Interaction of SK-human plasmin, SK-dog plasmin complexes with α2 antiplasmin and α2macroglobulin

THROMBOSIS RESEARCH 41; 671-680, 1986 0049-3848/86 $3.00 t .OO Printed in the USA. Copyright (c) 1986 Pergamon Press Ltd. All rights reserved.

INTERACTION OF SK-HUMAN PLASMIN, SK-DOG PLASMIN COMPLEXES WITH a2-ANTTPLASMIN AND aZ-MACROGLOBULIN ** ** and W. Ganz K.N.N. Reddy*, B. Cercek **, AS. Lew Department of BiochemGtry and Cancer Center, ** USC School of Medicine , Division of Cardiology, , Cedars-Sinai Medical Center and UCLA School of Medicine Los Angeie;, Ealifornia 90033

. . . (Received 31.10.1985; Accepted in original by Editor R.F. Doolittle)

form 10.12.1985

ABSTRACT In patients treated with streptokinase there is a rapid and significant decrease in the level of fibrinogen in the circulation. In dogs given streptokinase there is no such change in circulating fibrinogen. To find an explanation for this species difference in response to streptokinase, the inhibition of SK-human plasmin and SK-dog plasmin by soybean and a2-macroglobulin were compared in trypsin inhibitor, az -antiplasmin thisstudy. Soybean trypsin inhibitor completely blocked the hydrolysis of S-2251 substrate (D-val-L-leu-lys-p-nitroanilide) by SK-dog plasmin and had no effect on SK-human plasmin. atAntiplasmin, the physiologically important regulator of fibrinolysis, inhibited S-2251 hydrolysis by SK-dog plasmin but not the activity of SK-human plasmin. a2-Macroglobulin showed 100% inhibition of proteolytic activity and 50% inhibition of S2251 activity of SK-dog plasmin, and had no effect on SK-human plasmin. Studies with fresh human and dog plasma also showed that the SK-dog plasmin is rapidly inactivated by the 02-antiplssmin present in the plasma. The inactivation of SK-dog plasmin and not SK-human plasmin by plasma inhibitors explains the differences in the response of dog and humans to the administration of streptokinase.

INTRODUCTION Streptokinase (SK) is an effective therapeutic agent for dissolving blood clots in clinical practice. SK itself does not digest the fibrin in the clot. The enzyme responsible for the dissolution of fibrin clots, plasmin, is present in the blood as Key words: specificity.

Streptokinase,

plasmin,

a2-antiplasmin, 671

02-macroglobulin,

species

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INHIBITION OF SK-PLASMIN

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SK catalytically activates plasminogen to the inactive zymogen, plasminogen. plasmin. The unique mechanism of activation of plasminogen by SK was discovered by Reddy and Markus (1). SK reacts almost instantaneously with human plasminogen As a result of this complex formation, a to form an equimolar complex of SK-plgn. conformational change occurs and an active center which catalyzes the conversion of plasminogen to plasmin becomes exposed on the plasminogen moiety of the complex. The SK-p&n complex is then converted to a SK-p1 complex which has also the ability to activate plasminogen to plasmin. Significant differences have been found in the reaction of SK with plasminogen from different species (2,3,4,5). Our studies with dog plasminogen (3) had shown the following: 1) The extent of activation of dog plasminogen is dependent on the concentration of SK used for activation. 2) The rate of active site formation induced by SK is slow compared to human plasminogen. 3) Of great significance is the fact that when the SK-p@ complex is converted to the SK-p1 complex during the activation process, the SK in the latter complex is found to be in a modified form (3). This modification arises from proteolytic degradation of native SK of 47 kd to a 25.7 kd form of SK. As a result of this modification the SK-dog plasmin complex loses its ability to activate plasminogen to plasmin. In human patients treated with SK there is a rapid fall in the level of fibrinogen in the circulation (6). In dogs, SK administration does not result in the depletion of fibrinogen (7,8). This could possibly be due to 1) the rapid clearance from the circulation of SK-p1 before it could attack fibrinogen, or 2) the complex is neutralized by inhibitors in the circulation. In this study we had found that the SKdog plasmin is inhibited by the plasmin specific inhibitor, a2-antiplasmin and by a2macroglobulin.

MATERIALS

AND METHODS

Human and dog plasminogens were purified by the affinity chromatographic method of Deutsch and Mertz (9) as described previously (1). Out-dated human plasma was obtained from the Kenneth Norris, Jr. Cancer Hospital blood bank. Dog blood was collected into citrate (1:lO vol/vol) from mongrel dogs weighing from 2025 Kgs, anesthetized with morphium sulphate 1.5 mgs/Kg S.C. and sodium pentobarbital 30 mgs/Kg Iv. through an indwelling catheter in the left jugular vein. Highly purified SK was a generous gift of Behring Werke AG, Marburg, West Germany. a Antiplasmin (a -AP) was purchased from American Dignostica Inc., Greenwic k , (a2-Ml was kindly supplied by American Red Connecticut, U.5 .A. a2-Macroglobulin Cross Laboratories, Bethesda, Maryland, U.S.A. The peptide substrate S-2251 (D-val-L-leu-lys-p-nitroanilide) for plasmin assay was obtained from Helena laboratories, Beaumont, Texas, U.S.A. Soybean trypsin inhibitor (SBTI) was purchased from Sigma Chemical Co., St. Louis, Missouri, U.S.A. SK-human plasmin complex (SK-H.Pl) and SK-dog plasmin complex (SK-D.Pl) were prepared by activating the respective plasminogens with equimolar amounts of SK in 0.05M tris, 0.02M lysine buffer, pH 7.4, at room temperature (R.T.) for 20 minutes. The effect of inhibitors on the activity of SK-human and dog plasmin complexes were studied as follows: a -M was incubated with the complexes at R.T. for 30 min. Azocasein was added to &Ie reaction mixtures and digestion carried out at R.T. to minimize any dissociation of SK from the complex during the assay period. Azocasein incubation was carried out for 1 hr for SK-D.Pl and for 4 l-u for

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SK-H.Pl complex. SBTI was incubated with the enzyme complexes in 0.05 tris, 0.12M NaCl buffer, pH 7.4 for 10 min at R.T. After adding the tripeptide substrate S-2251, the rate of absorbance change at 405 nm was recorded at R.T. (10). a -AP interaction with the enzymes was similarly determined using S-2251 substra $e after incubating the enzymes with the inhibitor for 2 min at R.T. Enzyme inhibition by whole plasma was determined by the a2-antiplasmin assay method described in the Kabi technical bulletin. In control experiments human plasminogen was activated to plasmin with catalytic amounts of urokinase and this plasmin was used to standardize the activity of the inhibitor preparations.

RESULTS Effect

of SBTI on the activity

of SK-human plasmin

and SK-dog plasmin

complexes.

Equimolar complexes of SK-H.Pl and SK-D.Pl were treated with increasing amounts of SBTI and the reaction mixture assayed for proteolytic activity using azocasein as the substrate. The results in Fig. 1 show that both complexes are inhibited by SBTI. I

I

I

I

I

I

I

I

0.30

0.25 E z

0.20

% 5

0.15

!Z s: $

0.10 n

0.05 m

I I

0

10

I

I

20

30

I

40

I

50

I

I

I

60

70

80

SBTI, pgs FIG. 1 SBTI inhibition of the azocaseinolytic O-----O, and SK-dog plasmin ?? r

activity of SK-human (48.9 ugs).

plasmin

(47.5

ugs)

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When the SBTI enzyme reaction mixtures were assayed using S-2251 as the substrate a different result was obtained (Fig. 2). It can be seen that the activity of

I

I

0.2

0.4

I

I

12 , 10

,

._ E .6 a a 4

0.6

0.8

SBTI , pgs FIG. 2 SBTl inhibition of S-2251 substrate hydrolysis by SK-human _, and SK-dog plasmin o------O (2.49 Ugs).

plasmin

(2.37

I.lgs)

SK-D.Pl was inhibited by SBTl while the activity of SK-H.Pl was‘unaffected by SBTL Even in the presence of ten times the amount of SBTl needed to inhibit the SK-D.Pl, These results show that the smaller the SK-H.Pl complex retained its activity. synthetic tripeptide substrate is able to diffuse into the active site of SK-H.Pl even in the presence of SBTI, in contrast to the larger azocasein substrate. A much tighter binding of the SBTl to the SK-D.Pl does not allow S-2251 substrate to reach the active site of the enzyme.

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Effect of complexes.

INHIBITION OF SK-PLASMIN

a,-AP -

on

the

activities

of

SK-human

675

plasmin

and

SK-dog

plasmin

80 z 5: 60 F

\

0

Y *

40 \

0

20 \

0

0.5

O-0 1.0

1.5

2.0

2.5

3.0

3.5

arAP,pw FIG. 3 Hydrolysis of S-2251 substrate (2.18 dog plasmin o-------O antiplasmin.

by SK-human plasmin (2.34 ugs) 04, pgs) treated with increasing amounts

and SKof purified U2-

Fig. 3 shows the S-2251 hydrolytic activities of the two SK-p1 complexes when reacted with increasing amounts of purified human a -AP. The reaction of a -AP with the enzymes and the assay for enzyme activity ( !?-2251) was carried out at 2R _T. to minimize any dissociation of SK from the complexes. It can be seen that the SKComplete inhibition of S-2251 hydrolytic D.Pl reacted quite well with the inhibitor. activity is obtained when 2.1 ugs of a -AP was reacted with .2;18 ugs of SK-D.Pl. This compares very well with the talc Lzated value of 1.8 ugs of Ci2-AP required for complete inhibition on the basis of a mole-to-mole reaction. In another experiment twice equimolsr and four times dog plasminogen was activated with equimolar, equimolar amounts of SK for 20 min at R.T. The reaction mixtures were then treated with 02-AP and assayed for S-2251 activity. U2-AP inhibited all three preparations equally well indicating that inhibition does occur even in the presence of high levels of SK. In the control experiment SK-H.Pl was not inhibited by a2-AP in accordance with earlier reports (11,12).

INHIBITION OF SK-PLASMIN

676

I

25

I

50

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I

I

I

75

100

125

% a2-AP

FIG. 4 Effect of normal human plasma on S-2251 hydrolysis by SK-human plasmin 0 -0, and O------O SK-dog plasmin. Human plasma was diluted to contain different amounts of a2-antiplasmin (accordigg to KABI DIAGNOSTICA procedure), incubated with the enzymes for 20 set at 37 , and then assayed with the synthetic substrate.

Studies with whole human plasma also showed that the SK-D.Pl is inhibited very well by the a2-AP present in the plasma (Fig. 4). The activity of SK-H.Pl, as expected, was not Inhibited by the plasma. Similar results were obtained with fresh dog plasma. In the control experiment a preparation of SK-D.Pl had an activity of 0.170 h O.D./min at 405 nm against the S-2251 substrate. This activity was found to be reduced to a A O.D./min of 0.012 when the enzyme was incubated for 20 set at 37’ with dog plasma containing an equivalent of 125% a2-AP.

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Effect of complexes.

a2-M

INHIBITIONOF SK-PLASMIN

on

the

activities

I

of

SK-human

I

I

plasmin

I

677

and

SK-dog

plasmin

I

0.5

E

c 0.4

i w- 0.3 9 ts E 0.2 z 0.1

I

0

100

I

I

I

I

200

300

400

500

anM,

pw

FIG. 5 The inhibition of azocaseinolytic pgs) and SK-dog plasmin _ macroglobulin.

activity of SK-human plasmin O------O (71.2 (49 pgs) by increasing amounts of purified a2-

The azocaseinolytic activity of the two complexes treated with increasing amounts of a2-M is shown in Fig. 5. It can be seen that a2-M had no significant effect on the proteolytic activity of SK-H.Pl complex. The activity of SK-D.Pl, an however, was inhibited with increasing amounts of a -M. In a control experiment identical pattern of inhibition was obtained when ?I og plasminogen was activated with urokinase and then treated with a -M. The activity of SK-D.Pl towards S-2251 was also found to be inhibited approx. &?& by a2-M.

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DISCUSSION SK is used successfully as a thrombolytic agent in the treatment of thromboembolic disease (13). However, in patients treated with SK the levels of fibrinogen in the circulation decrease and stay depressed during the period of therapy. The reason for this fibrinogen depletion lies in the mechanisms involved in the SK activation of the fibrinolytic system. In human plasma, SK reacts rapidly with circulating plasminogen forming SKplgn complex which, in turn, is converted to the SK-P1 complex (I). This SK-P1 must reach the site of the clot, activate the plasminogen bound to the fibrin in the clot to plasmin, which then digests the clot. It is also likely that some free SK that reaches the clot will activate plasminogen in the clot by the sequence of reactions described The SK-P1 can and does activate systemic plasminogen to plasmin. The above. plasmin formed in the circulation is rapidly neutralized by the plasma inhibitor Q AP (11,lZ). Significantly, the SK-P1 complex is not inhibited by o -AP (16. Therefore, the presence of SK-P1 in the circulation leads to activation o2f plasminogen to plasmin and consumption of CX~-AP. This depletion of CX~-AP allows plasmin Thus, it was recently shown in cats (14) that to digest fibrinogen in the circulation. the decrease in fibrinogen and plasminogen caused by SK infusion was immediately inhibited by infusion of o.2-AP. Studies in dogs given SK show no decrease in the circulating levels of fibrinogen (7,8). Part of the explanation for this phenomenon comes from our earlier work (3). We had shown that 1) SK complexes slowly with dog plasminogen, and 2) the SK-dog plasmin complex cannot activate plasminogen to plasmin. Consequently, extensive activation of plasminogen and depletion of CL-AP does not occur in the circulation of the dog. However, the administered SK %oes form the SK-P1 complex in the circulation which should cause extensive degradation of fibrinogen since the half-life of this complex is 2.8 hr (15). In the present study we have found that the SK-D.Pl is rapidly and quantitatively inactivated by a2-AP. This, together with the fact that the SK-D.Pl has no plasminogen activator activity, can explain why fibrinogen remains unaltered in the circulation of dog treated with SK. a2-M also inhibited SK-D.Pl and not the activity of SK-H.Pl, although it was recently reported (16)that SK-H.Pl is inhibited by a -M. The lack of inhibition of SK-H.Pl by a2-M was reported earlier by Du 3 ekWojciechowska et al. (17) who suggested that differences in the reactivity of a -M -with proteases may be due to different rates of peptide hur!i cleavage in the iait region of the a2-M molecule (18). The results obtained in this study appear to There are significant differences in the enzymic properties of support this view. Plasmin does not have the ability to human plasmin and SK-H.Pl complex (19). activate plasminogen to plasmin while SK-H.Pl has this highly specific catalytic This may be reflected in the inability of SK-H.Pl to react with “2-M property. while SK-D.Pl which does not possess plasminogen activator .activity is easily inhibited by a -M. The main difference between SK-D.Pl and SK-H.Pl lies in the 2 nature of the SK present in these complexes. In the human system, the SK is present in the form of a 37 Kd polypeptide chain while in the dog it is further modified to a 25.7 Kd fragment. This structural difference in the SK of the SKplasmin complexes accounts for differences in activator activity and reactivity towards plasma inhibitors, and thus affect hemostasis profoundly - fibrinogen depletion in man, clot selectivity in the dog.

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ACKNOWLEDGEMENTS This study has been supported by a Grant-In-Aid from the American Heart Association, Greater Los Angeles Affiliate, and by a grant from the USC Faculty Research and Innovation Fund (K.N.N.R.). The authors are grateful to Juliana Yano and Ginny Cortes, R.N. for valuable assistance in this work.

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2.

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3.

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4.

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5.

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6.

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7.

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8.

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9.

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and MERTZ, E.T. Plasminogen: purification from chromatography. -Science ._-- ._ -170, 1095-1096, 1970.

human

FRIBERGER, P., KNOS, M., GUSTAVSON, S., AURIEL, L. and CLAESON, G. Methods for determination of plasmin, antiplasmin and plasminogen by means of substrate (S-2251). -_..-Haemostasis -....- -7, 138-145, 1978.

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11.

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12.

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14.

EINARSSON, M., MATTSON, C. and NILSSON, S. Effect on haemostasis of in cats treated with streptokinase. intravenous injection of a -antiplasmin Thromb. Res. 30 205-212, 1683. -. _ _’

15.

ROBBINS, K.C., SUMMARIA, L., WOHL, R.C. and BELL, W.R. The human plasmin-derived light (B) chain. Streptokinase complex: A second-generation thrombolytic agent. Thromb. Haemostas .-_.---- 50, 787-791, 1983.

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STEINER, J-P., BHATTACHARYA, zation of the a2-macroglobulin-plasmin

17.

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18.

BARRETT, A.J. and STARKEY, P.M. The interaction of U2-maaoglobulin with proteinases. Characteristics and specificity of the reaction, and a hypothesis concerning its molecular mechanism. Biochem. J. 133, 709-724, 1973.

19.

REDDY, K.N.N. and MARKUS, G. Esterase activities in the zymogen moiety of the streptokinase-plasminogen complex*. J. Biol. Chem. 249, 4851-4857, 1974.

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and

control

of

complex

fibrinolysis.

A.A.

with

Thromb.

Thromb-

P. and STRICKLAND, D.K. Charactericomplex. Fed. Proc. 44, 1430, 1985.