Competitive inhibition by adenosine 5'-triphosphate of the actions on human platelets of 2-chloroadenosine 5'-diphosphate, 2-azidoadenosine 5'-diphosphate and 2-methylthioadenosine 5'-diphosphate.

1 Adenosine 5'-diphosphate (ADP) induces human platelet aggregation and noncompetitively inhibits stimulated human platelet adenylate cyclase; these two effects are mediated by the same ADP receptor, at which adenosine 5'-triphosphate (ATP) is a competitive antagonist. 2 Two ADP analogues, 2-azidoadenosine 5'-diphosphate (2-azido-ADP) and 2-methylthioadenosine 5'-diphosphate (2-methylthio-ADP) have been reported to be more potent as inhibitors of adenylate cyclase than they are as aggregating agents, but no evidence has been presented that these actions are mediated solely by the ADP receptor. 3 We therefore tested the ability of ATP to inhibit the actions of these compounds and of another ADP analogue, 2-chloroadenosine 5'-diphosphate (2-chloro-ADP). 4 2-Chloro-ADP, 2-azido-ADP and 2-methylthio-ADP each induced aggregation and inhibited stimulated adenylate cyclase. Both of these actions were competitively inhibited by ATP (50 microM) with pA2 values similar to those previously found for inhibition by ATP of these effects of ADP. 5 The reported greater potency of 2-azido-ADP and of 2-methylthio-ADP as inhibitors of adenylate cyclase than as aggregating agents is therefore due only to their greater efficacy for this effect, not to some extra actions elsewhere.     

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)     

Differential inhibition by adenosine or by prostaglandin E1 of human platelet aggregation induced by adenosine 5'-O-(1-thiodiphosphate) and adenosine 5'-O-(2-thiodiphosphate).

Adenosine 5'-diphosphate (ADP) induces human platelet aggregation and inhibits stimulated adenylate cyclase. Adenosine 5'-O-(1-thiodiphosphate) (ADP-alpha-S) and adenosine 5'-O-(2-thiodiphosphate) (ADP-beta-S) act at the ADP receptor and achieve the same maximal rate of human platelet aggregation as each other. Adenosine and prostaglandin E1, which noncompetitively inhibit ADP-induced aggregation by stimulating adenylate cyclase, inhibit aggregation induced by ADP-x-S more than aggregation induced by ADP-beta-S. ADP-x-S, unlike ADP-beta-S and ADP itself, does not inhibit stimulated adenylate cyclase. This suggests that the inhibition of stimulated adenylate cyclase by ADP, although not a cause of aggregation, may be of physiological importance in reducing the effects of endogenous agents such as adenosine and prostaglandins (for example, prostacyclin) to which the platelet may be exposed.     

Uptake of 5-hydroxytryptamine by rat blood platelets and its inhibition by adenosine 5'-diphosphate.

1 The uptake of 5-hydroxytryptamine (5-HT) by rat blood platelets in citrated plasma was linear for at most 10 s and was substantially complete within 3 minutes. 2 Adenosine 5'-diphosphate (ADP) was a potent inhibitor of 5-HT uptake (Ki=0.38 muM) and kinetic analysis revealed that the inhibition was not competitive. 3 Inhibition of 5-HT uptake by ADP was abolished in the presence of prostaglandin E1 and 2-n-amylthio-AMP, which also inhibit the stimulant actions of ADP on blood platelets. 4 It is concluded that ADP could inhibit 5-HT uptake by changing the Na+/K+ distribution across the cell membrane, and the biological significance of this is discussed.     

Effects of suramin on contractions of the guinea-pig vas deferens induced by analogues of adenosine 5'-triphosphate.

1. Adenosine 5''-triphosphate (ATP) and some of its analogues contract the guinea-pig vas deferens, acting via receptors which have been classified as P2X-purinoceptors. We have recently shown, however, that the effects of ATP are enhanced, rather than inhibited, by the non-selective P2 antagonist, suramin, and that this enhancement could not easily be explained in terms of inhibition by suramin of the breakdown of ATP. We therefore investigated the effects of suramin on contractions induced by ATP analogues, to define the structure-activity relationships of the suramin-resistant response. 2. In the absence of suramin, the order of potency for ATP analogues was adenosine 5''-(alpha,beta-methylene)triphosphonate (AMPCPP) = P1,P5-diadenosine pentaphosphate (Ap5A) = adenosine 5''-tetraphosphate (Ap4) > adenosine 5''-O-(3-thiotriphosphate) (ATP gamma S) = adenylyl 5''-(beta,gamma-methylene) diphosphonate (AMPPCP) > P1,P5-diadenosine tetraphosphate (Ap4A) > adenosine 5''-O-(2- thiodiphosphate) (ADP beta S) > 2-methylthioadenosine 5''-triphosphate (MeSATP) > or = ATP > adenosine 5''-diphosphate (ADP). This is generally in agreement with previously reported structure-activity relationships in this tissue. 3. In the presence of suramin (1 mM), responses to Ap5A, Ap4A, AMPPCP, ADP beta S and ADP were abolished or greatly reduced, and contractions induced by AMPCPP, Ap4 and ATP gamma S were inhibited. Contractions induced by MeSATP however, like those induced by ATP itself, were not reduced, but at concentrations above 100 microM were enhanced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific inhibition of ADP-induced platelet aggregation by clopidogrel in vitro

1. The thienopyridine clopidogrel is a specific inhibitor of ADP-induced platelet aggregation ex vivo. No direct effects of clopidogrel (< or = 100 microM) on platelet aggregation in vitro have been described so far. 2. Possible in vitro antiaggregatory effects (turbidimetry) of clopidogrel were studied in human platelet-rich plasma and in washed platelets. 3. Incubation of platelet-rich plasma with clopidogrel (< or = 100 microM) for up to 8 h did not result in any inhibition of ADP (6 microM)-induced platelet aggregation. 4. Incubation of washed platelets with clopidogrel resulted in a time- (maximum effects after 30 min) and concentration-dependent (IC50 1.9+/-0.3 microM) inhibition of ADP (6 microM)-induced platelet aggregation. Clopidogrel (30 microM) did not inhibit collagen (2.5 microg ml(-1))-, U46619 (1 microM)- or thrombin (0.1 u ml(-1))-induced platelet aggregation. The inhibition of ADP-induced aggregation by clopidogrel (30 microM) was insurmountable indicating a non-equilibrium antagonism of ADP actions. The R enantiomer SR 25989 C (30 microM) was significantly less active than clopidogrel (30 microM) in inhibiting platelet aggregation (32+/-5% vs 70+/-1% inhibition, P < 0.05, n = 5). 5. In washed platelets, clopidogrel (< or = 30 microM) did not significantly reverse the inhibition of prostaglandin E1 (1 microM)-induced platelet cyclic AMP formation by ADP (6 microM). 6. The antiaggregatory effects of clopidogrel were unchanged when the compound was removed from the platelet suspension. However, platelet inhibition by clopidogrel was completely abolished when albumin (350 mg ml(-1)) was present in the test buffer. 7. It is concluded that clopidogrel specifically inhibits ADP-induced aggregation of washed platelets in vitro without hepatic bioactivation. Inhibition of ADP-induced platelet aggregation by clopidogrel in vitro occurs in the absence of measurable effects on the reversal of PGE1-stimulated cyclic AMP by ADP.     

Synergistic inhibition of thrombin-induced platelet aggregation by the novel nitric oxide-donor GEA 3175 and adenosine.

1. The influence of the novel nitric oxide-donor GEA 3175 on thrombin- and ionomycin-stimulated human platelets was investigated. The effect of GEA 3175 was compared with that of adenosine, an activator of platelet adenylyl cyclase. 2. GEA 3175 inhibited thrombin-induced secretion of ATP but did not affect aggregation; similar results were obtained with adenosine. 3. Thrombin-stimulated rises in the cytosolic free Ca2+ concentration, [Ca2+]i, were dose-dependently inhibited by GEA 3175 and adenosine. GEA 3175 and adenosine maximally reduced the initial rise in [Ca2+]i by 41% and 35%, respectively. 4. Simultaneous exposure to GEA 3175 and adenosine nearly abolished both the functional responses (i.e. aggregation and degranulation) and the rises in [Ca2+]i in thrombin-stimulated platelets. 5. Aggregation and increases in [Ca2+]i triggered in platelets by the Ca(2+)-ionophore ionomycin were only marginally affected by a combination of GEA 3175 and adenosine. 6. GEA 3175 potently increased the guanosine 3':5'-cyclic monophosphate (cyclic GMP) content in platelets but did not affect adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels. Adenosine did not increase either the cyclic AMP or the cyclic GMP levels in platelets. However, adenosine and GEA 3175 combined significantly elevated the platelet cyclic AMP content. 7. The results show that simultaneous exposure to GEA 3175 and adenosine promotes potent anti-aggregatory properties in platelets in vitro. The findings suggest that blockage of the cytosolic Ca(2+)-signal, which is probably mediated by an amplified cyclic nucleotide response, is an important event during the synergistic inhibition of thrombin-induced aggregation.     

川芎嗪抗剪切应力诱导血小板聚集的实验研究

目的：观测川芎嗪对剪切应力诱导血小板聚集（ＳＩＰＡ）的影响。方法：采用新研制的激光散射剪切装置观测川芎嗪及对照药Ａｓｐｉｒｉｎ在体内外对抗ＳＩＰＡ的作用。结果与结论：Ａｓｐｉｒｉｎ能抑制ＡＤＰ诱导的血小板聚集，但不能抑制ＳＩＰＡ。川芎嗪在体内（２５～５０ｍｇ·ｋｇ－１）体外（９．２～７３．４ｍｍｏｌ·Ｌ－１）均有显著抗ＳＩＰＡ作用（Ｐ＜０．０１），其机制待进一步研究。     

Effects of adenosine derivatives on human and rabbit platelet aggregation

Summary The inhibitory effects of several adenosine analogues, including the new A2-selective agonists 2-[p-(2-carboxyethyl)phenethylamino]-5-N-ethylcarboxamido-adenosine (CGS 21680) and 2-hexynyl-5-N-ethylcarbox-amidoadenosine (2-hexynyl-NECA), were investigated in vitro on human and rabbit platelet aggregation. The compounds examined inhibited ADP-induced platelet aggregation over a wide range of potency. The rank order of activity was similar between the two species thus showing that the rabbit is a useful animal model for studying the effects of adenosine derivatives on platelet aggregation. 2-Hexynyl-NECA was found to be the most potent adenosine compound of those currently available, having IC₅₀ values of 0.10 and 0.07 M in human and rabbit platelets, respectively. Conversely, the A1 agonists R(–)-N-⁶-(2-phenylisopropyl) adenosine (R-PIA), S(+)-N⁶-(2phenylisopropyl) adenosine (S-PIA) and 2-chloro-N⁶-cyclopentyl-adenosine (CCPA) were the least potent compounds with IC₅₀ values in the micromolar range. The potency of the compounds in inhibiting platelet aggregation was found to be highly correlated with their affinity for A2 receptors as measured using 3H-CGS 21680 binding in rat brain striatum.Correspondence to S. Dionisotti at the above address     

Structure-activity relationships of uridine 5'-diphosphate analogues at the human P2Y6 receptor

The structure-activity relationships and molecular modeling of the uracil nucleotide activated P2Y6 receptor have been studied. Uridine 5'-diphosphate (UDP) analogues bearing substitutions of the ribose moiety, the uracil ring, and the diphosphate group were synthesized and assayed for activity at the human P2Y6 receptor. The uracil ring was modified at the 4 position, with the synthesis of 4-substituted-thiouridine 5'-diphosphate analogues, as well as at positions 2, 3, and 5. The effect of modifications at the level of the phosphate chain was studied by preparing a cyclic 3',5'-diphosphate analogue, a 3'-diphosphate analogue, and several dinucleotide diphosphates. 5-Iodo-UDP 32 (EC50 = 0.15 microM) was equipotent to UDP, while substitutions of the 2'-hydroxyl (amino, azido) greatly reduce potency. The 2- and 4-thio analogues, 20 and 21, respectively, were also relatively potent in comparison to UDP. However, most other modifications greatly reduced potency. Molecular modeling indicates that the beta-phosphate of 5'-UDP and analogues is essential for the establishment of electrostatic interactions with two of the three conserved cationic residues of the receptor. Among 4-thioether derivatives, a 4-ethylthio analogue 23 displayed an EC50 of 0.28 microM, indicative of favorable interactions predicted for a small 4-alkylthio moiety with the aromatic ring of Y33 in TM1. The activity of analogue 19 in which the ribose was substituted with a 2-oxabicyclohexane ring in a rigid (S)-conformation (P = 126 degrees , 1'-exo) was consistent with molecular modeling. These results provide a better understanding of molecular recognition at the P2Y6 receptor and will be helpful in designing selective and potent P2Y6 receptor ligands.     

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.     

Inhibition by a stable analogue of adenosine triphosphate of platelet aggregation by adenosine diphosphate.

1 In citrated platelet-rich plasma, freshly prepared from rabbit blood, the velocity of platelet aggregation was within limits proportional to the log of the concentration of added adenosine diphosphate (ADP). 2 Addition of either adenosine triphosphate (ATP) or its beta,y-methylene analogue inhibited aggregation similarly except that the analogue was about half as potent as ATP. beta,y-Methylene ATP also reversed the optical effects associated with the shape change of platelets very similarly to ATP itself. 3 As beta,y-methylene ATP is not a substrate for nucleoside diphosphokinase, these observations do not support the proposition that inhibition of aggregation by added ATP is due to its utilization by the nucleoside diphosphokinase of platelets.     

Effects of adenosine analogs and adenine nucleotides on adenosine 5'-diphosphate-induced rat platelet aggregation

Various adenosine analogs and adenine nucleotides have been tested as inhibitors of ADP-induced aggregation of rat platelets. The potent inhibitors of human platelet aggregation, adenosine, 2-fluoroadenosine, 2-chloroadenosine, carbocyclic adenosine and N⁶-phenyl adenosine, had little effect on rat platelet aggregation (0–30 per cent inhibition). The effects of adenosine or its analogs on ADP-induced aggregation of cross-species platelet-rich plasmas (PRPs) (human platelets suspended in rat plasma or rat platelets in human plasma) were similar to those with the native PRPs, indicating that these species differences were due to intrinsic factors in the platelets and not in the plasma. When these analogs were tested in the presence of the cyclic AMP phosphodiesterase inhibitor papaverine, strong inhibiton of rat platelet ADP-induced aggregation was seen. 2′-Deoxyadenosine and 3′-deoxyadenosine were not inhibitory to ADP-induced aggregation of rat PRP even in the presence of papaverine. Adenosine 5′-tetraphosphate strongly inhibited both human and rat platelet aggregation. AMP, like adenosine, did not inhibit rat platelet aggregation but became strongly inhibitory in the presence of papaverine. This inhibitory effect was abolished by preincubating rat PRP with an adenylate cyclase inhibitor, 2′, 5′-dideoxyadenosine or adenosine deaminase. In the later case, however, if the adenosine deaminase inhibitor 2′-deoxycoformycin was included in the incubation mixture, the inhibition by AMP plus papaverine was similar to adenosine plus papaverine. About 50 per cent of [¹⁴C]AMP was converted to [¹⁴ C]adenosine in rat platelet-free plasma or PRP after a 10-min incubation. α,β-Methylene-ADP and β,γ-methylene-ATP (200 μM) inhibited rat platelet aggregation by 50 and 64 per cent, respectively. Cyclic AMP phosphodiesterase of rat and human platelets gave comparable K_m, and V_max values (K_m 0.53 and 0.21μM and V_max 6.0 and 6.7 pmoles/min/10⁷ platelets, respectively).     

Kinetic interaction of 5-AZA-2'-deoxycytidine-5'-monophosphate and its 5'-triphosphate with deoxycytidylate deaminase

5-AZA-2'-deoxycytidine-5'-monophosphate (5-AZA-dCMP) was tested as a substrate, and 5-aza-2'-deoxycytidine-5'-triphosphate (5-AZA-dCTP) was tested as an allosteric effector of purified spleen dCMP deaminase. Graphic analysis of the velocity of deamination of 5-AZA-dCMP versus its concentration gave a hyperbolic curve in which the estimated apparent Km was 0.1 mM. Since this curve was not sigmoidal and 5-AZA-dCMP at low concentrations stimulated the rate of deamination of the natural substrate, dCMP, it was proposed that the binding of 5-AZA-dCMP to the allosteric enzyme dCMP deaminase induced the R form. At substrate saturation, the rate of deamination of dCMP was 100-fold greater than that of 5-AZA-dCMP. dTTP inhibited the deamination of 5-AZA-dCMP with first-order kinetics. This inhibition was reversed by either 5-AZA-dCTP or dCTP. However, dCTP alone produced only a weak activation of the deamination of 5-AZA-dCMP in comparison to the potent activation when dCMP was the substrate. 5-AZA-dCTP was just as effective as dCTP for the allosteric activation of the deamination of dCMP. These results indicate that dCMP deaminase can play an important role in the metabolism 5-aza-2'-deoxycytidine nucleotides and may possibly modulate some of the pharmacological activity of this antimetabolite.     

Potentiation of aggregation and inhibition of adenylate cyclase in human platelets by prostaglandin E analogues.

1. The 16-phenoxy prostaglandin E analogue sulprostone consistently potentiates primary aggregation waves induced by adenosine 5'-diphosphate (ADP), PAF and 11,9-epoxymethano PGH2 (U-46619) in platelet-rich plasma from human donors. The effect is not blocked by the TP-receptor antagonists, EP 092 and GR 32191. The high potency of sulprostone (threshold concentration = 4-10 nM) and the weak block of sulprostone potentiation by the EP1-receptor antagonist, AH 6809 (pA2 = 4.3) suggest the involvement of EP3-receptors as opposed to EP1- or EP2-subtypes. 2. Eight prostaglandin E (PGE) analogues were compared against sulprostone for their effects on PAF-induced aggregation in human platelet-rich plasma (PRP) in the presence of GR 32191 and the DP-receptor antagonist, BW A868C. PGE2 and 11-deoxy PGE2-1-alcohol showed evidence of both potentiating and inhibitory actions and butaprost showed only inhibitory activity at high concentrations. The remaining analogues always elicited potentiation, with the following potency ranking: sulprostone = 16,16-dimethyl PGE2 > MB 28767 > misoprostol > GR 63779X = 17-phenyl-omega-trinor PGE2. The results again indicate that EP3- rather than EP1- or EP2-receptors are involved. However, relative potentiating potency could be affected by differences in plasma protein binding and the very high sensitivity of the human platelet to prostacyclin (IP)-receptor-mediated inhibition (IC50 for the specific IP-receptor agonist cicaprost = 0.8 nM).(ABSTRACT TRUNCATED AT 250 WORDS)