Mechanisms of anticoagulant effects of some sulphated polysaccharides

momoszs RESEAXCB 23; 7&l-747, 1932 !JO49-33c8/a2/240741-07303.00/0 Printed Copyright (c) 1982 Pergamon Press Ltd.

MECHANISMS

OF ANTICOAGULANT

in the YS4. Al1 rights reserved.

EFFECTS OF SOME SULPHATED

POLYSACCHARIDES

Lars-Olov Andersson, James Hoffmanx, Erik Holmer, Olle Larmx, Kjell Larssonxx and Gunilla Sijderstrijm Research and Development, Biochemistry, KabiVitrum AB, S-112 87 Stockholm, Sweden. 'Department of Chemistry and Molecular Biology, Swedish University of Agricultural Sciences, S-750 07 Uppsala, Sweden. XXPresent

address: The National Swedish Laboratory for Agricultural mistry, S-750 07 Uppsala, Sweden.

Che-

(Received 22.4.1982; in revised form 15.9.1982. Accepted by Editor U. Abildgaard) ABSTRACT The anticoagulant activity of a partially reduced sulphated alginic acid, a partially reduced aminated and sulphated alginic acid and sulphated guaran have been studied. The anticoagulant activities in the APTT assay were 28, 39 and 70 IU/mg respectively. None showed any activity in anti-factor X, assay. Studies on binding to Antithrombin III - Sepharose showed that sulphated guaran and a fraction of the aminated and sulphated alginic acid was bound, whereas no binding occurred with sulphated alginic acid. The inhibition of thrombin activity by these polysaccharides was studied in purified systems with or without added Antithrombin III, using both fibrinogen clotting and chromogenic peptide substrate assays. The two alginic acid preparations showed Antithrombin III-dependent inhibition of thrombin, whereas the sulphated guaran inhibits both by Antithrombin III-dependent and independent mechanisms.

INTRODUCTION Heparin has been used in anticoagulant therapy for more than forty years. Not only is heparin the oldest antithrombotic drug still in use, it also reHeparin exmains the most widely used agent for immediate anticoagulation. erts its main blood-anticoagulant activity by potentiating the inhibitory activity of Antithrombin III (AT). AT inhibits most of the serine proteases involved in the coagulation cascade including thrombin and factor X,. Heparin is a sulphated polysaccharide and contains (1 -+4)-linked a-hresidues idopyranosyluronic acid residues, 2-amino-2-deoxy-a-D-glucopyranosyl and a small portion of B-e-glucopyranosyluronic acid residues. The hexosamine and uranic acid residues are arranged alternately to form a linear chain. The residues are partially 0-sulphated; in addition, most of the 2-amino-2-deoxyg-glucose is N-sulphated, the remainder being N-acetylated (1). The antiKEYWORDS:

Heparin, Sulphated

Polysaccharides,

Anticoagulant,

Antithrombin

III

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coagulant activity of heparin has prompted studies on a number of other sulphated polysaccharides, semisynthetic and naturally occurring, aimed at finding substitutes for heparin and identifying the structural features that are connected with anticoagulant activity. A number of sulphated polysaccharides showing anticoagulant activity have been found (2-7) and a few have even been shown to be effective as antithrombotic agents in clinical use (8-9). In most cases, the anticoagulant activity is fairly low, however, and no compound has so far been found that is as active as heparin. In a recent study (lo), a series of sulphated polysaccharides with different structures were prepared and tested for anticoagulant activity in the activated partial thromboplastin time (APTT) and anti-factor Xa assays. The results obtained would imply that the prerequisites for significant anticoagulant activity in the APTT test are that the polysaccharide should have a degree of polymerization of at least 35, contain at least about one sulphate group per monomer unit and have a high proportion of sulphated primary hydroxyl functions. In the present study, three of the sulphated polysaccharides from the previous study have been selected and the mechanisms of their anticoagulant effect investigated.

MATERIALS

AND METHODS

Materials The polymers studied, prepared as described by Hoffman et al., (10) and Larm et al., (11) were a partially reduced, sulphated algin=id (SAA2), sulphmalginic acid containing approximately 20% glycosamine residues (SAA2N), sulphated guaran (SG) and, for comparison, heparin (KabiVitrum AB). The alginic acids contained approximately 50% glycuronic acid residues and the glycosamine residues were introduced by oxidation with bromine followed by reductive amination with ammonium acetate and sodium cyanoborohydride. The sulphation was performed as the final operation. Human AT was prepared according to Miller-Andersson et al., (12). Human thrombin had a specific activity of 2 650 NIH units/mg ansprepared as described previously (13). Bovine factor Xa was obtained from Diagnostic Reagents Ltd., Thame, England. Bovine fibrinogen was obtained from Behringwerke AB, Marburg, W. Germany. It was dissolved in 0.075 M Tris, pH 7.15 buffer to a concentration of 2.5 mg/ml. Chromogenic peptide substrates S-2222 and S-2238 were from Kabi Diagnostica, Stockholm, Sweden. Affinity chromatography Affinity chromatography on AT - Sepharose to study binding Of the various sulphated polysaccharides was performed as described et a1.,(14). Platelet-poor plasma was obtained by centrifugation by h-h-sson at 20 000 G of platelet-rich plasma prepared from fresh ACD blood. F nticoagulant activities of the sulphated polysaccharides were determined using the activated partial thromboplastin time (APTT) assay (13). The effects of the sulphated polymers on the inhibition of factor Xa were studied in plasma using substrate S-2222 (13). The effects of the polymers were compared with that of various dilutions of the Third International Standard of Heparin. The inhibition of thrombin in a purified system was studied using the following methods: 200 ~1 of a solution of sulphated polysaccharide (l-3 ug/ml) and 200 ~1 of AT (0.1 U/ml) solution in 0.05 M Tris, 7.5 mM Na2-EDTA, 0.175 M NaCl pH 8.4 buffer were mixed and prewarmed for 3 minutes at 370 C. Then 100 ~1 thrombin solution (6 NIH units/ml) were added. After 30 seconds, 100 vl of the incubation mixture was transferred to 300 ~1 of a fibrinogen solution prewarmed at 37OC and the clotting time was determined using a hook-type coagulation

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timer (Fibrometer, BBL, Maryland, USA). When the chromogenic peptide S-2238 was used as substrate, the concentration of the thrombin solution was 15 NIH units/ml and 100 ~1 of the incubation mixture were transferred to 300 ~1 of a 2 mM solution of S-2238. The reaction was stopped after 30 seconds through addition of 300 21 of 50% acetic acid. The absorbance at 405 nm was recorded. When experiments were performed in the absence of AT, the AT solution was replaced by buffer. All assays were performed in dublicate at three dilutions.

RESULTS Activity in APTT and anti-factor Xa assays The activity ot the polymers in the APTT and anti-factor Xa assays is shown in Table I. Whereas all polymers prepared are active in the APTT assay, none are active in the anti-factor Xa assay. In a previous study (10) it was shown that differences in the structure of alginic acids with respect to distribution of s-Q-mannopyranosyluronic and a-l-gulopyranosyluronic acid residues did not affect the APTT activity of the partially reduced and sulphated derivatives.

TABLE I Anticoagulant activities of some sulphated polysaccharides as measured by APTT and antifactor X, assays. Activity APTT Sulphated Aminated,

(IU/mg) Anti-factor

alginic acid (SAA2)

28 (26-29, n=4)

< 2

sulphated alginic acid (SAA2N)

39 (36-41, n=4)

< 2

70 (66-79, n=8)

< 2

Sulphated guaran (SG)

X,

Binding to Antithrombin III - Sepharose The anticoagulant activity of heparin is dependent on its binding and interaction with AT. By affinity chromatography on matrix-bound AT, heparin preparations can be divided into two fractions, one with very low anticoagulant activity and one with very high activity (14, 15). When this technique was applied to the sulphated alginic acid all the maWhen the sample of aminated alginic acid terial passed through the column. was applied, 11% of the material was bound at physiological ion strength and could be eluted by buffer containing 1.5 M NaCl. The eluted material showed a specific activity of 76 IU/mg in the APTT assay, which is approximately twice that of the starting material. When the sulphated guaran was applied to the AT-Sepharose column, all the material was bound and could be eluted with 1.5 M NaCl. Activity in thrombin-fibrinogen and thrombin-chromogenic peptide substrate =? o characterize the nature of the anticoagulant activity displayed by the various sulphated polysaccharides, their ability to inhibit the thrombin-fibrinogen reaction in a clotting system composed of purified reagents was studied. The study was performed both in the presence and absence of AT and the results were compared with those for heparin (see Fig. 1).

744

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SELPI-IATED POLYSACC’WRIDES

see

SG

No.5

SAA2

500

100

50 3 !,-:_:_: 1

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set

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1

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/US/ml

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Clotting time in a purified system versus polysacchaide concentration in the absence (A-A) and presence (U) of AT. Partially reduced sulphated alginate (SAA2), partially reduced, aminated and sulphated alginate (SAA2N), sulphated guaran (SG) and heparin. As expected, heparin only slightly affected the clotting time in the absence of AT. The same behaviour is found for the sulphated alginic acids. Addition of AT to the system strongly increases the coagulation inhibiting effect of heparin. Similar behaviour is found with the sulphated alginic acids although the effect is less pronounced. Sulphated guaran behaves differently as substantial inhibition is found even in the absence of AT. Addition of AT amplifies this inhibition. When the corresponding experiments were performed using S-2238 instead of fibrinogen as the thrombin substrate, somewhat different results were obtained Neither heparin nor any of the three other polysaccharides had any effect on In the presence of AT, all subthe thrombin activity in the absence of AT. stances showed activity and the rate for thrombin inhibition was enhanced. The specific activities of sulphated guaran, sulphated alginic acid and aminated sulphated alginic acid when compared to heparin were 24 IU/mg, 33 IU/mg and 34 IU/mg respectively.

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DISCUSSION The three sulphated polysaccharides studied all show anticoagulant activity in the activated partial thromboplastin time assay, whereas no activity is found in the anti-factor X, assay. Further, all show inhibiting activity when assessed with the thrombin-fibrinogen clotting time test, provided that AT is present. In the absence of AT, sulphated guaran still shows activity, although somewhat diminished, indicating that part of its anticoagulant activity is caused by interaction with the thrombin-fibrinogen reaction or the fibrin polymerization. Direct interaction with the active site of thrombin can be excluded since no effect is found upon the thrombin-chromogenic peptide substrate system. Partial dependency on AT has been indicated by Kindness et al., (16) for some other sulphated polysaccharides. Theactivity profiles of the polymers investigated here differ from that of heparin and thus their mechanisms of anticoagulant action must also to some extent differ from that of heparin. The anticoagulant activity of heparin at low heparin concentrations is almost completely dependent upon its interaction with and potentiation of AT (17). It has been shown (18) that the fraction of heparin displaying high anticoagulant activity contains one or several special hexasaccharide sequences which are responsible for the binding to AT. Binding of this type of heparin to AT induces a conformational change (19) in the protein and thereby accelerates its binding to the coagulation enzymes. In the inhibition of thrombin, factor IXa and factor XIa, there is also direct interaction between the enzyme and the heparin bound to AT, which further enhances the rate of the inhibition reactions (20). One of the sulphated polysaccharides studied, the sulphated guaran, is completely bound to matrix-bound AT, and is thus similar to HA-heparin in that Of the remaining two, one shows a minor degree of binding and the respect. other does not bind at all. However, both show strong dependence on AT in the studies on thrombin inhibition which indicates that there must be some interaction between them and AT, although too weak to be demonstrable by the affinity chromatography technique. In a previous study (ll), it was shown that from a sample of extensively modified alginic acid, it was possible to separate, using affinity chromatography on AT-Sepharose, a fraction that had high anticoagulant activity and contained significant amounts of glycosamine resiIn contrast to heparin and some other sulphated polysaccharides (4-7, dues. 15), none of the three substances significantly inhibited factor Xa. This is somewhat surprising, since the AT-mediated inhibition of thrombin indicated a The difference can be explained if it is assumed that heparin-like activity. the polymers, although bound, are unable to induce the conformational change The effects of the in AT molecule that favours inhibitor-enzyme reaction. thrombin-AT reaction, could then be due to a simultaneous binding of enzyme However, whereas this mechanism may and inhibitor to the sulphated polymer. constitute significantly to the potentiation of the thrombin-AT interaction by heparin, its role in the inhibition of factor Xa by AT is negligible. In conclusion, all three polymers give AT dependent inhibition of thrombin and one of them, sulphated guaran, also interacts directly with the thrombinThey all differ from heparin as no potentiation of infibrinogen reaction. hibition of factor X, is found.

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