The interaction of heparin with human plasmin

Int.

J. Biochem.

Printed

in Great

Vol. 15, NO. 6, pp. 871-874. Britain. All rights reserved

0020-71 lXi83/060871-04$03.00/O CopyrIght 0 1983 Pergamon Press Ltd

1983

THE INTERACTION HUMAN PAL I. BAUER’*,

MARIANNE

OF HEPARIN PLASMIN

POZSGAY~,

RAYMUND

WITH

MACHOVICH’.

PAL EL~DI~

and ISTVAN HORV~~TH~

‘Second Institute of Biochemistry, Semmelweis University Medical School and ZInstitute of Biochemistry, Debrecen University Medical School, H-1444 Budapest, P.O. Box 262, Hungary (Received 17 September 1982) Abstract-i. The interaction of heparin with human plasmin was investigated measuring plasmin activity and enzyme inactivation in the presence of heparin. Hydrolysis of synthetic substrates (H-o-Val-

Leu-Lys-pNA, H-D-Val-Phe-Lys-pNA and H-o-Pro-Phe-Lys-pNA) by plasmin was enhanced by heparin through an increase in k,,, values. 2. This effect was the consequence of a change of V,,, since K, values were not altered in the presence of heparin. The polysaccharide also enhanced the rate of enzyme inactivation using TLCK as an active site blocking reagent. 3. Furthermore, heparin increased the heat sensitivity of plasmin, when synthetic substrate H-D-ValLeu-Lys-pNA was used but it did not affect enzyme activity towards N-benzoyl-L-arginine-ethylester substrate. 4. The data show that microenvironmental conformation around the active center of plasmin is influenced by heparin.

in the present work we examined the interaction of heparin with plasmin. We demonstrate that heparin influences plasmin activity as well as its inactivation.

INTRODUCTION

One of the enhancing

main

functions

of heparin

in plasma

effect on the rate of proteinase

is its

inactivation

by

antithrombin III (Jackson & Nemerson, 1980; Machovich & Horvith, 1981). The action of heparin on the clotting enzymes results in the inhibition of blood coagulation. Heparin on the other hand interacts with the fibrinolytic system as well, i.e. it binds to plasminogen (Hatton & Regoeczi, 1974) and plasmin (Stiirzebecher & Markwardt, 1977; Hatton & Regoeczi, 1977; Smith & Sundboom, 1981; Machovich et ul., 1981) resulting in the acceleration of the rate of inactivation of plasmin by antithrombin III (Highsmith & Rosenberg, 1974; Smith & Sundboom, 198 1; Machovich et al., 198 1). Thus, heparin therapy is beneficial in the prevention of thrombosis, but once thrombus formed, it may exert disadvantageous effects by the inhibition of fibrinolysis. The interaction of heparin with the proteinases of blood coagulation and its role in their inactivation has been extensively studied (Jackson & Nemerson, 1980; Stiirzebecher & Markwardt, 1977; Smith & Sundboom, 1981; Li et al., 1974; Machovich, 1975; Gitel, 1975; Blask6 & Machovich, 1975; Fujikawa et ul., 1977). The most frequently used tools to investigate the nature of the interactions are measuring the effect of heparin on: enzyme activity towards synthetic substrates (Smith & Sundboom, 1981; Griffith et ul., 1979); the inactivation of enzymes by various synthetic inhibitors (Griffith et al. 1979a,b) and on heat inactivation of proteinases (Machovich & ArBnyi, 1978). The effect of heparin on the fibrinolytic system, especially on plasmin, is not so well known. Therefore,

* Address

for reprints and correspondence. 871

MATERIALS

AND

METHODS

Lysine-Sepharose 4B, phenyl-methylsulfonyl fluoride (PMSF) and N-benzoyl-L-arginine ethylester (BAEE) were purchased from Sigma Chem. Co., St Louis, U.S.A. H-DVal-Leu-Lys-p-nitoroanilide (S-225 I ; pNA) and streptokinase were the product of Kabi AB, Stockholm. Sweden. H-o-Val-Phe-Lys-pNA and H-o-Pro-Phe-Lys-pNA were the generous gifts of Dr R. Simonsson. N-a-tosyl-lysinechloromethyl-ketone (TLCK) was obtained from Serva GmbH, Heidelberg, FRG. Heparin (from bovine intestine with a specific activity of 165 U/mg) was the gift of G. Richter Pharmaceuticals. Plasminogen was isolated from human plasma by lysineSepharose 4B affinity chromatography as described by Deutsch & Mertz (1970). Plasminogen was activated by streptokinase as described elsewhere (Machovich er trl., 1981). The enzyme has a specific activity of 7.8CU;mg protein. In a part of the experiments plasmin produced by Kabi AB. was used (specific activity 15 CUsmg protein). Protein was measured by the method of Lowry er (11. (1951). Kinetic measurements towards synthetic substrates were performed in 50 mM Tris+HCl, pH 7.4, at 37’C. The concentration of plasmin was 200nM and that of the substrates 16.7-302 PM. The absorption of p-nitroaniline Iiberated from the substrates was recorded at 405 mm for 3 min (absorption coefficient 405 = 10.600. Szabb rt ul., 1980). Activity with S-2251 and BAEE was measured as described earlier (Machovich et a/., 19Xla; Machovich et ul.. 1981 b). Molar concentrations for plasmin and heparin were calculated using the respective molecular weights of 76.000 (Summaria et al., 1967) and 11,000 (Hillborn & Anastassiadis, 1971). Heat inactivation was carried out incubating 6.6pM plasmin in the absence or in the presence of heparin (4.5 or

PAL I.BAUER et al.

872 Table

I. Effect of heparin on plasmin anilide tripeptide Heparin (3 PM)

Substrate

activity measured substrates

(Z,

by synthetic

Vmax. 10’

kc,,

(msec- I)

(set- ‘)

H-D-Val-Leu-Lys-pNA

none added

0.33 0.33

0.60 0.83

0.30 0.42

H-D-Val-Phe-Lys-pNA

none added

0.01 I 0.011

0.60 0.77

0.30 0.38

H-D-Pro-Phe-Lys-pNA

none added

0.010 0.010

0.41 0.65

0.20 0.33

Plasmin activity was assayed as described in Materials and Methods. In these exoeriments olasmin uroduced by KABI AB was used in a concentratidn of 200 nM. .

45 FM) in 50 mM phosphate buffer, pH 7.4. The total volume was 400 ~1. At various times samples (25 ~1 for S-2251 or 50~1 for BAEE) were withdrawn and their remaining enzyme activity was determined. To study the effect of heparin on TLCK inactivation of plasmin, 6.6 PM plasmin was incubated with or without 2.7 mM of TLCK in the presence or in the absence of 9pM of heparin, in 50 mM phosphate buffer, pH 7.4. The remaining enzyme activity was assayed as described above. When the active site serine blocking agent PMSF was used as inactivator it was solved in abs. ethanol in a concentration of 287 mM. ‘Ten microlitres of inhibitor was added to 400 ~1 of incubating mixture at 0, 10 and 20 min and the withdrawn samples were centrifuged before measuring the remaining enzyme activity. The K, and V,,, values for plasmin with various synthetic substrates were determined using Lineweaver-Burk plots. The k,,, values were calculated as V,,, enzyme concentration ratio. The heat inactivation resulted in a biphasic plot in semilogarithmic representation. The k values were calculated from the first line having a higher slope.

lo-fold molar excess to plasmin enhanced the rate of heat inactivation when S-2251 was used as substrate, while there was no measurable heparin effect when plasmin denaturation was measured with BAEE substrate (Table 2). The rate enhancing effect of heparin was dependent from the ionic strength of the medium. When phosphate concentration used was 4-fold higher, k values were not changed by the addition of heparin (data not shown). InJluence of heparin active

site specific

on the inhibition reagents

of plasmin

with

Heparin enhanced the inhibition of plasmin by TLCK. Plasmin (6.6 PM) was incubated at room temperatures with or without TLCK (2.7mM) or with PMSF (21 mM). At various times samples were taken and their enzyme contents were assayed. As shown in Table 3 heparin accelerated the inactivation of plasmin by TLCK measured with either S-2251 or BAEE substrate. When PMSF was used as inactivator there was no heparin effect with either substrates.

RESULTS DISCUSSION effect of hepurin substrates

on the hydrolysis

of various

synthetic

For the determination of the effect of heparin on the amidolytic activity of plasmin, saturation kinetics of the enzyme with various synthetic substrates were

measured. The Lineweaver-Burk plots revaled that heparin in a molar ratio of 15: 1 to the enzyme, enhanced the I’,,,.,, values for all the substrates with Lys-pNA group, whereas the K, values did not change (Table 1). On the other hand, the V,,,,, values for Arg-pNA substrates were not altered by the addition of heparin (data not shown). Heat

inactivation

qf‘plusmin

in the presence

qf heparin

Plasmin (6.6pM) was incubated in the absence or in the presence of heparin (4.5 or 45 PM) at different temperatures. At various times samples were withdrawn and the remaining enzyme activity was determined. From the initial velocities, heat denaturation kinetics were measured. In semilogarithmic presentation a biophasic plot was obtained and k values (rate of inactivation) were calculated from the first phase. Heparin in equimolar concentration or in

Heparin plays an important role in the regulation of haemostasis. It enhances the rate of inactivation of proteinase of blood coagulation by antithrombin III. At the same time heparin may interact with the fibrinolytic system, too. The inactivation of Factor XII, can influence the Factor XII, dependent fibrinolysis.

Table

2. Effect of heparin

Substrate

S-225

I

BAEE

Plasmin determined

Heparin cont. (PM)

on the thermal plasmin Inactivation

denaturation

of

rate, k (min- ‘)

37°C

40°C

45°C

0 4.5 45

0.074 0.094 0.156

0.109 0.157 0.200

0.122 0.186 0.235

0 4.5 45

0.062 0.065 0.070

0.089 0.090 0.092

0.120 0.122 0. I25

concentration as described

was 6.6 pM. Enzyme

activity

in Materials and Methods.

was

Plasmin

and heparin

873

Table 3. Inactivation of plasmin by TLCK and PMSF in the presence of heparin

BLASK~) GY. & MACHOVICH R. (1975) Decreased heparin sensitivity of cyclodexanedione-modified Factor Xa.

Inactivation

DEUTSCH D. G. & MERTZ E. T. (1970) Plasminogen: purification from human plasma by affinity chromatography. Science 170, 1095-1097. FUJIKAWA K., KURACHI K. & DAVIS E. W. (1977) Characterization of bovine factor XIIa. Biocl~emistr) 16. 4182-4188. GI-~EL S. N. (1975) Evidence of catalytic role of heparin in anticoarulation reactions. Ado. cup. Med. Biol. 52, 243-24?. GRIFFITH M. J., KINGDON H. S. & LUFDBLAD R: L. (1979a) The interaction of heparin with human cc-thrombin effect on the hydrolysis of anilide tripeptide substrates. Archs

Thrombos.

rate, k (min-‘) TLCK

PMSF Heparin (PM)

S-225

I

BAEE

S-225

1

BAEE

0

0.027

0.013

0.044

0.032

9

0.027

0.013

0.083

0.080

Plasmin (6.6 PM) was incubated in the presence of TLCK (2.7 mM) or PMSF (3 x 2.9 pmol of PMSF added to the 400~1 incubation medium at 0, 10 and 20min) in 0.05 M phosphate buffer, pH 7.4. At various intervals, aliquots were taken and determined for remaining enzyme activity.

the other hand, plasmin is also inactivated by antithrombin III, which process is heparin dependent. Under normal circumstances plasmin is rapidly inactivated by a,-antiplasmin. The a2-antiplasmin concentration in plasma, however, is half of the plasminogen concentration. During some pathological conditions or when streptokinase or urokinase therapy is applied, the I,-antiplasmin pool may be exhausted. Aoki et al. (1979) have described congenital deficiency of antiplasmin. In this case heparin may effect plasmin activity by influencing the plasmin-antithrombin III reaction. Therefore, it is important to investigate the action of heparin on plasmin inactivation. It is known that heparin binds to plasmin tightly with a K, of 1 x IO-’ M (Smith & Sundboom, 1981). According to our findings heparin, through the interaction with the enzyme enhances the amidolytic activity of plasmin on three different synthetic substrates. Furthermore, heat inactivation of plasmin is also influenced by heparin when H-D-Val-Leus-LyspNA is used as substrate, but not with N-benzoyl-Larginine ethylester. These data suggest a microenvironmental change around the active center of enzyme, induced by binding of heparin. This is also supported by the fact that when heparin-plasmin interaction is destroyed by high ionic strength, the heparin effect on the heat inactivation of plasmin diminished. In addition, inactivation of plasmin by TLCK, a histidine reagent (but not by PMSF a serine blocking agent) is facilitated by heparin. The exact biochemical explanation of the heparin effect, however, needs further investigations. On

Acknowledgements-This work was supported by the Hungarian Ministry of Health (Grant No. 17/l-10/349 and TPB 70.270/82), We are highly indebted to Dr R. Simonsson for the kind gifts of substrates.

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874

PAL I. BAUER et al.

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