Anticoagulant properties of a fucoidan fraction

THROMBOSIS RESEARCH 64; 143-154,199l 0049-3848191 $3.00 + .OO Printed in the USA. Copyright (c) 1991 Pergamon Press pk. All rights reserved.

ANTICOAGULANT

PROPERTIES

OF A FUCOiDAN

COLLIEC,S.* , FISCHER, A.M.* * , TAPON-BRETAUDIERE, J.* DURAND, P.*

????

and

FRACTION

??

, BOISSON, C.* ,

JOZEFONVICZ, J* .

?? CNRS UA502, LRM, C.S.P., Universite Paris Nord, 93430 Villetaneuse, France. “‘Laboratoire d’Hematologie, C.H.U. Necker-Enfants Malades, 75730 Paris Cedexlli, France. ?? **lFREMER, 44037 Nantes Cedex 01, France. (Received 15.11.1990; accepted in revised form 29.7.1991 by Editor M.C. Boffa) ABSTRACT

Fuco’idans are a family of high molecular weight sulphated polysaccharides in the Mr range 8 x105 - 1 06, widely dispersed in brown seaweed cell wall. When extracted from several brown algae, they exhibit anticoagulant properties. The chemical degradation of a crude extract, from Pelvetia canaliculata, was undertaken to obtain a low molecular weight polysaccharide (Mr 20,000 + 5,000) with the purpose of a possible clinical use. Its anticoagulant potency was investigated through the inhibition of factor Ila and factor Xa in the presence of antithrombin III or heparin cofactor II. The degraded fucoi’dan revealed a potent antithrombin activity : studied in an antithrombin III depleted plasma or in the presence of purified heparin cofactor II, the fuco’idan was as efficient as heparin and dermatan sulphate on heparin cofactor II potentiation, at the same concentration by weight. In whole plasma or in the presence of the purified inhibitor, an anti-factor Ila activity mediated by antithrombin III was detected (30 times less potent than for heparin, on a weight to weight basis). In contrast, no anti-factor Xa activity was detected in the presence of the degraded fucoi’dan, under the same experimental conditions. These fucoldans, by-products of alginates preparation in the food and cosmetologic industries, are obtained easily. Thus, they may represent a cheap and easy source of a new type of anticoagulants.

UCTION Heparin has been used in anticoagulant therapy for more than forty years. Heterogeneous both in its chemical composition and its polysaccharide chains length, it is the oldest antithrombotic drug still in use and it remains the most widely used agent for immediate anticoagulation [l]. However, there is now a tremendous interest in research for sulphated polysaccharides showing anticoagulant properties [2-51. ---------------------------Key Words : antithrombin III, heparin Cofactor II, fucoi’dan, anticoagulant.

143

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ACTIVITIES OF FUCdiDAN

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Since 1930, numerous natural or synthetic heparin-like compounds have been produced which act via different mechanisms. Some of them potentiate the inhibition of thrombin (factor Ila) or factor Xa via antithrombin III (AT Ill), such as fractionated heparin [l], dextran sulphonate [6] or heparan sulphate [7,8] ; others inhibit thrombin via a heparin cofactor II (HC II) pathway, such as dermatan sulphate [8] , dextran sulphate [S] and pentosan polysulphate [lo]. The purpose of all these studies was to obtain new homogeneous antithrombotic drugs, better characterized than heparin, with lower bleeding complications, and a less expensive manufacturing cost. Our study is focused on sulphated homofucans, namely fuco’idans, extracted from the cell walls of marine brown algae, which exhibit anticoagulant properties roughly described in previous publications [ll-151. These studies based on routine coagulation assays never allowed the authors to elucidate the precise mechanism of the anticoagulant effect of these polysaccharides extracted from several different algae. However, in a recent publication, Church et al. [16] have determined the anti-factor Ila activity of a commercial nonfractionated fucoi’dan, extracted from Fucus vesiculosus . In the work reported here, we investigate the mechanism of the anticoagulant potency of a fucoi‘dan, extracted from Pelvefia canaliculata and degraded by a sulphuric acid treatment [15,17,18] to a low molecular weight, compatible with further in vivo experiments and clinical use.

Fucofdan was isolated from brown marine algae Pelvetia canaliculata as previously described [15,17] A very heterogeneous and high molecular weight fucoi’dan, in the Mr range 40,000-100,000, was degraded by an acid hydrolysis procedure [I 81 The degraded product was fractionated by gel filtration [18], and characterized by high performance steric exclusion chromatography (HPSEC) as previously described [15]. A fraction named F2, selected for its anticoagulant properties and low molecular weight (Mr 20,000 + 5,000), is now further investigated. Besides, a commercial crude fucoi’dan, extracted from Fucus vesiculosus by a modified method of Black et al. [lS], was obtained from Sigma, Saint Louis, U.S.A. Its molecular weight determined by HPSEC was Mr 108,000. Heparin (batch H108, 173 I.U./mg) was purchased from Sanofi Recherche - Centre Choay, Gentilly, France. Dermatan sulphate from bovine intestinal mucosa was kindly supplied by Dr. Mardiguian (Laboratoire Pharmuka, Gennevilliers, France). Human purified thrombin (1,500 NIH U/mg, 63% active by site titration using the method of Chase and Shaw) [20], chromogenic substrate Chromozym TH, purified HC II (10 Wmg) and Reptilase were purchased from Diagnostica Stago, Asnieres, France. Bovine factor Xa was from Diagnostic Reagents, London, U.K.. Purified human AT III (5 Ul/mg) was purchased from Centre Regional de Transfusion Sanguine, Lille, France. Chromogenic substrate S-2222 and protamine chloride were from Kabi-Vitrum SA, Stockholm, Sweden. The following reagents were prepared in our laboratory : human brain cephalin and human brain thromboplastin [21] ; platelet poor plasma (PPP), from a pool of freshly collected plasma from 15 healthy subjects ; AT III depleted PPP [22] and AT III free fibrinogen [21].

Prothrombin time using human brain thromboplastin, activated partial thromboplastin time (APTT), thrombin time (TT) performed with and without protamine chloride diluted in buffer, and Reptilase time were performed as previously described [S]. In these assays, fucoi’dan and heparin were tested, after dilution in PPP, at various concentrations ranging from 10 to 100 ug/ml for fucoTdan F2 and from 0.25 to 10 ug/ml for heparin. Buffer was used instead of polysaccharide solution for the control.

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ACTIVITIES OF FUCOIDAN

Thrombin Generation Test (TGT) was performed, in (10 and 30 ug/ml), heparin (0.3 &ml) or buffer for incubated with or without the polysaccharide solution diluted in CaC12 M/40 (0.8 ml) were added. At time

145

PPP, in the presence of fuco’idan F2 the control. PPP (0.8 ml) was first and 0.8 ml of human brain cephalin intervals, the amount of thrombin

generated was determined by adding 0.1 ml of the incubation mixture to 0.3 ml of a human fibrinogen solution (2 mg/ml). Each clotting time was converted into thrombin NIH units by using a standard curve obtained with serial dilutions of a standard thrombin (all measurements were performed in triplicate). The concentration of the generated thrombin was quantified as following : hx represents the maximum concentration of thrombin generated in the presence of the polysaccharide ; ho is the peak value of the thrombin generated in the absence of polysaccharide (buffer) ; the inhibition of the generated thrombin (I %), by the polysaccharide, is expressed by the following equation I % = 100 x (ho - hx)/ ho [21]. . . . of them rate of t&g&u I- In the presence of purified AT//l . Factor Ila inhibition by purified AT III, in the presence of fucordan or heparin, at various concentrations ranging from 0 to 10 ug/ml, was determined as following : 0.10 ml of fuco’ldan F2 or heparin solution or Tris buffer (0.05 M Tris/HCI buffer, pH 8.4, containing O.lM NaCI, 7.5 x10s3 M EDTA, 7.4 x10W6 M albumin) were preincubated with 0.15 ml of purified AT III (corresponding to a final concentration of 52 nM), or buffer (control) for 2 minutes at 37°C. Then, a solution of 0.15 ml of human factor Ila (final concentration 20 nM) was added. At various incubation times (0.25, 0.5, 1, 3 and 5 minutes), the residual factor Ila was determined at 37°C by adding 0.15 ml of Chromozym TH (1.5 x10-3

M). The extent of substrate amidolysis, measured by the initial rate A %05/min

was proportional to the amount of residual factor Ila, which was estimated from a standard curve established before each experiment. 2- In the presence of PPP . Factor Ila inhibition, in the presence of diluted PPP, was determined by the method described above. Diluted PPP at l/18 (corresponding to concentrations of 52 nM AT III and 28 nM HC II in the final mixture) was used instead of purified AT III. For these experiments, the concentration of polysaccharide required for a complete inhibition of the factor Ila added in the test system was chosen : fuco’idan F2 (10 ug/ml), heparin

(0.3 ug/ml).

3- ln the presence of AT 111depleted plasma . Factor Ila inhibition was determined using a l/10 diluted AT III depleted plasma (corresponding to a concentration of 52 nM HC II in the final mixture) instead of PPP. The concentration of heparin (0.3 ug/ml) required for a complete inhibition of factor Ila, in the presence of purified AT III or diluted PPP, was increased to 10 ug/ml when the assay was done in the presence of diluted AT III depleted plasma or purified HC II. Therefore, the same concentration of fucoYdan F2 or heparin (10 ug/ml) was Used.

4 In the presence of purified HC II . Factor Ila inhibition, in the presence of purified HC II l-. The instead of purified AT Ill, was established as described above in paragraph concentration of HC II in the final mixture was 52 nM. Fucoi’dan F2, heparin and dermatan sulphate were used at the same concentration

(10 pg/ml).

. . . n of the lnactlvatlon rate of f-r Ila bv AT III or HC II in the Draence of aa& polvsaccharidg The apparent second order rate constants of factor Ila inactivation, kapP were calculated

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ANTICOAGULANT

ACTIVITIES OF FUCdiDAN

Vol. 64, No. 2

from plots of residual factor Ila concentration versus time, in the presence of each inhibitor, AT III or HC II, at the polysaccharide concentration required for a complete inhibition of factor Ila as previously described by Jordan and toll [23]. The measurement of the initial rate of factor Ila inactivation allows us to determine the apparent second order rate constants by fitting the initial level of thrombin (To) as well as subsequent levels of residual thrombin (T) observed at varying times (t) to the following equation : -I-_ T where kapp is expressed

-kappt

L= To

in M-’ min-’ .

te of Xa in PPP The assay was performed as described for the inactivation rate of factor Ila with the : PPP was diluted l/l2 (with a concentration of 80 nM of AT III in the following modifications final mixture) ; factor Xa (12 U/ml) and S2222 (4 x10m3 M) were used instead of factor Ila and Chromozym TH. FucoTdan F2 was tested at two concentrations, 10 and 500 ug/ml ; fucoTdan Sigma at 500 ug/ml and heparin at 0.5 pg/ml.

The comparison between the effect of fuco’idan F2 and heparin, on routine coagulation assays, is presented in Table I. At the concentrations used, fucoi’dan has no effect on Reptilase time and only a weak effect on prothrombin time. On the other hand, like heparin, fucoTdan prolongs APTT and TT. The latter test is normalized by addition of protamine chloride (data not shown). In order to obtain the same prolongation as with heparin of these coagulation times, higher amounts of fucoldan are required (50 fold more on a weight to weight basis). Table I Effect of fucoi’dan and heparin on coagulation

assays.

Clotting time (seconds) Fucol’dan F2 concentration (@ml)

Prothrombin time

APTT

l-r

Reptilase time

0 10 25 30 50 100

13 14 15 15 15 18

56 70 100 120 155 220

25 40 130 >180 >180 >180

22 22 22 21 22 22

Heparin concentration @g/ml) 0.25 0.5 0.75 1 5 10

14 14 14 14 17 23

85 120 150 >200 >200 >200

90 >120 >120 >120 >120 >120

22 22 22 22 22 22

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ANTICOAGULANT

ACTIVITIES OF FUCdiDAN

147

Figure 1 shows the effect of fucoi’dan and heparin on the TGT. The percentage of inhibition of the generated thrombin was calculated for each concentration of each polysaccharide. The concentrations used were those that had a maximum effect ; fucoi’dan F2 : 10 and 30 ug/ml ; heparin : 0.3 ug/ml. For a fucoTdan concentration of 30 ug/ml, the percentage of inhibition of the generated thrombin is relatively close to that obtained for an heparin concentration of 0.3 ug/ml.

Fig 1. Effect of fucoi’dan F2 on a thrombin generation test in PPP. The results are expressed as the percentage of inhibition of the generated thrombin 100 x (ho-hx)/ho, with fucoldan 10 and 30 ug/ml (l///l) or heparin 0.3 ug/ml (l-1) (each histogram represents the mean values of three experiments). ho represents the peak value of the curve obtained without polysaccharide; hx represents the peak value of the curve obtained in the presence of fucoi’dan (or heparin). Table II Amount of inactivated factor Ila by purified AT III in the presence of each polysaccharide, fucordan F2 or heparin, at various concentrations (incubation time : 3 minutes). Polysaccharide concentration (CLg/ml) 0 0.05 0.1 0.3 1 5 10

Inactivated factor Ila (nM) Heparin 3.3 13.3 15.5 20 20 20 20

Fucoi’dan 3.3 3.3 4.6 7.3 8.3 12 18.6

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.ANJICOAGUlANT

ACTIVITIES

OF FUCOj’DAN

Vol. 64, No. 2

.. of t&t&n bv ~&u?.d AT III or PPP Various polysaccharide concentrations, ranging from 0 to 10 ug/ml, were tested, in the potentiation of.thrombin inhibition by purified AT III, at different incubation times. For f*ucoldan F2 at 10 ug/mt and heparin at 0.3 ug/ml, the plateau of inhibition is reached at 3 minutes incubation time (data not shown). Table II shows the amount of thrombin inhibited by AT III, in the presence of fuco’idan or heparin, at 3 minutes incubation time. As can be seen, polysaccharide concentrations of 0.3 ug/ml for heparin and 10 ug/ml for fucoi’dan are required to achieve a complete inhibition of factor Ila (20 nM). Then, to measure the factor Ila inactivation in diluted PPP, the same concentrations of polysaccharide were used. The effect of fucoi’dan F2 (10 ug/ml) and heparin (0.3 ug/ml) on the rate of thrombin inactivation, in diluted PPP or in a purified AT III solution, are shown on Fig 2.

(controi), the factor Ila is very slowly In the absence of sulphated polysaccharide inactivated by plasmatic or purified AT Ill. In the same way, in the absence of AT III, a fucoldan solution of 10 ug/ml inhibits weakly the factor Ila as does heparin (data not shown). Finally, in the presence of purified AT III or diluted PPP, the thrombin inhibition is strongly accelerated by a concentration of 10 Kg/ml of fucoi’dan F2 or 0.3 ug/ml of heparin. At the concentrations used (30 fold more for fucoi’dan than for heparin), both polysaccharides exhibit a similar effect, in the presence of purified AT Ill, while fuco’idan has less effect in the presence of diluted PPP. As a matter of fact, the plateau is reached after 5 minutes incubation time for fucoi’dan instead of 2 minutes for heparin.

0

loo

200

3OOsaconls

0

ImcuMlIonTlnE

A

100

200

300 seconds

lWCUbATlONIlflE

lOo-

E

0

100

200 ImuBAllcmmlE

3oosecocr)s

0

100

200

300

.:axbp

~mcue~r~omrh~

Fig 2. Thrombin inhibition in the presence of purified AT III (52 nM), diluted PPP (AT III 52 r&l, HC II 28 nM), diluted AT III depleted PPP (HC II 28 nM) and purified HC II (52 nM) ; without polysaccharide (control, +) ; with 10 ug/ml of fucoTdan F2 (F2, A ) ; with 10 ug/ml of heparin

(H,

0

) ; with 10 ug/ml of dermatan

sulphate

(OS, +

).

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ANTICOAGULANT

ACTIVITIES OF FUCO]DAN

149

.. of AT Ill-factor or hw The apparent second order rate constants which express the enhancement of AT Ill-factor Ila reaction by fucoi’dan F2, compared to heparin are summarized in Table III. The kapp value of factor ila inhibition

by AT III, 3 x lOa M“

min- l for fucoldan

F2 (10 ug/ml) is close to the

corresponding value, 8 x lOa M- ’ min -’ obtained for heparin (0.3 pglml). Besides, the value of the rate constant of thrombin inhibition by AT III, in the presence of fucoi’dan F2, is similar to the one of crude fucoi’dan Sigma calculated by Church et al. (161. Table III Apparent second order rate constants (kapp) of factor Ila inhibition by diluted PPP, purified AT III, AT Ill depleted PPP and purified HC II in the presence of fucoi’dan F2 or heparin or dermatan sulphate. kapp x 108 M-l min -’

Sulphated polysaccharide (i.&ml)

PPP

AT III

AT III depleted PPP

HC II

none

0.07

0.04

0.04

0.02

Heparin

5.00

8.00

2.00

4.00

0.30

3.80

(0.3) Heparin (10) Fucoidan F2 (10)

0.80

Dermatan sulphate (10)

-

3.00

1.oo

Inactivation rate of thrombin bv AT Ill depleted PPP or ourified HC II The effect of fucoTdan F2, compared to heparin and eventually dermatan sulphate, on factor Ila inhibition, in the presence of AT III depleted PPP or purified HC II solution, is shown on fig 2. At 10 ug/ml of F2, the plateau of thrombin inhibition by purified HC II (52 nM) is reached at 3 minutes incubation time (data not shown). Fucoi’dan F2 accelerates the inactivation of thrombin but more slowly than heparin in the AT III deficient plasma. On the other hand, the same inactivation of factor Ila as with heparin is obtained for fucoi;dan F2 in the presence of purified HC II which has no antithrombic activity by itself (control). A higher concentration of dermatan sulphate (>lO ug/ml) would be required to achieve a complete inhibition of factor Ila by purified HC II. Rate enhancement of HC II-fag& r The kapp values corresponding polysaccharide

.. I i to factor Ila-HC

at the same concentration

values of factor Ila inhibition min-l for fucordan dermatan sulphate.

II reaction

Ioh in the presence

of each

(10 ug/ml), are presented on Table III. The kapp

by HC II are of the same order of magnitude : 3.8 x 1O8 M- 1

F2, 4 x lOa M- 1 min-’

for heparin

and .l x lOa M- ’ min-’

for

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ACTIVITIES

OF FUCOIDAN

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Xa in the of PPP Different fucoTdan concentrations were tested, in the potentiation of factor Xa inhibition in PPP, at various incubation times. The inactivated factor Xa, expressed in units/ml is measured in the presence of two F2 fucoTdan concentrations, 10 ug/ml and 500 ug/ml respectively (data not shown). At a fucoi’dan concentration of 10 ug/ml, no significant difference is observed with the assay done in diluted PPP, in the absence of fucoi’dan (control). It is necessary to reach 500 ug/ml of fucoTdan F2 to observe a measurable factor Xa inactivation. Besides, the amount of inactivated factor Xa, at the plateau value, almost 7 units/ml, does not correspond to a complete inhibition of the amount of factor Xa added in the test (12 units/ml). Comparatively to fucoTdan F2, the crude fucoi’dan Sigma (FS), at 500 ug/ml, strongly accelerates the inactivation of factor Xa, in the presence of PPP, and the plateau value corresponds to the complete inactivation of factor Xa. For heparin, a complete inactivation of factor Xa, in diluted PPP, is observed for a concentration of 0.5 ugglml.

Dl=JS=N Fucoi’dans, sulphated polysaccharides primarily composed of a(l,2)L-fucose-4 sulphate units or homofucans, are constituents of the cell wall of brown algae [11,12,24]. They present anticoagulant properties which have been first roughly investigated, showing a prolongation of the recalcification time and thrombin time of a normal plasma in vitro and ex viva after intravenous injection in rabbits [1 l-13,16]. In the present study, we confirm first that a fucoi’dan, extracted from Pelvetia canaliculata and named fuco’idan F2, exhibits an anticoagulant activity, when measured on activated partial thromboplastin time and thrombin time. As a matter of fact, an equivalent prolongation of both clotting times can be obtained for fucoi’dan and heparin, with a concentration 50 times higher (on a weight to weight basis). Furthermore, our results indicate that fucordan F2 is a weak inhibitor of thrombin generation. In these conditions, the same percentage of inhibition is observed for 0.3 ug/ml of heparin and 30 ug/ml of fuco’idan F2. In the present investigation, we also study the enhancement of factor Ila and factor Xa inhibition by fucoi’dan F2, in the presence of either AT Ill or HC II. The action of HC II, a plasmatic factor Ila inhibitor, is catalysed by heparin, at higher concentrations (twenty fold) than those required for AT III. Besides, the factor Ila inhibition by HC II is specifically enhanced by another polysaccharide, dermatan sulphate [25]. In our experimental conditions, a fuco’idan F2 solution of 10 ug/ml (compared to a heparin solution of 0.3 ug/ml) is required to obtain a maximum factor Ila inhibition rate, of the same order of magnitude, in the presence of purified AT III (52 nM). The AT III concentration used (52 nM) was chosen to obtain a weak inactivation of factor Ila in the absence of polysaccharide versus the high increase of the factor Ila inactivation rate in the presence of fucoi’dan F2 or heparin. For the experiment done in the presence of HC II, an equivalent molar concentration (52 nM) of HC II was used while a higher concentration of heparin was clearly necessary to obtain a noticeable factor Ila inhibition rate. On the other hand, fuco’idan F 2 fraction and heparin, at the same concentration (10 ug/ml), exhibit comparable potentiation on the rate of factor Ila inhibition, in the presence of purified HC II (52 nM). The apparent second order rate constant of factor Ila inhibition by HC II, in the presence

of 10 uglml

of polysaccharide,

is slightly

lower for dermatan

min- ’ ) than for fucoiilan F2 (3.8 xl O8 M- ’ min- ’ ). Moreover, we note that the apparent second order rate constants of the presence of fuco’idan F2, are lower (4 or 10 fold) in plasma than purified inhibitors, in our experimental conditions. These important observed with heparin which is known to bind to many components interaction of fucoTdan with other species than the thrombin inhibitors importance.

sulphate

(1 O8 M- ’

thrombin inactivation in in the presence of the discrepancies are not in plasma. Thus the in plasma might be of

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151

Otherwise, like various glycosaminoglycans (GAGS) [8,24], natural or synthetic sulphated polysaccharides (2,6,7], fucoldan F2 does not show, in our experiments, a significant potentiation of factor Xa inhibition. As a matter of fact, a concentration of fucoldan F2, fifty times higher than the one required for a complete inhibition of factor Ila by AT III (or PPP), is not sufficient to achieve a complete inhibition of factor Xa. By comparison to our experiments, Church et al. [16], using a commercial crude fuco’idan from Fucus vesiculosus (Sigma), found a same enhancement on the rate of factor Ila inhibition by HC II, but a weaker inhibition mediated by AT III. These authors showed only a slight enhancement of the rate of factor Ila inhibition by AT III, in the presence of 30 pg/ml of the crude fucoi’dan : 5.7 x 1O7 M- ’ mine1 compared to 3.0 x 1O8 M- ’ min‘ ’ , in the presence of only 10 pg/ml of fucordan F2 in our experiments. On the other hand, Church et al. observed a weak factor Xa inhibition by AT III, ifi the presence of a very high fucoTdan concentration (500 pg/ml), corresponding to an acceleration of thirty five times when compared to the rate of factor Xa inhibition, in the absence of fucoi’dan. Using the same commercial fucoi’dan, at the same concentration (500 pg/ml), we obtain a similar inhibition, while fucoldan F2, at this high concentration, is not able to produce a noticeable inhibition. These discrepancies may be due to the higher molecular weight of the commercial fucoi;dan. In fact, the comparison between both fucoi’dans shows that the molecular weight is 20,000 + 5,000 daltons for fucoi’dan F2 and 100,000 daltons for fucoi’dan Sigma, determined in the same experimental conditions, in our laboratory, by an analytical high performance steric exclusion chromatography. Besides, the commercial fucoi’dan is very heterogeneous while the fraction F2 is homogeneous and presents a very narrow average molecular weight after a preparative steric exclusion chromatography of the degraded crude fucoi’dan [18]. The fuco’idan macromolecule presents mainly a(l,2) linked units of L-fucose-4-sulphate with branching or sulphate at position 3 and some additional units (D-xylose, D-galactose, and It has been shown in natural oligosaccharides [26,27] and synthetic uranic acid). pentasaccharide [26], that a 3-0-sulfated-D-glucosamine unit in position 4, which is claimed to be very important in the AT III binding sequence of heparin, is absent in the. fucordan structure. But a 3- or 4-0-sulphate-L-fucose unit described in some fucoIdans[24] could,be a binding site involved in the fucoTdan-AT III interaction. The comparison beiwer$ AT III and HC II catalysis in the presence of heparin, dermatan sulphate, or fucoi’dan allows us to specuiate on the mechanism of action of these polysaccharides. Factor Ila inhibition, by AT III, in the presence of heparin, requires the formation of a ternary complex, while factor Xa inhibition requires only a binary complex between heparin and AT III. The binding of heparin to AT III induces a change of intrinsic fluorescence of the inhibitor, related to a conformationa! change which is important for the enzyme inhibition. If we hypothesize that the formation of a ternary complex, between the enzyme, the inhibitor and the fucoi’dan, is necessary for the enzyme inhibition, we can imagine that factor Xa does not bind to the fucoi’dan while thrombin binds strongly to the polysaccharide, as described by Church et a/. [16]. Therefore the formation of a ternary complex implying factor Xa is impossible. Otherwise, no change of intrinsic fluorescence is observed when fucoldan binds to AT III, suggesting that no conformational change of the inhibitor is observed [16]. This fact may explain why the binding of the enzyme to the fuco’idan is essential for its inactivation by AT III or HC II. On the other hand, no specific oligosaccharide structure is required, in heparin, for binding and catalysis of HC II ; and a minimum of 6-7 disaccharide units in the macromolecules of dermatan sulphate are necessary to catalyse the inhibition of factor Ila by HC II [28]. However, it is not yet elucidated if a specific arrangement of sulphate groups is required for heparin-HC II interaction. FucoSdan may eventually exerts its potentiating effect in correlation with its high charge density. Further investigations, on a low molecular weight fucoi;dan fragment, will allow an approach of the mechanism of its catalytic action onto both plasmatic inhibitors, AT III and HC II. In conclusion, fuco’idan F2 exerts its anticoagulant activity by enhancing factor lla inhibition in the presence of either AT III or HC II. Among the anticoagulant sulphated

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OF FUCO’iDAN

polysaccharides, fucoTdan F2 appears to be the only one, as potent on the formation of an AT lllfactor Ila complex, as of an HC II-factor Ila complex. In fact, heparin requires much higher concentrations to enhance the activity of HC II than the one of AT III. On the contrary, pentosan polysulphate and dermatan sulphate are mainly active via an HC II pathway. Moreover, the absence of factor Xa inactivation by fucoi’dan F2, correlated with its low viscosity (compared to commercial fucoldan) and its high factor Ila inactivation, could be an important feature for a further clinical use, according to recent studies. Indeed, it has been demonstrated that low molecular weight heparin fragments, with high factor Xa inhibition and negligible factor Ila activity, are poor antithrombotic agents. Moreover some low molecular weight heparins, with a high anti-(factor Xa) to anti-(factor Ila) activity ratio, seem to act primarily via factor Ila inhibition [l]. FucoTdan, by-product of alginates preparation in the food and cosmetologic industries, is easily obtained, in great quantity. Thus, it may represent a cheap and easy source of a new type of anticoagulants. The in vitro studies show that fuco’idan F2 has antithrombic properties and are necessary to prove it, in thus could be a good antithrombotic agent ; in vim experiments an animal model. However, high amounts of fucoi’dan would be necessary to achieve these effects which are compatible with its solubility but we may have to deal with a low bioavailability, as observed for DS [29]. ACKNOWLEDGMENTS We thank C. Sternberg for her excellent assistance for clotting assays and M. Delvallee for her expert assistance in the preparation of the manuscript. Supported by Centre National de la Recherche Scientifique (CNRS) and by a grant from lnstitut Francais de Recherche pour I’Exploitation de la Mer (IFREMER).

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Correspondence and reprint requests should be sent : to Anne-Marie Fischer, MD, C.H.U. Necker-Enfants Malades, 156 rue de Vaugirard, 75730 Paris Cedex 15, France. Telephone (33-l) 47 83 33 03 ext : 359 - Fax : (33-i) 45 67 53 33.