inhibition of fibrinolytic activity of plasmin by suramin (antrypol) and trypan blue
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THROMBOSIS RESEARCH Volume 10, Pages 605-611. Pergamon Press, 1977. Printed in Gt. Britain.
INHIBITION OF FIBRINOLYTIC ACTIVITY OF PLASMIN By sum~m (ANTRYPOL) AND TRYPAN BLUE
LEONARD A. MOROZ The Harry Webster Thorp Laboratories, Division of Clinical Immunology, McGill University Clinic, Royal Victoria Hospital, 687 Pine Avenue West,
Montreal, Quebec, Canada H3A 1Al
(Received 7.1.1977; in revised form 22.2.1977. Accepted by Editor H.L. Nossel)
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).
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.
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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).
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.
MATERIALS AND METHODS
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.
Results are expressed as ng Assay tubes were coated with. approximately
1.5 ug 1251-fibrin with specific activity of 25,000 to 28,000 counts/min/ug fibrin.
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).
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,
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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.
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.
25 FIG. 1 Inhibition of fibrinolytic
f activity of spontaneously 2 active human plasmin c! (lOpg/ml) by suramin
5og (closed circles) and by etrypan blue (open circles). Points represent means of duplicate assays.
Ts Horizontal interrupted line and hatched area, mean f S.E.M. (four replicate determinations) for plasmin alone.
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1 FIG. 2
Inhibition of fibrinolytic -25 activity of plasmin,
f generated from plasminogen
= by urokinase, by suramin
7 and trypan blue. PL, -50 p plasmin (10 pg/ml); S,
s plasmin with suramin e, -3 M). T, plasmin with $iipan biue (lo-3 M).
-75 Results expressed as mean f S.E.M. for four replicate assays.
J 100 ts tT
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
TRYPSIN CHYMoTlrfPSlN CaJdaMsE PNONASE
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
plus suramin (lo-3 M); T, enzyme plus trypan Activities represent means of duplicate assays.
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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.
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).
Although it is said that suramin inhibits trypsin, in additio