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

THROMBOSIS RESEARCH 21; 593-601, 1981 Printed in the USA. All rights reserved.

0049-3848/81/060593-09$02.00/O Copyright (c) 1981 Pergamon Press Ltd

ENZYMATIC PROPERTIES OF PLASMINS CONVERTED FROM ACID-TREATED AND NATIVE GLU- AND LYS-PLASMINOGENS BY UROKINASE

Akikatu Takada and Yumiko Takada Department of Physiology, Hamamatsu University, School of Medicine, Hamamatsu-shi, Shizuoka-ken Japan, 431-31

(Received 20.1.1981. Accepted by Editor O.N. Ulutin. Received in final form by Executive Editorial Office 5.3.1981)

ABSTRACT 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 Gluplasmin, but Km value of Glu-plasmin was larger than Lys-plasmin. It can be concluded that plasmins formed from acidified plgs (Gluor Lys-plg) formed products from enzyme substrate complex faster than plasmins from non-acidified plgs.

INTRODUCTION Plasminogen exists in human plasma as a zymogen with glutamic acid as its N-terminal aminoacid (Glu-plg). Proteolytic cleavage of Glu-plg by plasmin results in the formation of plg with lysine as mainly its N-terminal aminoacid (Lys-pig). Glu-plg is known to change easily its conformation when its lysine binding sites (LBS) bind with lysine analogues (6-aminohexanoic acid or tranexamic acid) (1 - 4). Plasminogen also changes its conformation when it is treated with acid (5, 6). Key words: Glu-plasminogen, Lys-plasminogen, acid-treated plasminogen, tranexamic acid, S-2251 593

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ENZYMATICPROPERTIESOF PLASMXNS

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It has been shown that Glu-plg was better activated with UK in the presence of lysine analogues while Lys-plg did not change its activation rate in their presence (1, 3, 7, 8). Fibrin also enhanced the activation rate of Glu-plg but not Lys-plg by UK (9 - 11). Acid-treated and degraded plg was shown to decrease its activation rate in the presence of fibrin(8). In the present research we used acid-treated plg and indicated that plasmins formed from acid-treated plgs were more active with respect to their enzymatic activity compared to those formed from acid-non-treated plgs. MATERIALS AND METHODS Glu- lasmino en: Human plasma obtained from out-dated human blood (Red C-as dialyzed against 20 volumes of water at 4'C. After removing insolubles by centrifugation, dialyzed plasma was passed through lysine-Sepharose in the presence of aprotinin (Repulsofl), which was kindly provided by Mochida Seiyaku Co. Ltd., Tokyo, Japan and eluted by 6-aminohexanoic acid. Fractions containing plasminogen were further purified by using Sephadex G-200. Casein units were determined according to the method of Robbins and Sumnaria (12) L s- lasmino en:0.9 ml of 5.5 cu/ml of Glu-plg was incubated with 0.1 m* cu m of plasmin for 2 hours at 37°C. The mixture was then diluted 5 times with 0.1 M Tris-HCl, pH 7.4 to get 1 cu/ml of Lys-plg (= 1 cu/ml Lys-plg + 0.028 cu/ml plasmin).. Acid treatment of plasminogen : One ml of plasminogen was acidfied with 6 N HCl to pH 2 and incubated for 30 min at 37"C, then returned to pH 7.4 by using 3.5 N NaOH. As a control plg solution was added with buffer (0.1 M Tris-HCl, pH 7.4). Fibrinogen(Grade L, AB KABI, Stockholm, Sweden) was passed through lysine-Sepharose to remove plasminogen. Thrombin: Human thrombin was kindly provided by Green Cross Co. Ltd., Osaka, Urokinase was kindly provided by Kowa Co. Ltd., Tokyo, Japan. The UK preparation had high molecular weight UK (54,000) and low molecular weight UK (33,000) in 7 to 3 ration. International units were used. Tranexamic acid (trans-4-aminomethyl cyclohexane-1-carboxylic acid) was kindly provided by Daiich Seiyaku Co. Ltd., Tokyo, Japan. Hydrolysis of S-2251 (H-D-Val-Leu-Lys-pNA: AB KABI, Stockholm, Sweden): 0.5 ml of solution containing plasminogen, UK etc was incubated with 0.5 ml of S-2251 for various time intervals, and 1.5 ml of 50 % acetic acid solution was added to stop the reaction. OD405 of the mixture was measured. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemnli's method (13). Fig. 1 shows SDS-PAGE of native and acid-treated plasminogens (Glu- or Lys-form).

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93,000

-*

JiWZYMATIC PROPERTIESOF PLASMINS

~~-~_I-~-_/

??

89,000,*'

595

FIG. 1

90,000

‘**86vooo SDS-PAGE of various plasminogens

GlU

LYS

Glu-2 Lys-2

Glu = Glu-plg LYS = Lys-plg Glu 2 = acid-treated Glu-plg Lys 2 = acid treated LYs-Pig

RESULTS Hydrolysis of S-2251 by plasmins converted by UK from various plasminogens Fig. 2 shows the results of experiments in which 0.1 ml of 1 cu/ml of plasminogen, 0.3 ml of buffer and 0.1 ml of 10,000 u/ml of UK were mixed with 0.5 ml of S-2251 (100 rg) and incubated for various time intervals. Plasmin formed from acid-treated Glu-plg by UK (called acid-treated Glu-pl) hydrolyzed S-2251 more than plasmin formed from Glu-plg (called Glu-pl). Plasmin formed from acid-treated Lys-plg (called acid-treated Lys-pl) hydrolyzed S-2251 more than acid-treated Glu-pl and Lys-pl. Thus acid treatment of both Glu- and Lys-plgs resulted in more hydrolysis of S-2251 after their activation with UK than non-acidified Glu- and Lys-plgs. OD 05 rlys-plg:pH 2 ;;rGlu-plg:pH 2

0.5 I

FIG. 2 Hydrolysis of S-2251 by the mixture of various plgs with UK Lys-plg:pH2= Glu-plg:pH2=

5

acid-treated LYs-Pig acid-treated Glu-plg

10

Incubation time (min) Hydrolysis of S-2251 by plasmins obtained from Glu-plg and acid-treated Glu-plg preincubated with UK for various time intervals We now wanted to know if acid-treated plg was better activated by UK or if plasmin formed from acid-treated plg was enzymatically more active with respect to hydrolysis of S-2251.

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Fig. 3 shows the results of experiments in which 0.9 ml of 1.1 cu/ml of plg was incubated with 0.1 ml of 10,000 u/ml of UK for various time intervals. 0.1 ml of the mixture was taken and mixed with 0.4 ml of buffer and 0.5 ml of S-2251, and incubatedfor 3 min. Fig. 3 shows that acidtreated Glu-pl hydrolyzed more than Glu-pl after 10 to 30 min preincubation. Since there was no increase in the extent of hydrolysis of S-2251 after 10 min preincubation, all the plg molecules must have been activated by UK by 10 min. So larger extent of hydrolysis of S-2251 by acid-treated Glu-pl was due to its higher hydrolytic activity and not due to larger extent of activation of acid-treated Glu-plg by UK.

FIG. 3 Preincubation of Glu-plg and acid-treated Glu-plg with UK

Preincubation time (min) Effects of tranexamic acid on the activation of Glu- and Lys-plg by UK

Incubationtime: 10 min

Incubation time: 5 min

0.5 i .--a--a-.*cs._ 1 LYS-PI!& _

v 0.001

-+Lys-plg:pti 2

%

\li

'. t\

cGlu-plg

0.1

10

Cont. of tranexamicacid (mM)

FIG. 4

0.001

0.1

10

Cont. of tranexamic acid (mM) FIG. 5

Effects of tranexamic acid on the extent of hydrolysis of S-2251 by the mixture of plasminogens and UK

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ENZYMATICPROPERTIESOF PLASMINS

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We have previously shown that Glu-plg was better activated by UK in the presence of 1 mM of tranexamic acid and less activated in the presence of 10 mM or more (8). Since the activity of already formed plasmin did not change in the presence or absence of tranexamic acid (ll), the effects of tranexamic acid was on the activation rate of Glu-plg by UK, and not on the hydrolytic acitivity of already formed plasmin. 4 and 5 show the results of experiments in which plasminogen (0.1 k s 'was mixed with various concentrations of tranexamic acid, UK(lOO u) and S-2251 (lOOyg), and the mixture was incubated for 10 min (Fig. 4) or 5 min (Fig. 5). As shown in Fig. 4, hydrolysis of S-2251 by plasmin formed from Glu-plg increased much in the presence of about 1 mM of tranexamic acid while it did not change too much when Lys-plg was activated. When acid-treated Glu-plg was activated by UK in the absence of tranexamic acid, the extent of hydrolysis of S-2251 was more than that obtained in the use of native Glu-plg. The extent of hydrolysis of S-2251 by the mixture of acid-treated Glu-plg and UK increased in the presence of 1 mM of tranexamic acid. Hydrolysis of S-2251 was largest when acid treated Lys-plg was activated by UK in the presence and absence of tranexamic acid. Fig. 5 shows that hydrolysis of S-2251 by the mixture of acid-treated Glu-plg and UK was higher than that by the mixture of native Glu-plg and UK at any concentration of tranexamic acid, and that the extent of hydrolysis increased at about 1 mM of tranexamic acid. Effects of fibrinoqen and fibrin on the activation of native and acid-treated Glu-plq

0D405

OD

35 ,o+Glu:2+ fbg

0.5

.PGlu .

+ fn

,+Glu:

0.5

2 + fn

/

p*Glu + fbg

/*

/' /' ,' id /Glu . d

5

10

Incubation

15

time (min)

FIG. 6

5

10

15

Incubation time (min) FIG. 7

Effects of fibrin and fibrinogen on the extent of hydrolysis of S-2251 by UK activated plasminogens

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Native or acid-treated Glu-plg (0.1 cu) was mixed with 0.06 mg of fibrinogen in the presence or absence of thrombin ( 1 u) and UK (100 u), then 0.5 ml of S-2251 (100 rg) was added (total 1 ml ). Fig. 6 shows that Gluplg was most activated by UK in the presence of fibrin (0.06 mg of fibrinogen was three times 0.1 cu of plg on molar basis.). Fig. 7 shows that hydrolysis of S-2251 was more in the presence of fibrinogen than fibrin when acid-treated Glu-pig was activated by UK, but differences of the extent of hydrolysis of S-2251 in the presence or absence of fibrinogen and fibrin were small. Kinetics of hydrolytic activities of native and acid-treated Glu- and Lys-plg

1

2

4

6

0.5

810,

1

1.5

2 1 mg/cc

FIG. 8

FIG. 9

Lineweaver-Burk plot of plasmin formed from native or acid-treated pigs

by IJK

Glu- and Lys-plg was acidified and 1 cu of non-acidified or acidified plgs was mixed with 1,000 u of UK for 10 min at 37°C. 0.1 cu of plasmin obtained from above mixtures was added to S-2251 solution (total volume: 1 ml). Fig. 8 and 9 show double-reciprocal plots for Glu-plg and for Lysplg, respectively. Table 1 shows Km and Vmax values of these plasmins. Plasmins from acidified and non-acidified plgs had the same Km values regardless of Gluand Lys-forms. Plasmin from acidified plgs had higher Vmax values than plasmins from non-acidified plgs. Lys-plasmin seemed to have lower Km Values than Glu-plasmin.

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TABLE 1 Km, Vmax values of plasmins from various plasminogens Glu-plg*

Lys-p1g*

Glu-plg:Z**

Lys-plg:2**

Km (mg/cc)

0.190

0.125

0.190

0.125

Vmax(

0.157

0.158

0.305

0.202

OD405/min)

* One casein unit of plasminogen was activated by 1,000 u of UK and 0.1 casein unit of plasmin was mixed with S-2251. ** Plasminogen was acid-treated and activated by UK. DISCUSSION Plasminogen is a zymogen present in plasma which is converted to plasmin upon cleavage of the arginine valine bond by various activators such as UK (14). Plasminogen has a remarkable property to change its conformation under many conditions. Addition of lysine analogues such as 6-aminohexanoic acid and tranexamic acid (1 - 4), proteolytic cleavage of N-terminal peptide (15, 16) and acid treatment (5, 6) resulted in conformational changes. Lysine analogues are known to bind with lysine binding site (LBS) of the heavy chain part of plasminogen (17). Recently Lerch et al (18) indicated that plasminogen has five LBS and one of them is highly reactive with lysine (high affinity site). One high and one low affinity sites are shown to be present in the first kringle (18) and second to fourth kringles have each one low affinity site. Lerch et al (18) also proposed that a low affinity LBS in the first kringle is important in keeping a tight conformation of plasminogen since lysine in the N-terminal peptide binds with it. Binding of tranexamic acid to the low affinity LBS in the first kringle, or removal of this peptide by plasmin (Lys-plg) resulted in no linkage of the first kringle with N-terminal peptide, thus changing the conformation. Such plasminogen is shown to be better activated by activators. Acid treatment of plasminogen has first been employed by Kline (5) to purify plasminogen. Acid-treated plasminogen has been shown to be hardly sluble at neutral pH, suggesting the conformational change. Scully and Kakkar (6) and we (8) have indicated that the enzymatic activity of plasmin formed from acid-treated plg was higher. We wanted to know whether this is due to better activation of acid-treated plg by UK or higher enzymatic activities of plasmin formed from acid-treated plg. We (11) have previously shown that the addition of tranexamic acid to Glu-plg and UK resulted in higher extent of hydrolysis of S-2251 but addition to already formed plasmin hardly changed its hydrolysis of S-2251, thus suggesting that higher extent of hydrolysis of S-2251 in the presence of 1 mM of tranexamic acid was due to better activation of Glu-plg with its LBS bound with tranexamic acid. Fig. 1 shows that acid treatment of Glu- and Lys-plg did not change their molecular weights. Since plasmin formed from acid-treated Glu-plg hydrolyzed S-2251 more than plasmin formed from native Glu-plg, higher hydrolysis of S-2251 by acid-treated and UK-activated plg was not due to better activation of such plg. The presence of 1 mM of tranexamic acid resulted in enhanced hydrolysis of S-2251 by UK-activated Glu-plg, while the extent of hydrolysis

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ENZYMATICPROPERTIESOF PTASMINS

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of S-2251 by UK-activated Lys-plg was the same in the presence of 1 mM of tranexamic acid. Further increase in the concentration of tranexamic acid resulted in less hydrolysis of S-2251 by both plgs, which was due to less activation of Glu- and Lys-plg by UK at 10 mM of tranexamic acid (paper submitted). Figs. 4 and 5, especially Fig. 5 indicates that hydrolysis of S-2251 was higher by acid-treated and UK-activated Glu-plg than native Gluplg in the absence of tranexamic acid, but its hydrolysis by acid-treated and UK-activated Glu-plg increased at1 mM of tranexamic acid. This suggests that acid-treated Glu-plg still changes its conformation upon some of LBS binding with tranexamic acid. Further increase in the number of LBS binding with tranexamic acid results in another conformational change, leading to less activation rate by UK. Fig. 4 also shows that acid-treated Lys-plg was enzymatically more active than Lys-plg upon activation by UK. We have previously shown that Glu-plg was better activated by UK in the presence of fibrin than fibrinogen (7 - 11). Fig. 6 confirms those observations. Fig. 7 indicates that fibrinogen slightly increased the activation rate of acid-treated Glu-plg by UK, but fibrin increased the rate less than fibrinogen, which was previously reported by using acid-treated degraded plg. Fig. 8 and 9 show Lineweaver-Burkplots of Glu- and Lys-plg and acidtreated Glu- and Lys-plg. These plgs had been activated by UK and hydrolysis of S-2251 by plasmins was measured. Table 1 indicates the results. Acid-treated and native Glu-plg after activation by UK had the same Km values, and two kinds of Lys-plgs had also the same Km values. On the other hand, Glu- and Lys-plg after their activation by UK had the same Vmax value, but Km for Lys-plasmin was smaller than Km for Glu-plasmin. Acidified Gluplasmin had the largest Vmax value. From these data it can be suggested that the formation of enzyme substrate complex by plasmis formed from acidtreated plg (Glu or Lys) was the same with that by plasmins formed from acid-non-treated pigs, but product formation by the former was faster than the latter. Secondly the formation of enzyme substrate complex by Lys-plasmin was faster than that by Glu-plasmin, but there wasno difference concerning product formation.

In conclusion, acid treatment of Glu- and Lys-plg changed their conformations and activation of those conformationally changed plgs resulted in faster product formation from enzyme substrate complex than Glu-plasmin. REFERENCES 1. BROCKWAY, W.J. and CASTELLINO, F.J. Measurement of the binding of antifibrinolytic amino acids to various plasminogens. Arch. Biochim. Biophys. 151 194-199, 1972. -' 2. VIOLAND, B.N., BYRNE, R. and CASTELLINO, F.J. The effect of a-_-aminoacids on human plasminogen structure and activation. J. Biol.Chem. 253, 5395-5401, 1978. 3. MARKUS, G., EVERS, J.L. and HOBIKA, G.H. Comparison of some properties of native (glu) and modified (Lys) human plasminogen. J. Biol. Chem. 253, 733-739, 1978. 4. MARKUS, G., PRIORE, R.L. and WISSLER, F.C. The binding of tranexamic acid to native (Glu) and modified (Lys) human plasminogen and its effect of conformation. J. Biol. Chem. 254, 1211-1216, 1979.

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5. KLINE, D.L. The purification and crystallization of plasminogen (profibrinolysin). J. Biol. Chem. 204, 949-955, 1953. 6. SCULLY, M.F. and KAKKAR V.V. Method for the determination of plasminogen which discriminates between native and degraded plasminogen. Thrombosis B. 12, 1201-1205, 1978. 7. TAKADA, A., OHASHI, H. MATSUDA, H. and TAKADA, Y. Effect of tranexamic acid, cis-AMCHA, and 6-aminohexanoic acid on the activation rate of plasminogen by urokinase in the presence of clot. Thrombosis Res. 14, 915923, 1979. 8. TAKADA, A. and TAKADA, Y. Effects of L\)-aminoacidsand clot formation on the activation by urokinase of various plasminogen preparations. Thrombosis Res. 18, 167-176, 1980. 9. TAKADA, A. and TAKADA, Y. Effect of tranexamic acid, t-AMCHA, and its cis-isomer on the complement system in vitro and in vivo: Possible relationship between coagulation and complement systems. Thrombosis Res. 13, 193-205, 1978. 10. TAKADA, A., URANO, T. and TAKADA, Y. Influence of coagulation on the acti. vation of plasminogen by streptokinase and urokinase. Thrombos. Haemostas. 42 901-908, 1979. -' 11. TAKADA, A. and TAKADA, Y. Interaction of plasmin with o+macroglobulin and ti(Z-antiplasminin the presence and absence of tranexamic acid. Thrombosis Res. 18, 237-246, 1980. 12. ROBBINS, K.C. and SUMMARIA, L. Purification of human plasminogen and plasmin by gel filtration on Sephadex and chromatography on Diethylaminoethyl-Sephadex. J. Biol. Chem. 238, 952-962, 1963. 13. LAEMMLI, K.U. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685, 1970. The peptide chains 14. ROBBINS, K.C., SUMMARIA, L., HSIEH, B. and SHAH, R.J. of human plasmin. Mechanism of activation of human plasminogen to plasmin J. Biol. Chem. 242, 2333-2342, 1967. 15. CLAEYS, H. and VERMYLEN J. Physico-chemical and proenzyme properties of NH2-terminal glutamic acid and NH2-terminal lysine human plasminogen. Influence of 6-aminohexanoid acid. Biochim. Biophys. Acta 342, 351-359, 1974. 16. THORSEN, S., KOK, P. AND ASTRUP, T. Reversible and irreversible alterations of human plasminogen indicated by changes in susceptibility to plasminogen activators and in response to &-aminocaproic acid. Thrombos. Diathes. Haemorrh. -32, 325-340, 1974. 17. WALLEN, P. Chemistry of plasminogen and plasminogen activation. In Progress in Chemical Fibrinolysis and Thrombolysis J.F. Davidson, R.M. Rowan, M.M. Samama and P.C. Desnoyers (Eds.) New York, Raven Press, 1978, pp. 167-181. 18. LERCH, P.G., RICKLI, E.E., LERGIER, W. and GILLESSEN, D. Localization of individual lysine-binding regions in human plasminogen and investigations on their complex-forming properties. Eur. J. Biochem. 107, 7 - 13, 1980.