight of 89,000. When these plgs were activated by urokinase (UK) or streptokinase (SK) in the presence of S-2251 (H-D-Val-Leu-Lys-pNA), the hydrolysis of S-2251 by Glu-plg I activated by UK or SK was larger than that by Glu-plg II activated by UK or SK. The results of SDS-PAGE indicate that the conversion of Glu-plg I to plasmin by UK was faster than that of Glu-plg II. It may be concluded that Glu-plg I is activated better to plasmin by activators than Glu-plg II.     

Fluorescence spectrophotometric studies on the conformational changes induced by omega-aminoacids in two isozymes of Glu-plasminogen (I and II)

Glu-plasminogen I (Glu-plg I: with two carbohydrate chains) and Glu-plg II (with one carbohydrate chain) were separated by a gradient elution of 6 aminohexanoic acid (6AHA) through lysine-Sepharose. Each preparation was excited with ultraviolet light of wave length at 291 nm. The intensity of fluorescence was measured at 340 nm. The intensity of fluorescence increased to a small extent at 0.02 mM of tranexamic acid (t-x) for Glu-plg I and then quickly increased from 0.1 mM of t-x to reach the peak at 0.6 mM. The intensity of fluorescence for Glu-plg II started to increase at 0.2 mM to reach the peak at 0.7 mM. No small increase of fluorescence was observed at less than 0.2 mM of t-x for Glu-plg II. Kdobs of Glu-plg I for t-x and 6AHA were 0.34 mM and 1.35 mM, respectively, whereas Kdobs of Glu-plg II for t-x and 6AHA were 0.46 mM and 3.3 mM, respectively. When Glu-plg I and II were activated by urokinase (UK) and the hydrolysis of S-2251 was measured, the extent of hydrolysis increased in the presence of t-x and 6AHA. The rate of the increase of S-2251 hydrolysis (thus activation rate of Glu-plg I and II with UK) increased in parallel with increase in fluorescence intensity of Glu-plg I and II in the presence of omega-aminoacids. In conclusion, changes in the activation rate with UK and in fluorescence intensity were observed at lower concentrations of omega-aminoacids for Glu-plg I than for Glu-plg II.     

Effects of tranexamic acid on the conversion of Glu-plasminogen I and II to its Lys-forms

Glu-plasminogen (Glu-plg) was incubated with plasmin. It took more than 2 hr incubation for the conversion of Glu-plg to a modified form (Lys-plg) to take place. Especially the conversion of Glu-plg II to Lys-plg II by plasmin took place very slowly. On the other hand, the conversion of Glu-plg I to Lys-plg I took place faster than that of Glu-plg II. In the presence of 1 mM tranexamic acid, the conversion of both Glu-plg I and II to their Lys-forms by plasmin was accelerated and completed in 30 min incubation. Fifty percent increase in the rate of the conversion of Glu-plg I to Lys-plg I was observed in the presence of 0.18 mM tranexamic acid. For the conversion of Glu-plg II to Lys-plg II, larger concentration of tranexamic acid was needed. Another observation was that tranexamic acid protected the degradation of plasminogen by plasmin, indicating the involvement of the lysine binding sites (LBS) of plasmin in the proteolytic attack against plg.     

Potentiation of the activation of glu-plasminogen by streptokinase and urokinase in the presence of fibrinogen degradation products

When Glu-plasminogen was activated by streptokinase (SK), the presence of early degradation products of fibrinogen (FgDP) enhanced the rate of activation. When various fragments of FgDP were fractionated, FgDP-Y and E fragments had a potentiating activity while D fragment did not. When Glu-plasminogen was activated by urokinase (UK), the activation rate was enhanced by every fragment of FgDPs, i.e. X, Y, D and E. Although both D and E fragments seem to be able to bind with lysine binding sites of Glu-plasminogen, and to enhance the activation rate by UK, only binding of E fragment with plasminogen can result in enhancement of the activation of Glu-plasminogen by SK.     

Conversion of Glu-plasminogen to plasmin by urokinase in the presence of tranexamic acid

When Glu-plasminogen (Glu-plg) was incubated with urokinase (UK) in the presence of various concentrations of tranexamic acid and S-2251, the hydrolysis of S-2251 increased in the presence of 1 mM of tranexamic acid, but decreased in the presence of more than 10 mM of tranexamic acid. Use of SDS-polyacrylamide gel electrophoresis indicated: 1) Glu-plg was converted to plasmin better in the presence of 1 mM of tranexamic acid, but in the presence of more than 10 mM of tranexamic acid it was less converted. 2) Plg I (of higher molecular weight) was more easily converted than plg II by UK, but binding of tranexamic acid with lysine binding sites of plasminogen caused almost equal rate of activation of plg I and II.     

Plasmin inhibitors and fibrinogen breakdown during the initial phase of thrombolytic treatment--the problem of the alpha 2-antiplasmin determination

During the first 3 hr of thrombolytic treatment with porcine plasmin (p-PL) or streptokinase (SK) a rapid decrease of clottable fibrinogen with generation of large amounts of fibrin(-ogen) degradation products (FDP) are found. alpha 2-antiplasmin (alpha 2AP) is rapidly neutralized. Whereas in patients treated with SK more than half of the original plasminogen was consumed, its level remained unchanged during p-PL infusion. When alpha 2AP reaches values below some 20%, spontaneous amidolytic activity towards S-2251 representing either PL-alpha 2-macroglobulin- or SK-plasminogen-complex activity appears. This activity has to be considered in the alpha 2AP assay in order to avoid underestimation of this inhibitor.     

Activation of plasminogen by tissue activator is increased specifically in the presence of certain soluble fibrin(ogen) fragments

Tissue activator-mediated plasminogen activation is potentiated both by fibrin and by some soluble fibrin(ogen) fragments. The potentiating effect of the different fragments decreases in the order fibrin monomer greater than D-dimer greater than Y greater than D EGTA greater than Dcate greater than X. Fibrinogen and the fragments Ecate, E EGTA and E fibrin have almost no effect. The existence of a fibrin polymer is apparently not a prerequisite for this potentiating effect. The plasminogen activation by various urokinase preparations is not potentiated by fibrin and fibrin(ogen) fragments.     

Effects of N-terminal peptide of Glu-plasminogen on the activation of Glu-plasminogen and its conversion to Lys-plasminogen.

In order to analyze the mechanism of the intramolecular binding of the N-terminal peptide of Glu-plasminogen (Glu-plg) to its kringles, which results in its tight conformation, we synthesized peptides of the N-terminal portion of Glu-plg molecules and analyzed their effects on the activation of Glu-plg and its conversion to Lys-plasminogen (Lys-plg) by plasmin. Three peptides of Ala44-Lys50, Ala44-Glu51 and Ala44-Ser49 were synthesized in order to examine the effect of lysine residue in the peptide. Ala44-Lys50 and Ala44-Glu51 enhanced the activation of Glu-plg by urokinase, whereas the activation of Lys-plasminogen (Lys-plg) was slightly inhibited. The conversion of Glu-plg to Lys-plg by plasmin was also enhanced by these peptides. The results suggest that Ala44-Lys50 and Ala44-Glu51 worked on Glu-plg in a similar manner as lysine analogues by making its conformation looser. The third peptide Ala44-Ser49 did not have any effect on the activation of Glu-plg by urokinase or the conversion of Glu-plg to Lys-plg by plasmin. Ala44-Lys50 residue of Glu-plg is, therefore, strongly implicated as a candidate for the responsible site of the intramolecular binding in Glu-plg.