Anticoagulant activities of a pentosane polysulphate: Comparison with standard heparin and a fraction of low molecular weight heparin

THROMBOSIS RESEARCH 19; 455-463, 1980 0049-3848/80/160455-09502.00/0 Printed in the USA. All rights reserved. Copyright (c) 1980 Pergamon Press Ltd

ANTICOAGULANT ACTIVITIES OF A PENTOSANE POLYSULPHATE : COMPARISONWITH STANDARD HEPARIN AND A FRACTION OF LOW MOLECULARWEIGHT HEPARIN SORIA C., SORIA J . , RYCKEWAERTJ . J . , HOLMERE., CAEN J.P. Service des Professeurs CAEN et ROUSSELET, H6pital L a r i b o i s i 6 r e , Paris Service des Professeurs BILSKI-PASQUIER, SAMAMA, FABIANI, H6tel-Dieu, Paris Kabi A.B. Research Department, Stockholm, Sweden (Received 10.4.1980; in revised form 4.7.1980. Accepted by Editor V.V. Kakkar)

ABSTRACT The effect of pentosane po]ysulphate on the i n h i b i t i o n of thrombin and factor Xa and on the p a r t i a l thromboplastin time (PTT) was compared with that of low molecular weight (L.M.W.) heparin and standard heparin. Pentosane polysulphate showed s i m i l a r i t i e s with L.M.W. heparin in that factor Xa i n h i b i t i o n was to a r e l a t i v e l y higher extent potentiated than thrombin i n h i b i t i o n . The e f f e c t of pentosane polysulphate and heparin on i n a c t i v a t i o n of thrombin and factor Xa was completely antithrombin I I I (AT I I I ) dependant, while the effect on PTT appeared to be p a r t i a l l y independant of AT I I I . The quantity of pentosane polysulphate required to produce the equivalent effect of a given amount of standard heparin was greater in v i t r o than in vivo.

INTRODUCTION Many experiments have been performed on heparin f r a c t i o n s , separated according to t h e i r molecular weight. Large differences have been reported in the potency of such fractions when the anti-Xa a c t i v i t y results were compared with those of thrombin c l o t t i n g time and partial thromboplastin time (PTT) or a c t i vated p a r t i a l thromboplastin time (APTT). Lane et al ( i ) and Barrowcliffe (2) found that high molecular weight heparin while having the same anti Xa a c t i v i t y , was about 3 or 4 times more active than low-molecular weight heparin (L. ~!,W~eparin) in prolonging the PTT and Andersson et al (3) using heparin fractions. KEY-WORDS : heparin - L.MoW. heparin - pentosanep~ysulphate - antithrombin III.

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with high a f f i n i t y for antithrombin I I I (AT I I I ) found that L.M.W. heparin had as much as 12 times higher a c t i v i t y by anti Xa than by APTT. Lane et al ( I ) also showed that the antithrombin a c t i v i t y of heparin paralleled i t s prolongation of the p a r t i a l thromboplastin time and therefore suggested that the mechanisms by which heparin i n h i b i t s the thrombin c l o t t i n g of plasma and lengthens the p a r t i a l thromboplastin time are similar. In order to study the action of low molecular weight pentosane polysulphate on the coagulation system, we have compared i t s effect with that of standard heparin (Na and Ca s a l t s ) , and also with a low molecular weight fraction of heparin *. We measured anti-Xa a c t i v i t y , thrombin-inhibiting a c t i v i t y and anticoagulant a c t i v i t y in the PTT t e s t , with and without antithrombin I I I . Pentosane polysulphate is a sulphate polysaccharide extracted from brewer's yeast. The molecular weight, determined by high-pressure chromatography is 6,700 daltons (6,500 to 6,900). Its structure was analysed by Raveux and Brlot (4). I t is composed of chains of %-D-xylo-pyranose with sulfated groups on C2 and C3. At every tenth residue, a 4-0 methyl D glucuronic acid (with sulfated groups on C2 and C3) is associated in a lateral position in the chain. This substance has been used as an antithrombotic agent (5), but i t s action on the coagulation system is s t i l l poorly understood. MATERIAL AND METHODS The sodium and calcium salts of standard porcine mucosal heparin (MW : 14,900 daltons) were obtained from Choay. A heparin fraction of low molecular weight (5 - 8,000 daltons) determined by gel f i l t r a t i o n was a g i f t from Choay (6). The L.M.W. heparin was not high a f f i n i t y heparin for antithrombin I I I . Pentosane polysulphate (H~moclar, Clin-Midy), M.W. 6,500 to 6,900 (determined by gel f i l t r a t i o n ) was purchased from Clin-Midy. Antithrombin I I I depleted plasma was prepared by immuno adsorption of normal p l a t e l e t poor plasma using matrix - bound antibody to antithrombin I I I using Holmer's procedure (7). Cephalin was obtained from Stago laboratory (France). Purified human a n t i thrombin I I I (AT I I I ) , p u r i f i e d bovine factor Xa and the synthetic substrate for determination of Xa (S 2222) were obtained from Kabi. Cephalin treated bovine plasma was prepared using Yin's procedure (8). Prothrombinase was prepared by incubation f o r I0 min. of equal volumes of diluted c i t r a t e d human plasma adsorbed by aluminium hydroxide, diluted serum depleted of factor I I , cephalin and calcium 0.025 M (9). I) - In v i t r o studies Aliquots of Ca and Na heparin and pentosane polysulphate were diluted in 0.15 M NaCI and added to a pool of I0 normal platelet-poor plasmas (PPP) or to antithrombin I I I depleted plasma. Measurement of anti Xa a c t i v i t y using synthetic substrate : Experiments were performed on PPP containing various concentrations of heparin or pentosane polysulphate, or 0.15 M NaCI as control. The PPP was previously diluted I to 5 in Trizmal maleate buffer Sigma (20 x IO-3M, pH 7.4). To 0.2 ml of t h i s diluted plasma was added 0.i ml activated factor X at 7 ncat/ml ; the mixture was incubated at 37 ° C for 90 sec. The a c t i v i t y of residual factor Xa was measured by adding 0.2 ml IO-3M S 2222 according to the technique of Teien et al (i0) ; the mixture was incubated at 37 ° C for 150 seconds and the enzymatic reaction stopped by adding 0.3 ml 50 % acetic acid. Optical density -

~F-ITi-6-d'Fy given by Lormeau (Choay Laboratory) who is acknowledged

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(O.D.) was measured at 405 nm. A standard curve was obtained with the same procedure by replacing d i l u t e d plasma with 0.15 M NaCI and using factor Xa at various concentrations : 7, 3.5, 1.75 and 0.8 ncat/ml. The r e s u l t s were expressed as the percentage of f a c t o r Xa neutralized by the plasma in the presence of heparin or pentosane polysulpha~e. The same experiment was also carried out by replacing PPP with e i t h e r p u r i f i e d AT I I I (I00 % a c t i v i t y in comparison with normal plasma) or A T - I l l depleted plasma. The same technique was used with p u r i f i e d antithrombin I I I plasma.

instead of normal

C l o t t i n g assays : These were performed on the plasma pool in the presence of 0.15 M NaCI ( c o n t r o l ) , heparin or polysulphate of pentosane at various concentrations : I - thrombin c l o t t i n g time (9) in normal and AT I I I depleted plasma 2 - p a r t i a l thromboplastin time (PTT) (9) in normal and AT I I I depleted plasma 3 - measurement of the coagulation time of 0 . I ml of plasma (with NaCl 0.15 M, heparin or polysulphate pentosane) induced by 0 . I ml of d i l u t e d prothrombinase and 0 . i ml calcium chloride (0.025 M). 4 - measurement of anti Xa a c t i v i t y by a coagulation method : the incubation of Xa with plasma was carried out in the same conditions as in the assay f o r anti Xa a c t i v i t y of heparin and polysulphate of pentosane using a chromogenic substrate. The a c t i v i t y of f a c t o r Xa was assessed by measurement of the c l o t t i n g time ( i n sec.) of 0.2 ml of cephalin-treated bovine plasma added to 0 . I ml 0.025 M CaClo and 0 . I ml of the incubated mixture previously d i l u t e d I to I0 in 20 x i0-3~ Trizmal maleate buffer (pH 7.4). As in the technique using s y n t h e t i c substrate, r e s u l t s were expressed as the percentage of f a c t o r Xa i n a c t i v a t e d by heparin or pentosane polysulphate in plasma. 2) - In vivo studies The e f f e c t of an intramuscular i n j e c t i o n of i00 mg of pentosane polysulphate in 5 subjects was compared to t h a t of a subcutaneous i n j e c t i o n of 40 mg of calcium heparin. Coagulation tests were performed before the i n j e c t i o n of pentosane polysulphate and then r e g u l a r l y afterwards f o r 8 hours. The f o l l o wing tests were carried out : PTT, thrombin time and anti-Xa a c t i v i t y according to Yin et al (8). No bleeding tendency was noted a f t e r I.M. i n j e c t i o n of pentosane polysulphate. RESULTS In v i t r o experiments 1)

-

Effect o f standard heparin: low molecular weight heparin and pentosane polysulphate on the PTT of normal plasma and antithrombin l l l - d e p l e t e d plasma :

There was a logarithmic r e l a t i o n between the q u a n t i t y of standard sodium and calcium heparin or polysulphate of pentosane added to normal plasma and the PTT. As seen in f i g u r e la, the a c t i v i t y of Ca heparin was exactly the same as that of Na heparin. In c o n t r a s t , the low molecular weight heparin f r a c t i o n had very low a c t i v i t y . Up to i0 pg/ml; t h i s f r a c t i o n did not prolong the PTT. Pentosane polysulphate also lenghtened the PTT and 12 ~g were equivalent to 2 Ng of Na or Ca heparin.

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FIGURE

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I/ PTT (sec.) t" b = AT I I I free Log. scal~.'1 plasma

a = normal plasma PTT (sec.) Log. scale

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I 3 ~g/ml heparin i0 30 50 jug/ml pentosane polysulphate (final concentration) Coagulationof normal plasma with prothrombinase : effect of standard heparin or pentosane polysulphate

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In the anti-thrombin I I I depleted plasma, heparin at usual concentrations had very low anticoagulant a c t i v i t y as judjed by PTT. Whereas pentosane polysulphate had same kind of anticoagulant a c t i v i t y in normal and AT l l l - d e p l e t e d plasma (figure Ib). - Effect of standard heparin, low molecular weight f r a c t i o n of heparin and pentosane polysulphate on the thrombin c l o t t i n g time of normal plasma and AT l l l - d e p l e t e d plasma When heparin was added to normal plasma, the thrombin time was prolonged. This was observed clearly only i f the quantity of Na or Ca heparin was more than I ~g/ml. Beyond this amount, there was a logaritmic relationship between the quantity of heparin added to the normal plasma and the thrombin c l o t t i n g time. Ca and Na heparin had equal a c t i v i t y , while low molecular weight heparin had much lower a c t i v i t y : 2.5 ~g/ml of this fraction did not increase the thrombin time and I0 ~g/ml of the same fraction induced the same prolongation of thrombin time as 1.4 pg of standard heparin. Pentosane polysulphate also prolonged the thrombin time (Fig. 2). In this test 2 ~g of standard heparin were equivalent to 75 ~g of pentosane polysulphate. In addition, when polysaccharide sulfates were added to AT l l l - f r e e plasma (I0 ~g of heparin and 50pg of pentosane polysulphate) no increase in thrombin time was observed (Table I) TABLE I Reagent added

Thrombin time (sec)

NaCI 0.15 M Standard heparin (Na and Ca salts) I0 ~g/ml L.M.W. heparin i0 pg/ml Pentosane polysulphate 50 ~g/ml

20 20 21 20

Effect of heparin, L.M.W. heparin and polysulphate of pentosane on the thromb i n - c l o t t i n g time of an AT l l l - d e p l e t e d plasma. 3) - Effect of standard heparin and pentosane polysulphate on the c l o t t i n g time of plasma in the presence of prothrombinas e A logarithmic relationship was observed between the quantity of heparin and pentosane polysulphatein plasma and the prothrombinase c l o t t i n g time. In these conditions 2 pg of standard heparin were equivalent to 24 ~g of pentosane polysulphate (Figure 3). 4) - Anti-Xa a c t i v i t y of standard heparin, low molecular weight heparin and pentosane polysulphate (Figure 4) There was a direct correlation between the quantity of neutralized factor Xa and the concentration of heparin or pentosane polysulphate added to normal plasma. The results were the same whether the synthetic substrate method or the modified c l o t t i n g procedure were used. As observed in the preceding res u l t s , standard Na and Ca heparin had equal a c t i v i t y ; however, the low molecular weight heparin showed stronger a c t i v i t y (2 ~g of standard heparin had the same a c t i v i t y as 0.8 pg L.M.W. f r a c t i o n ) . Pentosane polysulphate also had anti Xa a c t i v i t y . In this t e s t , 2 Mg of standard heparin were equivalent to 20 Mg of pentosane polysulphate.

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FIGURE 4 Xa i n h i b i t i o n (in %)

Xa i n h i b i t i o n (in %) Synthetic substrate measurement

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Like heparin, pentosane polysulphate required the presence of antithrombin I I I to demonstrate anti Xa a c t i v i t y (Table I I ) : no anti Xa a c t i v i t y on synt h e t i c substrates was seen in the buffer system, or when heparin (I0 ~g/ml) or pentosane polysulphate (50 ~g/ml) were added to 1 ml of antithrombin l l l depleted plasma. TABLE II Agent tested (final concentration)

NaCl 0.15 M Heparin i pg/ml (Na s a l t ) 2 Ng/ml 10 ~g/ml Pentosane polysulphate 10 Ng/ml 30 ~g/ml 50 ~g/ml

Trizmal Buffer

0 0 0

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5 15 30

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Factor Xa i n h i b i t i o n (in per cent) in the presence of sodium heparin, and pentosane polysulphate (Measurement with synthetic substrate). The results of the in v i t r o experiments are summarized in table I I I . TABLE I I I

Test used

PTT Anti Xa a c t i v i t y Antiprothrombinase activity Antithrombin a c t i v i t y

Pentosane polysulphate (~g/ml f i n . concentration)

L.M.W. heparin (Hg/ml final concentration)

12 20 24

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75

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Concetrations (~g/ml) of pentosane polysulphate and L.M.W. heparin equivalent to the effect o~ standard heparin (2 ~g/ml) in 4 d i f f e r e n t tests. In vivo experiments The results obtained a f t e r i n j e c t i o n of standard Ca heparin and polysulphate of pentosane on anti Xa a c t i v i t y , and on the PTT, are shown in figure 5. No effect on thrombin time was observed a f t e r i n j e c t i o n of heparin or pentosane polysulphate. One mg of calcium heparin was found to be only s l i g h t l y less active than 3 mg pentosane polysulphate on anti Xa a c t i v i t y . In contrast, 3 mg pentosane polysulphate had a more potent e f f e c t that I mg of Ca heparin on the PTT.

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DISCUSSION Our results with heparin correlate with those already published by Barrowcliff e e t al (2), Lane et al (1) and Andersson et al (3). The low molecular weight f r a c t i o n we used had a stronger a c t i v i t y on anti Xa a c t i v i t y , but the action on thrombin time and on PTT were very small in comparison with standard heparin. The anticoagulant properties of pentosane polysulphate were studied with respect to anti Xa a c t i v i t y , thrombin i n h i b i t i n g a c t i v i t y and e f f e c t on PTT. I t was found that i t s anti Xa a c t i v i t y was greater than i t s thrombin i n h i b i t i n g a c t i v i t y , compared to standard heparin. In a s i m i l a r manner to heparin ( i i , 12), the anti Xa a c t i v i t y and thrombini n h i b i t i n g e f f e c t do not appear in the absence of AT I I I . In the presence of p u r i f i e d AT I I I (at the same concentration as in plasma) the anti Xa a c t i v i t y of pentosane polysulphate and of standard heparin was more pronounced than in normal plasma. This effect is probably due to partial neutralization of the polysaccharides by plasma components. In fact t h i s has been shown to be the case for heparin added to plasma (13, 14). The pentosane polysulphate also presented a strong effect on cephalin c l o t t i n g time (PTT), this l a t t e r a c t i v i t y being almost independant of antithrombin I I I . The same independance from AT I I I was also observed using high doses of standard heparin, as has been recently reported by Holmer et al (7). The e f f e c t of pentosane polysulphate was also observed in vivo. Given i n t r a muscularly, pentosane polysulphate had a more potent e f f e c t than in v i t r o on PTT and anti Xa a c t i v i t y when compared with calcium-heparin. Such a d i f f e rence between in vivo and in v i t r o effects does not seem to be related to heparin release after I.M. i n j e c t i o n of pentosane polysulphate, as i t was reported by Thomas et al (15) using a heparin analogue, but to a d i f f e r e n t metabolic clearance rate of the 2 drugs. Indeed the in vivo e f f e c t of pentosane polysulphate is higher on PTT when compared with a dose of heparin which induces the same anti Xa a c t i v i t y . In conclusion, there are certain points of s i m i l a r i t y between low-molecular weight heparin and pentosane polysulphate. They have a similar antithrombin I I I dependency for anti Xa and thrombin i n h i b i t i n g a c t i v i t i e s . Anti Xa a c t i v i t y is more pronounced that thrombin-inhibiting a c t i v i t y in comparison with standard heparin. In contrast, the e f f e c t of pentosane polysulphate on the PTT appear p a r t i a l l y independant of antithrombin I I I . In addition, the quantity of pentosane polysulphate required to produce an equivalent effect as i mg standard heparin, is greater in v i t r o than in vivo. Such a difference may be accounted for the d i f f e r e n t metabolic clearance of the 2 drugs. REFERENCES 1.

LANE D.A., MAC GREGOR I . R . , MICHALSKI R., KAKKARV.V. : Anticoagulant a c t i v i t i e s of four unfractionated and fractionated heparins. Thromb. Res. 12 : 257-271, 1978

2.

BARROWCLIFFET.W., JOHNSON E.A., EGGLETON C.A., KEMBALL-COOKG., THOMAS D. : Anticoagulant a c t i v i t i e s of high and low molecular weight heparin fractions. Br. J. Haematol. 41 : 573-583, 1979

3.

ANDERSSONL.O., BARROWCLIFFET.W., HOLMER E., JOHNSON E.A., SIMS G.E.C. Anticoagulant properties of heparin fractionated by a f f i n i t y chromatography on matrix-bound antithrombin I I I and by gel f i l t r a t i o n . Thrombos. Res. 9 : 575-583, 1976

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4.

RAVEUX R., GROS P., BRIOT ii. : Etude des polyesters s u l f u r i q u e s des xylanes n a t u r e l l e s e x t r a i t e s du bois de h@tre. I. Composition et s t r u c t u r e g@n@rale. B u l l . Soc. Chim. Fr. 33 : 2744-2749, 1966

5.

JOFFE S. : Prevention medicamenteuse des thromboses veineuses profondes post-op@ratoires. Une @tude comparative de l ' h ~ p a r i n a t e de calcium et du p o l y s u l f a t e de pentosaone. Archives Surg. 111, 37-40, 1976

6.

CHOAYJ . , LORi4EAU J.C., PETITOU 71., SINAY P., CASU B., PASQUA 0., TORRI G., GATTI G. : Anti Xa active heparin oligosaccharides. Thrombos. Res. (in press)

7.

HOLMERE., SODERSTROHG., ANDERSSON L.O. : Properties of antithrombin I I I depleted plasma. I. Effect of heparin. Thrombos. Res. 17 : 113-124, 1980

8.

YIN E.T., WESSLER S., BUTLER J. : Plamsa heparin : a unique, p r a c t i c a l submicrogramme s e n s i t i v e assay. J. Lab. C l i n . Hed. 81 : 298-305, 1973

9.

CAEN J.P., LARRIEU H.J., SAMAMA!I. : L'h@mostase - ~@thodes d ' e x p l o r a t i o n et diagnostic pratique. Expansion S c i e n t i f i q u e , vol. I , 1975

i0.

TEIEN A.N., LIE i,I., ABILGAARD U. : Evaluation of an amidolytic heparin assay method increased s e n s i t i v i t y by adding p u r i f i e d antithrombin I I I . Thrombos. Res. I0 : 399-410, 1977

11.

ALBIGAARD U. : Highly p u r i f i e d antithrombin I I I with heparin cofactor a c t i v i t y prepared by disc electrophoresis. Scand. J. Clin. Lab. Invest. 21 : 88-91, 1968

12.

ROSENBERGR.D. : Actions and i n t e r a c t i o n s of antithrombin and heparin. The New Engl. J. Med. 292 : 146-151, 1975

13.

ANDERSSONL.O., BARROWCLIFFE T.W., HOLHER E., JOHNSON E.A., SODERSTROH G. : Molecular weight dependency of the heparin potentiated i n h i b i t i o n of thrombin and activated f a c t o r X, e f f e c t of heparin n e u t r a l i z a t i o n in plasma. Thromb. Res. 15 : 531-541, 1979

14.

MacGREGORI . R . , LANE D.A., KAKKAR V.V. : Evidence f o r a plasma i n h i b i t o r of the heparin accelerated i n h i b i t i o n of f a c t o r Xa by antithrombin I I I . Biochim. Biophys. Acta 586 : 584-593, 1979

15.

THO~4AS D.P., BARROWCLIFFE T.W., JOHNSON E.A., STOCKS J . , DAWES J . , PEPPER D . S . : A heparin analogue that releases endogeneous heparin sulphate. V l l t h I n t e r n a t i o n a l Congress on Thrombosis and Haemostasis, Abstract I 000, London, 1979