Inhibition of thrombin by antithrombin III in the presence of certain glycosaminoglycans found in the mammalian aorta

THROXBOSIS QPergamon

RESEARCH

‘3:

655-670

Press Ltd.1978.

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in Great Britain GG4o-3848/78/1001_06jj

$02.00/O

INHIBITIONOF THROMBIN BY ANTITHROMBIN III IN THE PRESENCE OF CERTAIN GLYCOSAMINOGLYCANSFOUND IN THE MAMMALIAN AORTA M.W.C. Hatton, L.R. Berry and E. Rzgoec-i Plasma Protein Research Laboratory, Roan hf55, HcMas ter University Health Sciences Centre, Hamilton, Ontario, Canada 185 4J9

(Received 24.4.1978: in revised form 2.8.1978. Accented by Editor P.J. Gaffney)

ABSTRACT Chondroitin-4-sulphate, chondroitin-6-sulphate, dermatan sulphate, heparan sulphate and hyaluronic acid were compared with heparin in their abilities to influence the inactivationof bovine thrombin by rabbit antithrombinIII. The effect of the glycosaminoglycanson the enzymic activity of thrombinwas examined using the substrate a-Nbenzoyl arginine ethyl ester. Heparin, dermatan sulphate, heparan sulphate and, to a smaller extent, chondroitin-6-sulphate increased the esterase activity, whereas chondroitin-4-sulphate and hyaluronic acid had a negligible effect. Heparan sulphate and dermatan sulphate markedly acceleratedthe inactivationof thrombin by antithrombinIII, but chondroitin-4-sulphate, chondroitin-6-sulphate and hyaluronic acid did not significantlyaffect the reaction. The glycosaminoglycanswere adsorbed on Sepharose-lysineor polyacrylamide-ethylenediamineto test their ability to bind thrombin or antithrombin III under conditionswhere neither protein could directly bind to the conjugates. The binding of thrombin to immobilisedheparan sulphate or dermatan sulphate compared well with that to heparin. On immobilisedchondroitin-&sulphate,thrombin was retarded and on chondroitin-4-sulphateno thromhin binding was observed. In contrast to thrombin, antithrombinIII only bound to heparin: antithrombinIII did not bind to dermatan sulphate, heparan sulphate or the other glycosaminoglycans. AntithrombinIII inactivationof 1251-thrombinadsorbed to either heparan sulphate or dermatan sulphate produced a thrombin-antithrombin III complex which was spontaneouslyreleased from the glycosaminoglycan. However, no complex was recovered from imswbilisedheparin unless the column was eluted by 1M NaCl. These results support the view that heparan sulphate and dermstan sulphate could play a role similar to that of heparin in the inactivationof thrombin by antithrombinIII.

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INTRODUCTION

Earlier work in this laboratory showed that the sequence in which thrombin and antithrombinIII are exposed to immobilisedheparin determineswhether or not inactivationof the enzyme takes place (1,Z). Thus thrombin adsorbed to heparin exhibited increasedesterolytic activity and it readily attracted antithrombin III, whereas heparin-antithrombinIII failed to attract the enzyme. From these observationswe inferred that heparin, in agreementwith the propositionby Machovich et al. (3), accelerates the inactivationof thrombin by interactingwith the enzyme rather than antithrombinIII. The intima of mammalian blood vessels contains various glycosaminoglycans (4,5,6)which, because of their proximity to the plasma, could be of significance for the neutralizationof thrombin, provided they can interact with the enzyme in a heparin-likemanner. Here we report our relevant observations with five glycosaminoglycans,all of which are known to be present in the intima.

MATERIALS AND METHODS Glycossminoglycans Samples of chondroitin-4-sulphate(C4S), chondroitin-6-sulphate(C6S), hyaluronic acid (Hya), dermatan sulphate (Des), heparan sulphate (HeS) and heparin were National Heart Institute reference standards kindly provided by Dr. J.A. Cifonelli,Dept. of Pediatrics,University of Chicago. Preparation of bovine thrombin The procedure of Lundblad et al. (7) was modified as follows: Crude thrombin (Parke-DavisCo., Detroit, MI; 10,000 N.I.H. units) was dissolved in 5 ml 0.05M Na phosphatepH 6.5 and loaded on to a column (30 cm x 2 cm) of SP-Sephadex (PharmaciaCo., Montreal, Canada) equilibratedwith 0.05M Na phosphate pH 6.5 at room temperature. The column was washed with 400 ml of O.lM Na phosphate pH 6.5 at 50 ml/h. A gradient was applied (mixing chamber, 200 ml of 0.l.MNa phosphate pH 6.5; limit solvent, 0.3M Na phosphate pH 7.8) and a-thrombin,preceded by a B-thrombinshoulder, emerged after approx. 140 ml of the gradient, equivalent to 0.2251 Na phosphate. The athrombin peak was pooled and assayed by a plasma clotting method (8) relative to an internationalthrombin standard (9), obtained from the National

Vol.

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~NTITMHOMEXN

& GLYCOSAMINOGLYCAN

Institute for Biological Standards and Control, London, U.K.

657

The specific

activity was 2020 (r 140 S.E.M.) international units (IU) per mg using E28&%= 16.2 (10) to calculate protein concentration.

For all esterase activity

measurements, thrombln was buffered by 0.2M Na phosphate pH 8.0.

Amol.wt.

of 36,000 (11) was used to calculate molar ratios. Samples (0.5-l mg) of thrombin were iodinated with 1251 using iodine monochloride (12). protein.

Up to 2 atoms of iodine were substituted per molecule of

After labelling, the specific plasma clotting activity of bovine

thrombin was approximately 75-802 of the original preparation.

The chromato-

graphic behaviour of 1251-thrombin on SP-Sephadex was very similar to the unlabelled protein as measured by 280 nm absorbance and plasma clotting activity. Preparation of rabbit antithrombin III The method used has already been described for the corresponding human protein (1).

Contaminating heparin was removed from the preparation by pas-

sage through a small column (1 ml) of polyacrylamide-ethylenediamine (see below), equilibrated with 0.15M NaCl at room temperature, which efficiently adsorbed heparin (see Table l), but had no affinity for the protein.

Protein

concentration was calculated from absorbance measurements (280 nm) using the 1% values of E 110.5 and mol. wt. 62,300 for human antithrombin III (22). 280 125 Samples (0.8-l mg) of antithrombin III were labelled with I under similar conditions used for labelling thrombin.

No significant differences were ob-

served between labelled and unlabelled antithrombin III in their ability to form a complex with thrombin. Preparation of Sepharose-lysine and polyacrylamide-ethylenediamine Lysine was conjugated to Sepharose using the CNBr procedure of Cuatrecasas (13) as described before (14).

To estimate the amount of lysine, conjugated

samples (l-2 mg) of freeze-dried gel were hydrolysed by 1 ml 3.75M NaOH at 105' for 20 h in a sealed glass tube.

The hydrolysate was acidified by add-

ing 1 ml 6M HCl and 0.5 ml 1M citric acid.

After centrifuging, the lysine

content of the clear supernatant was determined using a Beckman 120C autoanalyser.

Batches 1 and 2 contained 225 and 279 umoles of lysine/g of dry gel

respectively. Polyacrylamide-ethylenediamine was prepared by the direct amino-ethylation method of Inman and Dintzis (15).

Biogel P-300 (Biorad Laboratories Richmond,

CA) and ethylenediamine (Fisher Scientific Co., Toronto, Canada) were heated

658

ANTITHFlOFt?3TN& GLYCOSAMINOGLYCAN

at 90%

vo1.13,xo.4

for 3 h using non-aqueous reaction conditions. The quantity of bound

ethylenediaminevas determined calorimetricallyby reacting weighed samples of the freeze-driedconjugatewith ninhydrin (16) at pH 5.5 in boiling water bath for various times (2-10 min) and comparing the colour yield (570 run)with appropriatestandards and unconjugatedBiogel P-300. The product contained 5520 pmoles ethylenediaminelgof dry conjugate. Measurementof thrombin activity To measure free thrombin activity, either the plasma clotting assay (see above) or an esterase method was used. In many experimentsboth procedures were used. A spectrophotometricesterase method (17) using a-N-benzoylarginine ethyl ester (Sigma; BAEE) was adapted in the followingways. To measure the thrombin activity eluted from a column, 0.5 ml BAEE (1 mg/ml in 820) and 2.6 ml O.lM Tris.HCl pH 8.0 were placed in a l-cm cuvette in the heated (37OC) cell-holderof a Unicam SP 500 spectrophotometer. The contents were warmed to temperature (approx. 2 min) before 0.1 ml thrombin was added, the reactants mixed and the reaction followed at 254 nm for 5 min.

In the experimentsde-

signed to study the influence of the glycosaminoglycanson the esterase activity of thrombin, 0.1 ml of a glycosaminoglycansolution (ranging from 0.1 ug/ ml to 1 mgfml) was added to 0.5 ml BAEE and 2.5 O.lM Tris.HCl pH 8.0 in the cuvette before the addition of 0.1 ml thrombfn (110 IV/ml). The experiments to determine the effect of glycosaminoglycanson the reaction between thrombin and antithrombinIII were undertaken as follows. The cuvette containing 2.4 ml O.lM NaCl, 0.1 ml thrombin (110 IU/mJ.)and 0.1 ml glycosaminoglycan (200 ug/ml) was warmed at 37'C. Then 0.1 ml antithrombinIII (0.21 mg/ml) was added, the stopwatchwas started and the contents rapidly mixed. After the required reaction time (l-4 min) 0.5 ml BAEE (warmed to 37OC) was added and the thrombin activity determined at 254 run. Control experimentswere made in which 0.15M NaCl replaced glycosaminoglycanand/or enzyme. For the detection of bound thrombin, 0.35 mg of a chromogenicsubstrate S-2160 (Ortho Diagnostics,Raritan, N.J.), dissolved in 0.5 ml Ringer's lactate was passed through the column and the effluent fractionsmeasured at 405 nm. The techniquehas been fully described previously (1,2). Turbidimetricestimation of glycosaminoglycansusing vrotamine sulphate To 0.5 ml of glycosaminoglycan(l-120 ug), 0.2 ml protamine sulphate (Sigma; 1 mg/ml in H20) was added and followed after 10 min by 1 ml O.lM arginine.HCland 2.3 ml of either 0.02M Na phosphate or 0.02M Tris.HCl at

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pH 7.8. The turbid solutionswere read at 470 run. The absorbancevaried directly with the concentrationof each glpcosaminoglycanup to 140 pg. Readings (1 cm cuvette) for 100 pg of glycosaminoglycanwere as follows: HeS, 0.405; Des, 0.645; heparin, 0.510; C6S, 0.285; c4s, 0.290; and Hya, 0.170. Column chromatographyon Sepharose-lysineand polyacrylandde-ethylenediamine In all experimentsinvolving these conjugates,Sepharose-lysineand polyacrylamide-ethylenedismine columns (approx.1.5 ml and 1.0 ml respectively were equilibratedwith Ringer's lactate at room temperature. All loads of glycosaminoglycans,thrombin and antithrombinIII were prepared in Ringer's lactate, conditionsin which neither thrombin nor antithrombinIII could bind to the conjugates. Each column was only used once. For gel filtrationof the thrombin-antithrombinIII complex, a column (53 cm x 2.2 cm) of Sephadex 6.200 (Pharmacia)was equilibratedwith O.OM Tris.HCl containing0.3N NaCl at pH 8.0. The loading volume was 2 ml and the flow rate at room temperaturewas 30 ml/h. Human serum albumin (mol. wt. 69,000) was obtained from BehringwerkeA.G. (Marburg-Lahn,G.F.R.). RFSULTS Effect of glycosaminoglycanson the BAEE esterase activity of thrombin Over the range 0.01 pg - 100 pg, Des, HeS and to a lesser extent C6S progressively increased the esterase activity but similar quantitiesof C4S and Hya did not significantlyaffect the reaction. These results, relative to the effect of heparin, are shown in Fig. 1. Effect of glycosaminoglycanson the inhibitionof thrombinby antithrombinIII Thrombin and antithrombinIII were incubatedwith and without the presence of a glycosaminoglycan. After an interval, ranging from 1 to 4 min, BABE was added and the residual thrombin activity determined. Fig. 2 gives the results obtained for a reaction mixture (2.7 ml) containing eleven IU (5.5 ug) thrombin, 20.9 pg antithrombinIII and 20 pg glycosaminoglycan. Using these reaction conditions the presence of DeS or HeS rapidly acceleratedthe inactivationalthough less efficientlythan a similar quantity of heparin. In comparison,C4S did not noticeably influencethe rate of thrombin inhibitionby antithrombinIII. Both C6S and Hya were slightly protective towards thrombin against the inhibitor during the early stage of reaction (up to 2 min) but, by 4 min the degree of thrombin inactivationapproached that of control.

660

ANTITHROMBIN

Vo1.13,No.4

& GLYCOSAMINOGLYCAN

-.-----J

HEPARIN

t

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-2

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-1

0

1

1

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2

LOGlO(GLYCOSAMINOGLYCAN) FIG. 1 Effect of glycosaminoglycanson the BABE esterase activity of bovine thrombin. The experimentaldetails are described in the Methods section. Glycosaminoglycanconcentrationis given as pg used in the reaction mixture (3.2 ml). The results from heparin have been reported before (1). ThrConwl

llwAT

III Control

INCUBATION WITH INHIBITOR (mid FIG.

2

Effect of glycosaminoglycanson the inactivationof thrombin by antithrombin III. The enzyme was measured by the BAEE method as described in the text. The thrombin (Thr) control values changed insignificantlyduring incubation for l-4 min at 3J°C and represent 100% activity. Results from the incubates are related to the relevant Thr control. 'Thr-AT111control' containedno glycosaminoglycan.

l-01

.

73,r;o.tr

XNTITHROMBIN

661

% GLYCOSAMINOCLYCAN

Affinity of glycosaminoglycansfor Sepharose-lysineand polyacrylamideethylenediamine In order to determinewhether the glycosaminoglycanscould bind either thrombin, antithrombin111 or the inactive complex, attempts were made to immobilise the glycosaminoglycanson Sepharose-lysineor polyacrylamideethylenediamine. Previous work from this laboratory (X3,2) had shown that affinity media possessing an amino group supported by an inert hydrocarbon chain, e.g. Sepharose-lysine,Sepharose-cadaverine, bound heparin efficiently. Several preparationsof Sepharose-lysinewere tested for their ability to bind the glycosaminoglycans. The results, (see Table 1) indicated that in the presence of Ringer's lactate, Des, HeS and C6S were adsorbed readily by Sepharose-lysine(especially batch 2), but C4S binding was considerablyless. Negligible amounts of Hya were adsorbed by the conjugate. By comparison,conjugatedethylenediaminefully retained heparin, C6S, HeS, DeS and Hya but only a small portion of C4S.

TABLE 1

Binding of Glycosaminoglycansto Sepharose-lysineand Polyacrylamide-ethylenediamine. Each column, equilibratedwith Ringer's lactate at room temperature, was loaded with 2.00 mg of each glycosaminoglycanin 1 ml Ringer's lactate. Any bound glycosaminoglycanwas step-elutedby 1M NaCl. Bound and unbound glycosaminoglycanwas estimated by the protamine sulphate method.

Quantity of Glycosaminoglycanbound to: Sepharose-lysine Glycosaminoglycan

c4s C6S DeS HeS Hya Heparin

Batch 2

Batch 1 % load

8.5 34.5 59.4 34.4 0 68.0

Sepharose-lysine

pg 170 690 1188 688 0 1360

% load

50.5 83.8 82.5 100
Polyacrylamideethylenediamine

ng

1009 1676 1650 2000 trace 2000

% load

13.0 100 100 100 100 100

ng 260 2000 2000 2000 2000 2000

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AF-Z-ITHROMBIX & GLYCOSA??NOGtYCAN

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Affinity of thrombin for immobilisedglycosaminoglycans For the initial experiment,coluxms of Sepharose-lysine(batch 1) were loaded with 2 mg of glycosaminoglycans(as described in Table l), and then, after washing with Ringer's lactate, with 75 N

of thrombin (1 ml).

The

quantity of thrombin which did not bind to the glycosaminoglycanon elution with Ringer's lactate (25 ml) was determined in the effluent fractionsby plasma clotting assay. The effluent from Des, HeS and heparin columns showed that no clotting activity passed through. In contrast, the thrombin load was totally recovered from the C4S column and approx. 81% from the C6S column. The latter retarded the enzyme. By loading S-2160 (0.35 mg), the bound thrombin was found to be active on the Des, HeS and heparin columns. The enzyme was recovered by eluting with 1M NaCl. Further experimentswere performed using Sepharose-lysine(batch 2) and 1251-thrombin(see Table 2). In experiment A, 2.0 mg of glycosaminoglycanwas followed by 200 IU of 1251-thrombin. The results closely resembled those experiments in which clotting activity had been measured. Experiment B was designed to measure thrombin-bindingcapacity

TABLE 2

125 I-Bovine Thrombin to GlycosaminoglycansAdsorbed to SepharoseBinding of h experimentA, 2.0 mg and in experiment B, 100 ug lysine (batch 2). of glycosaminoglycanwere loaded respectiy?iy, Each column was then eluted with Ringer's lactate (25 ml) before the I-thrombin load (200 IU in 1 ml Ringer's lactate). After eluting with SO ml Ringer's lactate, any bound enzyme was displaced by eluting with 1M NaCl. N.D. signifies 'not determined.'

12sI-thrombinRadioactivity Glycosaminoglycan

ExperimentA % not retained

Experiment B

4 displaced % not retained X displaced by lM NaCl

c4s C6S DeS HeS Heparin

>9a 83.8 <2 <2 <2

0 16.4 98.8 96.2 97.1

by lM NaCl ND ND 58.0 64.9 6.1

39.2 35.0 82.6

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dc GLYCOSAXINOGLYCXN

of Des, HeS and heparin. Each column received a load of 100 pg of glycosaminoglycan prior to 200 IU of 1251-thrombin. The DeS and HeS columns bound similar quantitiesof the enzyme (35-394)but significantlyless than the heparin column (83%). Similar experimentswere undertaken using polyacrylamide-ethylenediamine as the affinity matrix to immobilise the glycosaminoglycans(2.0 mg). Despite the great affinity of the glycosaminoglycansfor this matrix, 1251-labelled thrombin binding was greatly reduced in comparison to the results from the Sepharose-lysineexperiments. Using a load of 75 IU 1251-thrombinfor each glycosaminoglycancolumn, the percentages of radioactivitybound were measured as: c4s, < 1%; C6S, 6.3%; Des, 38.0%; HeS, 10.8% and heparin 50.5%. These results were matched by the plasma clotting activity passing through each column. Affinity of antithrombinIII for immobilisedglycosaminoglycans For these experiments,Sepharose-lysinewas used as the affinity medium. With the exception of heparin, which bound approximately60% of the protein

TABLE 3 Binding of antithrombinIII to Glycosaminoglycansadsorbed to Sepharose-lysine. Sepharose-lysinebatch 2 was used for C4S binding and batch 1 for the other glycosaminoglycans. After the glycosaminoglycanload (2.00 mg) the columns in experimentA were loaded with 26.8 ug 1251-antithrombinIII (in 1 ml Ringer's lactate) and those in experiment B with 0.725 mg unlabelled antithrombinIII. After washing with 50 ml Ringer's lactate any bound protein was displaced by 1M NaCl. The recovery of antithrombinIII in the effluent was determined by radioactivitymeasurement (experimentA) and protein concentration(experiment B). N.D. signifies 'not determined'.

Recovery of antithrombinIII load Glycosaminoglycans

Experiment A % not retained

c4s C6S DeS HeS Heparin

94.6 100.7 98.2 95.8 30.3

X displaced by lM NaCl 3.6 0 1.7 3.2 66.9

Experiment B 4:not retained

% displaced by lM NaCl

ND ND 99.0 96.5 46.1

0.5 2.7 53.8

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loaded, no other glycosaminoglycantested was able to bind significantquantities of antithrombin111 (Table 3). Glycosaminoglycanwas not displaced when antithrombinIII was loaded, but was recovered during elution with lM NaCl. Using polyacrylamide-ethylenediamine, only experimentswith heparin were performed. The quantity of 125I-antithrombinIII (26.8 ug) bound to the heparin column was negligible (4.2%). For this reason, columns of polyacrylamide-ethylenediamine have successfullybeen used as a simple, rapid method for removing traces of heparin from antithrombinIII preparations made by Sepharose-heparinchromatography. Inhibitionof glycosaatinoglycan-bound thrombin by antithrombinIII The fate of the thrombin-antithrombinIII complex was examined in the presence of HeS, DeS and heparin bound to Sepharose-lysine(Fig. 3) using reaction conditionstaken from an earlier study (2). Thus, by following 200 IU (2.8 nmoles) of bound l*'I-thrombinwith 4.8 nzoles of antithrombin III, full thrombin inactivationwas observed on the heparin and HeS columns but only 69% inactivationon the DeS column. In Fig. 3a the radioactivity measurementsshowed that, on applying antithrombinIII to the HeS column, most (79%) of the 125I-thrombinparted from the HeS. With DeS (Fig. 3b), 44% of the radioactivitywas displaced by antithrombinIII which was less than observed with HeS. In contrast, on the heparin column (Fig. 3c) the complex was tightly held unless lM NaCl was applied to displace all the bound components. The experimentwas repeated using similar quantities of DeS followed by 1251-thrombin. However, the bound enzyme was inactivatedby loading 13.4 nmoles of antithrombinIII. From BAEE esterase measurements,> 95% of the enzyme was inactivatedand radioactivitymeasurementsshowed that 80.1% of the 1251-thrombinwas displaced by the antithrombinIII load. The 1251-thrombin-antithrombin complex was further characterisedby Sephadex G.200 chromatography. The complex appeared as a broad peak (ve/Vo ratio: 1.77) which was eluted before human serum albumin (Ve/Vo ratio: 1.93). By using the method of Determann and Michel (19), a mol. wt. of apprOXi=telY 88,500 was calculated for the complex. DISCUSSION It has been known for a:number of years that arterial glycosaminoglycans possess anticoagulantproperties. Thus Kirk, in 1959, isolated acid mucopolysaccharides from human aorta and determined an anticoagulantactivity for the

Vo1.13,N0.4

ANTITHROMBIN

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b.

& \GLYCOSA.MINOGLYCAN

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665

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Des , 0.4

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- 0.3

20 -:

- 0.2 - 0.1 - 0.0

c.

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Heparin

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FRACTION NUMBER

FIG. 3 Inactivation of thrombin bound to glycosaminoglycan on Sepharose-lysine. The experiment compares the properties of heparin, HeS and DeS to promote the inactivation by antithrombin III. Each column (batch 2) was loaded with 2.0 mg of glycosaminoglycan and washed with Ringer's lactate. At fr. 1 and 11, 1251-thrombin (200 IV) and antithrombin 111 (0.29 mg) were applied respectively At fr. 23, the column was eluted by 1M NaCl. Fraction volume was 2.5 ml. After radioactivity measurements, BAEE esterase and protamine sulphate assays determined residual enzymk activity and glycosaminoglycan contents respectively a, 1251-thrombin radioactivy;a- - - -0, glycosaminoglycan (A470 nm). ?? -

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preparation (20). Later, Izuka and Murata (21) found that, of the acid glycosaminoglycansisolated from human blood vessels, DeS possessed the most powerful anticoagulantaction and that it was present in greater quantity in venous than in arterial tissues. Working with bovine aortae, Moritani and Ohta (5) isolated an anticoagulantactivity which was separated into two fractions. Separately the activitieswere weak but, on recombination,a preparationof high activity was obtained. One fraction was found to be plasma antithrombin III and the other contained acid mucopolysaccharidesof which DeS and HeS were the principle components. More recently, Teien et al. (22) have compared the anticoagulantactivities of many glycosaminoglycanswith heparin using an activated partial thromboplastintime test system. DeS and HeS possessed anticoagulantactivity but to equal that of heparin, approximately70-times the concentrationof each was required. The other glycosaminoglycanstested, including C4S, C6S and Hya, did not prolong the clotting time. The purpose of the present experimentswas to determine the mode of anticoagulantaction of these glycosaminoglycantypes in comparison to heparin. Heparin, DeS and HeS accelerated the inactivationof thrombin by antithrombin III whereas, C4S, C6S and Hya were ineffective. This result generally agrees with the observationsof Teien et al. (22). Thrombin binding studies showed that DeS and HeS compared well with heparin in their ability to bind the enzyme but the other glycosaminoglycanstested, C6S and C4S, had little or no affinity. In contrast, the antithrmnbin III binding experimentsclearly showed that, with the exception of heparin, binding of the inhibitor to the glycosaminoglycanswas negligible. However, on rechromatographyof antithrombin III, only approximately60X of the protein bound to the heparin column At present we cannot explain this poor affinity but the result closely compares with earlier observationsof Rosenberg and Damus (23) and from this laboratory (2). The BABE esterase activity of thrombin was significantlyincreasedby DeS and by HeS although less than the effect produced by heparin. In comparison, C6S had a weak effect and C4S or Hya had none. These findings are consistent with the conclusion that the accelerated inactivationof thrombin in the presence of certain glycosaminoglycans
Vol. 13*x0.4

ANTITHNOMBIN

acted as a cofactor of the inhibitor (23) but more recently mounting which

667

dc GLYCOS.LmNOGLYCAN

evidence

has been

suggests that the acceleratingeffect of heparin is brought

about by modifying the enzyme (24,1,2,25,26). A clear view was hampered by the awkward situation that heparin binds both thrombin and antithrombinIII. However, as shown here, DeS and HeS also possess significant cofactor activity despite their lack of affinity for antithrombinIII. Clearly, these glycosaminoglycansaccelerate the rate of reaction between thrombin and antithrombin by acting solely through the enzyme. On a weight basis, heparin was a more potent cofactor than EeS which, in turn, was more effective than DeS (Fig. 2). A similar trend is apparent from Fig. 3, DeS being the least effective of the three glycosaminoglycans. Although only glycosaminoglycanswhich bind thrombin are anticoagulant,the potency of a glycosaminoglycanis not solely a function of the amount of thrombin bound. This may be concluded from Table 2, which shows that the uptake of thrombin by heparin was approximatelytwice that by HeS or DeS (83% vs. 3539%). Yet, in view of the approximatelytwo-fold difference in mol. wt. (27) between heparin (11,000)and DeS (27,000)and HeS (25,000),the quantities of enzyme bound/moleculeof heparin, DeS and HeS were similar (0.25, 0.29 and 0.24 nmoles/nmolerespectively). This suggests that the mode of binding between thrombin and various glycosaminoglycansmay differ, and that these differences may be expressed in allostericeffects. Closer examinationof DeS and HeS in the inactivationprocess showed that, once formed, the thrombin-antithrombin III complex had no affinity for these immobilisedglycosaminoglycans(Fig. 3a,b). This situation differed from the behaviour of the complex on Sepharose-lysine-heparin (Fig. 3c) where dislodgement from the glycosaminoglycanwas not observed until elution with 1M NaCl was used. Recent reports, however, suggest that compared to antithrombinIII, the thrombin-antithrombinIII complex is less attracted to heparin (28,29), Conditions in our column system thereforewere probably not suited to observe this change. From radioactivitymeasurements,a maximum of only 80% of bound thrombin was displaced by antithrombinIII from the DeS and HeS columns although no significantenzyme activity remained in the column. To account for this difference (20%) an explanationcan possibly be found from the

loss

of some

clotting activity during the iodinationprocess. Despite losing 20-252 of the clotting activity 12'I-thrombinbound well to Sepharose-lysine-heparin, -DeS and -HeS as shown in Table 2.

It is likely therefore, that this fraction of

668

AN'l'ITHROMl3IN& GLYCOSAMINOGLYCAN

Vol. 13.No.4

1251-thrombinwhich has been inactivatedduring iodinationwill not interact with, and consequentlywill not become displaced by, antithrombinIII. The binding experimentshave relied on the affinity of the glycosaminoglycans for the cationic conjugates,Sepharose-lysineand polyacrylamideethylenediamine. Using Sepharose-lysine,Des, lies,heparin and, in part, C6S were able to interact well with thrombin but on using polyacrylamideethylenediaminethis property was greatly reduced. At present little is known about the interactionbetween glycosaminoglycansand conjugatesof this type. However, it can be seen that binding to Sepharose-lysineimproves as the amount of lysine conjugated to Sepharose increases (Table 1). the density of amine residues/g is With polyacrylamide-ethylenediamine, much greater than that for the Sepharose-lysinesand therefore interaction of a glycosaminoglycanwith the gel probably involves such a high proportion of the charged groups (0-sulphonyl,N-sulphonyl,-COOH) of the glycosaminoglycanthat few remain available to bind thrombin. For this reason few binding studies were made using polyacrylamide-ethylenediamine.

ACKNOWLEDGEMENTS Our grateful thanks to Mrs. Pat Taylor for technicalassistance,and Dr. Alan Horner (Dept. of Physiology, University of Toronto), for his advice and criticism. The work was supported by the M.R.C. of Canada and the Ontario Heart Foundation. REFERENCES 1.

HATTON, H.W.C. and REGOECZI, E.

The inactivationof thrombin and plasmin

by antithrombinIII in the presence of Sepharose-heparin. Thrombosis e.

10, 645, 1977.

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The effect of heparin adsorbed

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