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Biochimica et Biophysica Acta, 498 (1977) 215--222 © Elsevier/North-Holland Biomedical Press

BBA 28265 EFFECT OF HEPARIN MODIFICATION ON ITS ACTIVITY IN ENHANCING THE INHIBITION OF THROMBIN BY ANTITHROMBIN III

I. DANISHEFSKY, M. AHRENS and S. KLEIN Biochemistry Department, New York Medical College, Valhalla, N.Y. 10595 (U.S.A.)

(Received December 29th, 1976)

Summary Studies were conducted to determine the effect of modifying specific functional groups of heparin on its antithrombin III-enhancing activity. The derivatives employed were heparin methyl ester, heparinylglycine and N-desulfated heparin. The carboxyl-modified derivatives increase the rate of inhibition of thrombin by antithrombin III, although not to the same extent as heparin. N-Desulfated heparin is devoid of any activity. Heparin methyl ester is more p o t e n t than heparinylglycine in activating antithrombin III, as exhibited by its immediate effect on the thrombin-fibrinogen reaction. However, heparinylglycine is the more effective of the two, in increasing the rate of thrombin deactivation by antithrombin III. The results indicate that although free carboxyl groups of heparin are not crucial for its binding to antithrombin III, they are important for the combination of the latter with thromobin. In contrast, N-sulfates are critical for the interaction of heparin with antithrombin III.

Introduction The anticoagulant activity of heparin is ascribed to its effects on several steps in the sequential "cascade" process leading to the production of fibrin. It was demonstrated that maximal activity of this anticoagulant requires the presence of a blood c o m p o n e n t designated as heparin cofactor [ 1 ]. More recently, it was shown that the latter is identical with antithrombin III and that the effect of heparin is to accelerate the action of this natural inhibitor [2--4]. Since antithrombin III neutralizes thrombin, factors IXa, Xa and XIa, heparin effectively antagonizes the activities of these coagulation components [3--9]. Evidence has been presented that heparin binds to lysine residues of antithrombin III and it was postulated that this interaction produces a conformational change in the

216 latter which accelerates its activity against thrombin [4]. There is no information, however, concerning the functional group of heparin involved in its interaction with antithrombin III. We have studied the effects of functional group modifications on the anticoagulant activity of heparin. The uronic acid carboxyl groups were converted either to the methyl ester or to an amide with glycine [10,11]. Another derivative was one in which the sulfamino-sulfates were removed [12]. If a disaccharide section of heparin (omitting O-sulfate) is represented as I

glueosamine-NHSO~ i

uronic acid-COOH i

then derivatives are: ] glucosamine-NHSO~

i

glucosamine-NHSO• I uronic acid-COOCH3

uronic acid-CONHCH2COOH

i

]

i

N-Desulfated heparin

Heparin methyl ester

Heparinylglycine

I

glucosamine-NH2 uronic acid-COOH

I

Investigations on the action of these derivatives in inhibiting the clotting of plasma, revealed that their relative potencies are a function of the specific mode by which coagulation is induced [13,14]. This suggested that the mechanism of inhibition by heparin may not be identical for all the reactions in the coagulation sequence affected by heparin. Investigations were, therefore, initiated to study the activities of modified heparins on individual reactions of the clotting system. The present paper describes the effects of specific modifications on the interaction between antithrombin III and thrombin. Materials and Methods

Heparin. Beef lung heparin, 146 U.S.P. units per mg, generously supplied by the Upjohn Co., was purified further by fractionation of the quaternary ammonium complex [ 15]. Gel filtration of the material on Sephadex G-75 provided a fraction with an average molecular weight of 1.6 • 104 by the method of Iphantis [16]. This material had over 150% of the specific activity of the original heparin. Anal. (%): N, 2.12; S, 10.74; glucosamine, 25.26. Heparin modifications. Heparin methyl ester, heparinylglycine and N-desulfated heparin were prepared and characterized, as described previously [10--12]. Analyses for the preparations used in the present experiments, in terms of percentage by weight, are given below. Heparin methyl ester: N, 2.06; S, 10.67; glucosamine, 24.89; methoxyl, 4.60. Heparinylglycine: N, 3.35, S, 9.42; glucosamine, 22.19; glycine, 8.96. N-Desulfated heparin: N, 2.45; S, 8.73; glucosamine, 29.67. On Lhe basis of these data and titration results [10--12], the carboxyl groups were completely esterified in heparin methyl ester, and over 95% substituted by glycine in heparinylglycine, with no detectable loss of sulfate.

217

Fibrinogen. Human fubrinogen purchased from Calbiochem was purified, according to Laki [17].

Thrombin (EC 3.4.4.13). The thrombin fraction obtained by fractionation of Parke-Davis topical thrombin on D EAE-cellulose [18] was chromatographed on SP-Sephadex [19]. The product was then fractionated on phosphocellulose [20] and the active fractions were dialyzed against 0.15 M NaC1 in 0.1 M Tris • HC1, pH 7.5. The preparation showed a single band on sodium dodecyl sulfate gel electrophoresis and a specific activity of 2200 N.I.H. units per mg. Thrombin activity of the preparations was assayed by its action on fibrinogen [21], with National Institutes of Health thrombin lot 3B, as reference standard. Protein was determined by absorbance at 280 nm, with the appropriate corrections [22] and calculated, using an extinction coefficient, E~ am = 19.5 [23]. Antithrombin III. Antithrombin III was prepared from human plasma by treatment with BaCO3, heat defibrination, adsorption on aluminium hydroxide gel, elution with a m m o n i u m phosphate and affinity chromatography on heparin aminohexyl-agarose, as described previously [24]. The material employed in the present studies was that eluted from the affinity column with 1.0 M NaC1, subsequent to preliminary elution with 0.4 M NaC1. Assays were performed after dialysis against 0.15 M NaC1/0.1 Tris, pH 7.5. The inhibitory action of the antithrombin III on thrombin was determined in terms of progressive antithrombin [25] and as heparin cofactor activity [26]. The units, in these systems, are on the basis of defibrinated plasma which is assigned a value of 100 units per ml. [4]. Specific activity is expressed in terms of mg of protein, based on an extinction coefficient at 280 nm, E~ cm = 5.7 [27]. Accordingly, the preparations employed in the present studies had 690 units of activity per rag. Gel electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis with 10% gel were performed, according to the procedure of Weber and Osborn [28]. The runs were allowed to proceed for about 3 h, at 7 mA per tube. Coomassie Brilliant Blue was used for protein staining. Assay for heparin activity. The activities of heparin and derivatives were measured by their effects on the antithrombin III-mediated inhibition of thrombin. Antithrombin III (0.4--0.5 unit) was incubated for 1 min with 1 mg fibrinogen and limiting amounts of heparin or derivative in a total volume of 0.4 ml 0.15 M NaC1/0.01 M Tris buffer, pH 7.5.0.1 ml of thrombin (8--9 units per ml) were added and the clotting time was determined. The increase in clotting times (ACT), over those of blanks which did not contain heparin, were determined for several concentrations of heparin or derivatives. All assays were performed at least in quadruplicate. The activities of the derivatives were also compared with heparin in their effect in increasing the rate of progressive antithrombin action. Details of this procedure are given in the legend to Fig. 2. Results and Discussion The effects of heparin, heparinylglycine and heparin methyl ester on the heparin cofactor activity of antithrombin III, are shown in Fig. 1. In this system, the concentration of heparin and the derivatives are the limiting factors

218

30

2¢

IC ¢D

O

2

4

6

I

~ug HEPARIN OR DERIVATIVE

Fig. 1. E f f e c t of h e p a r i n a n d d e r i v a t i v e s o n h e p a r i n c o f a c t o r a c t i v i t y . 0.1 m l a n t i t h r o m b i n (4.3 u n i t s p e r m l ) w a s i n c u b a t e d at 3 7 ° C f o r 1 rain w i t h 0.2 m l of 0.5% f i b r i n o g e n a n d v a r y i n g a m o u n t s of h e p a r i n or d e r i v a t i v e in 0.1 ml. T h r o m b i n , 0.1 m l (8.8 u n i t s p e r m l ) w a s a d d e d a n d c l o t t i n g t i m e w a s m e a s u r e d . All s o l u t i o n s w e r e in 0 . 1 5 M NaC1/0.01 M Tris, p H 7.5. T h e i n c r e a s e in c l o t t i n g t i m e , ACT, is the d i f f e r e n c e b e t w e e n t h a t o b s e r v e d w i t h t h e test m i x t u r e s a n d t h o s e w h i c h did n o t c o n t a i n h e p a r i n or derivatives. A c o m p l e t e set of e x p e r i m e n t s , involving five o b s e r v a t i o n s f o r e a c h c o n c e n t r a t i o n , w a s p e r f o r m e d in 1 d a y w i t h t h e s a m e f i b r i n o g e n s o l u t i o n . E a c h p o i n t r e p r e s e n t s the m e a n w h i c h h a d a m a x i m u m d e v i a t i o n +- 4%. S i m i l a r results w e r e o b t a i n e d w h e n t h e c o m p l e t e set w a s r e p e a t e d f o u r times. ©, h e p a r i n ; e, h e p a r i n m e t h y l ester; ~, h e p a r i n y l g l y c i n e .

and the fibrinogen clotting time reflects the relative enhancement of antithrombin III activity. It is seen that heparin methyl ester is the more effective of the two derivatives. N-Desulfated heparin was completely inactive in this system. The effects of heparin or the derivatives were not augmented by increasing the time of preincubation of these materials with antithrombin III. Thus, interaction of antithrombin III with heparin or its modification is maximal within 1 min. By comparing the amounts required to achieve a given extension of clotting time, it was found that heparin methyl ester had 13% of the activity of heparin, whereas heparinylglycine had only 2.6%. The same derivatives were also compared with heparin for their activity in enhancing the progressive neutralization of thrombin by antithrombin III (Fig. 2). The degree of thrombin deactivation, after different time intervals, is reflected by the increase in time required to convert fibrinogen to fibrin; i.e. cloting time. It was necessary to limit the a m o u n t of heparin employed in these

219 9C 8C 7C 6C 5C 4G o 3C

S

~o

JAo

360

SECONDS OF PREINCUBATION

Fig. 2. E f f e c t s of heparin and its m o d i f i c a t i o n s in e n h a n c i n g the a c t i o n of a n t i t h r o m b i n . A s o l u t i o n c o m p o s e d of 0.4 m l a n t i t h r o m b i n ( 2 . 0 u n i t s ) , 0.1 m l t h r o m b i n ( 4 . 2 5 units) and 0.1 m l h e p a r i n ( 0 . 0 5 pg) or d e r i v a t i v e ( 0 . 1 0 pg) w e r e i n c u b a t e d a t 3 7 ° C . A f t e r a given i n t e r v a l , 0 . 1 - m l a l i q u o t s of the m i x t u r e w e r e a d d e d t o 0.3 m l of 0.5% f i b r i n o g e n and t h e c l o t t i n g t i m e w a s n o t e d . All s o l u t i o n s w e r e in 0 . 1 5 M NaCl/ 0 . 0 5 M Tris, p H 7.5. ©, h e p a r i n ; e, h e p a r i n m e t h y l ester; A h e p a r i n y l g l y c l n e ; A n o a d d i t i o n .

experiments, since greater amounts produced long clotting times which could not be determined accurately. The results with heparin m e t h y l ester and heparinylglycine are, therefore, compared with half the a m o u n t of heparin. It can be seen that preincubation with heparin for 150 s increases the clotting time by about 18 s over that effected with antithrombin alone. After a 300 s incubation, the difference is about 45 s. The methyl ester and glycine derivatives showed considerable activity but N-desulfated heparin was inactive. It should also be noted, from the slopes of the lines, that the activity of heparinylglycine is greater than that of heparin m e t h y l ester in this system. Moreover, preincubation for 300 s in the presence of 1.0 pg of heparinylglycine effected the neutralization of more thrombin than did 0.5 pg of heparin. In the heparin cofactor assay system, the activity of heparinylglycine was minimal. The effect of modified heparins on the thrombin-antithrombin III interaction can also be seen from the results of sodium dodecyl sulfate gel electrophoresis. Antithrombin III and thrombin migrate as single bands with apparent molecular weights of 63 000 and 33 000, respectively. Preincubation of the two components yields a new band which is attributed to the thrombin-inhibitor complex [4]. The complex cannot be detected after a 15 s incubation but is visible when the mixture is allowed of interact for at least 2 min. If heparin is added to the mixture of thrombin and antithrombin III, the complex appears within 15 s incubation time [4]. In the present studies, the effects with the heparin derivatives were compared with that of heparin, and with antithrombin III and thrombin alone (Fig. 3). It is seen that the m e t h y l ester and glycine derivatives accelerate the appearance of the thrombin-antithrombin III band, whereas N-desulfated heparin has no effect. Thus, heparinylglycine and heparin methyl ester operate by a mechanism similar to that of heparin; that is, they accelerate the rate of interaction between thrombin and antithrombin III.

220

Fig. 3. S o d i u m d o d e c y l s u l f a t e - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s p a t t e r n s s h o w i n g t h e e f f e c t of h e p a r i n and derivatives on the t h r o m b i n - a n t i t h r o m b i n III interaction. The reaction m i x t u r e for Tube 1 consisted of 0 . 4 8 3 m g of a n t i t h r o m b i n n l a n d 0 . 2 4 8 m g of t h r o m b i n in a t o t a l of 0.6 m l 0 . 1 5 N a C l / 0 . 1 M Tris, p H 7.5. A f t e r 15 s, a 0.1 m l a l i q u o t w a s a d d e d to 0.4 m l 0.01 M s o d i u m p h o s p h a t e b u f f e r , p H 7.0, c o n t a i n i n g 1% m e r c a p t o e t h a n o l a n d 1% s o d i u m d o d e c y l sulfate, a n d h e a t e d f o r 1 m i n a t 1 0 0 ° C . A 1 0 0 pl s a m p l e w a s a p p l i e d t o t h e 10% p o l y a c r y l a m i d e gel. E l e c t r o p h o r e s i s a n d staining p r o c e d u r e s are d e s c r i b e d in t h e exper i m e n t a l s e c t i o n . T h e t w o b a n d s f r o m b o t t o m t o t o p c o r r e s p o n d to t h r o m b i n (T) a n d a n t i t h r o m b i n I n ( A ) , r e s p e c t i v e l y . ( T h i s w a s f o u n d b y p r e l i m i n a r y e l e c t r o p h o r e s e s of t h e single p r o t e i n s . ) T h e i n c u b a t i o n m i x t u r e s f o r s a m p l e s 2, 3, 4, 5 c o n t a i n e d 18 pg of h e p a r i n , h e p a r i n y l g l y c i n e , h e p a r i n m e t h y l ester, Nd e s u l f a t e d h e p a r i n , r e s p e c t i v e l y , in a d d i t i o n t o t h e t w o p r o t e i n c o m p o n e n t s . T h e b a n d a b o v e t h a t of antit h r o m b i n I I I is a s c r i b e d to t h e t h r o m b i n - a n t i t h r o m b i n I I I c o m p l e x ( T A ) . S a m p l e s 6 a n d 7 c o n s i s t e d of the s a m e c o m p o n e n t s , as s a m p l e s 1 a n d 2, e x c e p t t h a t lesser a m o u n t s of t h r o m b i n w e r e e m p l o y e d (67 #g).

T he difference between the relative activities of heparin m e t h y l ester and heparinylglycine in the two assay systems indicates that there are two aspects in the effect o f heparin. One of these is binding of a n t i t h r o m b i n III to heparin. This is reflected by the heparin cof a c t or assay which measures the degree of immediate neutralization of t h r o m b i n by heparin-antithrombin III. The sulfamino-sulfates o f heparin are essential for its binding to antithrombin III but free carboxyl groups are n o t critical. Thus, heparin m e t h y l ester, with no carboxyls, has greater activity than heparinylglycine. The presence of the glycine residue on the latter pr obabl y introduces steric hinderance which decreases its relative binding to a n t i t h r o m b i n III. The ot h er action of heparin is to funct i on directly in the co m bi na t i on of ant i t hr om bi n III with thrombin. In this capacity it serves to increase the rate of interaction between the two proteins. This

221 process is measured by the enhancement of the progressive antithrombin effect. It is proposed that, in this reaction, the carboxyl groups in heparin play an important role. Thus, although heparinylglycine is not as effective as heparin methyl ester in activating antithrombin III, it is the more p o t e n t of the two in accelerating the reaction between thrombin and antithrombin III. The fact that heparin binds thrombin, as well as a number of other procoagulants, is well established [24,29--31]. Therefore, in addition to activating antithrombin III, heparin can function in the actual combination of the inhibitor with the serine protease coagulant. This rationale is consistent with our previous finding that specific heparin modifications do not have the same potencies, relative to heparin, for different sites of the coagulation cascade which are inhibited by antithrombin III [13,14]. This is conceivable, if heparin does not have the identical structural specificity for all the procoagulants affected by antithrombin III. Studies are currently in progress to determine functional group specificity of heparin in enhancing the anti-factor Xa activity of antithrombin III. Acknowledgements The authors wish to express their thanks to Dr. L.L. Coleman for samples of heparin and to Dr. Aronson for N.I.H. thrombin standards. In addition, we are indebted to R. Barone for his help in obtaining human plasma. These investigations were supported by Grant HL 16955, from the National Institutes of Health, U.S. Public Health Service. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

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27 Kurachi, K., Schmer, G., Hermodson, M.A., Teller, D.C. and Davie, E.W. (1976) Biochemistry 15, 368--373 28 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4406--4412 29 Gentry, P.W. and Alexander, B. (1973) Biochem. Biophys. Res. Commun. 50, 500--506 30 Li, E.H.H., Orton, C. and Feinman, R.D. (1974) Biochemistry 13, 5012--5017 31 Machovich, R. (1975) Biochim. Biophys. Acta 412, 13--17