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Title: The biochemical pharmacology of the human platelet ADP receptor
Author: Hall, David A.
ISNI:       0000 0001 3525 392X
Awarding Body: University of Surrey
Current Institution: University of Surrey
Date of Award: 1993
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The effects of a number of analogues of ADP and ATP were determined on increases in platelet cytosolic calcium concentration ([Ca2+]i). ADP, 2-methyIthio-ADP, adenosine 5'-O-(1-thiodiphosphate) and adenosine 5'-O-(2-thiodiphosphate) each induced increases in [Ca2+]i in a manner similar to their reported ability to induce platelet aggregation. The effects of these compounds were antagonized by ATP (a selective ADP receptor antagonist) and in the case of ADP this antagonism was shown to be competitive by Schild analysis. 2-chloro-ATP, adenosine 5'-O-(1-thiotriphosphate), adenosine 5'-O-(2-thiotriphosphate), P1,P5-diadenosine pentaphosphate and adenylyl 5'-(beta,gamma-methylene)diphosphonate all inhibited ADP-induced increases in [Ca2+]i in an apparently competitive manner. The pA2 values obtained for these compounds and for ATP as inhibitors of increases in [Ca2+]i showed a good correlation with those obtained for these compounds as inhibitors of aggregation. Adenosine 5,-(alpha,beta-methylene)triphosphonate and UTP were only weak inhibitors of ADP-induced increases in [Ca2+]i whilst 2-methylthio-ATP non-competitively inhibited the effect of ADP on platelet [Ca2+]i and these results are also consistent with the effects of these compounds on aggregation. This strong correlation between the effects of analogues of adenine nucleotides on platelet aggregation and on increases in [Ca2+]i confirms the causal relationship between these two actions of ADP and shows that they are mediated by the same receptor. The effects of suramin, an antagonist of P2X and P2Y receptors, were investigated on a number of platelet responses. In washed platelets suramin acted as an apparently competitive inhibitor of platelet aggregation causing parallel rightward shifts in the log concentration-response curve with no depression of the maximal response. However, the slope of the Schild plot was around 2 suggesting that the effect of suramin was not simply competitive. The value of suramin was 4.62. Suramin was also able to inhibit non-competitively aggregation induced by U46619 and by 5-hydroxytryptamine (5-HT). Suramin inhibited ADP-induced increases in [Ca2+]i in a similar manner to aggregation giving a Schild plot slope of ~2 and a pA2 value of 4.63. Again suramin was also able to inhibit non-competitively increases in [Ca2+]i induced by 5-HT. Suramin also inhibited the effect of ADP on prostaglandin E1 (PGE1)-stimulated adenylate cyclase, however, in this case the slope of the Schild plot was not significantly different from unity suggesting that the effect was competitive. The pA2 value of suramin was 5.09. Suramin was also able to inhibit the stimulation of adenylate cyclase by PGE1 These results show that suramin is an antagonist of the platelet ADP receptor, as it is of P2X and P2Y receptors, although it also has non-specific inhibitory effects. The effect of varying the extracellular divalent cation concentration was determined on platelet responses to ADP. There was a small but significant leftward shift in the log concentration-response curve to ADP in the presence of EGTA compared to that in the presence of both calcium and magnesium. This leftward shift was no longer significant when the log concentration-response curves were plotted against ADP3- rather than against total ADP concentration. There was also a marked increase in the pA2 value of ATP in the presence of EGTA compared to the presence of calcium and magnesium. However, when the pA2 value was determined for ATP4- there was no longer a significant difference in the pA2 under these different conditions. Similar results were obtained when the inhibition of PGE1-stimulated adenylate cyclase was compared in the presence of calcium and magnesium and in the presence of EGTA. These results suggest that, in common with other P2-purinoceptors, the platelet ADP receptor recognises the uncomplexed forms of adenine nucleotides. In studies on the effect of divalent cation concentration on ADP-induced inhibition of adenylate cyclase, ATP not only inhibited the effect of ADP but also appeared to enhance the accumulation of cAMP induced by PGE1. However, ATP was unable to induce an increase in adenylate cyclase activity in the absence of PGE1 and apyrase also enhanced the accumulation of cAMP induced by PGE1. This suggests that the effect of ATP was in fact due to inhibition of the effect of endogenous ADP released by platelets during preparation and storage rather than a true stimulation of adenylate cyclase. In binding studies, platelets showed both high and low affinity binding sites for [35S]-Sp-ATPaS. There were ~9000 high affinity sites per platelet with a KD value of 0.21 muM and ~24,000 low affinity sites with a KD value of 32 muM. This binding was not displaced by selective ligands at adenosine, alpha2, or 5-HT2 receptors and was not displaced by FSBA but was displaced by a number of competitive ADP receptor antagonists. The low affinity binding was not displaced by suramin. However, the ability of some of these ligands to displace bound ATPaS was not consistent with their effects in functional studies suggesting that neither of these binding sites is the ADP receptor.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biochemistry