The effect of polycations on the inhibitory action of antithrombin iii on thrombin in the absence and presence of polyanions

THROMBOSIS RESEARCH 43;

187-193,1986 0049-3848/86 $3.00t .OO Printed in the USA. Copyright (c) 1986 Pergamon Journals Ltd.

All rights reserved.

THE EFFECT OF POLYCATIONS ON THE INHIBITORY ACTION OF ANTITHROMBIN III ON THROMBIN IN THE ABSENCE AND PRESENCE OF POLYANIONS U. Schiele

and

C.C.

Heuck

UniversitZtskinderklinik, Zentrallabor Im Neuenheimer German Federal Republic 69 Heidelberg,

(Received

Feld

150,

21.10.1985; Accepted in revised form 16.4.1986 by Editor H.C. Godal)

ABSTRACT Polycations of biological and synthetic origin inhibit the action of AT III on thrombin activity. The effect is more pronounced with increasing molecular weight of branched polycations.Quantitatively protamine causes the same effect as quaternized polycations on the basis of charge equivalence.The accelerating effect of heparin or potassium polyvinylsulfate for the inhibitory action of AT III is abolished by charge equivalent amounts of polycation.The observations indicate a dual action of polycations in the heparin/AT III/thrombin interaction.

INTRODUCTION

Antithrombin III (AT III> is the most potent biological inhibitor of thrombin.The inhibiting action is accelerated by heparin.The postulated action of heparin as a true catalyst 11,2> was questioned because of a variety of other observations (3>.The effect of heparin is abolished by protamine (4) or aprotinine (5).In view of the opposite surface charge of the antagonists the abolishment of the heparin effect is assumed to result from intermolecular charge neutralization.Our present studies indicate that polycations interfere with the thrombinAT III interaction as well as the accelerating effect of highly charged polyanions. key words: polyelectrolyte.ionic interaction,polycations. 187

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EXPERIMENTAL PROCEDURES _---------------------Highly purified human alpha thrombin and human AT III was offered by Dr. Fughe,Behr-ingwerke AG,Marburg.Highly purified heparin (mw 14000) was a gift from Dr.Herr,Knoll AG,Ludwigshafen. Polydimethyl-diallylammonium chloride (PDDA,mw l-2 millionjwas from Chemviron,Eruxelles,N-methylglycolchitosane iodide !MGC, mw 17000) was received from ICN Pharmaceuticals,Plainview,USA. Protamine sulfate (mw 5000) and the other chemicals were from Merck GmbH,Darmstadt.Polyethylene imine with different molecular weight (PEI 10,mw 400; PEI 20,mw 1300; PEI 50,mw 4000; PEI 100, mw 10000; PEI 508,mw 25000; polymine P,mw 600000)was presented by Dr.Horn,EASF Aktiengesellschaft,Ludwigshafen.Polybren cl,5 dimethyl-1,5-diazaundec~ametliylene-polymethobromide~ was ordered from Ega-Chemie,Steinheim,potassium polyvinylsulfate (KPVS, mw 320000) from Serva GmbH,Heidelberg,Chromozym Th (Tos-glypro-ar,+-4-nitranilide acetate) from Eoehringer Mannheim,Mannheim. All reagents were used without further purification.!X)

Determination of surface charge of polyelectrolytes ---___--___---___---__---_-_----_~----_----_~----The number of charged residues of polyanions and polycations was determined by polyelectrolyte titration as previously described (6) at the appr-opriate pH as used in the subsequent experiments of thrombin activity determination.

Measurement of thrombin activity in the presence of AT III _---_-_----~~~--~_~_-_~~____~----_-----------_-----~___-~~ after preincubation with polycations -_____--___-----------------------Aliquot amounts of AT III (50 x10 E-12 M> were incubated with polycations (MGC ,Polybren ,PEI ,PDDA ,protamine ) in 550 ul Of TRIS/HCl buffer (0,05 molar,pH 8,4 containing 0,15 molar NaCl).The amount of polycation was calculated on the basis of charge equivalents (eqJ added to the solution.It varied between 0,5 and 6 x10 E-10 eq per incubation.After preincubating (5 x10 E-12 M > was added. for 5 min at 37 C thrombin Exactly 2 min. later Chromozym Th (4 xl0 E-10 M in 10~1) was added to start the measurement cf the activity of thrombin at 405 nm The total volume in each experiment was 790 ul.Control experiments were made without the addition of the polycation. (X) molecular weights of monomers of polyelectrolytes: PDDA:161,5; PEI:43; Polybren:194; MGC:419. ;KPVS:162

POLYCATIONS,AT III ANDTHROMBIN

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189

Measurement of thrombin activity in the presence of AT III and -----__--_----_-__--~---~_--_-------__------_--~-------------heparin or KPVS after preincubation with polycations ----~---------------~---~--_-___-_-_--_--__---_---_Thrombin (9 xl0 E-12 M> was incubated with heparin (195 iU/mg; 12,l ng =7,6x10 E-10 eq) or KPVS (12,3 ng =7,6x10 E-10 eq) in 500 ul of 0,05 molar THIS'HGl enriched with 0,15 molar- NaCl at pH E1,4 and 37 C.After 5 min. an aqueous solution of polycation was added.The amount of polycation varied ( 0,0x,1,2x,1,3x, ;!,4x,3,rjxand 9,5x 10 E-6 eq).After exactly 5 min. AT III (18 xl0 E-12 M in 10 ul of buffer) was added.After subsequent 5 min . the enzymatic reaction was started with Chromozym Th (4 xl0 E-10 M in 10 ~1). In parallel experiments thrombin was replaced by AT III and vice versa.

The effect of polycations on the AT III/thrombin reaction was investigated at an inhibitor concentration causing a 93% inbition of thrombin after 5 min.Linear quaternized polycations :PDDA,MGC and Polybren) relieve the inhibition by AT III uniformly and independent of their molecular weight and chemical structure
70-

I 2

Thr-ombin actiivity and polycations.

1 4

Fig.1 in the presence

I 6 ~10-‘~ Val Polycat ion

of AT III (molar ratio 1: 10)

POLYCATIONS, AT

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III AND THROMBIN

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However,the original thrombin activity is restored only by 58 to 78 %.Protamine acts like the synthetic polycations.Maximal effects are similar with the branched Polymin P,however the action of the nonquaternized polycation is more concentration dependent. Studies with PEI of smaller molecular weights also show a molecular weight dependent effect (fig.2)*With PEI 20 amounts twice as high as those for PEI 50,PEI 100 and PEI 500 are needed to cause the same degree of interference.PEI 10 inhibits the action of AT III only to a small extent.No effects are observed with tetraethylene pentamine and triethylene tetramine (not shown>. % of total Thrombin activity 80-

I I

2

I

4

I

6.16”Val

PEI

Fig.2 Thrombin activity in the presence of AT III (molar ratio 1:10> and PEI of different molecular weights. For the investigation of the effect of polycations on the action of heparin we chose a 1:l molar ratio for AT III/thrombin causing an inhibition of the thrombin activity after 5 min in the absence of heparin by 10 % and in the presence of heparin by 95 %. In parallel experiments we replaced heparin by the synthetic polyanion KPVS.The accelerating action of the polyanion was studied in the presence of MGC,PEI 50 and protamine at defined stoichiometrical charge equivalence.According to polyelectrolyte titration the degree of ionisation at pH 8,4 for MGC is 100 % (6), for PEI 50 it is 70 % (7) and for protamine it is 90 %.

Vol.

43,

No.

POLYCATIONS,

2

thrombin

AT

III AND THROMBIN

191

activity

% 100

+ pEl)+thrombin I

1

0,l 42

I

I

1,0

0,5

eq eq

PEI KPVS

% 100

+F

80

/I 0

60

+-+

/ I

0,l 0,2

0;s

+

LO 20

+/+ 0-O

o-0

O

0 ([thrombin+heparin] +PEl)+At-IIl

1

+ ([At-III+heparin] + PEI )+thrombin

1,0

eq PEI eq heparin

Fig.3 Thrombin activity in the presence of AT III and polyanions. Thrombin or AT III was preincubated with KPVS (upper diagram> or heparin (lower diagram>,Subsequently,PEI 50 was added. After 5 min. AT III was added to the thrombin solution to give a molar ratio 1:l with the enzyme or conversely thrombin was added to the AT III solution.The thrombin activity was started exactly 5 min. later by addition of Chromozym Th. The polycation/polyanion ratio is given on the basis of charge equivalence. The charge neutralisation of heparin and KPVS,respectively,by polycations abolishes the accelerating effect independent of the sequence of preincubation.The effect is identical independent of the chemical composition of the polycation. sition of the polycation.

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AND THROMBIN

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DISCUSSION ___---_---

The results of the study indicate that polycations interfere with the inhibitory action of AT III on thrombin.Obviously structural differences of the polycations are of minor importance as long as the high surface charge is maintained and the number of positively charged residues exceeds 25.The polycationic effect decreases however with small molecular weight polymers in parallel to the reduction of polymerisation.AT III has a negative surface charge under the experimental conditions and binds electrostatically to polycations (8,9>*Such an interaction does not occur between thrombin and polycations (S).This finding is further confirmed by the observation that the thrombin actity remains unchanged in the presence of polycations when AT III is absent-we therefore conclude that the inhibitory action of AT III is prevented through steric hindrance by polycations bound to the glycoprotein. The inhibitory effect of protamine and aprotinine on the accelerating action of heparin is commonly believed to result from ionic complex formation (lB,ll).Kitany et al. observed that protamine forms complexes with heparin (12).The molecular weight of these complexes is multiply higher than that of the isolated biopolymers.On the other hand they observed a recurrence of the slowly progressive AT III activity in a fraction with a molecular weight slightly above that of albumin,when protamine is added to a heparin-AT III solution.Therefore they concluded that protamine disrupts the heparin-AT III complex. Stone and Epstein found a charge ratio equal to 1 in stabile complexes between heparin and lysine-tyrosine copolymers (13). Correspondingly we observed a stoichiometric ionic binding between heparin and thrombin (9).Our present data indicate that independent of their chemical structure highly charged polycations interact stoichiomtrically with heparin and KPVS to form stabile ionic complexes.Such complexes do not exhibit a quasi-catalytic action like heparin alone (9). Obviously,the polycations compete with thrombin in the interaction with heparin In this light the abolishment of the accelerating action of heparin by charge neutralisation is not unexpected.It does not however not explain the dissociation of the nonionic interaction between heparin and AT III.

REFERENCES ---------1.

BJt)RK,I.and NORDENMAN,B.Acceleration of the reaction between thrombin and antithrombin III by non-stoichiometric amounts of heparin.Eur.J.Biochem,68, 507-511, 1976 ------------- --

2.

KOWALSKY,S. and FINLAY,TH.H.Heparin and the inactivation of thrombin by antithrombin III.Thromb.Res,l4, 387-397,1979 __-_------ __

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III AND THROMBIN

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3. HURST,R.E.,MENTER,I.M.,WEST,S.S.,SETTINE,J.M. and COYNE,E.H. Structural basis for the anticoagulant activity of heparin. Relationship to the number of charged groups. Biochemistry,l8, 4283-4287, 1979 ------------ -_ 4. JORPES,E,,EDMAN,P. and THANING,R.Neutralisation of heparin by protamine.Lancet, 3, 975-976, 1939 ------ 5. DAVIES,R.J.,LANE,D,A.,GREGOR,I.R. and PRESTON,F.E.The neutralisation of heparin by Trasylol.Thromb.Res,17, 533-537, 1980 ---------- -6. HORN,D.and HEUCK,C.C.Charge determination of proteins with polyelectrolyte titration.J.Biol.Chem,258, 1665-1670, 1983 ---------_- --7. HORN,D.in "Polymeric amines and ammonium salts"(GOETHALS,E.J., ed.) Pergamon Press,New York,pp. 333-344,1980 8. KERA,Y.and YAMASAWA,K.Isoelectric focusing pattern of human antithrombin III (AT III): Effects of heparin or thrombin on a micro heterogenous form of AT III, Electrophoresis, 3, 157-161, 1982 __--_--_--__-__ -_ o -1 HEUCK,C.C.,SCHIELE,U.,HORN,D.,FRONDA,D.and RITZ,E.The role of surface charge on the accelerating action of heparin on the antithrombin III- inhibited activity of alpha-thrombin. J.Biol.Chem,260, 4598-4603, 1985 ----_--_--- -_10.TAKAYAMA.T.,TAKAI.K.I.,KARIYAMA,R. and KANEMASA,Y.Colloid titration of heparin using catfioc ( polydiallyi-dimethyl ammonium chloride > as standard polycation. Anal,Biochem,88, 382-387, 1978 _---------_- _11

EDWARDS,H.E.,NAVARATNAM,S.,ALLEN,J.C. and PHILLIPS,G.O. The determination of the heparin neutralizing capacity of pr-otamineusing acridine orange fluorescence. Thromb.Haemostas,43, 108-111,1980 ---------------- --

1. 2 KITANI,T.,NAGARAJAN,S.C. and SHANBERGE,J.N.Effect of protamine on heparin-antithrombin III complexes. Thromb.Res,l7, 367-374,1980 -------_-- __ 13.STONE,A.L. and EPSTEIN,P.The aggregation of basic polypeptide residues bound to heparin.Biochim.Biophys.Acta,497, 298-306, _-_________--------- --1977