TxA2 receptor ligands in human platelets

THROMBOSIS RESEARCH 63; 239-248,199l 0049-3848/91 $3.00 + .OOPrinted in the USA. Copyright (c) 1991 Pergamon Press pk. All rights reserved.

12-HETE

INHIBITS THE BINDING OF PGHz/TXA2 LIGANDS IN HUMAN PLATELETS.

Pierre FONLUPT,

RECEPTOR

Martine CROSET and Michel LAGARDE.

Chimie Biologique

INSERM U205 - INSA B406, 69621 VILLEURBANNE

Cedex, FRANCE

(Received 17.7.1990; accepted in revised form 22.3.1991 by Editor M.C. Boffa) (Received by Executive Editorial Office 951991)

ABSTRACT

12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), the end-lipoxygenase product of arachidonic acid in platelets has been previously shown to prevent PGHz/TxAz-induced aggregation. From the present study, we show that 12-HETE inhibits the binding of [1251]-PTA-OH, a thromboxane antagonist, to platelet membranes with an IC,o of 8 PM. This value is in accordance with previously reported 12-HETE concentrations required to prevent the aggregation induced by TxA2 mimetics, the methano analogues of PGH2, U44069 and U46619. When [3H]-U44069 was used as a thromboxane agonist to label intact platelets, 12-HETE also inhibited its binding. We conclude that part of the inhibitory effect of 12HETE on PGHT/TxAz-induced aggregation might be the result of interacting with PGHz/TxA2 receptor sites.

INTRODUCTION

Arachidonic acid (20:4n-6) added exogenously to human blood platelets or released after agonist stimulation of these cells is metabolized by two oxygenation enzyme systems (1,2). The cyclooxygenase/thromboxane synthase pathway synthesizes prostaglandin endoperoxides (PGG2, PGH2)

Key words : 12-HETE,

PGHz/TxA2

receptor, 239

Binding,

Human Platelets.

240

12-HETE AND PGHcxA,

RECEPTOR

Vol. 63, No. 2

and thromboxane A2 (TxA2) (3) which induce platelet aggregation and secretion (4) and are vasoconstrictors (5). The lipoxygenase converts 20:4n-6 into 12(S)-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12HPETE), further reduced by the associated peroxidase into 12-hydroxy5,8,10,14-eicosatetraenoic acid (12-HETE) (6). The biological significance of these molecules in platelets is still unknown, although they have been reported as inhibitors of platelet aggregation induced by thrombin (7) collagen (7,8) and a stable PGHz/TxA2 analogue (9), when added exogenously to platelet suspensions. The potency of TxA2 and PGH2 to activate human platelets is thought to be mediated through their binding to a common receptor (10,ll). The binding of specific agonists of PGHz/TxA2 receptor, such as 15(S)-hydroxy-9a,l la-methanoepoxyprostadienoic acid (U46619) and 15(S)-hydroxy-9a, 11 a-epoxymethanoprostadienoic acid (U44069), to platelets induces a whole cascade of biological responses including platelet shape change, release reaction, intracellular Ca++ mobilization, fibrinogen receptor exposure and a first wave of aggregation (12,13,14). It also causes the platelet to synthesize PGH2 and TxA2 via arachidonic acid released from phospholipid stores, then initiating a secondary irreversible aggregation (15). We have previously reported that 12-HETE (9,16,17) can prevent U44069- and U46619-induced platelet aggregation but did not interfere with cyclooxygenase activity. Endogenous 12-HETE, whether it is allowed to attain PGHz/Tx A2 receptor, might consequently slow down the arachidonate-dependent platelet activation in a feed back mechanism. In this work, we found that 12-HETE alters [3H]-U44069 binding to isolated platelets as well as the binding of 9,11 -dimethylmethano11,12-methano16-.(3-iodo-4-hydroxypheny1)-13,14-dihydro-13-aza-15ap-o-tetranorTxA2 [*251]-PTA-OH, an antagonist of PGHz/TxA2 receptor (18), to platelet membranes.

MATERIALS

AND METHODS

Materials. [ I251]-PTA-OH (reference, IM254, 2000 Ci/mmol) was U.K. [3H]-U44069 (14 Ci/mmol) was a purchased from Amersham, generous gift from Dr R.L. Jones, University of Edinburgh, U.K. 4-[2(benzenesulfonylamino)-ethyl]-phenoxyacetic acid (BM 13177) was a gift FRG). 12-HETE was synthesized from Boehringer-Mannheim (Mannheim, by conversion of exogenous arachidonic acid by human platelets, according to the reference 9. U46619 and U44069 were provided by Upjohn Co. (Kalamazoo, MI).

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Preparation of olatelet suspensions, Human platelets were obtained from whole blood, collected into l/9 volume anticoagulant (citric acid/citrate/dextrose, pH 4.5), and taken from normal subjects free of drugs for at least 10 days. Platelet-rich plasma (PRP) was prepared by centrifugation of the blood for 15 min at lOOg, acidified to pH 6.4 with citric acid 0.15 M and immediately centrifuged at 9OOg for 10 min to pellet platelets (19). For studies on whole cells, platelets were resuspended into an ice cold Tyrode-HEPES buffer containing NaCl 136 mM, NaHC03 12 mM, NaH2P04 0.41 mM, KC1 2.7 mM, MgC12 1 mM, HEPES 5 mM and glucose 5.5 mM, pH 7.35) in order to obtain 1 to 1.5~ 109 platelets/ml. For further homogenization of the cells, they were resuspended in a Tyrode buffer containing EDTA 3 mM, pH 7.2 (20).

Prenaration of ulatelet membranes. Platelet suspension was pellets centrifuged at 4OC for 12 min at 1200g and the resulting resuspended into a sorbitol buffer (0.34 M sorbitol, 10 mM HEPES, pH 7.2). The suspension was sonicated with a sonicator (Branson Bl5) at setting 2 for 5 s and the homogenate centrifuged at 1200g for 10 min at 4°C. The supernatant was removed and the pellet resuspended and sonicated as above. The resulting supernatants were pooled and centrifuged at 13,000g for 20 min at 4°C to pellet a granule fraction. The supernatant was centrifuged at 100,OOOg for 90 min to pellet a mixed membrane fraction representing surface and intracellular membranes (20). 2 mg of microsomal protein were obtained per 1010 platelets. This fraction was rehomogenized into Tyrode HEPES buffer and kept at -80°C before use.

[*2511-PTA-OH binding. Binding was measured according to the reference 18. Briefly, 200 ul of platelet membranes, representing 85 ug of protein, (diluted 10 fold from stock) were incubated for 30 min at 37°C in Tris-HCl buffer 50 mM (pH 7.4) containing CaC12 1 mM, in a final volume of 400 ~1. At the end of the incubation period, 3 ml of cold Tris-HCl buffer, containing NaCl 100 mM, were added. Samples were filtered through GF/C filters (Whatman) and filters were washed 4 times with 3 ml of cold TrisHCl buffer before counting.

[3Hmbinding. Binding of [3H]-U44069 to whole platelets was measured according to Kattelman et al. (21). 1 ml of platelet suspension was incubated with [3H]-U44069 alone (final concentration : 70 nM) or with that concentration of [3H]-U44069 plus 1000 fold unlabelled U44069 for 5 min at 37OC. Samples were then applied to GF/C filters and washed as above prior counting.

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12-HETE AND PGHflxA,

RECEPTOR

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12-HETE effect on inositol phosnhate formation. Platelets (5x108 cells/ml) were labelled by 2 hour incubation at 37OC, with [3H]inositol (50 to 100 uCi/ml), in a Tyrode buffer containing 1 mM EGTA and 300 ng/ml prostacyclin. They were washed twice in a Tyrode buffer containing 5 mM inositol, 0.1 mM CaQ and 300 ng/ml prostacyclin. Platelets were finally resuspended in a Tyrode buffer supplemented with 0.1 mM CaC12 and 10 mM LiCI, pH 7.4 at 300 000 cells&l. Assays were performed by stimulating 500 ul of this cell suspension with 1 pM U46619, 2 min at 37OC. The reaction. was terminated by addition of 1 ml chloroform/methanol (1: 1, V/V) to the incubation medium and. inositol phosphates were extracted as previously described (22). 500 ul of the upper phase were mixed with 2 ml Hz0 and a 50% slurry of Dowex 1 (W/V) (0.5 ml; 100-200 mesh; x8) in the formate form. The resin was washed 5 times with 2 ml inositol solution (5 mM) and inositol phosphates were eluted by adding twice 0.5 ml of 0.1 M formic acid/l .O M ammonium formate solution. This allowed the elution of all species of inositol The radioactivity of assays was measured by liquid phosphates. scintillation counting.

RESULTS

AND DISCUSSION

12-HETE showed an inhibitory effect on [1251]-PTA-OH binding to platelet membranes with an IQ0 (concentration inhibiting 50 percent of the saturable binding) of 8 PM. We compared the 12-HETE efficiency to displace [*251]-PTA-OH binding to those of known PGHz/TxA2 receptor ligands. The agonist U46619 and the antagonist BM 13177 exhibited an ICso of 0.4 uM and 15 PM, respectively. Non saturable binding with PGHz/TxA2 ligand was high and reached 50 to 60% of total binding (18) (Fig 1). The inhibitory effect of 12-HETE might have been the result of a decrease of non-saturable binding instead of [1251]-PTA-OH displacement of specific sites. For this reason, we checked the inhibitory effect of 12HETE in the presence of U46619 (Fig 2). When U46619 was present at 0.5 uM (80% inhibition of saturable binding), adding 12-HETE induced an additional inhibition (95% inhibition of saturable binding). When U46619 was present at 5 uM (100% inhibition of saturable binding), 12-HETE did indicating that this lipoxygenase product not induce further inhibition, does not interfere with non-saturable binding.

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RECEPTOR

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Figure 1. Inhibitions of [ 1251]-PTA-OH binding by U46619 ( A ), BM 13177 ( v) and 12-HETE ( ?? ). determinations from 3 Data are represented as the mean of triplicate experiments for each compound as described in “MATERIALS AND METHODS”.

log (concentration,

M )

Figure 2. [1251]-PTA-OH binding in the absence (I) or in the presence of 0.5 ~.LM12-HETE ( ??), 10 pM 12-HETE ( a ), 0.5 pM U46619 + 10 pM 12HETE ( 4 ), 5 uM U46619 ( ? ?), 5 uM U46619 + 10 pM 12-HETE ( ??). Data are represented as the mean f SD of 6 determinations as described in “MATERIALS AND METHODS”

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RECEPTOR

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It has been reported that polyunsaturated fatty acids like 20:4n-6 and 8,11,14-eicosatrienoic acid, can inhibit U46619-induced platelet aggregation in the presence of aspirin (23). Therefore, we compared the respective efficiency of 12-HETE and 20:4n-6, at the same concentration, to displace [ I*51]-PTA-OH binding. Saturable binding values were decreased from 205 f 13 cpm (control) to 87 + 23 cpm (12-HETE, 8 pM ) but were not modified by adding 8 uM 20:4n-6 (231 + 20 cpm) (Mean f SD, n=lO). This is consistent with the observation that 20:4n-6 and 12HETE concentrations necessary for inhibiting U46619-induced aggregation of washed aspirinated platelets have been reported in range orders of 100 PM (23) and 5 pM (7,9,16), respectively. The binding of labelled agonists [3H]-U46619, [3H]-U44069 and antagonists [ **51]-PTA-OH has been shown to present different kinetic properties (24). Using [3H]-U44069, we investigated the effect of 12-HETB on the binding of this agonist to whole platelets. Both U44069 and 12HETE could inhibit [3H]-U44069 binding, 12-HETE being 10 to 50 fold less potent than U44069 in this respect (Table 1). This difference was in the same order of magnitude as the difference observed for their [I*511 -PTAOH binding site affinities.

Table 1. Inhibition of U44069 and 12-HETE. Saturable binding was U44069, (B): in presence Each value represents different blood samples.

I

[3H]-U44069

binding

to

washed

platelets

by

expressed as pmol/l09 cells. (A): in presence of of 12-HETE. the mean f SD of 6 determinations from two

Concentrations

of

unlabelled

products

0

0.21 FM

2.1 PM

21 PM

+ U44069 (A)

4.46 f 0.77

2.67 f 0.94

0.50 + 0.75

0 k 0.63

+ 12-HETE (B)

4.46 + 0.77

3.99 f 0.73

3.47 f 0.28

0 + 0.62

12-HETE was able to inhibit both bindings of an agonist and an antagonist to the PGHz/TxA2 receptor site. Moreover, the IQ0 value for inhibition of [1*51]-PTA-OH binding (8 pM) was in the same range order as the concentration which inhibited U46619- and U44069-induced platelet aggregation, as previously reported (7,9,16). Although [l*sI]-PTA-OH binds

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245

to a single PGHz/TxA2 receptor (25,26), U46619 would interact with two distinct subtype receptors (27). Recently, Takahara et al (27), using GR32191, a novel PGHz/TxA2 receptor antagonist, have reported evidence for the existence of two binding sites linked to distinct effector systems, in platelets. U46619 would induce platelet aggregation, serotonin release, inositol phosphate formation and protein kinase C activation by interacting with one binding site. Platelet shape change and cytosolic Ca++ increase would be essentially linked to U46619 interaction with the second site. Since the first site was associated with inositol phosphate production, we investigated whether 12-HETE inhibits the U466 1g-induced inositol phosphate formation. 1 uM of U46619 induced the formation of 48 It 17 fmol/assay inositol phosphates. Adding 10 uM of 12-HETE decreased the latter production to 7 f 21 fmol/assay. (Mean + SD of 6 separate experiments). This indicates that 12-HETE inhibits the binding of U46619 to the site associated with inositol phosphate release and platelet aggregation. Both platelet aggregation and binding on whole cells were performed in the absence of albumin, the major blood protein which binds unesterified fatty acids, of which 12-HETE. Although these results must be extrapolated with caution to the in vivo situation, the protein free model used in the present work was required to avoid the additional binding of 12-HETE and the thromboxane agonist to albumin. However, we may hypothesize that the affinity of both ligands to serum proteins would be much lower than to the platelet receptor sites, and presumably similar. It is then reasonable to assume that the relative affinity of 12-HETE and thromboxane agonist to the platelet sites, observed in the absence of plasma proteins, would be a relevant index of the physiological situation. We conclude that the interference of 12-HETE with PGH2 and/or TX A 2 binding sites might be one of the mechanism explaining its antiaggregatory effect, when added exogenously to platelet suspensions.

ACKNOWLEDGEMENTS This work was supported by INSERM. We thank Dr R-L. Jones for providing [3H]-U44069 and Boehringer Mannheim for the PGHz/TxA2 receptor site antagonists.

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