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.     

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.     

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.     

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 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 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.     

Inhibition by tranexamic acid of the conversion of single-chain tissue plasminogen activator to its two chain form by plasmin: The presence on tissue plasminogen activator of a site to bind with lysine binding sites of plasmin

The addition of tranexamic acid inhibited the conversion of single chain tissue plasminogen activator (sct-PA) to its two chain form (tct-PA) by plasmin. Increase in the concentrations of tranexamic acid resulted in more inhibition of the conversion, with 50 % inhibition being obtained at 0.251 mM of the concentration of tranexamic acid. The addition of the fragments of plasminogen such as kringle 1 to 3 (K1–3) and K4 resulted in the facilitation of the conversion of sct-PA to tct-PA by plasmin. The addition of tranexamic acid to the mixture of sct-PA and K4 inhibited the rate of the conversion of sct-PA by plasmin. These results suggest that binding of lysine binding sites of plasmin or plasminogen with sct-PA enhanced its conversion to tct-PA by plasmin, and that there is a site on a t-PA molecule to bind to plasminogen or plasmin.     

Demonstration of the presence of plasminogen activator in human small intestine

A cytosolic fraction of human small intestine was prepared. It contained esterase activity toward N-alpha-acetyl-lysine-methyl ester and amidolytic activities toward substrates S-2238, S-2288 and S-2251. In addition there was present a plasminogen activator activity which could cleave plasminogen to produce plasmin and the plasmin hydrolysed the same chromogenic substrates. Plasmin generation was also followed by a time-dependent hydrolysis of 125-I labeled plasminogen or monitored by fibrin-agar plate. The plasminogen activator was related to urinary urokinase immunologically. Anti-urokinase IgG cross-reacted with cytosolic fraction in double immunodiffusion. When the cytosolic fraction was electrophoresed in discontinuous polyacrylamide gel, two regions of hydrolytic activity toward the urokinase-specific substrate S-2444 were found. The activity of one of these regions could be completely inhibited by anti-urokinase while the other was not. The plasminogen activator was partially purified by ammonium sulfate precipitation and Concanavalin A-bound Sepharose chromatography.     

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.     

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.     

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.     

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.     

Effects of tranexamic acid and local fibrin deposition on fibrinolysis and granulation tissue formation in preformed cavities

The effects of locally deposited fibrin and of tranexamic acid-induced antifibrinolysis on forming granulation tissue were studied in the light of a recently developed method for treatment of postoperative fistulas by occlusion with a fibrin clot. Perforated teflon cylinders, either empty or fibrin-filled, were implanted subcutaneously in rats and extracted after 2 weeks. Fibrin deposition was found to stimulate granulation tissue ingrowth into the cylinders but it did not change the fibrinolytic activity in the granulation tissue. A significantly higher fibrinolytic activity was, however, found in the tissue fluid collected from the space between the granulation tissue and the implanted fibrin clot compared to tissue fluid from cylinders implanted empty. Tranexamic acid significantly reduced the fibrinolytic activity on the granulation tissue and delayed lysis of the implanted fibrin clot. It also reduced granulation tissue ingrowth but it did not abolish the positive effects of the clot on granulation tissue formation.     

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.     

Effects of tranexamic acid, CIS-AMCHA, and 6-aminohexanoic acid on the activation rate of plasminogen by urokinase in the presence of clot

When human plasma was mixed with 50 units of urokinase and clotted with thrombin in the presence of S-2251, the hydrolysis of S-2251 in the clot was higher than that in the plasma. The presence of 10 μM of tranexamic acid resulted in decrease in the activation rate of plasminogen in the clot but not in the plasma. Further increase in its concentration resulted in marked increase in plasminogen activation in both clot and plasma. The addition of 6-aminohexanoic acid or cis-AMCHA showed changes in the rate of plasminogen activation similar to that shown in the presence of tranexamic acid. Fifty μM of tranexamic acid totally inhibited urokinase induced clot lysis, while 1 mM of cis-AMCHA and 6-aminohexanoic acid totally inhibited clot lysis. The presence of 1 mM of these ω-aminoacids remarkably increased the activation of plasminogen by urokinase, but no clot lysis was observed.