2-Chloro N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate is a selective high affinity P2Y(1) receptor antagonist

1. We reported previously that bisphosphate derivatives of adenosine are antagonists of the P2Y(1) receptor and that modification of the ribose in these analogues is tolerated in the P2Y(1) receptor binding pharmacophore. 2. Here we delineate the pharmacological activity of one such non-nucleotide molecule, 2-chloro N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate (MRS2279), in which the ribose is replaced by a cyclopentane ring constrained in the (N)-conformation by a cyclopropane moiety. 3. MRS2279 antagonized 2MeSADP-stimulated inositol phosphate formation in turkey erythrocyte membranes with competitive kinetics (pK(B)=7.75). High affinity competitive antagonism by MRS2279 was also observed at the human P2Y(1) receptor (pK(B)=8.10) stably expressed in 1321N1 human astrocytoma cells. Antagonism was specific for the P2Y(1) receptor since MRS2279 had no effect on activation of the human P2Y(2), P2Y(4), P2Y(6), or P2Y(11) receptors by their cognate agonists. 4. MRS2279 also did not block the capacity of ADP to act through the Gi/adenylyl cyclase linked P2Y receptor of platelets to inhibit cyclic AMP accumulation. 5. In contrast, the P2Y(1) receptor is known to be obligatory in the process of ADP-induced platelet aggregation, and MRS2279 competitively inhibited ADP-promoted platelet aggregation with an apparent affinity (pK(B)=8.05) similar to that observed at the human P2Y(1) receptor heterologously expressed in 1321N1 cells. 6. Taken together these results illustrate selective high affinity antagonism of the P2Y(1) receptor by a non-nucleotide molecule that should prove useful for pharmacological delineation of this receptor in various tissues.     

Molecular cloning of the platelet P2T(AC) ADP receptor: pharmacological comparison with another ADP receptor, the P2Y(1) receptor

Platelet activation plays an essential role in thrombosis. ADP-induced platelet aggregation is mediated by two distinct G protein-coupled ADP receptors, Gq-linked P2Y(1), and Gi-linked P2T(AC), which has not been cloned. The cDNA encoding a novel G protein-coupled receptor, termed HORK3, was isolated. The HORK3 gene and P2Y(1) gene were mapped to chromosome 3q21-q25. HORK3, when transfected in the rat glioma cell subline (C6-15), responded to 2-methylthio-ADP (2MeSADP) (EC(50) = 0.08 nM) and ADP (EC(50) = 42 nM) with inhibition of forskolin-stimulated cAMP accumulation. 2MeSADP (EC(50) = 1.3 nM) and ADP (EC(50) = 18 nM) also induced intracellular calcium mobilization in P2Y(1)-expressing cells. These results show that HORK3 is a Gi/o-coupled receptor and that its natural ligand is ADP. AR-C69931 MX and 2MeSAMP, P2T(AC) antagonists, selectively inhibited 2MeSADP-induced adenylyl cyclase inhibition in HORK3-expressing cells. On the other hand, A3P5PS, a P2Y(1) antagonist, blocked only 2MeSADP-induced calcium mobilization in P2Y(1)-expressing cells. HORK3 mRNA was detected in human platelets and the expression level of HORK3 was equivalent to that of P2Y(1). These observations indicate that HORK3 has the characteristics of the proposed P2T(AC) receptor. We have also determined that [(3)H]2MeSADP binds to cloned HORK3 and P2Y(1). Competition binding experiments revealed a similarity in the rank orders of potency of agonists and the selectivity of antagonists as obtained in the functional assay. These results support the view that P2Y(1) functions as a high-affinity ADP receptor and P2T(AC) as a low-affinity ADP receptor in platelets.     

An elusive receptor is finally caught: P2Y(12'), an important drug target in platelets

Despite intensive research, the nucleotide P2 receptor that is involved in the aggregation and activation of platelets by ADP has remained elusive. However, now two research groups have independently identified a new platelet receptor of unexpected structure, P2Y(12), that acts with the P2Y(1) receptor to form the site of ADP activation and explains the multiple transduction mechanisms observed in response to ADP in platelets. Recent evidence also suggests that a third component, ATP action on the P2X(1) receptor ion channel, contributes to platelet activation.     

The P2Y(1) receptor closes the N-type Ca(2+) channel in neurones, with both adenosine triphosphates and diphosphates as potent agonists

The rat P2Y(1) nucleotide receptor, the P2Y subtype abundant in the brain, was heterologously expressed in rat superior cervical ganglion neurones by micro-injection of the receptor cRNA or cDNA. ADP inhibited the N-type Ca(2+) current by 64%, with EC(50) 8.2 nM, an action blocked competitively by the P2Y(1) receptor antagonist adenosine 3', 5'-bis-phosphate (K(i) 0.7 microM). 2-Methylthio-ADP inhibited the Ca(2+) current likewise, but with EC(50) 0.57 nM, giving the highest potency reported therewith for P2Y(1). Significantly, ATP and 2-methylthio-ATP were also agonists, the latter again at a very high potency (EC(50) 2.5 nM). We propose that this neuronal receptor, when present in brain at a high density as at synapses, can respond to very low concentrations of ATP and ADP as agonists, and that this would result in inhibition of N-type Ca(2+) currents and hence can reduce transmitter release or increase neuronal excitability.     

Inhibition of platelet function by administration of MRS2179, a P2Y1 receptor antagonist

The effects of a potent P2Y1 receptor antagonist, N6-methyl-2'-deoxyadenosine-3',5'-bisphosphate (MRS2179) on adenosine-5'-diphosphate (ADP)-induced platelet aggregation in vitro, ex vivo and on the bleeding time in vivo were determined. In suspensions of washed platelets, MRS2179 inhibited ADP-induced platelet shape change, aggregation and Ca2+ rise but had no effect on ADP-induced inhibition of adenylyl cyclase. Binding studies using the new radioligand [33P]MRS2179 showed that washed human platelets displayed 134+/-8 binding sites per platelet with an affinity (Kd) of 109+/-18 nM. Finally, intravenous injection of MRS2179 resulted in inhibition of rat platelet aggregation in response to ADP and prolonged the bleeding time, in rats or mice, as compared to controls. These results suggest this potent P2Y1 receptor antagonist to be a promising tool to evaluate the in vivo effects of pharmacologically targeting the P2Y1 receptor with a view to antithrombotic therapy.     

Two subtypes of G protein-coupled nucleotide receptors, P2Y(1) and P2Y(2) are involved in calcium signalling in glioma C6 cells

1. In glioma C6 cells, the stimulation of P2Y receptors by ADP, ATP and UTP initiated an increase in the intracellular Ca2+ concentration, in a process that involved the release of Ca2+ from InsP(3)-sensitive store and the capacitative, extracellular Ca2+ entry. The presence of external Ca2+ was not necessary to elevate Ca(2+). 2. The rank order of potencies of nucleotide analogues in stimulating [Ca2+](i) was: 2MeSADP > ADP > 2MeSATP = 2ClATP > ATP > UTP. alpha,beta-Methylene ATP, adenosine and AMP were ineffective. 3. ADP and UTP effects were additive, while actions of ATP and UTP were not additive on [Ca2+](i) increase. Similarly, cross-desensitization between ATP and UTP but not between ADP and UTP occurred. 4. Suramin, a non-specific nucleotide receptors inhibitor, antagonized ATP-, UTP- and ADP-evoked Ca2+ responses. PPADS, a selective antagonist of the P2Y(1) receptor-generated InsP(3) accumulation, decreased ADP-initiated Ca2+ response with no effect on ATP and UTP. 5. Pertussis toxin (PTX) reduced ADP- and ATP-induced Ca2+ increases. Short-term treatment with TPA, inhibited both ATP and ADP stimulatory effects on [Ca2+](i). 6. ADP inhibited isoproterenol-induced cyclic AMP accumulation. PTX blocked this effect, but PPADS did not. 7. RT - PCR analysis revealed the molecular identity of P2Y receptors expressed by glioma C6 cells to be both P2Y(1) and P2Y(2). 8. It is concluded that both P2Y(1) and P2Y(2) receptors co-exist in glioma C6 cells. ADP acts as agonist of the first, and ATP and UTP of the second one. Both receptors are linked to phospholipase C (PLC).     

Components of traditional Chinese medicine inhibit platelet aggregation by blocking ADP receptor subtypes P2Y_1 and P2Y_(12)

Platelets aggregation and thrombosis formation are major reasons of cardiovascular and cerebral vascular diseases.To develop new generative,potent and safe agents for inhibiting platelet aggregation and preventing above diseases are urgently required.Some traditional Chinese medicines of″Houxue Huayu″have been shown to inhibit platelet aggregation potently.In the present study the mechanisms and the molecular targets of puerarin,salvianolic acid B and the analogue of 3-n-butylphthalide,dl-PHPB were investigated and compared with ticlopidine.Four platelet aggregation inducers,ADP,arachidonic acid,collagen and thrombin were used in the study.It was found that puerarin and dl-PHPB specifically inhibited ADP induced platelet aggregation like ticlopidine did.However,salvianolic acid B inhibited both ADP and collagen induced platelet aggregations with similar potency.Due to existing two ADP receptor subtypes on platelets,P2Y1 and P2Y12,we studied the action of above compounds on the receptors and the signaling pathways.It was found that dl-PHPB decreased IP1 accumulation produced by ADP,but had no effect on IP1 level induced by m-3M3 FBS,an activator of PLC.M-3M3 FBS might attenuate the inhibitory effect of dl-PHPB on ADP-induced platelet aggregation.In addition,dl-PHPB did not affect cyclic AMP formation in platelets by ADP,which is different from P2Y12 antagonist ticlopidine.Puerarin showed the similar effects of dl-PHPB.Therefore,the actions of dl-PHPB and puerarin might be through P2Y1receptor-PLC-βpathway.Salvianolic acid B did not reduce the IP1 accumulation stimulated by ADP.It might act on the receptor subtype P2Y12.Our results suggest that components of Chinese herb medicine might be a resource for development of novel anti-platelet drugs.     

Attenuation of the P2Y receptor-mediated control of neuronal Ca2+ channels in PC12 cells by antithrombotic drugs

1. In PC12 cells, adenine nucleotides inhibit voltage-activated Ca(2+) currents and adenylyl cyclase activity, and the latter effect was reported to involve P2Y(12) receptors. To investigate whether these two effects are mediated by one P2Y receptor subtype, we used the antithrombotic agents 2-methylthio-AMP (2-MeSAMP) and N(6)-(2-methyl-thioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-dichloromethylene-ATP (AR-C69931MX). 2. ADP reduced A(2A) receptor-dependent cyclic AMP synthesis with half maximal effects at 0.1-0.17 micro M. In the presence of 30 micro M 2-MeSAMP or 100 nM AR-C69931MX, concentration response curves were shifted to the right by factors of 39 and 30, indicative of pA(2) values of 6.1 and 8.5, respectively. 3. The inhibition of Ca(2+) currents by ADP was attenuated by 10-1000 nM AR-C69931MX and by 3-300 micro M 2-MeSAMP. ADP reinhibited Ca(2+) currents after removal of 2-MeSAMP within less than 15 s, but required 2 min to do so after removal of AR-C69931MX. 4. ADP inhibited Ca(2+) currents with half maximal effects at 5-20 micro M. AR-C69931MX (10-100 nM) displaced concentration response curves to the right, and the resulting Schild plot showed a slope of 1.09 and an estimated pK(B) value of 8.7. Similarly, 10-100 micro M 2-MeSAMP also caused rightward shifts resulting in a Schild plot with a slope of 0.95 and an estimated pK(B) of 5.4. 5. The inhibition of Ca(2+) currents by 2-methylthio-ADP and ADPbetaS was also antagonized by AR-C69931MX, which (at 30 nM) caused a rightward shift of the concentration response curve for ADPbetaS by a factor of 3.8, indicative of a pA(2) value of 8.1. 6. These results show that antithrombotic drugs antagonize the inhibition of neuronal Ca(2+) channels by adenine nucleotides, which suggests that this effect is mediated by P2Y(12) receptors.     

Effects of phosphate-modified analogs of adenosine 5′-diphosphate and adenosine 5′-triphosphate at P2T-purinoceptors mediating human platelet activation by ADP

Adenosine 5′-diphosphate (ADP) induces human platelet aggregation and inhibits stimulated adenylate cyclase by an action at P_2T-purinoceptors. Both of these effects of ADP are inhibited competitively by ATP. Structure-activity relationships for phosphate-modified analogs of ADP and adenosine 5′-triphosphate (ATP) were studied by testing their effects on human platelet activation. Of the ADP analogs, only β,γ-imido-ADP (AMPNHP) induced platelet aggregation, but was a weak partial agonist (pA₅₀ 4.53). ADP-induced platelet aggregation was antagonized noncompetitively by β,γ-methylene-ADP (AMPCP) (pA₂ 3.2), β,γ-ethylene-ADP (AMPCCP) (pA₂ 4.42), and β,γ-difluoromethylene-ADP (AMPCF₂P) (pA₂ 4.77), and competitively by β,γ-dichloromethylene-ADP (AMPCCl₂P) (pA₂ 4.68). None of the ADP analogs inhibited prostaglandin E₁ (PGE₁)-stimulated adenylate cyclase, and ADP-induced inhibition of PGE₁-stimulated adenylate cyclase was unaffected by AMPNHP, AMPCP, or AMPCCP (100 μM), but was antagonized by AMPCF₂P (pA₂ 4.36) and AMPCCl₂P (4.24). ADP-induced platelet aggregation was antagonized competitively by the ATP analogs β,γ-difluoromethylene-ATP (AMP-PCF₂P) (pA₂ 4.55), β,γ-dichloromethylene-ATP (AMP-PCCl₂P) (pA₂ 4.42), and β,γ-imido-ATP (AMP-PNHP) (pA₂ 4.32) and non-competitively by 2-methylthio-β,γ-methylene-ATP (2-MeS-AMP-PCP), 2-methylthio-β,γ-difluoromethylene-ATP (2-MeS-AMP-PCF₂P), and 2-methylthio-β,γ-dichloromethylene-ATP (2-MeS-AMP-PCCl₂P). In summary, agonist activity at the human platelet P_2T-purinoceptor was extremely sensitive to alterations to the diphosphate chain of ADP, and only AMPNHP induced platelet aggregation. Increasing the electronegativity of the methylene group by halogen substitution increased the antagonist potency of the ADP analog AMPCP but resulted in little or no change in the antagonist potencies of the ATP analogs AMP-PCP and 2-MeS-AMP-PCP. © 1996 Wiley-Liss, Inc.     

The novel pyridoxal-5'-phosphate derivative PPNDS potently antagonizes activation of P2X(1) receptors

Pyridoxal-5'-phosphate-6-(2'-naphthylazo-6'-nitro-4',8'-disulfonat e) (PPNDS) potently antagonized P2X(1) receptor-mediated responses in rat vas deferens (pK(B)=7.43) and Xenopus laevis oocytes (pIC(50)=7. 84). It showed lower (up to 20,000-fold) inhibitory potency on ecto-nucleotidase in Xenopus oocytes and on P2Y(1) receptors in guinea-pig ileum (pA(2)=6.13). PPNDS did not interact with alpha(1A)-adrenoceptors, adenosine A(1) and A(2B), histamine H(1) and muscarinic M(3) receptors. Thus, PPNDS is a novel, specific P2 receptor antagonist and represents the pyridoxal-5'-phosphate derivative with the highest potency at P2X(1) receptors.     

Platelet aggregometry and receptor binding to predict the magnitude of antithrombotic and bleeding time effects of clopidogrel in rabbits

Target levels of ex vivo inhibition of platelet aggregation (IPA) induced by adenosine diphosphate (ADP) that produce clinically relevant effects of clopidogrel, a P2Y12 antagonist, are unclear. We examined standard and modified IPA and P2Y12 receptor occupancy as predictors of antithrombotic (% thrombus weight reduction) and bleeding time (BT, fold-increase over control) effects of clopidogrel in rabbit models of carotid artery thrombosis and cuticle bleeding, respectively. Standard and modified IPA with 20 microM ADP were measured in the absence and presence of partial P2Y1 blockade, respectively. Clopidogrel maximally produced standard IPA of 57% +/- 5%, antithrombotic effect of 85% +/- 1%, BT increase of 6.0 +/- 0.4-fold and P2Y12 receptor occupancy of 87% +/- 5%. Surprisingly, a clopidogrel dose that produced a low standard IPA of 17% +/- 4% and P2Y12 receptor occupancy of 39% +/- 5% achieved a significant antithrombotic activity of 55% +/- 2% with a moderate increase in BT of 2.0 +/- 0.1-fold. This underestimation of clopidogrel efficacy by standard IPA was improved by measuring either modified IPA or P2Y12 receptor occupancy. These results suggest that in clopidogrel-treated rabbits, low standard IPA is associated with significant antithrombotic effects. Moreover, modified IPA and P2Y12 receptor occupancy appear to better predict the magnitude of clopidogrel's efficacy compared with standard IPA, which may be a better predictor of BT.     

Activity of adenosine diphosphates and triphosphates on a P2Y(T) -type receptor in brain capillary endothelial cells

1. A P2Y (nucleotide) receptor activity in a clonal population (B10) of rat brain capillary endothelial cells is coupled to inhibition of adenylyl cyclase and has functional similarities to the P2Y(T) (previously designated 'P2T') receptor for ADP of blood platelets. However, the only P2Y receptor which was detectable in a previous study of B10 cells by mRNA analysis was the P2Y(1) receptor, which elsewhere shows no transduction via cyclic nucleotides. We have sought here to clarify these issues. 2. The inhibition of forskolin-stimulated adenylyl cyclase induced by purified nucleotides was measured on B10 cells. The EC(50) value for 2-methylthioADP (2-MeSADP) was 2.2 nM and, surprisingly, 2-MeSATP was an almost equally strong agonist (EC(50)=3.5 nM). ATP and 2-ClATP were weak partial agonists (EC(50)=26 microM and 10 microM respectively) and under appropriate conditions could antagonise the activity on 2-MeSADP. 3. A known selective antagonist of the platelet P2Y(T) receptor, 2-propylthioadenosine-5'-(beta,gamma)-difluoromethylene) triphosphonate (AR-C 66096), was a competitive antagonist of this B10 cell receptor, with pK(B)=7.6. That ligand is inactive at the P2Y(1) receptor in the same cells. Conversely, the competitive P2Y(1) receptor antagonists, the 3', 5'- and 2', 5'-adenosine bis-monophosphates, are, instead, weak agonists at the adenylyl cyclase-inhibitory receptor. 4. The inhibition of adenylyl cyclase by 2-MeSADP was completely abolished by pertussis toxin. 5. In summary, these brain endothelial cells possess a P2Y(T)-type receptor in addition to the P2Y(1) receptor. The two have similarities in agonist profiles but are clearly distinguishable by antagonists and by their second messenger activations. The possible relationships between the B10 and platelet P2Y(T) receptors are discussed.     

Pharmacological characterization of the human P2Y13 receptor

The P2Y13 receptor has recently been identified as a new P2Y receptor sharing a high sequence homology with the P2Y12 receptor as well as similar functional properties: coupling to Gi and responsiveness to ADP (Communi et al., 2001). In the present study, the pharmacology of the P2Y13 receptor and its differences with that of the P2Y12 receptor have been further characterized in 1321N1 cells (binding of [33P]2-methylthio-ADP (2MeSADP) and of GTPgamma[35S]), 1321N1 cells coexpressing Galpha16 [AG32 cells: inositol trisphosphate (IP3) measurement, binding of GTPgamma[35S]) and Chinese hamster ovary (CHO)-K1 cells (cAMP assay)]. 2MeSADP was more potent than ADP in displacing [33P]2MeSADP bound to 1321N1 cells and increasing GTPgamma[35S] binding to membranes prepared from the same cells. Similarly, 2MeSADP was more potent than ADP in stimulating IP3 accumulation after 10 min in AG32 cells and increasing cAMP in pertussis toxin-treated CHO-K1 cells stimulated by forskolin. On the other hand, ADP and 2MeSADP were equipotent at stimulating IP3 formation in AG32 cells after 30 s and inhibiting forskolininduced cAMP accumulation in CHO-K1 cells. These differences in potency cannot be explained by differences in degradation rate, which in AG32 cells was similar for the two nucleotides. When contaminating diphosphates were enzymatically removed and assay of IP3 was performed after 30 s, ATP and 2MeSATP seemed to be weak partial agonists of the P2Y13 receptor expressed in AG32 cells. The stimulatory effect of ADP on the P2Y13 receptor in AG32 cells was antagonized by reactive blue 2, suramin, pyridoxal-phosphate-6-azophenyl-2',4'disulfonic acid, diadenosine tetraphosphate, and 2-(propylthio)-5'-adenylic acid, monoanhydride with dichloromethylenebis (phosphonic acid) (AR-C67085MX), but not by N6-methyl 2'-deoxyadenosine 3',5'-bisphosphate (MRS-2179) (up to 100 microM). The most potent antagonist was N6-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-5'-adenylic acid, monoanhydride with dichloromethylenebis (phosphonic acid) (ARC69931MX) (IC50 = 4 nM), which behaved in a noncompetitive way. The active metabolite of clopidogrel was unable to displace bound 2MeSADP at concentrations up to 2 microM.     

Acyclic analogues of adenosine bisphosphates as P2Y receptor antagonists: phosphate substitution leads to multiple pathways of inhibition of platelet aggregation

Activation by ADP of both P2Y(1) and P2Y(12) receptors in platelets contributes to platelet aggregation, and antagonists at these receptor subtypes have antithrombotic properties. In an earlier publication, we have characterized the SAR as P2Y(1) receptor antagonists of acyclic analogues of adenine nucleotides, containing two phosphate groups on a symmetrically branched aliphatic chain, attached at the 9-position of adenine. In this study, we have focused on antiaggregatory effects of P2Y antagonists related to a 2-chloro-N(6)-methyladenine-9-(2-methylpropyl) scaffold, containing uncharged substitutions of the phosphate groups. For the known nucleotide (cyclic and acyclic) bisphosphate antagonists of P2Y(1) receptors, there was a significant correlation between inhibition of aggregation induced by 3.3 microM ADP in rat platelets and inhibition of P2Y(1) receptor-induced phospholipase C (PLC) activity previously determined in turkey erythrocytes. Substitution of the phosphate groups with nonhydrolyzable phosphonate groups preserved platelet antiaggregatory activity. Substitution of one of the phosphate groups with O-acyl greatly reduced the inhibitory potency, which tended to increase upon replacement of both phosphate moieties of the acyclic derivatives with uncharged (e.g., ester) groups. In the series of nonsymmetrically substituted monophosphates, the optimal antagonist potency occurred with the phenylcarbamate group. Among symmetrical diester derivatives, the optimal antagonist potency occurred with the di(phenylacetyl) group. A dipivaloyl derivative, a representative uncharged diester, inhibited ADP-induced aggregation in both rat (K(I) 3.6 microM) and human platelets. It antagonized the ADP-induced inhibition of the cyclic AMP pathway in rat platelets (IC(50) 7 microM) but did not affect hP2Y(1) receptor-induced PLC activity measured in transfected astrocytoma cells. We propose that the uncharged derivatives are acting as antagonists of a parallel pro-aggregatory receptor present on platelets, that is, the P2Y(12) receptor. Thus, different substitution of the same nucleoside scaffold can target either of two P2Y receptors in platelets.     

Effects of the P2-purinoceptor antagonist, suramin, on human platelet aggregation induced by adenosine 5'-diphosphate.

1. The effects of suramin, a trypanocidal drug which has been reported to be a P2-purinoceptor antagonist on smooth muscle, were investigated in human platelets, where adenosine 5'-diphosphate (ADP) induces aggregation by acting on a subtype of purinoceptors which has been called P2T. 2. Suramin (100 microM) had no inhibitory effect on ADP-induced platelet aggregation in plasma, even after 40 min incubation in the presence of bacitracin, a peptidase inhibitor, and did not affect the ability of adenosine 5'-triphosphate (ATP) (40 microM) to inhibit competitively ADP-induced aggregation. This lack of effect of suramin on platelets in plasma is probably due to its extensive binding to plasma proteins. 3. In washed platelets, suramin (50-400 microM) acted as an apparently competitive antagonist, causing parallel shifts to the right of the log concentration-response curve to ADP. No depression of the maximal response to ADP was observed at concentrations of suramin (50-150 microM) for which full log concentration-response curves to ADP could be obtained, but the slope of the Schild plot was around 2, indicating that this antagonism was not simply competitive. The apparent pA2 value for suramin, taken from this Schild plot, was 4.6. 4. Suramin (200-400 microM) also noncompetitively inhibited aggregation induced by U46619 (a thromboxane receptor agonist) or by 5-hydroxytryptamine in the presence of adrenaline (100 microM), and caused a depression of the maximal response to these agonists. This nonspecific effect of suramin may explain the high Schild plot slope obtained against ADP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antiaggregatory activity in human platelets of potent antagonists of the P2Y 1 receptor

Activation of the P2Y(1) nucleotide receptor in platelets by ADP causes changes in shape and aggregation, mediated by activation of phospholipase C (PLC). Recently, MRS2500(2-iodo-N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate) was introduced as a highly potent and selective antagonist for this receptor. We have studied the actions of MRS2500 in human platelets and compared these effects with the effects of two acyclic nucleotide analogues, a bisphosphate MRS2298 and a bisphosphonate derivative MRS2496, which act as P2Y(1) receptor antagonists, although less potently than MRS2500. Improved synthetic methods for MRS2500 and MRS2496 were devised. The bisphosphonate is predicted to be more stable in general in biological systems than phosphate antagonists due to the non-hydrolyzable CP bond. MRS2500 inhibited the ADP-induced aggregation of human platelets with an IC(50) value of 0.95 nM. MRS2298 and MRS2496 also both inhibited the ADP-induced aggregation of human platelets with IC(50) values of 62.8 nM and 1.5 microM, respectively. A similar order of potency was observed for the three antagonists in binding to the recombinant human P2Y(1) receptor and in inhibition of ADP-induced shape change and ADP-induced rise in intracellular Ca(2+). No substantial antagonism of the pathway linked to the inhibition of cyclic AMP was observed for the nucleotide derivatives, indicating no interaction of these three P2Y(1) receptor antagonists with the proaggregatory P2Y(12) receptor, which is also activated by ADP. Thus, all three of the bisphosphate derivatives are highly selective antagonists of the platelet P2Y(1) receptor, and MRS2500 is the most potent such antagonist yet reported.