Mechanism of thrombin inhibition by heparin cofactor II in the presence of dermatan sulphates, native or oversulphated, and a heparin-like dextran derivative

~iomoterioh

16 (1997)

359-366

Elsevier Science Limited Printed in Great Britain. All rights reserved 0142-9612/97/$17.00 0

0142-9612(95)00355-X

1997

Mechanism of thrombin inhibition bv heparin cofactor II in the presence oi dermatan sulphates, native or oversulphated, and a heparin-like dextran derivative Raoui M. Maaroufi* , Marcel Jozefowicz+ , Jacqueline Tapon-Bretaudike”, Jacqueline Jozefonvicz+ and Anne-Marie Fischer* ‘Laboratoire d’HfSmato/gie, CHlJ Necker-Enfants Malades, 149, rue de SBvres. 75743 Paris; f Laboratoire Recherche sur /es MacromolBcules, lnstitut Galilke, Avenue J.B. CIBment, 93430 Villetaneuse, France The kinetics native

of thrombin

carboxymethyl, groups initial

inhibition

(DS), or oversulphated (CMDBS),

data obtained

has been studied

rate constant

and KPS,HC 4.3 x lo-’ results

inhibition

content

II. A better

protease.

Unlike

heparin,

thrombin

inhibition,

and thrombin.

DS at concentrations

rapidly

The reaction

forms

10v5 M, whereas

CMDBS

data suggest dissociation

the rate constant

of the reaction

indicate

that CMDBS

tute a new class Keywords:

exerts

constant

dermatan

complexes

thrombin

by the absence

of this complex

which

drugs. sulphate,

effect 0

through

strongly

affinity

in which

reactive

heparin

rate of

between

DS

the biospecific

towards

concentrations

HC II than

equal

to or

with the fibrinogen-thrombin

for the protease complex

a unique

with the

the reaction

formation

model

DSSl-HC

interaction

not modify

(KPS

HC II (k) was 1.7 x lOeM-

1997 Elsevier

dextran,

a faster

is more

for CMDBS

to interfere

with

in the case of DSS 2,

of complex

fit a kinetic

M

min-’

Knowing

of both complexes

than 10e5 M does

has a strong

and the

11.5% for DSS2, these

may favour

of the CMDBS-thrombin

its catalytic

of anticoagulant

Heparin,

only

towards

unchanged

was found

which

and DSSP, respectively.

for HC II is increased

with thrombin

that CMDBS

II. The computed

model

II complex

(k). A KPs,+,c of 9.6 x lo-’

with

for CMDBS

rate remained

The

of the experimental

described

compared

can be explained a complex

for DSSl

with

functional

KPS,HC 2.1 x 10e6 M, k 1.1 x 10” M-’

found

higher

data obtained

protease. These

affinity

sulphates,

substituted

concentration.

Analysis

a previously

of 7.8%,

of these

derivative

interaction.

were

conformation

dextran

than

min-’

the reactivities

The experimental

polysaccharide levels.

II complex

for DS, whereas

per disaccharide

a fact which

using

of dermatan

derivative

(K Ps,Hc) of the polysaccharide-HC

increases

the free higher

found

that the polysaccharide

the oversulphation

II and DSS2-HC

of the sulphated

by the polysaccharide-HC

were

dextran

benzylamide-sulphonate

set at equimolar

constant

M, k 1.4 x 10” M-’

has a sulphur

indicate

were

and DSS2 was performed

and a k of 4.5 x 10’ Mm’ mini’

whereas

as a function

of the dissociation

of thrombin

II (HC II) in the presence

and carboxymethyl

concentrations

for DS, DSSl

computation

that DSSl

cofactor

carboxymethyl-benzylamide

HC II and thrombin

allows

by heparin

(DSS 1 and DSS 2) and a biospecific

de

mechanism

l$

and no affinity .~ mm ‘. These

of action

Science

Limited.

cofactor

II, thrombin

for HC

) was 2.4 x lo-‘M

All rights

and

findings

and may constireserved

Received 11 July 1995; accepted 5 December 1995

Dermatan sulphate (DS) increases the rate at which heparin cofactor II (HC II) inactivates thrombin. However, higher DS concentrations than those requested for heparin, on a weight basis, are needed to produce the same effect on the thrombin-HC II reaction”‘. DS and heparin may exert their catalytic activity by

interacting with the same N-terminal domain in the HC II molecule3f4. Yamagishi et ~1.~ and Tollefsen et ~1.~claimed that DS forms with thrombin and HC II a ternary complex preceding the generation of the covalent thrombin-HC II complex (template mechanism). Yamagishi et ~1.~ and Verhamme and Jackson7” also reported that DS bound preferentially to thrombin and subsequently formed a ternary complex with HC II.

Correspondence to Dr A.-M. Fischer. 359

Biomaterials

1997,

Vol. 18 No. 4

Thrombin

360

The oversulphation of native DS provided compounds which had higher catalytic properties than the native material with regard to the thrombin-HC II reactiong-13. It was also suggested that these catalytic properties depended on both the level of sulphation and the oversulphation procedure usedr3. The substitution of dextran with carboxymethyl (CM), carboxymethyl-benzylamide (B) and carboxymethyl-benzylamide-sulphonate (S) functional groups confers to the CMDBS type polysaccharides biospecific properties which depend upon the substitution rate, the molar mass and the final chemical composition. Indeed, specific binding sites for the functional domains of specified proteins are formed along the polysaccharide chains as a result of random substitution by suitable functional groups14,15. Subsequently, it was reported that some members of the CMDBS family exhibited anticoagulant properties involving plasma inhibitors of the coagulation process. Some of these dextran derivatives, with a known chemical composition, had an antithrombin (AT)-mediated activity. However, they exhibited a weaker specific activity (up to 17 UImg-‘) by comparison with heparin (150UImg-‘). At high concentrations, they were also found to exhibit an antithrombin effect which was independent of AT: this effect was, however, reversible and of lower importance than the AT-mediated effect16’17. The aim of this study was to elucidate the mechanism of catalysis of the thrombin-HC II reaction, in the presence of native DS, of two oversulphated DS derivatives and of a biospecific dextran derivative. Equimolar initial inhibitor and protease concentrations were used while varying the polysaccharide concentration. Experimental data were analysed by use of the previously described model, in which the catalyst binds to the inhibitor (or protease) to form a complex which reacts rapidly with the protease (or inhibitor).

MATERIALS AND METHODS Proteins Purified human thrombin (3300 NIH U rng-‘) was purchased from Sigma, St. Louis, USA; purified human heparin cofactor II, Chromozym-TH and Reptilase FTH50 were obtained from Diagnostica Stago, Asnieres, France.

Polysaccharides Unfractionated dermatan sulphate (intestinal bovine mucosa, 6% sulphurldisaccharide) and its two oversulphated derivatives DSSl and DSS2 (7.8% and 11.5% sulphurldisaccharide, respectively) were kindly provided by Mr. Mardiguian, Pharmuka, Gennevilliers, France. Pullulan standards ranging from 5 800 to 853 OOOgmoll’ with a polydispersity index of 1.10 (Pullulan Kit) were purchased from Polymer Laboratories, Shropshire, UK. CMDBS (46 000 gmoll’, 2.3 UImg-‘, an anticoagulant activity assessed from a calibration curve established with unfractionated heparin) was a dextran derivative substituted with Biomaterials

1997, Vol. 18 No. 4

inhibition

by heparin

cofactor

II: R.M.

Maaroufi et al.

carboxymethyl23% carboxymethyl, 83% benzylamide and 13% carboxymethyl-benzylamidesulphonate functional groups. Its preparation was carried out as previously described”.

Chemical oversulphation of dermatan sulphate Dermatan sulphate was oversulphated by reaction with a sulphur trioxide trimethylamine complex according to two different procedures13.

Heparin removal from DS samples 25 mg of DS was diluted in 10mL NaNO, 0.24M, CH3 COOH 1.8M and left for 80min at room temperature. The solution was dialysed 24 h against 500mL of tris buffer (Tris HCl, 0.05 M, pH8.4 albumin 0.5 gL_l).

Glycosaminoglycans

molar mass determination

The molar mass determination of the glycosaminoglycans used was achieved by analytical high performance steric exclusion chromatography using a Si 300 Diol column (marble diameter = 10pm). 50 PL of reference polymer or studied polysaccharide sample were injected. Molar masses before and after treatment with nitrous acid were assessed from calibration curves which were obtained with reference polymers (pullulans) of different known molar masses.

Assays of biological activity Thrombin was assayed by its amidolytic activity on a chromogenic substrate (Chromozym-TH) and the initial rate of amidolysis measured at 405nm as previously described18.

Measurement of inhibitor concentration The inhibitor stock solution concentration was evaluated by incubating HC II with an excess of thrombin at saturating levels of dermatan sulphate. The amount of HC II was then proportional to the part of thrombin which was inhibitedI’.

Kinetic evaluation of thrombin-inhibitor tion

interac-

The molar equivalence between thrombin and HC II was established by neutralizing a known amount of thrombin with increasing levels of inhibitor, in the presence of saturating amounts of dermatan sulphate. The decrease of the enzyme activity was a linear function of the inhibitor concentration, and extrapolation to 100% inactivation was used to compute the level of HC II equimolar to thrombin”.

Kinetics of thrombin inhibition in the presence of polysaccharide The kinetics of the thrombin-HC II interaction was studied by determining the residual enzyme activity as a function of the incubation time of the protease with the inhibitor in the presence of various polysaccharide concentrations”.

Thrombin

inhibition

by heparin

II: R.M. Maaroufi

cofactor

et a/.

Fibrinogen clotting activity of thrombin or reptilase in the presence of the polysaccharides

RESULTS

The fibrinogen clotting time in the presence of thrombin or reptilase was measured in the absence or presence of various DS, DSSl, DSSZ, CMDBS or heparin concentrationsr8.

Theoretical All experimental data were analysed using a previously described and discussed model18, lg in which the polysaccharide (PS) binds quickly to either the inhibitor (I) or thrombin (E). The complex formed, PSI or PSE, rapidly reacts with the free protease or inhibitor, respectively, in a second step which is rate-limiting. This leads in both cases to the formation of an inactive inhibitor-thrombin complex and release of the free polysaccharide according to: (a)

PS+HC

&,

PSHCfE

L

PS+E.HC

where KpS.HCis the dissociation constant of PSHC and k is the second-order rate constant of free thrombin inhibition by PSHC, eventually followed by: PS+E

mKPS.E

PSE+PSHC

k:

PS+E.HC

where Kps,E is the dissociation constant of PSE and k’ is the second-order rate constant of polysaccharide-bound thrombin inhibition by PSHC. (b)

PS + E &

PSE+HC

k

k ?!f+

Molar mass determination of the native DS and its oversulphated derivatives DSSl and DSS2 Molar masses of treated and non-treated samples were 50 100 g mall’ and 47 800gmol11 for DS, 50 lOOgmol-’ and 512OOgmoll’ for DSSl and 51200gmol-’ and 50 100 g mall’ for DSS2, respectively. Taking into consideration that the molar polydispersity index of these glycosaminoglycans was not modified (approximately 1.193, 1.170 and 1.082 for DS, DSSl and DSS2, respectively), these results indicate a negligible depolymerization upon nitrous acid treatment. Kinetic studies were performed on the treated glycosaminoglycans alone whose molar mass values were used for computation of the kinetic constants.

Thrombin inactivation by heparin cofactor II in the presence of native DS The inhibitor and the protease were both set at equimolar initial concentrations (Cr = Cz = lo-‘M). The residual thrombin [Erlt was measured for various incubation times t and for each initial polysaccharide concentration (Crs) ranging from lo-‘M to 6.4 x 10-4M. The reciprocal of the residual enzyme [ETlt was plotted versus the incubation time t for each Cps value (Figure 1). The curves l/[ET], =_f(t) were linear and indicated that the total

7 n

4.2 X IO-8 M

0

4.2 X IO-7 M

.

4.2 X IO-6 M

PS+E.HC

where k is the second-order rate constant of polysaccharide-bound thrombin inhibition by the free inhibitor. The kinetic model considers that the total protease (Er) (free (E) and/or bound to polysaccharide (PSE)) is inactivated by the total inhibitor (IT) (free (HC) or bound to polysaccharide (PSHC)). The total reaction is subsequently considered as a bimolecular reaction: Ir+Er

361

E.1

h

0 4.2X IO-5 M

E

-

W

z \2 rl

where kappis the apparent second-order rate constant of the total reaction. The total reaction rate is then written as follows: I

(1) For each PS concentration (Cps), kappis the slope of the curve l/[E,], =f(t) obeying the second-order equation:

A-2 - k”pp. t [ET], is the residual enzyme concentration the reaction time t.

at the end of

0

2

4

6

8 10 12 14 16 18 20

Incubation time (set) Figure 1 Thrombin inactivation by HC II in the presence of native DS. Reaction mixtures containing final equimolar thrombin and HC II concentrations of lOma M were incubated with DS at various concentrations. The residual enzyme ET was measured at time intervals by a chromogenic assay. Results for four DS concentrations ranging from 4.2 x lo-’ M to 4.2 x lo-’ M are presented.

Biomaterials

1997, Vol. 18 No. 4

Thrombin

362

50

? 15 40-

inhibition

by heparin

cofactor

II: R.M. Maaroufi

et al.

higher the Cps, the more the initial equilibrium was displaced towards the polysaccharide-HC II (PSHC) complex formation:

$

& 3000 2

5

20-

PS+HC

&%z!&

Kps,wc is

the dissociation and is expressed as:

complex

lo-

K

Figure 2 Thrombin-HC II reaction rate as a function of DS concentration. The apparent second-order rate constant of thrombin inhibition was plotted versus DS concentrations: k app = f(C&. For each Cos, kapp was the slope of the corresponding l/ET = f(t) curve (see Figure 7). (0) represent the experimental data (mean f SD), and the solid curve a computer fit of our data to Equation 5.

reaction was second-order whatever the Crs (see Equation 2). For a given Crs, the slope of the corresponding curve l/[Er], =f(t) is an experimental value of kapp.kapp was subsequently plotted versus Cps (Figure 2). The graph obtained showed a significant increase in kappas Ces increased to 10m5 M, as observed for the heparin effect on the thrombin-HC II reaction in the same heparin concentration rangel’. When Cps was about 10m5M, maximal value of (4.5 i 0.2) x ~JMmw~n_a ; at optimal concentrations, the native DS accelerated the thrombin-HC II reaction 7500-fold by comparison with the non-catalysed reaction (k, = 6 x lo5Mm1 mini’). The reaction rate remained unchanged for higher DS concentrations, whereas a decrease in the reaction rate had been previously observed at heparin concentrations higher than 10Y5 Ml’. In the latter case, the formation of a heparin-thrombin complex had been shown to be involved in the decrease of the reaction rate. A previous study of the adsorption of DS on sepharose-concanavalin A-HC II indicated that a fraction of the native DS bound to HC II with high affinity”. The DS was found unable to prolong the fibrinogen clotting time in the presence of thrombin, whatever the concentration used (Figure 3). These findings altogether suggest that the formation of a DSHC II complex is involved in reaction rate enhancement. They also indicate that DS has no affinity for the protease, which is in agreement with the fact that the thrombin inhibition rate reaches a plateau at DS concentrations higher than 10e5M. According to the previously described kinetic model, DS quickly forms with HC II a complex which is more reactive than the free inhibitor towards thrombin, whereas there is no interaction between DS and thrombin. The formation of a DS-HC II complex thus accounts for the catalysis of thrombin inhibition which is illustrated by the sharp increase in kappobserved in the increasing part of the curve kaPP= f (C,,). The Biomaterials 1997, Vol. 18 No. 4

PSHC

PSI, ” HC =

constant

of

the

WA

PSHC

(3)

[PSHC],

The total reaction is subsequently considered to be reduced to the sum of the non-catalysed reaction and the reaction of PSHC with free thrombin, whose rate constants were k. and k, respectively: HC+E

k

-%

PSHC + E 5

E.HC

(A)

E,HC+PS

03)

Taking these considerations into account, equation of the total reaction rate is expressed by: d[EJ, ___ dt

= k.

[HC],

[El, + k. [PSHC],

the

[El,

We can assume that the PS concentration is constant and equal to Ces given that the amount of PS bound to

Polysaccharide concentration (M) Figure 3 Effect of heparin, DS, DSSl, DSS2 and CMDBS on fibrinogen clotting in the presence of thrombin. 0.3mL of 5NIH U ml-’ thrombin was mixed with 0.1 mL buffer or polysaccharide, at concentrations ranging from 10mgM to 1O-4 M, and then fibrinogen 2g L-’ was added before recording the clotting time.

Thrombin

inhibition

by heparin

cofactor

II: R.M. Maaroufi et a/.

HC II is negligible when compared with CPs. Subsequently, according to Equation 3, the development of Equation 4 and then the identification of its terms with those of the total reaction (Equation I), we obtain the expression of the experimentally measured koppas a function of KPSzHc,k,,, k, and Cps: k OPP

ko .&.Hc =

KPS.HC

+k.c,s +

(5)

CPS

KPs,Hc and k were calculated from the experimental values obtained at the corresponding CPs values ranging from lo-'M to 4.2 x 10w5M. Kpsswc and k were (9.6 f 0.2)x 1O-7M and (4.5f 0.3)x 10'Mm’ min-’ , respectively. As can be seen from Figure 2, the theoretical curve deduced from the kinetic model is in good agreement with the experimental data, for all the DS concentrations used.

Thrombin inactivation by heparin cofactor II in the presence of oversulphated derivatives DSSl and DSS2 The oversulphation effect on DS affinity towards HC II was assessed using two oversulphated derivatives DSSl and DSS2 under the same experimental used for the native DS conditions as those (C, = CE = 10e8 M). The residual thrombin [Erlt was measured for various incubation times t, for polysaccharide concentrations (Cps) ranging from 4 x lomgM to 4 x 1oe7M. For lack of oversulphated derivatives, it has not been possible to test higher concentrations. The reciprocal of the residual enzyme was plotted versus the incubation time t for each !!T’tvalue. PS The curves l/[Er], =f(t) (Figure 4) were linear and suggested, as previously stated for the native DS, that the total reactions were second-order at any concentra-

363

tion used for both DSSl and DSS2. The slopes of these curves are the total reaction rate constants kopp,which are subsequently plotted versus DSSl and DSS2 concentrations, respectively (Figure 5). kappwas higher when the oversulphated derivatives were used at the same concentrations as the native DS. Furthermore, kapp was even higher wen DSS2 rather than DSSl was used. It is noteworthy that neither DSSl nor DSS2 significantly affected the fibrinogen clotting time in the presence of thrombin when they were used at the concentrations employed for the kinetic study (Figure

3).

The same mechanism could then account for the catalytic effect of the DS and oversulphated derivatives on the thrombin-HC II reaction. A glycosaminoglycanHC II complex quickly forms and subsequently reacts with thrombin. This complex is more reactive than free HC II towards the protease. The oversulphation of DS did not alter its mode of action but only increased its effectiveness. The same theoretical Equation 5 gave the expression of kopp as a function of Cps and the corresponding thermodynamic and kinetic constants, which were subsequently determined in the same manner as previously done for the native DS. KPS,HC was (2.1f 0.5)x 10m6M and (4.3 f 0.5) x 1oe7M for DSSl and DSS2, respectively, whereas k was (1.1 f 0.5)x lOloM-’ mine1 and (1.4 f 0.5) x 10’OM-lmin-’ for DSSl and DSSB, respectively. On the one hand, these data indicate that DSSl affinity for HC II is not statistically different from that of the native DS. Higher DSSl concentrations than those used in the kinetic study significantly prolonged the fibrinogen clotting time in the presence of thrombin (Figure 3), thereby predicting a possible competition between HC II and thrombin for DSSl, as previously On the other hand, DSS2 predicted for heparin”. affinity for the inhibitor is significantly increased. The reactivities of DSSl-HC II and DSS2-HC II.

r A 4. 10-8 M

(B)

(A)

0

30

60

90

120

150

Incubation time (set)

180

0

10

20

30

40

Incubation time (set)

Figure 4 Thrombin inactivation by HC II in the presence of DSSl or DSSP. Reaction mixtures containing final equimolar HC II and thrombin concentrations of 1Om8M were incubated with either DSSl (panel A) or DSS2 (panel 6) at concentrations ranging from 4 x lo-’ M to 4 x lo-’ M. The residual enzyme ET was measured at time intervals by a chromogenic assay. Biomaterials 1997, Vol. 18 No.

4

Thrombin

364

inhibition

1.8

by heparin

cofactor

r

1.6

10Y

lo-”

10’

Polysaccharide

lo-”

lo-”

concentration

10.”

II: R.M. Maaroufi

et al.

n

5. 10-8 M

0

5. 10-7 M

l

5. 10-6 M

0

5. 10-5 M

1o.3

(M)

Figure 5

Thrombin-HC II reaction rate as a function of DS, DSSl and DSSP concentrations. The apparent second-order rate constant of thrombin inhibition was plotted versus DS concentrations: k,, = f (CDs). For each Cus, k,, was the slope of the corresponding l/ET = f(t) curve (see Figure 4). (0), (A) and (m) represent the experimental data (mean i SD) for each DS, and the solid curve a computer fit of our data to Equation 5.

60

respectively, however, towards thrombin are, comparable and at the same time higher than that of the native DS (k = (4.5 f 0.3) x 10’ M-l min-‘).

Thrombin inactivation by heparin cofactor II in the presence of CMDBS The effect of CMDBS upon the thrombin-HC II reaction was investigated for polysaccharide concentrations (Crs) ranging from 10mgM to 10m4M, while using the inhibitor and enzyme at equimolar initial levels (Cr = Cz = lOma M). The reciprocal of the residual thrombin l/[Er], was plotted against the reaction time interval t for each CMDBS concentration (Figure 6). The curves l/[Er]( = f(t) are linear and suggest that the total reaction is second-order (see Equation 2). The slopes of the above curves are the total reaction rate constants kOppcorresponding to the CMDBS concentrations employed. kapp was subsequently plotted versus C rs. As can be seen from Figure 7, k,, increase significantly as Cps is raised to 10e5 M. When Cps is 10m5 M, the reaction rate is maximal and has a value of 1.6 x lOa M-l min-‘. CMBDS accelerates the thrombinHC II reaction 260-fold by comparison with the noncatalysed reaction (kO = (6.0 f 2) x lo5 M-’ min-‘) and appears less effective than native DS or heparin. The rate of the reaction remains unchanged for CMDBS concentrations higher than 10m5M, as previously observed in the case of native DS. It can thus be suggested that either the inhibitor or the protease alone interacted with CMDBS in the catalytic mechanism. Otherwise, CMDBS caused, contrary to DS, a large prolongation of the fibrinogen clotting time in the presence of thrombin without any effort in the presence of reptilase (Figure 3). This, in turn, suggested that there was a CMDBS-thrombin complex formation which strongly interfered with the thrombin-fibrinogen interaction. The higher the the more polysaccharideCMDBS concentration, enzyme (PSE) complex is formed: Biomaterials

1997, Vol. 18 No. 4

80

Incubation time (set) Figure 6

Thrombin inactivation by HC II in the presence of CMDBS. Reaction mixtures containin final equimolar HC II and thrombin concentrations of lOA were incubated with CMDBS at various concentrations. The residual enzyme ET was measured at time intervals by a chromogenic assay. Results for four CMDBS concentrations ranging from 5 x lo-’ M to 5 x 10m5 M are presented.

E+PS

&

, PSE

KP~,E is the dissociation expressed as follows: K

constant

PSI,. [El, PS.E=

of PSE

and

is

(6)

[PSE],

[PSI,, [El, and [PSE], were PS, E and PSE concentrations at reaction time t. In agreement with the above finding, we favoured the hypothesis according to which CMDBS quickly forms a complex with thrombin which would be more reactive than the free protease with regard to HC II. We postulated that the total reaction was the sum of both the non-catalysed reaction and PSE reaction with the free inhibitor: E+HC PSE+HC

h

+

E.HC

(A)

L

E.HC+PS

(C)

The equation of the total reaction rate is expressed and developed, taking into account (A), (C) and Equation 6 altogether with the assumption that the polysaccharide concentration is constant and equal to Cps. Further identification with Equation I provides the expression of kopp as a function of KP~,E, kO, k, and Ces:

Thrombin

366

8

thrombin, for both DSSl and DSSZ, yet without changing the involved mechanism, for the glycosaminoglycan concentrations used in the kinetic study. It is noteworthy that the increase observed in the k value is linked to a stronger affinity for HC II in the case of DSSZ, whereas it was not the case for DSSl. In both cases, however, the inhibitor bound to the oversulphated derivative might exhibit a conformation more favourable to interaction with thrombin than the inhibitor bound to the native DS. The differences observed between DSSl and DSSZ might be due to the sulphation rate or to the oversulphation procedure used. The present conclusions are in agreement with previous observations which indicated that oversulphation enhanced the DS anticoagulant properties11-13 mainly through the thrombin-HC II reaction13. Contrary to DS and heparin, CMDBS exhibited an unusual catalytic effect since the initial step of the catalytic mechanism was in this case the formation of a polysaccharide-protease complex. These findings suggest that catalysis of thrombin inhibition by such a compound belonging to the family of biospecific dextran derivatives, tested at low inhibitor and enzyme concentrations, mainly involves interaction of the CMDBS-thrombin complex with HC II. Considering this antithrombin mechanism, CMDBS could constitute a member of a new family of anticoagulant agents.

15

ACKNOWLEDGEMENT

16

towards

This work was supported by DRED - Universite V and Fondation pour la Recherche Medicale.

9

10

11

12

13

14

Paris 17

REFERENCES Tollefsen, D. M., Petska, C. A. and Monafo, W. J., Activation of heparin cofactor II by dermatan sulfate. J Biol Chem,

9183,258,

6713-6716.

Tollefsen, D. M., Activation of heparin cofactor II by heparin and dermatan sulfate. Nouv Rev Fr Hematol, 1984,26,

19

233-237.

Van Deerlin, V. M. D. and Tollefsen, D. M., The Nterminal acidic domain of heparin cofactor II mediates the inhibition of cc-thrombin in the presence of glycosaminoglycans. JBiol Chem, 1991,266, 20223-20231. Rogers, S. J., Pratt, C. W., Whinna, H. C and Church, F. C., Role of thrombin exosites in inhibition by heparin cofactor II. JBiol Chem, 1992, 267, 3616-3617. Yamagishi, R., Koide, T. and Sakuragawa, N., Binding of heparin or dermatan sulfate to thrombin is essential for the sulfated polysaccharide-accelerated inhibition of thrombin by heparin cofactor II. FEBS Lett, 1987, 225,109-112.

Tollefsen,

18

20

21

22 D. M., Peacock,

M. E. and Monafo, W.J.,

Molecular size of dermatan sulfate oligosaccharides required to bind and activate heparin cofactor II. J Biol Chem,1986,261,8854-8858. Verhamme, I. M. A. and Jackson, C. M., Glycosaminoglycan-catalyzed inactivation of thrombin by heparin cofactor II: Differences between the dermatan sulfate and heparin catalyzed reactions. Thromb Haemost, 1991, 65(6), Abstract no 392: 786.

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1997,Vol.18 No. 4

by heparin

cofactor

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et al.

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