Inhibition of fibrinolytic activity of plasmin by suramin (antrypol) and trypan blue

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<ul><li><p>THROMBOSIS RESEARCH Volume 10, Pages 605-611. Pergamon Press, 1977. Printed in Gt. Britain. </p><p>INHIBITION OF FIBRINOLYTIC ACTIVITY OF PLASMIN By sum~m (ANTRYPOL) AND TRYPAN BLUE </p><p>LEONARD A. MOROZ The Harry Webster Thorp Laboratories, Division of Clinical Immunology, McGill University Clinic, Royal Victoria Hospital, 687 Pine Avenue West, </p><p>Montreal, Quebec, Canada H3A 1Al </p><p>(Received 7.1.1977; in revised form 22.2.1977. Accepted by Editor H.L. Nossel) </p><p>ABSTRACT Using a 125 I-fibrin solid phase assay, the trypanocidal agent, suramin (Antrypol), was found to inhibit the fibrinolytic activity of purified plasmin and of streptokinase-activated normal human plasma. Inhibition was observed at concentrations (lo-3 M) known to inhibit several activation and control reactions of the complement system. Suramin did not inhibit the activities of collagenase, trypsin, chymotrypsin, pronase, pepsin, elastase or papain at similar molar concentrations of inhibitor and enzyme. The structurally related trypanocidal agent, trypan blue, inhibited the activities of both plasmin and collagenase (or a proteinase contaminant in the collagenase preparatibn). </p><p>INTRODUCTION --- </p><p>The proenzyme, plasminogen, is abundant in plasma, and mechanisms exist for its conversion to its active form, plasmin, a proteolytic enzyme of broad substrate specificity (1). In addition to its well-known fibrinolytic activity (2), and its ability to activate factor XII (3) and kininogens (41, plasmin, under appropriate experimental conditions, has several effects on the complement system, all based on its proteolytic function. These include activation-of Cl (5), C3 (6) and C5 (71, and substitution for factor D of the alternate pathway (8). The physiological importance of these effects of plasmin remain to be established, and specific measures are rarely taken to exclude the participation of plasmin in complement activation or control mechanisms dependent upon proteolytic cleavage. </p><p>605 </p></li><li><p>606 INHIBITION BY SURAMIN Vol.lO,No.4 </p><p>Suramin (Antrypol) is a common reagent in complement technology. Documented effects of this agent include inhibition of the C8 and C9 steps of immune lysis (9), competitive inhibition of interactions between EACl and C4, and between EACl4 and C2 (lo), inhibition of interaction of EA with Cl and of EAC142 with C3-C9 (ll), and inhibition of C3b inactivator (9,ll). </p><p>In view of the foregoing, it seems worthwhile to record that suramin inhibits the fibrinolytic (proteolytic) action of plasmin at concentrations known to influence complement reactions. In addition, similar effects of the structurally related but older trypanocidal agent, trypan blue, are described. </p><p>MATERIALS AND METHODS </p><p>The preparation of electrophoretically homogeneous human plasminogen and plasmin from plasma by lysine-Sepharose affinity chromatography, together with details of the 1251-fibrin solid phase radiometric assay for fibrinolytic activity, have been described in detail previously (12,13). The assay is based on release of radioactive fibrin degradation products by enzyme incubated in polystyrene tubes coated with 1251-fibrin. Spontaneously active plasmin (14), urokinase-activated plasmin, streptokinase-activated plasma, or other proteolytic enzymes were assayed (reaction volume, 0.2 ml) in 1251-fibrin tubes for 30 minutes at 37O , with or without addition of suramin (Antrypol, Imperial Chemical Industries, Cheshire, England) or trypan blue (Allied Chemical, Morristown, New Jersey) at the concentrations indicated. The buffer used throughout was 0.015 M Tris, 0.15 M NaCl, pH 7.4 (Tris-NaCl buffer). 1251-fibrin lysed. </p><p>Results are expressed as ng Assay tubes were coated with. approximately </p><p>1.5 ug 1251-fibrin with specific activity of 25,000 to 28,000 counts/min/ug fibrin. </p><p>Urokinase-activated plasmin was prepared by incubation (30. minutes, 370) of fibrinolytically inactive plasminogen (final concentration, 10 pg/ml) with urokinase (40 CTA units/ml), kindly provided by Dr. J. Donahoe, Abbott Laboratories, North Chicago, Illinois. Streptokinase-activated plasma was prepared by prior incubation (30 minutes, 370) of normal human plasma with streptokinase (40 unitstml). Streptokinase was 'in the form of Varidase (streptokinase-streptodornase, for intramuscular use, Lederle, Montreal, Canada). Plasma was prepared from heparinized venous blood (heparin, 10 units/ml) by preliminary centrifugation at 800 x g for 20 minutes, followed by further centrifugation of the supernatant plasma at 10,000 x g for 10 minutes (14). </p><p>Other proteolytic enzymes were tested for fibrinolytic activity, with and without suramin or trypan blue, at molar concentrations of enzyme equivalent to that of 10 ug plasmin/ml. The following enzymes were studied (concentrations in parentheses): trypsin, bovine pancreas (2.7 ug/ml), d-chymotrypsin, bovine pancreas (2.7 ).lg/ml), collagenase, clostridiopeptidase A (13.4 pg/ml), papain,(2.7 pg/ml) and pepsin, swine stomach mucosa (4.0 ug/ml), all from Worthington Biochemical Corporation, </p></li><li><p>Vol.lO,No.4 INHIBITION BY SUBAMIN 607 </p><p>Freehold, New Jersey; elastase, hog pancreas (3.3 pg/ml), and pronase, protease from Streptomyces griseus type VI (3.3pg/ml), from Sigma Chemical Company, St. Louis, Missouri. </p><p>HESULTS </p><p>At a concentration of 10W3 M (1430 yg/ml), which compares with concentrations which inhibit several complement reactions (250 to 1000 ug/ml) (g-11), suramin produced 48% inhibition of the fibrinolytic activity of spontaneously active plasmin (10 ug/ml) (Fig. 1). This plasmin concentration represents fibrinolytic activity which is approximately twenty-fold greater than that of unstimulated normal plasma(l3). Trypan blue was even more effective as a plasmin inhibitor. At a concentration of 10-3 M, it resulted in 64% inhibition of spontaneously active plasmin (Fig. 1) and 80% inhibition of urokinase-activated enzyme (Fig. 2). Inhibition by suramin was also demonstrable with plasmin generated by streptokinase treatment of normal human plasma. In a representative experiment, fibrinolytic activity of activated plasma alone was 365 ng 1251-fibrin lysed, and of plasma with suramin (lo-3 M), 179 ng fibrin lysed, representing inhibition of 48%, which was similar to that observed with purified plasmin (Fig. 1 and 2) at enzyme concentrations yielding fibrinolytic activities comparable to those of streptokinase-treated plasma. </p><p>IO </p><p>25 FIG. 1 Inhibition of fibrinolytic </p><p>f activity of spontaneously 2 active human plasmin c! (lOpg/ml) by suramin </p><p>5og (closed circles) and by etrypan blue (open circles). Points represent means of duplicate assays. </p><p>Ts Horizontal interrupted line and hatched area, mean f S.E.M. (four replicate determinations) for plasmin alone. </p><p>loo </p></li><li><p>608 INHIBITION BY SURAMIN Vol.lO,No.4 </p><p>600 </p><p>i </p><p>1 FIG. 2 </p><p>Inhibition of fibrinolytic -25 activity of plasmin, </p><p>f generated from plasminogen </p><p>= by urokinase, by suramin </p><p>7 and trypan blue. PL, -50 p plasmin (10 pg/ml); S, </p><p>s plasmin with suramin e, -3 M). T, plasmin with $iipan biue (lo-3 M). </p><p>-75 Results expressed as mean f S.E.M. for four replicate assays. </p><p>J 100 ts tT </p><p>Fibrin is lysed by other proteinases in addition to plasmin. Since protein binding has been offered as one explanation of the action of suramin (15), the effects of suramin and trypan blue on fibrinolytic activities of other proteinases were examined (Fig. 3). In addition to plasmin, only collagenase (or a possible proteinase contaminant in the collagenase preparation) was inhibited by trypan blue, and suramin did not inhibit the activity of the collagenase preparation. Fibrinolysis by trypsin, chymo- trypsin, and pronase (Fig. 3), as well as by pepsin, elastase and pepsin (not shown) was not inhibited by either agent. In some cases, e.g. trypsin and chymotrypsin, suramin and trypan blue resulted in increased fibrinolysis, although neither agent produced nonspecific release of radioactive substrate in the assay </p><p>TRYPSIN CHYMoTlrfPSlN CaJdaMsE PNONASE </p><p>FIG. 3 Effects of suramin and trypan blue on fibrinolytic activities of plasmin and other proteinases, tested at eguimolar concentrations of enzyme (plasmin, 10 pg/mll. C, control values with enzyme </p><p>plus suramin (lo-3 M); T, enzyme plus trypan Activities represent means of duplicate assays. </p></li><li><p>Vol.lO,No.4 INHIBITION BY SUBAMIN 609 </p><p>DISCUSSION </p><p>In one study, suramin (5 x 10m4 M) failed to inhibit streptokinase-induced lysis of a standard fibrin clot by serum, a finding which was interpreted as failure to inhibit either activation of plasminogen or the activity of plasmin generated (16). However, suramin, at the same concentration, did inhibit the esterolytic activity of both streptokinase-activated serum and of partially ljurified plasmin, suggesting that the failure to inhibit in the fibrin clot assay might reflect the relative insensitivity of that method. The present observations, made with a radiometric assay at least ten times more sensitive than clot lysis methods (121, indicate that suramin does inhibit the proteolytic (fibrinolytic) activity of highly purified plasmin. </p><p>This inhibitory effect of suramin may be relevant to analysis of plasmin-complement interactions, particularly the physiological action of plasmin in both complement activation, and control. It is also pertinent to the observation that preparations of C3b inactivator may contain suramin-sensitive proteolytic activities distinct from the factor generally considered responsible for functional inactivation of C3b (17). There may be effects of suramin additional to its known protective effect on cell-bound C3b, the substrate for C3b inactivator (18). </p><p>Although it is said that suramin inhibits trypsin, in addition to other non-proteolytic enzymes (e.g. hexokinase, urease, succinic dehydrogenase), this statement is based on the observation that suramin at 10-5 to 10-4 M, inhibits the ability of trypsin, papain, rennin and pepsin to promote clotting of milk at pH 4.8 (19). In the present study, made at relatively physiological pH and salt concentration, trypsin was not inhibited by suramin. </p><p>Trypan blue, commonly employed as a reagent for determining cell viability, is teratogenic, an action attributed to inhibition of lysosomal enzymes (20). In addition, it inhibits the nonspecific cytotoxicity of activated macrophages for heterologous tumor cells, a phenomenon which has been explained on a similar basis (21). Since one known product of activated macrophages is a plasminogen activator (22), the present finding that trypan blue inhibits plasmin suggests a possible role for that enzyme in the macrophage cytotoxicity effect. </p><p>ACKNOWLEDGMENTS </p><p>We are indebted to Miss Eloisa Hervas and _Mr. King Hei Man for their expert technical assistance. This study was supported by grants from the Medical Research Council of Canada and the Canadian Arthritis Society. </p></li><li><p>610 INHIBITION BY SU-RAMIN Vol.lO,No.4 </p><p>REFERENCES </p><p>1. KAPLAN, A.P. and AUSTEN, K-F. The fibrinolytic pathway of human plasma: isolation and characterization of the plasminogen proactivator. J. Exptl. Med. 136,1378, 1972. _- </p><p>2. SHERRY, S. Fibrinolysis. Ann. Rev. Med. 19,247, 1968. </p><p>3. KAPLAN, A.P. and AUSTEN, K.F. A prealbumin activator of prekallikrein. II. Derivation of activators of prekallikrein from active Hageman factor by digestion with plasmin. J. Exptl. Med. 133, 696, 1971. </p><p>4. HABAL, F.M., BURROWES, C.E. and MOVAT, H.Z. Generation of kinin by plasma kallikrein and plasmin and the effect of al-antitrypsin and antithrombin III on the kininogenases. In: Kinins, Pharmacodynamics and Biological Roles. F. Sicuteri, N. Back and G.L. Haberland (Eds.), Plenum Press, New York, London, 1976. </p><p>5. RATNOFF, O.D. and NAFF, G.B. The conversion of C'ls to C'l esterase by plasmin and trypsin. J. Exptl. Med. 125, 337, 1967. </p><p>6. WARD, P.A. A plasmin-split fragment of C'3 as a new chemotactic factor. J. Exptl. Med. 126,189, 1967. </p><p>7. ARROYAVE, C.M. and MELLER-EBERHARD, H.J. Interactions between human C5, C6 and C7 and their functional significance in complement-dependent cytolysis. J. Immunol. 111, 536, 1973. </p><p>8. BRADE, V.A., NICHOLSON, A., BITTER-SUERMANN, D. and HADDING, U. Formation of the C3-cleaving properdin enzyme on zymosan. Demonstration that factor D is replaceable by proteolytic enzymes. J. Immunol. 113, 1735, 1974. </p><p>9. TAMURA, N. and NELSON, R.A., JR. Three naturally occurring inhibitors of components of complement in guinea pig and rabbit serum. J. Immunol. 99, 582, 1967. - </p><p>10. FONG, J.S.C. and GOOD, R.A. Suramin - a potent reversible and competitive inhibitor of complement systems. Clin. exp. Immunol._ 10, 127, 1973. </p><p>11. LACHMANN, P-J., ELIAS, D.E. and MOFFETT, A. Conglutinin and immunoconglutinins. In: Biological Activities of Complement. D.G. Ingram (Ed.), Basel, Karger, 1972, p. 204. </p><p>12. MOROZ, L.A. and GILMORE, N.J. A rapid and sensitive 1251-fibrin solid phase fibrinolytic assay for plasmin. Blood 46,543, 1975. </p><p>13. MOROZ, L.A. and GILMORE, N.J. Fibrinolytic activity in normal blood and plasma: Evidence for significant mechanisms independent of the plasminogen-plasmin system. Blood 48_, 531, 1976. </p></li><li><p>Vol.lO,No.4 INHIBITION BY SURAMIB 611 </p><p>14. </p><p>15. </p><p>16. </p><p>17. </p><p>18. </p><p>19. </p><p>20. </p><p>21. </p><p>22. </p><p>MOROZ, L.A. and GILMORE, N.J. Mechanisms involved in enhancement of fibrinolysis by chloroform. Blood 48, 777, - .w 1976. </p><p>TOWN, B.W., WILLS, E-D., WILSON, E.J. and WORMALL, A. Studies on suramin. 8. The action of the drug on enzymes and some other proteins. General considerations. Biochem. J. 47, 149, 1950. </p><p>EISEN, V. and LCVEDAY, C. Effects of suramin on complement, blood clotting, fibrinolysis and kinin formation. Brit. J. Pharmacol. 49, 678, 1973. </p><p>GITLIN, J.D., ROSEN, F.S. and LACHMANN, P.J. The mechanism of action of the C3b inactivator (conglutinogen-activating factor) on its naturally occurring substrate, the major fragment of the"third component of complement (C3b). J. Exptl. Med._ 141, 1221, 1975. </p><p>LACHMANN, P.J., NICOL, P. and ASTON, W.P. Further studies on the C3b inactivator or conglutinogen activating factor (KAF). Immunochemistry 10, 695, 1973. </p><p>TOWN, B.W., WILLS, E.D., WILSON, E.J. and WORMALL, A. Studies on suramin. 9. The action of the drug on some enzymes. Biochem. J 47 158, 1950. &amp; -' </p><p>BECK, F., LLOYD, J.B. and GRIFFITHS, A. Lysosomal enzyme inhibition by trypan blue: a theory of teratogenesis. Science 157, 1180, 1967. </p><p>HIBBS, J.B., JR. Heterocytolysis by macrophages activated by bacillus Calmette-Guerin: lysosome exocytosis into tumor cells. Science 184:468, 1974. </p><p>DNKELESS, J.C., GORDON, S. and REICH, E. Secretion of plasminogen activator by stimulated macrophages. J. Exptl. Med. 139;834, 1974. _- </p></li></ul>

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