Stanley Heptinstall

Inhibition of ADP-induced intracellular Ca2+ responses and platelet aggregation by the P2Y12 receptor antagonists AR-C69931MX and clopidogrel is enhanced by prostaglandin E1.

P2Y(12) antagonists such as clopidogrel and AR-C69931MX inhibit aggregation by antagonizing the effects of ADP at P2Y(12) receptors on platelets. Agents such as PGE(1) also inhibit aggregation by stimulating adenylate cyclase to produce cAMP, which interferes with Ca(2+) mobilization within the cell. Since one facet of P2Y(12) receptors is that they mediate inhibition of adenylate cyclase by ADP, it might be expected that P2Y(12) antagonists would interact with PGE(1). We have explored the effects of PGE(1) and AR-C69931MX singly and in combination on ADP-induced intracellular Ca(2+) ([Ca(2+)](i)) responses and aggregation. PGE(1) alone caused parallel dose-dependent inhibition of [Ca(2+)](i) and aggregation responses. AR-C66931MX alone caused only partial inhibition of [Ca(2+)](i) despite a marked inhibitory effect on aggregation. Combinations of PGE(1) with AR-C66931MX were found to act in synergy to reduce both [Ca(2+)](i) and aggregation. This effect was confirmed in patients with acute coronary syndromes by studying the inhibitory effects of PGE(1) on [Ca(2+)](i) and aggregation before and after clopidogrel. In summary, we have shown that P2Y(12) antagonists interact with natural agents such as PGE(1) to provide more effective inhibition of [Ca(2+)](i) and platelet aggregation. This would contribute to the effectiveness of P2Y(12) antagonists as antithrombotic agents in man.

Adenosine Diphosphate Induced Platelet Aggregation and Release Reaction in Heparinized Platelet Rich Plasma and the Influence of Added Citrate

Summary. Platelets in heparinized platelet‐rich plasma (PRP) from 54 volunteers were examined for their ability to aggregate and to release tritiated 5‐hydroxy tryptamine (3H‐5HT) in response to adenosine diphosphate (ADP). Release of 3H‐5HT was considerable in 26 of the preparations. When release was high, lower concentrations of ADP were required to induce irreversible aggregation. Second phase aggregation reflected extensive release reaction. Citrate always augmented the release induced by ADP.

Leukocyte count and leukocyte ecto-nucleotidase are major determinants of the effects of adenosine triphosphate and adenosine diphosphate on platelet aggregation in human blood

ADP induces platelet aggregation in human whole blood and platelet-rich plasma (PRP). ATP induces aggregation in whole blood only; this involves leukocytes and is mediated by ADP. Here we studied ATP- and ADP-induced aggregation in patients with raised leukocyte counts (mean 46.2 × 103 leukocytes/µl). Platelet aggregation was measured by platelet counting. ATP, ADP and metabolites were measured by HPLC. Aggregation to ADP (1–10 µM) and ATP (10–100 µM) was markedly reduced, but to ATP (1000 µM) was enhanced (all p < 0.001). Aggregation to ADP in PRP was normal. Increasing the leukocyte count in normal blood reproduced the findings in the patients. Adding leukocytes (either MNLs or PMNLs) to normal PRP enabled a response to ATP and caused marked inhibition of ADP-induced aggregation. Breakdown of ATP or ADP to AMP and adenosine in leukocyte-rich plasma was rapid (t1/2 = 4 min) and far higher than in cell-free plasma or PRP. With ATP there was also formation of ADP, maximal at 4 min. The presence of the ectonucleotidase NTPDase1 (CD39) was demonstrated on MNLs (all of the monocytes and a proportion of the lymphocytes) and all PMNLs by flow cytometry. We conclude that leukocytes provide a means of dephosphorylating ATP which enables ATP-induced aggregation via conversion to ADP, but also convert ADP to AMP and adenosine. Platelet aggregation extent is a balance between these activities, and high white cell counts influence this balance.

Detection of P2Y14 protein in platelets and investigation of the role of P2Y14 in platelet function in comparison with the EP3 receptor

mRNA encoding the recently discovered P2Y14 receptor has been reported in platelets, but the presence of P2Y14 receptor protein and its functionality have not been studied.If P2Y14 is expressed along with P2Y1 and P2Y12 receptors it may have a role in haemostasis. It was the objective of this study to investigate the presence of the P2Y14 receptor in platelets and its role in platelet function.The effects of the agonist UDP-glucose were compared with those of sulprostone, a selective EP3 receptor agonist. Expression of P2Y14 receptor was investigated by immunoblotting and confocal microscopy. Platelet aggregation in platelet-rich plasma (PRP) and whole blood was measured using light absorbance and platelet counting. VASP phosphorylation was investigated using flow cytometry. Immunoblotting provided evidence for P2Y14 receptor protein and microscopy confirmed its presence on platelets. Despite this, UDP-glucose (up to 100 μM) did not induce platelet aggregation in either PRP or whole blood, and did not potentiate aggregation induced by other agonists.P2Y14 did not substitute for P2Y12 in experiments using the P2Y12 antagonist AR-C69931. No effect of UDP-glucose was seen on adenylate cyclase activity as measured byVASP phosphorylation. In contrast, sulprostone acting via the EP3 receptor promoted platelet aggregation with effects on adenylate cyclase activity. EP3 also partially substituted for P2Y12 receptor. We have demonstrated the presence of P2Y14 receptor protein in platelets, but no contribution of this receptor to several measures of platelet function has been observed. Further studies are necessary to determine whether the P2Y14 receptor in platelets has any functionality.

Acyl derivatives of coenzyme A inhibit platelet function via antagonism at P2Y1 and P2Y12 receptors: A new finding that may influence the design of anti-thrombotic agents

We have performed a detailed investigation of the effects on platelet function of coenzyme A (CoA) and several acyl-CoAs. Platelet aggregation was measured by turbidimetry and by platelet counting; platelet shape change was measured using light scattering; P-selectin, Ca2+ mobilization and vasodilator-stimulated phosphoprotein (VASP) phosphorylation were measured by flow cytometry. The compounds investigated inhibited ADP-induced platelet aggregation; those with saturated acyl groups containing 16-18 carbons were most effective. The effects of palmitoyl-CoA (16:0) were studied in depth. It inhibited platelet shape change and Ca2+ mobilization brought about by ADP (but not other agonists) indicating antagonism at P2Y1 receptors, and also inhibited ADP-induced P-selectin expression. Effects of palmitoyl-CoA on the platelet aggregation and Ca2+ mobilization induced by several different agonists and agonist combinations were compared with those of MRS 2179 (a P2Y1 antagonist) and AR-C69931 (a P2Y12 antagonist), and were consistent with palmitoyl-CoA acting mainly at P2Y1 but also with partial antagonism at P2Y12 receptors. Antagonism at P2Y12 receptors was confirmed in studies of VASP-phosphorylation. Palmitoyl-CoA did not act as an antagonist at P2X1 receptors. The results are discussed in relation to the possibility that acyl-CoAs may contribute as endogenous modulators of platelet function and might serve as lead compounds for the design of novel antithrombotics.