The vitamin k-antagonism of salicylate and warfarin

THROMBOSIS RESEARCH 42; 727-736, 1986 0049-3848/86 $3.00 t .OO Printed in the USA. Copyright (c) 1986 Pergamon Journals Ltd. All rights reserved.

THE VITAMIN K-ANTAGONISM OF SALICYLATE AND WARFARIN Roncaglioni+, M.M.W. Ulrich, A.D. Muller, B.&M. Soute, M.A.G. de Boer-van den Berg and C. Vermeer Department of Biochemistry, University of Limburg, P.O. Box 616, 6200 MD Maastricht, The Netherlands t From the Istituto di Ricerche Farmacologiche "Mario Negri", Via Eritrea 62, 20157 Milan, Italy * To whom all correspondence should be addressed

M.C.

Accepted by Editors-in-Chief (Received by Editor S. Magnusson 14.6.1984; B. BlombZck and A.L. Copley 2.4.1986)**

ABSTRACT When administered in high dosages, salicylate acts as a vitamin K-antagonist: it induces a decrease of the plasma concentration of the Gla-containing coagulation factors and an accumulation of microsomal substrates for vitamin K-dependent carboxylase in the liver and in the lung. In vitro the drugs inhibit the DTT-dependent reductases which mediate the reduction of vitamin K epoxide and vitamin K quinone. NADH-dependent reductase and vitamin K-dependent carboxylase are not inhibited.

INTRODUCTION On several occasions it has been reported that aspirin and salicylate have a direct effect on the plasma level of the vitamin K-dependent coagulation factors (l-4). In this respect these drugs mimic the coumarinderivates, which are frequently used as anticoagulants in patients during thrombogenic episodes. The coumarin-derivatives (e.g. warfarin) primarily inhibit the enzyme(s) that play(s) a role in the reduction of vitamin K epoxide and vitamin K quinone into vitamin K hydroquinone, which is the coenzyme for vitamin K-dependent carboxylase. Up till now three endoplasmic reductases have been reported: vitamin K epoxide reductase, DTT-dependent vitamin K quinone reductase and NADH-dependent vitamin K quinone reductase (5-9). We have compared the in vivo effects of warfarin and salicylate on the plasma level of the coagulation factors and on the accumulation of endogenous substrates (e.g. clotting factor precursors) in the liver and in

Key words: Vitamin K, warfarin, l(-carboxyglutamicacid

carboxylase,

reductase

salicylate,

** The acceptance of this comnunication by the Editors-in-Chief is exceptional and caused by the utter negligence of the Editor, to whom it was originally sulzmitted.. 727

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the lung. Using the hepatic carboxylase/reductase enzyme complex, we have also studied the in vitro carboxylation of the synthetic substrate Phe-Leu-Glu-Glu-Leu in the presence of various forms of vitamin K and either NADH or DTT, and we have measured how salicylate influences these reactions. Because in the in vivo experiments groups of animals were compared, the rat was the experimental animal of choice in our investigations, the results of which are presented in this paper.

METHODS Chemicals. Vitamin K was obtained from Sigma (Saint Louis, USA) and vitamin K hydroquinone 2nd vitamin K epoxide were prepared as described earlier (10). The various forms of vitamin K were solubilized in 7.5% Triton X-100. For in vivo experiments we used solubilized vitamin K (Konakion) from Hoffmann-La Roche (Basel, Switzerland). The synthetif substrate Phe-Leu-Glu-Glu-LeulhF L E E L) was obtained from Vega Biochemi(60 Ci/mol) from Amersham International cal Co (Tucson, USA) and NaH CO (Amersham, UK). Triton X-100, NA&, warfarin and dithiothreitol (DTT) were from Packard purchased from Sigma (Saint Louis, USA) and Picofluor-15 lnstruments (Warrenville, USA). All other chemicals were from Merck (Darmstadt, FRG). Experimental animals. For our in vivo experiments (sections A and B) we used male rats of the Lewis strain weighing about 200 grams each. After treatment with the drugs (as indicated) the animals were killed under ether anestesia, blood was taken by heart puncture and collected in 0.1 M trisodium citrate (10% v/v). The livers and lungs were removed, washed with icecold buffer (0.2 M KCl, 50 mM Tris-HCl, pH 7.4, 1 mM EDTA and 30% ethylene glycol) and used for the preparation of microsomes (11). For our in vitro experiments we used microsomes from normal, non-treated animals. The vitamin K-dependent Measurement p$ carboxylase activity. incorporation of "CO, was measured in,Q.25 ml reaction mixtures containing 0.4 M KCl, 50 mM Tris-HCl, 12% 4 mg microsomal protefns, 0.01 mCi NaHi4C0 ADH, FLEEL, vitamin K, warfarin ethylene glycol, 0.3% Triton X-100. DTT, i' and salicylate were added as indicated and the mixtures were incubated at 25 'C. The reaction was stopped with 1 ml 5% trichloroacetic acid and in order to remove the last traces of non-bound label the samples were boiled for 1 min before they were counted in Picofluor. Measurement of blood coagulation factors. Rat standard plasma and rat brain thromboplastin were prepared from 30 animals and stored at -30 'C before use (12). The coagulation factors were measured in one-stage coagulation assays, using either artificial prothrombin-deficient plasma (13) or human congenital factor deficient plasmas (for factors V, VII and X).

RESULTS

A. The effect of warfarin coagulation factors.

and

salicylate

on

the

plasma

level

of

blood

The effect of warfarin and salicylate was compared in 9 groups of 5 rats. One group served as a control and four groups were treated with various amounts of salicylate (as indicated). The drug was administered by

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a peritoneal injection and because of the short half-life time of salicylate in blood, the injections were repeated every 6 h. The rats in the last four groups were treated with different amounts of warfarin which were injected in single dosages. After 20 h blood was taken by a heart puncture and the livers and lungs were removed for the detection of accumulated substrates for carboxylase (see section B). The results of this experiment are summarized in table I. Both drugs induced a decrease of the

TABLE I Dose-response Relation Between Salicylate, Warfarin and Clotting Factors

Drug administered None salicylate, 4x 50 mg/kg salicylate, 4x100 mg/kg salicylate, 4x200 mg/kg salicylate, 4x200 mg/kg + vitamin K, 1x20 mg/kg warfarin, 1x0.2 mg/kg warfarin, 1x0.5 mg/kg warfarin, 1x1.0 mg/kg warfarin, 1x1.0 mg/kg + vitamin K, 1x20 mg/kg

Plasma level of clotting factor (X) F VII FX FV F II 100+15 lOO+lO 100+12 100-t9 9G 9 67+ 7 8G7 lOOTl1 7G8 377 5 517 6 104710 52+ 5 29T4 3G5 947 - 8 98+11 96+10 97+11 109+ 8 _ 78-l8 3G4 22T2

40t 5 12T 2 ST _ 1

78+10 327 3 ST - 2

85+10

97+ - 6

85+9

94+ 7 98TlO 9612 100+11 _

Salicylate and warfarin were dissolved in 0.15 M NaCl, pH 7.5 and injected in 1 ml samples. Vitamin K (if added) was mixed with the drugs shortly before injection. The data are the means (+ S.E.M.) of five different animals, each of which was tested in duplicate. Further details are described in the text.

plasma level of the vitamin K-dependent coagulation factors, whereas the level of factor V remained unchanged. The effects of salicylate and warfarin could be completely reversed by vitamin K, which was mixed with the drugs before injection. Furthermore warfarin appeared to be a much more potent inhibitor than salicylate is. The same effect was observed when we compared the plasma from patients receiving anticoagulant therapy and that from patients receiving high dosages of aspirin (6-10 grams daily for 2 weeks). In these studies (data not shown) it was striking that only in the plasma from dicoumarol-treated patients we were able to detect descarboxyprothrombin. The aspirin-induced decrease of the plasma prothrombin level was not accompanied by a simultaneous appearance of descarboxyprothrombin. B. The effect of warfarin and salicylate in liver and in lung. From the rats described in section A we also obtained the livers and lungs for the production of tissue homogenates. From these homogenates we prepared the microsomal fractions in which vitamin K-dependent carboxylase is known to be present, together with non-carboxylated substrate proteins

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such as precursors of the vitamin K-dependent clotting factors (5). The amounts of carboxylatable proteins were lgstimated in these preparations by and having the carboxylation adding vitamin K hydroquinone and NaH CO reaction proceed until completion. The resul3ts of this experiment are shown in table II and it is obvious that like warfarin, salicylate induced an

TABLE II Dose-response

Relation

Drug administered

Between Substrates

Salicylate, Warfarin for Carboxylase.

Carboxylation in liver 1 285 7 852 8 542 10 960

and

Microsomal

of endogenous substrates in lung 208 520 847 1 230

(dpm)

none salicylate, 4x 50 mg/kg salicylate, 4x100 mg/kg salicylate, 4x200 mg/kg salicylate, 4x200 mg/kg + vitamin K, 1x20 mg/kg 857 12 ____--------------------_---____-__-__-------_-_--_-_-_____________~_~~~--warfarin, lx 0.2 mg/kg 5 430 535 warfarin, lx 0.5 mg/kg 7 083 987 warfarin, lx 1.0 mg/kg 10 280 1 783 warfarin, lx 1.0 mg/kg + vitamin K, 1x20 mgfkg 725 126 ____________________-----------------------_-______~~_~_~~~~~~~~~~~~~~~---The livers and lungs from each group of animals described in the legend to table I were combined and used for the preparation of washed microsomes. These microsomes were tested in the carboxylase assay in the presence of 10 mM DTT and 0.5 mM vitamin K hydroquinone. All incubations were performed for 1 h at 25 'C. The data are the means of duplicate experiments.

accumulation of carboxylatable proteins in the hepatic microsomes as well as in the microsomes from lung. The accumulation was dose dependent and the effect of both drugs was efficiently counteracted by a single dose of vitamin K. C. The effect of warfarin and salicylate on in vitro carboxylating

systems.

required for carboxylase Vitamin K hydroquinone is the coenzyme activity in vitro. When vitamin K quinone or vitamin K epoxide are used instead, these forms of vitamin K have first to be reduced by reductase, which is also present in the microsomes, and which may even be complexed to or associated with carboxylase (14). Regarding these crude microsomes as a carboxylating enzyme complex (thus containing at least carboxylase and one or more reductases) we have determined its apparent and V for vitamin K hydroquinone, vitamin K quinone and for vitamin 3 epoxidneax(table III). Since it has been described, that the reduction of vitamin K may occur in the presence of either DTT or NADH (5), the various kinetic constants were

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VITAMIN K-ANTAGONISTS

TABLE III Kinetic

Coenzyme

Constants

of

CarboxylasejReductase Conditions.

Reductant

Measured

for )

Under

Various

Vm,, (dpm/min)

none 0.150 3 710 vitamin K hydroquinone 0.020 6 945 vitamin K hydroquinone DTT 0.110 3 985 vitamin K hydroquinone NADH 0.025 4 445 vitamin K quinone DTT 0.125 335 vitamin K quinone NADH 0.020 3 267 DTT vitamin K epoxide NADH vitamin K epoxide _--____----_~~~---~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~----~~------of the carboxylating enzyme complex were The apparant determined fr>tahzd i!%?al carboxylation rates at various vitamin K concentrations, in the presence of 4 mM F L E E L and either DTI (10 mM) or NADH (2 mM). Carboxylase was obtained from the livers of normal, non-treated animals.

determined in the presence of each reductant. No reductant was required when vitamin K hydroquinone was used as a coenzyme in the carboxylation Furthermore the reaction, although DTT induced a marked decrease of the KM* DTT-dependent carboxylasefreductase complex is active in the presence of vitamin K quinone as well as vitamin K epoxide. The NADH-dependent carboxylaselreductase complex, however, cannot use vitamin K epoxide as a coenzyme, whereas its KM for vitamin K quinone is rather high. We also measured the extent of inhibition of the various reactions by either warfarin or salicylate. The vitamin K concentration that we used in these experiments was 4 times the KM and the results are shown in fig. 1.

5 10 kSALICVLATE1hM)

The activity of carboxylase/reductase in the presence of increasing concentrations of inhibitor. The inhibition by warfarin (left panel) and salicylate (right panel) was measured in the presence of 0.6 mM vitamin K hydroquinone (O--0),0.08 mM vitamin K epoxide f 10 mM DTT (o-_O), 0.1 mM vitamin K quinone + 10 mM DTT (H-w) and 0.5 mM vitamin K quinone + 2 mM NADH ( o----O>. The experiments were performed with microsomes from normal, non-treated animals in the presence of 4 mM F L E E L and the incubation time was 1 h at 25 'C in all cases. FIG. 1

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It was confirmed Lhat warfarin strongly inhibits the UTT-dependent carboxylation in the presence of vitamin K quinone as well as vitamin K epoxide. The two other reactions (in the presence of either vitamin K hydroquinone or vitamin K quinone + NADH) were only slightly affected by warfarin. Similar results were obtained with salicylate: the DTT-dependent systems are much more sensitive for salicylate than is the NADH-dependent one, whereas hardly any effect was measured on the vitamin K hydroquinone-driven reaction. Moreover we observed that the effects of the two drugs were cumulative over a wide range of concentrations: the inhibition of carboxylase by mixtures of warfarin and salicylate was similar to the sum of the effects of each drug separately fig. 2). We have also checked whether increased vitamin K concentrations might help to overcome the inhibitory

Cumulative inhibition of carboxylase by warfarin and salicylate. The effect of warfarin on the initial carboxylation rate was measured in the absence (0-O) and presence (O--O) of 7.5 mM salicylate. The reaction was performed in the presence of 10 mM DTT and 0.1 mM vitamin K quinone. FIG. 2

2

100

! a

I

g a +

0

0

50_.

P 5

r3

0

.

. .’

0

.

.

5 5 ii a

0

1

0 [VITAMIN

K

QUINONE]

2 (mM)

Relative inhibition of carboxylaselreductase in the presence of increasing concentrations of vitamin K quinone. Reaction mixtures containing microsomes from normal animals, 10 mM DTT, 4 mM F L E E L and either warfarin (0.02 mM, - ) or salicylate (5 mM,o-_O) were incubated in the presence of varying concentrations of vitamin K for 1 h at 25 'C. The relative carboxylation rates of the inhibited reactions are plotted as a percentage of the non-inhibited reaction. FIG. 3

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733

effect of warfarin and salicylate in a DTT-dependent system. For that reason we prepared reaction mixtures containing a fixeP4 concentration of inhibitor and varying amounts of vitamin K quinone. The CO incorporation in these mixtures was compared with that in similar non-inh?bited mixtures and expressed as a percentage of the non-inhibited reaction. The data are summarized in fig. 3, and it is obvious that under our in vitro conditions vitamin K quinone does not antagonize the effects of warfarin and salicylate. Similar results (data not shown) were obtained with vitamin K epoxide. We have also tried to reverse the inhibition of the NADH-dependent enzyme complex. The experiments were performed at either 10 mM warfarin or 20 mM salicylate. Under these conditions the residual enzyme activity was 47% in the presence of warfarin and 62% in that of salicylate. In none of these cases the inhibition of carboxylase/reductase could be counteracted by adding vitamin K quinone or vitamin K epoxide. At present we do not have an explanation for this discrepancy between the in vivo and the in vitro situation.

DTLd. KO

Gla

;I,0

DTL.

\1/,K

I

NADH + H+

The vitamin K cycle. Abbreviations used are: KO, vitamin K epoxide; K, vitamin K quinone; KH2, vitamin K hydroquinone; DTT dithiothreitol (reE$ei form); DTT , dithiothreitol (oxidyzed ?:&a).

NAD’

.

FIG. 4

KH, Glu + CO2 + 0,

DISCUSSION For more than 50 years it has been known that coumarin derivatives such as warfarin may act as vitamin K-antagonists (15). Their mode of action is based on the inhibition of the vitamin K-dependent carboxylation of glutamic acid residues (16) and results for instance in a decreased plasma level of the coagulation factors II, VII, IX and X (17). The effect of aspirin has remained less clear thus far: on one hand it strongly inhibits the blood platelet aggregation (2), on the other hand also a direct interaction of aspirin and salicylate with the production of the vitamin K-dependent coagulation factors has occasionally been reported (l-4). We have investigated the latter effect in more detail by comparing the vitamin K-antagonism of salicylate with that of warfarin. The results of these experiments were, that in vivo both drugs induce a decrease of the plasma level of the vitamin K-dependent coagulation factors, which is reversible by the administration of vitamin K. In parallel, endogenous substrates for carboxylase accumulate in liver and in lung in a dose-dependent way during the treatment with both, warfarin and salicylate. Whereas in man and in many animal species coumarin-derivatives induce the appearance of noncarboxylated descarboxy factors in plasma (17,18), the same could not be ,jemonstratedfor aspirin. This is in agreement with the data reported by

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Owens and Cimino (19,20) who showed that perfused rat livers are able to produce normal clotting factors and that warfarin and salicylate both inhibit the synthesis of the vitamin K-dependent factors. Whereas in the presence of warfarin the clotting factors were excreted as biologically inactive antigens (presumably descarboxyfactors), no excretion of abnormal coagulation factors could be demonstrated by these authors after the administration of salicylate. At present we do not have an explanation for this discrepancy between the two drugs. We have also studied the inhibition of the vitamin K-dependent enzymes in a more detailed way in in vitro experiments. In liver three vitamin K-metabolites are known: vitamin K hydroquinone, quinone and epoxide, and it has been proposed several years ago that these metabolites are interconverted in a cyclic way (21). This is represented in a schematical way in fig. 4. In step 1 vitamin K epoxide is reduced by KO-reductase and up till now dithiols such as DTT are the only reductants known to stimulate this reaction. In step 2 vitamin K quinone is reduced further into the hydroquinone and in this step both, DTT and NADH may serve as coenzymes for the reducing enzyme system(s). So either K-reductase may use both reductants as a cofactor or two different enzyme systems, each with its own cofactor, are present. In step 3 finally the vitamin K hydroquinone is used as a coenzyme for vitamin K-dependent carboxylase. During the formation of a Gla-residue it is converted again into the epoxide (22,23) rhus coillpletingthe cycle. So only in the presence of DTT vitamin K can be recycled on the cdrboxylaselreductase complex and only under these conditions carboxylase has a low apparent s for vitamin K, regardless whether we used the hydroquinone, the quinone or the epoxide (table III). In the absence of a reductant (vitamin K hydroquinone) or in the presence of NADH (vitamin K quinone) no recycling is possible, so high apparent s values were observed. It is also clear from table III, that the differences between the maximal reaction rates of the various carboxylating systems are small except in the case where we used vitamin K quinone + NADH. Using this combination of coenzymes to be about lo-fold lower than in all other cases. This we found the V might be cau.&e for instance, by a slow association rate of NADH ontXJ reductase, but other explanations are possible. Doubtlessly the DTT-dependent reductases have the highest sensitivit-, for warfarin. This is in agreement with the observation that vitamin /_ epoxide is accumulated during in vivo warfarin treatment (5). The effect of salicylate resembled that of warfarin: the DTT-depender' reductases (fig. 4, steps 1 and 2a) were inhibited stronger than was the NADH-dependent reductase (step 2b), whereas the vitamin K hydroquinonedriven reaction was not inhibited. This is in contrast to the work presented by Hildebrandt and Suttie (4), who claimed that salicylate exclusively inhibits the NADH-dependent reductase. These authors could not explain, however, why in vivo salicylate induced the accumulation of vitamin K-epoxide in liver, whereas such an accumulation is merely expected on the basis of our results. It is also very clear that both, in vivo and in vitro, salicylate is only a weak inhibitor of the carboxylase/reductase system, so that only at very high dosages (4-6 g daily) the effect is measurable in man. Since the effect of salicylate is cumulative to the effect of warfarin, however, it is to be expected that much lower dosages will interfere with the oral anticoagulant therapy. This therapy comprises a reduction of the normal vitamin K-dependent clotting factor concentration in plasma up to 5-10% of normal by the administration of coumarin-derivatives. Aspirin-induced fluctuations in these levels might have serious results for patients undergoing the therapy, whereas similar fluctuation would even not be measurable in normal volunteers.

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ACKNOWLEDGEMENTS The authors wish to thank Dr. H.C. Hemker for his support with respect to the clinical aspects of this work and for kindly reviewing this manuscript, Dr. M.P. van Dieijen-Visser for supplying us with patient plasmas, Dr. G. de Gaetano for sharing with us his expert knowledge about aspirin and Mrs. M. Molenaar-van de Voort for typing this manuscript. This research was supported by grant MD 82145 from the Thrombosestichting Nederland. M.C. Roncaglioni is a recipient of the European Fellowship 1982-1983.

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of sodium rat liver.

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