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.     

The enhanced activation of Glu-plasminogen by urokinase in the presence of fibrin or des A fibrin as measured by the release of B beta peptide and FDP

Fresh plasma was incubated either with urokinase (UK) alone or the mixture of UK and human thrombin or Reptilase. In a purified system Glu-plasminogen (Glu-plg) was incubated with fibrinogen or fibrinogen plus thrombin in the presence of UK. At intervals, aprotinin was added to stop the reactions and the amounts of B beta peptide and fibrin(ogen) degradation products (FDP, FgDP) were measured by radioimmunoassay and enzyme immunoassay, respectively. Results obtained by using plasma showed that fibrin was degraded faster than fibrinogen upon the addition of UK to the plasma or plasma clotted with thrombin. B beta peptide was released faster from the clot than plasma. The clot formation caused by Reptilase (release of fibrinopeptide A) was accompanied by increase in the release of B beta 1-42 from des A fibrin (fibrin without fibrinopeptide A). In a purified system, fibrin was degraded faster than fibrinogen upon the activation of Glu-plg by UK. These results may correlate well with the observation that Glu-plg was activated better by UK in the presence of fibrin than fibrinogen.     

The activation of Glu- and Lys-plasminogens by streptokinase: effects of fibrin, fibrinogen and their degradation products

Studies were performed on the activation of a native form of human plasminogen (Glu-plg) or its degraded form (Lys-plg) by streptokinase (SK) in the presence of fibrin, fibrinogen, SK-potentiator, fragment D or E. When Glu-plg (0.1 microM) was activated by 0.5 u/ml of SK in the presence of 100 micrograms of S-2251 and 0.1 microM of fibrin, fibrinogen or their degradation products (potentiating agents), fibrin enhanced the rate of the hydrolysis of S-2251 to the largest extent. Fragments D and E only slightly enhanced it. The order of effectiveness of enhancement was fibrin greater than SK-potentiator greater than fibrinogen greater than D greater than E. When Lys-plg (0.1 microM) was activated by 0.5 u/ml of SK in the presence of potentiating agents, SK-potentiator enhanced the hydrolysis of S-2251 to the largest extent. The enhancement was far less in comparison to the enhancement of the hydrolysis by Glu-plg and SK. The measurement of delta OD405/min at the time of 50% hydrolysis of the substrate was performed in order to compare the effects of concentrations of potentiating agents. The maximum enhancement was obtained at almost an equimolar ratio of plasminogen and fibrin. Fifty percent enhancement was obtained at 0.05 microM for SK-potentiator, 0.072 microM for fibrinogen, 0.21 microM for D and 0.35 microM for E. Fibrin caused the largest extent of enhancement among other potentiating agents. These results may indicate that a trimolecular complex between SK, plasminogen and potentiating agents hydrolyzes S-2251 more effectively than SK-plasminogen complex, thus a trimolecular complex being a better activator than SK-plasminogen complex. Although D and E enhanced only slightly the rate of hydrolysis of S-2251 at equimolar ratio to plasminogen, increase in their concentration resulted in the same extent of enhancement as shown in the presence of fibrinogen or SK-potentiator.     

Activation pathway of glu-plasminogen to lys-plasmin by urokinase

When Glu-plasminogen (Glu-plg) was incubated with plasmin for various time intervals, and the mixture was activated by urokinase (UK), the activation rate increased gradually as incubation time increased. The presence of fibrin not only enhanced the activation rate of Glu-plg but also that of proteolytically modified form to some extent. The results of SDS-PAGE indicate that the release of N-terminal peptides from Glu-plg or Glu-plasmin takes place gradually when the concentration of plg was about 1 μM, and that Glu-plasmin I of larger molecular weight is more slowly converted to Lys-plasmin than Glu-plasmin II of smaller molecular weight. The amounts of carbohydrate moieties on the heavy chain of plasmin may influence the release of N-terminal peptide from Glu-plasmin. Kinetic studies indicate that Lys-plasmin has smaller Km than Glu-plasmin, thus the former being better enzymatically than the latter.     

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.     

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.     

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.     

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.     

Fluorescence polarization and spectropolarimetric studies on the conformational changes induced by ω-aminoacids in two isozymes of glu-plasminogen (I and II)

Conformational changes of two isozymes of Glu-plasminogen(Glu-plg I and II) induced by ω-aminoacids were studied by using fluorescence polarization and spectropolarimetry. The rotational relaxation times (P_h) of FITC labeled Glu-plg I and II decreased in the presence of 6-aminohexanoic acid (6AHA) or tranexamic acid (t-x), which may mean increase in Brownian motion of FITC labeled region (possibly N-terminal region) of Glu-plg I and II when 6AHA or t-x binds with lysine binding sites (LBS) of these plasminogens. Glu-plg II seems to have longer rotational relaxation time compared to that of Glu-plg I, which may mean smaller extent of Brownian motion of FITC labeled region of Glu-plg II in comparison to that of Glu-plg I. The far ultraviolet circular dichroism (CD) spectra indicate that there may be some difference in the polypeptide backbone between Glu-plg I and II, possibly more of β-structure and less of random coil structure in Glu-plg II in comparison to Glu-plg I The presence of 6AHA or t-x gave rise to larger change of the negative ellipticity at around 208 nm in Glu-plg I in comparison to its change in Glu-plg II, which may mean the larger extent of conformational change of Glu-plg I induced by 6AHA or t-x than that of Glu-plg II.     

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.     

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.     

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.     

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.     

Autoradiographic and immunoblotting analyses of the activation pathways of plasminogen by urokinase and tissue plasminogen activator in the plasma or clotted plasma

The activation pathway of Glu-plasminogen (Glu-plg) was analyzed by using immunoblotting and autoradiography. Glu-plg was slowly activated to Glu-plasmin by urokinase (UK) and tissue plasminogen activator (t-PA) in the plasma. Significant amounts of a complex of alpha 2AP with plasmin were found. On the other hand, UK and t-PA rapidly activated Glu-plg in the clotted plasma. The major form of plasmin was Lys-form, and significant amounts of alpha 2M complexed with plasmin were found. A complex of alpha 2AP with plasmin was not so significantly formed in the clot, confirming ineffective inhibition of plasmin by alpha 2AP in the presence of fibrin. Glu-plg was directly activated by UK or t-PA to Glu-plasmin and further to Lys-plasmin in the plasma or clotted plasma.     

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 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.     

Degradation of Glu- and Lys-plasminogen by elastase in the presence or absence of tranexamic acid

Glu-plasminogen (Glu-plg) was degraded by elastase in the presence or absence of tranexamic acid. Glu-plg was degraded faster in the presence of tranexamic acid. Increase in the concentration of tranexamic acid resulted in increase in the appearance of degradation products, reaching a plateau level at 1 mM of tranexamic acid. Fifty percent increase in the concentration of one of degradation products was obtained at 0.22 mM of tranexamic acid, which is similar to a dissociation constant (Kd) of low affinity lysine binding sites (LBS) with tranexamic acid. As to the degradation rate of two isozymes of Glu-plg (Glu-plg I and II), Glu-plg II containing one carbohydrate chain was degraded faster than Glu-plg I containing two carbohydrate chains. Comparison of the degradation rates of Glu-plg and Lys-plg indicated that Lys-plg was degraded faster.     

Release of N-terminal peptides from glu-plasminogen by plasmin in the presence of fibrin

The rate of plasmin-catalyzed Glu-plasminogen to Lys-plasminogen conversion was faster in the presence of fibrin than in its absence when assayed by SDS-polyacrylamide gel electrophoresis followed by quantification of stained bands by densitometer. Experiments with the isozymes Glu-plasminogen I and Glu-plasminogen II showed that plasmin catalyzed formation of Lys-plasminogen II was especially facilitated in the presence of fibrin.     

Release of B beta peptides from fibrinogen or fibrin in the presence of alpha 2 antiplasmin

When Glu-plasminogen (plg) was activated by urokinase (UK) in the presence of fibrinogen or fibrin, B beta peptides (B beta 1-42) were released faster from fibrinogen than from fibrin (B beta 15-42). These results were contrary to faster release of B beta 15-42 from fibrin in the UK-activated clotted plasma in comparison to the release of B beta 1-42 from UK-activated plasma. The addition of plasma or lysine-Sepharose pass through fraction to the above system resulted in faster release of B beta peptides from fibrin than fibrinogen. The addition of alpha 2 antiplasmin (alpha 2AP) to the mixture of Glu-pig, UK and fibrinogen or fibrin resulted in faster release of B beta peptides from fibrin than from fibrinogen. These results indicate that fibrin protected plasmin from inactivation by alpha 2AP, leading to cleavage of Arg(42)-Ala(43) bond in beta-chain of fibrin which seems to be less susceptible to plasmin than the same bond in fibrinogen.