Jackie R. Glenn

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.

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.

Adenosine Derived From ADP Can Contribute to Inhibition of Platelet Aggregation in the Presence of a P2Y12 Antagonist

Objective—To investigate whether adenosine diphosphate (ADP)–derived adenosine might inhibit platelet aggregation, especially in the presence of a P2Y12 antagonist, where the effects of ADP at the P2Y12 receptor would be prevented. Methods and Results—Platelet aggregation was measured in response to thrombin receptor activator peptide by platelet counting in platelet-rich plasma (PRP) and whole blood in the presence of ADP and the P2Y12 antagonists cangrelor, prasugrel active metabolite, and ticagrelor. In the presence of a P2Y12 antagonist, preincubation of PRP with ADP inhibited aggregation; this effect was abolished by adenosine deaminase. No inhibition of aggregation occurred in whole blood except when dipyridamole was added to inhibit adenosine uptake into erythrocytes. The effects of ADP in PRP and whole blood were replicated using adenosine and were directly related to changes in cAMP (assessed by vasodilator-stimulated phosphoprotein phosphorylation). All results were the same irrespective of the P2Y12 antagonist used. Conclusion—ADP inhibits platelet aggregation in the presence of a P2Y12 antagonist through conversion to adenosine. Inhibition occurs in PRP but not in whole blood except when adenosine uptake is inhibited. None of the P2Y12 antagonists studied replicated the effects of dipyridamole in the experiments that were performed.

Mode of action of P2Y12 antagonists as inhibitors of platelet function

P2Y(12) receptor antagonists are antithrombotic agents that inhibit platelet function by blocking the effects of adenosine diphosphate (ADP) at P2Y (12)receptors. However, some P2Y(12) receptor antagonists may affect platelet function through additional mechanisms. It was the objective of this study to investigate the possibility that P2Y(12) antagonists inhibit platelet function through interaction with G-protein-coupled receptors other than P2Y(12) receptors. We compared the effects of cangrelor, ticagrelor and the prasugrel active metabolite on platelet aggregation and on phosphorylation of vasodilator-stimulated phosphoprotein (VASP). We compared their effects with those of selective IP, EP4 and A2A agonists, which act at Gs-coupled receptors. All three P2Y(12) antagonists were strong inhibitors of ADP-induced platelet aggregation but only partial inhibitors of aggregation induced by thrombin receptor activating peptide (TRAP) or the thromboxane A2 mimetic U46619. Further, after removing ADP and its metabolites using apyrase and adenosine deaminase, the P2Y(12) antagonists produced only minor additional inhibition of TRAP or U46619-induced aggregation. Conversely, the Gs-coupled receptor agonists always produced strong inhibition of aggregation irrespective of whether ADP was removed. Other experiments using selective receptor agonists and antagonists provided no evidence of any of the P2Y(12) antagonists acting through PAR1, TP, IP, EP4, A2A or EP3 receptors. All three P2Y (12)antagonists enhanced VASP-phosphorylation to a small and equal extent but the effects were much smaller than those of the IP, EP4 and A2A agonists. The effects of cangrelor, ticagrelor and prasugrel on platelet function are mediated mainly through P2Y(12)receptors and not through another G-protein-coupled receptor.

P2Y12 and EP3 antagonists promote the inhibitory effects of natural modulators of platelet aggregation that act via cAMP

Several antiplatelet drugs that are used or in development as antithrombotic agents, such as antagonists of P2Y12 and EP3 receptors, act as antagonists at Gi-coupled receptors, thus preventing a reduction in intracellular cyclic adenosine monophosphate (cAMP) in platelets. Other antiplatelet agents, including vascular prostaglandins, inhibit platelet function by raising intracellular cAMP. Agents that act as antagonists at Gi-coupled receptors might be expected to promote the inhibitory effects of agents that raise cAMP. Here, we investigate the ability of the P2Y12 antagonists cangrelor, ticagrelor and prasugrel active metabolite (PAM), and the EP3 antagonist DG-041 to promote the inhibitory effects of modulators of platelet aggregation that act via cAMP. Platelet aggregation was measured by platelet counting in whole blood in response to the TXA2 mimetic U46619, thrombin receptor activating peptide and the combination of these. Vasodilator-stimulated phosphoprotein phosphorylation (VASP-P) was measured using a cytometric bead assay. Cangrelor always increased the potency of inhibitory agents that act by raising cAMP (PGI2, iloprost, PGD2, adenosine and forskolin). Ticagrelor and PAM acted similarly to cangrelor. DG-041 increased the potency of PGE1 and PGE2 as inhibitors of aggregation, and cangrelor and DG-041 together had more effect than either agent alone. Cangrelor and DG-041 were able to increase the ability of agents to raise cAMP in platelets as measured by increases in VASP-P. Thus, P2Y12 antagonists and the EP3 antagonist DG-041 are able to promote inhibition of platelet aggregation brought about by natural and other agents that raise intracellular cAMP. This action is likely to contribute to the overall clinical effects of such antagonists after administration to man.

The role of prostanoid receptors in mediating the effects of PGE3 on human platelet function

The role of prostanoid receptors in mediating the effects of PGE3 on human platelet function -

Platelet function reduces significantly during the morning

To the EditorIn a recent Letter-to-the-Editor [1], Wiens et al.described significant reductions in platelet functionbetween 8am and 2pm in a group of 15 younghealthy volunteers. Platelet function was measuredby determining the levels of expression of P-selectin(CD62P), glycoprotein 53 (CD63) and activatedglycoprotein IIb/IIIa (PAC-1 binding) on plateletsusing flow cytometry. All three measures decreasedboth on unstimulated platelets and on plateletsfollowing stimulation with ADP (4mM). Similarresults were obtained during fasting and afterconsumption of a high fat meal. Reductions inplatelet function were accompanied by a smallreduction in platelet count.Here we report the results of our own studies inwhich we measured platelet function at differenttimes of the day. The first study was performed onblood samples from a group of older people (n¼89;32 men, 57 women; mean age¼53, range 45 to 70).Measurements were performed first at 8am after anovernight fast, and again at 11am and 1pm.Measurements were made of ADP (3mM) andcollagen (1mg/ml)-induced platelet aggregation incitrated platelet-rich plasma using light absorbanceaggregometry following platelet count adjustment to250000/ml. A PAP-4 Aggregometer (from Bio/data,Hatboro, USA; operating at 37 C) was used andextreme care was taken to ensure the proceduresused at the different times of day were identical andthat fresh preparations of the stimulants were usedeach time. Results for ADP and collagen wereexpressed as maximum aggregation (%) and lagtime (seconds) respectively. Results obtained at thedifferent timepoints were compared using a pairedt-test (two-tailed).Platelet aggregation to ADP and to collagenchanged significantly in the direction of reducedplatelet aggregation between 8am and 11am(Table I, Figure 1ab) with no further change between11am and 1pm (Table I). The maximum extent ofaggregation induced by ADP was increased in theearly morning, and the lag time leading to collagen-induced aggregation was shorter. It should be noted,however, that there were greater differences in extentof platelet aggregation between different people thanbetween measurements performed on two bloodsamples from the same person in the early and latemorning. We did not detect any change in blood cellcounts including platelet counts over the three bloodsamples that were taken (results not shown).The second study also involved healthy volunteers,this time a group of people (n¼32; 20 men, 12women; mean age¼37, range 18 to 63). Bloodsamples were taken at 8am after an overnight fast,and again at 12pm. Here we measured P-selectin onunstimulated platelets and on platelets stimulatedwith ADP (10mM). The assay used was a standar-dized procedure that we have developed specificallyto provide quantitative data that is both accurate andreproducible and that can be used routinely as ameasure of platelet activation. P-selectin was mea-sured by flow cytometry using antibodies to CD62P-FITC and results were expressed as median fluores-cence (mf, arbitrary units). The results obtained atdifferent times of day were compared using a pairedt-test (two-tailed).As can be seen in Table II and Figure 1(c) and (d),the levels of P-selectin expressed on platelets wereindeed highly reproducible in blood taken from thesame people at different times of day, both inunstimulated platelets and in platelets stimulatedwith ADP. Nevertheless, in both cases, there was a

Raised levels of CD39 in leucocytosis result in marked inhibition of ADP-induced platelet aggregation via rapid ADP hydrolysis

We observed previously that the extent of ADP-induced platelet aggregation in blood from patients with leucocytosis is markedly reduced. We obtained evidence that this is via enhanced ADP metabolism consequent to the high leucocyte count, and speculated that ecto-NTPDase CD39 on leucocytes may be involved. Here we have investigated the association between ADP-induced platelet aggregation, ADP metabolism and expression of ecto-NTPDase CD39 on leucocytes in patients with leucocytosis. Six patients with leucocytosis were compared with six normal controls. Platelet aggregation was measured using platelet counting. ADP metabolism was analysed by HPLC. CD39 on leucocytes from each volunteer and patient was measured by flow cytometry and is presented as the CD39 fluorescence index (CD39FI, the sum of the product of CD39 median fluorescence and cell number for each leucocyte subtype). Compared with the controls, all patients displayed markedly reduced platelet aggregation to ADP in whole blood, markedly enhanced metabolism of ADP to AMP in whole blood, and increased leucocyte CD39FI. The increased CD39FI was due to either a high number of CD39+ve lymphocytes or a high number of CD39+ve neutrophils. In contrast, the measures of aggregation and ADP metabolism performed in platelet-rich plasma from the patients were similar to those obtained for the controls. There was an inverse correlation between ADP-induced aggregation in whole blood and CD39FI, and between the time taken to achieve complete removal of ADP from blood and CD39FI. For two patients with very high CD39FI (60,000 cf 1500 for controls) ADP-induced aggregation was abolished. Reduced aggregation, enhanced ADP metabolism and a raised CD39FI returned to normal in one patient following successful chemotherapy. It is concluded that ADP-induced platelet aggregation in leucocytosis is reduced as a result of enhanced ADP metabolism due to raised levels of leucocyte-associated CD39.

How does measurement of platelet P-selectin compare with other methods of measuring platelet function as a means of determining the effectiveness of antiplatelet therapy?

Abstract Measurement of P-selectin on activated platelets as a means of measuring platelet function utilizing the technology described here has the advantage of not requiring immediate access to specialist equipment and expertise. Blood samples are activated, fixed, stored, and transported to a central laboratory for flow cytometric analysis. Here we have compared P-selectin with other more traditional approaches to measuring platelet function in blood and/or platelet-rich plasma (PRP) from patients with acute coronary syndromes on treatment for at least 1 month with either aspirin and clopidogrel or aspirin with prasugrel. The comparators were light transmission aggregometry (LTA), VerifyNow and Multiplate aggregometry (for determining the effects of aspirin) and LTA, VerifyNow and Multiplate together with the BioCytex VASP phosphorylation assay (for the P2Y12 antagonists). The P-selectin Aspirin Test revealed substantial inhibition of platelet function in all but three of 96 patients receiving aspirin with clopidogrel and in none of 51 patients receiving aspirin and prasugrel. The results were very similar to those obtained using LTA. There was only one patient with high residual platelet aggregation and low P-selectin expression. The same patients identified as “non-responders” to aspirin also presented with the highest residual platelet activity as measured using the VerifyNow system, although not quite as well separated from the other values. With the Multiplate test only one of these patients clearly stood out from the others. The results obtained using the P-selectin P2Y12 Test in 102 patients taking aspirin and clopidogrel were similar to the more traditional approaches in that a wide scatter of results was obtained. Generally, high values seen with the P-selectin P2Y12 Test were also high with the LTA, VerifyNow, Multiplate, and BioCytex VASP P2Y12 Tests. Similarly, low residual platelet function using the P2Y12 test was seen irrespective of the testing procedure used. However, there were differences in some patients. Prasugrel was always more effective than clopidogrel in inhibiting platelet function with none of 56 patients (P-selectin and VerifyNow), only 2 of 56 patients (Multiplate) and only 3 of 56 patients (Biocytex VASP) demonstrating high on-treatment residual platelet reactivity (HRPR) defined using previously published cut-off values. The exception was LTA where there were 11 of 56 patients with HRPR. It remains to be seen which experimental approach provides the most useful information regarding outcomes after adjusting therapies in treated patients.