Susan C. Fox

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.

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.

DG-041 inhibits the EP3 prostanoid receptor--a new target for inhibition of platelet function in atherothrombotic disease.

Receptors for prostanoids on platelets include the EP3 receptor for which the natural agonist is the inflammatory mediator prostaglandin E2 (PGE2) produced in atherosclerotic plaques. EP3 is implicated in atherothrombosis and an EP3 antagonist might provide atherosclerotic lesion-specific antithrombotic therapy. DG-041 (2,3-dichlorothiophene-5-sulfonic acid, 3-[1-(2,4-dichlorobenzyl)-5-fluoro-3-methyl-1H-indol-7-yl]acryloylamide) is a direct-acting EP3 antagonist currently being evaluated in Phase 2 clinical trials. We have examined the contributions of EP3 to platelet function using the selective EP3 agonist sulprostone and also PGE2, and determined the effects of DG-041 on these. Studies were in human platelet-rich plasma or whole blood and included aggregometry and flow cytometry. Sulprostone enhanced aggregation induced by primary agonists including collagen, TRAP, platelet activating factor, U46619, serotonin and adenosine diphosphate, and enhanced P-selectin expression and platelet–leukocyte conjugate formation. It inhibited adenylate cyclase (measured by vasodilator-stimulated phosphoprotein phosphorylation) and enhanced Ca2+ mobilization. It potentiated platelet function even in the presence of aspirin and/or AR-C69931 (a P2Y12 antagonist). DG-041 antagonized the effects of sulprostone on platelet function. The effect of PGE2 on platelet aggregation depended on the nature of the agonist and the concentration of PGE2 used as a consequence of both pro-aggregatory effects via EP3 and anti-aggregatory effects via other receptors. DG-041 potentiated the protective effects of PGE2 on platelet aggregation by inhibiting the pro-aggregatory effect via EP3 stimulation. DG-041 remained effective in the presence of a P2Y12 antagonist and aspirin. DG-041 warrants continued investigation as a potential agent for the treatment of atherothrombosis without inducing unwanted bleeding risk.

The role of prostanoid receptors in mediating the effects of PGE2 on human platelet function

The effects of prostaglandin E2 (PGE2) on platelet function are believed to be the result of opposing mechanisms that lead to both enhancement and inhibition of platelet function. Enhancement of platelet function is known to be via EP3 receptors linked to Gi and inhibition of adenylyl cyclase. However, the receptors involved in inhibition of platelet function have not been fully defined. Here we have used measurements of platelet aggregation, calcium signaling and P-selectin expression to assess platelet function induced by platelet activating factor (PAF), thrombin receptor activating peptide (TRAP-6) and the thromboxane A2 mimetic U46619 respectively, to determine the effects of PGE2 and of selective prostanoid receptor agonists on platelet function. Their effects on vasodilator-stimulated phosphoprotein (VASP) phosphorylation were also determined. We also assessed the ability of selective prostanoid receptor antagonists to modify the effects of PGE2. The agonists and antagonists used were iloprost (IP agonist), ONO-DI-004 (EP1 agonist), ONO-AE1-259 (EP2 agonist), sulprostone (EP3 agonist), ONO-AE1-329 (EP4 agonist), CAY10441 (IP antagonist), ONO-8713 (EP1 antagonist), DG-041 (EP3 antagonist) and ONO-AE3-208 (EP4 antagonist). Using the agonists available to us we demonstrated that EP3, EP4 and IP receptors elicit functional responses in platelets. The EP3 receptor agonist promoted platelet aggregation, calcium signaling and P-selectin expression and this was associated with a reduction in VASP phosphorylation. Conversely agonists acting at IP and EP4 receptors inhibited platelet function and this was associated with an increase in VASP phosphorylation. The effects on platelet function and VASP phosphorylation of the selective prostanoid receptor antagonists used in conjunction with PGE2 were consistent with PGE2 interacting with EP3 receptors to enhance platelet function and with EP4 receptors (but not IP receptors) to inhibit platelet function. This is the first demonstration of the involvement of EP4 receptors in platelet responses to PGE2.

Enhanced platelet aggregation and activation under conditions of hypothermia

The effects on platelet function of temperatures attained during hypothermia used in cardiac surgery are controversial. Here we have performed studies on platelet aggregation in whole blood and platelet-rich plasma after stimulation with a range of concentrations of ADP, TRAP, U46619 and PAF at both 28 degrees C and 37 degrees C. Spontaneous aggregation was also measured after addition of saline alone. In citrated blood, spontaneous aggregation was markedly enhanced at 28 degrees C compared with 37 degrees C. Aggregation induced by ADP was also enhanced. Similar results were obtained in hirudinised blood. There was no spontaneous aggregation in PRP but ADP-induced aggregation was enhanced at 28 degrees C. The P2Y12 antagonist AR-C69931 inhibited all spontaneous aggregation at 28 degrees C and reduced all ADP-induced aggregation responses to small, reversible responses. Aspirin had no effect. Aggregation was also enhanced at 28 degrees C compared with 37 degrees C with low but not high concentrations of TRAP and U46619. PAF-induced aggregation was maximal at all concentrations when measured at 28 degrees C, but reversal of aggregation was seen at 37 degrees C. Baseline levels of platelet CD62P and CD63 were significantly enhanced at 28 degrees C compared with 37 degrees C. Expression was significantly increased at 28 degrees C after stimulation with ADP, PAF and TRAP but not after stimulation with U46619. Overall, our results demonstrate an enhancement of platelet function at 28 degrees C compared with 37 degrees C, particularly in the presence of ADP.

PGE1 and PGE2 modify platelet function through different prostanoid receptors.

There is evidence that the overall effects of prostaglandin E(2) (PGE(2)) on human platelet function are the consequence of a balance between promotory effects of PGE(2) acting at the EP3 receptor and inhibitory effects acting at the EP4 receptor, with no role for the IP receptor. Another prostaglandin that has been reported to affect platelet function is prostaglandin E(1) (PGE(1)), however the receptors that mediate its actions on platelet function have not been fully defined. Here we have used measurements of platelet aggregation and P-selectin expression induced by the thromboxane A(2) mimetic U46619 to compare the effects of PGE(1) and PGE(2) on platelet function. Their effects on vasodilator-stimulated phosphoprotein (VASP) phosphorylation, as a marker of cAMP, were also determined. We also investigated the ability of the selective prostanoid receptor antagonists CAY10441 (IP antagonist), DG-041 (EP3 antagonist) and ONO-AE3-208 (EP4 antagonist) to modify the effects of the prostaglandins on platelet function. The results obtained confirm that PGE(2) interacts with EP3 and EP4 receptors, but not IP receptors. In contrast PGE(1) interacts with EP3 and IP receptors, but not EP4 receptors. In both cases the overall effects on platelet function reflect the balance between promotory and inhibitory effects at receptors that have opposite effects on adenylate cyclase.

A longitudinal study of platelet behaviour and thromboxane production in whole blood in normal pregnancy and the puerperium

Summary. A longitudinal study of platelet behaviour (platelet aggregation and release reaction) in whole blood and of serum thromboxane B2 production was performed before, during and after normal pregnancy. The response of platelets to arachidonic acid and to adrenaline was significantly increased in the third trimester. Six weeks after delivery, values were still modestly increased but return to non‐pregnant values was complete by 12 weeks. Serum thromboxane B2 production was unchanged throughout pregnancy and the puerperium.

Measurement of platelet P-selectin for remote testing of platelet function during treatment with clopidogrel and/or aspirin.

There is great interest in assessing the efficacy of treatment with clopidogrel and aspirin in patients with cardiovascular disease using procedures that can be used in a remote setting. Here we have established methods to assess the effects of clopidogrel and aspirin on platelets based on measurements of platelet P-selectin. Platelets were stimulated in whole blood by adding the combination of adenosine diphosphate and the TXA2 mimetic U46619 (ADP/U4, designed to assess P2Y12 inhibition) or the combination of arachidonic acid and epinephrine (AA/Epi, designed to assess COX-1 inhibition). The stimulated samples were then fixed using a fixative solution that provides stability for at least 9 days, and sent to a central laboratory for analysis of P-selectin by flow cytometry. Measurements were performed in blood from healthy volunteers and patients with cardiovascular disease. The inhibitory effects of clopidogrel and aspirin were assessed ex vivo and the effects of the direct acting P2Y12 antagonist cangrelor and aspirin were assessed in vitro. Measurements of platelet aggregation were also performed for comparison. In healthy volunteers clopidogrel ex vivo and cangrelor in vitro markedly inhibited P-selectin expression induced by ADP/U4. Aspirin did not inhibit and did not interfere with the effects of clopidogrel or cangrelor using this test. There was very little overlap of results obtained in the absence and presence of clopidogrel or cangrelor. In contrast, over half of 42 patients with cardiovascular disease did not respond well to clopidogrel treatment, although cangrelor was still effective. Aspirin markedly inhibited P-selectin expression induced by AA/Epi. Clopidogrel had much less effect and did not interfere with the effects of aspirin. There was no overlap of results obtained in the absence and presence of aspirin. Aspirin provided near-complete inhibition in 29 of 30 patients with cardiovascular disease. Aggregometry measurements agreed well with the P-selectin data obtained ex vivo following both clopidogrel and aspirin treatment. It is concluded that measurements of P-selectin performed on fixed blood samples following platelet stimulation in whole blood in a remote setting can be used effectively to monitor the effects of clopidogrel and aspirin.

Neonatal platelet reactivity and serum thromboxane B2 production in whole blood: the effect of maternal low dose aspirin.

Objectives Concern has been expressed about possible neonatal side effects after the use of maternal anti‐platelet agents in pregnancy, particularly low dose aspirin treatment. We have studied neonatal platelet behaviour using whole blood techniques, and assessed the neonatal effect of the maternal ingestion of 60 mg aspirin daily.

Platelet reactivity and serum thromboxane B2 production in whole blood in gestational hypertension and pre-eclampsia

Objective— To determine the nature and extent of changes in platelet reactivity in gestational hypertension and pre‐eclampsia (using whole blood techniques which may be more physiological than those previously employed).