THROMBOSIS RESEARCH 60; 9-l 8,199O 0049-3848/90 $3.00 + .OO Printed in the USA. Copyright (c) 1990 Pergamon Press pk. All rights reserved.
ANTICOAGULANT ACTIVITY OF A BACTERIAL GLYCOPEPTIDE
Akoum*, A., Josefonvicz**, J. and Sigot*, M. * Laboratoire de Biologie Cellulaire Experimentale, Universite de Technologie de Compiegne BP 233,60206 Compiegne, France. **Laboratoire de Recherches sur les Macromolecules, CNRS, UA 502, Universite Paris-Nord, Avenue J.B. Clement, 93430 Villetaneuse, France. (Received
23.11.1988;
accepted
in revised form 57.1990
by Editor Y. Sultan)
ABSTRACT The anticoagulant properties of myxalin, a glycopeptide secreted by a Gram negative bacterium strain (Mvxococcus xanthus) are studied and compared to those of heparin. This soluble material exhibits an anticoagulant activity which implies the inhibition of some serine proteases, thrombin and factor Xa. In the presence of normal and antithrombin III-depleted plasma, myxalin inhibits the amidolytic activity of thrombin on synthetic chromogenic substrate as a function of its concentration, but fails to increase thrombin inactivation significantly in the presense of purified AT III. However, crossed immunoelectrophoresis data suggests that its antithrombic effect is mainly mediated by binding to the enzyme, rather than to AT III and probably differs from the catalytic activity of heparin which requires the presense of AT III. The anticoagulant process occurs without degradation of fibrinogen and can be neutralized by protamine.
INTRODUCTION It has been reported that certain bacteria (StaDhvlococcus enidermidis) produce a slime possessing anticoagulant properties (1). Other semi-synthetic substances, such as that prepared from a sulphated polysaccharide found in the culture medium of an arthrobacter strain, also possess an anticoagulant effect (2). Mvxococcus xanthus, a Gram-negative non pathogenic bacterium growing in the environment and having the ability to undergo a morphogenetic multicellular development upon nutrient starvation, produces numerous extracellular molecules (3). A glycopeptide with blood anticoagulant activity has recently been found in the culture medium of growing cells of M. xanthus (4). This glycopeptide called “Myxalin” has been isolated and purified on small and large scales (5). The present paper describes the anticoagulant properties of myxalin compared to heparin, and its interactions with AT III and thrombin by crossed immunoelectrophoresis. Key words : Glycopeptide - bacteria - anticoagulant - thrombin - factor Xa
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Materials Myxalin was purified from the culture supematant of Mvxococcus xanthus strain CM01 1 and characterized as described previously (4). The sulphate content was determined by the method described by Dodgson (6). The principal characteristics of the sample of myxalin used in this study are reported in Table 1. TABLE 1 Some Characteristics of Myxalin sugars
%ofd.w.
Aminoacids %ofd.w.
Sulphate Content %ofd.w.
70
2-3
27
Molecular Weight 6300
Isoelectric Point 3.6
Hog intestine heparin (H 108) with specific anticoagulant activity of 173 international units per mg (IU/mg) was obtained from Institut Choay, Paris, France, It was diluted in distilled water at 10 IU/ml and kept at 4°C. Human platelet-poor plasma (PPP) stored at -8O’C, was thawed and kept at 37’C before use. Human thrombin (1094 NIH U./mg-CNTS, France) was diluted in Michaelis buffer and kept at 4’C. Reptilase (Stago, France) was diluted in distilled water and kept at 37’C. A cephalin and kaolin activator solution (Stago, France) was reconstituted according to the Stago instruction manual and kept at 4’C. Neoplastin (Stago) was dissolved in distilled water and incubated at 37’C before use. Factor Xa, AT III and plasma substrate (Hepaclot ; Stago, France) were dissolved in adequate volumes of distilled water and incubated at 4°C. Human antithrombin III free fibrinogen (CRTS, Lille, France) solution was prepared at 4g/l in 0.15 M NaCl. Protamine chlorhydrate solution (Kabi Vitrum, France) was diluted in Michaelis buffer at different concentrations and stored at 4’C. Reagents for amidolytic tests were obtained from Kabi-Vitrum, France. Anti-human AT III rabbit serum (Assera III) and anti-human prothrombin rabbit serum (Assera II) were obtained from Stago, France. Clotting methods Measurements of Activated Partial Thromboplastin Time (APIT), were carried out in PPP (0.1 ml) using a Cephalin-Kaolin suspension (0.1 ml) and 0.025 CaC12 (0.1 ml) to initiate coagulation according to the conditions recommended by the manufacturer. The sample was assayed at different dilutions. In order to measure the Prothrombin Time (PT), 0.2 ml of thromboplastin (Neoplastin) was added to 0.1 ml of PPP in the presence of heparin or myxalin (0.1 ml) at different concentrations, and the clotting time was recorded. Thrombin Time (TT) was measured using bovine thrombin (56.4 NIH U/mg - Hoffman La Roche, Basel, Switzerland) by adding 0.1 ml thrombin solution (20 NIH U./ml in assay) as previously described (7) to either 0.2 ml PPP or 0.2 ml human fibrinogen solution (4 g/l). Thrombin time on fibrinogen was also performed by adding a protamine hydrochloride solution ii.; n$ at different concentrations to the solutions of fibrinogen (0.1 ml) and heparin or myxalin
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Reptilase clotting time was likewise measured on fibrinogen. Anti-Xa activity was measured by a clotting assay using the Hepaclot kit from Diagnostica Stago, France. Clotting times were recorded automatically at 37°C by a Dade KC1 Coagulometer. All the assays were performed in triplicate. Before each test, the sample was dissolved in Michaelis buffer solution (Sodium chloride 100 mM, Sodium acetate 25 mM, Sodium diethyl-malonylurea 25 mM, hydrochloric acid, pH 7.35). Amidolvtic methods The thrombin inactivation assay was undertaken by mixing a 100 ~1 solution of myxalin or heparin at different concentrations, with 200 l.~lof tris-EDTA buffer and 100 ~1 of normal plasma diluted 10 times, AT III (0.1 III/ml) or AT III-depleted plasma diluted 1 in 10. After incubating at 37 “C for 5 min, 100 ~1 of thrombin solution (0.7 U/ml) were added. 5 min later, 100 l.tl of the synthetic substrate solution (S-2238, 0.75 pmole/ml) were added to the above mixture and amidolysis was stopped after 3 min by the addition of 250 l.tl of 50% acetic acid. The absorbance was measured at 405 nm. Activity was expressed by the following equation : Thrombin inhibition (%) = (OD control - OD samole) 100. OD control In the control mixture 100 l.~lof the tris-EDTA buffer were used instead of the heparin or myxalin solution. Each assay was carried out in triplicate. Antithrombin III (AT III) depleted plasma was prepared from human plasma as described by Barrowcliffe et al (8). This plasma contained no residual immunologically-detectable antithrombin III (less than 1%).
Crossed immunoelectrophoresis of AT III was carried out using the method described by Barrowcliffe (9). Heparin was incorporated in the gel at a concentration of 0.1 mg/ml while myxalin was used at 5 mg/ml in the gel. Purified AT III was diluted to 1 IU/ml and a 10 ~1 sample was applied to the slide. Crossed immunoelectrophoresis of thrombin was carried out using the same method as reported above except that a 12 ~1 sample was applied to the slide. It contained 4 l.~lof thrombin (500 U/ml) either with 8 ~1 of Verona1 tris-glycine buffer (pH 8.8) or with myxalin (25 mg/ml) or heparin (12.5mg/ml) in the same buffer solution. RESULTS Anticoagulant uronertv of mvxalin Table 2 summarizes the results obtained with heparin and myxalin at low and high concentrations. To observe a significant anticoagulant activity, a lo-fold higher concentration of myxalin was used compared to the concentration of heparin. The APTT, which explores the intrinsic pathway of blood coagulation, was increased with heparin and with the higher concentration of myxalin. In contrast, the PT, which explores the extrinsic pathway, was not modified. At the higher myxalin concentration, the thrombin time was increased when measured both on normal plasma and fibrinogen i.e., in the presence and absence of antithrombin III respectively. When the test was performed at the lower myxalin concentration (5 pg/ml), no effect was observed. An equivalent result was observed with heparin at its lower concentration of 0.58 pgjml.
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It was shown that the PPP reptilase clotting time was not increased in the presence of myxalin. As a result, the increase in thrombin clotting time is not to be attributed to some fibrinogen degradation. TABLE 2 Effect of Myjtalinon CoagulationTests Myxalin Concentrations @g/ml)
5
Heparin 50
0.58
Activated Partial Thromboplastin Time (APTT) (s)
65.0 f 3.4
93.5 f 4.1
Prothrombin Time (PT) (s)
16.6 f 0.4
16.8 f 0.2
Thrombin Time (TT) (s)
5.3 f 0.3
Thrombin Time measured on Fibrinogen (s)
13.0 It 1.0
Reptilase Clotting Time (s)
ND
Michaelis buffer 5.8
440.0 f 8.0
67.5 f 4.3
16.5 f 0.5
16.6 f 0.3
16.7 f 0.2
12.3 f 0.5
6.5 k 0.6
16.3 f 0.4
5.3 f 0.2
101 5 6
13.7 f 0.7
22.9 f 0.4
12.5 It 0.5
58.0 + 1.7
58.4 f 2.9
56.0 f 3.3
95.3 f 2.1
ND
N.D. : Not determined Values are given as mean f SD
Antithrombin activitv of mvxalin and effect of protamine, The antithrombin activity of myxalin compared to that of heparin was first determined by measuring the thrombin clotting times of PPP in the presence of increasing amounts of these two anticoagulants. According to the procedure described by Mauzac et al. (lo), the specific anticoagulant activity of myxalin and of heparin “a” is expressed as the number of NIH units of thrombin inactivated by one milligram of compound. The quantity of inactivated units of thrombin are determined from a
CONCENTRATION
(&ml)
FIG. 1 Inhibition of thrombin activity by myxalin (0) and heparin (A) at various concentrations in the presence of platelet poor plasma. The curves permit comparison of the level of thrombin inactivation by the two anticoagulant compounds. Values are expressed as mean f SD.
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PROPERTIES OF MYXALIN
13
calibration curve. The relation between the quantity of inactivated units of thrombin and the concentration of the anticoagulant compound is reported in Figure 1. From these curves we have determined that 1 mg of myxalin inactivates 280 NIH U of thrombin. Under the same conditions, heparin exhibits an anticoagulant activity of 3,100 NIH U of thrombin per mg of mucopolysaccharide, i. e. about eleven times more. In order to check the degree of AT III dependence of myxalin antithrombic activity, we studied its effect on the amidolytic activity of thrombin in the presence of normal titrated plasma, AT III depleted plasma and purified human AT III. As shown in Figure 2, myxalin inhibited the thrombin amidolytic activity as a function of its concentration. Plasma was a necessary cofactor of the myxalin activity. In the presence of purified AT III, only low antithrombic activity of myxalin was observed. However, myxalin was distinctly less AT III-dependent than that of heparin assayed under the same conditions.
0
MY XALIN
II 10 (&ml)
2 CON&NT&ION
FIG. 2 Inhibition of the thrombin amidolytic activity by heparin (a) and myxalin (b) in the presence of normal plasma (o), AT III-depleted plasma (I) and purified AT III (4). Values are expressed as mean + SD. TABLE 3 Effect of Protamine on the Thrombin Clotting Time on Fibrinogen in the Presence of MyxaIin and Heparin Thrombin Clotting Time on Fibrinogen in the Presence of Protamine Concentration in the Assay
_________________________________________-_------_____________________..__________.-__---.
WmU Myxalin w
0 16 32 40 53 160 200
33.9 * 0.4 33.0 + 0.3 29.8 + 0.5 27.0 f 0.5 25.1 + 0.3 18.7 + 0.6 13.2 + 0.4
Heparin (s)
39.4 f 0.2 38.7 It 0.3 33.4 * 0.3 21.5 f 0.5 15.4 It 0.8 11.0 ?I 0.6 10.3 f 0.3
Values are given as mean + SD., Michaelis buffer : 10.5 (s). Myxalin and heparin are used at concentrations of 50 l.@nI and 20 pg/ml respectively.
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ANTICOAGULANT PROPERTIES OF MYXALIN
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In order to evaluate the effect of a possible inhibitor of myxalin’s anticoagulant activity, thrombin clotting times were measured in the presence of a range of concentrations of protamine, an inhibitor of heparin. As shown in Table 3, the thrombin clotting time for myxalin was slightly normalized and like it appears to be sensitive to a protamine inhibitory effect.
hepain,
AT III
Thrombin
+ Myxalin
+ Myxalin
+ Heparin
+ Heparin
FIG. 3 Crossed immunoelectrophoresis ( 1st dimension left to right). AT III, plus gel concentration of either 5 mg/ml myxalin or 0.1 mg/ml heparin. Thrombin, 4 l.~lof thrombin (500 U/ml) mixed wih either 8 l.tlof myxalin (25 mg/rnl), or heparin (12.5 mg/rnl).
The results of crossed immunoelectrophoresis with AT III and thrombin are shown in Figure 3. Compared to heparin, myxalin did not show an appreciable shift in the peak when it was incorporated in the gel at a concentration of 5 mg/ml, i. e. 50 times the heparin concentration. This indicates that there is no significant binding between myxalin and AT III. However, when mixed with thrombin at a concentration of 25 mg/ml, myxalin did cause an appreciable shift of the peak, producing a similar effect to heparin when used at a high concentration (12.5 mtr/ml).
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Anti FXa activitv of mvxalin The Anti-FXa activity of myxalin compared to heparin is shown in Figure 4. This activity was measured in the presence of normal plasma and purified AT III.
10'
0
30
CONd&TRATIO&~ml) FIG. 4 of factor Xa activity by myxalin (0) and heparin (A) at different concentrations using Hepaclot reagents. The residual activity of factor Xa is measured by the clotting time of blood plasma in the presence of purified AT III. Inhibition
Myxalin had no effect on factor Xa activity at concentrations up to 2 pg/ml. At higher concentrations inhibition of human factor Xa was observed; 1 l.tg/ml having a similar effect to 0.07 ltg/rnl of heparin. About 14 times more myxalin than heparin was required for equivalent inhibition of human factor Xa using the particular conditions of this study.
DISCUSSION We have confirmed that myxalin has an anticoagulant effect on blood by demonstrating that it increases activated partial thromboplastin time and thrombin time, and inhibits thrombin amidolytic activity, and factor Xa activity. When compared to heparin however, myxalin is a weaker anticoagulant in vitro. At low concentrations (< 2 pg/ml), myxalin causes no change in factor Xa activity. At higher concentrations (-7 l.tg/ml) it begins to inactivate thrombin whether associated with platelet-poor plasma or fibrinogen, Moreover, this thrombin inhibition is lower in plasma than in AT III-free fibrinogen solution. This result seems to indicate a direct inhibition of thrombin by myxalin. Even so, the presence of some plasma component(s) which might limit the inhibiting effect of myxalin is still one possible explanation. “Non specific” binding with some plasma protein(s) has already been described as a probable factor affecting the anticoagulant property of pentosan polysulphate in plasma in vitro (12). However, the amidolytic tests suggest that the inhibition of thrombin by myxalin occurs mainly through an AT III-independent pathway. This explains our observations of high antithrombic activity of myxalin when exposed to AT III-depleted plasma. Moreover, the results illustrated in Figure 2 suggest that the effect of myxalin on thrombin inhibition when combined with AT III is not significant compared to that of heparin. It is well known that heparin possess a high degree of structural specificity which enables it to bind with high affinity to AT III (12). This AT III-heparin complex so formed rapidly neutralizes several clotting serine proteases
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PROPERTIES OF MYXALIN
(13). The results of our crossed immunoelectrophoresis studies show no evidence of significant binding between myxalin and AT III, even at high concentrations. In contrast, evidence to support significant binding between myxalin and thrombin has been found.
Like heparin and other glycosaminoglycans, the anticoagulant activity of myxalin can be neutralized by protamine. This phenomenon is probably due to the formation of a complex between this polycation and negative charges on the heparin and myxalin molecules. The results in Table 3 suggest that myxalin is less sensitive than heparin to protamine. Recent in vivo experiments using Wessler’s thrombosis model in rabbits (unpublished results), have also demonstrated that myxalin when used at 200 pg/Kg i. v. bolus gives a similar antithrombotic effect to that of heparin at 66 pg/Kg i. v. bolus. The anticoagulant property of myxalin probably depends upon its chemical composition. We have already shown that its sugar moiety is rich in glutamic acid (4). Sulphate groups are also present in various proportions (2-3 % of the dry weight). The role of the carboxylic and sulphate functional groups has been shown to be essential in the mechanism of heparin anticoagulant activity (14). It has also been reported that natural or synthetic materials substituted with carboxylic groups and other functional groups, such as sulphates and sulphonates, exhibit anticoagulant activity (15). Myxalin contains carboxylic groups from glutamic acid, the major aminoacid of the molecule (22-25 % of the dry weight) as well as sulphate groups. their combined presence can therefore account for its anticoagulant behaviour. Further investigations will be necessary to improve our understanding of the mechanism of antithrombotic activity, as well as the structure/function relationship of the myxlin molecule. Sulphate polysaccharides are widely distributed in higher organisms. However, only few papers have reported their presence in microorganisms (16, 17, 18). Recently, two anticoagulant agents have been isolated. The first was found in the culture medium of Arthrobacter species and has fibrinolytic activity (19). The second was isolated from the slime produced by Stauhvlococcus euidermidis (1). Myxalin differs from both these two anticoagulant compounds in terms of its composition. A number of sulphated polysaccharides have been reported to exhibit anticoagulant activity. One of these is pentosan polysulphate. This heparinoid has a low anticoagulant effect compared to heparin. At low concentrations, it potentiates inhibition of thrombin and factor Xa in the presence of purified AT III. However, these effects are mediated by binding to the enzymes, rather than to AT III (11). Our results suggest that myxalin antithrombin activity is due mainly to direct binding with thrombin itself. The action of myxalin towards HC II (Heparin Cofactor II) requires further investigation. This protease inhibitor has been found to contribute to the anticoagulant effect of heparin particularly at high concentrations (20, 21). HC II has also been reported to be the main cofactor of anticoagulant activity for some sulphated glycosaminoglycans such as dermatan sulphate (22,23) and pentosan sulphate (24). ACKNOWLEDGMENT
Special nada)
thanks
for his
to
helpful
Professor
M.W. King
(University
of
Laval,
Ca-
collaboration.
REFERENCES 1.
BYKOWSKA, K., LUDWICKA, A., WEGRZYNOVICZ, Z., LOPACIUK, S. and KOPEE, M. Anticoagulant properties of extracellular slime substance produced by Staphylococcus epidermidis. Thromb. and Haemost. 54.853-856, 1985.
2.
KUMADA, T., INOUE, K., KORENAGA, H. and ABIKO, Y. Antithrombic and thrombolytic effects of galactan polysulfate (DH 6322) in rats. Thromb. Res. 39, 21-28, 1985.
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3.
KAISER, D., MANOIL, C. and DWORKIN, M. Myxobacteria : cell interactions, genetics and development. Ann. Rev. Microbial, 33.595639, 1979.
4.
AKOUM, A., VIJAYALAKSHMI, M. A., CARDON, P., FOURNET,B., SIGOT, Iv!. and GUESPIN-MICHEL, J. F. Myxococcus xanthus produces an eXtraCe!!U!ar glycopeptide that displays blood anticoagulant properties. Enz, and Microb. Technol. 2, 426-429, 1987.
5.
AKOUM, A., VIJAYALAKSHMI, M. A. and SIGOT, M. Scale-up of “myxalin” purification by a pseudoaffinity method using a radial flow column. Chromatomaphia 28, 157-160, 1989.
6.
DODGSON, K. S. Determination of inorganic sulphate in studies on the enzymatic and non-enzymatic hydrolysis of carbohydrate and other sulphate esters. Biochem. J, z, 312-319, 1961.
7.
MAUZAC, M., AUBERT, N. and JOSEFONVICZ, J. Antithrombic activity of some polysaccharide resins. Biomaterials 3,221-224, 1982.
8.
BARROWCLIFFE, T. W. and EGGLETON, C. A. Studies of anti-Xa activity in human plasma. I : Comparison of a fast-acting inhibitor with antithrombin Ill. Thromb. Res. 27, 399-411, 1981.
9.
BARROWCLIFFE, T. W. Studies of heparin binding to antithrombin Ill by crossed immunoelectrophoresis. Thromb. Haemostas. 42, 1434-1445, 1980
10.
MAUZAC, M. and JOSEFONVICZ, J. Anticoagulant activity of dextran derivatives. Part I : synthesis and characterization. Biomaterials 5, 301-304, 1984.
11.
FISCHER, A. M., BARROWCLIFFE, T. W. and THOMAS, D. P. A comparison of pentosan polysulphate (SP54) and heparin I : Mechanism of action on blood coagulation. Thromb. Haemostas, (Stuttgart) 47, 104108, 1982.
12.
LINDAHL, U., BACKSTROM, G., HOOK, M., THUNBERG, L., FRANSSON, L-A. and LINKER, A. Structure of the antithrombin Ill binding site in heparin. Proc. Nat, Acad, Sci, USA 76, 3198-3202, 1979.
13.
ROSENBERG, R. D. and DAMUS, P. S. The purification and mechanism of action of antithrombin-heparin cofactor. J. Biol. Chem . 248, 6490-6505, 1973.
14.
KAZATCHKINE, M. D., FEARON, D. T., METCALFE, D. D., ROSENBERG, R. D. and AUSTEN, K. F. Structural determinants of the capacity of heparin to inhibit the formation of the human amplification C3 convertase. J. Clin. Invest. 67,223-228, 1981.
15.
JOZEFOWICZ, M. and JOSEFONVICZ, J. Antithrombogenic polymers. Pure and ADO!. Chem. 56, 1335-1344, 1984.
16.
KONCEVICZ, M. The presence of sulphated polysaccharide Halobacterium halobium. Biochem. J. 130,4Op-41p, 1972.
17.
DARBY, G. K., JONES, A. S., KENNEDY, J. F. and WALKER, R. T. Isolation and analysis of the nucleic acids and polysaccharides from Clostridium welchii.. J. Bacterial. 103, 159-165,197O.
18.
STEBER, J. and SCHLEFER, K. H. Halococcus moorhuae : A sulfated heteropolysaccharide as the structural component of the bacterial cell wall. Arch. Microbio!. 105, 173-177, 1975.
in the cell envelopes of
18
19.
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PROPERTIES OF MYXALIN
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KUMADA, T. and ABIKO, Y. Fibrinolytic action of a new semi-synthetic polysaccharide sulfate, Galactan polysulfate (DH6322), in the rat. Thromb. Res. 39, 9-19, 1985.
20.
TOLLEFSEN, D. M. and BLANK, M. K. Detection of a new heparin-dependent inhibitor of thrombin in human plasma. J. Clin, Invest. 68,589-596, 198 1.
21.
TOLLEFSEN, D. M., MAJERUS, D. W. and BLANK, M. K. Heparin cofactor II. Purification and properties of heparin-dependent inhibitor of thrombin in human plasma. ,I. Biol. Chem, 257, 2162-69, 1982.
22.
BRIGINSHAW, G. F. and SHANBERGE, J .N. Identification of two distinct cofactors in human plasma-separation and partial purification. Arch, Biochem. Bioohvs. 161, 683-690, 1974.
23.
TOLLEFSEN, D. M., PESTKA, C. A. and MONAFO, W. J. Activation of heparin cofactor II by dermatan sulfate. J. Biol. Chem. 25&,6713-16, 1983.
24.
SCULLY, M. F. and KAKKAR, V. V. Identification of heparin cofactor II as the prirciIma1glsma cofactor for the antithrombin activity of pentosan polysulphate (SP 54). o b. s, 36, 187-194, 1984.
ANTICOAGULANT ACTIVITY OF A BACTERIAL GLYCOPEPTIDE
Akoum*, A., Josefonvicz**, J. and Sigot*, M. * Laboratoire de Biologie Cellulaire Experimentale, Universite de Technologie de Compiegne BP 233,60206 Compiegne, France. **Laboratoire de Recherches sur les Macromolecules, CNRS, UA 502, Universite Paris-Nord, Avenue J.B. Clement, 93430 Villetaneuse, France. (Received
23.11.1988;
accepted
in revised form 57.1990
by Editor Y. Sultan)
ABSTRACT The anticoagulant properties of myxalin, a glycopeptide secreted by a Gram negative bacterium strain (Mvxococcus xanthus) are studied and compared to those of heparin. This soluble material exhibits an anticoagulant activity which implies the inhibition of some serine proteases, thrombin and factor Xa. In the presence of normal and antithrombin III-depleted plasma, myxalin inhibits the amidolytic activity of thrombin on synthetic chromogenic substrate as a function of its concentration, but fails to increase thrombin inactivation significantly in the presense of purified AT III. However, crossed immunoelectrophoresis data suggests that its antithrombic effect is mainly mediated by binding to the enzyme, rather than to AT III and probably differs from the catalytic activity of heparin which requires the presense of AT III. The anticoagulant process occurs without degradation of fibrinogen and can be neutralized by protamine.
INTRODUCTION It has been reported that certain bacteria (StaDhvlococcus enidermidis) produce a slime possessing anticoagulant properties (1). Other semi-synthetic substances, such as that prepared from a sulphated polysaccharide found in the culture medium of an arthrobacter strain, also possess an anticoagulant effect (2). Mvxococcus xanthus, a Gram-negative non pathogenic bacterium growing in the environment and having the ability to undergo a morphogenetic multicellular development upon nutrient starvation, produces numerous extracellular molecules (3). A glycopeptide with blood anticoagulant activity has recently been found in the culture medium of growing cells of M. xanthus (4). This glycopeptide called “Myxalin” has been isolated and purified on small and large scales (5). The present paper describes the anticoagulant properties of myxalin compared to heparin, and its interactions with AT III and thrombin by crossed immunoelectrophoresis. Key words : Glycopeptide - bacteria - anticoagulant - thrombin - factor Xa
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Materials Myxalin was purified from the culture supematant of Mvxococcus xanthus strain CM01 1 and characterized as described previously (4). The sulphate content was determined by the method described by Dodgson (6). The principal characteristics of the sample of myxalin used in this study are reported in Table 1. TABLE 1 Some Characteristics of Myxalin sugars
%ofd.w.
Aminoacids %ofd.w.
Sulphate Content %ofd.w.
70
2-3
27
Molecular Weight 6300
Isoelectric Point 3.6
Hog intestine heparin (H 108) with specific anticoagulant activity of 173 international units per mg (IU/mg) was obtained from Institut Choay, Paris, France, It was diluted in distilled water at 10 IU/ml and kept at 4°C. Human platelet-poor plasma (PPP) stored at -8O’C, was thawed and kept at 37’C before use. Human thrombin (1094 NIH U./mg-CNTS, France) was diluted in Michaelis buffer and kept at 4’C. Reptilase (Stago, France) was diluted in distilled water and kept at 37’C. A cephalin and kaolin activator solution (Stago, France) was reconstituted according to the Stago instruction manual and kept at 4’C. Neoplastin (Stago) was dissolved in distilled water and incubated at 37’C before use. Factor Xa, AT III and plasma substrate (Hepaclot ; Stago, France) were dissolved in adequate volumes of distilled water and incubated at 4°C. Human antithrombin III free fibrinogen (CRTS, Lille, France) solution was prepared at 4g/l in 0.15 M NaCl. Protamine chlorhydrate solution (Kabi Vitrum, France) was diluted in Michaelis buffer at different concentrations and stored at 4’C. Reagents for amidolytic tests were obtained from Kabi-Vitrum, France. Anti-human AT III rabbit serum (Assera III) and anti-human prothrombin rabbit serum (Assera II) were obtained from Stago, France. Clotting methods Measurements of Activated Partial Thromboplastin Time (APIT), were carried out in PPP (0.1 ml) using a Cephalin-Kaolin suspension (0.1 ml) and 0.025 CaC12 (0.1 ml) to initiate coagulation according to the conditions recommended by the manufacturer. The sample was assayed at different dilutions. In order to measure the Prothrombin Time (PT), 0.2 ml of thromboplastin (Neoplastin) was added to 0.1 ml of PPP in the presence of heparin or myxalin (0.1 ml) at different concentrations, and the clotting time was recorded. Thrombin Time (TT) was measured using bovine thrombin (56.4 NIH U/mg - Hoffman La Roche, Basel, Switzerland) by adding 0.1 ml thrombin solution (20 NIH U./ml in assay) as previously described (7) to either 0.2 ml PPP or 0.2 ml human fibrinogen solution (4 g/l). Thrombin time on fibrinogen was also performed by adding a protamine hydrochloride solution ii.; n$ at different concentrations to the solutions of fibrinogen (0.1 ml) and heparin or myxalin
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PROPERTIES OF MYXALIN
11
Reptilase clotting time was likewise measured on fibrinogen. Anti-Xa activity was measured by a clotting assay using the Hepaclot kit from Diagnostica Stago, France. Clotting times were recorded automatically at 37°C by a Dade KC1 Coagulometer. All the assays were performed in triplicate. Before each test, the sample was dissolved in Michaelis buffer solution (Sodium chloride 100 mM, Sodium acetate 25 mM, Sodium diethyl-malonylurea 25 mM, hydrochloric acid, pH 7.35). Amidolvtic methods The thrombin inactivation assay was undertaken by mixing a 100 ~1 solution of myxalin or heparin at different concentrations, with 200 l.~lof tris-EDTA buffer and 100 ~1 of normal plasma diluted 10 times, AT III (0.1 III/ml) or AT III-depleted plasma diluted 1 in 10. After incubating at 37 “C for 5 min, 100 ~1 of thrombin solution (0.7 U/ml) were added. 5 min later, 100 l.tl of the synthetic substrate solution (S-2238, 0.75 pmole/ml) were added to the above mixture and amidolysis was stopped after 3 min by the addition of 250 l.tl of 50% acetic acid. The absorbance was measured at 405 nm. Activity was expressed by the following equation : Thrombin inhibition (%) = (OD control - OD samole) 100. OD control In the control mixture 100 l.~lof the tris-EDTA buffer were used instead of the heparin or myxalin solution. Each assay was carried out in triplicate. Antithrombin III (AT III) depleted plasma was prepared from human plasma as described by Barrowcliffe et al (8). This plasma contained no residual immunologically-detectable antithrombin III (less than 1%).
Crossed immunoelectrophoresis of AT III was carried out using the method described by Barrowcliffe (9). Heparin was incorporated in the gel at a concentration of 0.1 mg/ml while myxalin was used at 5 mg/ml in the gel. Purified AT III was diluted to 1 IU/ml and a 10 ~1 sample was applied to the slide. Crossed immunoelectrophoresis of thrombin was carried out using the same method as reported above except that a 12 ~1 sample was applied to the slide. It contained 4 l.~lof thrombin (500 U/ml) either with 8 ~1 of Verona1 tris-glycine buffer (pH 8.8) or with myxalin (25 mg/ml) or heparin (12.5mg/ml) in the same buffer solution. RESULTS Anticoagulant uronertv of mvxalin Table 2 summarizes the results obtained with heparin and myxalin at low and high concentrations. To observe a significant anticoagulant activity, a lo-fold higher concentration of myxalin was used compared to the concentration of heparin. The APTT, which explores the intrinsic pathway of blood coagulation, was increased with heparin and with the higher concentration of myxalin. In contrast, the PT, which explores the extrinsic pathway, was not modified. At the higher myxalin concentration, the thrombin time was increased when measured both on normal plasma and fibrinogen i.e., in the presence and absence of antithrombin III respectively. When the test was performed at the lower myxalin concentration (5 pg/ml), no effect was observed. An equivalent result was observed with heparin at its lower concentration of 0.58 pgjml.
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ANTICOAGULANT
PROPERTIES OF MYXALIN
Vol. 60, No. 1
It was shown that the PPP reptilase clotting time was not increased in the presence of myxalin. As a result, the increase in thrombin clotting time is not to be attributed to some fibrinogen degradation. TABLE 2 Effect of Myjtalinon CoagulationTests Myxalin Concentrations @g/ml)
5
Heparin 50
0.58
Activated Partial Thromboplastin Time (APTT) (s)
65.0 f 3.4
93.5 f 4.1
Prothrombin Time (PT) (s)
16.6 f 0.4
16.8 f 0.2
Thrombin Time (TT) (s)
5.3 f 0.3
Thrombin Time measured on Fibrinogen (s)
13.0 It 1.0
Reptilase Clotting Time (s)
ND
Michaelis buffer 5.8
440.0 f 8.0
67.5 f 4.3
16.5 f 0.5
16.6 f 0.3
16.7 f 0.2
12.3 f 0.5
6.5 k 0.6
16.3 f 0.4
5.3 f 0.2
101 5 6
13.7 f 0.7
22.9 f 0.4
12.5 It 0.5
58.0 + 1.7
58.4 f 2.9
56.0 f 3.3
95.3 f 2.1
ND
N.D. : Not determined Values are given as mean f SD
Antithrombin activitv of mvxalin and effect of protamine, The antithrombin activity of myxalin compared to that of heparin was first determined by measuring the thrombin clotting times of PPP in the presence of increasing amounts of these two anticoagulants. According to the procedure described by Mauzac et al. (lo), the specific anticoagulant activity of myxalin and of heparin “a” is expressed as the number of NIH units of thrombin inactivated by one milligram of compound. The quantity of inactivated units of thrombin are determined from a
CONCENTRATION
(&ml)
FIG. 1 Inhibition of thrombin activity by myxalin (0) and heparin (A) at various concentrations in the presence of platelet poor plasma. The curves permit comparison of the level of thrombin inactivation by the two anticoagulant compounds. Values are expressed as mean f SD.
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ANTICOAGULANT
PROPERTIES OF MYXALIN
13
calibration curve. The relation between the quantity of inactivated units of thrombin and the concentration of the anticoagulant compound is reported in Figure 1. From these curves we have determined that 1 mg of myxalin inactivates 280 NIH U of thrombin. Under the same conditions, heparin exhibits an anticoagulant activity of 3,100 NIH U of thrombin per mg of mucopolysaccharide, i. e. about eleven times more. In order to check the degree of AT III dependence of myxalin antithrombic activity, we studied its effect on the amidolytic activity of thrombin in the presence of normal titrated plasma, AT III depleted plasma and purified human AT III. As shown in Figure 2, myxalin inhibited the thrombin amidolytic activity as a function of its concentration. Plasma was a necessary cofactor of the myxalin activity. In the presence of purified AT III, only low antithrombic activity of myxalin was observed. However, myxalin was distinctly less AT III-dependent than that of heparin assayed under the same conditions.
0
MY XALIN
II 10 (&ml)
2 CON&NT&ION
FIG. 2 Inhibition of the thrombin amidolytic activity by heparin (a) and myxalin (b) in the presence of normal plasma (o), AT III-depleted plasma (I) and purified AT III (4). Values are expressed as mean + SD. TABLE 3 Effect of Protamine on the Thrombin Clotting Time on Fibrinogen in the Presence of MyxaIin and Heparin Thrombin Clotting Time on Fibrinogen in the Presence of Protamine Concentration in the Assay
_________________________________________-_------_____________________..__________.-__---.
WmU Myxalin w
0 16 32 40 53 160 200
33.9 * 0.4 33.0 + 0.3 29.8 + 0.5 27.0 f 0.5 25.1 + 0.3 18.7 + 0.6 13.2 + 0.4
Heparin (s)
39.4 f 0.2 38.7 It 0.3 33.4 * 0.3 21.5 f 0.5 15.4 It 0.8 11.0 ?I 0.6 10.3 f 0.3
Values are given as mean + SD., Michaelis buffer : 10.5 (s). Myxalin and heparin are used at concentrations of 50 l.@nI and 20 pg/ml respectively.
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ANTICOAGULANT PROPERTIES OF MYXALIN
Vol. 60, No. 1
In order to evaluate the effect of a possible inhibitor of myxalin’s anticoagulant activity, thrombin clotting times were measured in the presence of a range of concentrations of protamine, an inhibitor of heparin. As shown in Table 3, the thrombin clotting time for myxalin was slightly normalized and like it appears to be sensitive to a protamine inhibitory effect.
hepain,
AT III
Thrombin
+ Myxalin
+ Myxalin
+ Heparin
+ Heparin
FIG. 3 Crossed immunoelectrophoresis ( 1st dimension left to right). AT III, plus gel concentration of either 5 mg/ml myxalin or 0.1 mg/ml heparin. Thrombin, 4 l.~lof thrombin (500 U/ml) mixed wih either 8 l.tlof myxalin (25 mg/rnl), or heparin (12.5 mg/rnl).
The results of crossed immunoelectrophoresis with AT III and thrombin are shown in Figure 3. Compared to heparin, myxalin did not show an appreciable shift in the peak when it was incorporated in the gel at a concentration of 5 mg/ml, i. e. 50 times the heparin concentration. This indicates that there is no significant binding between myxalin and AT III. However, when mixed with thrombin at a concentration of 25 mg/ml, myxalin did cause an appreciable shift of the peak, producing a similar effect to heparin when used at a high concentration (12.5 mtr/ml).
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PROPERTIES
OF MYXALIN
15
Anti FXa activitv of mvxalin The Anti-FXa activity of myxalin compared to heparin is shown in Figure 4. This activity was measured in the presence of normal plasma and purified AT III.
10'
0
30
CONd&TRATIO&~ml) FIG. 4 of factor Xa activity by myxalin (0) and heparin (A) at different concentrations using Hepaclot reagents. The residual activity of factor Xa is measured by the clotting time of blood plasma in the presence of purified AT III. Inhibition
Myxalin had no effect on factor Xa activity at concentrations up to 2 pg/ml. At higher concentrations inhibition of human factor Xa was observed; 1 l.tg/ml having a similar effect to 0.07 ltg/rnl of heparin. About 14 times more myxalin than heparin was required for equivalent inhibition of human factor Xa using the particular conditions of this study.
DISCUSSION We have confirmed that myxalin has an anticoagulant effect on blood by demonstrating that it increases activated partial thromboplastin time and thrombin time, and inhibits thrombin amidolytic activity, and factor Xa activity. When compared to heparin however, myxalin is a weaker anticoagulant in vitro. At low concentrations (< 2 pg/ml), myxalin causes no change in factor Xa activity. At higher concentrations (-7 l.tg/ml) it begins to inactivate thrombin whether associated with platelet-poor plasma or fibrinogen, Moreover, this thrombin inhibition is lower in plasma than in AT III-free fibrinogen solution. This result seems to indicate a direct inhibition of thrombin by myxalin. Even so, the presence of some plasma component(s) which might limit the inhibiting effect of myxalin is still one possible explanation. “Non specific” binding with some plasma protein(s) has already been described as a probable factor affecting the anticoagulant property of pentosan polysulphate in plasma in vitro (12). However, the amidolytic tests suggest that the inhibition of thrombin by myxalin occurs mainly through an AT III-independent pathway. This explains our observations of high antithrombic activity of myxalin when exposed to AT III-depleted plasma. Moreover, the results illustrated in Figure 2 suggest that the effect of myxalin on thrombin inhibition when combined with AT III is not significant compared to that of heparin. It is well known that heparin possess a high degree of structural specificity which enables it to bind with high affinity to AT III (12). This AT III-heparin complex so formed rapidly neutralizes several clotting serine proteases
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ANTICOAGULANT
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PROPERTIES OF MYXALIN
(13). The results of our crossed immunoelectrophoresis studies show no evidence of significant binding between myxalin and AT III, even at high concentrations. In contrast, evidence to support significant binding between myxalin and thrombin has been found.
Like heparin and other glycosaminoglycans, the anticoagulant activity of myxalin can be neutralized by protamine. This phenomenon is probably due to the formation of a complex between this polycation and negative charges on the heparin and myxalin molecules. The results in Table 3 suggest that myxalin is less sensitive than heparin to protamine. Recent in vivo experiments using Wessler’s thrombosis model in rabbits (unpublished results), have also demonstrated that myxalin when used at 200 pg/Kg i. v. bolus gives a similar antithrombotic effect to that of heparin at 66 pg/Kg i. v. bolus. The anticoagulant property of myxalin probably depends upon its chemical composition. We have already shown that its sugar moiety is rich in glutamic acid (4). Sulphate groups are also present in various proportions (2-3 % of the dry weight). The role of the carboxylic and sulphate functional groups has been shown to be essential in the mechanism of heparin anticoagulant activity (14). It has also been reported that natural or synthetic materials substituted with carboxylic groups and other functional groups, such as sulphates and sulphonates, exhibit anticoagulant activity (15). Myxalin contains carboxylic groups from glutamic acid, the major aminoacid of the molecule (22-25 % of the dry weight) as well as sulphate groups. their combined presence can therefore account for its anticoagulant behaviour. Further investigations will be necessary to improve our understanding of the mechanism of antithrombotic activity, as well as the structure/function relationship of the myxlin molecule. Sulphate polysaccharides are widely distributed in higher organisms. However, only few papers have reported their presence in microorganisms (16, 17, 18). Recently, two anticoagulant agents have been isolated. The first was found in the culture medium of Arthrobacter species and has fibrinolytic activity (19). The second was isolated from the slime produced by Stauhvlococcus euidermidis (1). Myxalin differs from both these two anticoagulant compounds in terms of its composition. A number of sulphated polysaccharides have been reported to exhibit anticoagulant activity. One of these is pentosan polysulphate. This heparinoid has a low anticoagulant effect compared to heparin. At low concentrations, it potentiates inhibition of thrombin and factor Xa in the presence of purified AT III. However, these effects are mediated by binding to the enzymes, rather than to AT III (11). Our results suggest that myxalin antithrombin activity is due mainly to direct binding with thrombin itself. The action of myxalin towards HC II (Heparin Cofactor II) requires further investigation. This protease inhibitor has been found to contribute to the anticoagulant effect of heparin particularly at high concentrations (20, 21). HC II has also been reported to be the main cofactor of anticoagulant activity for some sulphated glycosaminoglycans such as dermatan sulphate (22,23) and pentosan sulphate (24). ACKNOWLEDGMENT
Special nada)
thanks
for his
to
helpful
Professor
M.W. King
(University
of
Laval,
Ca-
collaboration.
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2.
KUMADA, T., INOUE, K., KORENAGA, H. and ABIKO, Y. Antithrombic and thrombolytic effects of galactan polysulfate (DH 6322) in rats. Thromb. Res. 39, 21-28, 1985.
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17
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4.
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7.
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9.
BARROWCLIFFE, T. W. Studies of heparin binding to antithrombin Ill by crossed immunoelectrophoresis. Thromb. Haemostas. 42, 1434-1445, 1980
10.
MAUZAC, M. and JOSEFONVICZ, J. Anticoagulant activity of dextran derivatives. Part I : synthesis and characterization. Biomaterials 5, 301-304, 1984.
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FISCHER, A. M., BARROWCLIFFE, T. W. and THOMAS, D. P. A comparison of pentosan polysulphate (SP54) and heparin I : Mechanism of action on blood coagulation. Thromb. Haemostas, (Stuttgart) 47, 104108, 1982.
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LINDAHL, U., BACKSTROM, G., HOOK, M., THUNBERG, L., FRANSSON, L-A. and LINKER, A. Structure of the antithrombin Ill binding site in heparin. Proc. Nat, Acad, Sci, USA 76, 3198-3202, 1979.
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ROSENBERG, R. D. and DAMUS, P. S. The purification and mechanism of action of antithrombin-heparin cofactor. J. Biol. Chem . 248, 6490-6505, 1973.
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KAZATCHKINE, M. D., FEARON, D. T., METCALFE, D. D., ROSENBERG, R. D. and AUSTEN, K. F. Structural determinants of the capacity of heparin to inhibit the formation of the human amplification C3 convertase. J. Clin. Invest. 67,223-228, 1981.
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JOZEFOWICZ, M. and JOSEFONVICZ, J. Antithrombogenic polymers. Pure and ADO!. Chem. 56, 1335-1344, 1984.
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KONCEVICZ, M. The presence of sulphated polysaccharide Halobacterium halobium. Biochem. J. 130,4Op-41p, 1972.
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DARBY, G. K., JONES, A. S., KENNEDY, J. F. and WALKER, R. T. Isolation and analysis of the nucleic acids and polysaccharides from Clostridium welchii.. J. Bacterial. 103, 159-165,197O.
18.
STEBER, J. and SCHLEFER, K. H. Halococcus moorhuae : A sulfated heteropolysaccharide as the structural component of the bacterial cell wall. Arch. Microbio!. 105, 173-177, 1975.
in the cell envelopes of
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KUMADA, T. and ABIKO, Y. Fibrinolytic action of a new semi-synthetic polysaccharide sulfate, Galactan polysulfate (DH6322), in the rat. Thromb. Res. 39, 9-19, 1985.
20.
TOLLEFSEN, D. M. and BLANK, M. K. Detection of a new heparin-dependent inhibitor of thrombin in human plasma. J. Clin, Invest. 68,589-596, 198 1.
21.
TOLLEFSEN, D. M., MAJERUS, D. W. and BLANK, M. K. Heparin cofactor II. Purification and properties of heparin-dependent inhibitor of thrombin in human plasma. ,I. Biol. Chem, 257, 2162-69, 1982.
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BRIGINSHAW, G. F. and SHANBERGE, J .N. Identification of two distinct cofactors in human plasma-separation and partial purification. Arch, Biochem. Bioohvs. 161, 683-690, 1974.
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TOLLEFSEN, D. M., PESTKA, C. A. and MONAFO, W. J. Activation of heparin cofactor II by dermatan sulfate. J. Biol. Chem. 25&,6713-16, 1983.
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SCULLY, M. F. and KAKKAR, V. V. Identification of heparin cofactor II as the prirciIma1glsma cofactor for the antithrombin activity of pentosan polysulphate (SP 54). o b. s, 36, 187-194, 1984.
