Biochimica et Biophysica Acta, 412 (1975) 13-17

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA 37185 M E C H A N I S M OF A C T I O N OF H E P A R I N T H R O U G H T H R O M B I N ON BLOOD C O A G U L A T I O N

RAYMUND MACHOVICH Postgraduate Medical School, First Department of Medicine, 1389 Budapest (Hungary)

(Received May 7th, 1975)

SUMMARY It has been suggested that heparin can affect blood coagulation through thrombin, i.e. the binding of heparin to thrombin induces a conformational change in the enzyme, facilitating a complex formation between thrombin and antithrombin (Machovich, R., Blask6, Gyo and Palos, L. (1975) Biochim. Biophys. Acta 379, 193-200). This hypothesis seems to have been proved. Modification of arginine residues in thrombin did not result in decreased thrombin activity and decreased sensitivity to antithrombin, whereas the heparin sensitivity of the enzyme and the thrombin-antithrombin reaction were diminished.

INTRODUCTION It is generally believed that heparin affects blood coagulation through antithrombin-III. However, heparin binding to thrombin (at a site distinct from its active centre) has also been demonstrated by different methods [1, 2]. As the thrombinheparin interaction is tight with an average constant of 10 -7 M [2] and is stronger than the heparin-antithrombin binding, it was suggested that heparin may also affect blood coagulation through thrombin [3]. Namely, the binding of heparin to thrombin can induce a conformational change of the enzyme resulting in an accelerated inactivation by antithrombin. Since recently even the importance of antithrombin-heparin interaction was doubted [4], this new theory about the effect of heparin on the thrombinantithrombin reaction seems to be reasonable. Furthermore, thrombin proved to exist in multiple forms with respect to heparin sensitivity [5], therefore the mechanism of a conformational change of the enzyme induced by heparin has become an important question. MATERIALS AND METHODS Thrombin activity was assayed in a 0.25 ml reaction mixture containing 10/tmol sodium phosphate buffer, p H 7.4, and 500/zg fibrinogen (KABI, Grade L. human, lyophilized). Clotting time was measured at 37 °C with the Hyland Clotek System. Thrombin activity, expressed in N.I.H. units, was calculated from a standard

14 calibration curve established by U.S. Standard Thrombin (Human, Lot H-I) and demonstrated on a semilogarithmic scale. Thrombin was partially purified from Topostasin (Hoffman-La Roche) by DEAE-cellulose chromatography according to the method of Yin and Wessler [6]. The specific activity of thrombin was about 150 N.I.H. units/mg. Protein was determined according to the method of Lowry et al. [7]. Antithrombin-III was purified as published elsewhere [3]. Antithrombin activity was measured in a 0.2 ml reaction mixture containing 10 #mol sodium phosphate buffer, pH 7.4, and the appropriately diluted thrombin protein, antithrombin protein with or without heparin (G. Richter Pharm., Ltd), respectively. After incubation at 37 °C for varying periods of time, 500 #g fibrinogen solution in a 0.05 ml volume was added and clotting time measured with the Hyland Clotek System. Treatment of protein with cyclohexanedione was carried out as published by Patthy and Smith [8]. 1.5-2 mg thrombin protein was incubated in plastic tube at 37 °C in 4 ml of reaction mixture containing 0.2 mmol cyclohexanedione (Koch-Light Laboratories Ltd) and 0.2 mmol sodium borate buffer, pH 9.0. After 6 min incubation, the reaction mixture was gel filtered on a Sephadex G-25 column equilibrated with 0.05 M sodium phosphate buffer, pH 7.4 (Tm-thrombin). As a control, the same procedure was also carried out without cyclohexanedione (Ts-thrombin). RESULTS

Effect of antithrombin and heparin on modified thrombin Arginine residues of thrombin were modified by cyclohexanedione in sodium borate buffer at pH 9.0. The reaction mixture was gel filtered on a Sephadex G-25 column equilibrated with 0.05 M sodium phosphate buffer, pH 7.4 (Tm-thrombin). As a control, the same procedure was also carried out without cyclohexanedione (Ts-thrombin). As the specific activity of thrombin was not changed after a 6 min treatment and the clotting times were altered in a similar manner with the dilution of both forms of thrombin (Ts- and Tm-thrombin), it can be assumed that the active centre (the proteolytic activity) of Tm-thrombin was not changed. Ts-thrombin alone or with heparin were not inactivated at 37 °C in a 5 min incubation. In the presence of antithrombin, about 50 ~ of enzyme activity was inhibited in a 2 rain incubation (Fig. 1). If heparin was also in the reaction mixture, the rate of inactivation increased and about 90 ~ of the activity was already lost in a 0.5 min incubation. These kinetics of thrombin inactivation are in agreement with the kinetics of the so-called progressive antithrombin-III found in the literature. Tm-thrombin behaved differently. Tm-thrombin alone or with heparin also showed no inactivation in a 5 min incubation. However, in the presence of antithrombin the rate of inactivation was equivalent to the Ts-thrombin inactivation (Fig. 2). That is, 50 ~ of Tm-thrombin activity was inhibited by antithrombin alone in a 2 min incubation. Although, heparin, at the same or twice as high concentration as was used for Ts-thrombin inactivation, resulted in an increased thrombin inactivation in the first minute, it was a very slight effect compared with Ts-thrombin inactivation. During further incubation even this moderate effect was diminished, Tm-thrombin acted toward antithrombin as if heparin were not present. On the basis of these

15

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Fig. I. Inhibition of Ts-thrombin activity by antithrombin and heparin. 20/~g Ts-thrombin protein was incubated either alone, or with 0.05/~g heparin, or with 200/~g antithrombin protein, or with antithrombin plus heparin, under the conditions as detailed in Materials and Methods. Results are demonstrated on a semilogarithmic scale. (3--(3, Ts-thrombin alone; ~ , Ts-thrombin plus heparin; ~ - - ~ , Ts-thrombin plus antithrombin; &--~,, Ts-thrombin plus antithrombin plus heparin.

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Fig. 2. Inhibition of Tm-thrombin activity by antithrombin and heparin. 25/~g Tm-thrombin protein was incubated either alone, or with heparin, or with 200/~g antithrombin protein, or with antithrombin plus heparin, under the conditions as detailed in Materials and Methods. Results are presented on a semilogarithmic scale. 0 - - ( 3 , Tm-thrombin alone; O - - ~ , Tm-thrombin plus heparin; rq--[~, Tm-thrombin plus antithrombin; M--R, Tm-thrombin plus antithrombin plus heparin (0.05/zg); ~ - - ~ , Tm-thrombin plus antithrombin plus heparin (0.1/~g).

16 kinetics, it may be supposed that the heparin effect in the first minute can be attributed to a contamination by Ts-thrombin (not every molecule of thrombin was modified with cyclohexanedione). As to the structure of modified thrombin, since the effect of heparin on Tmthrombin and antithrombin reaction was diminished in a phosphate-buffer system, it may be suggested that a conformational change of the enzyme induced by heparin is protected without neutralizing the positive charge of arginine. Whether this specific interaction of cyclohexanedione with arginines of thrombin inhibits heparin binding or whether heparin can be bound to modified thrombin without inducing a conformational change, needs further examination.

Heat inactivation of Ts-thrombin and Tm-thrombin Thrombin can be inactivated easily at 54 °C. As it can be seen from Fig. 3, about 70 % of Ts-thrombin activity was already lost in a 2 min incubation. However,

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Fig. 3. Heat inactivation of Ts-thrombin and Tm-thrombin. 240 #g Ts-thrombin or 260/~g Tmthrombin protein was incubated either alone or with 100 #g heparin, respectively, in 0.55 ml volume containing 20/~mol sodium phosphate buffer, pH 7.4, in glass tubes. Varying periods of time 0.05ml aliquots of reaction mixture were taken, determined for clotting time as detailed in Materials and Methods. Results are demonstrated on a semilogarithmic scale. ~ - - ~ , Ts-thrombin alone; i,--~,, Ts-thrombin plus heparin; ~ 3 - - ~ , Tm-thrombin alone; I - - I I , Tm-thrombin plus hel3arin.

the inactivation of Ts-thrombin decreased in the presence of 200 #g heparin per ml. Even after a 10 min incubation, the remaining thrombin activity was equivalent to the thrombin activity after 2 rain incubation without heparin. Tm-thrombin acted in another way. It showed a higher stability at 54 °C than Ts-thrombin, namely, it behaved as if heparin had been present in the reaction mixture. That is, even after a 10-rain incubation about 30 % of the initial activity of Tm-thrombin remained. Heparin did not essentially influence these kinetics of Tmthrombin inactivation at the concentration mentioned above. The moderate protection can be attributed to a contamination of Tm-thrombin by Ts-thrombin.

17 DISCUSSION According to the findings, at least three conformations of thrombin can be proposed. One is a basic form, another is a conformation of thrombin induced by heparin and a third, a structure formed after modification of its arginines. The second and the third forms are stable against heat inactivation but only the second conformation is sensitive to antithrombin. Although the belief that heparin acts on blood coagulation through antithrombin is generally accepted [9, 10], i.e. heparin is bound to antithrombin inducing an "allosteric modification in it" Ill], the inactivation of thrombin by antithrombin accelerated with heparin can now be explained by another way too. It is not of minor importance from the point of view of either the biochemical mechanism and the regulation of blood coagulation, or from the medical experience as well, (as under normal conditions, thrombin does not exist, while antithrombin has a permanent level in blood circulation), which process takes place in plasma. The findings presented in this paper suggest that heparin affects blood coagulation primarily through thrombin, since heparin loses its accelerating effect on thrombin-antithrombin reaction if the arginines of thrombin are modified by cyclohexanedione. These results raise another question too, namely whether there is an in vivo possibility for the conversion of a heparin-sensitive conformation of thrombin into a non-sensitive form. If so, it can give an explanation to the findings presented elsewhere [5], i.e. the heparin-sensitive and non-sensitive forms of thrombin may take place without a change in the amino acid sequence of the enzyme. ACKNOWLEDGEMENTS We are grateful to Dr L. Patthy for his advice on the modification of arginine residues. We thank Mrs Therese Fazekas and Mrs ,~gnes Himer for their excellent assistance. REFERENCES 1 2 3 4 5 6 7

Machovich, R., Blask6, Gy. and P~los, L. (1974) Abstr. 9th. Meet. FEBS, Budapest, 1974, p. 375 Li, H. E., Orton, C. and Feinman, D. R. (1974) Biochemistry 13, 5012-5017 Machovich, R., Blask6, Gy~ and P~los, L. (1975) Biochim. Biophys. Acta 379, 193-200 Marciniak, E. (1974) J. Lab. Clin. Med. 84, 344-356 Machovich, R. (1975) Biochim. Biophys. Acta 400, 62-68 Yin, E. T. and Wessler, S. (1968) J. Biol. Chem. 243, 112-117 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 8 Patthy, L. and Smith, L. E. 0975) J. Biol. Chem. 250, 557-564 9 Yin, E. T., Wessler, S. and Stoll, J. P. (1971) J. Biol. Chem. 246, 3712-3719 10 Rosenberg, R. and Damus, P. (1973) J. Biol. Chem. 248, 6490-6505 ll Damus, P., Hicks, M. and Rosenberg, R. (1973) Nature 246, 355-357

Mechanism of action of heparin through thrombin on blood coagulation.

It has been suggested that heparin can affect blood coagulation through thrombin, i.e. the binding of heparin to thrombin induces a conformational cha...
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