S. Heptinstall

The central role of the P2T receptor in amplification of human platelet activation, aggregation, secretion and procoagulant activity

Adenosine diphosphate (ADP) is an important platelet agonist and ADP released from platelet dense granules amplifies responses to other agonists. There are three known subtypes of ADP receptor on platelets: P2X1, P2Y1 and P2T receptors. Sustained ADP‐induced aggregation requires co‐activation of P2Y1 and P2T receptors. AR‐C69931MX, a selective P2T receptor antagonist and novel antithrombotic agent, was studied to characterize further the function of the P2T receptor. The roles of the P2Y1 receptor and thromboxane A2 were assessed using the selective P2Y1 antagonist A2P5P and aspirin respectively. Aggregation was measured by whole blood single‐platelet counting and platelet‐rich plasma turbidimetry, using hirudin anticoagulation. Dense granule release was estimated using [14C]‐5‐hydroxytryptamine (HT)‐labelled platelets. Ca2+ mobilization, P‐selectin expression, Annexin V binding and microparticle formation were determined by flow cytometry. P2T receptor activation amplified ADP‐induced aggregation initiated by the P2Y1 receptor, as well as amplifying aggregation, secretion and procoagulant responses induced by other agonists, including U46619, thrombin receptor‐activating peptide (TRAP) and collagen, independent of thromboxane A2 synthesis, which played a more peripheral role. P2T receptor activation sustained elevated cytosolic Ca2+ induced by other pathways. These studies indicate that the P2T receptor plays a central role in amplifying platelet responses and demonstrate the clinical potential of P2T receptor antagonists.

RANDOMISED DOUBLE-BLIND PLACEBO-CONTROLLED TRIAL OF FEVERFEW IN MIGRAINE PREVENTION

The use of feverfew (Tanacetum parthenium) for migraine prophylaxis was assessed in a randomised, double-blind, placebo-controlled crossover study. After a one-month single-blind placebo run-in, 72 volunteers were randomly allocated to receive either one capsule of dried feverfew leaves a day or matching placebo for four months and then transferred to the other treatment limb for a further four months. Frequency and severity of attacks were determined from diary cards which were issued every two months; efficacy of each treatment was also assessed by visual analogue scores. 60 patients completed the study and full information was available in 59. Treatment with feverfew was associated with a reduction in the mean number and severity of attacks in each two-month period, and in the degree of vomiting; duration of individual attacks was unaltered. Visual analogue scores also indicated a significant improvement with feverfew. There were no serious side-effects.

Differential inhibition by low-dose aspirin of human venous prostacyclin synthesis and platelet thromboxane synthesis.

The capacity of venous tissue for prostacyclin synthesis was determined in 68 patients undergoing surgery for removal of varicose veins. A single dose of aspirin (81 mg or 300 mg) taken 14 h preoperatively strongly inhibited its synthesis, and the effect of 300 mg was still evident 48 h after ingestion. A single dose of 40 mg aspirin taken 14 h preoperatively had no effect on prostacyclin synthesis. The capacity of blood platelets to synthesise thromboxane (measured as malondialdehyde) was determined in volunteers before and at various times after ingestion of 300 mg or 40 mg aspirin. Both doses had an inhibitory effect that lasted for at least 96 h. The length of time for which the amount of thromboxane synthesised was insufficient to support platelet aggregation and the platelet release reaction depended on both the donor and the dose of aspirin. If prostacyclin and thromboxane are important in the pathogenesis of thrombosis, then doses of aspirin much lower than those used previously should be tested. The long-lasting effect of 300 mg aspirin on both venous tissue and platelets indicates that this dose is unlikely to produce a favourable prostacyclin/thromboxane balance.

Comparison of the pharmacodynamic effects of the platelet ADP receptor antagonists clopidogrel and AR-C69931MX in patients with ischaemic heart disease.

We compared the antiplatelet effects of clopidogrel and the intravenous platelet P2Y 12 receptor antagonist AR-C69931MX, which acts on the same receptor as clopidogrel by a different and reversible mechanism and, unlike clopidogrel, is active in vitro . Thirteen patients with acute coronary syndromes entered into a phase II study of intravenous AR-C69931MX (Group 1) and eight patients undergoing intracoronary stent implantation and treated with clopidogrel (Group 2) were studied using a whole blood single-platelet counting aggregation assay. Group 2 patients were also studied using turbidimetry with ADP and TRAP as agonists and whole blood [ 14 C]5HT release to study dense granule secretion in response to ADP, collagen and TRAP. In Group 2 studies, a therapeutic concentration of AR-C69931MX was added in vitro before and after clopidogrel administration. AR-C69931MX in Group 1 achieved greater inhibition of ADP-induced platelet aggregation than clopidogrel in Group 2 and AR-C69931MX in vitro added to the effects of clopidogrel on ADP-induced aggregation. AR-C69931MX but not clopidogrel inhibited TRAP-induced aggregation and granule secretion and AR-C69931MX had a more consistent inhibitory effect on collagen-induced responses. In conclusion, therapeutic administration of clopidogrel moderately inhibits platelet P2Y 12 receptor activation and substantially greater P2Y 12 receptor blockade can be achieved with AR-C69931MX.

EXTRACTS OF FEVERFEW INHIBIT GRANULE SECRETION IN BLOOD PLATELETS AND POLYMORPHONUCLEAR LEUCOCYTES

Extracts of feverfew (Tanacetum parthenium) inhibited secretory activity in blood platelets and polymorphonuclear leucocytes (PMNs). Release of serotonin from platelets induced by various aggregating agents (adenosine diphosphate, adrenaline, sodium arachidonate, collagen, and U46619) was inhibited. Platelet aggregation was consistently inhibited but thromboxane synthesis was not. Feverfew also inhibited release of vitamin B12-binding protein from PMNs induced by the secretagogues formyl-methionyl-leucyl-phenylalanine, sodium arachidonate, and zymosan-activated serum. Feverfew did not inhibit the secretion induced in platelets or PMNs by the calcium ionophore A23187. The pattern of the effects of the feverfew extracts on platelets is different from that obtained with other inhibitors of platelet aggregation and the effect on PMNs is more pronounced than has been obtained with very high concentrations of non-steroidal anti-inflammatory agents.

Effects of P2Y 1 and P2Y 12 receptor antagonists on platelet aggregation induced by different agonists in human whole blood

ADP plays a major role in the amplification of platelet aggregation induced by other platelet agonists. ADP initiates platelet activation via the P2Y 1 receptor and amplifies platelet activation via the P2Y 12 receptor. Using the selective P2Y 1 receptor antagonist A2P5P and the selective P2Y 12 receptor antagonist AR-C69931MX, we assessed the relative contributions of P2Y 1 receptor and P2Y 12 receptor activation to platelet aggregation in hirudin-anticoagulated whole blood induced by PAF, 5HT, epinephrine, TRAP, streptokinase, U46619 and collagen. The effects of aspirin were assessed concurrently. A2P5P and AR-C69931MX variably inhibited aggregation induced by most of the agonists studied, whereas aspirin only inhibited aggregation induced by streptokinase and collagen. In some experiments, A2P5P and AR-C69931MX yielded additive inhibition of aggregation. All three antagonists interacted synergistically to inhibit collagen-induced aggregation. These studies demonstrate that P2Y 1 receptor activation plays a significant role in amplifying aggregation induced by agonists other than ADP, in addition to the established roles of P2Y 12 receptor activation and thromboxane A 2 synthesis.

The reversible P2Y12 antagonist cangrelor influences the ability of the active metabolites of clopidogrel and prasugrel to produce irreversible inhibition of platelet function

Summary.  Background: Agents that act as antagonists at P2Y12 ADP receptors on platelets are in use (clopidogrel), and in development for use (cangrelor and prasugrel), in patients with cardiovascular disease. Cangrelor is a direct‐acting reversible antagonist being developed for short‐term infusion; clopidogrel and prasugrel are oral prodrugs that provide irreversible inhibition via transient formation of active metabolites. At the cessation of cangrelor infusion, patients are likely to receive clopidogrel or prasugrel as a means of maintaining antiplatelet therapy. Objectives: To apply an experimental in vitro approach to investigate the possibility that cangrelor influences the ability of the active metabolites of clopidogrel and prasugrel to inhibit ADP‐mediated platelet function. Methods: The effects of cangrelor and the active metabolites of clopidogrel (C‐AM) and prasugrel (P‐AM) on platelet function were assessed by ADP‐induced platelet P‐selectin expression in whole blood. The method involved rapid removal of the antagonists by dilution, and measurement of residual platelet inhibition. Results: Cangrelor, C‐AM and P‐AM markedly inhibited P‐selectin expression. The effect of cangrelor, but not of C‐AM and P‐AM, was reversible following antagonist removal. Preincubation of blood with cangrelor prior to addition of C‐AM or P‐AM reduced the ability of metabolites to irreversibly antagonize P2Y12. Irreversible inhibition was maintained when blood was preincubated with metabolites prior to cangrelor. Conclusions: Cangrelor influences the ability of the active metabolites of clopidogrel or prasugrel to inhibit platelet function irreversibly. Careful consideration should be given to the timing of administration of an oral P2Y12 antagonist following cangrelor infusion.

Effects of a selective inhibitor of thromboxane synthetase on human blood platelet behaviour.

Abstract The selective thromboxane synthetase inhibitor UK-34787 always inhibited thromboxane and malondialdehyde generation in intact platelets but had diverse effects on platelet behaviour. At concentrations similar to those required to inhibit thromboxane synthesis, UK-34787 inhibited arachidonate-induced aggregation and release reaction in platelet rich plasma from some, but not all, individuals. On this basis, individuals were designated “responders” or “non-responders”. Low concentrations of UK-34787 inhibited platelet responses to collagen but only a high concentration had any effect on platelet responses to adrenaline or adenosine diphosphate.

Cocoa flavanols and platelet and leukocyte function: recent in vitro and ex vivo studies in healthy adults.

There is growing interest in possible beneficial effects of specific dietary components on cardiovascular health. Platelets and leukocytes contribute to arterial thrombosis and to inflammatory processes. Previous studies performed in vitro have demonstrated inhibition of platelet function by (−)-epicatechin and (+)-catechin, flavan-3-ols (flavanols) that are present in several foods including some cocoas. Also, some modest inhibition of platelet function has been observed ex vivo after the consumption of flavanol-containing cocoa products by healthy adults. So far there are no reports of effects of cocoa flavanols on leukocytes. This paper summarizes 2 recent investigations. The first was a study of the effects of cocoa flavanols on platelet and leukocyte function in vitro. The second was a study of the effects of consumption of a flavanol-rich cocoa beverage by healthy adults on platelet and leukocyte function ex vivo. Measurements were made of platelet aggregation, platelet-monocyte conjugate formation (P/M), platelet-neutrophil conjugate formation (P/N), platelet activation (CD62P on monocytes and neutrophils), and leukocyte activation (CD11b on monocytes and neutrophils) in response to collagen and/or arachidonic acid. In the in vitro study several cocoa flavanols and their metabolites were shown to inhibit platelet aggregation, P/M, P/N, and platelet activation. Their effects were similar to those of aspirin and the effects of a cocoa flavanol and aspirin did not seem to be additive. There was also inhibition of monocyte and neutrophil activation by flavanols, but this was not replicated by aspirin. 4′-O-methyl-epicatechin, 1 of the known metabolites of the cocoa flavanol (−)-epicatechin, was consistently effective as an inhibitor of platelet and leukocyte activation. The consumption of a flavanol-rich cocoa beverage also resulted in significant inhibition of platelet aggregation, P/M and P/N, and platelet activation induced by collagen. The inhibitory effects were related to their flavanol content. There was also inhibition of monocyte and neutrophil activation, but here it was concluded that cocoa constituents other than flavanols may contribute to the inhibition that was observed. It can be concluded that cocoa flavanols, their metabolites and possibly other cocoa constituents can modulate the activity of platelets and leukocytes in vitro and ex vivo. The research suggests that the consumption of certain cocoa products may provide a dietary approach to maintaining or improving cardiovascular health.

A Comparison of the Effects of an Extract of Feverfew and Parthenolide, a Component of Feverfew, on Human Platelet Activity In-vitro

Abstract— Extracts of the herb feverfew inhibit human blood platelet aggregation and secretion induced by a number of agents in‐vitro and this may relate to the beneficial effects of feverfew in migraine. We previously identified several compounds with antisecretory activity in human blood platelets using adrenaline as the stimulant. In the present study, we have compared the inhibitory activity of one of these compounds, parthenolide, with that of crude feverfew extract. The effects of both on [14C]5‐HT secretion from platelets and on platelet aggregation induced by a number of different stimulants were determined. The activating agents studied included the phorbol ester PMA, ADP, arachidonic acid, collagen, the thromboxane mimetic U46619, the calcium ionophore A23187, the diacylglycerol analogue OAG and adrenaline. The results show that there are marked similarities between the effects of feverfew extract and of parthenolide on both [14C]5‐HT secretion and platelet aggregation, which is consistent with the effects of feverfew extract on platelets being brought about by parthenolide or similar compounds in the extract. Only in one case, when A23187 was used as the stimulatory agent, was there any discrepancy, which may have been due to materials in the extract other than parthenolide. Both feverfew extract and parthenolide were more effective as inhibitors of the [14C]5‐HT secretion and aggregation induced by some agents and not others, and were most effective as inhibitors of the [14C]5‐HT secretion (but not the aggregation) induced by PMA. This suggests that the effects of feverfew/parthenolide on the protein kinase C pathway warrants further study.