PROSTAGLANDINSLEUKOTRIENES ANDESSENTIALFATTYACIDS

Hypothesis for the Receptors of Human Blood Platelet Aggregation and Its Inhibition by Structure-Activity Relationship M. Ojima. T. Tokuhiro Depurtnzetzt oj’Medicinu1 Research

ad Injbrmation. Osuku .Wl( Japan (Reprint requests to MO)

Ono Pharmaceuticd

Company Ltd. -7 1-S Doshomcrchi. Chuoku,

We tried to clarify the size and the common charge distribution of the inhibition or stimulation of human platelet aggregation by structure-activity relationship. Numerou: inhibiting and stimulating agents were able to enter the receptors. Inhibitory receptor had recess of 14 x 12SA in diameter. Stimulatory receptor had recess of 11 x 12A in diameter. In the recess, there were three charges, two negative and one positive in the inhibitory receptor, and one negative and two positive in the stimulatory receptor, respectively. Charge distributions and conformation of inhibiting or stimulating agents were similar for the inhibitory agents, prostaglandin Iz (PGIJ, PGD,, PGEi adenosine and isoproterenol and conformation of the stimulating agents, thromboxane AZ(TXA&, platelet activating factor (PAF), adenosine diphosphate (ADP) and adrenaline. Each molecule had 3-10 inhibiting and stimulating conformations. The ratio of the number of conformations for inhibition and stimulating of platelet aggregation was highest for PGI? which showed the strongest inhibitory activity. TXAz was opposite in both respects.

ABSTRACT.

INTRODUCTION

theory is correct, PG compounds which eliminate the component causing aggregation should be strong antiaggregatory agents. We sought for the essential structures for inhibiting and stimulating platelet aggregation by the investigation of structure-activity relationships.

ONO-747 was the first prostaglandin

(PG)E, compound (17-ethyl-trans-A?-PGE,) (1) to have an antithrombotic action in patients after revascularization (2). Because of adverse inflammatory reactions, further development of this compound as a medicine was limited. With the aim of getting an antithrombotic effect with high specificity. we investigated the receptors that can inhibit and induce human platelet aggregation. Many receptors have been reported, e.g. receptors for PGI, (3), PGD, (4), adenosine (5). thromboxane (TX)A, (6), platelet activating factor (PAF) (7), adenosine phosphate (ADP) (8) etc.. and the number is still increasing. However concerning the number of receptors of the cell membrane, only 3000 particles with about lOOA/flm? are observed electronmicroscopically (9) in spite of the report that there are 100-2000 receptors per 1 agent per 1 cell on the basis of receptor binding studies (3-8) and the length of platelet cell membrane is about 3 p (30 OOOA) but the length of molecule in inhibiting or stimulating agents are about 1OA or more. By the study of agonist/receptor/ cyclic adenosine monophosphate (CAMP), it has been proposed that PGI, and PGE, bind to receptors which both inhibit and induce platelet aggregation (10). If the

METHOD We estimated the structure-activity relationship of PGs and the relative molecules using HGS molecular model, in which both holding and non-holding of intramolecular hydrogen bond (H-bond) were taken into consideration. The H-bond in the molecule was estimated only when two negative atoms holding the hydrogen atom were straight. The charges in the molecule were calculated by the methods of CND0/2 (11). MNDO ( 12) and the Hiickel’s extension method (13). The methods for measurement of human platelet aggregation and the CAMP were the same as described elsewhere (1, 14, 15). Inhibitory activity for the human platelet aggregation was calculated as 1.0 for the concentration of PGEi to give SO% inhibition for the platelet aggregation by ADP (6 pg/ml).

RESULTS AND DISCUSSION Platelet aggregation

Date received 32 January 1992 Date accepted 2 April 1993

Acetylcholine 69

(Ach) induces canine platelet aggregation

70

Prostaglandins

Leukotrienes

and Essential Fatty Acids

(16), contracts many smooth muscles and releases various chemical agents. Thrombosthenin, a platelet smooth muscle, is thought to be related to platelet aggregation (17). We have also reported a similar hypothesis that platelet aggregation would be induced when the ATPase of the smooth muscle is inhibited by ADP. This ADP is released by ATPase contained in smooth muscle which is activated by the aggregatory agents (14). The density of negative charge caused mainly by sialic acid in the plasma membrane decreases. The relaxation of membrane occurs because of inhibition of ATPase of smooth muscle caused by ADP, and platelet aggregation is induced by the increase of energy of collision between platelet and platelet (14). TXA2 (18). PGHl (18), adrenaline (19), nicotine (20), ADP (21) and PAF (22) also release ADP from human platelet and can induce platelet aggregation. In TXAz and PGH,, intramolecular H-bond exists between OH group at CIs position and COOH group at C, position by computer analysis (23) and Fourier-

transform infrared (m-IR) spectra (24). We also examined intramolecular H-bond between double bond at C,. position and COO- group at C, position because 1. carboxyl group completely exists as COO- in biological pH 2. it makes stronger intramolecular H-bond than COOH group (24) 3. the H-bond can be straight between double bond at C5 position and COO- group at C, position 4. carbon atom at C5 position has the weak negative charge ( 12) 5. the recess of receptor for adrenaline produced by cloning (25) and for Ach obtained by structureactivity relationship (26) are small. The charge distribution common to TXA7, PGH,, PAF, adrenaline and ADP was obtained by referring to Beckett’s muscarine receptor model, the results of which are shown in Figure 1. We estimated that the oxygen which does not contribute to H-bond in COOH plays a

PAF

adrenaline

diagrammaticrepresentationof receptor in platelet

ADP

aggregation

Eeckett’s Muscarine Receptor ( ileum 1

Fig. 1

Common charge distribution

in stimulatory

agents of platelet aggregation.

Human Blood Platelet Aggregation

role of methyl group in Ach (Fig. 1). Adrenaline and PAF have methyl group. The number of charge distribution in the triangle in the structures of aggregatory agents was two for TXA*, adrenaline and PAF, and three for PGH*, respectively. The supposed receptor of platelet aggregation had a recess of 11~12A in diameter and one negative and two positive charges in the recess (Fig. 1). We reported that PGs, PAF, leukotriene (LT) D, and histamine can completely enter the Beckett’s muscarine receptor of ileum and uterus (27). When PGs which have an inhibitory action of the platelet aggregation were administered in high dose, they antagonized TXA-, agonists or antagonists by TXA2 receptor binding study of the cloned (28), purified (6) and solubilized (29) human platelet membrane. PGs such as 6-keto-PGE,, PGD,. PGE, and PGE, could form intramolecular H-bond between OH group at CIs position in o-chain and COO- group at C, position in achain. It was the same conformation as the computer analyzed conformation of TXA2 and PGH? (23). PGDz and PGE, could also have an intramolecular H-bond between double bond at Cs position in a-chain and COO- group at C, position in a-chain as same as the conformation of TXA? and PGHz obtained in molecular model (Fig. 2). By this reason, PGD, and PGE? were thought to easily antagonize TXAl agonist and antagonist by the analysis of ligand binding study (6, 28, 29). In PGI,, PGI,. 6-keto-PGE,, PGE,, and PGE,! which inhibit platelet aggregation. H-bond between OH group

Fig. 2

Structure-activity

relationship

in stimulation

of platelet aggregation

and Its Inhibition by Structure-Activity

Relationship

71

at C, , position in cyclopentane ring and COO- group in a-chain could be formed. In PGD?, H-bond could be formed between OH group at Cq position in cyclopentane ring and COO- group in a-chain. In PG13, intramolecular H-bond could be formed between double bond at C,# position and COO- group in a-chain. These conformations were different from those of TXAl and PGH2 (Figs 1 & 2). The antagonistic action between substances could be thought to take place when the total force of static electricity of ionic bond and van der Waals bond approached each other and also if the receptor size was uniform. The antagonism between PG and other substances may not take place if the total force of electricity of van der Waals’ force between receptor and substance is very different, even if the ionic charges of two molecules coincide. The isotopically labeled PG12. PGD,. PGE,, PAF, adrenaline and ADP may be able to bind the purified TXAl receptor, because such substances could enter the receptor obtained by structure-activity relationship.

Inhibition

of platelet aggregation

George (30) and Hoyland (13) reported that PGE, has a common feature to N-n-propyl-norepinephrine, a padrenergic compound; the distance between OH groups of the ring and side chain in both PGE, and N-n-propylnorepinephrine is about .5A. w-Chain in PGE, and Nalkyl group in P-adrenergic compound are similar.

by prostaglandins.

72

Prostaglandins

Leukotrienes

and Essential Fatty Acids

Therefore, they assumed that PGE, showed the pharmacological action like P-adrenergic agonist. Their finding by computer analysis was also confirmed by our study, when there was no intramolecular H-bond. However if there was an intramolecular H-bond between COOgroup at C, position and OH group at C,, position, we found that PGE, and P-adrenergic agonist had the same three charges, two positive and one negative, when the former gave three conformations while the latter gave only one (Fig. 3). PGEi, PGI,, adenosine and isoproterenol are also common to each other in increasing CAMP of platelet (10, 15, 31). P-Adrenergic receptor obtained by cloning increases CAMP by the addition of isoproterenol and also PGE, (32). In the P-adrenergic receptor mutant that has Asparagine in place of aspartic acid 79, its affinity with isoproterenol becomes considerably weak, but the affinity with PGE, is only a little weak. This phenomenon can be explicable by that the P-adrenergic receptor mutant would have a conformational effect in its receptor recess by its Asparagine 79. PGEi, which can take more conformational change than isoproterenol, could be considered to cause a stronger affinity with the

diagrammatic of platelet

Activity in inhibition of platelet aggregation The inhibitory activities, and the inhibitory and stimulative conformations of PGI,, PGI,, 6-keto-PGE,, PGD,, PGE,, PGEp and 15-keto-PGE, are shown in Table 1,

adenosine

isoproterenol

of receptor

P-adrenergic receptor mutant by this conformational change. The conformations of PGE, or PG12 to inhibit platelet aggregation which was estimated by structureactivity relationship, coincided with Strader’s p-adrenergic receptor model obtained by cloning (25). It was considered that two atoms with positively charged hydrogen atom OH group at C,, position and carbon atom at Cb position, and the atom with negative charge of COOgroup at CI position in PGI, molecule, bound to COOgroup of aspartic acid 113 and OH group of serine 204 or 207 in the receptor. The P-adrenergic receptor model coincided with the four conformations in PG12 and the three in PGE,. The recess of inhibitory receptor obtained by structure-activity relationship had a hole of 14 x 12.5A in diameter (Fig. 3).

supposed model between PGI,

representation

end B - receptor

in inhibition aggregation

8-

Fig. 3

KC

Common charge distribution

in inhibitory

adrenergic

receptor

: Strader,

agents of platelet aggregation.

C. D.

(1989)

Human Blood Platelet Aggregation

Table 1 Correlation between inhibitory activities in human platelet

aggregation

and Its Inhibition by Structure-Activity

and conformations

Platelet aggregation Conformation

Conformation Number of coincidence in triangular charge distribution

Number of coincidence distribution

Total

Total

No hydrogen bond

4 5 5 4 3 4 0

4 5 6 7 5 8 2

0 0 2 1 1 1 I

(PGE,=I.Ol

PGIZ PGI, 6-keto-PGE, PGDL PGEI PGE2 15-keto-PGE,

38 22 4.3 1.9 1.0 0.006

Hypothesis for the receptors of human blood platelet aggregation and its inhibition by structure-activity relationship.

We tried to clarify the size and the common charge distribution of the inhibition or stimulation of human platelet aggregation by structure-activity r...
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