Antithrombotic and haemorrhagic effects of synthetic and naturally occurring thrombin inhibitors

THROMBOSIS RESEARCH 43; 613-620, 1986 0049-3848/86 $3.00 t .OO Printed in the USA. Copyright (c) 1986 Pergamon Journals Ltd. All rights reserved.

ANTITHROMBOTIC AND HAEMORRHAGIC EFFECTS OF SYNTHETIC AND NATURALLY OCCURRING THROMBIN INHIBITORS

B. Kaiser and F. Markwardt Institute of Pharmacology and Toxicology, Medical Academy Erfurt, DDR-5010 Erfurt, G.D.R.

(Received 2.6.1986; Accepted in original form 11.6.1986 by Editor J. Hladovec)

ABSTRACT The effects of the synthetic thrombin inhibitor Na-(2-naphthylsulfonylglycyl)-4-amidinophenylalanine piperidide (fiNas-Gly-(pAm)Phe-Pip) and the naturally occurring inhibitors hirudin and heparin on the bleeding time were studied in mice and rats by the method of transection of the tail tip and of standardized incision of the tail. With both methods the thrombin inhibitors prolonged the bleeding time in dependence on the dose and the plasma concentration obtained. The transection bleeding time was influenced by the inhibitors in a similar manner, whereas in the case of incision of the tail heparin caused a more pronounced effect than hirudin and DNas-Gly(pAm)Phe-Pip. Comparison of the antithrombotic actions of the inhibitors with their effects on the bleeding time showed that, in contrast to the selective thrombin inhibitors hirudin and BNas-Gly-(pAm) Phe-Pip, antithrombotically effective doses of heparin induced a clear prolongation of bleeding time. INTRODUCTION Naturally occurring and synthetic inhibitors of the coagulation enzyme thrombin are potential drugs for the control of blood coagulation (1, 2). Hirudin, the selective thrombin inhibitor from medicinal leeches, and Na-(2_naphthylsulfonylglycyl)-4-amidinophenylalanine piperidide, the most potent synthetic competitive inhibitor of thrombin described so far (3), have been shown to exert pronounced anticoagulant and Key words: Thrombin inhibitors Bleeding time

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antithrombotic actions (4, 5, 6, 7, 8, 9). The anticoagulant heparin which exerts its inhibitory effect on the clotting enzyme indirectly via a potentiation of the inhibitory function of the endogenous antithrombin III is an effective antithrombotic agent, but its major side effect when administered therapeutically is bleeding. Therefore, the antithrombotic properties of naturally occurring and synthetic thrombin inhibitors were studied in relation to their effects on the bleeding time as a reliable test for measuring the influence of drugs on primary haemostasis.

MATERIALS AND METHODS Experimental animals: Mice (strain "AB/MCR", 20 - 25 g body weight) and rats (Wistar strain "Jena", 250 - 350 g body weight) were kept under conventional conditions. Anaesthesia was carried out with 1.5 g ethyl urethane/kg intraperitoneally. Thrombin inhibitors: Hirudin, specific activity 15,000 AT-U/mg, was prepared according to (IO). Heparin sodium (5,000 I.U./ml) was obtained from SPOFA, Prague, Czechoslovakia. For calculation on a weight basis an activity of 150 I.U./mg corresponding to mean specific activities of commercial preparations was assumed. Na-(2-naphthylsulfonylglycyl)-4-amidinophenylalanine piperidide (BNas-Gly-(pAm)Phe-Pip) was synthesized by WAGNER et al. (11). For intravenous administration the inhibitors were dissolved in physiologic saline, the solution of ONas-Gly-(pAm)Phe-Pip contained 5 to IO per cent dimethylformamide. Stasis-induced venous thrombosis: In anaesthetized rats venous clotting thrombi were induced using the method of WESSLER et al. (12). A segment of the jugular vein of 15 to 20 mm in length was exposed and dissected free of surrounding tissue. Glass contact-activated human serum was given via the femoral vein in a dose of 1.32 ml/kg. The jugular vein segment was ligated at both ends 30 s after serum administration. After IO min the segment was removed and the thrombi formed were evaluated macroscopically after rinsing out the contents of the segment into a sodium citrate-filled Petri-dish. Thrombus size was graded by a score: 0 = fluid blood; 1 = one or several small clots; 2 = not fully occluding thrombus; 3 = fully occluding thrombus. For calculation of relative thrombus score the mean thrombus size in an inhibitor-treated group was divided by the mean thrombus size in the control group. The inhibitors were given as intravenous infusion starting 30 min before serum administration over the whole period of thrombus formation. Bleeding time in rats: Bleeding time in anaesthetized rats was measured after standardized incision of the tail (13). The amount of blood escaped from the incision wound was determined by conductometry. The tail of the rat was superfused with saline solution (1.54 mmol/l, 37 "C) and the conductivity of the solution was measured in a flow-through-cell and con-

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tinuously recorded. At the ventral part of the tail a standardized incision (length 5 mm, depth 1 mm) was made without injuring a tail vein or artery. The bleeding time was determined from the course of the conductivity vs time curve. The thrombin inhibitors were administered as intravenous infusion starting 30 min before incision of the tail. In each animal the individual control value was determined at a proximal part of the tail 15 min before the infusion of the inhibitor. Bleeding time in mice: Bleeding time in conscious mice was measured after both transection of the tail tip and standardized incision of the tail (13). The tail was transected 0.5 mm from the tip using a razor blade. The tail was placed in vertical position in isotonic saline solution (37 "C) immediately after injury. Bleeding time was defined as the time from the transection of the tail tip until complete cessation of bleeding. The thrombin inhibitors studied were injected intravenously 5 min before transection of the tail tip. The bleeding time at standardized incision of the tail was measured as described above. The incision of the tail was made 15 min after intravenous injection of the inhibitors and the change of conductivity of the saline solution flowing round the tail was recorded. Determination of plasma concentrations of the inhibitors: The concentrations of the inhibitors in plasma were determined using the thrombin clotting time method.-Citrated blood (1 part of 0.11 mol/l trisodium citrate + 9 parts of blood) was drawn from the cannulated carotid artery in rats and by cardiac puncture in mice. After centrifugation (10 min at 2,500 g for rat and at 1,000 g for mouse plasma) the plasma thrombin times were measured: A volume of 0.1 ml of plasma was mixed with 0.1 ml of Tris buffer (0.1 mol/l, pH 7.5); after addition of 0.05 ml of thrombin solution (20 NIH-U/ml in physiologic saline for rat and 10 NIH-U/ml for mouse plasma) the time until a firm clot was formed was measured. Concentrations of the inhibitors in the ex vivo samples were estimated by means of calibration curves obtained with plasma to which defined amounts of the inhibitors were added.

RESULTS Intravenous injection of glass contact-activated human serum into rats followed by stasis in a segment of the jugular vein led to the formation of typical clotting thrombi. Intravenous infusions of hirudin, heparin and BNas-Gly-(pAm)Phe-Pip reduced significantly the size of thrombi or prevented thrombus formation. These antithrombotic effects were clearly dosedependent (Fig. I). The doses and resulting plasma concentrations of the inhibitors required for prevention of stasisinduced venous thrombosis are shown in Table I. In order to study the relation between antithrombotic and haemorrhagic effects of the inhibitors the bleeding time in rats after standardized incision of the tail was investigated using antithrombotically equieffective doses. As shown in

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Table I the administration of doses of hirudin and DNas-Gly(pAm)Phe-Pip which prevented thrombus formation caused no significant prolongation of bleeding time. In contrast, the bleeding time was significantly prolonged after infusion of corresponding doses of heparin. Doubling of the antithrombotically effective doses of hirudin and RNas-Gly-(pAm)Phe-Pip prolonged the bleeding time by about 50 to 60 % of the control value. In case of heparin an increase in bleeding time of more than 500 % (range 160 to 1,300 %) was found.

l.O-

t 0.1

I

1 Dose

[pg

I

1

10

100

/kg - min]

FIG. 1 Dose-response curves of hirudin ( ??), heparin (I ) and BNas-Gly-(pAm)Phe-Pip (or) in stasis-induced venous thrombosis in rats (n = 6) TABLE I Influence of antithrombotically equieffective doses and plasma concentrations of thrombin inhibitors on bleeding time in rats (n = 5) Inhibitor

Hirudin

Dose (pg/kg.min) 2.6

Plasma con-, centration (ug/ml) 0.39

13.3

7.87

26.6 DNas-Gly(pAm)Phe-Pip

z;*: .

+ 6;:: f

5.3 Heparin

Prolongation of bleeding time (X, ? +_ S.D.)

0.70

3'0:::

45.2 =_ 27.4 512.3 - 492.6 19.9 $ 50.6 -

17.1 18.6

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In mice the dose-dependency of the inhibitors on the bleeding time was studied after transection of the tail tip and standardized incision of the tail. It could be shown that the intravenous injection of the inhibitors prolonged the bleeding time in mice after transection of the tail tip in dependence on the dose and the plasma concentration obtained (control value 1.38 - 0.37 min, n = 71). The plasma concentration-response curves for the effect of hirudin, heparin and fiNas-Gly-(pAm)Phe-Pip on the transection bleeding time showed nearly the same course (Fig. 2).

r

OS

I

1

10

1 Plosma concentration

[pg ImlJ

FIG. 2 Plasma concentration-response curves of hirudin ( ??), heparin (I) and fiNas-Gly(pAm)Phe-Pip (4) for bleeding time in mice at transection (closed symbols) and incision (open symbols) of the tail (x + S.E.M., n = 10-18) The bleeding time in mice after standardized incision of the tail was alst prolonged by the thrombin inhibitors (control value 2.51 - 0.63 min, n = 16). The effect of hirudin and BNas-Gly-(pAm)Phe-Pip in this model was similar to or nearly identical with that on the transection bleeding time, whereas heparin showed a more pronounced prolongation of bleeding time (Fig. 2). At the highest dose of heparin used (plasma level of about 13 pg/ml) no cessation of bleeding occurred until 60 min after the incision. A comparison of plasma concentrations of the inhibitors

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required for prevention of venous thrombosis in rats with plasma levels which caused doubling of the bleeding time in mice showed that antithrombotically effective plasma concentrations of heparin would induce a clear prolongation of bleeding time, which was not observed on administration of hirudin or the synthetic thrombin inhibitor (Table II). TABLE II Comparison of equieffective plasma concentrations of thrombin inhibitors for prevention of stasis-induced venous thrombosis (ct, in rats) and bleeding time cc,, in mice) Inhibitor

Plasma concentration Prevention of venous thrombosis $1

Doubling of bleeding time (transection) (Cbt'

(ug/ml)

Ratio

Ratio

Doubling Ct:cbt Ct:cbi of bleeding time (incision) ccbi>

Hirudin

0.39

0.67

0.72

1:1.7

1:1.9

Heparin

7.87

5.33

3.67

1:0.7

1:0.5

BNas-Gly(pAm)Phe-Pip

0.70

1.60

1.60

1:2.3

1:2.3

DISCUSSION The bleeding time is an in vivo measurement for evaluating the effects of drugs on primary haemostasis. The methods of transection and standardized incision of the tail used in this study differ from each other with regard to the main components participating in the formation of the respective haemostatic plug. Prolongation of bleeding time following transection of the tail tip indicates mainly coagulation defects while that following incision of the tail is sensitive to changes of platelet number and function provided that only small vessels in the dermis and subcutaneous tissue are cut (13, 14). Upon transection of the tail the thrombin inhibitors studied prolonged the bleeding time in a dose-dependent manner. It is likely that this effect of the compounds tested is caused by the inhibitory action on the coagulation enzyme thrombin and the following inhibition of the thrombin-catalyzed fibrinogen conversion. On the other hand it could be shown that after incision of the tail heparin caused a more pronounced prolongation of bleeding time than hirudin and BNas-Gly(pAm)Phe-Pip. These haemorrhagic properties of heparin may be related to its anticoagulant effect, but other factors may also be important. Heparin interacts with platelets and it is possible that the haemorrhagic effects are related in part to an effect on platelet function. The mechanism of the heparinplatelet interactions remains uncertain. Different investigators have obtained varying in vitro results, ranging from

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heparin-induced aggregation, to potentiation of aggregation induced by other agents, or to an actual inhibition of platelet aggregation (15, 16, 17). In order to assess the relevance of the effect of thrombin inhibitors on the bleeding time this action was compared with their antithrombotic efficacy. In stasis-induced venous thrombosis in which predominantly plasmatic coagulation factors are activated leading to generation of the coagulation enzyme thrombin the inhibitors were able to prevent thrombus formation in a dose-dependent manner. Antithrombotically effective doses of hirudin and DNas-Gly-(pAm)Phe-Pip did not cause a significant prolongation of bleeding time, whereas heparin did. A possible explanation for the difference between antithrombotic and haemorrhagic effects of hirudin and bNas-Gly-(pAm)Phe-Pip would be that for the inhibition of the thrombin-platelet reaction higher concentrations of the inhibitors are required than for that of the thrombin-fibrinogen reaction (18). From the results presented it may be concluded that, in contrast to heparin, hirudin and the synthetic thrombin inhibitor are not expected to exert haemorrhagic complications during administration.

REFERENCES 1.

MARKWARDT. F. Hirudin as an inhibitor of thrombin. In: Methods in Enzymology, Vol. 19: Proteolytic Enzymes. G. E. Perlmann and L. Lorand (Eds.). New York, London: Academic Press, 1970, pp. 924 - 932.

2.

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3.

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4.

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5.

MARKWARDT, F., NOWAK, G., STURZEBECHER, J., GRIESSBACH, U., WALSMANN, P. and VOGEL, G. Pharmacokinetics and anticoagulant effect of hirudin in man. Thrombos. Haemost., 52, 160 - 163, 1984.

6.

MARKWARDT, F., NOWAK, G. and HOFFMANN, J. Comparative studies on thrombin inhibitors in experimental microthrombosis. Thrombos. Haemost. 2, 235 - 237, 1983.

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7.

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8.

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9.

KAISER, B. and MARKWARDT, F. Experimental studies on the antithrombotic action of a highly effective synthetic thrombin inhibitor. Thrombos. Haemost. (in press).

10.

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11.

WAGNER, G., VOIGT, B., VIEWEG, H., MARKWARDT, F. und STURZEBECHER, J. Verfahren zur Herstellung von Na-arylbzw. Na-heteroarylsulfonylaminoacylierten Amidinophenylalaninamiden. DDR-Wirtschaftspatent 155954, 3. 2. 1981.

12.

WESSLER, S., REIMER, S. M. and SHEPS, M. C. Biologic assay of a thrombosis-inducing activity in human serum. J. Appl. Physiol. l4, 943 - 946, 1959.

13.

DEJANA, E., VILLA, S. and deGAETAN0, G. Bleeding time in rats: A comparison of different experimental conditions. Thrombos. Haemost. 48, 108 - 111, 1982.

14.

WESTER, J., SIXMA, J. J., GEUZE, J. J. and van der VEEN, J. Morphology of the early hemostasis in human skin wounds. Influence of acetylsalicylic acid. Lab. Invest. 2, 298 - 311, 1978.

15.

SALZMAN, E. W., ROSENBERG, R. D. SMITH, M. H., LINDON, J. N. and FAVREAU, L. Effects of heparin and heparin fractions on platelet aggregation. J. Clin. Invest. 5, 64 - 73, 1980.

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HIRSH, J. Heparin induced bleeding. Nouv. Rev. Fr. Hematol. 2, 261 - 266, 1984.

17.

BRACE, L. D. and FAREED, J. An objective assessment of the interaction of heparin and its fractions with human platelets. Semin. Thromb. Hemost. 11, 190 - 198, 1985.

18.

GLUSA, E., HOFFMANN, A. and MARKWARDT, F. Influence of benzamidine derivatives on thrombin-induced platelet reactions. Folia Haematol. (Lpz.) 12, 98 - 106, 1982.